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This file is part of the following work:

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.

Access to this file is available from:

https://doi.org/10.4225/28/5acd39d9bf34e

Copyright © 2017 Tiange Liu.

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Development of a Mouse Model to Evaluate the Degree of Immunological Cross-reactivity between Prawns and Abalone

Thesis submitted by

Tiange LIU

B.Sc.(Agr.)

For the degree of Doctor of Philosophy

in the College of Public Health, Medical and Veterinary Sciences

James Cook University

Townsville, Australia

March 2017

Statement of contribution

I

STATEMENT ON THE CONTRIBUTION OF OTHERS

Nature of Assistance

Contribution Name and Affiliation

Intellectual support

Project plan and

development

• Prof. Andreas Lopata, James Cook University

• Dr. Severine Navarro, James Cook University

• Dr. Paul Giacomin, James Cook University

Editorial support • Prof. Andreas Lopata, James Cook University

• Dr. Severine Navarro, James Cook University

Grant proposal

writing • Prof. Andreas Lopata, James Cook University

Financial support

Research • ARC Future Fellowship for Prof. Andreas Lopata

• The Australian Institute of Tropical Health and Medicine research grant

Conference travel

• The Australian Institute of Tropical Health and Medicine

• Women Scientists Travel Award, Beijing Genomics Institute (BGI)

• Grant-in aid conference funding, College of Public Health, Medical and Veterinary

Stipend

• China Scholarship Council (CSC) PhD Scholarship

• James Cook University Postgraduate Scholarship

• College Scholarship, College of Public Health, Medical and Veterinary Sciences, James Cook

University

Write-up support • Doctoral Completion Grant, College of Public

Health, Medical and Veterinary Sciences, James

Cook University

Data collection

Human blood

sample collection

• Prof. Jennifer Rolland, Monash University

• Prof. Robyn O’Hehir, Monash University and The Alfred Hospital

Technical support

Recombinant

protein assay • Dr. Sandip Kamath, James Cook University

• Dr. Shruti Saptarshi, James Cook University

Mouse sample

analysis • Dr. Severine Navarro, James Cook University

• Dr. Paul Giacomin, James Cook University

Mass spectrometry • Mr. Roni Nugraha, James Cook University

Statement of contribution

II

CONTRIBUTION OF OTHERS IN CHAPTERS

Chapter 1. Introduction

T. Liu, S. Navarro, A.L. Lopata, Current advances of murine models for food allergy,

Molecular immunology, 70 (2016) 104-117.

Prof. Andreas L. Lopata

(AL), James Cook University

Dr. Severine Navarro (SN),


TL and AL co-developed the aim of the review article. The

figures and tables were generated by TL with editorial inputs

from AL and SN.

Chapter 2. Establishment of a mouse model for prawn allergy



Dr. Severine Navarro (SN),


TL, SN and AL co-developed the research question. TL

performed the experimental work and data analysis. SN

provided assistance with mouse sample evaluation. The

figures and tables were generated by TL with the technical

assistant from SN and editorial input from AL.

Chapter 3. Evaluation of the potential immunological and clinical cross-reactivity between the whole heated extracts of Black tiger prawn (Penaeus monodon) and Jade tiger abalone (Haliotis laevigata × Haliotis rubra)



Dr. Paul Giacomin (PG),


TL, PG and AL co-developed the research question. TL

performed the experimental work and data analysis. PG

provided assistance with mouse sample evaluation. The

figures and tables were generated by TL with the editorial

input from AL.

Chapter 4. The application of recombinant tropomyosins from Black tiger prawn (Penaeus monodon) and Jade tiger abalone (Haliotis laevigata × Haliotis rubra) in the development of mouse models for shellfish allergy



Dr. Sandip Kamath (SK),


TL and AL co-developed the research question. TL performed

the experimental work and data analysis. SK provided

assistance with the immunoblots. The figures and tables were

generated by TL with editorial input from AL.

Chapter 5. Evaluation of the potential immunological and clinical cross-reactivity between tropomyosin from Black tiger prawn (Penaeus monodon) and Jade tiger abalone (Haliotis laevigata × Haliotis rubra)



TL and AL co-developed the research question. TL performed

the experimental work and data analysis. The figures and

tables were generated by TL with the editorial input from AL.

Acknowledgements

III

ACKNOWLEDGEMENTS

Years ago, achieving a PhD degree overseas was a seemingly unreachable dream for me.

I could never have imagined that CSC could provide me such a valuable opportunity to

study in Australia. This unique experience has changed and reshaped me. I wish to

express my heartfelt gratitude to all the amazing people who accompanied me on my

journey.

Firstly, I would like to thank my primary supervisor Prof. Andreas Lopata. Without his

unwavering support and advice, I would not have been able to complete this PhD and this

thesis would never had been written. I thank him for giving me enormous support at every

essential stage of this PhD. His guidance has had a profound effect on my growth of a

scientist and person. I also would like to thank my co-supervisor Dr. Severine Navarro.

Her extensive knowledge and rigorous scientific attitude is impressive. Receiving her

supervision at Cairns AITHM was a very lucky turning point of my PhD. Another co-

supervisor, Dr. Sandip Kamath, has become a role model for me as a PhD student. I

admire him for his diligence, modesty and devotion, and appreciate his help with many

aspects of my PhD studies, especially the learning of molecular techniques.

I am also particularly grateful to four people for their timely and selfless help in my

darkest days. Prof. James Burnell is a super-warmhearted and wise mentor. With his

guidance, I started my first real independent experiment with confidence and passion.

Even after he retired, his support is still with me. I will always remember his clear glance

and his firm tone when I was having difficulties: “keep the faith, and I believe you can

Acknowledgements

IV

make it”. Dr. Paul Giacomin offered me extensive technical and intelligent support in

my mouse experiments. I truly thank him for being there for me during this PhD’s

rollercoaster-ride. I greatly appreciate the advice from my research student monitor

A/Prof. Jeffrey Warner while completing my PhD studies. I feel so blessed because of

his understanding, patience and wisdom. I also need to thank A/Prof. Xiaoqin Sun from

Northwest Agriculture & Forestry University for using her personal experiences to paint

the picture of what success for me might look like.

I am very fortunate to be a part of the Molecular Immunology Group, and thank every

member for their guidance, supports and friendship during all these years: Mr. Roni

Nugraha, Dr. Aya Taki, Dr. Martina Koeberl, Dr. Shanshan Sun, Mr. Thimo Ruether,

Mr. Juan Stephen and Dr. Elecia Johnston. Also, I would like to thank everyone in the

AITHM Loukas team, especially Prof. Alex Loukas and Ms. Mel Campbell, who made

me feel welcome and supported when I resettled in a different working environment. I

want to give many hugs to Ms. Geraldine Buitrago, Ms. Linda Jones, Mr. Luke Becker,

Mr. Rafid Al-Hallaf, Ms. Stephanie Ryan, Ms. Sally Troy and Mr. Atik Susianto for

their willingness to lend me a hand on my busiest days in the laboratory.

I would like to express my sincere gratitude to the academic and administrative staff in

the College of Public Health, Medical and Veterinary Sciences: Prof. Alan Baxter, Dr.

Margaret Jordan, Mr. Benjamin Crowley, Ms. Tina Cornell, Ms. Candi Lee, Mr.

David Jusseaume, Dr. Ray Layton, Ms. Melissa Hines and Mrs. Maree Hines. Every

success of my work has been based on their immense assistance. Also, I would like to

sincerely acknowledge the CSC, GRS, college and AITHM for their administrative and

Acknowledgements

V

financial support.

Finally, and most importantly, I am so thankful for every second of life because of my

loving family. My parents are my best friends. Their unconditional love makes me

courageous to fly thousands of kilometres away alone for a new life but I have never felt

I am on my own. Many special thanks to my boyfriend, Liqiang, who as a doctor of

engineering knows the allergy mouse models even more than I do now. Thanks for being

so caring, considerate, good-natured, smart, and coming all the way to Australia for me.

He makes me the luckiest but also a better person who is happy to strive to deserve him.

Acknowledgements

VI

This thesis is dedicated to my beloved parents, younger brother,

grandmother, and Liqiang.

Thesis abstract

VII

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

Thesis abstract

VIII

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

Thesis abstract

IX

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-

Thesis abstract

X

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.

Table of contents

XI

TABLE OF CONTENTS

STATEMENT ON THE CONTRIBUTION OF OTHERS ......................................... I

CONTRIBUTION OF OTHERS IN CHAPTERS ..................................................... II

ACKNOWLEDGEMENTS ........................................................................................ III

ABSTRACT ................................................................................................................. VII

LIST OF FIGURES ................................................................................................. XVII

LIST OF TABLES .................................................................................................... XXII

LIST OF ABBREVIATIONS ................................................................................. XXIV

CHAPTER 1: INTRODUCTION .................................................................................. 1

1.1 Allergy and allergen .................................................................................................. 2

1.2 Food allergy ............................................................................................................... 5

1.3 Cross-reactivity in food allergy ................................................................................ 5

1.3.1 Characteristics of cross-reactive allergens ........................................................... 7

1.3.2 Cross-reactive allergens in shellfish ..................................................................... 8

1.4 Mouse models for food allergies ............................................................................ 19

1.4.1 Mouse strain ....................................................................................................... 25

1.4.2 Mouse gender ..................................................................................................... 28

1.4.3 Route of sensitisation ......................................................................................... 31

1.4.4 The food allergen .................................................................................................. 7

1.4.4.1 Milk allergens in mouse studies ................................................................... 41

1.4.4.2 Peanut allergens in mouse studies ................................................................ 43

1.4.4.3 Shellfish allergens in mouse studies ............................................................ 45

1.4.5 Advances of food allergy mechanisms in mouse studies ................................... 47

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XII

1.4.6 Conclusion .......................................................................................................... 49

1.5 Summary and research synopsis of the thesis ...................................................... 50

1.6 References ................................................................................................................ 37

CHAPTER 2: ESTABLISHMENT OF A MOUSE MODEL FOR PRAWN

ALLERGY ..................................................................................................................... 79

2.1 Introduction ............................................................................................................. 80

2.2 Aims .......................................................................................................................... 83

2.3 Animals, materials, and methods ........................................................................... 84

2.3.1 Animals ............................................................................................................... 84

2.3.2 Preparation of shellfish extracts ......................................................................... 84

2.3.2.1 Preparation of heated Black tiger prawn extracts ........................................ 84

2.3.2.2 Protein concentration estimate ..................................................................... 85

2.3.3 Establishment and evaluation of mouse models in shellfish allergies ............... 85

2.3.3.1 Mice sensitisation and challenge with allergens .......................................... 85

2.3.3.2 Measurement of antibodies and mast cell protease-1 in serum ................... 86

2.3.3.3 Cell stimulation with allergens .................................................................... 89

2.3.3.4 Flow cytometry ............................................................................................ 91

2.3.3.5 Preparation of jejunum protein extracts ....................................................... 92

2.3.3.6 Measurement of cytokines ........................................................................... 93

2.3.3.7 Histology ...................................................................................................... 95

2.3.3.8 Measurement of allergen-specific IgA antibody .......................................... 95

2.3.3.9 Statistical analysis ........................................................................................ 96

2.4 Results ...................................................................................................................... 97

2.4.1 Experiment #1 .................................................................................................... 97

2.4.1.1 Experimental design ..................................................................................... 97

2.4.1.2 Induction of antibody and mast cell protease-1 in sera ................................ 99

2.4.1.3 Production of cytokines by splenocytes in vitro ........................................ 101

2.4.1.4 Production of cytokines in jejunums .......................................................... 104

2.4.2 Experiment #2 .................................................................................................. 107

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XIII

2.4.2.1 Experimental design ................................................................................... 107

2.4.2.2 Induction of serum antibody responses ...................................................... 109

2.4.2.3 Eosinophil and basophil responses in blood .............................................. 112

2.4.2.4 Secretion of allergen-specific IgA in jejunum ........................................... 116

2.4.3 Experiment #3 .................................................................................................. 118

2.4.3.1 Experimental design ................................................................................... 118

2.4.3.2 Serum antibodies and mast cell protease-1 ................................................ 120

2.4.3.3 Production of cytokines by spleens and mesenteric lymph nodes ............. 122

2.4.3.4 Induction of eosinophilia in spleens .......................................................... 124

2.4.3.5 Jejunum histology ...................................................................................... 125

2.5 Discussion .............................................................................................................. 127

2.6 References .............................................................................................................. 131

CHAPTER : EVALUATION OF THE POTENTIAL IMMUNOLOGICAL

CROSS-REACTIVITY BETWEEN THE WHOLE HEATED EXTRACTS OF

BLACK TIGER PRAWN (Penaeus monodon) AND JADE TIGER ABALONE

(Haliotis laevigata × Haliotis rubra) ........................................................................... 135

3.1 Introduction ........................................................................................................... 136

3.2 Aims ........................................................................................................................ 138

3.3 Animals, materials, and methods ......................................................................... 139

3.3.1 Animals ............................................................................................................. 139

3.3.2 Preparation of heated extracts of prawns, abalone and cow’s milk .................. 139

3.3.3 Induction and evaluation of mouse models for shellfish allergies ................... 139

3.3.3.1 Mice sensitisation and challenge ............................................................... 139

3.3.3.2 Measurement of antibodies and mast cell protease-1 in serum ................. 142

3.3.3.3 In vitro stimulation of splenocytes with shellfish extracts ......................... 142

3.3.3.4 Flow cytometry .......................................................................................... 143

3.3.3.5 Measurement of cytokines ......................................................................... 143

3.3.3.6 Measurement of allergen-specific IgA antibody ........................................ 143

Table of contents

XIV

3.3.3.7 Statistical analysis ...................................................................................... 143

3.3.3.8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis .................... 144

3.3.3.9 Mouse immunoblotting .............................................................................. 144

3.3.3.10 Mass spectrometric identification of mouse IgG reactive proteins ......... 145

3.4 Results .................................................................................................................... 147

3.4.1 Induction of serum antibodies .......................................................................... 147

3.4.2 Protein separation and identification of antibody binding proteins ................. 152

3.4.3 Production of cytokines by stimulated splenocytes ......................................... 155

3.4.4 Induction of eosinophil and basophil in spleen and blood ............................... 158

3.4.5 Secretion of IgA antibody in jejunums ............................................................. 161

3.5 Discussion .............................................................................................................. 163

3.6 References .............................................................................................................. 167

CHAPTER : THE APPLICATION OF RECOMBINANT TROPOMYOSINS

FROM BLACK TIGER PRAWN (Penaeus monodon) AND JADE TIGER

ABALONE (Haliotis laevigata × Haliotis rubra) IN THE DEVELOPMENT OF

MOUSE MODELS FOR SHELLFISH ALLERGY ................................................ 172

4.1 Introduction ........................................................................................................... 173

4.2 Aims ........................................................................................................................ 176

4.3 Animals, materials and methods .......................................................................... 177

4.3.1 Cloning and cDNA sequencing of tropomyosin from Jade tiger abalone ........ 177

4.3.1.1 Total RNA isolation ................................................................................... 177

4.3.1.2 cDNA transcription and PCR amplification of tropomyosin ..................... 177

4.3.1.3 Cloning of the coding region of tropomyosin into pProEX HTb vector ... 178

4.3.1.4 Transformation of DH12S competent cells and sequencing of the plasmid DNA ....................................................................................................................... 180

4.3.1.5 Transformation of BL21 competent cells .................................................. 180

4.3.2 Expression and purification of recombinant tropomyosins .............................. 181

4.3.2.1 Culturing of BL21 Escherichia coli cells and auto-induction of the recombinant protein expression ............................................................................. 181

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XV

4.3.2.2 Purification of recombinant tropomyosins of Jade tiger abalone and Black tiger prawn ............................................................................................................. 182

4.3.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ..... 182

4.3.4 Patient sera ....................................................................................................... 183

4.3.5 Patient immunoblotting .................................................................................... 183

4.3.6 Mouse immunoblotting .................................................................................... 184

4.3.7 Mass spectrometric identification .................................................................... 184

4.3.8 Amino acid sequence alignment of prawn and abalone tropomyosins ............ 184

4.3.9 Animals ............................................................................................................. 186

4.3.10 Preparation of shellfish extracts and recombinant tropomyosins ................... 186

4.3.11 Induction and evaluation of mouse models in shellfish allergies ................... 186

4.3.11.1 Mouse sensitisation and challenge ........................................................... 186

4.3.11.2 Detection of serum antibodies and mast cell protease-1 .......................... 188

4.3.11.3 In vitro stimulation of splenocytes and measurement of cytokines ......... 188

4.3.11.4 Flow cytometry ........................................................................................ 188

4.3.11.5 Statistical analysis .................................................................................... 188

4.4 Results .................................................................................................................... 189

4.4.1 Jade tiger abalone and Black tiger prawn tropomyosin sequence comparison 189

4.4.2 Identification of human IgE reactivity to abalone and prawn tropomyosin ..... 191

4.4.3 Serum antibodies and mast cell protease-1 ...................................................... 195

4.4.4 Identification of mouse IgG binding proteins .................................................. 198

4.4.5 Production of cytokines by stimulated and unstimulated splenocytes ............. 200

4.4.6 Secretion of IgA antibody in mouse jejunum ................................................... 203

4.4.7 Induction of eosinophil and basophil in circulating blood and spleen ............. 204

4.5 Discussion .............................................................................................................. 206

4.6 References .............................................................................................................. 209

CHAPTER 5: EVALUATION OF THE POTENTIAL IMMUNOLOGICAL

CROSS-REACTIVITY BETWEEN TROPOMYOSIN FROM BLACK TIGER

PRAWN (Penaeus monodon) AND JADE TIGER ABALONE (Haliotis laevigata ×

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XVI

Haliotis rubra) .............................................................................................................. 215

5.1 Introduction ........................................................................................................... 216

5.2 Aims ........................................................................................................................ 218

5.3 Animals, materials, and methods ......................................................................... 219

5.3.1 Animals ............................................................................................................. 219

5.3.2 Preparation of shellfish extracts and recombinant tropomyosins ..................... 219

5.3.3 Induction and evaluation of mouse models in shellfish allergies ..................... 219

5.3.3.1 Mice sensitisation and challenge ............................................................... 219

5.3.3.2 Detection of serum specific antibody and mast cell protease-1 ................. 220

5.3.3.3 In vitro re-stimulation of splenocyte and quantification of cytokines ....... 221

5.3.3.4 Flow cytometry .......................................................................................... 222

5.3.3.5 Measurement of intestinal specific IgA antibody ...................................... 222

5.3.3.6 Statistical analysis ...................................................................................... 223

5.4 Results .................................................................................................................... 224

5.4.1 Antibody response and mMCP-1 levels in serum ............................................ 224

5.4.2 Release of cytokines by allergen restimulated splenocytes in vitro ................. 227

5.4.3 Induction of eosinophil and basophil in circulating blood and spleen ............. 230

5.4.4 Secretion of mucosal IgA antibody in jejunum ................................................ 231

5.5 Discussion .............................................................................................................. 233

5.6 References .............................................................................................................. 237

CHAPTER 6: GENERAL DISCUSSION AND FUTURE DIRECTIONS ........... 241

APPENDIX .................................................................................................................. 248

List of figures

XVII

LIST OF FIGURES

Figure 1.1 Humoral and cellular mechanisms of IgE-mediated allergic inflammation

against food allergens .................................................................................................... 6

Figure 1.2 The proportional prevalence of self-reported food allergen sources of

children and adults in the United States ...................................................................... 38

Figure 2.1 Protocol of inducing shellfish allergy in a mouse model ........................... 98

Figure 2.2 Levels of serum antibodies including hBTP-specific IgE (A), IgG1 (B) and

IgG2a (C), and mMCP-1 (D) ...................................................................................... 99

Figure 2.3 Production of cytokines by unstimulated splenocytes: IL-4 (A), IL-5 (B),

IL-6 (C), IL-9 (D), IL-10 (E), IL-13 (F), IL-17A (G), IL-23 (H), mMCP-1 (I) and

IFN-gamma (J) .......................................................................................................... 101

Figure 2.4 Production of cytokines by hBTP-stimulated splenocytes: IL-4 (A), IL-5

(B), IL-6 (C), IL-9 (D), IL-10 (E), IL-13 (F), IL-17A (G), IL-23 (H), mMCP-1 (I) and

IFN-gamma (J) .......................................................................................................... 103

Figure 2.5 Production of cytokines in homogenised jejunum: IL-4 (A), IL-5 (B), IL-

10 (C), IL-17A (D), IL-23 (E), mMCP-1 (F) and IFN-gamma (G) .......................... 105

Figure. 2.6 Optimised sensitisation and oral challenge protocol, on the basis of

Experiment #1, to establish the mouse model for shellfish allergy ........................... 108

Figure. 2.7 Levels of total serum IgE on Day 25 (A), 31 (B) and 34 (C), and hBTP-

specific IgE on Day 34 (D) ........................................................................................ 110

List of figures

XVIII

Figure. 2.8 Levels of serum hBTP-specific IgG1 (A) and IgG2a (B) on Day 34 ..... 111

Figure. 2.9 A: Representative contour plots showing the gating strategy of viable

eosinophils, FcεRI-basophils and IgE-binding basophils. B: The representative

contour plots of control and treatment groups, showing circulating eosinophils,

FcεRI-basophils and IgE-binding basophils. C, D and E: The levels of eosinophils,

FcεRI-binding basophils, and mean fluorescence intensity (MFI) of IgE antibody

binding on blood basophils from three mouse groups sensitised with different doses of

hBTP as well as the PBS/alum control mice ...................................................... 112-114

Figure 2.10 The level of hBTP-specific IgA in the jejunum ..................................... 116

Figure 2.11 Optimised sensitisation and oral challenge protocol, based on previous

Experiments #1 and 2, to establish the mouse model for prawn allergy ................... 119

Figure 2.12 Levels of serum total IgE antibody in each mouse group at four time-

points (A), serum hBTP-specific IgE antibody (B), IgG2a antibody (F) and mMCP-1

on Day 34 (C), as well as serum hBTP-specific IgG1 antibody on Day 31 (D) and

Day 34 (E) .......................................................................................................... 120-121

Figure 2.13 Cytokine IL-5, IL-13 and IFN-gamma released by hBTP-stimulated

spleens (A, B and C) and MLNs (A’, B’ and C’) ...................................................... 123

Figure 2.14 The number of eosinophil in spleens ..................................................... 124

Figure 2.15 Representative histology of tissue stained with hematoxylin and eosin

demonstrating vasodilation and eosinophilic infiltration into the jejunum tissues ... 125

List of figures

XIX

Figure 3.1 Experimental design in the sensitisation and control groups ................... 141

Figure 3.2 Levels of serum hBTP-specific IgE antibody in the hBTP-sensitised mice

(A), hAbal-specific IgE antibody in the hAbal-sensitised mice (B), and the allergen

recognition of IgE antibody in hBTP/hBTP group (C) and hAbal/hAbal group (D),

and the total IgE antibody (E) ............................................................................ 147-147

Figure 3.3 Levels of serum specific IgG1 antibody in the hBTP-sensitised mice (A)

and hAbal-sensitised mice (B), and the IgG1 reactivity to hBTP and hAbal in the sera

from hBTP/hBTP group (C) and hAbal/hAbal group (D) ......................................... 149

Figure 3.4 Levels of serum hBTP-specific IgG2a antibody in the hBTP-sensitised

mice (A), hAbal-specific IgG2a antibody in the hAbal-sensitised mice (B), and the

allergen recognition of IgG2a antibody in hBTP/hBTP group (C) and hAbal/hAbal

group (D) ................................................................................................................... 151

Figure 3.5 SDS-PAGE protein analysis (A) and immunoblot for analysing the cross-

reactivity of IgG in the sera from hBTP/hBTP group and hAbal/hAbal group to the

heated extracts of abalone and prawns (B) ................................................................ 152

Figure 3.6 Production of IL-5 (A and A’), IL-13 (B and B’), IFN-gamma (C and C’)

by hBTP or hAbal-stimulated splenocytes ................................................................ 155

Figure 3.7 Proportions of eosinophil and IgE-binding basophil in blood (A and B) and

spleen (A’ and B’) ...................................................................................................... 158

Figure 3.8 Levels of the intestinal hBTP-specific IgA antibody in hBTP-sensitised

List of figures

XX

mice (A), hAbal-specific IgA antibody in hAbal-sensitised mice (B), and the allergen

recognition of IgA antibody in the hBTP/hBTP group (C) and hAbal/hAbal group (D)

................................................................................................................................... 161

Figure 4.1 The experimental design of the mouse model for shellfish allergy ......... 187

Figure 4.2 The Amino acid sequence alignment of tropomyosins from Black tiger

prawn (Penaeus monodon), Genbank protein ID ADM34184.1, and Jade tiger abalone

(Haliotis laevigata × Haliotis rubra), Genbank protein ID APG42675.1 ................ 190

Figure 4.3 SDS-PAGE Coomassie stain (A1 and B1) and IgE immunoblot using sera

from 12 shellfish allergic patients of heated abalone extracts (A2), heated prawn

extracts (A3), recombinant abalone tropomyosin (B2) and recombinant prawn

tropomyosin (B3) ...................................................................................................... 191

Figure 4.4 Production of serum allergen-specific IgE (A and B), total IgE (C) and

mast cell protease-1 (mMCP-1) (D) .......................................................................... 195

Figure 4.5 Production of serum allergen-specific IgG1 (A and B) and allergen-specific

IgG2a (C and D) ........................................................................................................ 196

Figure 4.6 Immunoblot analysis of the IgG reactivity to the heated extracts of abalone

(A) and prawns (B) .................................................................................................... 198

Figure 4.7 Production of cytokines by stimulated splenocytes ................................. 200

Figure 4.8 Production of cytokines by unstimulated splenocytes ............................. 202

Figure 4.9 Secretion of intestinal hBTP-specific IgA (A) and hAbal-specific IgA (B)

List of figures

XXI

................................................................................................................................... 203

Figure 4.10 Proportions of eosinophil and IgE-binding basophil in blood (A and B)

and spleen (A’ and B’) ............................................................................................... 204

Figure 5.1. Experimental procedures of sensitisation and oral challenge in seven

mouse groups ............................................................................................................. 220

Figure 5.2 Levels of serum IgE antibody (A and B) and mMCP-1 (E) and reactivity of

IgG1 antibody (C and D) against heterologous tropomyosin ............................. 225-226

Figure 5.3 Production of cytokines by allergen-restimulated splenocytes ................ 227

Figure 5.4 Eosinophil and IgE bound basophil in circulating blood (A and B) and

spleen (A’ and B’) ...................................................................................................... 230

Figure 5.5 Levels of allergen-specific IgA in jejunum .............................................. 232

Figure 6.1 Molecular phylogenetic tree of tropomyosins from different invertebrate

species inferred using Neighbour-Joining method .................................................... 247

List of tables

XXII

LIST OF TABLES

Table 1.1 Current allergen databases ............................................................................. 3

Table 1.2 The risk of cross-reaction to other foods in patients with specific food

allergy .......................................................................................................................... 12

Table 1.3 The risk of cross-reaction to foods in patients allergic to non-food ............ 15

Table 1.4 Identified and characterised allergens of shellfish....................................... 17

Table 1.5 Sequence identify of shellfish tropomyosins (TMs) .................................... 18

Table 1.6 A summary of 26 different mouse models for food allergy established

within the past two decades, categorised by sensitisation and challenge routes ......... 21

Table 1.7 The most common anaphylaxis scoring system used in published studies of

food allergy mouse models .......................................................................................... 25

Table 1.8 Comparison of the immune responses of mouse models for peanut

sensitisation via the oral or nasal route ....................................................................... 36

Table 1.9 Characteristics of the major identified allergenic proteins derived from milk,

peanut and shellfish, and their usage in the establishment of food allergy mouse

models ......................................................................................................................... 39

Table 3.1 IgG antibody reactive proteins identified by SDS-PAGE using mass

spectrometry technique .............................................................................................. 153

Table 4.1 Demographics of 12 shellfish allergic patients and one non-atopic control

List of tables

XXIII

donor .......................................................................................................................... 185

Table 4.2 IgE antibody reactive proteins identified by SDS-PAGE using mass

spectrometry technique .............................................................................................. 192

List of abbreviations

XXIV

LIST OF ABBREVIATIONS

°C – Degree Celsius

μl – Microlitre

AH – Aluminum hydroxide

AK – Arginine kinase

ASCIA – Australasian Society of Clinical Immunology and Allergy

BAA – Basophil activation assay

BLAST – Basic Local Alignment Search Tool

BSA – Bovine serum albumin

BT – Body temperature

BW – Body weight

CD – Cluster of differentiation

cDNA – Complementary deoxynucleic acid

CMP – Cow’s milk protein

CNP – Cashew nut protein extracts

CPE – Crude peanut extracts

CT – Cholera toxin

Da – Daltons

DBPCFC – Double blinded placebo controlled food challenges

EBBE – Enterocyte brush border enzymes

EC – Epicutaneous

E. coli – Escherichia coli


XXV

ELISA – Enzyme-linked immunosorbent assay

EU – European Union

FBS – Fetal bovine serum

FcεR – The high affinity immunoglobulin E receptor

FSC- A, H or W – Forward scatter-area, height or width

g – Gravity

GWP – Ground whole peanut

hAbal – Heated Jade tiger abalone (Haliotis laevigata × Haliotis rubra))

hBTP – Heated Black tiger prawn (Penaeus monodon)

HRP – Horseradish peroxidase

IFN – Interferon

IG – Intragastric

IgE – Immunoglobulin Isotype E

IgG – Immunoglobulin Isotype G

IgA – Immunoglobulin Isotype A

IL – Interleukin

IN – Intranasal

IP – Intraperitoneal

IV – Intravenous

kDa – Kilodaltons

l - Litre

M – Molar


XXVI

MFI – Mean fluorescence intensity

mg – Milligram

MHC – Major histocompatibility complex

ml – Millilitre

MLC – Myosin light chain

MLN – Mesenteric lymph node

mMCP-1 – Mouse mast cell protease-1

MS – Mass spectrometry

NA – Not applicable

NCBI – National Center for Biotechnology Information

ND – Not determined

ng – Nanogram

ns – Not significant

NT – Not tested

OVA – Ovalbumin

Ovm – Egg-allergen ovomucoid

PBMC – Peripheral blood mononuclear cell

PBS – Phosphate buffered saline

PC – Pilchard extracts

PCR – Polymerase chain reaction

pg – Picogram

PN – Peanut


XXVII

PT – Pertussis toxin

PVDF – Polyvinylidene difluoride

r – Recombinant

RNA – Ribonucleic acid

RPM – Revolutions per minute

RT-PCR – Reverse transcriptase – polymerase chain reaction

SC – Subcutaneous

SEB – Staphylococcal-enterotoxin

SCBP – Sarcoplasmic calcium binding protein

SDS – Sodium dodecyl sulphate

SEM – The standard error of the mean

Siglec F – Sialic acid-binding immunoglobulin-like lectin F

SPT – Skin prick test

SSC-A, H or W – Side scatter-area, height or width

SUC – Sucralfate

TBS – Tris buffered saline

TF – Transcription factor

Th – T helper

TIM – Triose phosphate isomerase

TLR – Toll-like receptor

TM – Tropomyosin

TnC – Troponin C


XXVIII

Treg – T regulator cells

CHAPTER

INTRODUCTION

Published in part:

T. Liu, S. Navarro, A.L. Lopata, Current advances of murine models for food allergy,


Chapter 1

2

1.1 Allergy and allergen

Allergy is often defined as the adverse immune reactions mediated by abnormally

produced Immunoglobulin E (IgE) antibody to certain substances that most of the people

tolerate. The substances that could trigger allergic responses are termed allergens and the

most common allergens are pollens, dust mites, mold spores, pet dander, foods, insect

stings and medicines. The allergic diseases that can significantly impact the quality of life

include asthma, allergic rhinitis (hay fever), food allergies and atopic dermatitis. Over the

recent two decades, there have been a greatly increasing number of reports of allergic

diseases in Australia, food allergies in particular [1, 2]. The 2013 Allergy and Immune

Diseases in Australia (AIDA) Report and the 2014 Allergy in Australia report released

by the Australasian Society for Clinical Immunology and Allergy (ASCIA) stated that

almost 20% of Australians are suffering allergic diseases, and by 2050 the number of

patients affected by allergic diseases in Australia will increase by 70% to 7.7 million [3].

Allergy has become a very essential issue of human health causing constant concerns.

Both environmental factors and genetic associations probably contribute to allergy

diseases. The development of Genome-Wide Association Studies (GWAS) promotes the

identification of a substantial number of susceptibility loci associated with allergic

disease [4, 5]. The hygiene hypothesis is one of the popular but controversial theories

explaining the relatively high prevalence of allergic disease in the industrialised society

compared with the developing world. Briefly, the hygiene theory proposes that during the

childhood, people who live in a hygiene-improved environment have less chance to be

exposed to pathogens, such as bacteria and viruses. Consequently, the immature immune

system could not receive appropriate stimulations from microbes to develop a strong T

helper 1 (Th1) pattern of immune responses, while pro-allergic responses of T helper 2

Chapter 1

3

(Th2) may not be counterbalanced in the immunological frame. Thus, predisposition to

allergy might result from the low infection during childhood [6].

Table 1.1 Current allergen databases [7, 8].

Category Name URL (http://) Biological database

Allergome http://www.allergome.org

Informall http://www.farrp.unl.edu/resources/gi-fas/informall

Allallergy http://www.allallergy.net

Molecular database

Allermatch http://www.allermatch.org

Allergen online http://allergenonline.com

Allergen database for food safety (ADFS)

http://allergen.nihs.go.jp/ADFS/

Structural Database of Allergenic Proteins

http://fermi.utmb.edu/SDAP

During the last few decades, the rapid advances of genomic and proteomic technologies

have promoted the discovery of allergenic proteins based on their sequences, biological

functions, immunological potentials and three-dimensional structures. The relative

information to allergens is organised and stored at database systems (Table 1.1). A better

knowledge of the characteristics of allergens is very necessary for the development of

allergen-specific immunotherapy and the understanding of immunological mechanisms

of allergic diseases. The very first allergen characterised was the food allergen found in

cod fish, namely Gad c 1, a parvalbumin protein [9].

The first engineered recombinant allergens is Dol m 5 from the white-faced hornet

(Dolichovespula maculata) [10], and during the last two decades, a greatly increasing

number of recombinant allergens with molecular and immunological properties

comparable with their native counterparts have been produced using recombinant DNA

Chapter 1

4

technology [11]. The advances of recombinant allergens provide novel perspectives to

study two major topics in molecular and clinical allergology: diagnosis and

immunotherapy. The availability of recombinant allergens promotes the development of

component-resolved diagnostics, that means precise identification of the disease-eliciting

allergens and thus the individual patient’s sensitisation profiles [12]. Another introduced

concept is component-resolved immunotherapy or specific immunotherapy (SIT) in

which hypoallergenic allergen derivatives with reduced allergenicity are generated and

used for inducing immuno-modulation in patients with allergic diseases [13-16].

Chapter 1

5

1.2 Food allergy

Food allergy is an important public health issue in industrial countries due to the

increasing prevalence and the potential life-threatening consequence, effecting up to 8%

of children and 4% of adults [17-19]. Food allergies are also becoming increasingly

important in developing countries [20], particularly to more exotic food sources. Food

allergy or hypersensitivity can be defined as adverse reactions by the immune system to

food compounds that are allergens. The most common pattern of food allergy is known

as type 1 hypersensitivity, mediated by IgE antibody. Allergic mechanisms consist of two

phases: the sensitisation and the effector phases (Figure 1.1). Food allergens are taken up

by antigen-presenting cells (APCs), processed and presented to T helper (Th)

lymphocytes. In sensitised individuals, Th2 responses activate B cells to produce antigen-

specific IgE antibody after the exposure to allergens. During the effector phase, enhanced

Th2-type pattern of immunological responses to the same allergens can induce clinical

symptoms after subsequent exposures. The central allergic inflammation events include

the activation of mast cells, eosinophils and basophils, and the release of histamine and

Th2-type cytokines [21, 22]. The exact symptoms of IgE-mediated food allergy usually

relate to the exposure routes to allergens, as shown in Figure 1.1, and may include itchy

flushing skin, diarrhea and/or vomiting, respiratory distress, or even fatal anaphylaxis

[23]. I focus on IgE-mediated food allergy in this research project.

So far, there are still many questions to be investigated about the mechanisms, multiple

risk factors and events leading to sensitisation towards food compounds [19, 24].

Unfortunately, currently all therapeutic approaches for food allergy are still in clinical

phase I-III and patients can only protect themselves through strict avoidance of all

allergen-containing food products [25]. However, many important questions about food

allergy cannot be answered by in vitro molecular experiments alone, and an appropriate

Chapter 1

6

in vivo experimental platform becomes an urgent need. Mechanistic research or

modifications of experimental conditions using human studies cannot be easily performed

in patients. Moreover, the amount and types of tissue samples that can be collected from

patients with food allergy are very restricted. Therefore, animal models become very

valuable research tools of choice. Without taking risks on human lives, animal models

help us getting more insights into three major aspects of food allergy: 1) the evaluation

of food allergens, 2) studies on immunological responses and mechanisms and 3) the

development of preventive and therapeutic treatments [26].

Figure 1.1 Humoral and cellular mechanisms of IgE-mediated allergic inflammation

against food allergens (Modified and adapted from [27, 28]).

IgE antibody production

Allergen processing

Antigen processing cell

T-lymphocyte

B-lymphocyte

Mast

Initial exposure

SeafoodAllergen

IgE receptorst cell

Subsequent exposure

Cellular barrier

Ca2

Mediatorrelease

Clinical EffectsRhinitisAsthmaUrticariaProtein contact dermatitis

NoseLung

dn Skin

Gut

Chapter 1

7

1.3 Cross-reactivity in food allergy

Allergenic proteins derive from a high variety of natural sources and are capable to result

in Th2 immune responses mediated by high-affinity IgE antibody and/or provoke clinic

effects in sensitised individuals. The phenomenon that the IgE antibody can recognise or

bind to allergens from a different source as to the sensitising inducer is called cross-

reactivity. The risk of cross-reactivity to foods in susceptible populations is illustrated in

Table 1.2 and 1.3 [29-33]. In food allergy, the cross-reactivity between milk from

different animals including cow, goat and buffalo, between eggs from different birds, or

between different species of legumes are very frequent. Except for the classic food

allergies where the primary sensitisation is believed to occur in the gastrointestinal tract,

many cases of food allergies are resulting from immunological cross-reactivity with

inhalant allergens, such as tree pollen, natural latex and house dust-mite.

1.3.1 Characteristics of cross-reactive allergens

The allergens that cause cross-reactivity often have certain homology, especially the

similarity of the antibody-binding regions. The effecter cells may not be activated if the

allergen with sequence similarity of amino acid less than 70% [29]. In general, 50% or

less of identity can rarely lead to cross-reactivity, although similar protein folds might be

generated [34]. The research on the cloning and sequencing of allergens can provide the

molecular basis to help to predict this cross-reactivity. In addition to the amino acid

sequence similarity, the shared tertiary structure of allergenic proteins can also contribute

to cross-reactivity. X-ray diffraction or NMR has been used to identify the three-

dimensional structures of some allergens within the recent two decades. From the

perspective of the molecular classification only, most allergens belong to a small number

of structural or functional protein families [31]. However, the roles of their biochemical

Chapter 1

8

functions e.g. the enzymatic activity of some allergens in the allergic mechanisms remain

largely unknown [35]. The closer the taxonomic distance between different allergen

sources are, this higher the possibility of clinical and immunological cross-reactivity

might be [29].

1.3.2 Cross-reactive allergens in shellfish

Shellfish, combining crustacean and mollusks, is one of the big eight groups of foods

responsible for around 90% of food allergies [36]. Unlike milk allergy, shellfish allergy

often persists through the entire lifetime and is often not “outgrown”. The prevalence of

shellfish allergy is increasing considerable with the global growing consumption of

seafood [37, 38]. A small amount of shellfish can cause serious clinical reactions and

patients have variable symptoms ranging from mild rashes and hives to fatal anaphylactic

shock [39]. The adverse immune responses occur mainly when people ingest shellfish.

Patients with asthma, dermatitis and urticaria can also be found in the domestic and in the

occupational environment through handling of shellfish and inhalation of cooking vapors

[40, 41]. Many patients cross-react to more than one shellfish species, especially within

crustacean [42-44]. Several pan-allergens, mainly tropomyosin (TM) and arginine kinase

(AK), can contribute to cross-reactivity among shellfish and with other inedible

invertebrates such as mites, parasites and cockroaches [45-47].

TM has been identified as the major allergen in a variety of shellfish species (Table 1.4).

Over 60% of people with shellfish allergy are sensitised and react to TM [47]. Within the

crustacean, IgE cross-reactivity to TM is very frequent [43, 44, 48]. Also, inhibition

immunoblotting data showed patients with crustacean allergy had IgE cross-reactivity to

TM from cephalopods (cuttlefish, squid and octopus) [49], gastropods (abalone, turban

shell, Middendorf’s buccinum and whelk), and bivalves (mussel, clam, cockle, oyster and

Chapter 1

9

scallop) [50, 51]. TMs are coiled-coil parallel dimers that form a head-to-tail polymer

along the length of actin filaments, and the best-known function of TM is binding to actin

to regulate contractions of skeletal muscle and the heart [52]. The molecular weight of

TM ranges from 34 kDa to 38 kDa. It is a highly heat stable, water soluble, and IgE

reactivity is due with mainly to linear IgE epitopes. Heated shellfish species still contain

allergenic TM [53], nevertheless combining thermal and high-pressure treatments may

accelerate the digestion of TM in the gastrointestinal tract and reduce the allergenicity of

TM [54, 55]. As a highly conserved protein, the amino acid sequence identity of TM

seems over 89% within the crustacean, but much lower between crustacean and molluscs

(Table 1.5). The different degrees of TM’s primary structure can help to explain the IgE-

mediated cross-reactivity among various shellfish species.

Importantly, TM is also the major suspect for hypersensitivity against inhaled arthropods

(i.e. cockroach, house dust mites and even nematodes), and their cross-reactivity with

shellfish. Several tropomyosins have been officially acknowledged by the WHO/IUIS

allergen nomenclature subcommittee (www.allergen.org), including Bla g 7 from

Blattella germanica (German cockroach), Blo t 10 from Storage mite (Blomia tropicalis),

Lep d 10 from Storage mite (Lepidoglyphus destructor), Der f 10 from American house

dust mite (Dermatophagoides farinae), Der p 10 from European house dust mite

(Dermatophagoides pteronyssinus), Tyr p 10 from Mold mite (Tyrophagus

putrescentiae), Ani s 3 from Herring worm (Anisakis simplex), and Onch v from Filarial

nematode (Onchocerca volvulus). More than 80% of the amino sequence of prawn TM is

identical with mite TM and cockroach TM. Shared IgE epitopes of TM were

demonstrated among prawn, house dust mite and cockroach as well [56]. Clinical

relevance of shellfish-mites cross-reactivity was reported in a population of Orthodox

Jews with no prior exposure to shellfish. The subjects allergic to house dust mite could

Chapter 1

10

cross-react to shrimp tropomyosin [57]. A study from Italy illustrated the ingestion of

snail could probably cause asthmatic symptoms in patients allergic to house dust mite,

whereas TM had only minor contribution to the cross-reactivity between snail and house

dust mite [58].

AK is believed to be another cross-reactive allergen widely distributed among

invertebrates. AK is a 40-42 kDa phosphotransferase that catalyses the reversible

phosphorylation of high-energy phosphate from arginine phosphate to ADP to form ATP,

thereby regenerating and maintaining high ATP during bursts of cellular activity in

invertebrates [59, 60]. According to the Allfam database

(http://www.meduniwien.ac.at/allfam), AK has been identified in six crustacean species

(mainly prawns), one mollusk species (octopus), house dust mite and cockroach. The IgE

cross-reactive homologs of AK has been found in several invertebrate species such as

mite, cockroach, lobster, king prawn, and mussel [61].

Currently, in addition to TM and AK, four allergenic proteins are identified particularly

in crustacean (Table 1.4). Myosin light chain (MLC), troponin C (TnC), and sarcoplasmic

calcium binding proteins (SCBPs) are from the EF hand superfamily, and these proteins

derived from crustaceans [62-64], such as Pen m 3, Hom a 3, Pen m 6, Hom a 6, Cra c 4,

Lit v 4, were demonstrated to bind to human IgE antibody. Moreover, the allergenicity of

triosephosphate isomerase (TIM) has been demonstrated in Brown shrimp (Crangon

crangon), American house dust mite (Dermatophagoides farinae), Freshwater crayfish

(Archaeopotamobius sibiriensis), and German cockroach (Blattella germanica). The

biochemical function of TIM is to catalyse the interconversion of dihydroxyacetone

phosphate and glyceraldehyde-3-phosphate in the glycolytic and gluconeogenic pathways

[65, 66]. As a ubiquitous enzyme, TIM is highly conserved among both eukaryotes and

prokaryotes with a molecular weight of 28 kDa [67]. Similar to MLC, TnC and SCBPs,

Chapter 1

11

TIM has not been well studied and there is a lack of evidence showing the potential

contribution of these allergens to shellfish cross-reactivity [47].

Identification and characterisation of shellfish allergens are essential for detailing

immunological mechanisms of cross-reactivity, but also developing precise diagnostics

and therapies. Further investigation is needed to determine the cross-reactive IgE and

cellular epitopes of allergens from different shellfish species and inhalant invertebrate

allergen sources. In vivo research is still limited to confirm the clinical relevance of in

vitro IgE cross-reactivity in shellfish allergy.

Chapter 1

12

Table 1.2 The risk of cross-reaction to other foods in patients with specific food

allergy.

•

•

•

•

•

•

•

Chapter 1

13

•

•

•

•

•

•

•

Chapter 1

14

•

•

•

•

•

Chapter 1

15

Table 1.3 The risk of cross-reaction to foods in patients allergic to non-food.

•

•

•

•

•

Chapter 1

16

•

•

•

•

Chapter 1

17

Table 1.4 Identified and characterised allergens of shellfish [47].

Chapter 1

18

Table 1.5 Sequence identify of shellfish tropomyosins (TMs). Amino acid sequence of TM from each species is obtained from National Center

for Biotechnology Information (NCBI) database and sequence alignment is performed using Clustal Omega.

Chapter 1

19

1.4 Mouse models for food allergies

Mice have some unique characteristics that contribute to its much wider use to study

human biology than any other organisms. Importantly, the mouse has many physiological,

genetic and immunological similarities to humans. The overall structure of the immune

system of mice and humans are very similar [91], in spite of the necessary consideration

of discrepancies between mouse and human innate and adaptive immune system [92].

Mouse strains, which are well characterised provide an abundance of genetic resource

due to its nature and a string of unique technological strengths. It allows intensive genetic

manipulations, such as forward genetics, reverse genetics, transgenesis, targeted

mutations and knock-ins/outs [93]. With the application of these genetic engineering

approaches on mice, a great number of humanised mouse strains with targeted

modifications become available; hence, more hypotheses on human diseases can be

tested. Moreover, as selective breeding is necessary in the production of disease models,

the mouse, which has a short generation cycle, is an appropriate option.

The evaluation criteria of successfully establishing a mouse model for food allergy is the

extent to which mice mimic human allergic reactions, including clinical symptoms as

well as humoral and cellular responses to food allergens. To date, a large diversity of

protocols to establish mouse models for food allergy have been published as illustrated

in Table 1.6.

The comprehensive analysis of 26 established protocols highlight several important

elements, including the mouse genders, genetic background of mouse strains, routes of

sensitisation, nature of food allergens and usage of adjuvants that could impact the

performance of a mouse model for food allergy.

In the following sections the detailed influence of each key impact factor is discussed on

Chapter 1

20

the levels and patterns of the induced allergy-like immune disorders in mice as well as

the most important scientific highlighted.

Chapter 1

21

Table 1.6 A summary of 26 different mouse models for food allergy established

within the past two decades, categorised by sensitisation and challenge routes.

Mouse strain,

gender and age

Sensitisation Challenge

Evaluation Ref. Route Time points (Day)

Food dose Adjuvant or solvent

Route Time points (Day)

Dose

BALB/c 8-12 week Female

IP

0

16

10µg OVA 20µg OVA

Alum PBS

IG

26 50mg OVA Anaphylaxis score (see Table 1.7) IgE, IgG2a Histamine Proliferation Cytokine Histology of jejunum In vivo IFN-γ neutralisation

[94]


0

14

2µg OVA 1µg OVA

AH PBS

18 1mg OVA IgE Histamine (whole gut tissue) Anti-cytokine treatment

[95]


0, 14 50µg OVA x2 1mg alum 21, 22, 23, 24, 25, 26

CEW x6 (dose was not mentioned)

Anaphylaxis score (fur, feces) IgE, IgG2a, IgG1 Cytokine (MLN) Histology of jejunum Weight change BAA

[96]

BALB/c Male

0, 14

50µg OVA x2 4mg alum 28-(43)

50mg OVA in saline x6, every 3 days

Anaphylaxis score Diarrhea score IgE, IgG2a, IgG1 Cytokine (MLN) mMCP-1 BT Histology of intestine

[97]

BALB/c (H-2b) 2 month Female

1, 14 60µg OVA OR heated OVA x2

1mg AH 28-49 15mg OVA x10 at 2–3 days intervals

IgE, IgG2a, IgG1, IgA (serum) Cytokine Histology of jejunum Histamine (fecal) Diarrhea mMCP-1 FACs (Treg in spleen & MLN) Activities of EBBE in jejunum

[98]

BALB/c 6 week Female BALB/c 3 week Female BALB/c 3 week Female

IP 1,25 IG 1, 6, 12, 18, 24, 30 IG 1, 6, 12, 18, 24, 30

100µg WPE x2 5mg homogenised WPE x6 5mg CMP x6

Alum 0.6µg CT 0.6µg CT

- - IgE, IgG2a, IgG1 Cytokine Histology of intestine Histamine (fecal)

[99]

Chapter 1

22

BALB/c 8 week Female

I.P. 0, 56 IG 0, 56

50μL caviar extract (40mg/mL) 150μL caviar extracts (20mg/mL)

100μL aluminium hydroxide 2 mg sucralfate

125μL caviar extract (40mg/mL) 125μL caviar extract (40mg/mL)

Skin test IgE, IgG2a, IgG1 Proliferation Electron microscop (antrum mucosa)

[100]

BALB/c 8-10 week Female IL-4–/– and IL-4Rα–/–

Week 0 and Week 8

2 larvae x2

-

Week 11 5mg Anisakis protein extract

Anaphylaxis score IgE, IgG2a, IgG2b, IgG1 (serum) Cytokine Histology of lung mMCP-1 (lung)

[101]

C3H/HeJ 6 week Male IP

2, 3, 4 weekly injections

100µg A.s

PT (300ng) + Alum (1mg)

IV

Week6 IV 200µg OR IG 2mg

Anaphylaxis score IgE, IgG2a, IgG1 Histamine Cytokine

[102]


IP

0, 7, 14 50μg raw or cooked PC

1.5% alum

IN

21, 22, 23 100μg raw PC, cooked PC, purified PC parvalbumin, or rCyp c1.01

IgG1, IgG2b, IgE eosinophil peroxidase (lung) Cytokine Histology of lung BAL

[103]

BALB/c 6-8 week Female Wide type and IL-4Rα–/–

0, 21

2 larvae x2

-

42, 43, 44 0.1mg Anisakis protein extract

IgE, IgG2a, IgG2b, IgG1 (serum) Cytokine Histology of lung BAL Airway hyperresponsiveness

[104]

BALB/c 6-8 week Male

IG

Once a week for 5 weeks

OVA 0.2mg in 0.3ml saline x5

20µg CT

IG

Week6 OVA (No dose mentioned)

Diarrhea BT IgE Cytokine mMCP-1 Histology of jejunum

[105]

C3H/HeJ 3 week Female


1.0 mg/g BW homogenised CM

0.3μg / BW (g) CT

Week6 1x CM (No dose mentioned)

Anaphylaxis score

[106]

C3H/HeOuJ 4-5 week Female

0, 7, 14, 21 200mg BLG x4

10µg CT 0.2mol/L NaHCO3

28 100mg BLG Anaphylaxis score Vascular leakage IgE, IgG1, IgG2a, and/or IgA BT(ear) Cytokine (IL-10) Proliferation

[107]



0.01mg, 0.1mg, 1.0mg, or 2 mg/ BW (g) CM

0.3μg / BW (g) CT

Week7

2 doses of CM (30mg/mouse) given 30 minutes apart

Anaphylaxis score Vascular leakage IgE Histamine PAC Cytokine Histology of intestine & lung & ear Skin test

[108]

Chapter 1

23

C3H/HeJ 3 week Female and BALB/c


Homoge--nised CM 1mg/g BW x5

0.3μg / BW (g) CT

Week6

33mg

Cytokine Anaphylaxis score IgE Histamine

[109]

C3H/HeJ 5 week Female and BALB/c

Once a week for 5 weeks + Two boosts at 2-week intervals

10mg PNx5 + 50mg of protein-CT x2

20µg CT 1.5% NaHCO3

Week14 50mg Cytokine Anaphylaxis score IgE Histamine

[109]


0, 7, 21 5mgx3 PE

10µg x3 CT

Week11 (Week4-week11, herbal formula or water)

10mg CPE Anaphylaxis score IgE, IgG, IgA Histamine Proliferation Cytokine BT Histology of ear

[110]

BALB/c 4- to 8-week Female Or C57Bl/6 mice


100 μgx8 OVA OR 100μgx8 WPE

10µg CT or various SEB (better in eosinophil and score) 10µg SEB

Week9 5mg Blood eosinophil quantification Cytokine Anaphylaxis score IgE, IgG2a, IgG1 Histamine Real time PCR Histology of jejunum BT

[111] However, [112]


Weekly for 5 weeks; boosted at week6 and week8

Ground peanut (10 mg x5; 50 mg x2)

20μg CT Week14, week18 (after IG treatment of sham)

200mg Anaphylaxis score FACs (blood basophil numbers and peritoneal mast cell numbers and FceRI expression) BT

[113]


0, 7 5mgx2 (higher score& IgE) or 25mgx2 GWP

10µg x2 CT

21 (week3) Week 5

10 mg CPE divided into two doses at 30- to 40-minute intervals; Mice surviving – re-challenged

Anaphylaxis score Vascular leakage IgE Histamine PCA Proliferation Histology of ear

[114]



100µg pST x4

10µg CT Week5 600µg pST IgE, IgA (fecal) Real Time (cytokine and TF expression of intestinal tissue) Anaphylaxis score Histamine (fecal) IgE, IgG2a, IgG1 FACs (T cell subpopulations)

[115]

BALB/c mice 6–8 week Female

IG

Twice per week for 4 weeks

1mg of Ovm x2x4

10 µg CT

IP

Week11 (After 4wk-PBS oral treatment)

20mg Ovm Serum histamine Cytokine Anaphylaxis score IgE, IgG2a, IgG1 IgA (fecal) FACs (Treg cells in blood)

[116]

C57BL/6 6–8 week Female

Once a week for 1, 2, 3, or 4 weeks

1mg PN (from PN butter)

10µg CT 2 weeks after the last sensitisation

5mg CPN Anaphylaxis score Vascular leakage IgE, IgG2a, IgG1 Histamine and leukotriene levels BT

[117]

Chapter 1

24

Late-phase peritoneal &cutaneous responses to CPE Cytokine

C3H/HeJ, C57BL/6 genetic modification


200µgx7 BSA

10µg x7 CT

56 1mg BSA Anaphylaxis score Vascular leakage IgE, IgG2a, IgG1 Histamine Spleen cell transfer FACs Cytokine

[118]

BALB/c mice 6–8 week Female

Other

IP 0, 7, 21, 28, 35

50µg DIII x5 2.0 mg AH

IP

42 1mg DIII +alum Cytokine Anaphylaxis score IgE, IgG2a, IgG1 Histamine

[13]

BALB/c 6–8 week Female

IG 0, 7, 14, 21, 28, 35, 42 and 49,

0.2mg OVA x8

SUC Or Alum

-

- - skin test IgE, IgG2a Histamine Cytokine

[119]

BALB/c 6–8 week Female

Cutaneous exposure to allergen with a 4- day interval for 4 weeks

0.05, 0.5 or 1mg CNP

-

IG

Day6 following the 4th exposure

15mg Cytokine Anaphylaxis score IgE, IgG2a, IgG1 Histamine Histology of intestinal tissues BT

[120]

AKR/J 4–5 week Male

0, 8, 16-IG 1, 9, 17-IP

1mg x3 0.5µgx3 CPE

CT AH

IP

24 200µg of r-Arah2 protein

Anaphylaxis score Vascular leakage IgE, IgG2a Histamine

[121]

BALB/c 8 week

SC 4-wk intervals

10μg parvalbumin mutant X3

AH

- - IgG1 and IgE

[122]

Chapter 1

25

Table 1.7 The most common anaphylaxis scoring system used in published studies

of food allergy mouse models [113, 116, 121]

Score Anaphylactic symptoms

0 No symptoms

1 Scratching and rubbing around the nose and head

2 Puffiness around the eyes and mouth, pilar erecti, reduced activity and/or decreased activity with increased respiratory rate

3 Wheezing, labored respiration, and cyanosis around the mouth and tail

4 Slight or no activity after prodding, or tremor and convulsion 5 Death

1.4.1 Mouse strain

Genetic susceptibility seems to have a close association with the development of allergic

diseases, however no particular genes have been identified predisposing humans to food

allergy [123]. Considering the unfeasibility of studying allergy risk factors directly on

human subjects, mouse strains with defined genetic background become very useful in

this field. Under the control of genetic variations and living environment, mice are

essential for a better understanding of multi-mediator involved mechanisms underlying

the allergic responses.

BALB/c mice are generally believed to be a Th2-baised strain, however reported to be

resistant to the induction of hypersensitivity to cow’s milk or peanut, in contrast to the

C3H/HeJ strain [109]. In this study conducted by Morafo et al., three-week-old mice of

both strains were sensitised orally using a mixture of peanut or cow’s milk with cholera

toxin (CT) as a mucosal adjuvant. After one oral challenge, C3H/HeJ mice presented mild

clinical reactions and a significantly increase of serum antigen-specific IgE and plasma

histamine, while BALB/c mice only generated a high level of IgE antibody to peanut, but

not cow’s milk. No anaphylactic symptoms could be observed in BALB/c mice.

Chapter 1

26

Therefore, BALB/c mice, often used in modeling allergic diseases induced by systemic

allergen sensitisation, might be not sensitive enough to mimic human allergic responses

when orally sensitised. In addition, Morafo etc. found the higher expression level of IL-

4 and IL-10 in C3H/HeJ only, but significantly increased Th1 cytokines in BALB/c,

which suggests that C3H/HeJ and BALB/c mice may develop different immunologic

response patterns to food allergens.

C3H/HeJ mice are very often used to develop models for peanut-allergy, because

C3H/HeJ mice are anaphylaxis-susceptible. This characteristic is probably due to a Toll-

like receptor (TLR)-4 mutation in C3H/HeJ mice that blocked intestinal LPS

(lipolysaccharide) signaling [124]. Both C3H/HeJ and C57BL/10ScNHsd strain with

TLR4-deficiency showed more Th2-biased reactivity to peanuts by means of oral

sensitisation with peanuts and CT as adjuvant. In contrast, the same administration of

peanuts in MHC-matched or congenic controls (C3HeB/FeJ, C3H/HeOuJ, CBA/J,

C57BL/10SnJ and C57BL/6J) were not able to induce the increased level of antigen-

specific IgE and histamine, or any anaphylactic symptoms [125]. In addition, TLR-4

deficiency was not sufficient to confer BALB/c mice with proper clinical reactions to

peanut [126]. Accordingly, more differences than only the TLR-4 genetic mutation

between C3H/HeJ and BALB/c mice contribute to the different susceptibility to food

allergies.

AKR/J mice were successfully used in a peanut allergen-induced murine anaphylactic

model to monitor the new immunoprophylactic strategy [121]. However, following the

same protocol of the well-established anaphylaxis model for milk allergy using C3H/HeJ

by Li et al.[108], this research group failed to generate an appropriate allergic phenotype

using AKR/J or CBA/J mice. The different results in AKR/J mice indicate the great

diversity of genetic susceptibility to different food allergies in mice, similar to the fact

Chapter 1

27

that only some human individuals getting sensitised to food allergens [109]. The same

conclusion was reached when comparing C3H/HeOuJ mice and C3H/HeJ mice.

C3H/HeOuJ mice were a negative control strain when induced with peanut [125],

however were good allergy responders as demonstrated for the establishment of a β-

lactoglobulin-induced anaphylaxis model [107]. Peanut-treated C3H/HeJ mice were

observed to have more severe clinical symptoms than β-lactoglobulin-treated C3H/HeJ

mice [126]. It is worth noting that in the studies of comparing murine susceptibilities to

different foods, mice should be sensitised and/or challenged with the same amount of

respective food allergens. Thus crude extracts of foods containing multiple allergenic

proteins with various allergic efficacies are not well suited for these studies.

C57BL/6 mice are generally regarded as a less Th2-prone strain than BALB/c due to a

relatively weak allergic response to allergens. However, with an advantage of the

enormous genetic variants available, C57BL/6 mice could become a more convenient

platform targeting essential elements of the immune system involved in the pathogenic

mechanisms. Moreover, a recently established asthma model might provide a more

facetted comparison between C57BL/6 and BALB/c mice [127]. In this study, BALB/c

mice showed a greater level of inflammation in lung tissue, including the increased

number of mast cells and concentration of IL-4, IL-5 and IL-13, which validate the bias

of BALB/c to Th2. While, surprisingly, C57BL/6 exhibited much more pro-inflammatory

cytokines and eosinophilic inflammation in bronchoalveolar lavage fluid (BALF) than

BALB/c mice, which may reflect a mixed pattern of Th1/Th2.

In conclusion, the genetic background of the chosen mouse strain can affect

hyperresponsiveness and allergic inflammation in the murine model. The selection of

experimental mouse strains is very important for the characterisation of allergic diseases,

and greatly dependent on the allergen types and the target phase or features of particular

Chapter 1

28

allergic diseases.

1.4.2 Mouse gender

Gender has been widely believed as one of the potential risk factors in the development

of food allergy. Gender preference in food allergy could also possibly be affected by age.

A typical pattern is found in some investigations on the basis of a large number of subjects

in peanut allergy or tree nut allergy. In a telephone survey of 13, 493 participates in the

United States [128], generally no significant difference of peanut or tree nut allergy

sensitisation rates were found among different age groups. However, among children’s,

the prevalence of peanut or tree nut allergy was much higher among males than females,

while the female/male ratio of adult patients with peanut or tree nut allergy reached almost

2. The same trend was also observed in asthmatic patients [24]. Overall, the male

predominance of allergic diseases occurred in children but the situation was reversed in

female predominance among adults. Accordingly, the gender effect on allergy prevalence

is possibility associated with some of the physiological and immunological changes

during different growth stages. In contrast the Multiple Logistic Regression Models

constructed on the basis of 38,480 children in the United State could not identify gender

as a significantly related characteristic with food allergy [129]. Furthermore very similar

prevalence rates of peanut allergy or cashew allergy were reported among males and

females in 141 English children [130].

Some studies on animal models have also compared the influence of different mouse

genders on allergic responses. In a model of Brown Norway rats [131], repeated oral

dosing with OVA provoked a significantly higher level of OVA-specific IgE and IgG

antibodies and a much higher incidence rate among female rats compared to males. Two

studies on BALB/c mouse models of airway hyperreactivity supporting this gender

Chapter 1

29

preference [132, 133]. In these studies, a more severe lung inflammation in female or

castrated males as compared to uncastrated males was detected, which included a higher

level of antigen-specific IgE antibody and IL-4 cytokines, the upregulation of CD4+ cells

and a remarkable lung peribronchial infiltration of lymphocytes and eosinophil.

Compared to males, less regulatory T cells were detected in female lungs, which

suggested a relatively weak protection in female lungs against allergen challenges, hence

worsening the inflammatory responses. In addition to the deficient regulatory T cells, a

lower level of testosterone in females and castrated males were speculated to contribute

to a stronger airway inflammation. Moreover, estrogen is another sex hormone that has

been well studied for its effects on gender difference in allergic diseases [134]. Recent

experimental evidence was derived from a passive systemic anaphylaxis mouse model.

This study revealed the regulatory function of estrogen in the production of endothelial

nitric oxide synthase and nitric oxide without impacting mast cells, contributing to an

increased anaphylaxis severity in female mice [135].

Although the majority of mouse models of food allergy have been conducted on female

mice, so far, some male mouse models have also succeeded in providing novel insights

into food allergy, particularly the development of more specific remedies for a better

immunological regulation of allergic diseases. For example, in a CT-dependent ingestion

model using male adult BALB/c mice [105], a unique subpopulation of tolerogenic B

cells in intestine tissues was characterised. More importantly, in the effector phase of food

allergy, this cell strain was found to be able to control the activation of murine effector

T-cells through the expression of transforming growth factor (TGF)-β and to inhibit Th2

inflammation of murine intestine tissues greatly after adoptive transfer. Another recent

male mouse model highlighted rapamycin as a potential preventive and therapeutic drug

in food allergy [97]. In the preventive part of this study, male mice with the administration

Chapter 1

30

of rapamycin prior to oral challenge of OVA showed that mast cell responses were locally

(intestine) and systemically (serum mouse mast cell protease-1) reduced and

consequently less severe allergy-related symptoms were observed. In addition the

therapeutic treatment with, rapamycin dramatically decreased the levels of antigen-

specific IgE antibody as well as several cytokines including IFN-γ, IL-4, IL-13, IL-9, IL-

10, and IL-17.

To summarise, regardless of possible gender preference in human food allergy, female

mice may be a better choice for the model establishment of severe airway allergic

conditions, while male mice can be well employed in studies of food allergy prevention

and therapies. The few published investigations did not explain the reasons of mouse

gender preference, and male mice should not automatically be excluded to develop a

mouse model for food allergy. Further research to link the physiological and

immunological characteristics of the two genders with different applications in food

allergy will be necessary.

Chapter 1

31

1.4.3 Route of sensitisation

The first phase of the IgE-mediated allergy cascade responses is sensitisation, which is

represented by the development of antigen-specific IgE antibodies after exposure to an

allergen. People with food allergy actually have often difficulty recognising their routes

of sensitisation because no symptoms are experienced at this early stage. Tree-nut specific

IgE antibody was detected in more than 50% of the children patients with tree nut allergy

who did not realise their history of exposure [136]. In the same study, a similar situation

was seen among patients with peanut allergy. The first acute reactions to peanut occurred

at a median age of 24 months, which suggested one or multiple exposure(s) that sensitised

most individuals at an earlier stage. However, 72% of young patients thought the event

that led to initial allergic reactions was their first exposure to peanuts. In fact, allergens

potentially obtain access to lymphoid organs via three main routes: ingestion, inhalation

and skin contact. Subsequent allergen exposure is boosting the production of antigen-

specific IgE antibody. Different sensitisation routes have been established in animal

models of food allergy, including intragastric (IG), intraperitoneal (IP), epicutaneous

(EC), intranasal (IN) and subcutaneous (SC) administration. Because the clinical

allergenic potency of proteins and immunological responses can vary greatly depending

on the exposure route and the fate of the allergen, selecting a reasonable sensitisation

route is very critical in developing the best possible mouse model for food allergy.

The gastrointestinal tract is believed to be the most common exposure sites to food

allergens. As a result, oral delivery of allergens into the gastrointestinal tract of animals

is the most widely used route of sensitisation in food allergy models. However, similar as

humans, animal models have an innate tendency to develop oral tolerance to a myriad of

food proteins [137]. Consequently, some adjuvants are often required to overcome oral

tolerance, as for example CT which is a well-known mucosal adjuvant. However, the

Chapter 1

32

involvement of CT does not reflect usual human pathogenic mechanism of food allergy.

CT is an endotoxin produced by Vibrio cholera. The non-toxic subunit B of CT links to

the allergen of interests and may have the capacity to bind to the receptor on intestinal

epithelial cells, which increases allergen uptake [138, 139]. It has been reported that CT

is able to enhance Th2-biased responses and suppress Th1-mediated responses in some

autoimmune diseases [140] and delayed-type hypersensitivity [141]. In addition to the

modification of T-cell responses, the imbalance of intestinal fluid balance has been

suggested to contribute to CT actions as well [142]. However, to date, the exact

mechanisms through which CT strengthens the murine Th2 immune response still

remains unclear. Staphylococcal enterotoxin B (SEB) is another utilised adjuvant that

could bypass the state of tolerance by oral administration together with food allergens in

mice. SEB is known as causing food poisoning, but could act as a super-antigen to

virtually stimulate Th2 lymphocyte activities in food allergy models. In particular, unlike

CT/allergen-sensitised BALB/c mice, intense eosinophilia is presented in the blood and

intestinal tissues of SEB/allergen sensitised BALB/c mice, which is also a hallmark of

pathological characteristics seen in patients with food allergy [111]. Furthermore, some

studies suggested an association of SEB with the induction of other allergic diseases, such

as atopic dermatitis and chronic rhino sinusitis. Therefore, SEB might be a better option

of adjuvants to enhance the effectiveness of murine sensitisation with food allergens.

However, there is still no evidence to show the necessary involvement of SEB as an

environmental risk factor in the development of human food allergy.

Instead of breaking the oral tolerance, Bowman evaluated the allergenic potency of

different foods by the failure to induce tolerance in adult C3H/HeJ mice [143]. In this

study, mice orally exposed to food extracts without adjuvants received subsequently IP

treatment of food extracts. The tolerance indicator was the decreased production of

Chapter 1

33

antigen-specific serum IgE in “tolerant” mice compared to those without feeding of

extracts. The experiment results showed that foods containing non-allergens (turkey,

spinach) were more tolerising than foods containing strong allergens (peanut, Brazil nut),

while foods containing resolving allergy (egg white) were most likely tolerant. From this

perspective, murine immunologic tolerance to foods can mimic human oral tolerance in

many ways.

In addition, an adjuvant-free mouse model study of rice allergy demonstrated the

effectiveness of repeated IG gavage to elicit hypersensitivity in BALB/c mice [144]. The

typical allergic responses of food allergy include mild clinical symptoms, antigen-specific

IgE antibody response and small intestine inflammation were all detectable in this mouse

model of rice allergy, which placed this model the first successful gastrointestinal mouse

model in the absence of adjuvants.

The intraperineal route (IP) of sensitisation has a relatively high efficiency to

systemically trigger the production of mouse circulating IgE and IgG antibody. Alum is

frequently used to increase the sensitivity to allergens through establishing allergen sites

of high concentration to stimulate macrophages. The clinical utility of alum in human has

been approved [145]. However, the usage of alum in food allergy model is still in doubt

because of its non-physiological nature. Therefore, in the absent of alum, systemic (IP)

administration of proteins in mice has been applied, but only for the purpose of screening

novel allergens [146-148]. Different inherent allergenic potentials of proteins could be

identified by the measurement of IgE antibody responses in the high IgE responder, the

BALB/c mouse.

The epicutanous route (EC) of sensitisation has been applied to establish adjuvant-free

BALB/c models. Allergens were applied to an area of the mouse back or belly skin with

Chapter 1

34

hair removed, and then this area was covered with a non-latex, non-occlusive bandage for

a short period, followed by a longer resting period. The sensitisation was conferred by

repeated transdermal exposure. Mouse models of hazelnut [149], cashew nut [120],

Anisakis [150] and milk whey protein allergy [151] have been established using this

approach. These studies published the successful induction of antigen-specific IgE

antibody, Th2-biased cytokines and clinical responses in dose-dependent manners. In

another example of an adjuvant-free mouse model for hazelnut allergy, similar to

persistent human hazelnut allergy, BALB/c mice could respond to transdermal challenges

of hazelnuts after a longer resting period after transdermal sensitisation. This study

discovered mechanisms underlying the long-term hypersensitivity with long-lasting

memory response of murine antigen-specific IgE antibody and splenocyte IL-4 responses

[152].

The intranasal route (IN) of sensitisation is often applied for aerosolised allergens to

elicit food allergy. An investigation in 2007 showed 1/4 of all allergic reactions were due

to hidden allergens, with 35% of fish-allergic patients and 22% of egg-allergic reactions

caused by allergens hidden in other foods or food vapours [153]. Subsequently it was

demonstrated by Kirstein et al [104] that also food borne parasite allergens can generate

allergic sensitisation through allergen inhalation. Acute allergic airway hyperreactivity

was also reported in peanut-allergic patients who inhaled airborne peanut at school or on

commercial airlines. Moreover, IgE reactivity to multiple spice allergens was found in

spice mill workers exposed to high concentration (>10 mg/m3) of inhalable spice dust

[154]. Therefore, the immune reaction to aerosolised food allergens has been an essential

aspect of food allergies in both domestic and working environments [155]. Another

related issue for attention is the immunological cross-reactive allergens between food and

inhalants increase the complexity of food allergy mechanisms. The muscle protein

Chapter 1

35

tropomyosin (TM) is an example of a highly cross-reactive pan-allergen. TM is the most

allergenic protein known, and has been identified in over 150 species, derived from a few

distinct animal groups [40], including ingested shellfish, inhaled insect debris (e.g.

cockroaches) and the most abundant allergen source, house dust-mites. TM exhibits a

high degree of immunological cross-reactivity and consequently is responsible for

sensitising over 30% of the world-population. Therefore inhalation of environmental

allergens can play a considerable role in either sensitisation or effector phases to

contribute to food allergies.

However, airway exposures to allergens are usually overlooked in the establishment of

food allergy models. Only a few intranasally food-challenged mouse models have been

published and even less models with airway sensitisation. In a recent study of fish allergy,

a mouse model was established for a better understanding of work related inhalant allergy

during food-processing [103]. After IP sensitisation, pilchard-sensitised mice received

intranasal challenges of the same allergens. Increased levels of antigen-specific IgE

antibody, Th2 biased cytokines of IL-4, IL-5 and IL-13, as well as the lung inflammation

and cells infiltration, but no allergy-related clinical symptoms were reported in this

model. A comparative study of respiratory sensitisation and oral sensitisation in peanut

allergy provided one of the very few published nasally sensitised model using C57BL/6

mice [156]. However this study used CT conjunct with peanut extracts to orally initiate

the immune responses. In Table 1.8 the main differences of two models are highlighted,

that were sensitised by two mucosal routes and nasally challenged. Additionally, many

mouse models of allergic diseases, such as allergic rhinitis [157] and asthma [158], are

often developed with aerosolised OVA as allergen using a similar protocol, which could

be good references for the establishment and analysis of novel inhalant models of food

allergy.

Chapter 1

36

Table 1.8 Comparison of the immune responses of mouse models for peanut

sensitisation via the oral or nasal route [156].

Chapter 1

37

In the effector phase of food allergy, the airway exposure to food allergen seems less

common than via the IG route. Also, the airway sensitisation has been much less studied.

Therefore, a mouse model that receives nasal sensitisation but oral challenges might align

better with some of the diagnosed food allergy patients with no clear exposure histology.

The subcutaneous route (SC) of allergens has been also applied in the establishment of

some animal model, which are not discussed in this review due to causing skin

inflammation and are unlikely to be acceptable for human applications [159].

1.4.4 The food allergen

In food allergy, there are eight groups of foods people are more commonly allergic to,

which are egg, milk, peanut, tree nut, soy, wheat, fish and shellfish. Figure 1.2 is based

on a national survey of self-reported food allergy in 20, 686 individuals in the United States

between 2007-2010 [160]. There seem to be three leading causes for food allergy, namely

milk, peanut and shellfish in both children and adults. In contrast to mouse studies of food

allergy, the most intensively investigated food allergen sources are egg (mainly due to the

wide usage of OVA as allergen), milk and peanut. As we are advancing the identification

and characterisation of more allergens, mapping the IgE binding epitopes and the

generation of recombinant proteins, more food allergy complications could be explored

based on more specific investigations. An increasing number of mouse models have been

developed with the introduction of different specific allergenic proteins, see Table 1.9. In

addition, the allergenicity of processed foods has been discussed in some mouse model

studies. Here, the most recent findings for milk, shellfish and peanut allergies are

summarised.

Chapter 1

38

Figure 1.2 The proportional prevalence of self-reported food allergen sources of

children and adults in the United States [160].

Children

Total prevalence of child food allergy: 6.53%

Adults

Total prevalence of adult food allergy : 9.72%

milk eggpeanutfishtree nutshellfishsoywheatothers

Chapter 1

39

Table 1.9 Characteristics of the major identified allergenic proteins derived from

milk, peanut and shellfish, and their usage in the establishment of food allergy

mouse models (http://allergenonline.com, http://www.ncbi.nlm.nih.gov/) [40, 161-

165].

Food source

Allergen name and nature Molecular

weight (kDa)

Representative mouse models

implicated

Milk

Milk NA [166], [167] and see more in Table 1.6

Processed milk NA

Major allergenic proteins

α -Lactalbumin 14.2 ND

β -Lactoglobulin 18.3 [168]

Bovine serum albumin 66.3 [169]

Immunoglobulin 160 ND

Lactoferrin 80 [170]

αs1-casein 23.6 [171]

αs2-casein 25.2 ND

β -casein 24 ND

κ -casein 19 ND

Peanut

Peanut extracts NA [156], [126] and see more in Table 1.6

Processed peanut extracts NA [172], [99]


Lipid transfer protein

Ara h 9 9.8 ND

Oleosin Ara h 10 Ara h 11

16 14

ND

Profiling Ara h 5 15 ND

α-amylase NA 16 ND

Chapter 1

40

Conglutin (2S albumin)

Ara h 2

Ara h 6

Ara h 7

16.6

18.0

15

[114], [173], [174]

Reference for Ara h 7 is ND.

PR-10, Bet v 1 family

member Ara h 8 16.9 ND

Agglutinin NA 31 ND

Cupin (vicilin-type, 7S globulin)

Ara h 1

63.5 [173], [114], [125]

Cupin (11S globulin, glycinin)

Ara h 3

Ara h 4

60

61

[173] Reference for Ara h 4 is

ND.

Phospholipase D

NA 91 ND

Conarachin I NA 142-295 ND

α-arachin NA 120-600 ND

Shellfish

Shellfish extracts NA ND

Processed shellfish extracts NA

ND


Tropomyosin 34-39 Sand shrimp

(Metapenaeus ensis): [175]

Arginine kinase 40 Mud crab

(Scylla paramamosain): [176]

Myosin light chain 20 ND

Sarcoplasmic calcium-binding protein

20 ND

Troponin C 16.8 ND

Myosin heavy chain 208-400 ND

α-actine 41 ND

Triosphosphate isomerase 28 ND

SERCA/Ca2+ATPase 122.2 ND

Abbreviations: NA = Not applicable, ND = Not determined.

Chapter 1

41

1.4.4.1 Milk allergens in mouse studies

Milk allergy is the most common food allergy in human’s early life. 2%-3% of children

younger than two years of age are suffering from cow’s milk allergy and 60% of them

showed IgE mediated (type I) allergic responses. A recent study from Australia reported

up to 10% of infants with IgE confirmed allergy to milk [177] Although up to 87% of

infants could outgrow IgE-mediated milk allergy at three years of age [178], some

individuals could not outgrow their allergy, probably due to the deficient development of

CD4+CD25+ Treg cells in their childhood [179]. Moreover, IgE- and IgG-binding

epitopes on αs1-, β- and κ-casein have been mapped with a recognition difference

between patients with a persistent and transient histories of cow’s milk allergy [180].

There are a variety of allergenic proteins contained in milk with varying structural and

functional differences. However, the majority of mouse models of milk allergy have been

developed with the use of whole milk or β-lactoglobulin, and the involvement of other

milk proteins in the induction of allergic responses have been poorly studied.

Cow’s milk consists of more than 25 different proteins, which can be divided into two

major categories, whey and whole casein, accounting for approximately 18% and 82% of

milk proteins respectively. BALB/c mice sensitised with modified milk of a casein/whey

protein ratio of 40:60 showed a significantly lower diarrhea incidence and plasma

histamine levels [181]. Therefore the casein fraction might be more allergenic than the

whey fraction of milk. The whole casein fraction is comprised of four proteins, αs1-, αs2-

, β-, and κ-casein with little primary structure homology. The αs1-casein is the most

abundant milk proteins and its allergenicity is frequently observed, whereas not a single

protein could be identified as the dominant allergen in milk. Different binding specificity

and intensity of sera IgE antibodies to multiple milk proteins, regardless of the amount of

Chapter 1

42

these proteins in milk, were recognised in 20 documented paediatric patients with milk

allergy [182].

Although some mouse models have been successfully established using whole milk [99,

106, 108], very limited published models were established using single milk casein

protein. There is one systematically induced BALB/c model with αs1-casein from goat’s

or sheep's milk (GSM), which is highly homologous to cow’s milk. The severity of

murine allergic reactions, including clinical symptoms and plasma levels of IgE antibody

and mouse mast cell protease-I, was greatly dependent on the dose of αs1-casein [171].

This dose-dependent manner suggests the important responsibility of αs1-casein in milk

allergy.

In the whey fraction of milk, the two main allergenic proteins are β-lactoglobulin and α-

lactalbumin. β-lactoglobulin has been recently studied using several different mouse

models and some interesting studies may contribute to our knowledge of milk allergy

therapeutics and mechanisms. By contrast, α-lactalbumin has been rarely studied in vivo.

In 2011, Morin successfully established an adjuvant free mouse model of milk allergy by

IP sensitisation of β-lactoglobulin in germ-free BALB/c mice [168]. In contrast, IP

administration of whole casein or heated β-lactoglobulin could not induce antigen-

specific IgE antibody responses in either germ-free mice or conventional mice, which

indicates a lower allergenicity of whole casein or heated β-lactoglobulin compared to

untreated β-lactoglobulin. Regarding to the heat effects on the allergenicity of β-

lactoglobulin, an in vitro evaluation by indirect competitive ELISA reported that heat

treatment could increase the allergenicity of β-lactoglobulin and also α-lactalbumin, with

a peak at 90°C, but dramatically decreased binding reaction at higher temperature (up to

120°C) [183]. Another important conclusion from Morin’s adjuvant free mouse model of

β-lactoglobulin allergy was that germ-free mice might have advantages over conventional

Chapter 1

43

mice when used in experimental models for food allergy. Because mice raised and

maintained under axenic (germ-free) conditions lack normal intestinal microbiota,

commensal organisms in particular, leading to a defective immune system [184]. The

complete development of commensal organisms in gastrointestinal tract after birth may

reduce the susceptibility of individuals to milk allergy. This theory is supported by

another mouse model. The pretreatment of transfected Lactococcus lactis with the

capacity of producing IL-10 was demonstrated to have a protective effect on C3H/HeOuJ

mice before oral sensitisation with a mixture of β-lactoglobulin and CT [107]. Other milk

proteins such as serum albumin and lactoferrin are also known milk allergens, however

require more animal models for a better comparison of their allergenic potentials and

further understanding the mechanisms of milk allergy.

1.4.4.2 Peanut allergens in mouse studies

In recent decades there has been an increasing number of reported life-threatening

anaphylactic shock caused through exposure to peanuts and most patients don’t outgrow

peanut allergy [185]. As a result, peanut allergy is regarded as one of the most dangerous

food allergy. So far, eleven Ara h-type allergenic proteins have been identified with the

capacity to bind specific IgE antibody from sera of peanut allergy patients, and a majority

of them are peanut storage proteins [163]. Ara h 1, Ara h 2 and Ara h 3 have been widely

considered as the main peanut allergens with strong allergenic potency with more than

50% of patients reacting to these three allergens. Ara h 5 and Ara h 8 might be the main

allergens cross-reacting with a variety of pollen derived allergens [165, 186, 187].

However, the results from in vivo assessments of the allergenicity of peanut allergens

seems to be conflicting. Orally or systematically induced peanut-sensitised C3H/HeJ

mice developed a stronger IgE, IgG1 and IgG2a antibodies response to purified Ara h 1

Chapter 1

44

and Ara h 3 as compared to Ara h 2 and Ara h 6 [173]. Nevertheless, another orally

sensitised C3H/HeJ model indicated the predominant allergenicity of Ara h 2 and Ara h

6 from roasted peanuts[174]. Ara h 2 and Ara h 6 are 2S albumin and seem to have more

digestion-resistant properties than other proteins in peanuts. In this study, the depletion

of Ara h 2 and Ara h 6 in peanut remarkably reduced the severity of murine allergy-like

anaphylactic reactions after IP challenges. Moreover, Ara h 2 and Ara h 6 had similar

desensitisation effects as the whole peanut extracts in an immunotherapy evaluated in

mice. In addition, the presence of Ara h 1 and Ara h 2 specific IgE antibodies was also

observed in an orally induced C3H/HeJ model using crude peanut extracts [114].

Moreover, both Ara h 1 and Ara h 2 were able to stimulate proliferations of spleen cells

from sensitised mice, although not as effective as whole peanut extracts. Unfortunately,

other possible peanut allergens were not assessed in the same study as comparisons.

Non-allergenic components in foods may contribute to the phenomenon that whole

peanut extracts could be used to sensitise mice most effectively, in contrast to any of the

individual peanut allergens. Fats, carbohydrate and non -allergenic proteins are very

common ingredients of foods. The role of these components in the development peanut

allergy has been rarely studied using murine immune system. The influence of peanut fat

on the murine immune activation has been recently highlighted [188]. Peanut extracts or

purified peanut proteins were SC injected into the hind footpad of BALB/c mice, and one

week later popliteral lymph nodes (PLN) collected for analysis. Peanut extracts, but not

purified proteins including Ara h 1, 2 and 6 were able to induce PLN cell proliferation,

cytokine production (IL-4, IL-10 and IFN-γ) in restimulation of PLN cell culture and

activation of APCs. Compared with the peanut extracts containing 0.2% fat, the peanut-

oil emulsion with 3.5% fat appeared to induce even stronger immune responses, which

suggested peanut fat had a capacity to modify the allergenicity of peanut. In spite of the

Chapter 1

45

limited pathogenic relevance of PLN assay to food allergy, this study highlights that the

factors affecting food allergenicity are not confined to antigenic proteins. The potential

influence of different non-allergen food components or food-processing methods on food

allergenicity can be analysed in detail using mouse models.

Novel immunotherapies, mainly through the desensitisation effects of engineered peanut

allergens, have been also analysed using murine immunity models. With the modification

of the main IgE binding sites, recombinant peanut allergens rAra h 1, 2 and 3 had the

potential to generate a much lower IgE antibody production than wild-type Ara h 1, 2 and

3. Thus, a frequent administration of rAra h 1-3 mixture in peanut/CT-sensitised mice

could reduce the level of anaphylactic responses to oral challenges. Interestingly, the SC

co-administration of heat-killed Listeria monocytogenes (HKLM) [189] or the rectal co-

administration of Escherichia coli [172] with rAra h 1-3 could dramatically enhances the

effectiveness of desensitisation in the C3H/HeJ mouse model. Antigen-specificTh2

responses appeared to be shifted to a more Th1 responses. Unfortunately, the immune

mechanisms in which HKLM and Escherichia coli itself may play as potential bacterial

adjuvants are largely unknown.

1.4.4.3 Shellfish allergens in mouse studies

With the increasing consumption of shellfish, over recent years the prevalence of shellfish

allergies has been increasing in many industrialised as well as developing countries [40,

190, 191]. Similar to peanut allergy, shellfish allergy often persists throughout adulthood

and seems to be the most common food allergy in adults. A number of proteins have been

identified as shellfish allergens (see Table 1.4 and 1.9). They are species-specific with

different degrees of sequence homology considering the large number of shellfish species.

Consequently, although various techniques have been used to characterise and quantify

Chapter 1

46

shellfish allergens [27, 192], many of the shellfish allergens are still poorly studied at the

molecular and immunological level [38]. Mouse models have not been frequently applied

in the clinical evaluation of allergenic shellfish components and the development of

therapeutic interventions for shellfish allergy.

In the few model systems published, the major allergen tropomyosin (TM) has been

considered as a good representative for sensitisation to shellfish, because the muscle

protein tropomyosin is the most prominent pan-allergen responsible for allergic reactions

to shellfish. For example, TM of the Brown shrimp (Penaeus aztecus) Pen a 1 is

responsible for more than 75% of the shrimp-specific IgE antibody reactions [193] and

TM has been the main allergenic protein identified in a variety of mollusks species [53,

194, 195]. Thus, TM becomes a key allergen to better understand the cross-reactivity

between a wider range of invertebrate species and also a suitable candidate allergen to be

used in the induction of in vivo models for shellfish allergy. Particularly, shrimp

tropomyosin accounts for approximately 20% of all shrimp proteins and the amino acid

sequence identity among various edible shrimp species is 95%-100% [192, 196]. In 2008,

shrimp tropomyosin was initially used in oral sensitisation with CT as mucosal adjuvant

in C3H/HeJ mice and elicited significantly increased titers of serum TM-specific IgE and

IgG1 antibody [175]. Oral challenge with high doses of purified TM triggered an

appropriate level of anaphylactic reactions. The TM re-stimulated splenocytes from

sensitised donor mice released significantly higher levels of IL-4, IL-13 and IFN-γ than

control mice, which confirmed the Th1/Th2 pattern of immune reactions in shrimp

allergy. In the same year, oral sensitisation with the recombinant TM rMet e 1 plus CT

also successfully induced hypersensitivity in BALB/c mice [197]. Unfortunately, no cell-

mediated immune responses and local inflammations were assessed in these two studies.

Recently, Shi et al. established a mouse model of mud crab (Scylla paramamosain) TM

Chapter 1

47

allergy in BALB/c mice by systematic sensitisation [176]. Oedema and lymphocytic

infiltration of the jejunum was observed in TM/alum treated mice. On the basis of this

mud crab TM model, the potential immunoregulatory effect of isolate sulfated

polysaccharide from Porphyra haitanensis (PHPS) was investigated. Repeated

systematic treatments of PHPS either before or after tropomyosin sensitisation could

skew a mixed Th1/Th2 immune pattern to more Th1 responses to TM, suggesting a

potential medical application of PHPS in crustacean allergy.

Different processing methods may impact on shellfish allergenicity [53], however few

mouse model studies have explained this topic. A very recent study of a IG mouse model

revealed that high hydrostatic pressure, in combination with thermal treatment could

significantly reduce the allergenicity of shrimp TM [55]. Such studies provide more

perspectives for food processing and storage technology targeting people allergic to

shellfish.

However, except for these animal models using TM, there are no animal models

developed for the other shellfish allergens. Despite the importance of TM, particularly as

a highly cross-reactive protein in IG and inhalant allergies, there are no published in vivo

studies, warranting a detailed analysis of the mechanisms underlying cross-reactivity to

TM derived from various allergen sources. The development of mouse models for

shellfish allergy and cross-reactivity could greatly improve the specificity and accuracy

of clinical diagnosis and medical advises on allergen avoidance.

1.4.5 Advances of food allergy mechanisms in mouse studies

The detailed immunological analysis of a mouse model not only plays a very necessary

role to evaluate the level of murine responses to allergens, but also reveals more

knowledge of the molecular mechanism underlying allergic reactions to food. For

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48

example, a recent study found that insulin-like growth factor (IGF) 2 could stimulate Treg

cell proliferation and improve Treg cell function in vivo. The IP administration of IGF2

and/or the adoptive transferring of Antigen (OVA)–specific Treg cells in an oral

OVA/CT-sensitised mouse model significantly reduced the serum IgE responses and the

inflammation in intestine [198]. More molecular components that may play a regulatory

role in the allergic pathway can be discovered using mice with target gene modified. The

genetic depletion of sphingosine-kinase (SphK) 1 and 2 in mice could alter the production

of Sphingosine-1-phosphate (S1P), thus suppressing the murine antibody responses to

OVA in oral sensitisation with acid-suppression and subsequently the accumulation of

CD4+ effector cells [199]. However, the OVA specific IgE and IgG1 level through IP

sensitisation of OVA mixed with Al(OH)3 was not affected by the absence of SphK 1 and

2. Therefore, SphK1 and 2 seems to play an important role in the regulation of CD4+ T

cells leading to intestinal allergies.

Furthermore, in a study of mouse model for peanut allergy, the provirus integration site

for Moloney murine leukemia virus (Pim) 1 kinase and downregulated expression of

Runt-related transcription factor (Runx) were found to contribute to the development of

intestine allergic responses via promoting Th2 and Th17 cell differentiation and

expansion [200]. The same research team also reported that the activation of Cyp11a1

may lead to peanut induced intestine anaphylaxis [201].

Chapter 1

49

1.4.6 Conclusion

The availability of different mouse models greatly improves our knowledge on food

allergies. The selection of different establishment parameters can contribute to the

sensitisation and effector mechanisms of murine immunity. The majority of published

mouse studies could reflect some of the essential features of human food allergy, such as

boosted levels of antigen-specific IgE antibody in serum and Th2 cytokines in tissue and

re-stimulated spleenocytes. However, the variability of mouse models increases the

difficulty to compare the different in vivo studies on food allergens and food allergy. Such

mouse studies might more easily conclude controversial views. Moreover, the

consistency of mouse models between laboratories has to be validated.

There are still many food allergens and hypotheses of therapeutic or preventive strategy

to be analysed using in vivo platforms. Further research on the role of non-allergen

components of food and environmental factors in the induction of allergic responses will

help to develop a more comprehensive understanding of food allergy.

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1.5 Summary and research synopsis of the thesis

In this chapter, a detailed review is provided on the current advances of food allergens,

cross-reactive allergens of shellfish in particular, as well as the establishment and

applications of murine models for food allergy. This introduction compares the

differential approaches in developing appropriate murine models for food allergy and

details specific findings for three major food allergen sources, peanut, milk and shellfish.

The work undertaken in this PhD thesis describes in-depth investigations on the

development of a good shellfish allergy mouse model, and subsequent evaluations of

immunological cross-reactivity between crustacean and molluscs species. Immunological

cross-reactivity to the pan-allergen tropomyosin has been particularly studied in murine

models.

The chapters of the thesis have the following specific aims and are outlined as follows:

The aim of Chapter 2 was to develop a novel shellfish allergy model. A series of

optimisation experiments using different mouse strains, sensitisation doses and mouse

sacrifice time-points were performed to elicit appropriate allergy-like humoral and

cellular responses in experimental animals. The mouse model was established with

predominant Th2 responses to Black tiger prawn (Penaeus mondon) using BALB/c mice.

The aims of Chapter 3 were to assess the immunological cross-reactivity between prawns

and abalone, but also to recognise the specific allergens responsible for cross-reactivity.

The heated allergen extracts of two commonly consumed shellfish species in the Asia-

Pacific region, Black tiger prawn (Penaeus mondon) and Jade tiger abalone (Haliotis

laevigata x Haliotis rubra) were generated and subsequently the immunological cross-

reactivity between these extracts at both humoral and cellular levels comprehensively

analysed using the established mouse model. Cross-reactive allergens between Black

Chapter 1

51

tiger prawn and Jade tiger abalone were identified on molecular level through IgE

immunoblotting and mass spectrometry.

The aims of Chapter 4 were to prepare recombinant tropomyosins from Black tiger

prawn and Jade tiger abalone and retained allergenicity as compared to natural

tropomyosin. This chapter details the identification and characterisation of tropomyosins

from Black tiger prawn and Jade tiger abalone. A detailed amino acid sequence analysis

was conducted to reveal the molecular basis of IgE cross-reactivity. Recombinant

tropomyosins of prawn and abalone were expressed and furthermore, their allergenicity

was confirmed by inducing allergy-like inflammatory responses in mice.

The aim of Chapter 5 was to investigate the potential immunological cross-reactivity

between prawn and abalone tropomyosins in detail using the mouse model. The roles of

antibodies, cytokines and effector cells in this immunological cross-reactivity was

thoroughly demonstrated.

The final Chapter 6 summarises the major findings of this thesis, but also elucidates the

essential contribution of these findings towards a better understanding of the detailed

cross-reactivity mechanisms in food allergy and improved diet managements for

susceptible populations to shellfish allergy.

Chapter 1

52

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CHAPTER

ESTABLISHMENT OF A MOUSE MODEL

FOR PRAWN ALLERGY

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80

2.1 Introduction

Shellfish are good sources of protein and have become a very popular option in human

diets. Shellfish are a very large and diverse group of invertebrates including more than

50,000 crustacean species and 100,000 mollusk species. The most widely consumed

shellfish species are prawn, lobster, crab, abalone, oyster, squid, clam, cockle, snail and

mussel [1]. With the worldwide increasing consumption of shellfish, reports of people

with allergies to shellfish have increased significantly in recent decades. Shellfish allergy

has been reported as the most common food allergy in adults in the United States [2, 3].

However, there is still no cure for shellfish allergy available, and prevention of allergic

reaction relies currently on avoidance. Furthermore, the sensitisation phase and effector

mechanisms of shellfish allergy have not been fully understood.

Mouse models are very useful tools for studying several key aspects of allergic diseases,

including the understanding of the humoral and cellular mechanisms, the development of

preventive and therapeutic strategies and the in vivo evaluation of the allergenic potential

of proteins [4-6]. In food allergies, many mouse models have been developed, especially

for sensitisation to ovalbumin and peanut-induced allergies. However, mouse models

characterising allergic responses against shellfish components are still very limited.

Current models of food allergy have been developed using a high variety of protocols [6].

These models usually consist of sensitisation phase and subsequent challenge phase,

while the corresponding exposure route and allergen dose at each phase differs greatly in

the different induction protocols. Also, mouse strains, allergen sources and sensitisation

adjuvants are very essential parameters to be considered carefully because of their

potential influence on the phenotype of murine allergic responses.

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81

Many allergy studies have utilised the BALB/c mouse strain, which is believed to have a

mainly Th2 focused immune response. In contrast, C57BL/6 mice are less used in food

allergy research, probably due to the relatively weak allergic responses to a few allergens

[7]. However, the exact association of the genetic background with the susceptibility to

food allergy remains unclear and the comparison between BALB/c and C57BL/6 mice

seems to be similar in their mechanisms of asthma inflammation [8]. Taking in

consideration the enormous genetic variants available in C57BL/6 mice, I investigated

and compared in this study both mouse strains with their response to prawn extracts.

Black tiger prawn (Penaeus monodon) is one of the most essential commercial products

of the seafood industry in Southeast Asia and is widely distributed in the Indo-West-

Pacific region. The major allergens of Black tiger prawn (BTP) have been well-studied

using immunochemical approaches and advanced mass spectroscopy [9, 10]. Therefore,

BTP is a good representative shellfish species used to develop a mouse model for food

allergy to shellfish.

In patients, the identification of the sensitisation route and specific dose of allergen in the

sensitisation phase is often very difficult to establish. The possible routes of sensitisation

with food allergens are the gastrointestinal tract, skin contact or via airways [11].

Although there is no evidence to show the predominant sensitisation site in patients with

allergies to shellfish, the majority of reported mouse models for food allergies utilise the

oral route, using cholera toxin (CT) as a super antigen to break the murine oral tolerance.

However, the interference of CT does not reflect the usual pathogenesis of human food

allergies and the mechanisms through which CT enhances murine Th2-biased responses

remains to be described [12]. Unlike the oral sensitised models, some allergy models use

the systemic sensitised route. The immune response is improved by using alum through

better absorption of the allergens. Alum can help by delivering higher concentrations of

Chapter 2

82

allergens, where the allergenic proteins are slowly released to antigen stimulation cells

and enhance the allergen presentation to Th2 cells [13]. Alum is also one of the most

common adjuvants used for human immunisations [14]. In the current project, to establish

the shellfish-allergy model, I chose to administrate the allergens initially via

intraperitoneal (IP) sensitisation with shellfish whole protein extract mixed with alum.

Furthermore, to establish the best dose of sensitisation, different amounts of protein

extracts (10-500 μg) were compared in their sensitisation potential.

This chapter was aimed to establish a novel shellfish allergy model to mimic the allergic

sensitisation of patients to shellfish. The mouse model was optimised after comparing

different allergen concentration during IP sensitisation and subsequent oral challenge. In

particular, two different mouse strains were compared for their humoral and cellular

responses to heated Black tiger prawn protein extracts to establish the best protocol for a

novel murine model for shellfish allergy.

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83

2.2 Aims

The aims of Chapter 2 were:

(1) To design the best induction protocol of a mouse model for prawn allergy.

(2) To characterise the mouse model of prawn allergy in detail by assessing the

immunological responses including serum antibodies, splenic cytokines and

granulocyte, circulating granulocytes, as well as the pathology of the jejunum.

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84

2.3 Animals, materials, and methods

2.3.1 Animals

C57BL/6 mice and BALB/c mice (7 or 8-week-old females) were housed at the Cairns

campus, building E4 Animal Facility under specific pathogen-free conditions, and

maintained on a commercial diet purchased from Specialty Feeds. The experiments were

approved by the animal ethics committee from James Cook University (Animal ethics

approval number A2108).

2.3.2 Preparation of shellfish extracts

2.3.2.1 Preparation of heated Black tiger prawn extracts

Black tiger prawns (Penaeus monodon) were purchased from local seafood shops. To

mimic the most common cooking methods for human consumption, the whole prawns

were washed with Milli-Q water and subsequently heated in 1 x PBS (phosphate buffered

saline) at 100 °C for 20 min. Then the outer shell was removed and the abdominal muscles

shredded into small pieces and homogenised in 200 ml PBS using gentleMACS Octo

Dissociator (Miltenyi Biotec). The protein slurry was mixed overnight at 4 °C.

Supernatant was collected into new Falcon tubes after centrifugation at 13,000 RPM for

30 min using Beckman Coulter Avanti J-26 XP. The heated Black tiger prawn extracts

(hBTP) were filtered through 0.22 μm membrane filter (Millipore) before used for

intraperitoneal (IP) injections. The concentration of proteins was estimated using Pierce

BCA Protein Assay Kit (Thermo Fisher Scientific) and adjusted with PBS. Heated

shellfish extracts could be added into Amicon Ultra-15 centrifugal filters (REF

UFC900396) and concentrated by centrifugation. The aliquots of prawn extracts were

named “hBTP" and stored at -80 °C until further use.

Chapter 2

85

2.3.2.2 Protein concentration estimate

Following the manufacturer’s instruction, Pierce BCA Protein Assay Kit (Thermo Fisher

Scientific) was used for the estimation of proteins. Briefly, 10 μl of Albumin (BSA)

Standards were 1:2 serially diluted in microcentrifuge tubes with PBS buffer. The

concentration of the highest standard was 2 mg/ml and the blank standard is PBS buffer

only. 10 μl of neat or diluted samples were prepared as well. Solution A and B were mixed

well at 50:1. 200μl of the mixture of Solution A and B were added into each tube of BSA

standards or samples, following by incubation at 55-60 °C for 15 min in a water bath.

After a quick spinning down of all the tubes, 100 μl of reaction from each tube was

transferred into 96 well plates. When the reaction cooled to room temperature, the

microplate was read at 562 nm in a spectrophotometer (POLARstar Omega microplate

reader). The standard curve was generated by plotting the average for each BAS standard,

corrected with the reading for the Blank and calculated in mg/ml. The protein

concentration of each unknown sample was determined using the standard curve.

2.3.3 Establishment and evaluation of mouse models in shellfish allergies

2.3.3.1 Mice sensitisation and challenge with allergens

2 mg of Imject™ Alum Adjuvant (Thermo Fisher Scientific) was added to the hBTP in

150 μl PBS and mixed on a suspension mixer (Ratek) for 30 min, so alum could

effectively absorb the allergen proteins.

Mice received three intraperitoneal (IP) injections of the allergen and alum mixture on a

weekly basis. Mice were fasted overnight but had unlimited access to water before the

intragastric (IG) challenge. On the challenge dates, all allergen-sensitised mice were

orally fed on the allergens in PBS, while the control mice received PBS only. After each

oral challenge, mice were visually monitored for 60 min. 24 hours after the last oral

Chapter 2

86

challenge, mice were sacrificed and for further analysis samples isolated from blood,

spleen, stool, and jejunum.

Every experimental design is shown in the first figure under each experimental section of

Chapter 2; Figure 2.1 in Experiment #1, Figure 2.6 in Experiment #2, and Figure 2.11 in

Experiment #3.

2.3.3.2 Measurement of antibodies and mast cell protease-1 in serum

Isolation of mouse serum

Mice were euthanised by CO2 asphyxiation and cardiac puncture was conducted to

harvest blood. Mouse blood was collected in clean microcentrifuge tubes and cooled at 4

°C for 1 hour. The blood was then centrifuged at 13,000 RPM for 20 min to separate

serum from blood clot. Serum was transferred into new tubes and stored at -20 °C for

future analysis.

Serum immunoglobulin levels

hBTP specific IgE, IgG1 and IgG2a antibody were measured in serum by sandwich

enzyme-linked immunosorbent assay (ELISA). Briefly, BD Falcon 96-well Flat-bottom

ELISA Plates (Becton Dickinson) were coated with 100 μl of 20 μg/ml hBTP, wrapped

with an adhesive plastic and incubated overnight at 4 °C. Coated plates were flicked over

a sink to remove the liquid. The remaining drops were discarded by patting the plate on

clean paper towels. Next, coated plates were blocked with 200 μl of 10% fetal bovine

serum (FBS) in PBS. The plates were wrapped with an adhesive plastic and incubated at

room temperature for one hour or 4 °C overnight. The liquid was removed and 50 μl of

individual serum samples, standards and blank (PBS only) were added to the plates. In

IgE and IgG2a plates, neat or 1:2 diluted serum was loaded. For IgG1 specific plates,

serum was diluted 1:10 with PBS. The plates were incubated at room temperature for two

Chapter 2

87

hours or preferably 4 °C overnight for maximal sensitivity. The serum was discarded or

recovered into clean microplates to be reused for other purposes. Plates were washed three

times by filling the wells with PBS/0.05% Tween20. 50 μl of 1:500 diluted detection

antibody (Biotin Rat anti-mouse IgE, Biotin Rat anti-mouse IgG1 or Biotin Rat anti-

mouse IgG2a respectively, Becton Dickinson) with PBS-2% FBS was added to each well.

The plates were covered with an adhesive plastic and incubated for 1.5 hours at room

temperature. Plates were washed five times with PBS-0.05% Tween 20. 50 μl of

streptavidin-HRP was added into each well, following with incubation in dark at room

temperature for 30 min. The plates were washed five times with PBS-0.05 % Tween 20.

Liquid was discarded and made sure no air bubbles were remaining in the wells. TMB

substrate (Becton Dickinson) was warmed up to room temperature before use. Equal

volumes of Substrate Reagent A and Substrate Reagent B were freshly mixed before each

experiment. 50 μl of the mixed substrate was loaded into each well to develop the desired

colour reaction. Normally this step would take 1 to 2 minutes but longer for the detection

of specific IgE. The last reagent added is 50 μl of 1 M HCl per well to stop the reaction.

As a result, the colour changed from blue to yellow. The plates were read at 450 nm using

a POLARstar Omega microplate reader (BMG LABTECH). The average 450 nm

absorbance measurement of the Blank standard replicates was subtracted from the 562

nm measurements of all other individual standards and unknown samples.

Total IgE antibody was measured in serum by sandwich ELISA. The basic protocol was

the same as the measurement of specific IgE, except that the coating solution was purified

rat anti-mouse IgE antibody (Becton Dickinson) diluted 1: 500 and serum diluted at1:10.

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88

Serum mouse mast cell protease-1 level

Serum mouse mast cell protease (mMCP)-1 level was detected using Mouse MCPT-1

(mMCP-1) ELISA Ready-SET-Go reagent set (eBioscience) according to the

manufacturer protocol. Briefly, BD Falcon 96-well Flat-bottom ELISA Plates (Becton

Dickinson Pty Ltd) were coated with 100 μl of 1 x MCP-1 capture antibodies which was

diluted with 1 x coating buffer. The plates were sealed and incubated overnight at 4 °C.

Coated plates were washed three times. During every wash, the wells were filled with

wash buffer, incubated for 1 min before discarding liquid. Next, coated plates were

blocked with 200 μl of 1 x Assay Diluent for 1 hour at room temperature. The top standard

concentration (15,000 pg/ml) was prepared by adding 15 μl of Standard to 1 ml of

ELISA/ELISPOT Diluent. 2-fold serial dilutions of the top standards with 1 x Assay

Diluent were performed. Eight points in total were needed to produce the standard curve.

50 μl of samples and standards were added to the wells following by two-hour incubation

at room temperature or overnight at 4 °C. After five washes with wash buffer, the 50 μl

of 1 x detection antibody was loaded into each well and the plates were incubated for one

hour at room temperature. After that, the plates were washed five times before 30 min

incubation with 50 μl of 1 x streptavidin-HRP at room temperature. The ELISA plates

were washed seven times and then 50 μl of substrate solution was added for another 15-

min incubation at room temperature. 50 μl of 1 M HCl was used to stop the reaction. Then

the plates were read at 450 nm and the values of samples were calculated on the basis of

the standard curve.

Chapter 2

89

2.3.3.3 Cell stimulation with allergens

Preparation of single cell suspension

Sing cell suspension of spleens and mesenteric lymph nodes (MLNs) was prepared for

flow cytometry analysis or cell culture. For a sterile cell suspension preparation, all the

instruments were soaked in ethanol or autoclaved.

Firstly, 2 ml of the cell wash buffer was added into a culture plate on ice for the organ

collection. Organs with wash buffer were then transferred into gentleMACS tubes

(Miltenyi Biotec). The tubes were placed onto the gentleMACS Octo Dissociator

(Miltenyi Biotec Australia) and a program of 55 sec mincing was applied. To prepare

cells for culture use, the subsequent organ processing was conducted in Class II Biosafety

Cabinet (ESCO). The minced organs flowed through 70 μm cell strainers (Biologix

Group Limited) and the un-minced pieces were scratched against a mesh using a clean 2

ml syringe plunger. About 13 ml of cell wash buffer was used to rinse the cell strainer,

and the suspended cells from each mouse were collected individually into a clean 15 ml-

size conical tube. After centrifugation at 1300 RPM for 10 min at room temperature, the

supernatant was removed. Cell pallets from spleens were resuspended with 1 ml of Red

Blood Lysis Buffer (Sigma). Following with 1-min incubation, 13 ml of wash buffer was

added and cell suspension was centrifuged at 1300 RPM again for 7 min. The splenocyte

pallet was resuspended with 8 ml complete culture medium and ready for cell counting.

In contrast, MLN did not undergo red blood lysis and MLN cell pallet was resuspended

with 2 ml of complete culture medium. All the single cell suspension was kept on ice.

Chapter 2

90

Cell counting

10 μl of suspended cells from each spleen or MLN was loaded individually into the well

of a 96-well plate. 10 μl of 0.4% trypan blue solution (Sigma) was mixed up with each

sample. 10 μl of each strained cell sample was loaded into a chamber of the counting slice

(BIO-RAD). The number of live cells was read and recorded using the TC20 Automated

Cell Counter (BIO-RAD). The cell concentration was accordingly adjusted with the

complete culture medium, and ready for cell stimulation and flow cytometric analysis.

In vitro cell stimulation with allergens

In Chapter 2, Experiment #1 and #2, one million cells (from spleen or MLN) in 100 μl of

the complete culture medium were seeded into each well of a 96-well U-bottom cell

culture plate (FALCON). 100 μl of 80 μg/ml allergen solution (hBTP) was added into

cells to stimulate the cytokine production. The unstimulated cells were only incubated

with 100 μl of the culture medium. All the cell culture plates were placed in 5% CO2 at

37°C using SANYO CO2 Incubator Model MCO-18AIC (UV) Dual Stack. 72 hours later,

the culture plates were centrifuged at 1500 RPM for 15 min, and the supernatant was

collected and stored at -80 °C for cytokine analysis.

A few modifications of the protocol were made in Experiment #3. 0.5 million cells were

stimulated with 40 μg/ml hBTP. Moreover, the positive control was obtained by

incubating 0.5 million cells with 100 μl of 1 μg/ml anti-CD3/anti-CD28.

Chapter 2

91

2.3.3.4 Flow cytometry

Preparation of single cell suspension of spleens and MLNs has been described

above in Section 2.3.3.3 above.

Isolation of peripheral blood mononuclear cell (PBMC) from the whole blood

Blood was harvested from mouse heart. Two to three drops of mouse blood were collected

into a microcentrifuge tube and immediately mixed well with 20 μl of 1,000 unit/ml

heparin as the anticoagulant. The cells were incubated with 1 ml of Red Blood Lysis

Buffer (Sigma) for 5 min, and subsequently centrifuged at 1300 RPM at 4 °C for 10 min.

The cell pallets were re-suspended with 200 μl of Fluorescence-activated cell sorting

(FACS) buffer. Cell suspension was transferred into a 96-well plate, ready for cell surface

immunofluorescence staining.

Cell surface immunofluorescence staining

The populations of eosinophils and basophils in PBMC (Experiment #2), spleen and

MLN (Experiment #3) were analysed by means of flow cytometry. The stain panel

included 7 fluorescence-conjugated antibodies (Jomar Life Research), including Anti-

Mouse CD170 (Siglec F) PerCP-eFluor 710, Anti-Mouse CD3 APC-eFluor 780, Anti-

Mouse IgE FITC, Anti-Mouse CD11b PE, Anti-Mouse CD49b (Integrin alpha 2) APC,

Anti-Mouse CD19 APC-eFluor 780, Anti-Mouse Fc epsilon Receptor I alpha (FcεR1)

eFluor 450. These antibodies were 1:300 diluted with FACS buffer and mixed together.

All the volume of PBMC or 0.5 x 106 cells from each spleen or MLN were prepared in

the 96-well plates, and centrifuged at 1500 RPM for 3 min at 4 °C. The plates were flicked

to remove the liquid and gently patted on paper tissues. 200 μl of FACS buffer was added

to wash the cells. Subsequently, 20 μl of Fc block was added to resuspend cells, following

with incubation for 5 min at room temperature. Next, the cells were incubated with 50 μl

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of the antibody mix in dark for 30 min on ice. 150 μl of FACS buffer was added to wash

away the redundant antibodies. After centrifugation at 1500 RPM for 3 min, the cell

pallets were resuspended with 200 μl of Fixation buffer (Jomar Life Research), and

incubated on ice for 20 min in dark. Cells were centrifuged and finally resuspended in

200 μl of FACS buffer. The cell plates were kept in dark and stored at 4 °C, ready to be

analysed using BD FACS Canto II.

2.3.3.5 Preparation of jejunum protein extracts

Mouse duodenum is about 4-5 cm in 8-week-old mice, starting from the connection point

with the lateral stomach. Mouse jejunum locates next to duodenum. In this study two 1-

2 cm segments of jejunums were harvested. The first segment was used for protein

extraction following the protocol below. The second segment for producing

histopathological slides, which was described in Section 2.3.3.7.

Firstly, mouse jejunum was briefly washed with PBS buffer in a clean petri dish. The

washed jejunum was placed in a round-bottom microcentrifuge tubes with a metal bead

and 500 μl PBS on ice. Jejunums were homogenised at 4 °C under the frequency of 25/sec

for 5 min using TissueLyser II (QIAGEN 08-08569), following with centrifugation at 13,

000 RPM for 5 min at 4 °C. The supernatant was then transferred in a new tube and stored

at -80 °C for the analysis of cytokines or the measurement of allergen-specific IgA

antibody.

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2.3.3.6 Measurement of cytokines

Cytometric Bead Array (CBA)

Both the samples from cell re-stimulation experiments and protein extracts of jejunums

were briefly centrifuged to make sure only the supernatant was pipetted for CBA assays.

Firstly, the panel of beads was calculated and prepared. 0.5 μl of each BD CBA Flex Set

capture or detection bead was taken for each sample. Every vial of beads needs to be

vortexed thoroughly before opening. Ten different beads targeting cytokines IL-4, IL-5,

IL-6, IL-9, IL-10, IL-13, IL-17, IL-23, IFN-gamma and MCP-1, were included in the

panel. CBA wash buffer, assay diluent and capture diluent were filtered through sterile

0.22 μm membranes before use. The diluent volume was calculated based on using 50 μl

of diluted mixed beads per sample (well). Ten types of beads were mixed well together

with diluent by vortex. A serial 1:2 dilution was performed in microcentrifuge tubes

starting from the pre-mixed top standard. A total of 9 standard tubes (60 μl per tube) were

prepared with filtered assay diluent.

Next came the plate protocol. 96-well plates were wet with 100 μl of filtered wash buffer.

The liquid was discarded by hard flick and patting on clean paper towels. 50 μl of diluted

capture beads was added into each well. 50 μl of the standards and samples were added

into each well for one-hour incubation at room temperature. Subsequently, 50 μl of

diluted detection beads was added into each well and the plates were incubated in dark

for one hour. The supernatant of each well was removed by gentle flick following with

centrifugation at 2,000 RPM for 2 min. 150 μl of filtered wash buffer was added into each

well and the plates of samples were acquired immediately using BD FACS Canto II. The

minimum detection levels for each cytokine were: IL-6, 1.4 pg/ml; IL-9, 10.7 pg/ml;

MCP-1, 2.7 pg/ml; IL-23, 1.04 pg/ml; IL-17, 0.95 pg/ml; IL-4, 0.3 pg/ml; IL-5, 0.9 pg/ml;

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IL-10, 9.6 pg/ml; IL-13, 2.4 pg/ml; and IFN-gamma, 0.5 pg/ml. When the calculated

cytokine concentration was below the given sensitivity, it was treated as undetectable.

Enzyme-linked immunosorbent assay (ELISA)

Levels of mouse cytokines including IL-5, IL-13 and IFN-gamma were measured using

Mouse ELISA Ready-SET-Go reagent sets (eBioscience) and following the protocols

from the manufacturer. Briefly, 96-well Flat-bottom ELISA Plates (Becton Dickinson)

were coated with 100 μl of 1 x corresponding cytokine (IL-5, IL-13 or IFN-gamma)

capture antibody which was diluted with 1 x coating buffer. The plates were sealed and

incubated overnight at 4 °C. Coated plates were washed three times with PBS/0.05%

Tween 20. Next, coated plates were blocked with 200 μl of 1 x Assay Diluent for 1 hour

at room temperature. The top standard concentration was 500 pg/ml for IL-5 and IL-13,

and 2000 pg/ml for IFN-gamma, which was prepared by adding corresponding cytokine

Standard to ELISA/ELISPOT Diluent. 2-fold serial dilutions of the top standards with 1

x Assay Diluent were performed. Eight points in total were needed to produce standard

curve. 50 μl of samples and standards were added into wells following by two-hour

incubation at room temperature or overnight at 4 °C. After five washes with wash buffer,

the 50 μl of 1 x detection antibody was loaded into each well and the plates were incubated

for one hour at room temperature. After that, the plates were washed five times before 30

min incubation with 50 μl of 1 x Avidin-HRP at room temperature. The ELISA plates

were washed seven times and then 50μl of TMB was added for another 15 min incubation

at room temperature. 50 μl of 1 M HCl was used to stop the reaction. Then the plates were

read at 450 nm and the values of samples were calculated on the basis of standard curve.

The detection limit was: IL-5 and IL-13, 4pg/ml; and IFN-gamma, 15 pg/ml. When the

calculated cytokine concentration was below the given sensitivity, it was treated as

undetectable.

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2.3.3.7 Histology

After mice were sacrificed, lungs and jejunums were collected for histopathological

examination. See “Preparation of jejunum protein extracts” for the method of harvesting

jejunums. The second 1-2 cm segment of jejunum was carefully cut open and washed

with PBS in a petri dish. The washed jejunum was preserved in 10% formalin at 4 °C for

24 hours and then transferred into 70% ethanol for long-time storage. The lung and

oesophagus samples were processed the same way after isolation. Then, the preserved

samples were shipped to QIMR Berghofer (Brisbane, Australia) to produce haematoxylin

and eosin (H&E) stained histopathological slides.

2.3.3.8 Measurement of allergen-specific IgA antibody

The protocol to detect the level of IgA antibody was similar with the measurement of IgE

antibody and mMCP-1. Briefly, 100 μl of 20 μg/ml hBTP was seeded into each well of

96-well ELISA plates (Becton Dickinson) and incubated at 4 °C overnight. The hBTP-

coated ELISA plates were flicked, washed with PBS/0.05% Tween 20 and patted on clean

paper towels. Subsequently the ELISA plates were blocked for one hour at room

temperature. The ELISA plates were washed once and then 50 μl of neat jejunum extracts

and PBS as blank were added into plates to incubate overnight at 4 °C. After that, plates

were washed three times with PBS/0.05% Tween 20. 50 μl of 1:500 diluted biotin anti-

mouse IgA antibody (Biolegend) was added to each well and plates were incubated for

1.5 hours at room temperature. Next, plates were washed five times with PBS/0.05%

Tween 20. 50 μl of 1 x streptavidin-HRP was added into wells, following with incubation

in dark at room temperature for 30 min. the plates were washed five times with

PBS/0.05% Tween 20. 50 μl of substrate was loaded into each well. Once the desired

colour reaction was developed, 50 μl of 1 M HCl was added on top of each well. The

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plates were read at 450 nm using POLARstar Omega microplate reader (BMG

LABTECH).

2.3.3.9 Statistical analysis

To compare the difference between every two groups of mice, the two-tailed Mann-

Whitney U test was performed using Prism 6.0. Data are presented as mean values with

the standard error of the mean (SEM). p values of less than 0.05 were considered

statistically significant.

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2.4 Results

2.4.1 Experiment #1:

Different sensitisation doses and time-points in C57BL/6 mice

2.4.1.1 Experimental design

Animals: C57BL/6 mice (8-week-old females) were obtained from the Animal Resource

Centre, Australia. Mice were housed at the Cairns campus, building E4 Animal Facility

under specific pathogen-free conditions, and maintained on a commercial meat free diet

purchased from Specialty Feeds. The experiments were approved by the animal ethics

committee from James Cook University (Animal ethics approval number A2108).

Mice sensitisation and challenge with allergens: Mice received three intraperitoneal (IP)

injections of 10 μg of hBTP mixed with 2 mg of alum in Group A, 20 μg of hBTP mixed

with alum in Group B, or PBS with alum in Group D. Injection proceeded on Days 0, 7

and 14. Group C received only one injection with 50 μg of hBTP on Day 0. Mice were

fasted overnight but had access to water before oral challenge. On Day 21 and Day 24,

all hBTP-sensitised mice were fed with 100 μg of hBTP in PBS, while Control mice PBS

only. The experimental design is shown in Figure 2.1. 24 hours after the last oral challenge,

mice were sacrificed, and mouse blood, spleen, jejunum were isolated.

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Figure 2.1 Protocol of inducing shellfish allergy in a mouse model. The schedules for

IP sensitisation and oral challenge are shown. Each group comprised of six mice.

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2.4.1.2 Induction of antibody and mast cell protease-1 in sera

Figure 2.2 Levels of serum antibodies including hBTP-specific IgE (A), IgG1 (B) and

IgG2a (C), and mMCP-1 (D). Sera were obtained on the sacrifice day from C57BL/6

mice immunised with different doses of hBTP or with PBS only. mMCP-1 and hBTP-

specific IgE, IgG1 and IgG2a levels were determined by sandwich ELISA. Sera were neat

for the measurement of IgE antibody and mMCP-1; 1: 10,000 for IgG1 antibody and

1:100 for IgG2a antibody. Data are reported as mean ± SEM (n = 6) for each group. p

< 0.05, and p < 0.01. Each dot represents an individual mouse.

*

**

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To determine the induction of allergy-like responses, I investigated the generation of

allergen-specific immunoglobulins (Ig) after weekly IP administration of hBTP with alum.

As shown in Figure 2.2.A, the levels of circulating hBTP-specific IgE were low in all

hBTP-treated mice, indicating that the C57BL/6 mice were insufficiently sensitised. IgG1

and IgG2a antibody are also indicative parameters of immune responses. Figure 2.2.B

and C demonstrate that all hBTP-treated mice were able to provoke significantly

increased levels of allergen-specific IgG1 and IgG2a antibodies in comparison with PBS

control mice. No allergen-specific IgE antibody and IgG1 antibody were induced in

control mice injected with PBS mixed with alum.

Mast cells are one of the essential effector cells leading to allergic inflammation. The

level of mMCP-1 enzyme is therefore regarded in this study as the marker of mast cells

activation and degranulation. mMCP-1, classified as a β-chymase, is believed to be

predominantly expressed in intestinal mucosal mast cells. mMCP-1 can therefore be used

as an indicator of IgE-mediated intestinal allergic responses and might be related with a

variety of inflammatory events including permeability changes and leucocyte infiltration.

In this case, Figure 2.2.D demonstrates the increased level of circulating mMCP-1 in all

hBTP-sensitised mice. Comparing with one IP injection with 50 μg of hBTP, the

administration of three IP injections of 20 μg or 10 μg of hBTP could induce significantly

higher levels of serum mMCP-1 in mice.

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2.4.1.3 Production of cytokines by splenocytes in vitro

Figure 2.3 Production of cytokines by unstimulated splenocytes: IL-4 (A), IL-5 (B),

IL-6 (C), IL-9 (D), IL-10 (E), IL-13 (F), IL-17A (G), IL-23 (H), mMCP-1 (I) and

IFN-gamma (J). The splenocytes were cultured in the absence of hBTP for three days.

The levels of cytokines were determined by CBA. Data are reported as mean ± SEM (n

= 6) for each group. p < 0.05, and p < 0.01. Each dot represents an individual mouse.* **

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To determine if the local cellular activation could drive an allergen-specific Th2-cellular

immune response, secreted cytokines were measured in supernatants of splenocytes re-

stimulated in vitro. Single cell isolated from spleens were incubated for 72 h with culture

medium only or 100 μl, 80 μg/ml of hBTP. IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A,

IL-23, mMCP-1 and IFN-gamma contained in the supernatant of cell culture were

measured by CBA.

Comparing with hBTP-re-stimulated splenocytes, unstimulated splenocytes that were

incubated with completed cell culture medium only produced lower levels of cytokines,

except for mMCP-1 and IFN-gamma, shown in Figure 2.3. In unstimulated cultures,

levels of Th2 cytokines in hBTP-treated mice were not significantly different from those

in control mice, except for IL-17A from the mouse group of 10 μg x 3. IL-17A is believed

to be involved in inflammation, but the contribution of IL-17A to allergic diseases

remains controversial [15, 16]

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Figure 2.4 Production of cytokines by hBTP-stimulated splenocytes: IL-4 (A), IL-5

(B), IL-6 (C), IL-9 (D), IL-10 (E), IL-13 (F), IL-17A (G), IL-23 (H), mMCP-1 (I) and

IFN-gamma (J). The splenocytes were cultured with hBTP for three days. The levels of

cytokines were determined by CBA. Data are reported as mean ± SEM (n = 6) for each

group. p < 0.05, and p < 0.01. Each dot represents an individual mouse.* **

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As demonstrated in Figure 2.4, in the supernatant of hBTP-restimulated splenocytes,

multiple cytokines including IL5, IL-6, IL-10, IL-17A and IL-13 presented statistically

higher levels than the PBS control group, although the production of IL-4 and IL-9 was

still very low. IL-4, IL-5, IL-10, IL-6, IL-9 and IL-13 are classic Th2-associated cytokines.

IL-23 seemed to be a non-relevant cytokine to prawn allergy as its production was low

by either unstimulated or hBTP-re-stimulated splenocytes (Figure 2.3.H and Figure

2.4.H).

Comparing the different treatment groups, the mice that received one IP administration

of 50 μg of hBTP on Day 0 had stronger responses against hBTP as demonstrated through

higher expression levels of IL-5, IL-6, IL-9, IL-13 and IL-17A. Moreover, only

splenocytes from mice that received 50 μg of allergens released a significantly higher

level of IFN-gamma than control mice after re-stimulation with hBTP (Figure 2.4.J).

2.4.1.4 Production of cytokines in jejunums

To explore the immune responses of the intestine of allergen challenged mice, the levels

of the cytokines in the total extracts of mouse jejunums were also investigated. As shown

in Figure 2.5, the levels of all cytokines and mMCP-1 were very low and IL-6, IL-9 and

IL-13 were not detectable in any jejunum extracts. The levels of IL-17A and IL-10 in the

jejunums from mice treated with 50 μg of allergens were marginally higher than other

mouse groups. However, Figure 2.5 demonstrates that none of these detectable cytokines

displayed a significantly higher level in hBTP-sensitised mice as compared to PBS

control mice. In contrast to cytokines, the levels of jejunum-based MCP-1 in the 10 μg x

3 group and 20 μg x 3 group were significantly higher than the control group.

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Figure 2.5 Production of cytokines in homogenised jejunum: IL-4 (A), IL-5 (B), IL-

10 (C), IL-17A (D), IL-23 (E), mMCP-1 (F) and IFN-gamma (G). The total protein

extracts of jejunums in PBS were prepared to measure the levels of mMCP-1 and

cytokines by CBA. Data are reported as mean ± SEM (n = 6) for each group. p < 0.05,

and p < 0.01. Each dot represents an individual mouse.

*

**

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The aim of this study is to generate a mouse model that can exhibit key characteristics of

IgE-mediated adverse immune responses to shellfish allergens. In this study, increased

levels of serum IgG1 and mMCP-1 were detected, as well as elevated levels of multiple

Th2 cytokines in hBTP-treated mice as compared to PBS control mice. IgE antibodies

play a pivotal role in allergic conditions in humans as well as in mice. However, in this

study only low levels of elevated allergen-specific IgE antibody were detected in the sera

of hBTP-sensitised C57BL/6 mice. Furthermore, no significant Th2 cytokine responses

were observed in jejunum extracts.

To summarise, the results of this study indicate that the current protocol of sensitisation

and challenge with prawn extracts cannot effectively prime sufficient Th2 responses,

which are required for an appropriate in vivo research tool. C57BL/6 may not be a good

mouse strain responding to prawn allergens. Three doses of 10 μg or 20 μg of hBTP can

trigger a higher level of circulating IgG antibodies and mMCP-1, while one dose of 50

μg of hBTP confers a stronger production of cytokines and mMCP-1 as demonstrated by

re-stimulated splenocytes.

Based on the results in Experiment #1, in the next experiment (Experiment #2) two

modifications on the experimental protocol were made to achieve higher Th2 responses.

Firstly, the mouse strain was changed to BALB/c mice that have been previously used for

studying egg and milk allergy [17, 18]. Secondly, to induce sufficient allergic responses,

different doses of IP sensitisation were compared and the doses of oral exposure were

increased in BALB/c mice.

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Increased sensitisation doses and oral challenges in BALB/c mice


Animals: BALB/c (8-week-old females) were obtained from the Animal Resource

Center, Australia. Mice were housed at the Cairns campus, building E4 Animal Facility

under specific pathogen-free conditions, and maintained on a fish meal free diet

purchased from Specialty Feeds. The animal ethics committee from James Cook

University approved the experiment.

Mice sensitisation and challenge with allergens: Every group consisted of six BALB/c

mice. Mice received three IP injections of different doses of hBTP mixed with 2 mg of

alum in Group A, B and C. Each sensitisation dose of hBTP on each injection day (Day

0, 7 and 14) consisted of 50 μg in Group A, 200 μg in Group B, and 500 μg in Group C.

All hBTP-sensitised mice were orally challenged with 1 mg of hBTP starting from Day

21. A two-day interval was introduced between oral challenges and a total of five

challenges were performed. Group D consisted of control mice injected with 200 μl of

PBS mixed with 2 mg of alum on Day 0, 7 and 14, and subsequently orally challenged

with 200 μl of PBS for five times on the same challenge dates as hBTP-sensitised mice

in Group A, B and C. Mice were fasted overnight but had unlimited access to water before

every oral challenge.

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The experimental design is shown in Figure 2.6. 24 hours after the last oral challenge,

mice were sacrificed. Blood was isolated one day after the 2nd challenge (Day 25), one

day after the 4th oral challenge (Day 31) and also on the sacrifice day (Day 34).

Figure 2.6 Optimised sensitisation and oral challenge protocol, on the basis of

Experiment #1, to establish the mouse model for shellfish allergy. The schedules of

IP sensitisation, oral challenge and the sample collection are shown. Each group

comprised of six mice.

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2.4.2.2 Induction of serum antibody responses

To track down the dynamic changes of serum IgE antibody, mouse serum was isolated at

different time-points. Figure. 2.7.A, B and C demonstrate that the ranks of the levels of

total IgE serum antibody among different mouse groups remained the same on Day 25,

31 and 34. Mice that received the IP sensitisation with 50 μg of hBTP had the highest

levels of IgE antibody in their sera. There is no significant difference between the other

two hBTP-treated mouse groups, whilst the mean value of IgE antibody levels in sera

from the lower-dose sensitised group (200 μg x 3 of hBTP) seems slightly higher than

that in sera from mice sensitised with 500 μg x 3 of hBTP. This trend of lower

sensitisation dose of hBTP provoking higher polyclonal IgE antibody responses was

consistent with the data of hBTP-specific IgE antibody (Figure 2.7.D).

After mice were orally challenged twice in Figure.2.7.A, the 50 μg x 3 group had

significantly higher levels of total IgE antibody than all the other mouse groups, while

there was no statistical difference between higher-dose-sensitised mice (200 μg x 3 or

500 μg x 3) and control mice in the generation of total IgE antibody.

With the increase of oral exposure to hBTP, the levels of total IgE antibody in sera from

mice sensitised with 200 μg x 3 and 500 μg x 3 was significantly elevated compared to

the PBS control (Figure 2.7.B, C and D), but still less than the level of serum total IgE

antibody in the 500 μg x 3 group. The tendency that the lower IP dose elicited a higher

serum antibody response was confirmed through the results of allergen-specific IgE

antibody (Figure 2.7.D).

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Figure 2.7 Levels of total serum IgE on Day 25 (A), 31 (B) and 34 (C), and hBTP-

specific IgE on Day 34 (D). The levels of total IgE at three different time-points and the

hBTP-specific IgE levels in the sacrificed mice were determined by sandwich ELISA.

Sera were diluted 1:10 for the measurement of total IgE and neat for the measurement of

hBTP-specific IgE. Data are reported as mean ± SEM (n = 6) for each group. p < 0.05,


*

**

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Next, as shown in Figure 2.8, the levels of hBTP-specific IgG1 and IgG2a in sera

harvested on mouse sacrifice day were measured. IgG1 antibody in PBS control mice was

undetectable, while hBTP treatment induced significantly increased levels of specific

IgG1 antibody. The levels of hBTP-specific IgG1 in mice receiving sensitisation with 50

μg x 3 of hBTP and 200 μg x 3 of hBTP were significantly higher than mice sensitised

with 500 μg x 3 of hBTP. The pattern of hBTP-specific IgG2a antibody level among the

four mouse groups were consistent with the IgE antibody responses in sera.

Figure 2.8 Levels of serum hBTP-specific IgG1 (A) and IgG2a (B) on Day 34.

Levels of hBTP-specific IgG1 and IgG2a were determined by sandwich ELISA. Sera

were diluted 1:1000 for measuring hBTP-specific IgG1 and neat for measuring hBTP-

specific IgG2a. Data are reported as mean ± SEM (n = 6) for each group. p < 0.05, and

p< 0.01. Each dot represents an individual mouse.

*

**

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112

2.4.2.3 Eosinophil and basophil responses in blood

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Figure 2.9 A: Representative contour plots showing the gating strategy of viable

eosinophils, FcεRI-basophils and IgE-binding basophils. B: The representative

contour plots of control and treatment groups, showing circulating eosinophils,

FcεRI-basophils and IgE-binding basophils. C, D and E: The levels of eosinophils,

FcεRI-binding basophils, and mean fluorescence intensity (MFI) of IgE antibody

binding on blood basophils from three mouse groups sensitised with different doses

of hBTP as well as the PBS/alum control mice. The populations of eosinophils,

basophils and MFI of basophil-binding IgE in blood were determined by flow cytometry

and the cell populations in plots were analysed by FlowJo software. Data in the scatter

plots are reported as mean ± SEM (n = 6). p < 0.05, and p < 0.01. In panel C, D and

E, each dot represents an individual mouse.

Abbreviations: SSC-A, H or W=side scatter-area, height or width, FSC-A, H or W

=forward scatter-area, height or width, Siglec F=sialic acid-binding immunoglobulin-like

lectin F, CD=cluster of differentiation, and FcεRI=the high-affinity IgE receptor.

* **

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Basophils and eosinophils are central effector cells in allergic inflammation, as well as in

innate and adaptive immunity. Therefore, in this study, the percentage of eosinophils,

basophils and the amount of IgE that bind on basophils in blood was investigated. As

shown in Figure 2.9.C, the eosinophil frequency (%) in mice sensitised with 50 μg x 3 of

hBTP (3.667 ± 0.5027) and 200 μg x 3 of hBTP (3.232 ± 0.4556) was marginally higher

than that in PBS/alum treated mice (2.942 ± 0.4816). FcεRI is the high-affinity IgE

binding receptor, expressed on mast cells and basophils. The expression level of human

basophil FcεRI expression usually correlates directly with the serum IgE antibody level,

and binding of IgE antibody stabilises the receptor at the cell surface [19]. In this study,

Figure 2.9.E shows the proportions of FcεRI-basophils in mice receiving IP

administration of 50 μg x 3 of hBTP (3.437 ± 0.1384) and 200 μg x 3 of hBTP (3.435 ±

0.1767) were much higher than those in mice sensitised with 500 μg x 3 of hBTP (2.228

± 0.2544), while not significantly different from those in control mice (2.7 ± 0.5628).

Figure 2.9.E also illustrates that MFI of the IgE antibody bound to basophils in hBTP-

treated mice increased dramatically as compared to the control mice, indicating the

recruitment and activation of basophils in this shellfish allergy model.

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2.4.2.4 Secretion of allergen-specific IgA in jejunum

Figure 2.10 The level of hBTP-specific IgA in the jejunum. hBTP-specific IgA was

determined by sandwich ELISA using neat protein extracts. Data are reported as mean ±

SEM for each group. p < 0.05, and p < 0.01. Each dot represents an individual mouse.

Next, to evaluate the mucosal inflammation in intestine caused by allergen extracts, we

measured the hBTP-specific IgA antibody in homogenised jejunum. Figure 2.10 shows

that mice sensitised with the lower dose of hBTP (50 μg x 3 and 200 μg x 3) had

significantly high levels of hBTP-specific IgA antibody in their jejunums, comparing with

control mice. Nevertheless, while there was no big difference between 500 μg x 3 treated

mice and PBS control mice. The highest level of IgA antibody against hBTP was found

in the lowest dose group of mice (50 μg x 3). This noticeable trend of IgA antibody levels

among mice sensitised with different doses of hBTP was correlated with the levels of

serum IgE antibody.

* **

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117

Taken together, the level of serum IgE and IgG antibody responses against hBTP in four

groups of mice were correlated with the results of blood eosinophils and IgA antibody

production in jejunum tissue. The lowest dose of hBTP (50 μg x 3) seems to be capable

to elicit stronger IgE antibody mediated allergic responses as compared to higher

concentrations. Based on this negative correlation between the increased IP sensitisation

dose and the level of Th2 responses, in the next experiments the sensitisation capacity of

lower IP doses in BALB/c mice was investigated to further optimise the induction

protocol of this shellfish allergy model.

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Lower sensitisation doses in BALB/c mice


Animals: BALB/c (8-week-old females) were obtained from the Australian Phenomics

Facility, Australian National University, Australia. Mice were housed at the Cairns

campus, building E4 Animal Facility under specific pathogen-free conditions, and

maintained on a fishmeal free diet purchased from Specialty Feeds. The usage of mice

was approved by James Cook University animal ethics committee (Animal ethics

approval number A2108).

Mice sensitisation and challenge with allergens: Every group consisted of six female

BALB/c mice. Mice received three IP injections of different doses of hBTP mixed with

2 mg of alum in Group A, B, C and D (Figure 2.11). Each sensitisation dose of hBTP on

each injection day (Day 0, 7 and 14) was 10 μg in Group A, 20 μg in Group B, 50 μg in

Group C, and 200 μg in Group D.

All hBTP-sensitised mice were orally challenged with 1 mg of hBTP starting from Day

21. Two-day time interval was set up between oral challenges and a total of five

challenges were performed. The control mice in Group E were injected with 200 μl of

PBS mixed with 2 mg of alum on Day 0, 7 and 14, and subsequently challenged with 200

μl of PBS for five times on the same challenge dates as hBTP-sensitised mice in Group

A, B, C and D.

Blood was collected on Day 20, 25, 31 and 34. One day after the 5th oral challenge, Day

34, mice were sacrificed. Spleens, mesenteric lymph nodes (MLNs), jejunums and stools

were isolated.

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Figure 2.11 Optimised sensitisation and oral challenge protocol, based on previous

Experiments #1 and 2, to establish the mouse model for prawn allergy. The schedules

of IP sensitisation and oral challenge are shown. Every hBTP-sensitised group comprised

of six mice, and the control group of five mice.

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2.4.3.2 Serum antibodies and mast cell protease-1

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121

Figure 2.12 Levels of serum total IgE antibody in each mouse group at four time-

points (A), serum hBTP-specific IgE antibody (B), IgG2a antibody (F) and mMCP-

1 on Day 34 (C), as well as serum hBTP-specific IgG1 antibody on Day 31 (D) and

Day 34 (E). The time-points involved in this experiment were one day before the 1st oral

challenge (Day 20), one day after two oral challenges (Day 25), one day after four oral

challenges (Day 31) and mouse sacrifice day (Day 34). Antibody and mMCP-1 levels

were determined by ELISA. Sera were diluted for each measurement: 1:10 for total IgE,

1: 100,000 for allergen-specific IgG1, and neat for allergen-specific IgE, IgG2a as well

as mMCP-1. Data are reported as mean ± SEM of each group (n = 6 in hBTP-sensitised

groups, and n = 5 in PBS control group). p < .05, and p < 0.01. Each dot represents

an individual mouse.

The fluctuation of total IgE antibody levels is shown in Figure 2.12 A. During the first

four oral challenges with hBTP, there was a steep upward trend in the level of total IgE

antibody in the sera from 10 μg x 3 and 20 μg x 3 groups. Subsequently, the serum total

IgE antibody level dropped on Day 34. From the start of the oral exposure with hBTP,

the mean levels of circulating total IgE antibody remained to be the highest in the 10μg x

* **

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3 group. For example the IgE antibody response on Day 31 (one day after four

challenges), significantly increased levels of serum total IgE antibody were observed in

all the groups of hBTP-treated mice, as compared with control mice. The 10 μg x 3 mouse

group presented a significantly higher level of serum total IgE antibody than the 50 μg x

3 mouse group and the 200 μg x 3 mouse group. Consistently, the relatively high level of

IgG1 antibody (Figure 2.12.D and E) and MCP-1 (Figure 2.12.C) were also detected in

sera from the 10 μg x 3 mouse group. Some of BALB/c mice from the 10 μg x 3, 50 μg

x 3 and 200 μg x 3 mouse groups seemed to be able to produce high levels of IgG2a

antibody against hBTP, whereas individual variation in these groups was observed

(Figure 2.12.F).

2.4.3.3 Production of cytokines by spleens and mesenteric lymph nodes

As shown in Figure 2.13, the cytokines IL-5, IL-13 and IFN-gamma in both re-stimulated

spleens and mesenteric lymph nodes (MLNs) were very low or undetectable in the control

mice. MLNs and spleens isolated from mice that received the highest sensitisation dose

of 200 μg x 3 produced much lower concentration of IL-5, IL-13 and IFN-gamma as

compared to mice sensitised with lower doses of hBTP. The expression of Th2-associated

IL-5 and IL-13 was the highest in the 10 μg x 3 hBTP-sensitised mice, although in this

group the level of IL-5 in spleen was similar in the 20 μg x 3 group, and IL-13 in MLNs

similar in the 50 μg x 3 group. In contrast IFN-gamma responses to hBTP stimulation in

MLNs and spleens was not higher in the 10 μg x 3 as compared to the 20 μg x 3 and 50

μg x 3 groups. These data suggest that IP sensitisation of hBTP/alum promotes a

predominantly Th2-biased systemic cytokine response, especially the lower IP doses of

10 μg x 3 hBTP.

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Figure 2.13 Cytokine IL-5, IL-13 and IFN-gamma released by hBTP-stimulated

spleens (A, B and C) and MLNs (A’, B’ and C’). Single cells from spleen and were

cultured with hBTP for three days and subsequently the supernatant collected for the

cytokine measurements. Levels of cytokines were determined by ELISA. The supernatant

was diluted 1:10 for IL-13, and 1:2 for IFN-gamma and IL-5. Data are reported as mean

± SEM (n = 6 in hBTP-sensitised groups, and n = 5 in PBS control group). p < 0.05,


*

**

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2.4.3.4 Induction of eosinophilia in spleens

Figure 2.14 The number of eosinophil in spleens. The population of eosinophils in

spleens was determined by flow cytometry. Data are reported as mean ± SEM ((n = 6 in

hBTP-sensitised groups, and n = 5 in PBS control group). p < 0.05, and p < 0.01.

Each dot represents an individual mouse.

We found increased eosinophils in spleens from all the hBTP-treated animals (Figure

2.14). The total number of eosinophils in mice with 10μg x 3 IP sensitisation (2.996 x 106

± 945913) was the highest, comparing with those in the 20 μg x 3 group (1.182 x 106 ±

333057), 50 μg x 3 group (1.401 x 106 ± 353893) and 200 μg x 3 group (1.001 x 106 ±

245632).

×

×

×

×

* **

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2.4.3.5 Jejunum histology

Considerable vasodilation and capillary congestion (Figure 2.15.B) was demonstrated in

intestinal tissue as compared to the intestinal tissues from PBS control mice (Figure

2.15.A). Figure 2.15.D demonstrates that the jejunum of hBTP/alum-sensitised animals

had inflammatory cell infiltrate that contained a number of eosinophils that were not

observed in control animals (Figure 2.15.C). Eosinophils were predominately located in

the lamina propria. Infiltration of eosinophilic was only examined in the 10 μg x 3 IP

sensitisation group.

Figure 2.15 Representative histology of tissue stained with hematoxylin and eosin

demonstrating vasodilation and eosinophilic infiltration into the jejunum tissues.

White arrows indicate the vasodilation and capillary congestion (panel B) and eosinophils

(panel D) in 10μg x 3 hBTP treated mice, as well as the similar locations in control

jejunum for comparison. (panel A and C).

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To summarise, IP sensitisation of hBTP/alum and subsequent oral challenges of hBTP

succeeded in promoting a dominant IgE and IgG1 antibody responses against allergen,

mast cell activation, spleen Th2 cytokine production and eosinophilia, as well as intestinal

eosinophilic infiltration. Sensitising BALB/c mice with 10 μg x 3 of hBTP three times on

a weekly basis and subsequently four oral administrations of 1mg of hBTP can be an

appropriate procedure to establish a novel mouse model of prawn allergy.

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2.5 Discussion

So far, a number of experimental models of food allergy have been developed targeting

total extracts or single allergen of peanut, milk or egg. In contrast, mouse models studying

shellfish allergy are very much lacking [6]. Therefore, the aim of this chapter was to

design a suitable mouse model for shellfish allergy to investigate the immunological

mechanisms underlying and to create a superior immunisation protocol.

For this purpose, I have chosen hBTP as the model allergen. The Black tiger prawn (BTP)

(Penaeus monodon) is one of the most widely consumed prawn species. TM is the most

abundant allergen in BTP and has over 95% amino acid sequence identity with TMs from

other prawn species [9, 20]. Moreover, BTP allergens have been well characterised using

biochemical and immunological technology [9, 10]. Therefore, BTP is a very good

representative of edible crustacean species source for the establishment of an animal

model. The heating process used in this study does mimic the human dietary habits. In

addition, this process seems also to concentrates allergenic proteins [10]. Consequently,

immunisation mice with hBTP should be the most natural but also very effective method

when imitating the development of shellfish allergy in human. In this chapter, my results

with BALB/c mice have confirmed that hBTP have the capacity to systematically induce

prominent Th2 antibody responses, charactersied by significantly increased serum titers

of hBTP-specific IgE and IgG1 antibodies.

To achieve a better production of IgE and IgG1 antibodies in sensitised mice, I started to

use BALB/c mice that are believed to be more Th2-biased than C57BL/6, and optimised

a few important parameters of the induction protocol. I found that firstly a relatively low

allergen dose in IP sensitisation administrated repeated (10 μg x 3) in BALB/c mice could

induce a striking serum elevation of polyclonal IgE antibody (Experiment #2 and #3). It

was reported previously in BALB/c mice that only low dosage of OVA feeding triggered

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IgE-mediated food allergy [21]. High doses of allergens induced rather tolerance than

sensitisation [22, 23]. Nevertheless, rather low dose of allergens could not help further

more production of IgE in oral sensitisation [24]. This is consistent with my finding that

10 μg x 3 and 20 μg x 3 of hBTP as the sensitisation induced a similar level of IgE

antibody in Experiment #3.

It is worth noting that the dose-dependent manner of sensitisation or tolerance can be

affected by the nature of selected allergenic protein sources [23] but also probably the

mouse strain utilised [6, 25]. It is apparent that the exact dose-associated mechanism of

murine sensitisation cannot be completely revealed by any single study, and its relevance

with human studies needs to be very carefully investigated before interpretation.

The duration of the induction phase is another essential condition to modulate in the

model establishment because of the antibody fluctuation over time. I discovered a

appropriate frequency of oral challenges with hBTP in Experiment #3. The production of

polyclonal IgE in sera increased during the first four challenges with hBTP and on Day

32 reached the peak as compared with Day 15, Day 25 and Day 34. Therefore, Day 32

was chosen as the best date of mouse sacrifice and organ harvest to show sufficient

humoral responses against the prawn allergens. Interestingly, the changing amount of

serum IgE antibody with challenge duration time has previously been reported in patients

with egg allergy [26]. Therefore, together with the inbred mouse strain, allergen and

allergen dose mentioned above, the key time-points of administrations can also help to

determine the immune responsiveness in a mouse models. Thus, to study different food

allergies, the combination of all these parameters involved in the development of a mouse

model may often require adjustment after optimisation.

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Apart from measuring serum antibodies, activities of related murine immune cells were

also comprehensively evaluated in this chapter. The release of IgE antibody by B cells is

a T cell dependent phenomenon, requiring the expression of Th2 cytokines. In this study,

hBTP successfully stimulated monocytes from spleen and MLN to produce remarkable

increased levels of IL-5 and IL-13 in vitro. Functionally, IL-5 and IL-13 attract and

activate eosinophils and basophils to react to allergens leading to tissue eosinophilia and

mast cell activation [19]. Evidences of the participation of eosinophils in inflammatory

process have been observed in Experiment #3. The number of splenic eosinophils

increased greatly and an increased number of eosinophils in the jejunum was also

observed. Basophils can be activated once circulating IgE antibody binds on surface

FcεRI. Basophils share several features and functions with mast cells, contributing to

allergic inflammation, such as the expression of Th2 cytokines and histamine by IgE-

binding basophils [19]. In Experiment #2, all hBTP-treated mice displayed significantly

higher IgE binding to basophils in blood, which suggested the recruitment of basophil in

prawn allergy. Mast cells are central to proinflammatory functions in allergic diseases. In

this chapter, elevated levels of mMCP-1 were identified in circulating blood and

homogenised jejunum, which indicates the activation of mast cells and their trafficking

to inflammation sites.

Because the intestine is the major allergen exposure site in hBTP challenges, and any

food allergy via ingestion, the detailed inflammatory conditions in the intestine is also

very important in prawn allergy. In the mouse model I developed in this chapter, the

intestine inflammation induced in hBTP-sensitised and challenged mice was

characterised by the increased allergen-specific IgA antibody, mMCP-1, and the presence

of vasodilation and a small number of eosinophil aggregation in jejunum. Activated

intestinal mast cells release histamine to lead to vasodilation, thus permitting eosinophils

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to transfer from the blood and localised in the jejunum. Eosinophilia in intestinal tissue

and blood was reported previously in a mouse model on egg allergy [12]. Accumulation

of eosinophils demonstrated in the intestine of my mouse model was moderate and there

was not severely damaged area of jejunum. This could also be one of reasons why no

diarrhea was observed after oral challenges with hBTP. IgA antibody is usually secreted

locally after foreign proteins enter the intestine including allergens. The exact role of IgA

in food allergy is however still unclear. IgA may be able to prevent allergen from

connecting with epithelial cells to further eliminate the allergens, or IgA may help the

uptake of the allergen by dendritic cells, thereby reducing the proinflammatory response

[27].

In summary, I have shown a good induction protocol to develop a mouse model to study

prawn allergy. Mice IP sensitised and IG challenged with hBTP display an allergic

inflammation characterised by enhanced levels of circulating IgE and IgG1 antibodies,

local production of predominant Th2 cytokines, increased eosinophil circulation and IgE

bound basophils, as well as the activation of mast cells. This model should be an

interesting tool to detect the immunological cross-reactivity among shellfish allergy, and

to further investigate the major allergens responsible for shellfish cross-reactivity.

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2.6 References

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comprehensive review, Clinical reviews in allergy & immunology, 49 (2015) 203-216.


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[3] S.H. Sicherer, H.A. Sampson, Food allergy, Journal of Allergy and Clinical

Immunology, 117 (2006) S470-S475.

[4] M.K. Oyoshi, H.C. Oettgen, T.A. Chatila, R.S. Geha, P.J. Bryce, Food allergy:

Insights into etiology, prevention, and treatment provided by murine models, Journal of




33 (2003) 1586-1594.

[6] T. Liu, S. Navarro, A.L. Lopata, Current advances of murine models for food allergy,


[7] Y. Zhang, W. Lamm, R.K. Albert, E.Y. Chi, W.R. Henderson Jr, D.B. Lewis,

Influence of the route of allergen administration and genetic background on the murine

allergic pulmonary response, American journal of respiratory and critical care medicine,

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[8] M.M. Gueders, G. Paulissen, C. Crahay, F. Quesada-Calvo, J. Hacha, C. Van Hove,

K. Tournoy, R. Louis, J.-M. Foidart, A. Noël, Mouse models of asthma: a comparison

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spectrometry, 24 (2010) 2462-2470.


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[11] M.F. Jeebhay, T.G. Robins, M.E. Miller, E. Bateman, M. Smuts, R. Baatjies, A.L.

Lopata, Occupational allergy and asthma among salt water fish processing workers,

American journal of industrial medicine, 51 (2008) 899-910.

[12] K. Ganeshan, C.V. Neilsen, A. Hadsaitong, R.P. Schleimer, X. Luo, P.J. Bryce,

Impairing oral tolerance promotes allergy and anaphylaxis: a new murine food allergy

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[13] R. Brunner, E. Jensen-Jarolim, I. Pali-Schöll, The ABC of clinical and experimental

adjuvants—a brief overview, Immunology letters, 128 (2010) 29-35.

[14] L. Tomljenovic, C. A Shaw, Aluminum vaccine adjuvants: are they safe?, Current

medicinal chemistry, 18 (2011) 2630-2637.

[15] A.M. Nanzer, E.S. Chambers, K. Ryanna, D.F. Richards, C. Black, P.M. Timms,

A.R. Martineau, C.J. Griffiths, C.J. Corrigan, C.M. Hawrylowicz, Enhanced production

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a glucocorticoid-independent fashion, Journal of Allergy and Clinical Immunology, 132

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[16] G. Herberth, C. Daegelmann, S. Röder, H. Behrendt, U. Krämer, M. Borte, J.

Heinrich, O. Herbarth, I. Lehmann, IL 17E but not IL 17A is associated with allergic

sensitization: results from the LISA study, Pediatric Allergy and Immunology, 21 (2010)

1086-1090.

[17] C. Bohnen, A. Wangorsch, S. Schülke, H. Nakajima Adachi, S. Hachimura, M.

Burggraf, Y. Süzer, A. Schwantes, G. Sutter, Z. Waibler, Vaccination with recombinant

modified vaccinia virus Ankara prevents the onset of intestinal allergy in mice, Allergy,

68 (2013) 1021-1028.

[18] K. Adel Patient, H. Bernard, S. Ah Leung, C. Creminon, J.M. Wal, Peanut and

cow's milk specific IgE, Th2 cells and local anaphylactic reaction are induced in Balb/c

mice orally sensitized with cholera toxin, Allergy, 60 (2005) 658-664.

[19] K.D. Stone, C. Prussin, D.D. Metcalfe, IgE, mast cells, basophils, and eosinophils,

Journal of Allergy and Clinical Immunology, 125 (2010) S73-S80.

[20] M. Koeberl, S.D. Kamath, S.R. Saptarshi, M.J. Smout, J.M. Rolland, R.E. O'Hehir,

A.L. Lopata, Auto-induction for high yield expression of recombinant novel isoallergen

tropomyosin from King prawn (Melicertus latisulcatus) for improved diagnostics and

immunotherapeutics, Journal of immunological methods, 415 (2014) 6-16.

[21] S.C. Diesner, R. Knittelfelder, D. Krishnamurthy, I. Pali-Schöll, L. Gajdzik, E.

Jensen-Jarolim, E. Untersmayr, Dose-dependent food allergy induction against

ovalbumin under acid-suppression: a murine food allergy model, Immunology letters, 121

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[22] R.M. Helm, R.W. Ermel, O.L. Frick, Nonmurine animal models of food allergy,

Environmental health perspectives, 111 (2003) 239.

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[23] J. Strid, M. Thomson, J. Hourihane, I. Kimber, S. Strobel, A novel model of

sensitization and oral tolerance to peanut protein, Immunology, 113 (2004) 293.

[24] A. Wakabayashi, M. Utsuyama, T. Hosoda, K. Sato, Induction of immunological

tolerance by oral, but not intravenous and intraportal, administration of ovalbumin and

the difference between young and old mice, The journal of nutrition, health & aging, 10

(2006) 183.

[25] X.-m. Li, B.H. Schofield, C.-K. Huang, G.I. Kleiner, H.A. Sampson, A murine model

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Immunology, 103 (1999) 206-214.

[26] L.P. Shek, L. Soderstrom, S. Ahlstedt, K. Beyer, H.A. Sampson, Determination of

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and hen's egg allergy, Journal of Allergy and Clinical Immunology, 114 (2004) 387.

[27] N.J. Mantis, N. Rol, B. Corthésy, Secretory IgA's complex roles in immunity and

mucosal homeostasis in the gut, Mucosal immunology, 4 (2011) 603-611.

CHAPTER

EVALUATION OF THE POTENTIAL IMMUNOLOGICAL

CROSS-REACTIVITY BETWEEN THE WHOLE HEATED

EXTRACTS OF BLACK TIGER PRAWN

(Penaeus monodon) AND JADE TIGER ABALONE

(Haliotis laevigata × Haliotis rubra)

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3.1 Introduction

Crustaceans are well known allergen sources in susceptible individuals. The

hypersensitivity usually presents as wheezing dyspnoea, erythema, gastrointestinal

distress, urticaria, angioedema and anaphylaxis. Similar symptoms have been also

described in patients consuming mollusks such as abalone [1], limpet [1, 2] and squid [3].

As compared to crustacean allergy, mollusc allergy has been less studied and recently

been of particular interest. The general impression to the public is that the cross-reactivity

among different shellfish species is very frequent. Consequently, people who react to one

of shellfish species will have to avoid all the seafood, which greatly restrict their nutrition

sources [4, 5].

The immunological cross-reactivity between prawns and abalone has been reported in

human studies. For example, in a cohort study performed in Hong Kong, among 70

positive shellfish skin tests, 27 (38.6%) patients were sensitive to both crustaceans and

mollusks. A strong association between sensitivity to prawns and to abalone was

predicted: the subjects with a positive skin test to prawns had a probability of 0.39 to react

to abalone, and the subjects with a positive skin test to abalone had a probability of 0.45

to react to prawns [6]. Also, in a study of 81 Korean patients with a positive skin test

against abalone extracts, ELISA inhibition tests on the serum IgE antibody from these

patients showed significant dose-dependent inhibitions by prawn extracts [7]. However,

the reactions of neither the skin nor the in vitro serum IgE equal to the real pathological

process of allergy, and the accuracy of these tests in predicting the clinical significance is

in debate [8, 9].

Within recent three decades, there has been a growing interest in the design and

establishment of appropriate mouse models and their potential integration into safety

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assessment paradigms [10-16]. In the 2001 report from the joint Food and Agricultural

Organisation of the United Nations and the World Health Organization (FAO/WHO)

Expert Consultative Committee, an essential conclusion was that the accumulated

evidence has suggested some animal models may provide very useful information for the

determination of allergenic potential of foods (http://www.who.int/foodsafety/en/).

Researchers can control the exposure conditions of experimental animals and collect

samples from multiple allergy-related organs and tissues. These unique advantages of

mouse models allow a thorough investigation on the mechanisms underlying the prawn-

abalone cross-reactivity.

In the current chapter, two popular shellfish species were employed for the study of

prawn-abalone cross-reactivity: Black tiger prawn (Penaeus monodon) (BTP) and Jade

tiger abalone (Haliotis laevigata × Haliotis rubra) (Abal). BTP is one of the crustacean

species that are widely consumed in the Asian-Pacific region [17]. In Chapter 2, the

heated extracts of BTP were demonstrated to induce Th2 predominant immune responses.

Abal is one of the three main types of abalone farmed in Australia, and is based on

hybridisation between Australia’s Blacklip (Haliotis rubra) and Greenlip abalone

(Haliotis laevigata). The aquaculture facility of Jade tiger abalone is now expanding and

the interests of investment and research are growing due to the rapidly increasing

demands from the global market, especially in China and Southeast Asia [18].

Nevertheless, the current research only focuses on improvements of the aquaculture

breeding, while the allergenicity of Abal has not been investigated yet. In this chapter,

mice were treated with the heated extracts of these two species separately for the

induction of allergy-like reactions. The immunological cross-reactivity between BTP and

Abal was comprehensively evaluated at both humoral and cellular levels.

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3.2 Aims


(1) To evaluate the degree of the potential immunological cross-reactivity between

heated Black tiger prawn (Penaeus monodon) and Jade tiger abalone (Haliotis

laevigata × Haliotis rubra) in vivo.

(2) To identify the potential cross-reactive allergens between Black tiger prawn and

Jade tiger abalone.

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3.3.1 Animals

BALB/c mice (8-week-old females) were housed at the Cairns campus, building E4

Animal Facility under specific pathogen-free conditions, and maintained on a commercial

vegetarian diet purchased from Specialty Feeds. The animal ethics committee from James

Cook University approved the experiments project (Animal ethics approval number

A2108).

3.3.2 Preparation of heated extracts of prawns, abalone and cow’s milk

Black tiger prawns (Penaeus monodon) and Jade tiger abalone (Haliotis laevigata ×

Haliotis rubra) were purchased from the local seafood shops. The heated extracts of

Black tiger prawn (hBTP) and Jade tiger abalone (hAbal) were prepared and the

concentration of the total protein in the heated extracts was estimated using the same

methods that were previously described in 2.3.2. Likewise, the cow’s milk (Dairy

Farmers) was heated to boil for 20 min and the protein content was measured using Pierce

BCA Protein Assay Kit (Thermo Fisher Scientific). The protein concentration of the

heated milk was adjusted with PBS buffer before used in the mouse experiment.

3.3.3 Induction and evaluation of mouse models for shellfish allergies

3.3.3.1 Mice sensitisation and challenge

The basic procedure of the mouse intraperitoneal (IP) sensitisation and the oral challenge

was described in Chapter 2, Section 2.3.3.1. In the current chapter, two independent

mouse experiments were conducted (n = 5 or 6), and the allergen sources to treat mice

were modified as demonstrated in Figure 3.3.1. In Group 1, 2 and 3, BALB/c mice

received three IP injections of the hBTP and alum (Thermo Fisher Scientific) mixture on

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Day 0, 7 and 14, while BALB/c mice in Group 4, 5 and 6 were IP injected with hAbal

and alum at the same time-points. Mice were fasted overnight but had unlimited access

to water before gavage. On Day 21, 24, 27 and 30, the three groups of hBTP-sentisised

or hAbal-sensitised mice were orally challenged with 1 mg of the hBTP, hAbal or hMilk

(the nonrelated proteins). The control mice were IP injected with PBS and alum mixture

and subsequently orally challenged with PBS only. 24 hours after the last oral challenge,

all the mice were sacrificed and the samples collected from blood, spleen and jejunum for

further analysis.

In this chapter, each mouse group was named as follows: “hBTP/hMilk group” was the

group of hBTP-sensitised, hMilk-gavaged mice, “hBTP/hBTP group” was the group of

hBTP-sensitised, hBTP-gavaged mice, “hBTP/hAbal group” was the group of hBTP-

sensitised, hAbal-gavaged mice, “hAbal/hMilk group” was the group of hAbal -sensitised,

hMilk-gavaged mice, “hAbal/hAbal group” was the group of hAbal -sensitised, hAbal-

gavaged mice, and “hAbal/hBTP group” was the group of hAbal -sensitised, hBTP-

gavaged mice.

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Figure 3.1 Experimental design in the sensitisation and control groups. Each group

comprised of five to six BALB/c female mice.

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142

3.3.3.2 Measurement of antibodies and mast cell protease-1 in serum

The mouse serum was isolated and then the total IgE antibody, mMCP-1, as well as the

allergen-specific IgE, IgG1 and IgG2a antibodies were measured by means of sandwich

ELISA. The protocols were previously mentioned in Chapter 2, Section 2.3.3.2. In the

current chapter, the major modification of the ELISA protocol was that the coating protein

extracts involved not only hBTP but also hAbal (100 μl of 20 μg/ml extracts per well of

the 96-well ELISA plate). The coated plates were blocked with 10% FBS in PBS and then

incubated with mouse sera at 4°C overnight for the maximal binding. The serum was 1:2

diluted for the specific IgE plate, 1:10 diluted for the total IgE plate and the specific IgG2a

plate, and 1:106 diluted for the specific IgG1 plate. The plates were washed with

PBS/0.05% Tween 20 and subsequently, the corresponding detection antibody was added

and incubated in the ELISA plates for 1.5 h at room temperature. Next, the plates were

incubated with streptavidin-HRP in dark for 30 min following with the incubation with

TMB substrate. After the desired colour reaction was developed, 50μl of 1M HCl per well

was added to stop the reaction. The absorbance of the samples was detected at 450 nm.

3.3.3.3 In vitro stimulation of splenocytes with shellfish extracts

The preparation of the single cell suspension of mouse splenocytes was previously

demonstrated in Chapter 2, Section 2.3.3.3, and in this chapter, a few modifications were

made in the cell stimulation assay. Splenocytes (0.5 million cells/well) were cultured with

100 μl of 40 μg/ml hBTP or hAbal for 72 h in 5% CO2 at 37°C using the SANYO CO2

Incubator Model MCO-18AIC (UV) Dual Stack. With the absence of shellfish extracts,

the splenocytes incubated with the culture medium only were labelled as un-stimulated

splenocytes. The positive control was the splenocytes stimulated with 1 μg/ml anti-

CD3/anti-CD28 for 72 h.

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The protocol of cell surface immunofluorescence staining was previously described in

Chapter 2, Section 2.3.3.4. Peripheral blood mononuclear cells and splenocytes were

stained with a mixture of seven fluorescence-conjugated antibodies (Jomar Life

Research), including Anti-Mouse CD170 (Siglec F) PerCP-eFluor 710, Anti-Mouse CD3

APC-eFluor 780, Anti-Mouse IgE FITC, Anti-Mouse CD11b PE, Anti-Mouse CD49b

(Integrin alpha 2) APC, Anti-Mouse CD19 APC-eFluor 780, and Anti-Mouse Fc epsilon

Receptor I alpha (FcεR1) eFluor 450. BD FACS Canto II was used for analysing the

stained cell populations.

3.3.3.5 Measurement of cytokines

The supernatant was isolated after the three-day culture of splenocytes and ELISA was

performed for the measurement of the levels of mouse cytokines including interleukin

(IL)-4, IL-5, IL-13 and interferon (IFN)-gamma following the protocols described

previously in Chapter 2, Section 2.3.3.6.

3.3.3.6 Measurement of allergen-specific IgA antibody

Jejunums were homogenised at 4°C and subsequently sandwich ELISA was conducted

to measure the hAbal or hBTP-specific IgA antibody. The detailed protocol was

previously described in Chapter 2, Section 2.3.3.5 and 2.3.3.8.


Two-tailed Mann-Whitney U test was performed using Prism 6.0. The data shown in this

chapter are representative of two independent experiments, and presented as mean values

with the standard errors of the mean (SEM). p values of less than 0.05 were considered

statistically significant.

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3.3.3.8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) was

performed to visualise the protein repertoire in the prepared sample, which was described

previously [19]. Twenty μl of the protein sample was heated in Laemmli buffer with

dithiothreitol at 100°C for 5 min and then spined down briefly. The supernatant was

loaded onto a 12% bis-acrylamide gel. Precision Plus Protein™ Dual Colour Standards

(Bio-Rad) was used as a molecular weight marker. Protein bands were separated by

electrophoresis at 80V for 20 min and then 170V for 1 h (when the tracker dye reached

the gel base) using a Mini-Protean Tetra Cell electrophoresis system (Bio-Rad). The

separated proteins were visualised by staining the SDS-PAGE gel with Commassie

brilliant blue R250 (Bio-Rad).

3.3.3.9 Mouse immunoblotting

35 μg of hAbal and hBTP was heated and loaded onto a 12% SDS-PAGE gel, following

with the electrophoretic separation at 170 V until the tracker dye reached the bottom of

the gel. In the next step, resolved proteins were transferred from the gel to a nitrocellulose

membrane, and subsequently the membrane was blocked with 5% skim milk. The

membrane was incubated with the pooled mouse serum (1:100 diluted) using a slot blot

apparatus (Idea Scientific) for 1 h at room temperature, following with the incubation

with 1:15, 000 diluted IRDye 800CW Goat anti-Mouse IgG polyclonal antibody (LI-

COR) for 1 h at room temperature to visualise the IgE antibody reactivity.

Chapter 3

145

3.3.3.10 Mass spectrometric identification of mouse IgG reactive proteins

The selected protein bands were excised from the SDS-PAGE plate. The in-gel tryptic

digestion procedure was performed on gel slices using the protocol developed by Jason

Mulvenna. The gel slice was incubated 200 mM ammonium bicarbonate and 50%

acetonitrile) at 37°C for 45 min. Subsequently, the destained gel slice was dried, reduced

with 20 mM dithiothreitol and 25 mM ammonium bicarbonate at 65 °C for 1 h, alkylated

in dark with 50 mM iodoacetamide and 25 mM ammonium bicarbonate at 37 °C for 40

min, and then digested with 20 ng/μl trypsin enzyme overnight. After that, the supernatant

of digestion reaction was isolated. The protein pellet was incubated with 50 μl of 0.1%

trifluoroacetic acid (TFA) and subsequently washed twice. Finally, all the supernatant

was combined together and concentrated for mass spectrometric (MS) analysis.

MS analysis of the digested peptides was performed at the Bio21 Molecular Science &

Biotechnology Institute using the protocol described as follows. LC MS/MS was carried

out on a LTQ Orbitrap Elite (Thermo Scientific) with a nanoESI interface in conjunction

with an Ultimate 3000 RSLC nanoHPLC (Dionex Ultimate 3000). The LC system was

equipped with an Acclaim Pepmap nano-trap column (Dionex-C18, 100 Å, 75 μm x 2

cm) and an Acclaim Pepmap RSLC analytical column (Dionex-C18, 100 Å, 75 μm x 50

cm). The tryptic peptides were injected to the enrichment column at an isocratic flow of

5 μL/min of 3% v/v CH3CN containing 0.1% v/v formic acid for 5 min before the

enrichment column was switched in-line with the analytical column. The eluents were

0.1% v/v formic acid (solvent A) and 100% v/v CH3CN in 0.l% v/v formic acid (solvent

B). The flow gradient was (i) 0-5min at 3% B, (ii) 5-25 min, 3-25% B (iii) 25-27 min, 25-

40% B (iv) 27-29 min, 40-80% B (v) 29-31 min at 80% B (vii) 31-32 min, 80-3% B and

(viii) 32-38 min at 3% B. The LTQ Orbitrap Elite spectrometer was operated in the data-

dependent mode with nanoESI spray voltage of 1.8kV, capillary temperature of 250°C

Chapter 3

146

and S-lens RF value of 55%. All spectra were acquired in positive mode with full scan

MS spectra from m/z 300-1650 in the FT mode at 240,000 resolutions. Automated gain

control was set to a target value of 1.0e6. Lock mass of 445.120025 was used. The top 20

most intense precursors were subjected to rapid collision induced dissociation (rCID)

with normalised collision energy of 30 and activation q of 0.25. Dynamic exclusion with

of 30 seconds was applied for repeated precursors. MS data was analysed using the

Mascot search engine offered by Matrix Science. The non-redundant protein sequence

database, Swiss-Prot, was used for searching matching peptides to identify protein

samples.

Chapter 3

147

3.4 Results

3.4.1 Induction of serum antibodies

Total IgE

Control

hBTP/hMilk

hBTP/hBTP

hBTP/hAbal

hAbal/hMilk

hAbal/hAbal

hAbal/hBTP

0.0

0.2

0.4

0.6

OD

450n

m **

** **

E

Chapter 3

148

Figure 3.2 Levels of serum hBTP-specific IgE antibody in the hBTP-sensitised mice

(A), hAbal-specific IgE antibody in the hAbal-sensitised mice (B), and the allergen

recognition of IgE antibody in hBTP/hBTP group (C) and hAbal/hAbal group (D),

and the total IgE antibody (E). Levels of IgE antibody were determined by sandwich

ELISA. Data are reported as mean ± SEM (n = 5) and are representative of two

independent experiments. p< 0.05, and p< 0.01. Each dot represents an individual

mouse.

This mouse study was undertaken to investigate whether hBTP-sensitised mice can

develop allergic responses after oral challenges with hAbal, and vice versa. hBTP-

sensitised, hBTP-challenged mice (named “hBTP/hBTP”) and hAbal-sensitised, hAbal-

challenged mice (named “hAbal/hAbal”) were positive controls. hBTP-sensitised, hAbal-

challenged mice (named “hBTP/hAbal”) and hAbal-sensitised, hBTP-challenged mice

(named “hAbal/hBTP”) were examined thoroughly for the potential immunological

cross-reactivity.

To investigate the levels of antibody cross-reactivity, the production of the serum IgE

(Figure 3.2), IgG1 (Figure 3.3) and IgG2a (Figure 3.5) was assessed by ELISA. The mice

that received IP sensitisation with hBTP or hAbal and subsequent gavages with milk

produced a significantly increased level of polyclonal IgE antibody or specific IgE

antibody as compared to the control mice. Notably, the oral challenges of hAbal could

elicit the hBTP-sensitised mice to generate IgG1 antibody but not IgE antibody. In

contrast, Figure 3.2.B demonstrates that hAbal-sensitised mice had increased IgE

antibody after oral challenges with hBTP. Nevertheless, Figure 3.3.B shows no

significant difference of hAbal-specific IgG1 antibody between hAbal/hMilk group and

hAbal/hAbal group, which may indicate the less relevance of the IgG1 antibody

production to shellfish allergy as compared to IgE antibody production in this model, or

* **

Chapter 3

149

the production of IgG1 antibody at the current time-point of serum harvest was not

suitable for evaluating IgG1 cross-reactivity.

Figure 3.3 Levels of serum specific IgG1 antibody in the hBTP-sensitised mice (A)

and hAbal-sensitised mice (B), and the IgG1 reactivity to hBTP and hAbal in the

sera from hBTP/hBTP group (C) and hAbal/hAbal group (D). Levels of IgG1

antibody were measured by sandwich ELISA. Data are reported as mean ± SEM (n = 5)

and are representative of two independent experiments. p < 0.05, and p < 0.01.

Abbreviation: ns = Not significant. Each dot represents an individual mouse.

hBTP-specific IgG1

OD

450n

m

Control

hBTP/hMilk

hBTP/hBTP

hBTP/hAbal0.0

0.1

0.2

0.3

0.4

0.5

**

*

*

ns

Allergen recognition of IgG1

OD

450n

m

Anti-hAbal Anti-hAbal Anti-hBTP0.00

0.05

0.10

0.15

hBTP/hBTP serumControl serum

hAbal-specific IgG1

OD

450n

m

Control

hAbal/hMilk

hAbal/hAbal

hAbal/hBTP

0.0

0.1

0.2

0.3

0.4

ns

ns

Allergen recognition of IgG1

Anti-hBTP Anti-hBTP Anti-hAbal0.0

0.1

0.2

0.3

OD

450n

m

**

hAbal/hAbal serumControl serum

ns

A B

C D

* **

Chapter 3

150

To further study the capacity of IgE and IgG1 antibody to react to the heterologous

shellfish extracts, the serum from hBTP/hBTP group was loaded into hAbal-coated solid

phase and the serum from hAbal/hAbal mice into hBTP-coated solid phase. It is apparent

that IgE antibody could bind to the heterologous extracts but at a significantly lower level

as compared to binding to the inducer extracts (Figure 3.2.C and D). In this ELISA assay,

the serum IgG1 antibody from hAbal/hAbal group and hBTP/hBTP group seemed very

specific to the corresponding sensitisation extracts of shellfish (Figure 3.3.C and D).

In addition, the production of serum IgE antibody in hAbal/hAbal group was markedly

higher than in hBTP/hBTP mouse group (Figure 3.2 A, B and E), the reasons for which

remains unclear. The hBTP and hAbal might differ in the aspects of the actual content of

allergens contained and the total allergenicity, which could probably affect the induction

of circulating IgE antibody.

Also, the increased levels of IgG2a antibody were demonstrated in the sera from hBTP

or hAbal-sensitised mice (Figure 3.4.A and B). IgG2a, as a Th1-associated isotype

antibody, was able to recognise the heterologous extracts at a relatively low level in

contrast to the IgG2a responses to the inducer extracts (Figure 3.4.C and D).

Chapter 3

151

Figure 3.4 Levels of serum hBTP-specific IgG2a antibody in the hBTP-sensitised

mice (A), hAbal-specific IgG2a antibody in the hAbal-sensitised mice (B), and the

allergen recognition of IgG2a antibody in hBTP/hBTP group (C) and hAbal/hAbal

group (D). Levels of IgG2a antibody were determined by sandwich ELISA. Data are

reported as mean ± SEM (n = 5) and are representative of two independent experiments.

p < 0.05, and p < 0.01. Abbreviation: ns = Not significant. Each dot represents an

individual mouse.

hBTP-specific IgG2a

Control

hBTP/hMilk

hBTP/hBTP

hBTP/hAbal0.0

0.5

1.0O

D45

0nm

** ***

Allergen recognition of IgG2a

Anti-hAbal Anti-hAbal Anti-hBTP0.00

0.25

0.50

0.75

1.00

OD

450n

m

hBTP/hBTP serumControl serum

ns

hAbal-specific igG2a

OD

450n

m

Control

hAbal/hMilk

hAbal/hAbal

hAbal/hBTP

0.00

0.05

0.10

0.15

0.20

Allergen recognition of IgG2a

Anti-hBTP Anti-hBTP Anti-hAbal0.00

0.02

0.04

0.06

0.08

OD

450n

m

**

hAbal/hAbal serumControl serum

A B

C D

* **

Chapter 3

152

3.4.2 Protein separation and identification of antibody binding proteins

Figure 3.5 SDS-PAGE protein analysis (A) and immunoblot for analysing the cross-

reactivity of IgG in the sera from hBTP/hBTP group and hAbal/hAbal group to the

heated extracts of abalone and prawns (B). In panel A (left), the red stars label the

selected protein bands for mass spectrometric identification.

Chapter 3

153

Table 3.1 IgG antibody reactive proteins identified by SDS-PAGE using mass

spectrometry technique. The sequence coverage is denoted as percentage. IgG reactive

proteins have been matched to known allergens using Uniport entry names.

Chapter 3

154

SDS-PAGE analysis, immunoblot using anti-mouse IgG antibody and mass spectrometric

(MS) analysis were performed to identify the antibody reactive proteins in hAbal and

hBTP, and the antibody cross-reactivity (Figure 3.5).

In hAbal, four major bands were observed in a SDS-PAGE gel (Figure 3.5.A) and selected

for MS identification. The following proteins were detected: tropomyosin (TM) in the 36

kDa and 37.6 kDa regions, TM and troponin T at 41.5 kDa, and arginine kinase (AK) at

43 kDa (Table 3.1). Importantly, Figure 3.5.B shows that these four bands in hAbal were

IgG reactive. Notably, hBTP-specific IgG could bind to the protein at 37 kDa which was

identified as abalone TM.

In hBTP, two major protein bands were IgG reactive and identified as prawn TM and AK.

However, in hBTP immunoblot, there was no IgG binding visualised using the sera from

hAbal-treated mice, indicating the very limited antibody cross-reactivity between heated

prawns and abalone.

Chapter 3

155

3.4.3 Production of cytokines by stimulated splenocytes

Figure 3.6 Production of IL-5 (A and A’), IL-13 (B and B’), IFN-gamma (C and C’)

by hBTP or hAbal-stimulated splenocytes. The splenocytes were cultured with hBTP

or hAbal for three days. The levels of cytokines were determined by ELISA. Data are

reported as mean ± SEM (n = 5) and are representative of two independent experiments.

p < 0.05, and p < 0.01. Abbreviation: ns = Not significant. * **

Chapter 3

156

To illuminate if cellular cross-reactivity could be triggered by the local activation with

shellfish extracts, the cytokine secretion by stimulated splenocytes was analysed as well.

In the groups of hBTP or hAbal-sensitised mice, no matter subsequently gavaged with

the inducer extracts or hMilk, their splenocytes produced the similar levels of IL-5 and

IL-13 after stimulation with the inducer extracts in vitro (Figure 3.6.A, A’, B, and B’). In

contrast, the oral gavages with inducer extracts significantly reduced the expression of

IFN-gamma by re-stimulated splenocytes, as demonstrated in Figure 3.6.C and C’. There

was not statistical difference between the levels of IFN-gamma generated by control

splenocytes and those by shellfish-sensitised splenocytes, which confirmed the Th2-

associated reactivity predominated the allergy model.

Interestingly, at the cytokine (IL-5 or IL-13) level, the splenocytes from hBTP/hAbal and

hAbal/hBTP responded differently to the corresponding extracts that were used in oral

challenge. Figure 3.6.A and B show that after the splenocytes from hBTP/hAbal group

were stimulated with hAbal in vitro, IL-5 (5.293 ± 2.79 pg/ml) and IL-13 (23.12 ± 3.648

pg/ml) were detected but at a significantly lower level, as compared to the expression of

IL-5 (307 ± 74.02 pg/ml) and IL-13 (183.7 ± 43.37 pg/ml) by the hBTP-stimulated

splenocytes from hBTP/hBTP group. Nevertheless, the splenocytes from hAbal/hBTP

could respond to hBTP as stimulus through releasing the similar levels of IL-5 and IL-13

in vitro, as compared to the hAbal-stimulated splenocytes from hAbal/hAbal group

(Figure 3.6.A’and B’). Furthermore, the stronger cytokine responses of hAbal-sensitised

mice to heterologous extracts as compared to hBTP-sensitised mice were also evidenced

by the data from the hMilk-gavaged mice. Figure 3.6.A and B illustrates that when the

splenocytes from hBTP/hMilk mice was cultured with hAbal, which was the first time

for these cells exposed to hAbal, the levels of IL-5 or IL-13 were not upregulated. By

comparison, the level of IL-13 expressed by hBTP-stimulated splenocytes from

Chapter 3

157

hAbal/hMilk group was significantly higher as compared to the hBTP-stimulated control

(Figure 3.6.B’).

Chapter 3

158

3.4.4 Induction of eosinophil and basophil in spleen and blood


and spleen (A’ and B’). The cell populations in blood and spleen were analysed by flow

cytometer. Data are reported as mean ± SEM (n = 5) and are representative of two

independent experiments. p < 0.05, and p < 0.01. Abbreviation: ns = Not significant.

Eosinophil

Control

hBTP/hMilk

hBTP/hBTP

hBTP/hAbal

hAbal/hMilk

hAbal/hAbal

hAbal/hBTP

0.0

0.7

1.4

2.1

2.8

Freq

uenc

y (%

)

** ** **

**ns

IgE-basophil

Control

hBTP/hMilk

hBTP/hBTP

hBTP/hAbal

hAbal/hMilk

hAbal/hAbal

hAbal/hBTP

0.0

0.7

1.4

2.1

2.8

Freq

uenc

y (%

) ***

**

Eosinophil

Freq

uenc

y (%

)

Control

hBTP/hMilk

hBTP/hBTP

hBTP/hAbal

hAbal/hMilk

hAbal/hAbal

hAbal/hBTP

0

2

4

6

8 ns

ns

*

** **

IgE-basophil

Freq

uenc

y (%

)

Control

hBTP/hMilk

hBTP/hBTP

hBTP/hAbal

hAbal/hMilk

hAbal/hAbal

hAbal/hBTP

0

1

2

3 **

**

**

ns

ns

Spleen

BloodA B

A' B'

* **

Chapter 3

159

As shown in Figure 3.7.A, in the blood from hBTP/hAbal group, eosinophil accounted

for 5.412 ± 1.924 % of live cells with the statistical significance of the difference (p =

0.0159) from the proportion of circulating eosinophil in hBTP/hMilk group. The

proportion of blood eosinophil in the group of hBTP/hBTP was significantly higher as

compared to hBTP/hMilk group (p = 0.0079) but not to hBTP/hAbal group (p = 0.5317).

This result indicates the capacity of hBTP-sensitised eosinophil to react to hAbal.

However, regarding to the proportion of splenic eosinophil, there was no statistical

difference between hBTP/hAbal group and hBTP/hMilk group (or control group) (Figure

3.7.A’). Among the three groups of hAbal-sensitised mice, hAbal-gavaged mice had a

significantly larger proportion of eosinophil in circulating blood (Figure 3.7.A) and

spleen (Figure 3.7.A’) as compared to hMilk-gavaged mice. In blood, hAbal/hBTP group

had 2.086 ± 0.2479 % of eosinophil, and as compared to the other two groups of hAbal-

sensitised mice the difference was not statistically significant (Figure 3.7.A). However,

in spleen, hAbal/hBTP group had 1.336 ± 0.06 % of eosinophil that was significantly

lower as compared to hAbal/hAbal group (Figure 3.7.A’). The immune effector cells may

prefer trafficking towards the inflammatory sites after oral exposure to shellfish extracts,

which leading to the less frequency of eosinophils in spleen as compared to blood as

demonstrated.

Figure 3.7.B and B’ show that in both spleen and circulating blood, the percentage of IgE-

basophils was considerably increased in every shellfish-sensitised group as compared to

the control. There was no significant difference in the frequency of IgE-basophil among

the three groups of hBTP-sensitised mice. This result may indicate that at the current

time-point of the sample harvest, the oral challenges performed in this study cannot

greatly promote the development or activation of IgE-basophils. Consistent with the

results of eosinophil, the percentage of IgE-basophils was the highest in hAbal/hAbal

Chapter 3

160

group among the three groups of hAbal-sensitised mice. In contrast to hAbal/hAbal group,

hAbal/hBTP group presented a similar level of circulating IgE-basophils (Figure 3.7.B)

but a notably lower level of splenic IgE-basophils (Figure 3.7.B’).

Chapter 3

161

3.4.5 Secretion of IgA antibody in jejunums

Figure 3.8 Levels of the intestinal hBTP-specific IgA antibody in hBTP-sensitised

mice (A), hAbal-specific IgA antibody in hAbal-sensitised mice (B), and the allergen

recognition of IgA antibody in the hBTP/hBTP group (C) and hAbal/hAbal group

(D). The level of IgA antibody was determined by sandwich ELISA using the neat protein

extracts of jejunums. Data are expressed as mean ± SEM (n = 5) and are representative

of two independent experiments. p < 0.05, and p < 0.01. Abbreviation: ns = Not

significant. Each dot represents an individual mouse.

* **

Chapter 3

162

To elucidate if cross-reactivity could be induced in the gastrointestinal mucosa, jejunums

were isolated and the secretion of specific IgA antibody was measured. As demonstrated

in Figure 3.8.A and B, the gavage of milk did not result in intestinal IgA responses. As

compared to hMilk-gavaged mice, the mean levels of the specific IgA antibody from the

positive control groups (hBTP/hBTP group or hAbal/hAbal group) were higher but the

difference was not statistically significant (p = 0.1508 between hBTP/hMilk group and

hBTP/hBTP group; p = 0.1508 between hAbal/hMilk group and hAbal/hAbal group). The

IgA response of hBTP-sensitised mice to hAbal (0.2434 ± 0.08373) seemed higher than

that to hMilk (0.115 ± 0.03446), but still in a statistically insignificant manner.

Next, hBTP/hBTP group and hAbal/hAbal group were selected for particularly analysing

the degree of IgA responses against the heterologous extracts on an immobilised surface.

The reactivity of the IgA antibody from hBTP/hBTP group to hAbal could not be

evidenced (Figure 3.8.C), while importantly, it seems that the IgA antibodies with high

affinity from hAbal/hAbal group could recognise hBTP. Nevertheless, it is worth noting

that the secretory IgA from hAbal/hAbal group could only bind to heterologous allergen

at a limited level in vitro. These results suggest the immunodominant peptides in hBTP

and hAbal have distinct IgA-binding regions.

Chapter 3

163

3.5 Discussion

Many patients with allergies to certain shellfish species are interested to learn if other

shellfish species needs to be strictly avoided. Unnecessary elimination diets can lead to

potentially hazardous health situations [20]. The immunological cross-reactivity between

different crustacean species seems more frequently reported than that between crustacean

and molluscs [21, 22]. Skin prick test and serum IgE detection have been employed to

predict the clinical significance of prawn-abalone cross-reactivity but the accuracy

remains unverified [8, 9]. Double blind placebo controlled food challenge (DBPCFC) is

considered as the gold standard to prove or rule out allergy to a specific food. To arrive

at a more definitive diagnosis, DBPCFC can sometimes be performed on patients by

allergists [23, 24]. However, during DBPCFC, patients who have been already diagnosed

with certain food allergies are fed with related foods that may cause severe reactions. To

this end, mouse models are very useful research tools to study the immunological cross-

reactivity without taking risk on human health. Substantial evidence can be collected from

this in vivo platform under designed experimental conditions to help to evaluate of the

allergenicity of shellfish species of interest but also to comprehensively reveal the multi-

organ involvement during immunological cross-reactivity [11]. Moreover, the cross-

reactivity studies using human samples have problems in determining the true

sensitisation. The use of animal models has overcome this problem, because the exposure

history of each experimental animal can be well designed and controlled. This is almost

impossible to achieve in human studies.

In Chapter 2, a suitable mouse model for prawn allergy was developed, and subsequently

in the current chapter, this model was used for examining the potential immunological

cross-reactivity between the heated extracts of Black tiger prawn (hBTP) and Jade tiger

abalone (hAbal) in detail. In this study of prawn-abalone cross-reactivity, BALB/c mice

Chapter 3

164

were IP sensitised with hBTP or hAbal and subsequently orally exposed to heterologous

extracts. Although clinical symptoms were not elicited during the development of this

model, the changes of essential immunological elements, such as serum antibodies,

splenic cytokines, intestinal IgA antibody, and eosinophils and basophils, were very

helpful to predict the potential clinical significance of the immunological cross-reactivity.

Firstly, in this work, I observed that the antibodies in sera or intestines had a very limited

capacity to bind to heterologous extracts (hBTP or hAbal). Moreover, the splenocytes

could express very low or little IL-5 and IL-13 at their first time of exposure to

heterologous extracts. This potentially indicates the immunological cross-reactivity

between the heated extracts of prawns and abalone is at a low level and thus the clinical

symptoms caused by prawn-abalone cross-reactivity are unlikely to occur or to be severe

in susceptible people. However, importantly, it is evidenced that more oral exposures of

the heterologous extracts may increase the Th2 responses (IL-5 and IL-13) in the

susceptible subjects. Therefore, even if no severe symptoms occur after eating abalone,

the patients with allergy to prawns may still need to avoid the very frequent exposure to

abalone, and vice versa.

In this study, another interesting finding is that as compared to hBTP-sensitised animals,

the hAbal-sensitised animals could react to homologous extracts at a higher level of IgE,

IgE-binding basophils and Th2-associated cytokines. Furthermore, the IgE and cellular

cross-reactivity also seems more significant in the hAbal-sensitised animals. This is the

first study that demonstrates the influence of sensitisation allergen sources (Black tiger

prawn or Jade tiger abalone) on the severity of the immunological responses to the

homologous or heterologous shellfish that are subsequently ingested.

Chapter 3

165

Here it must be recognised that this model has not been validated for a definitive

evaluation of the allergenicity of shellfish. Also, allergy is a complex disease, and the

susceptibility to allergenic foods can differ from individuals, depending on the genetic

predisposition, environmental factors and exposure history [14]. Therefore, it is unlikely

that a single mouse study could predict the exact degree of human allergic reactions to

different foods i.e. shellfish. Nevertheless, the prudent utility of well-designed animal

models, in tandem with the other scientific approaches to identify and characterise

allergenic molecules, would ultimately improve the assessment of food allergenicity and

related cross-reactivity.

In shellfish allergy, the most well studied pan-allergen is tropomyosin (TM). TMs from

crustacean species including prawns [19, 25-27], crabs [25], crayfishes [28], lobsters [28],

and krills [29] have been reported reactive to IgE antibody from patient sera. Also, the

evidence for the identification of TM as an essential allergen has been reported in

molluscs including cephalopods, gastropods and bivalves, such as octopus [30], disc

abalone, turban shell, whelk, bloody cockle, Japanese oyster, Japanese cockle, surf clam,

horse clam, razor clam, short-neck clam [31]. Moreover, importantly, TM is believed to

be responsible for shellfish cross-reactivity due to its highly conserved amino acid. The

accumulated data on the primary structures shows that crustacean tropomyosin shared

very high sequence identity (over 89%) with each other, 80-82% with mites, but only 55-

65% with molluscs (See Chapter 1). The data in the current chapter confirm that TM is

not only a major IgG reactive allergen in prawn allergy and abalone allergy, but also a

key suspect for the prawn-abalone cross-reactivity. Therefore, in the next chapter, TM

was investigated in particular using recombinant DNA technology for the in vivo

assessment of its allergenicity. In Chapter 5, its responsibility in the immunological cross-

Chapter 3

166

reactivity between Black tiger prawn and Jade tiger abalone was further evaluated in a

mouse model.

Chapter 3

167

3.6 References

[1] A. Morikawa, M. Kato, K. Tokuyama, T. Kuroume, M. Minoshima, S. Iwata,

Anaphylaxis to grand keyhole limpet (abalone-like shellfish) and abalone, Annals of

allergy, 65 (1990) 415.

[2] T. Carrillo, F. De Castro, M. Cuevas, J. Caminero, P. Cabrera, Allergy to limpet,

Allergy, 46 (1991) 515-519.

[3] M. Wiszniewska, D. Tymoszuk, A. Pas-Wyroślak, E. Nowakowska-Świrta, D.

Chomiczewska-Skóra, C. Pałczyński, J. Walusiak-Skorupa, Occupational allergy to squid

(Loligo vulgaris), Occupational medicine, 63.4 (2013) 298-300.

[4] S. Emanuel, D. Hawarden, ABC of allergy-hypersensitivity to seafood and fish,

Current Allergy & Clinical Immunology, 29 (2016) 118-120.

[5] B. Pereira, C. Venter, J. Grundy, C.B. Clayton, S.H. Arshad, T. Dean, Prevalence of

sensitization to food allergens, reported adverse reaction to foods, food avoidance, and

food hypersensitivity among teenagers, Journal of Allergy and Clinical Immunology, 116

(2005) 884-892.

[6] A.Y. Wu, G.A. Williams, Clinical characteristics and pattern of skin test reactivities

in shellfish allergy patients in Hong Kong, Allergy and asthma proceedings, OceanSide

Publications, 25.4 (2004) 237-242.

[7] J.H. Choi, S.H. Yoon, Y.J. Suh, C.H. Suh, D.H. Nahm, Y.K. Kim, K.U. Min, H.S.

Park, Measurement of specific IgE to abalone (Haliotis discus hannai) and identification

of IgE binding components, Journal of Asthma, Allergy and Clinical Immunology, 23

(2003) 349-357.

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[8] J.D. Kattan, J. Wang, Allergen component testing for food allergy: ready for prime

time?, Current allergy and asthma reports, 13 (2013) 58-63.

[9] S.H. Sicherer, R.A. Wood, Allergy testing in childhood: using allergen-specific IgE

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(2009) 634-641.

CHAPTER

THE APPLICATION OF RECOMBINANT

TROPOMYOSINS FROM BLACK TIGER PRAWN

(Penaeus monodon) AND JADE TIGER ABALONE (Haliotis

laevigata × Haliotis rubra) IN THE DEVELOPMENT OF

MOUSE MODELS FOR SHELLFISH ALLERGY

Chapter 4

173

4.1 Introduction

According to the Australasian Society of Clinical Immunology and Allergy (ASCIA)

2016 report, 4-10% of children and approximately 2% adults have been affected by food

allergy and the rate of anaphylaxis based on hospital admissions has been increasing

dramatically over the last decade in Australia, USA and UK. Type I hypersensitivity is

the most frequent pattern of food allergy, the adverse reactions of which are caused by

forming the complex of specific IgE antibody and food allergens. Crude extracts of foods

are often applied in the conventional diagnosis and immunotherapy, thus only the food

sources that patients are allergic to can be identified [1, 2]. Within recent two decades,

the advances of molecular cloning techniques allow researchers to reveal the exact

composition of allergenic sources through generating and characterising recombinant

proteins [3]. To date, multiple food allergens have been identified, cloned and

characterised at a molecular and biochemical level, such as Gly m 1 and Gly m 3 in soy

allergy, Bos d 5 in milk allergy, Gal d 1, 2 and 3 in egg allergy [4], and Ara h 1, Ara h 2,

Ara h 3, Ara h 4, Ara h 5, Ara h 6 and Ara h 7 in peanut allergy [5].

Currently, the major applications of recombinant allergens include the studies of

component-resolved diagnostics, allergen-specific immunotherapies and

immunopathologic mechanisms [1, 6]. The recombinant protein that can be used for these

studies has to be validated for its immunological allergenicity. The conventional

approaches include the test of their reactivity with patient IgE antibody in vitro, and the

monitoring of specific cellular immune responses (i.e., T-cell responses and specific

basophil activation) against recombinant allergens in vitro [7]. However, neither IgE

antibody nor immune cells alone are equal to the whole immunological process of allergy.

Moreover, there is a lack of clinical relevance in in vitro research. Since recombinant

components cannot be easily applied in human research due to the potential risk on human

Chapter 4

174

health, to the end of this question, mouse models can be very useful tools to study

recombinant allergens. Substantial evidence for the humoral and cellular responses can

be provided by experimental animals for a comprehensive evaluation of the potential

allergenicity of any recombinant allergen of interests. To date, several recombinant food

allergens, such as carp parvalbumin Cyp c1.01 [8, 9], modified peanut allergens Ara h 1,

Ara h 2, and Ara h 3 [10] and hypoallergenic ovomucoid [11], have been used in mouse

studies, greatly contributing to the development of precise diagnosis or

immunomodulatory strategies.

In shellfish allergy, an increasing number of recombinant allergens have been produced

within the recent two decades, such as recombinant tropomyosin (rTM) from Greasyback

shrimp [12], Black tiger prawn [13], Brown shrimp [14], Blue swimmer crab [15], Coral

swimmer crab [16], Pacific oyster [17], Variously coloured abalone, Noble scallop and

Asian green mussel [18], sarcoplasmic calcium-binding protein from Pacific white

shrimp [19] and Whiteleg shrimp [20], arginine kinase derived from Mud crab [21], and

myosin light chain derived from Black tiger prawn [22]. However, these recombinant

allergens have been barely applied in mouse studies to examine their allergenic properties

but also their potentials to induce immunological cross-reactivity in shellfish allergy [23].

In Chapter 3, the immunological cross-reactivity between the heated protein extracts of

Jade tiger abalone (Haliotis laevigata × Haliotis rubra) and Black tiger prawn (Penaeus

monodon) has been thoroughly evaluated, and tropomyosin (TM) has been suspected as

the major cross-reactive allergen. To further investigate TM for its responsibility in the

prawn-abalone cross-reactivity, the allergenic properties of TM were to be investigated

and the availability of rTMs from Jade tiger abalone and Black tiger prawn would be very

important. Therefore, in the current chapter, TMs from Black tiger prawn and Jade tiger

abalone were expressed as His-tagged fusion recombinant proteins in Escherichia coli.

Chapter 4

175

Subsequently, their allergenicity was tested with serum of allergic patients and the

established mouse model, but also compared with the allergenicity of corresponding

heated extracts of shellfish in mice.

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176

4.2 Aims


(1) To clone, express and purify the recombinant tropomyosins from Jade tiger

abalone (Haliotis laevigata × Haliotis rubra) and Black tiger prawn (Penaeus

monodon).

(2) To evaluate the allergenic property of the recombinant tropomyosins from Jade

tiger abalone and Black tiger prawn using human sera and a mouse model.

(3) To investigate the antibody cross-reactivity to tropomyosin from Jade tiger

abalone and Black tiger prawn.

(4) To identify human IgE reactive proteins in heated extracts of Jade tiger abalone

and Black tiger prawn.

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4.3 Animals, materials and methods

4.3.1 Cloning and cDNA sequencing of tropomyosin from Jade tiger abalone

4.3.1.1 Total RNA isolation

Alive Jade tiger abalone (Haliotis laevigata × Haliotis rubra) (Abal) were purchased

from a local seafood shop (Townsville, Australia) and transported to the laboratory on

ice. All the equipment involved in RNA extraction was treated with RNaseZap™ (Life

Technologies) before start. Briefly, 100 mg of the fresh foot muscle from abalone was

collected and homogenised in 1 ml of TRIzol® reagent (Life Technologies). After the

incubations with 0.5 ml of chloroform and 100% isopropand and the centrifugation at 12,

000 x g, RNA was precipitated in an RNase-free tube, and subsequently washed with 1

ml of 75% ethanol. The washed RNA pallet was air dried by incubation at 60°C for 5-10

min and subsequently resuspended in 30 μl of RNase-free water. The resuspended RNA

was incubated at 60°C for 10-15 min before storage at -20°C or any downstream

applications. The concentration and purity of RNA was tested using a Nanodrop ND-

1000 spectrophotometer (New England Biolabs).

4.3.1.2 cDNA transcription and PCR amplification of tropomyosin

cDNA was produced from RNA by reverse transcription-polymerase chain reaction (RT-

PCR) using Tetro cDNA Synthesis kit (Bioline). Firstly, the extracted RNA was treated

with RQ1 RNase-Free DNase (Promega) to remove DNA residue. According to the

protocol of Tetro cDNA Synthesis kit, briefly, 20 μl of the priming premix was prepared

by mixing up total RNA (4 μg), Oligo (dT)18 (1 μl), 10 M dNTP mix (1 μl), RNase

inhibitor (1 μl) and Tetro reversetranscriptase (200 unit). The premix was incubated at

45°C for 30 min. Subsequently, the reaction was stopped by heating at 85°C for 5 min

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178

and then cooled down on ice. The cDNA was ready to be processed as the template to

PCR.

The coding region of tropomyosin (TM) from Jade tiger abalone was amplified in a 25 μl

PCR system. A pair of primers (TM-Abal-F and TM-Abal-R) were designed on the basis

of the published mRNA of TM from Haliotis discus discus (GenBank accession no.

AB444939.1), and used in PCR:

(Forward) TM-Abal-F

5’-GCCTGAATTCTGGATGCCATCAAGAAGAAGATG-3’

(Reverse) TM-Abal-R

5’-GCACAAGCTTTTAATAACCAGCCAACTCCGCGAA-3’

The PCR reaction contained 12.5 μl of GoTaq® DNA Polymerase (Promega, Australia),

0.6 μl of each primer, and 3 μl of synthesised cDNA. The PCR program included 94°C

for 2 min, followed by 35 cycles of 94°C for 30 s, 60°C for 1 min and 72°C for 45 s, and

subsequently a final extension at 72°C for 7 min. The PCR product was analysed on a 2%

agarose gel.

4.3.1.3 Cloning of the coding region of tropomyosin into pProEX HTb vector

The PCR product was purified using Wizard® DNA Clean-Up system (Promega).

Briefly, the PCR product was added to DNA clean-up resin and pushed into a Wizard®

Minicolumn. The DNA-binding Minicolumn was washed with 2 ml of 80% isopropanol

and centrifuged for 2 min. Subsequently, 50 μl of warm water was applied to the

Minicolumn. At last, the DNA was eluted by centrifugation for 20 s. The purified PCR

product is ready for the double digestion with restriction enzymes.

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pProEX HTb vector, which was kindly provided by Professor James Burnell at James

Cook University, were used for both DNA clone and protein expression. Both the pProEX

HTb vector (260 ng) and the purified PCR product (310 ng) were digested with two

restriction enzymes EcoR I ((New England Biolab, 24 unit) and Hind III (New England

Biolab, 10 unit). The mixture of digestion reaction (20 μl) was incubated at 37°C for 1 h

and then terminated by adding 10 μl of 6 x loading buffer. Subsequently, the digested

PCR product was purified using Wizard® DNA Clean-Up system, and here named “pure

digested TM-Abal”. The digested pProEX HTb vector was purified from a low-melting

agarose gel using Wizard® PCR Preps DNA kit (Promega). Briefly, the digested pProEX

HTb vector was loaded into a 1% low-melting agarose gel and 80-voltage electrophoresis

was conducted for 30 min. Subsequently, the desire band was excised and weighed (less

than 300 mg). The gel slice was melt after 70°C incubation and then thoroughly mixed

with 1 ml of the resin. The resin/DNA mix was pushed into the Minicolumn and washed

with 2 ml of 80% isopropanol. The resin was dried by centrifugation at 10, 000 x g for 2

min. 50 μl of pre-heated water (65-80°C) was added to the Minicolumn to elute DNA.

One min later, the Minicolumn was centrifuged for 20 s. Finally, the DNA fragment was

collected in a new tube and was named “pure digested pProEX HTb”.

Pure digested TM-Abal and pure digested pProEX HTb were ligated using T4 DNA

Ligase (Promega). The ligation reaction consisted of 45 ng of pure digested pProEX HTb,

15 ng of pure digested TM-Abal, 1 μl of 10 x ligase buffer and 0.3 μl of 3 unit/μl T4 DNA

Ligase. The reaction mixture was incubated at room temperature for 3 h and then used

immediately for the cell transformation.

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4.3.1.4 Transformation of DH12S competent cells and sequencing of the plasmid

DNA

The chemically competent E. coli cells named DH12S, kindly provided by Dr. Bernhard

Waltenspiel at James Cook University, were prepared and used to clone the recombinant

DNA of tropomyosin from Jade tiger abalone. Briefly, the reaction mix of ligation was

gently added into DH12S cells, and the cells were thawed on ice for 20 min.

Subsequently, this mixture received a heat shock at 42°C for 60 s, and then transferred on

ice for 2 min. The entire reaction mix was spread onto the surface of LB agar plate made

with 100 μg/ml ampicillin, following with overnight incubation at 37°C. Several colonies

were picked up separately and tested for the insertion of TM-Abalone into the vector by

means of colony PCR. The colonies with positive PCR results were inoculated into 4 ml

of LB broth with 100 μg/ml ampicillin and incubated on a shaking platform overnight at

37°C. Subsequently, the recombinant DNA was extracted out of DH12S cells using

AxyPrep Plasmid Miniprep Kit (Corning) and the instructions of manufactures were

followed. Finally, the purified recombinant DNA of TM-Abal-pProEX HTb was sent to

Macrogen Inc, South Korea for sequencing of the open reading frame of Abal-TM.

4.3.1.5 Transformation of BL21 competent cells

TM-Abal-pProEX HTb was transformed into another chemically competent cell strain

BL21 for auto-induction of the recombinant protein of Abal-TM following a similar

protocol of transformation of DH12S competent cells. After inoculation, the glycerol

stock of positive clones was prepared and stored at -80°C.

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4.3.2 Expression and purification of recombinant tropomyosins

4.3.2.1 Culturing of BL21 Escherichia coli cells and auto-induction of the

recombinant protein expression

The DNA of tropomyosin from Black tiger prawn (Penaeus monodon) (TM-BTP) that

was inserted into vector pProEX HTb was labeled as TM-BTP-pProEX HTb. The E. coli

cells (BL21) that carried the DNA of TM-BTP were kindly provided by Dr. Sandip

Kamath at James Cook University. The method of auto-induction was based on a previous

study performed by F.W. Studier [23] and some modifications developed by Dr. Martina

Koeberl [24]. 50 μl of freshly cultured E. coli that contained the plasmid DNA of TM-

BTP-pProEX HTb or TM-Abal-pProEX HTb was transferred into an autoclaved baffled

flask with 250 ml of ZYP-5052 medium, following with incubation at 37°C. To optimise

the harvest time-point, small aliquots were collected at different time-points for testing

the OD600nm. When OD600nm reached 2.0-2.3, the bacterial culture was centrifuged at 5, 000

RPM for 3 min at 4°C, and subsequently the supernatant was collected. Cell pallets from

every 50 ml of the cell culture were re-suspended with 2 ml of the extraction buffer,

following with four times of French pressure cell. Cell lysate was collected and

centrifuged at 25, 000 x g for 20 min. 10 ml of fresh extraction buffer was added to re-

suspend cell pallets and subsequently the second batch of supernatant was collected. The

procedure of re-suspension with new extraction buffer, centrifugation and collection of

the supernatant was performed in total of five times. Sodium dodecyl sulphate -

polyacrylamide gel electrophoresis (SDS-PAGE) analysis was conducted to determine

the supernatant for further purification.

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4.3.2.2 Purification of recombinant tropomyosins of Jade tiger abalone and Black

tiger prawn

To purify the generated recombinant TM (rTM) from the crude lysate, immobilised metal

ion affinity chromatography (IMAC) was conducted using HisPur Nickel (Ni)-

Nitrilotriacetic Acid (NTA) metal-chelate Resin. Firstly, 3 ml of the dispersed resin was

added into an empty Pierce spin column to prepare a HisPur Ni-NTA Spin Column.

Subsequently, 1 ml of the crude lysate extract was mixed with 1 ml of equilibration buffer,

and this mixture was filtered through a 0.22 μm membrane. In the next step, the filtered

extract was loaded onto an equilibrated HisPur Ni-NTA Spin Column with the bottom

plugged. The sample of cell lysate extract was mixed with a HisPur Ni-NTA beads on an

orbital shaker at 4°C for at least 30 min, following with centrifugation to remove the flow-

through. Then the resin was washed with 3 ml of wash buffer and the flow-through was

collected for analysing the presence of rTM by a SDS-PAGE gel. At last, rTM was eluted

with elution buffer and dialysed into fresh PBS buffer. The purified recombinant proteins

were concentrated using spin columns, analysed using SDS-PAGE gel and their

sequences confirmed by mass spectrometry. Pierce BCA Protein Assay was performed to

quantify the concentration of rTM.

4.3.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was conducted to examine the protein repertoire in the prepared sample, the

protocol of which was previously described in Chapter 3. In the current chapter, the

analysed protein samples were heated Black tiger prawn (hBTP), heated Jade tiger

abalone (hAbal), rTM-BTP and rTM-Abal.

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4.3.4 Patient sera

Twelve subjects with a confirmed clinical history of shellfish allergy and one healthy

subject as a negative control were recruited by The Alfred Hospital, Allergy Clinic,

Melbourne, Victoria, Australia [25, 26]. Allergen specific IgE was quantified using the

ImmunoCAP system (Thermo Scientific) at The Alfred Hospital by Prof. Robyn

O’Hehir’s group.

Table 4.1 provides the demographic details of these patients. James Cook University’s

Ethics committee granted the ethics approval for this research (Project number H4313) in

collaboration with The Alfred Hospital (Project number 192/07) and Monash University’s

Ethics Committees (MUHREC CF08/0225).

4.3.5 Patient immunoblotting

35 μg of hAbal and hBTP, and 25 μg of rTM-Abal and rTM-BTP were heated at 95°C

for 5 min before loaded onto a 12% SDS-PAGE gel separately. Subsequently, the

electrophoretic separation was conducted at 170 V until the tracker dye reached the

bottom of the gel. In the next step, resolved proteins were transferred from the gel to an

activated PVDF or a nitrocellulose membrane, and subsequently the membrane was

blocked with 5% skim milk. The blocked membrane was incubated with the patient serum

(1:10 diluted) using a slot blot apparatus (Idea Scientific) overnight at 4°C, following

with the incubation with 1:10, 000 diluted rabbit anti-human IgE polyclonal antibody

(DAKO Corporation) for 1 h at room temperature. Subsequently, the membrane was

incubated with 1:10, 000 diluted goat-anti-rabbit IgG HRP conjugate (Promega) to

visualise the IgE antibody reactivity [24, 26].

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4.3.6 Mouse immunoblotting

The protocol of mouse immunoblot was described previously in Chapter 3, Section 3.3.5.

The serum that was examined in this chapter was pooled from the group of rTM-BTP

sensitised, hBTP gavaged mice (rTM-BTP/hBTP group) and the group of rTM-Abal

sensitised, hAbal gavaged mice (rTM-Abal/hAbal group), respectively.

4.3.7 Mass spectrometric identification

To identify the antibody reactive proteins in hBTP and hAbal, and to confirm the

expressed protein was TM, in-gel tryptic digestion of the selected bands in a SDS-PAGE

gel was performed for mass spectrometric (MS) analysis, following the protocols that

were described previously in Chapter 3, Section 3.3.6.

4.3.8 Amino acid sequence alignment of prawn and abalone tropomyosins

The amino acid sequences of TMs from Black tiger prawn (HM486525) and Jade tiger

abalone were compared using the MEGA ClustalW alignment. The amino acid sequence

of TM from Black tiger prawn was obtained from the database of National Centre for

Biotechnology Information (NCBI).

Chapter 4

185

Table 4.1 Demographics of 12 shellfish allergic patients and one non-atopic control donor.

Note: NT=Not tested.

Chapter 4

186

4.3.9 Animals

8-week-old female BALB/c mice were kept at the Cairns campus, building E4 Animal

Facility under specific pathogen-free conditions. Mice were fed with a commercial

vegetarian diet purchased from Specialty Feeds. The animal ethics committee from James

Cook University approved the experiments (Animal ethics approval number A2108).

4.3.10 Preparation of shellfish extracts and recombinant tropomyosins

Heated extracts of Black tiger prawn and Jade tiger abalone were prepared using the

method previously described in Chapter 2, Section 2.3.2. The concentration of heated

shellfish extracts and purified rTMs were estimated and adjusted for mouse studies.

4.3.11 Induction and evaluation of mouse models in shellfish allergies

4.3.11.1 Mouse sensitisation and challenge

As shown in Figure 4.1, from Day 0 to Day 14, mice were IP sensitised weekly with a

mixture of 10 μg of the allergen(s) and 2 mg of alum. Allergens were the heated shellfish

extracts (hBTP or hAbal) or the recombinant tropomyosin (rTM-BTP or rTM-Abal). On

Day 21, 24, 27 and 30, hBTP or rTM-BTP sensitised mice were orally gavaged with 1mg

of hBTP, while hAbal or rTM-Abal sensitised mice were orally gavaged with 1mg of

hAbal. Mice were fasted overnight but had unlimited access to water before every gavage.

In contrast, the control mice were treated with a mixture of PBS only with alum during

the sensitisation period, and subsequently gavaged with 200 μl of PBS on Day 21, 24, 27

and 30. One day after the forth gavage, all the animals were sacrificed and the samples

from blood, spleens and jejunums were isolated for further analysis.

During the gavage phase, the heated shellfish extracts were used rather than rTMs. The

reason was that the required amount of proteins for repeated gavages greatly exceeded

Chapter 4

187

the production capacity of the current expression system, although the auto-induction

method used in this work has been regarded as an optimised approach for high yield

production of recombinant proteins [25].

In this thesis, each mouse group was named as follows: “hBTP/hBTP group” was the

group of hBTP-sensitised, hBTP-gavaged mice, “hAbal/hAbal group” was the group of

hAbal -sensitised, hAbal -gavaged mice, “rTM-BTP/hBTP group” was the group of rTM-

BTP-sensitised, hBTP-gavaged mice, and “rTM-Abal/hAbal group” was the group of

rTM-Abal-sensitised, hAbal -gavaged mice.

Figure 4.1 The experimental design of the mouse model for shellfish allergy. Each

group comprised of six BALB/c female mice.

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188

4.3.11.2 Detection of serum antibodies and mast cell protease-1

The serum isolation and sandwich ELISA were performed following the protocols that

have been described in Chapter 2. The only modification made in the current ELISA was

that the serum samples from hBTP-gavaged mice were loaded to the hBTP-coated ELISA

plates, and the serum samples from hAbal-gavaged mice were loaded to the hAbal-coated

ELISA plates for the measurement of allergen-specific IgE, IgG1 and IgG2a.

4.3.11.3 In vitro stimulation of splenocytes and measurement of cytokines

Spleens were isolated and single cell suspension prepared as previously described in

Chapter 2. 0.5 million cells were cultured with hBTP (4 μg) or hAbal (4 μg) for 72 h in

5% CO2 at 37°C. The splenocytes cultured with 1 μg/ml anti-CD3 and anti-CD28 were

considered as the positive control for this assay.


The single cell suspension of blood and spleen was prepared as described in Chapter 2,

Section 2.3.3.4. The surface staining panel consisted of 7 fluorescence-conjugated

antibodies (Jomar Life Research), including Anti-Mouse CD170 (Siglec F) PerCP-eFluor

710, Anti-Mouse CD3 APC-eFluor 780, Anti-Mouse IgE FITC, Anti-Mouse CD11b PE,

Anti-Mouse CD49b (Integrin alpha 2) APC, Anti-Mouse CD19 APC-eFluor 780, Anti-

Mouse Fc Fc Epsilon Receptor I alpha (FcεR1) eFluor 450.


Two-tailed Mann-Whitney U test was performed using Prism 6.0. The data shown in this

chapter are presented as mean values with the standard error of the mean (SEM). p values

of less than 0.05 were considered statistically significant.

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189

4.4 Results

4.4.1 Jade tiger abalone and Black tiger prawn tropomyosin sequence comparison

Jade tiger abalone and Black tiger prawn TM were cloned and analysed by cDNA

sequencing to compare primary structures (Figure 4.2). The amino acid sequence of

abalone TM was deduced from the generated cDNA and published under the Genbank

accession number KX961689. The alignment of amino acid sequence shows 63.4%

amino acid sequence identity between prawn and abalone TM. The amino acids variations

between these two TMs distributed throughout the whole sequence. Eight IgE-binding

epitopes of prawn TM has been previously predicted and characterised by by Reese et al

2005 [28]. Figure 4.1 demonstrates only one potential IgE-binding epitope was identical

as compared to abalone TM.

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190

Figure 4.2 Amino acid sequence alignment of tropomyosins from Black tiger prawn (Penaeus monodon), Genbank protein ID

ADM34184.1, and Jade tiger abalone (Haliotis laevigata × Haliotis rubra), Genbank protein ID APG42675.1. The amino acids marked

with yellow boxes are variations in the two tropomyosin sequences; orange boxes indicate the eight IgE binding regions (I – VIII) previously

identified by Reese et al [28].

Black tiger prawn (Penaeus monodon) M D A I K K K M Q A M K L E K D N A M D R A D T L E Q Q N K E A N N R A E K S E E E V H N L Q K R M Q Q L E N D L D Q V 60

Jade tiger abalone (Haliotis laevigata x Haliotis rubra) M D A I K K K M L A M K M E K E N A V D R A E Q N E Q K L R D T E E Q K A K I E E D L N N L Q K K C A N L E N D F D N V 60

Black tiger prawn (Penaeus monodon) Q E S L L K A N I Q L V E K D K A L S N A E G E V A A L N R R I Q L L E E D L E R S E E R L N T A T T K L A E A S Q A A 120

Jade tiger abalone (Haliotis laevigata x Haliotis rubra) N E Q L Q E A M T K L E T S E K R V T E M E Q E V S G T T R K I T L L E E D L E R N E E R L Q T A T E R L E E A S K A A 120

Black tiger prawn (Penaeus monodon) D E S E R M R K V L E N R S L S D E E R M D A L E N Q L K E A R F L A E E A D R K Y D E V A R K L A M V E A D L E R A E 180

Jade tiger abalone (Haliotis laevigata x Haliotis rubra) D E S E R G R K V L E S R S L A D D E R I D Q L E A Q L K E A K Y I A E D A E R K Y D E A A R K L A I T E V D L E R A E 180

Black tiger prawn (Penaeus monodon) E R A E T G E S K I V E L E E E L R V V G N N L K S L E V S E E K A N Q R E E A Y K E Q I K T L T N K L K A A E A R A E 240

Jade tiger abalone (Haliotis laevigata x Haliotis rubra) A R L E A A E A K I L E L E E E L K V V G N N M K S L E I S E Q E A S Q R E D S Y E E T I R D L T Q R L K D A E N R A T 240

Black tiger prawn (Penaeus monodon) F A E R S V Q K L Q K E V D R L E D E L V N E K E K Y K S I T D E L D Q T F S E L S G Y 284

Jade tiger abalone (Haliotis laevigata x Haliotis rubra) E A E R T V S K L Q K E V D R L E D E L L A E K E K Y K A I S D E L D Q T F A E L A G Y 284

Chapter 4

191

4.4.2 Identification of human IgE reactivity to abalone and prawn tropomyosin

Figure 4.3 SDS-PAGE Coomassie stain (A1 and B1) and IgE immunoblot using sera

from 12 shellfish allergic patients of heated abalone extracts (A2), heated prawn

extracts (A3), recombinant abalone tropomyosin (B2) and recombinant prawn

tropomyosin (B3). Red stars indicate the selected protein bands for mass spectrometric

identification. Abbreviations: hAbal = heated abalone extracts, hBTP = heated prawn

extracts, rTM-Abal = recombinant abalone tropomyosin, rTM-BTP = recombinant prawn

tropomyosin, 1 – 12 = patient sera, and N = negative control (healthy individual).

Chapter 4

192

Table 4.2 IgE antibody reactive proteins identified by SDS-PAGE using mass

spectrometry technique. The sequence coverage is denoted as percentage. IgE reactive

proteins have been matched to known allergens using Uniport entry names.

Chapter 4

193

To study the allergenicity of TM and identify the IgE reactive proteins in hBTP and hAbal,

IgE immunoblot was performed using patient sera. In the immunoblots of hBTP and

hAbal (Figure 4.3.A), the strongest IgE binding appeared at about 37 kDa, and the

corresponding protein was previously identified as TM (Chapter 3, Table 3.1). 7 of 12

patients showed positive IgE binding to the identified abalone TM and 9 of 12 recognised

the identified prawn TM. Also, TMs from Jade tiger abalone and Black tiger prawn were

generated as recombinant proteins in E. coli to analyse the pattern of IgE recognising TM

in allergy patients. As illustrated in Table 4.2, the MS analysis has confirmed that the

expressed protein is TM, with the sequence coverage of 54% for rTM-BTP and 73% for

rTM-Abal, respectively. The serum IgE from 5 of 8 patients could bind to rTM-Abal and

9 of 12 recognised rTM-BTP. The patients with positive IgE binding to the rTM were all

able to recognise the identified natural TM, which confirms the allergenic property of

rTM and the MS identification of TM in the total protein extracts. Furthermore, the IgE

reactivity to abalone TM was only reported in the patient sera with IgE reactivity to prawn

TM (patient NO.1, 2, 5, 6, 7 and 8). This result indicates the IgE cross-reactivity between

prawn and abalone TM in these patients. The registration of TM from Jade tiger abalone

has been officially approved by World Health Organization and International Union of

Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee and the

allergen name is Hal l 1.

Figure 4.3.A shows that the pattern of human IgE reactivity in the sera from multiple

patients including subject 5, 6, 7 and 8 seemed very consistent with the results of mouse

IgG immunoblots performed in Chapter 3 (Figure 3.4 and Table 3.1). In addition to TM,

MS analysis demonstrates the other potential reactive proteins were arginine kinase and

troponin T (Chapter 3, Table 3.1). In addition, subject 5 displayed a unique pattern of IgE

binding to several proteins in hBTP and hAbal. MS identification shows that they were

Chapter 4

194

very likely to be myosin heavy chain and paramyosin located at the region between 120

kDa and 140 kDa. (Table 4.2).

Chapter 4

195

4.4.3 Serum antibodies and mast cell protease-1

Figure 4.4 Production of serum allergen-specific IgE (A and B), total IgE (C) and

mast cell protease-1 (mMCP-1) (D). Levels of mMCP-1 and IgE antibody were

determined by sandwich ELISA. Data are expressed as mean ± SEM (n = 6). p< 0.05,

and p< 0.01. Each dot represents an individual mouse. Abbreviation: ns = Not

significant.

Control

hBTP/hBTP

rTM-B

TP/hBTP0.0

0.2

0.4

0.6

OD

450n

m

**

**

hBTP-specific IgE

ns

OD

450n

m

Control

hAbal/hAbal

rTM-A

bal/hAbal

0.0

0.2

0.4

0.6**

***

hAbal-specific IgE

Total IgE

Control

hBTP/hBTP

rTM-B

TP/hBTP

hAbal/hAbal

rTM-A

bal/hAbal

0.0

0.2

0.4

0.6

OD

450n

m

**

** **

ns

A B

C D mMCP-1ng

/ml

Control

hBTP/hBTP

rTM-B

TP/hBTP

hAbal/hAbal

rTM-A

bal/hAbal

0

5000

10000

15000 ***

*

ns

**

*

**

Chapter 4

196

Figure 4.5 Production of serum allergen-specific IgG1 (A and B) and allergen-

specific IgG2a (C and D). Levels of IgG1 and IgG2a antibody were determined by

sandwich ELISA. Data are expressed as mean ± SEM (n = 6). p< 0.05, and p< 0.01.

Each dot represents an individual mouse.

* **

Chapter 4

197

To investigate whether recombinant tropomyosin could elicit Th2-associated humoral

responses, in this experiment, mice were sensitised with the recombinant tropomyosin

(rTM-BTP or rTM-Abal), and their serum antibodies and mMCP-1 were analysed. As

demonstrated in Figure 4.4 and Figure 4.5, specific IgE antibody and IgG1 antibody were

increased significantly in all the groups of sensitised mice. Figure 4.4.A and C show that

the IgE antibody in rTM-BTP-sensitised mice was at a very similar level as compared to

the mice that were sensitised with hBTP. In contrast, as demonstrated in Figure 4.4.B, the

sensitisation with rTM-Abal induced a remarkably lower level of IgE as compared to

hAbal. The data of serum total IgE antibody was consistent with the result of specific IgE,

except that there was no statistically significant difference between the control and rTM-

Abal/hAbal group. In contrast, comparing with heated extracts of shellfish, recombinant

tropomyosins induced significantly higher levels of serum specific IgG1 antibody.

Moreover, the results of serum mMCP-1 confirmed that rTM-BTP and hBTP might have

a similar level of allergenicity but hAbal could provoke stronger responses of mast cells

as compared to rTM-Abal (Figure 4.4.D). Specific IgG2a as a Th1-associated antibody

was only detectable in the sera from the mice that were sensitised with rTMs (Figure

4.5.C and D).

Chapter 4

198

4.4.4 Identification of mouse IgG binding proteins

Figure 4.6 Immunoblot analysis of the IgG reactivity to the heated extracts of

abalone (A) and prawns (B).

Chapter 4

199

To confirm the induction of allergen-specific antibody in this mouse model, IgG

immunoblot was performed using the sera from rTM-BTP/hBTP group and rTM-

Abal/hAbal group. The strong IgG binding to the protein at 37 kDa was observed in rTM-

sensitised mice, and this protein has been previously identified as TM by MS analysis in

Chapter 3, Table 3.1. Moreover, IgG antibody from rTM-Abal sensitised mice could

recognise TM-BTP, but the IgG reactivity was less strong as compared to binding to TM-

Abal, and vice versa. This result indicates the IP injection of the mixture of rTM and

adjuvant can successfully trigger the production of TM-specific antibody, and there is

antibody cross-reactivity between prawn and abalone TM.

Chapter 4

200

4.4.5 Production of cytokines by stimulated and unstimulated splenocytes

Figure 4.7 Production of cytokines by stimulated splenocytes. The splenocytes in

hBTP/hBTP group and rTM-BTP/hBTP group were cultured with hBTP, and the

splenocytes in hAbal/ hAbal group and rTM-Abal/hAbal group were cultured with hAbal

for three days in vitro. ELISA was preformed to determine the levels of IL-5 (A and A’),

IL-13 (B and B’) and IFN-gamma (C and C’). Data are expressed as mean ± SEM (n =

6). p< 0.05, and p< 0.01. Each dot represents an individual mouse. Abbreviation: ns

= Not significant.

pg/m

l

Control

hBTP/hBTP

rTM-B

TP/hBTP0

500

1000

1500

2000 ns

pg/m

l

Control

hBTP/hBTP

rTM-B

TP/hBTP0

500

1000

1500 **

**

ns

pg/m

l

Control

hBTP/hBTP

rTM-B

TP/hBTP0

500

1000

1500

pg/m

l

Control

hAbal/hAbal

rTM-A

bal/hAbal

0

200

400

600

800**

pg/m

l

Control

hAbal/hAbal

rTM-A

bal/hAbal

0

200

400

600

**** ns

pg/m

l

Control

hAbal/hAbal

rTM-A

bal/hAbal

0

200

400

600

800

IL-5

IL-13

IFN-gamma

A

B

C

A'

B'

C'

* **

Chapter 4

201

To study the pattern of cytokines systemically induced by sensitisation with rTMs,

splenocytes were collected and stimulated with hBTP or hAbal in vitro. The data in Figure

4.7 C and C’ show that the splenocytes from control mice, no matter stimulated with

allergens or not in vitro, expressed a considerable level of IFN-gamma (i.e. 169.8 ± 78.01

pg/ml in Figure 4.7.C’) but very low or undetectable levels of Th2-type cytokines. In

contrast, as illustrated in Figure 4.7. A, A’, B and B’, after the stimulation with hBTP or

hAbal, the splenocytes from all the allergen-treated mice could release greatly increased

levels of Th2-related cytokines including IL-5 and IL-13 but not Th1-type cytokine IFN-

gamma, as compared to the control. This result confirms that the increase of Th2-

associated cytokines can be an important hallmark of allergic responses. In this work,

both rTMs could elicited a significant production of IL-5 and IL-13. This finding indicates

the allergenic potent of rTMs and consequently supports the application of rTM in the

future mouse studies. Importantly, there was no statistically significant difference

between the amount of IL-5 and IL-13 produced by hBTP-sensitised splenocytes and

rTM-BTP-sensitised splenocytes (Figure 4.7.A and B). However, Figure 4.7.A’ and B’

show that as compared to the stimulated splenocytes from hAbal-sensitised mice, the

splenocytes from rTM-Abal-sensitised mice released lower levels of IL-13 (267.4 ± 52.56

pg/ml, p = 0.093) and IL-5 (169.5 ± 35.25 pg/ml, p = 0.0022).

Chapter 4

202

Figure 4.8 Production of cytokines by unstimulated splenocytes. The splenocytes

were cultured with the absence of allergens for three days in vitro. ELISA was preformed

to determine the levels of IL-5 (A), IL-13 (B) and IFN-gamma (C). Data are expressed as

mean ± SEM (n = 6). p< 0.05, and p< 0.01. Each dot represents an individual mouse.

Unstimulated splenocytes produced limited Th2-associated cytokines, although increased

IL-13 or IL-5 could still be detected in some of the sensitised splenocytes. For example,

as compared to the control group, IL-13 was increased in rTM-Abal/hAbal group (Figure

4.8. B), and IL-5 was increased in hAbal/hAbal group (Figure 4.8.A). These

upregulations of the cytokine concentration in unstimulated splenocytes might result from

the remaining cytokines within splenocytes that had not been totally secreted yet in vivo.

IL-5 pg

/ml

Control

hBTP/hBTP

rTM-B

TP/hBTP

hAbal/hAbal

rTM-A

bal/hAbal

0

10

20

30

40*

IFN-gamma

pg/m

l

Control

hBTP/hBTP

rTM-B

TP/hBTP

hAbal/hAbal

rTM-A

bal/hAbal

0

500

1000

1500

2000 *IL-13

pg/m

l

Control

hBTP/hBTP

rTM-B

TP/hBTP

hAbal/hAbal

rTM-A

bal/hAbal

0

50

100

150*

A B C

* **

Chapter 4

203

4.4.6 Secretion of IgA antibody in mouse jejunum

Figure 4.9 Secretion of intestinal hBTP-specific IgA (A) and hAbal-specific IgA (B).

Levels of IgA antibody in the protein extracts of jejunums were determined by sandwich

ELISA. Data are expressed as mean ± SEM (n = 6). p< 0.05, and p< 0.01. Each dot

represents an individual mouse. Abbreviation: ns = Not significant.

The intestinal IgA antibody against hBTP was not significantly increased in the groups

of hBTP/hBTP or rTM-BTP/hBTP, although a few good responders were observed.

Interestingly, by comparison, hAbal tended to be a more offending food, since the level

of secretary IgA antibody against hAbal in the jejunums from hAbal-sensitised mice was

significantly higher as compared to the control mice. The lower secretion of IgA in rTM-

Abal/hAbal group as compared to hAbal/ hAbal group was clearly consistent with the

results of serum IgE levels.

hBTP-specific IgA O

D45

0nm

Control

hBTP/hBTP

rTM-B

TP/hBTP0.0

0.5

1.0

1.5ns

nshAbal-specific IgA

OD

450n

m

Control

hAbal/hAbal

rTM-A

bal/hAbal

0.0

0.5

1.0

1.5

** *

A B

* **

Chapter 4

204

4.4.7 Induction of eosinophil and basophil in circulating blood and spleen


and spleen (A’ and B’). The populations of eosinophil and basophil in blood and spleen

were determined by flow cytometer and the statistics were analysed by FlowJo software.

Data are expressed as mean ± SEM (n = 6). p< 0.05, and p< 0.01. Each dot

represents an individual mouse.

* **

Chapter 4

205

The healthy murine blood and spleen usually does not contain appreciable numbers of

eosinophil and basophil. As shown in Figure 4.10, the blood or spleen from control mice

contained a small number of eosinophils (accounting for less than 2% of living cells), and

limited IgE-binding basophils (accounting for less than 2% of non-T non-B cells). By

comparison, the proportions of eosinophils and basophils were increased in the blood and

spleen from all the four groups of sensitised mice. Except for the circulating eosinophils

in hBTP/hBTP group, the upregulation of eosinophils and basophils in the blood and

spleen from other sensitised mice was statistically significant. There was no statistically

significant difference in the frequency of these two cell populations between hBTP-

sensitised mice and rTM-BTP-sensitised mice, or between hAbal-sensitised mice and

rTM-Abal-sensitised mice.

Chapter 4

206

4.5 Discussion

Shellfish allergy is amongst the most common food allergies especially for adults. The

large number and high diversity of shellfish species including crustacean and molluscs

warrant massive investigations on the identification of allergens and the evaluation of

allergenicity for precise diagnosis and immunopathogenic research. The highly conserved

muscle protein tropomyosin (TM) has been the most frequently reported shellfish allergen

and is very likely to be cross-reactive among different invertebrates. Recombinant TMs

(rTMs) derived from multiple crustacean species and mites have been produced and their

allergenic potentials were confirmed by the reactivity of patient IgE in vitro. However,

rTMs from many commonly consumed mollusc species are still not available. Moreover,

the capacity of rTMs to provoke Th2-associated responses in vivo has not been previously

evaluated.

In the present study, TM from Jade tiger abalone (Haliots. laevigata × Haliots. rubra)

(TM-Abal) was for the first time completely sequenced, cloned and expressed as a His-

tagged recombinant protein with a strong IgE reactivity. rTM from Black tiger prawn

(Penaeus monodon) (rTM-BTP) was also generated and its allergenicity confirmed with

the serum of allergic patients. AllFam database of allergen families shows TMs from Disk

abalone (Haliotis discus) and Variously coloured abalone (Haliotis diversicolor) have

been reported as the major allergens (http://www.meduniwien.ac.at/allfam/search.php).

In this chapter, TM from Jade tiger abalone was for the first time evidenced as an essential

allergen using patient sera and officially registered as Hal l 1 by WHO/IUIS Allergen

Nomenclature Sub-committee. Also, very importantly, both prawn and abalone rTMs

could successfully elicit murine hypersensitivities. The strong reactivity of mouse rTM-

specific IgG to TM from heated extracts further confirmed the generated rTM had very

similar immunological characteristics as natural TM. As reported previously, the higher

Chapter 4

207

level of IgG1 that was elicited by rTMs as compared to heated protein extracts might be

resulted from the presence of non-Th2-associated type of IgG1 that could be functionally

similar as IgG2a [29]. The increased IgG2a in rTM-sensitised mice might be caused by

the remaining lipopolysaccharides (LPS) derived from the expression system of rTMs.

TM is the most abundant and heat-stable prawn allergen inducing allergic sensitisation

[13, 30-32]. TM-BTP has been previously identified as immunologically reactive proteins

using immunochemical techniques [26, 31] and characterised by means of mass

spectrometry (MS) [30]. In this chapter, TM has been evidenced again as a major allergen

in hBTP using both patient sera and a mouse model. Firstly, the mouse IgG reactivity and

human IgE reactivity to TM were the strongest among the total proteins of heated prawn

and abalone. Secondly, as compared to hBTP, rTM-BTP was able to induce similar levels

of the circulating allergen-specific IgE antibody, splenic IL-5 and IL-13, and eosinophils

and basophils in both blood and spleen. In contrast, the allergenicity of prawn TM seemed

much lower as compared to the allergenicity of hAbal. Among the different proteins in

hAbal, TM showed a relatively high content and strong antibody reactivity. In hAbal,

four major protein bands were reactive with patient IgE but also mouse IgG reactive, and

the identified proteins were TM, arginine kinase and troponin T in MS.

TM has been considered as the major cross-reactive allergen among different

invertebrates [33-36]. The sequence identity of TM from various crustacean species is

more than 89% [13]. Therefore, IgE cross-reactivity to crustacean TM is very frequently

reported. In this chapter, the similarity of amino acid sequence between prawn TM and

abalone TM is 63.4% as demonstrated. As compared to abalone TM, only one out of eight

potential IgE-binding regions of prawn TM is identical, which may indicate the limited

level of clinical cross-reactivity to TM from prawns and abalone. However, it may not be

accurate to predict the degree of cross-reactivity based on the primary structure only. The

Chapter 4

208

exact influence of amino acid variations on the structure of B cell and T cell epitopes still

needs to be investigated in the future. Here, in the mouse model, I found the IgG antibody

with high affinity could react to the heterologous TM, consistent with the fact that seven

patients with prawn allergy exhibited strong IgE reactivity to both prawn TM and abalone

TM in immunoblots. Due to the cross-reactive nature of abalone TM, monoclonal

antibody with specificity to abalone was able to recognise crustacean species including

shrimp, lobster and crab, as evidenced by Lu et al [37]. Interestingly, abalone TM, as

Suzuki et al. demonstrated by inhibition immunoblotting and inhibition ELISA, might

have cross-reactivity with another abalone allergen paramyosin [38]. After comparing the

identified IgE reactive proteins in these two species, I found that arginine kinase might

also contribute to the immunological cross-reactivity between Black tiger prawn and Jade

tiger abalone, and myosin heavy chain might be a minor allergen in abalone and prawns.

Taken together, in the current chapter, rTMs from prawn and abalone were successfully

generated and used for the immunisation of mice. With the availability of rTMs with

allergenic potentials, more scientific hypotheses can be tested for a better understanding

of immunological mechanisms, and the development of component resolved diagnosis

and allergen-specific immunotherapies in the future.

In this chapter, the antibody cross-reactivity to TM between Black tiger prawn and Jade

tiger abalone was revealed using human sera and mouse sera. In the next chapter, the

degree of immunological cross-reactivity between prawn and abalone TM was

comprehensively evaluated at both humoral and cellular levels using a mouse model.

Chapter 4

209

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[29] E.L. Faquim-Mauro, R.L. Coffman, I.A. Abrahamsohn, M.S. Macedo, Cutting

edge: mouse IgG1 antibodies comprise two functionally distinct types that are

differentially regulated by IL-4 and IL-12, The Journal of Immunology, 163 (1999)

3572-3576.




spectrometry, 24 (2010) 2462-2470.




[32] S. Sahabudin, R. Misnan, Z.H.M. Yadzir, J. Mohamad, N. Abdullah, F. Bakhtiar, S.

Murad, Identification of major and minor allergens of black tiger prawn (Penaeus

monodon) and king prawn (Penaeus latisulcatus), DOI (2013).

[33] P.S. Leung, W.K. Chow, S. Duffey, H.S. Kwan, M.E. Gershwin, K.H. Chu, IgE

reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for

tropomyosin as the common allergen, Journal of Allergy and Clinical Immunology, 98

(1996) 954-961.



(2009) 634-641.

[35] R. Ayuso, G. Reese, S. Leong-Kee, M. Plante, S. Lehrer, Molecular basis of

arthropod cross-reactivity: IgE-binding cross-reactive epitopes of shrimp, house dust mite

Chapter 4

214

and cockroach tropomyosins, International archives of allergy and immunology, 129

(2002) 38.

[36] R.C. Aalberse, Shrimp serology: we need tests with more and less cross-reactivity,

Journal of Allergy and Clinical Immunology: In Practice, 3 (2015) 530-531.

[37] Y. Lu, T. Oshima, H. Ushio, K. Shiomi, Preparation and characterization of

monoclonal antibody against abalone allergen tropomyosin, Hybridoma and

hybridomics, 23 (2004) 357-361.

[38] M. Suzuki, Y. Kobayashi, Y. Hiraki, H. Nakata, K. Shiomi, Paramyosin of the disc

abalone Haliotis discus discus: Identification as a new allergen and cross-reactivity with

tropomyosin, Food chemistry, 124 (2011) 921-926.

CHAPTER

EVALUATION OF THE POTENTIAL IMMUNOLOGICAL

CROSS-REACTIVITY BETWEEN TROPOMYOSIN

FROM BLACK TIGER PRAWN (Penaeus monodon) AND

JADE TIGER ABALONE (Haliotis laevigata × Haliotis rubra)

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5.1 Introduction

Shellfish is one of the common food sources eliciting allergic reactions. Prawn is the most

frequently studied animal group that belongs to the class crustacean. Many patients with

prawn allergy can also react to other crustacean mollusc species [1-3], which might result

from co-sensitisation or cross-reactivity. The co-existence of different antibodies (mainly

IgE antibody) reacting with different allergens is often termed co-sensitisation, which is

developed through exposure to multiple allergens, whereas antibody cross-reactivity

refers to the antibody induced by one allergen can also react to another allergen that shares

similar sequence/epitopes/structures [4]. Usually cross-reactivity is suspended when pan-

allergen is found in different sources. Tropomyosins have been considered as the

prominent animal-derived pan-allergen family [5]. Between the years 2003 to 2016, 16

different tropomyosins have been identified as food allergens and officially registered by

the World Health Organization and International Union of Immunological Societies

(WHO/IUIS) Allergen Nomenclature Sub-committee (www.allergen.org). The

allergenicity of tropomyosin derived from BTP and Jade tiger abalone has been

confirmed by patient IgE reactivity and the mouse model established in Chapter 4. Under

this circumstance, tropomyosin has been implicated to be responsible for the prawn-

abalone cross-reactivity.

As an evolutionally conserved protein, invertebrate tropomyosin has varying degrees of

homology, which indicates the potentially different levels of the clinical cross-reactivity

to TM derived from different allergen sources, while vertebrate tropomyosins are non-

allergenic. The major sources of allergenic tropomyosins are crustaceans (prawn, lobster,

crab, and crawfish), arachnids (house dust mites), insects (cockroaches), and molluscs

(abalone, oyster and squid) [6]. The high degree of sequence identity among crustacean

tropomyosin suggests that tropomyosin is the common major allergen in crustaceans.

Chapter 5

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Unlike the high incidence of IgE cross-reactivity between crustacean tropomyosins [7, 8],

the immunological relationship between crustacean and molluscs is in debate, due to the

very low amino acid sequence identity of tropomyosins [9-11]. In Chapter 4, the sequence

alignment of tropomyosin from BTP and Jade tiger abalone showed only 63.4% of amino

acid sequence identity, but also the reported IgE-binding regions were distinct. Although

the molecular basis of tropomyosin can help to predict the level of immunological cross-

reactivity, its clinical relevance is still unknown. Allergic cross-reactivity is often defined,

but actually not restricted to, IgE cross-reactivity [12, 13]. Consequently, more

comprehensive investigation is in need to reveal the detailed immunological mechanisms

of true cross-reactivity to tropomyosin at both humoral and cellular levels.

The current chapter detailed the murine responses of serum antibody, circulating and

splenic eosinophil’s and basophil’s, intestinal antibody, as well as cytokines responses to

the heterologous tropomyosin allergens. In this chapter, a new mouse model was

developed by modifying the model described previously in Chapter 3. The potential

immunological cross-reactivity was evaluated using this new model whereby the major

suspect allergen tropomyosin was utilised instead of the crude protein extracts of prawn

or abalone containing a mixture of allergens and non-allergenic molecules.

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5.2 Aims

The aim of Chapter 5 was to investigate the degree of immunological and clinical cross-

reactivity between prawn tropomyosin and abalone tropomyosin through comparing the

humoral and cellular responses caused by homologous and heterologous allergens in a

mouse model.

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5.3.1 Animals

BALB/c mice, 8-week-old females, were raised at the Cairns campus, building E4 Animal

Facility under specific pathogen-free conditions. Mice were fed with a commercial

vegetarian diet purchased from Specialty Feeds. James Cook University animal ethics

committee approved the usage of animals in this project (Animal ethics approval number

A2108).

5.3.2 Preparation of shellfish extracts and recombinant tropomyosins

Heated extracts of Black tiger prawn (Penaeus monodon) and Jade tiger abalone (Haliotis

laevigata × Haliotis rubra) were prepared using the method described in Chapter 2,

Section 2.3.2. The generation and purification of rTMs were described in Chapter 4,

Section 4.3.1 and 4.3.2. Both rTMs were used during the sensitisation phase and heated

shellfish extracts for the oral challenges.

5.3.3 Induction and evaluation of mouse models in shellfish allergies

5.3.3.1 Mice sensitisation and challenge

Three groups of mice (n=5 or 6) were IP injected with 10 μg of rTM-BTP mixed with 2

mg of alum, and another three groups of mice IP injected with 10 μg of rTM-Abal mixed

with 2 mg of alum, on Day 0, 7 and 14 (see figure 5.1). From Day 21, mice started

receiving oral gavage with shellfish extracts or heated milk (hMilk) as nonrelated food.

The three groups of mice sensitised to the same allergen were orally challenged with

different allergenic foods; hMilk, hBTP, or hAbal. During a two-day interval mice were

gavaged in a total of four times and 24 h later mice were sacrificed. Before each gavage,

mice were fasted overnight but had unlimited access to water. The sham treated mice

Chapter 5

220

were only treated with PBS. Spleen, blood, jejunum and stool were harvested and

processed for further analysis.

Figure 5.1. Experimental procedures of sensitisation and oral challenge in seven

mouse groups. Each group comprised of five to six mice.

5.3.3.2 Detection of serum specific antibody and mast cell protease-1

Specific IgE, IgG1, IgG2a were measured in serum samples by means of ELISA as

described previously in Chapter 2. Briefly, high-binding ELISA solid phase plates were

coated with recombinant proteins rTM-BTP or rTM-Abal overnight at 4°C. ELISA plates

were blocked and then incubated with 50 μl of mouse sera overnight at 4°C. Next, the

wells were incubated with biotin rat anti-mouse IgE, IgG1 and IgG2a antibody at room

temperature for 1.5 h, washed with PBS/0.05% Tween 20 and subsequently incubated

with Streptavidin-HRP for 30 min at room temperature. After 7 washes with PBS/0.05%

Tween 20, TMB substrate was added as a visualising reagent. 50 μl of 1 M HCl was

added to stop the colour reaction. The absorbance was measured at 450 nm. As described

in Chapter 2, the procedure of detecting MCP-1 was similar as the protocol above but

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221

using a commercial Mouse MCPT-1 (mMCP-1) ELISA Ready-SET-Go reagent set

(eBioscience).

5.3.3.3 In vitro re-stimulation of splenocyte and quantification of cytokines

Mice were sacrificed one day after the last oral challenge and spleens were removed.

Single cell suspension was prepared and restimulated as described previously in Chapter

2 with the modification of stimulus allergens. In this assay, 0.5 million splenocytes in 100

μl of culture medium were cultured with 100 μl of 40 μg/ml recombinant allergens (rTM-

BTP or rTM-Abal), or culture medium only, or 1μg/ml a mix of anti-CD3 and anti-CD28,

for 72 hours in 5% CO2 at 37°C using SANYO CO2 Incubator Model MCO-18AIC (UV)

Dual Stack.

Culture supernatant was isolated after centrifugation and cytokines including IL-5, IL-13

and IFN-gamma contained in the supernatant were measured using Mouse ELISA Ready-

SET-Go reagent sets (eBioscience). The manufacturer’s protocol was followed and

described previously in Chapter 2. Briefly, the ELISA plates coated with the cytokine

capture antibody were blocked with 1 x Assay Diluent provided in the reagent set. The

plates were incubated with diluted samples and standards overnight at 4°C, following by

a two-hour incubation with the detection antibody at room temperature, and then

incubated for 30 minutes with Avidin-HRP at room temperature. Plates were washed

thoroughly prior to loading TMB. 1 M of HCl was added to stop the colour reaction.

Absorbance was determined at 450 nm and cytokines were quantified based on the

generated standard curve.

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222


Mononuclear cells from blood and spleen were prepared and analysed by flow cytometry

as described in Chapter 2. To stain eosinophil and IgE-basophil, seven fluorescence-

conjugated antibodies (Jomar Life Research), including Anti-Mouse CD170 (Siglec F)

PerCP-eFluor 710, Anti-Mouse CD3 APC-eFluor 780, Anti-Mouse IgE FITC, Anti-

Mouse CD11b PE, Anti-Mouse CD49b (Integrin alpha 2) APC, Anti-Mouse CD19 APC-

eFluor 780, Anti-Mouse Fc epsilon Receptor I alpha (FcεR1) eFluor 450, were mixed as

a panel and each antibody was 1:300 diluted with FACS buffer. At first, all the volume

of PBMC or 0.5 x 106 splenocytes were washed in a 96-well plate, and incubated with Fc

block buffer for 5 min, follows by incubation with the stain mix in the dark for 30 min on

ice. Unbound antibodies were removed by washing the plate containing cells with FACS

buffer, before cell fixation for 20 min on ice. Cells were resuspended into FACS buffer

before analysis using BD FACS Canto II.

5.3.3.5 Measurement of intestinal specific IgA antibody

Total proteins extracted from mouse jejunums were analysed for allergen-specific IgA

antibody by sandwich ELISA described previously in Chapter 2. The only revision of the

protocol was rTM-BTP or rTM-Abal used as the coating allergens in this experiment.

Briefly, coated plates were blocked with 10% FBS in PBS and incubated with 50 μl of

neat jejunum extracts overnight at 4°C. Biotin anti-mouse IgA (Biolegend, USA) was

added to detect IgA antibody, following with incubations of Streptavidin-HRP and

subsequent TMB substrate at room temperature. 1 M HCl was applied as the stop solution.

Finally, the optical density at 450 nm was measured using the POLARstar Omega

microplate reader (BMG LABTECH).

Chapter 5

223


Two-tailed Mann-Whitney U test was performed to analyse the difference of every two

related groups of data using Prism 6.0. Data are presented as mean ± SED from one

representative experiment out of two similar experiments. p values of less than 0.05 were

regarded statistically significant.

Chapter 5

224

5.4 Results

5.4.1 Serum antibodies and mast cell protease-1

To investigate humoral immune responses of immunised mice, mouse serum was isolated

for measuring circulating IgE antibody at the time-point of sacrifice (Day 31). As shown

in milk-gavaged mice, IP sensitisation with rTM conferred a significant rise of allergen-

specific IgE antibody (Figure 5.2.A and B). Comparing with rTM-BTP/hMilk group, oral

challenges with hAbal promoted rTM-BTP-sensitised mice to produce more IgE antibody,

although the rise of specific IgE antibody was more statistically significant after oral

challenges with hBTP. By comparison, there is no statistical increase of IgE antibody in

the blood of rTM-Abal-sensitised mice after gavage with hBTP, and only gavage of hAbal

triggered a strong increase in serum specific IgE.

To further study whether serum antibody can co-recognise abalone TM and prawn TM,

ELISA was performed and heterologous TM was coated on the solid binding surface to

test the serum collected from mice without previous exposure to heterologous TM. As

demonstrated in Figure 5.2.C and D, some IgG1 antibody was able to bind heterologous

TM, whereas the extracts containing homologous TM could clearly cause much stronger

IgG1 responses. These data indicate the importance of primary sensitization that may

determine the severity and specificity of antibody responses.

Next, circulating mMCP-1 was quantified to show the involvement of mast cells during

the allergic reaction in different mouse groups. Comparing mice sensitised with the same

allergen but gavaged differently, the highest titer of mMCP-1 was detected in the positive

control groups in which mice received oral challenges with extracts containing the

homologous allergen, including the rTM-BTP/hBTP group and the rTM-Abal/hAbal

group. There was no significant difference of mMCP-1 between PBS sham mice and TM-

Chapter 5

225

sensitised milk-gavage mice, which suggested the low participation of mast cells during

the sensitisation phase of allergy.

Chapter 5

226

Figure 5.2 Levels of serum IgE antibody (A and B) and mMCP-1 (E) and reactivity

of IgG1 antibody (C and D) against heterologous tropomyosin. Mouse serum was

collected on sacrifice day from BALB/c mice. Levels of specific IgE and IgG1 antibody

were determined by sandwich ELISA. Data are reported as mean ± SEM (n = 5) for each

mouse group. p < 0.05, and p< 0.01. Each dot represents an individual mouse.

Abbreviation: ns = Not significant. Note: Different scales of Y axis are used for the

antibody responses.

* **

Chapter 5

227

5.4.2 Release of cytokines by allergen restimulated splenocytes in vitro

Figure 5.3 Production of cytokines by allergen-restimulated splenocytes. Splenocytes

were cultured with rTM-BTP or rTM-Abal for three days in vitro. ELISA was performed

to determine the levels of IL-5, IL-13 and IFN-gamma. Data are reported as mean ± SEM

(n = 5) for each mouse group. p < 0.05, and p < 0.01. Each dot represents an

individual mouse. Abbreviation: ns = Not significant.

IL-5

pg/m

l

Control

rTM-B

TP/hMilk

rTM-B

TP/hBTP

Control

rTM-B

TP/hMilk

rTM-B

TP/hAbal0

200

400

600

800

1000

rTM-BTP restim

**ns

ns

ns

rTM-Abal restim

IL-5

Control

rTM-A

bal/hMilk

rTM-A

bal/hAbal

Control

rTM-A

bal/hMilk

rTM-A

bal/hBTP

0

100

200

300

400

pg/m

l

rTM-BTP restim

**

ns

ns

*

rTM-Abal restim

IL-13

pg/m

lContro

l

rTM-B

TP/hMilk

rTM-B

TP/hBTP

Control

rTM-B

TP/hMilk

rTM-B

TP/hAbal0

500

1000

1500

2000

rTM-BTP restim

**

ns

ns

****

rTM-Abal restim

IL-13

pg/m

l

Control

rTM-A

bal/hMilk

rTM-A

bal/hAbal

Control

rTM-A

bal/hMilk

rTM-A

bal/hBTP

0

500

1000

1500

rTM-BTP restim

**

**

**ns

ns

**

rTM-Abal restim

IFN-gamma

pg/m

l

Control

rTM-B

TP/hMilk

rTM-B

TP/hBTP

Control

rTM-B

TP/hMilk

rTM-B

TP/hAbal0

1000

2000

3000

4000

5000

rTM-BTP restim

*

nsns

*ns

rTM-Abal restim

IFN-gamma

pg/m

l

Control

rTM-A

bal/hMilk

rTM-A

bal/hAbal

Control

rTM-A

bal/hMilk

rTM-A

bal/hBTP

0

1000

2000

3000

4000

5000

rTM-BTP restim

****

ns**

rTM-Abal restim

rTM-BTP sensitised mice

rTM-Abal sensitised mice

A B C

A' B' C'

* **

Chapter 5

228

Th2-cytokine producing cells in spleen were restimulated with the recombinant allergen

for three days in vitro. Figure 5.3 illustrates that the elevation of IL-5 and IL-13 is a vital

immunological event caused by sensitisation, since the high concentrations of these two

cytokines appeared in allergen-sensitised milk-gavaged mice. Splenocytes from mice

treated with PBS only released neither IL-5 nor IL-13. The presence of TM-Abal in the

culture of rTM-BTP-sensitised splenocytes resulted in increased IL-5 and IL-13 in the

medium (Figure 5.3.A and B), and similar responses were observed when rTM-BTP was

used to restimulate rTM-Abal-sensitised splenocytes (Figure 5.3.A’ and B’). There was

no statistical difference between the level of cytokines (IL-5 or IL-13) secreted by

splenocytes from rTM-BTP/hBTP and rTM-BTP/hAbal. However, the Th2-cytokine

responses of rTM-Abal-sensitised splenocytes to rTM-BTP were much weaker than

responses to the homologues rTM-Abal, resulting in a 90.9% lower IL-5 level and 75.6%

lower IL-13 level. Splenocytes from rTM-BTP/hAbal mice and rTM-Abal/hBTP mice

seemed to present different degrees of cytokine cross-reactivity, which was consistent

with the results of serum IgE antibody (Figure 5.2).

It is worth noting that, as described previously in Chapter 4, rTM-sensitised mice were

orally challenged with hBTP or hAbal in which the contents of natural TM contained

might be different. To exclude the possible influence of different doses of TM delivered

orally to mice on the degree of immunological cross-reactivity, dedicated analysis of two

groups of rTM-sensitised hMilk-gavaged mice might provide more evidence for better

evaluation of the cross-reactivity. In this cell restimulation assay, splenocytes from rTM-

BTP/hMilk mice were for the first time exposed to rTM-Abal after the sensitisation phase

and successfully secreted Th2 cytokines, although the concentration of IL-5 was 93% less

and the concentration of IL-13 was 79% less than the cytokine levels detected in the

culture of rTM-BTP/hMilk splenocytes with rTM-BTP. Similarly, the cytokine

Chapter 5

229

producing cells specific to rTM-Abal, without confronting rTM-BTP before, could cross-

react to rTM-BTP presented in culture medium but at a limited level.

Comparing with sham splenocytes, all the sensitised splenoctes with either rTM-BTP or

rTM-Abal released significantly higher levels of Th1 cytokine IFN-gamma, when

cultured with either rTM-BTP or rTM-Abal (Figure 5.3.C and C’). Since considerable

amount of IFN-gamma was detected in both rTM-BTP/hMilk group and rTM-Abal/hMilk

group, the Th1-cytokine producing cells were very likely initially activated during IP

sensitisation with rTM in which lipopolysaccharides (LPS) was present.

Chapter 5

230

5.4.3 Induction of eosinophil and basophil in circulating blood and spleen

Figure 5.4 Eosinophil and IgE bound basophil in circulating blood (A and B) and

spleen (A’ and B’). Cell populations stained with fluorescence-conjugated antibodies

were analysed by flow cytometer. Data are reported as mean ± SEM for each mouse group

(n=5). p < 0.05, and p < 0.01. Abbreviation: ns = Not significant. * **

Chapter 5

231

The patterns of eosinophil and basophil accumulation in allergic pathologies were

investigated by means of flow cytometry. As demonstrated in Figure 5.4.A and B, the

mean proportions of circulating eosinophil and IgE bound basophil were as expected the

highest in positive control groups, and there was no significant rise of these two cell

populations in blood after rTM-sensitised mice were gavaged with shellfish extracts

containing heterologous TM. Moreover, there was no statistical difference of circulating

eosinophil between the positive group and hMilk-gavaged negative group (rTM-

BTP/hBTP vs rTM-BTP/hMilk, and rTM-Abal/hAbal vs rTM-Abal/hMilk). In contrast

to blood, the accumulation of splenic eosinophil and basophil elicited by cross-reactivity

was clearly observed, especially eosinophilia promoted by oral gavage with hAbal in

mice sensitised with rTM-BTP. Also, by comparing rTM-BTP/hMilk group and rTM-

Abal/hMilk group in Figure 5.4.B and B’, rTM-Abal seemed to provoke more IgE-

binding basophils than rTM-BTP as a sensitisation allergen, although levels of serum

specific IgE (Figure 5.2.A and B) and the in vitro expression of IL-13 (Figure 5.3.B and

B’) were similar in these two groups.

5.4.4 Secretion of mucosal IgA antibody in jejunum

Mucosal responses against heterologous allergen were also studied through measuring

the level of secretory IgA antibody in jejunums. Figure 5.5 demonstrates that mice

gavaged with homologous allergens seemed to secret more intestinal IgA (0.7483 ±

0.4104 in rTM-BTP/hBTP group, and 0.4291 ± 0.1712 in rTM-Abal/hAbal group) than

milk-gavaged mice (0.3009 ± 0.08907 in rTM-BTP/hMilk group, and 0.1883 ± 0.04527

in rTM-Abal/hMilk group), however this was statistical not significance. IgA producing

cells in jejunums from sensitised mice barely reacted to heterologous allergens that were

delivered by gastrointestinal route in vivo or coated on ELISA plates in vitro.

Chapter 5

232

Figure 5.5 Levels of allergen-specific IgA in jejunum. Homogenised jejunums were

tested in sandwich ELISA for comparing levels of IgA antibody in different mouse groups.

Data are reported as mean ± SEM (n = 5) for each mouse group. p< .05, and p< .01.

Each dot represents an individual mouse. Abbreviation: ns = Not significant. Note:

Different scales of Y axis are used for the antibody responses.

Control

rTM-BTP/hMilk

rTM-BTP/rTM-BTP

rTM-BTP/rTM-Abal0.0

0.5

1.0

1.5

2.0

2.5

rTM-BTP sepcific IgA

OD

450n

m

p=0.0556

Control

rTM-Abal/hMilk

rTM-Abal/hAbal

rTM-Abal/hBTP0.0

0.5

1.0

1.5

OD

450n

m

rTM-Abal sepcific IgA

p=0.0952

Anti-rTM-Abal

Anti-rTM-Abal

Anti-rTM-BTP

0.0

0.7

1.4

2.1

2.8

Allergen recognising of IgA in jejunum

OD

450n

m

ns

ns

Control jejunum rTM-BTP/hBTP jejunum

Anti-rTM-BTP

Anti-rTM-BTP

Anti-rTM-Abal

0.0

0.5

1.0

1.5O

D45

0nm

Allergen recognising of IgA in jejunum

ns

ns

Control jejunum rTM-Abal/hAbal jejunum

A

C

B

D

* **

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233

5.5 Discussion

Although allergic reactions to molluscs species are generally thought to be acquired by

primary sensitisation through ingestion, molluscs allergy may also occur in patients

sensitised to cross-reactive allergen sources, such as crustacean species, and vice versa.

Tropomyosin is believed to be the major cross-reactive allergen, widely distributed in a

high variety of shellfish species and other inhalant allergen invertebrates [6, 14-19].

Cross-reactivity between crustacean and molluscs species seem to be less frequent than

within crustacean, which may partially result from the lower degrees of amino acid

homology between crustacean and molluscs TM [10]. In Chapter 3 I described the

immunological cross-reactivity between the total protein extracts of Black tiger prawn

and Jade tiger abalone. In the current chapter, the immunological cross-reactivity to TM

from Black tiger prawn and Jade tiger abalone was evaluated in detail to investigate

whether TM was the major culprit causing the prawn-abalone cross-reactivity.

Determination of the true sensitisation is an obstacle to studies of cross-reactivity in

allergy patients. The presence of allergenic structures shared by abalone TM and prawn

TM has been investigated in vitro using human IgE [19] and monoclonal antibodies [20].

Results showed that serum IgE from nine patients with documented anaphylaxis to

shrimp could all react to abalone (Haliotis diveriscolor) TM and pre-absorption of the

tested sera with recombinant shrimp TM, Met e 1, completely blocked their IgE reactivity

to abalone TM. Lu’s group generated monoclonal antibody specific to Japanese abalone

(Haliotis discus) that was observed also to cross-react to Black tiger prawn TM [20].

Although cross-reactive IgE epitopes between abalone TM and prawn TM seems to exist,

their clinical relevance remains to be revealed. It is important to note that serologic IgE

reactivity is only part of immunological pathways leading to clinical hypersensitivity

reactions [19, 21]. The reactivity of important mediator cells located at relevant organs

Chapter 5

234

should also be addressed. Without involving patients and possible health risks, mouse

models in allergies are a very useful in vivo tool to provide valuable information about

allergic mechanisms [22]. In the current study, a mouse model was designed for the

evaluation of immunological cross-reactivity to recombinant TM from Black tiger prawn

and Jade tiger abalone, at both humoral and cellular levels. Based on the data I collected

from thes mouse studies, I could demonstrate that mice sensitised with prawn TM could

react to heated abalone extracts at a considerable level through increased IgE antibody,

Th2 cytokines and splenic eosinophils. In contrast the gavage with prawn extracts could

not elicit Th2-responses in abalone-TM-sensitised mice. These results suggest that most

immunodominant allergens from Black tiger prawn TM might share similar allergenic

structure with those from Jade tiger abalone TM. However, some allergenic structures of

abalone TM may be unique and make abalone-TM-sensitisited individuals unlikely to

react to TM from Black tiger prawn. Moreover, circulating effector cells were not

significantly increased in groups where either hBTP or hAbal were gavaged as

heterologous allergens, which suggests the restricted level of in vivo immunological

cross-reactivity between prawn TM and abalone TM.

In the mouse model I employed, the sensitisation with alum as adjuvant promoted the

production of IgE and IgG1 antibody. Only a small portion of TM-BTP or TM-Abal

specific IgG1 reacted to heterologous TM in vitro. TM-BTP-specific IgE was increased

after mice gavaged hAbal. In contrast, abalone-TM specific IgE was not induced by the

gavage with hBTP in the abalone-TM-sensitised mice. This study demonstrates for the

first time, using an in vivo platform, that abalone TM may include more immunoreactive

epitopes than those shared by prawn and abalone TM, in contrast to prawn TM which

seem to be less reactive.

Chapter 5

235

In this mouse model, lymphocytes in the spleen displayed a typical Th2 cytokines profile,

which made a critical systemic contribution to the allergic cross-reactivity. IL-5 is well

known for enhancing the growth, differentiation, effector function and migration of

eosinophils [23, 24]. IL-13 is another critical cytokine for the induction of intestinal

allergy [25], and can support the class switching towards IgE [26]. Shifting away from a

Th2-pattern of immune responses through reducing the production of IL-5 and IL-13

could be a promising therapeutic strategy for allergy patients [27, 28]. In this work, TM-

sensitised murine splenocytes produced only low levels of IL-5 and IL-13, when they

were for the first time exposed to the heterologous TM in vitro. The reactivity of the

splenocytes from mice that were already gavaged with heterogous allergens was similar

against heterologous TM. In comparison, in Chapter 3, murine splenocytes more

frequently exposed to heterologous shellfish extracts could release more IL-5 and IL-13.

This indicates that with the increasing exposure incidence of sensitised individuals to

homologous but also heterologous shellfish, other allergens than tropomyosin may play

an increasingly important role in inducing immunological cross-reactivity. Arginine

kinase is one of these potential candidates as it has been reported to be a cross-reactive

allergen between crustacean and edible insect [29], but also between prawn and mite [30].

Currently, there is a lack of evidence on immunological cross-reactivity of other shellfish

minor allergens, such as sarcoplasmic calcium binding protein, troponin C and

triosephosphate isomerase, among crustacean, molluscs or other invertebrates [31].

In conclusion, the mouse model in this study showed evidence of antibody, cytokine and

effector cell involvement in the immunological cross-reactivity between prawn TM and

abalone TM. Results obtained from this work may be useful for the development of

precise diagnosis and new immunotherapies for susceptible individuals. Further studies

Chapter 5

236

are warranted to identify other potential cross-reactive allergens or the B and T cell

immunodominant epitopes.

Chapter 5

237

5.6 References

[1] K.H. Waibel, B. Haney, M. Moore, B. Whisman, R. Gomez, Safety of chitosan

bandages in shellfish allergic patients, Military medicine, 176 (2011) 1153-1156.

[2] D.P. Steensma, The kiss of death: a severe allergic reaction to a shellfish induced by

a good-night kiss, Mayo Clinic Proceedings, Elsevier, 78 (2003), 221-222.

[3] R.M. Kandyil, C.M. Davis, Shellfish allergy in children, Pediatric Allergy and

Immunology, 20 (2009) 408-414.

[4] W. Li, Z. Zou, Cross-Reactivity, Allergy Bioinformatics, (2015) 67-92.

[5] O.E. McKenna, C. Asam, G. R Araujo, A. Roulias, L.R. Goulart, F. Ferreira, How

relevant is panallergen sensitisation in the development of allergies?, Pediatric Allergy

and Immunology, 27 (2016) 560-568.

[6] G. Reese, R. Ayuso, S.B. Lehrer, Tropomyosin: An invertebrate pan–allergen,

International Archives of Allergy and Immunology, 119 (1999) 247-258.

[7] C. Lemiere, A. Desjardins, S. Lehrer, J. L Malo, Occupational asthma to lobster and

shrimp, Allergy, 51 (1996) 272-273.

[8] Y. Zhang, H. Matsuo, E. Morita, Cross-reactivity among shrimp, crab and scallops in

a patient with a seafood allergy, The Journal of dermatology, 33 (2006) 174-177.

[9] A.L. Lopata, C. Zinn, P.C. Potter, Characteristics of hypersensitivity reactions and

identification of a unique 49 kd IgE-binding protein (Hal-m-1) in abalone (Haliotis

midae), Journal of allergy and clinical immunology, 100 (1997) 642-648.


40 (2010) 850-858.

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238

[11] A.L. Lopata, M.F. Jeebhay, Airborne seafood allergens as a cause of occupational

allergy and asthma, Current allergy and asthma reports, 13 (2013) 288-297.

[12] B. Jahn-Schmid, A. Radakovics, D. Lüttkopf, S. Scheurer, S. Vieths, C. Ebner, B.

Bohle, Bet v 1 142-156 is the dominant T-cell epitope of the major birch pollen allergen

and important for cross-reactivity with Bet v 1–related food allergens, Journal of allergy

and clinical immunology, 116 (2005) 213-219.

[13] B. Jahn-Schmid, M. Hauser, N. Wopfner, P. Briza, U.E. Berger, R. Asero, C. Ebner,

F. Ferreira, B. Bohle, Humoral and cellular cross-reactivity between Amb a 1, the major

ragweed pollen allergen, and its mugwort homolog Art v 6, The Journal of Immunology,

188 (2012) 1559-1567.




PLoS One, 8 (2013) e67487.




[16] Å.M. DeWitt, L. Mattsson, I. Lauer, G. Reese, J. Lidholm, Recombinant

tropomyosin from Penaeus aztecus (rPen a 1) for measurement of specific immuno

globulin E antibodies relevant in food allergy to crustaceans and other invertebrates,

Molecular nutrition & food research, 48 (2004) 370-379.

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(2009) 634-641.




[19] P.S. Leung, W.K. Chow, S. Duffey, H.S. Kwan, M.E. Gershwin, K.H. Chu, IgE

reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for

tropomyosin as the common allergen, Journal of Allergy and Clinical Immunology, 98

(1996) 954-961.

[20] Y. Lu, T. Oshima, H. Ushio, K. Shiomi, Preparation and characterization of

monoclonal antibody against abalone allergen tropomyosin, Hybridoma and

hybridomics, 23 (2004) 357-361.

[21] F. Ferreira, T. Hawranek, P. Gruber, N. Wopfner, A. Mari, Allergic cross

reactivity: from gene to the clinic, Allergy, 59 (2004) 243-267.



[23] S.C. Bischoff, Food allergy and eosinophilic gastroenteritis and colitis, Current

opinion in allergy and clinical immunology, 10 (2010) 238-245.

[24] A. Mishra, S.P. Hogan, E.B. Brandt, M.E. Rothenberg, IL-5 promotes eosinophil

trafficking to the esophagus, The Journal of Immunology, 168 (2002) 2464-2469.

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[25] M. Wang, K. Takeda, Y. Shiraishi, M. Okamoto, A. Dakhama, A. Joetham, E.W.

Gelfand, Peanut-induced intestinal allergy is mediated through a mast cell–IgE–FcεRI–

IL-13 pathway, Journal of Allergy and Clinical Immunology, 126 (2010) 306-316.

[26] M.C. Berin, S. Sicherer, Food allergy: mechanisms and therapeutics, Current opinion

in immunology, 23 (2011) 794-800.

[27] J. Wang, H.A. Sampson, Treatments for food allergy: how close are we?,

Immunologic research, 54 (2012) 83-94.

[28] Y.-H. Yang, B.-L. Chiang, Novel approaches to food allergy, Clinical reviews in

allergy & immunology, 46 (2014) 250-257.

[29] C. Srinroch, C. Srisomsap, D. Chokchaichamnankit, P. Punyarit, P. Phiriyangkul,

Identification of novel allergen in edible insect, Gryllus bimaculatus and its cross-

reactivity with Macrobrachium spp. allergens, Food chemistry, 184 (2015) 160-166.

[30] C. Gámez, M.P. Zafra, M. Boquete, V. Sanz, C. Mazzeo, M.D. Ibáñez, S. Sánchez

García, J. Sastre, V. Pozo, New shrimp IgE binding proteins involved in mite seafood

cross reactivity, Molecular nutrition & food research, 58 (2014) 1915-1925.

[31] A.L. Lopata, J. Kleine-Tebbe, S.D. Kamath, Allergens and molecular diagnostics of

shellfish allergy, Allergo Journal International, 25 (2016) 210-218.

CHAPTER

GENERAL DISCUSSION AND FUTURE DIRECTIONS

Chapter 6

242

Shellfish is one of the essential nutrition sources in human diets. With the increasing

consumption of shellfish across the world, unfortunately, within recent decades, the

incidence of shellfish allergy has been significantly more frequent. So far, there is still no

cure for shellfish allergy. The best treatment strategy for susceptible individuals is to

strictly avoid all shellfish-related foods. The clinical cross-reactivity among crustacean

species has been frequently reported and the major cross-reactive allergen has been

identified as tropomyosin. By comparison, the mollusc allergens are much less studied

and the clinical and immunological cross-reactivity between crustacean and mollusc

species is largely unknown. The current approaches to assess the allergenic potential of

shellfish proteins and the clinical significance of allergic reactions include in vitro tests

of patient serum IgE and basophil activation, as well as skin prick testing. However, their

clinical relevance is often lacking and thus their implication for prescribing food

avoidance is in debate. Currently, a number of mouse models have been used in studies

of food allergies to egg, peanut and milk. Controlling allergen exposure using mice can

be a very useful in vivo tool to provide new insights on the understanding of

immunological mechanisms, assessment of allergen sources and the development of

diagnostics and immunotherapeutics.

The work presented in this PhD thesis aims at evaluating the immunological cross-

reactivity between Black tiger prawn (Penaeus monodon) and Jade tiger abalone (Haliotis

laevigata × Haliotis rubra). These two shellfish species are widely consumed in the Asia-

Pacific region. In chapter 2, a novel mouse model of shellfish allergy was developed and

characterised. In Chapter 3, the heated protein extracts of prawns and abalone were used

in this model for the detection of potential immunological cross-reactivity between the

different allergens. Moreover, the major cross-reactive allergen was identified to be

tropomyosin. Consequently, in Chapter 4, the amino acid sequence alignment was

Chapter 6

243

performed between prawn and abalone tropomyosins. Subsequently, recombinant

tropomyosins from prawns and abalone were generated and assessed for their allergenic

potentials using patient sera and the developed mouse model. Finally, in chapter 5, the in

vivo immunological cross-reactivity to tropomyosins from prawns and abalone was

thoroughly evaluated in mice.

Food allergy is a complex chronic disease and IgE, mast cells, basophils, eosinophils,

cytokines, and intestinal IgA are essential components of the allergic inflammation. The

novel mouse model, established in Chapter 2, successfully mimicked these key hallmarks

of allergic reactions to the heated prawn extracts. A serial optimisation was conducted for

the establishment of a better mouse model. BALB/c mice showed a higher baseline of

antibody production as compared to C57BL/6 mice, which makes BALB/c mice a better

mouse strain for allergy research. Also, these result confirm the influence of genetic

backgrounds on the susceptibility to shellfish allergy. For the intraperitoneal sensitisation

dose 3 x 10 μg of prawn extracts was selected as this promoted the highest levels of serum

IgE as compared to higher doses.

Human IgE immunoblots, mouse IgG immunoblots, SDS-PAGE analysis and mass

spectrometric analysis were performed to identify the prawn and abalone allergens.

Tropomyosin was demonstrated to be the most abundant but also the major antibody

reactive protein in the heated extracts of Black tiger prawn. In contrast, the protein

tropomyosin was a smaller proportion in the total protein content of heated abalone

extracts, but still generates the strongest antibody reactivity as demonstrated using patient

and mouse sera. In addition to tropomyosin, other potential allergens with a less

significant allergenic potency were also detected, including arginine kinase, myosin

heavy chain, paramyosin and Troponin T. More interestingly, the mouse IgG antibody

response to prawn extracts detected also the natural tropomyosin in the heated abalone

Chapter 6

244

extracts, which was confirmed by mass spectrometry.

Consequently, for a better understanding of the allergenic capacity and the cross-reactive

properties of tropomyosin, the primary structure of tropomyosins from prawns and

abalone were compared, and recombinant proteins expressed and tested using patient sera

and experimental animals. Notably, prawn tropomyosin shared 63.4% amino acid

sequence identity with abalone tropomyosin and only one of eight previously predicted

prawn IgE epitopes is identical in abalone tropomyosin.

This is the first time the molecular basis of the immunological cross-reactivity to

tropomyosin from Black tiger prawn and Jade tiger abalone has been reported.

Importantly, serum IgG from recombinant-tropomyosin-sensitised mice could bind to the

heterologous tropomyosin, which confirms the antibody cross-reactivity between prawn

and abalone tropomyosins. Regarding the allergenicity of recombinant tropomyosin from

prawns and abalone, both recombinant tropomyosins showed a very strong capacity to

bind to human IgE. This is the first time to evidence tropomyosin from Jade tiger abalone

as an important allergen. Furthermore, the allergenic potential of recombinant

tropomyosin from Black tiger prawn seemed very similar as compared to the total heated

extracts of prawns when used in the induction of allergic responses in mice. Nevertheless,

compared to heated abalone extracts, recombinant tropomyosin from Jade tiger abalone

elicited less serum IgE, mast cell protease-1, intestinal IgA and splenic IL-5, indicating

the noticeable contributions of other allergens (e.g. arginine kinase) to the total

allergenicity of abalone.

The in vivo immunological cross-reactivity between the heated extracts of prawns and

abalone or between tropomyosins from prawns and abalone was comprehensively

evaluated in Chapter 3 and Chapter 5, respectively. Overall, the immunological cross-

reactivity at both humoral and cellular aspects was observed at certain degrees between

Chapter 6

245

either the heated extracts or tropomyosins of these two species as demonstrated in mice.

The capacity of serum and intestinal antibodies to bind heterologous extracts was reduced

as compared to homologous extracts. However, the results of splenic cytokine stimulation

illustrated that the frequent exposure of heterologous extracts seemed to increase the level

of Th2-associated reactions in the sensitised mice. However, increased exposure of

heterologous tropomyosins did not promote the production of Th2 cytokines, indicating

that other allergens might contribute to the overall allergenicity. In contrast, with the

continual gavages of heterologous extracts, the Th2-associated responses to the other

shellfish allergens might be enhanced. Another important finding is the different

allergenic properties between the heated protein extracts and tropomyosins from prawns

and abalone. As compared to the heated prawn extracts, mice that were sensitised with

heated abalone extracts were more susceptible to either homologous or heterologous

allergen stimulation. This might be correlated with the findings in Chapter 5 that abalone

tropomyosin may contain a more complex panel of immunodominant epitopes as

compared to prawn tropomyosin. This is concluded from the results where the gavage of

abalone extracts elicited significant IgE production in prawn-tropomyosin-sensitised

mice, rather than vice versa.

Overall, in this PhD thesis, a very useful in vivo experimental platform was established

to study shellfish allergy. This platform was successfully applied for assessing

immunological shellfish cross-reactivity and the allergenicity of recombinant

tropomyosins from prawns and abalone. Tropomyosin has been demonstrated to be the

central cross-reactive allergen in shellfish allergy. The work present in this thesis provides

an important contribution towards better diagnostics and prescribing therapeutic

elimination diets in patients with shellfish allergy.

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Future directions

(1) Epitope mapping of shellfish allergens

In-depth identification and characterisation of B and T cell epitopes of major allergens

can be very helpful for studying many aspects of food allergy, including the assessment

of food allergenicity, and the development of improved diagnosis/prognosis and

immunotherapies. In this thesis, the responsibility of tropomyosin in the prawn-abalone

cross-reactivity has been evidenced. As shown in Figure 6.1, tropomyosin is widely

distributed in several distinct animal groups, and thus the susceptible patients need

medical advice about the potential clinical cross-reactivity between these shellfish species.

The immunodominant epitopes of tropomyosin, including the cross-reactive properties,

can be identified as candidate biomarkers to predict the occurrence or the degree of

immunological and clinical cross-reactivity between shellfish and other allergenic

invertebrates. The appropriate application of these biomarkers in skin prick test, serum

IgE detection and the induction of animal models may decrease the need for oral

challenges. Accurate recommendations of cross-reactive shellfish species from the same

phyla that could be safely consumed could avoid overly restrictive diets. Moreover, the

identification of species-specific epitopes of tropomyosin can be very useful for precise

diagnosis of true sensitisation to a specific allergen source. Other potential cross-reactive

allergens in shellfish and their informative epitopes should also be further investigated.

Based on the identification of cross-reactive epitopes, characterisation of allergen

molecules by X-ray crystallography and site-directed mutagenesis can be a promising

strategy for the generation of specific allergen vaccination.

Chapter 6

247

Figure 6.1 Molecular phylogenetic tree of tropomyosins from different invertebrate

species inferred using Neighbour-Joining method. Clustering of species from the same

phyla is demonstrated on the right side of the figure.

(2) Validation of animal models

Protein extracts of more shellfish species and the derived allergen molecules should be

prepared and applied in both patient tests and mouse model inductions. Hence, according

to the data of patient tests, animal models can be optimised for improved clinical

relevance, through adjusting the animal genetics, sensitisation doses, nature of allergens

and routes of allergen exposure. For the further validation of animal models, it is also

recommended to test whether the animal model could respond to specific treatments to

relieve allergic symptoms, such as antihistamines, corticosteroids and bronchodilators, or

promising therapeutic strategies, with clinical efficacy. Subsequently, the establishment

of animal models needs to be standardised, which would promote their wider acceptance

and applications in studies of shellfish allergy.

APPENDIX

BUFFERS AND SOLUTIONS

Appendix

249

1.1 General buffer

20 x Phosphate buffered saline (PBS)

Na2HPO4 ------------------------------------ 28.8 g

KH2PO4 --------------------------------------- 4.8 g

NaCl ------------------------------------------ 160 g

KCl ---------------------------------------------- 4 g

Milli-Q H2O ------------------------------------ 1 l

Mix to dissolve and adjust pH to 7.2. Autoclave or filter through 0.45μm membrane for

long-time storage. Dilute 1:20 with Milli-Q water before use and adjust pH if necessary.

1.2 ELISA buffers

2% Fetal bovine serum (FBS) (antibody dilution buffer)

FBS --------------------------------------------- 2 ml

1 x PBS -------------------------------------- 100 ml

10% FBS (block buffer)

FBS -------------------------------------------- 10 ml

1 x PBS -------------------------------------- 100 ml

PBS/0.05% Tween 20 (wash buffer)

Tween 20 ------------------------------------- 2.5 ml

1 x PBS ------------------------------------------- 5 l

1M HCl (stop buffer)

38% HCl -------------------------------------- 8.3 ml

Milli-Q H2O --------------------------- up to 100 ml

Appendix

250

1.3 Cell culture buffers

Cell wash buffer

RPMI-1640 ---------------------------------- 500 ml

FBS ---------------------------------------------- 5 ml

Complete culture medium

RPMI-1640 ---------------------------------- 500 ml

FBS -------------------------------------------- 50 ml

2-mercaptoethanol -------------------------- 500 μl

HEPES (1M) --------------------------------- 10 ml

Gluta-max (100 x) ---------------------------- 5 ml

Penicillin/Streptomycin Solution (P/S) ---- 5 ml

1.4 Flow cytometry

Fluorescence-activated cell sorting (FACS) buffer

Bovine serum albumin ------------------------- 1 g

Sodium azide ------------------------------------ 1 g

1 x PBS ------------------------------------ up to 1 l

1.5 SDS-PAGE solutions

Solution B

Tris-HCl (2 M, pH 8.8) ---------------------- 75 ml

10% SDS in Milli-Q H2O --------------------- 4 ml

Milli-Q H2O ------------------------------------21 ml

Appendix

251

Solution C

Tris-HCl (1 M, pH 6.8) ---------------------- 50 ml

10% SDS in Milli-Q H2O --------------------- 4 ml

Milli-Q H2O ------------------------------------46 ml

5 x Protein sample loading buffer

Tris-HCl (1 M, pH 6.8) ---------------------- 0.6 ml

50% Glycerol ------------------------------------ 5ml

10% SDS ---------------------------------------- 2 ml

Dithiothreitol (1 M) ---------------------------- 1 ml

1% Bromophenol blue ------------------------- 1 ml

Milli-Q H2O ----------------------------- up to 10 ml

12% SDS-PAGE gel recipe

Resolving gel

40% 29:1 Acrylamide -------------------------- 6 ml

Solution B ---------------------------------------- 5 ml

Milli-Q H2O ------------------------------------ 8.9 ml

10% Ammonium persulphate ---------------- 100 μl

TEMED ------------------------------------------- 10 μl

Stacking gel

40% 29:1 Acrylamide ----------------------- 0.93 ml

Solution C -------------------------------------- 2.5 ml

Milli-Q H2O ------------------------------------ 6.5 ml

10% Ammonium persulphate ---------------- 100 μl

TEMED ------------------------------------------- 10 μl

Appendix

252

1 x Gel Electrophoresis running buffer

Tris ------------------------------------------------ 3 g/l

Glycine ---------------------------------------- 14.4 g/l

SDS ------------------------------------------------ 1 g/l

Milli-Q H2O ---------------------------------- up to 1 l

SDS-PAGE gel destaining solution

Methanol (AR grade) ------------------------- 500 ml

Glacial acetic acid ----------------------------- 100 ml

Milli-Q H2O ------------------------------------ 400 ml

1.6 Immunoblotting Buffers

Transfer buffer

Tris ------------------------------------------- 1.164 g

Glycine ---------------------------------------- 0.58 g

10% SDS -------------------------------------- 750 μl

Methanol --------------------------------------- 40 ml

Milli-Q H2O --------------------------- up to 200 ml

Blocking buffer

Skimmed Milk powder -------------------------- 5 g

Milli-Q H2O --------------------------- up to 100 ml

Antibody dilution buffer

Skimmed Milk powder -------------------------- 1 g

Milli-Q H2O --------------------------- up to 100 ml

Appendix

253

1.7 DNA clone buffers

LB broth

Tryptone -------------------------------------- 10 g/l

NaCl ------------------------------------------- 10 g/l

Yeast extract ----------------------------------- 5 g/l

Milli-Q H2O ------------------------------- up to 1 l

Mix to dissolve and autoclave before use for bacterial culture.

LB plates

Tryptone -------------------------------------- 10 g/l

NaCl ------------------------------------------- 10 g/l

Yeast extract ----------------------------------- 5 g/l

Agar ------------------------------------------- 15 g/l

Milli-Q H2O ------------------------------- up to 1 l

Ampicillin stock

Ampicillin ---------------------------------------- 1 g

70% Ethanol ---------------------------------- 10 ml

Filter sterilised before use.

Glycerol stocks

Fresh overnight culture --------------------- 700 μl

50% sterile glycerol in Milli-Q H2O ------ 300 μl

Appendix

254

1.8 Protein expression buffers

ZY

Peptone ------------------------------------------ 10 g

Yeast extract -------------------------------------- 5 g

Milli-Q H2O -------------------------------- up to 1 l

20 x NPS

(NH4)2SO4 ------------------------------------ 16.5 g

KH2PO4 ----------------------------------------- 34 g

NaHPO4 ---------------------------------------35.5 g

Milli-Q H2O --------------------------------- 250 ml

50 x 5052

Glycerol --------------------------------------- 125 g

Glucose --------------------------------------- 12.5 g

α-Lactose --------------------------------------- 50 g

Milli-Q H2O --------------------------------- 365 ml

MgSO4 (1 M)

MgSO4.7H2O ------------------------------ 24.65 g

Milli-Q H2O -------------------------------- 100 ml

FeCl3 (1000 x)

HCl (0.1 M) ---------------------------------- 81 ml

FeCl3.6H20 ------------------------------------- 2.7 g

Milli-Q H2O -------------------------- up to 100 ml

Autoclave every component above before generating ZYP-5052 medium.

Appendix

255

ZYP-5052 medium

ZY -------------------------------------------- 928 ml

MgSO4 (1 M) ---------------------------------- 1 ml

FeCl3 (1000 x) --------------------------------- 1 ml

50 x 5052 -------------------------------------- 20 ml

20 x NPS --------------------------------------- 50 ml

Ampicillin (100 mg/ml) --------------------- 0.5 ml

Extraction buffer

Tris ------------------------------------------- 3.0285 g

NaCl ------------------------------------------- 17.53 g

Imidazole -------------------------------------- 0.068 g

HCl ------------------------------------------ till pH 8.0

Milli-Q H2O ---------------------------------- up to 1 l

1.9 Protein Purification Buffers

Phosphate buffer

Na2HPO4 ----------------------------------------- 1.44 g

KH2PO4 ------------------------------------------- 0.24 g

NaCl ----------------------------------------------- 17.5 g

KCl -------------------------------------------------- 0.2 g

Milli-Q H2O ------------------------------------------- 1 l

Mix to dissolve and adjust pH to 7.4.

Stock solution

Imidazole --------------------------------------- 0.068 g

Appendix

256

Phosphate buffer --------------------------------- 50 ml

Equilibration buffer

Stock solution ------------------------------------- 1 ml

Milli-Q H2O ------------------------------- up to 50 ml

Wash buffer

Stock solution ------------------------------------- 5 ml

Milli-Q H2O ------------------------------- up to 50 ml

Elution buffer

Stock solution ------------------------------------ 30 ml

Milli-Q H2O ------------------------------- up to 50 ml

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