Hypoallergenic Mutants of Ana O 2, a Major Cashew Allergen & Identification and Characterization of Tree Nut Allergens

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2008 Hypoallergenic Mutants of Ana O 2, a Major Cashew Allergen & Identification and Characterization of Tree Nut Allergens Pallavi Dattatray Tawde

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FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

HYPOALLERGENIC MUTANTS OF ANA O 2, A MAJOR CASHEW

ALLERGEN

IDENTIFICATION AND CHARACTERIZATION OF TREE NUT

ALLERGENS

Pallavi Tawde

A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Spring Semester, 2008

The members of the Committee approve the Dissertation of Pallavi Tawde defended on

Date, April 4th 2008.

______Kenneth H. Roux Professor Directing Dissertation

______Shridhar K. Sathe Outside Committee Member

______Thomas C.S. Keller III Committee Member

______Michael Blaber Committee Member

______Robert Reeves Committee Member

Approved:

______Timothy S. Moerland, Chair, Department of Biological Science

______Joseph Travis, Dean, College of Arts and Sciences

The Office of Graduate Studies has verified and approved the above named committee members

ACKNOWLEDGEMENTS

I especially wish to extend my gratitude to my mentor Dr. Kenneth Roux for his patience and confidence in my intellectual and personal abilities. His astute guidance, assiduous attention to detail, and valuable criticism of experimental strategies and results made this experience both professionally rewarding and personally enjoyable. I would like to thank the members of my committee Drs. Shridhar Sathe, Michael Blaber, Tom Keller, and Robert Reeves for their guidance, encouragement, valuable inputs, and willingness to support me throughout the different stages of my graduate school career. In addition to those at FSU, I would like to express my gratitude to Dr. Suzanne Teuber, who not only generously provided me with serum samples but also allowed me to conduct a crucial part of my dissertation analyses in her lab at UC, Davis. Many thanks to Sarah Comstock for her generous help with the basophil assay. Certainly, a great deal of credit is due to those around me from whom I derive a great deal of support. I would therefore like to thank all the former and current Roux lab members. My sincere gratitude to my friends and colleagues--Jason Robothom, Henry Grise, Leanna Willison, Bodhana Dhole on whom I could rely for thoughtful conversations or good laughs. I especially thank Jason Robotham for his friendship and advice. Thanks to Fang Wang for getting me initiated with the cloning and molecular biology techniques. During the course of my graduate studies many people have in one way or another contributed to its completion. My sincere thanks to all the personnel that made the completion of this project possible, including the Analytical lab, the Sequencing lab, the Cloning Facility, KLB and the Medical school tissue culture and flow cytometry facilities. My deepest gratitude extends to Rani Dhanarajan and Margaret Seavy for their huge help with protein purification, cloning, protein expression and the nitty gritty details of molecular biology. The training and invaluable technical assistance provided by both Rani and Margaret helped me immensely. Also, I tender my sincere appreciation to Ruth Didier for providing me with invaluable training and technical assistance with flow cytometry. Many thanks to Greg Hoffman for his technical help with homology modeling of proteins.

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I am eternally indebted to the Department of Biology for giving me this opportunity and providing generously the financial support for me to pursue academic endeavors and advance my career. My special thanks to Manacy Pai for her tremendous support and enthusiasm that has enriched both my personal and professional endeavors. Finally, my largest debt is to my family--I am thankful to my parents for encouraging and supporting me to pursue my goals. Without their unconditional love and unwavering confidence, this dissertation and more importantly, this journey wouldn’t have been possible.

TABLE OF CONTENTS

List of Tables ...... vi

List of Figures ...... vii

Abstract ...... ix

1. TYPE I (IGE) - FOOD HYPERSENSITIVITY AND TREE NUT ALLERGY 1

2. CLONING AND CHARACTERIZATION OF PROFILIN (PRU DU 4), A CROSS-REACTIVE ALMOND (PRUNUS DULCIS) ALLERGEN

Introduction ...... 24

Methods ...... 25

Results ...... 32

Discussion ...... 40

3. ALMOND AND WALNUT 60S RIBOSOMAL PROTEIN P2: A NEW CLASS OF IGE-BINDING FOOD PROTEIN WITH FUNGAL AEROALLERGEN CROSS-REACTIVITY

Introduction ...... 43

Methods ...... 44

Results ...... 49

Discussion ...... 59

4. IDENTIFICATION OF PISTACHIO 7S AND 11S GLOBULIN AND DETECTION OF CROSS-REACTIVITY WITH HOMOLOGOUS CASHEW ALLERGENS

Introduction ...... 62

Methods ...... 65

Results ...... 70

Discussion ...... 90

5. MUTATIONAL ANALYSIS OF ANA O 2, A MAJOR CASHEW ALLERGEN DESIGNING HYPOALLERGENIC MUTANTS OF ANA O 2

Introduction ...... 96

Methods ...... 101

Results ...... 108

Discussion ...... 127

CONCLUSION ...... 134

FUTURE DIRECTIONS ...... 136

APPENDICES ...... 137

REFERENCES ...... 139

BIOGRAPHICAL SKETCH ...... 164

LIST OF TABLES

2.1 Clinical characteristics of almond-allergic subjects ...... 26

2.2 Profilins displaying sequence homology to almond profilin ...... 34

3.1 Clinical characteristics of almond and/or walnut allergic subjects...... 45

3.2 Homology comparisons between almond, walnut, fungal, and human 60S acidic ribosomal P2 proteins...... 51

3.3 IgE reactivity of almond and/or walnut allergic sera with rPru du 5, rJug r 5, and rFus c 1 analyzed by non-reducing dot-blot and ELISA...... 53

4.1 Clinical characteristics of pistachio and/or cashew-allergic subjects...... 65

4.2 Vicilins demonstrating the greatest homology to Pis v 3 ...... 77

4.3 IgE-reactive linear epitopes identified on Ana o 1 ...... 78

4.4 Sequences demonstrating the greatest homology to Ana o 2 ...... 84

4.5 Linear IgE reactive epitopes of Ana o 2…………………………………. 85

5.1 Linear IgE-reactive epitopes identified on Ana o 2...... 110

5.2 Immunoreactivity of Ana o 2 specific mAbs, with Ana o 2 wt and mutants by ELISA...... 119

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LIST OF FIGURES

2.1 Nucleotide and deduced amino acid sequence of rPru du 4, clone 1 (AY081850), showing start and stop codons...... 33

2.2 Immunoreactivity of rabbit anti-maize profilin polyclonal antibody total almond extract, rPru du 4 and inhibition immunoblot of almond extract with allergic patients sera...... 35

2.3 Dot-blot analysis of nPru du 4, rPru du 4, and almond extract with almond allergic patient’s sera ...... 36

2.4 ELISA analysis of specific IgE reactivity of sera from patients with almond allergy to almond extract, native and recombinant Pru du 4...... 37

2.5 Inhibition dot-blot with patient #34 serum with rPru du 4, nPru du 4, and almond extract...... 38

2.6 Detection of almond profilin isoforms on 2-DE immunoblot of almond with rabbit anti-maize profilin polyclonal antibody (anti-ZMPro5 ...... 39

2.7 Inhibition dot blot demonstrating cross-reactivity between nPru du 4 and rye grass pollen nRGPP ...... 39

3.1 Almond 60S RP rPru du 5 nucleotide and amino acid sequence...... 50

3.2 Amino acid sequence alignment of almond rPru du 5 and the two isoforms of walnut rJug r 5 ...... 51

3.3 Phylogenetic analysis of 60S RPs and cross-comparison of allergenic 60S RPs with human 60S ribosomal protein ...... 52

3.4 Inhibition dot-blot demonstrating cross-reactivity between rPru du 5, rJug r 5, and rFus c 1...... 54

3.5 IgE inhibition ELISA using rPru du 5, rJug r 5, and rFus c 1 ...... 54

3.6 Multiple sequence alignment of rPru du 5, rJug r 5, and rFus c 1...... 55

3.7 Dot-blot analysis of almond, walnut, and fungal 60S RPs with rabbit anti-rPru du 5 and effect of reducing conditions...... 56

3.8 Inhibition dot-blot demonstrating the presence of native 60S RPs in

viii

almond and walnut extracts ...... 57

3.9 Inhibition IgE ELISA determining the relative concentrations of native 60S RPs in almond and walnut extracts...... 58

4.1 Nucleotide sequence of the Ana o 1 cDNA clones Ana o 1.0101 and Ana o 1.0102...... 70

4.2 Amino acid sequence of the Ana o 1 cDNA clones...... 71

4.3 SDS-PAGE and Coomassie blue stain of cashew and pistachio extracts... 72

4.4 Immunoblotting profile of cashew and pistachio extracts with human cashew allergic sera, and rabbit anti-cashew polyclonal antisera...... 73

4.5 2-DE immunoblot of cashew extract probed with pooled human cashew allergic sera...... 74

4.6 2-DE immunoblot of pistachio extract probed with pooled human cashew allergic sera...... 75

4.7 Nucleotide and derived amino acid sequence of Pis v 3 cDNA ...... 76

4.8 Amino acid sequence alignment of rAna o 1 and rPis v 3...... 79

4.9 IgE reactivity of cashew-allergic patients’ sera to rAna o 1 and rPis v 3 by dot-blot.. ………………………………………………………………….. 80

4.10 Inhibition dot-blot with rAna o 1 and rPis v 3 and human cashew allergic sera...... 81

4.11 Inhibition immunoblot of pistachio protein extract probed with pooled cashew and tree nut allergic patient sera, pre-absorbed with rAna o 1, or with rPis v 3...... 81

4.12 Dot-blot analysis of anti-cashew rAna o 1 mAbs probed with rAna o 1 and rPis v 3...... 82

4.13 Nucleotide and derived amino acid sequence of Ana o 2 cDNA...... 83

4.14 Nucleotide and derived amino acid sequence of Pis v 2 cDNA...... 87

4.15 Amino acid sequence alignment of rAna o 2 and rPis v 2...... 88

4.16 Dot-blot immunoblotting of rAna o 2 and rPis v 2 with Ana o 2 specific mAbs...... 90

5.1 Amino acid sequence of the Ana o 2 coding region (AAN76862)...... 108 5.2 Linear epitopes mapping of Ana o 2 with pooled human cashew allergic sera...... 109

5.3 Alanine scanning mutagenesis of immunodominant IgE reactive Ana o 2 linear epitopes...... 111

5.4 Expression of Ana o 2 mutants as observed by SDS-PAGE stained by Coomassie Blue ...... 112

5.5 IgE-binding capacity of the Ana o 2 mutants in comparison with the wild-type protein using six cashew allergic sera by ELISA...... 113

5.6 IgE immunoblot analysis of wt Ana o 2 and mutant Q110A with six cashew allergic patient sera...... 114

5.7 Inhibition ELISA with Ana o 2 wt and muatnts ...... 115

5.8 Dot-blot analysis of rAna o 2 wt and six combinatory Ana o 2 mutants with six individual cashew allergic sera...... 116

5.9 Effect of mutations on the reactivity of Ana o 2 mAbs by ELISA...... 118

5.10 Flow cytometry dot-plot using pt#1 serum IgE and reactivity of Ana o 2 wt, Ana o 2 mutant ...... 120

5.11 Flow cytometry dot-plot using pt#4 serum IgE and reactivity of Ana o 2 wt, Ana o 2 mutants...... 121

5.12 Flow cytometry dot-plot using pt#6 serum IgE and reactivity of Ana o 2 wt, Ana o 2 mutants...... 123

5.13 Comparison of CD63 expression of basophils triggered with Ana o 2 mutants with respect to Ana o 2 wt...... 124

5.14 The CD spectra of Ana o 2 wt and mutants...... 125

5.15 Surface epitope mapping of the Ana o 2 trimer model...... 126

5.16 The highlighted three immunodinant IgE reactive epitopes of Ana o 2 shown on the homology model...... 126

5.17 The point mutations in the three immundominant epitopes depicted in the Ana o 2 homology model...... 127

ABSTRACT

Allergic diseases are a heterogeneous group of type I IgE-mediated hypersensitivity reactions affecting more than 25% of the world's population of developed countries becoming a major clinical and public health issue. According to the American Academy of Allergy, Asthma and Immunology (AAAAI), allergies affect as many as 40-50 million people in the United States, making it the sixth most common cause of chronic illness. Recent studies have estimated that food allergies occur in 6 - 8% of children under 3 years of age and in 4% of adults in the US population. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) proposed that milk, shellfish, egg, fish, peanut, soybean, tree nuts and wheat are eight major sources of food allergens and the causes of most food allergies. Peanut, tree nuts, and seafood allergies predominate in adults, whereas milk and egg allergies are important in children. Tree nut allergies, in particular, affect about 0.5% of the US population. The commonly consumed tree nuts in the US include walnuts, almonds, cashews, pistachios, and pecans, all of which are allergenic to predisposed indviduals of the consuming population. The major allergenic proteins associated with tree nut allergies include the seed storage proteins belonging to the 7S and 11S globulin, and 2S albumin gene families. Complementary DNA expression libraries were created from English walnut, cashew and almond. To identify the almond profilin, almond and walnut 60S ribosomal protein P2, 7S and 11S globulin genes in cashew and pistachio nuts, the libraries were used either directly as targets for degenerate primers in PCR ‘fishing’ experiments or transferred to nitrocellulose membranes and screened with tree nut allergenic patient sera. Upon identification and amplification of the respective coding genes, they were modified with restriction enzymes, ligated into an expression vector, and expressed as fusion proteins. These purified fusion proteins were used to screen for tree nut-allergic patient IgE-reactivity in direct immunoblots and to identify the native counterparts of these proteins in crude nut extracts via inhibition immunoblots and/or inhibition ELISA. Information on the linear epitopes and homology modeling of 7S and 11S globulins from cashew and pistachio would provide structural insight into the issue of serological and clinical cross-reactivity between these two nuts from the Anacardiaceae family.

The 11S globulins represent a family of most abundant seed storage proteins, and have been shown to be the most allergenic (i.e., react with high percentage of patients sera) of the seed storage proteins (allergenic in cashew, pistachio, almond, walnut, Brazil nut, and hazelnut). Linear IgE-reactive epitopes on the primary amino acid sequence of major cashew allergen and protein --11S globulin, Ana o 2 were located by overlapping synthetic peptide analysis using cashew allergic sera. Additionally, alanine scanning mutagenesis of the immunodominant IgE reactive linear epitopes was used in the identification of key amino acid residues critical for IgE binding. The identified critical amino acids would serve as targets to create hypoallergenic variants of Ana o 2, by introducing point mutations of those critical amino acids using site- directed mutagenesis in the Ana o 2 cDNA and unaltering the overall structural fold of the molecule. The biological potential of the hypoallergenic engineered Ana o 2 mutants would be analyzed using an ex vivo basophil activation assay. Thus, by reducing the anaphylactic potential of Ana o 2 while preserving T cell epitopes, we could potentially use these hypoallergens for allergen specific immunotherapy in cashew sensitized individuals.

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CHAPTER 1

FOOD HYPERSENSITIVITY AND TREE NUT ALLERGY

Food allergy is an adverse immunological response, classified as type I hypersensitivity to allergenic molecules present in foods resulting in elevated specific IgE and displays a range of clinical reactions within minutes of consuming the offending foods. The range of disorders encompasses acute, potentially chronic and fatal reactions mainly affecting the skin, respiratory, and gastrointestinal (GI) tract.1;2 The type I allergic hypersensitivity represents a significant health problem in industrialized countries and affects almost 500 million people worldwide.3 From a rare condition 40 or 50 years ago, the prevalence of allergy has increased dramatically over the past several decades affecting many aspects of society and the lives of many individuals. However the underlying causes of this increase are not yet fully understood. Most type I food allergies appears in the first 2 years of life and occurs in 6–8% of infants.4 As their immune systems mature (by 5 years or so), 80% of allergic infants lose their food allergies.5 Recent epidemiologic studies suggest that nearly 4% of Americans are afflicted with IgE-mediated food allergies, prevalence much higher than appreciated in the past.1;2;6;7 The source of food allergens range from fruits and vegetables to meats; the big 8 foods have been identified as the most frequent human food allergens and account for 90% of food allergies.6 They include fish, shellfish, soybean, wheat, egg, milk, peanuts, and tree nuts causing majority of all allergic reactions to foods. Data from a voluntary registry of peanut-and tree nut- allergic US patients shows that 62% of those reporting allergy to tree nuts list sensitivity to walnut and 44% report sensitivity to cashew.7 Allergies to milk, eggs, wheat and soy in infants are usually outgrown by age three; however allergies to fish, shellfish, peanuts and tree nuts often persist throughout life.8 It has been estimated that peanut and tree nuts cause over 90% of fatalities in the US.9 Peanut and tree nut allergies are typically severe and fatal, and are characterized by high frequency of life-threatening anaphylactic reactions and typically lifelong persistence.3 Additionally there is also a potential for frequent accidental dietary contamination.4 It has been reported recently that as many as 20% of children outgrow peanut allergy and a subset of children with tree nut allergy outgrow their allergy over time, although the resolution rate appears to be less as that seen in peanut allergy.10

The dietary proteins that are sampled by the GI immune system generally evoke a state of specific immunological unresponsiveness termed oral tolerance (OT).11 However, when this immune tolerance is abrogated, adverse reactions to food proteins arise. Adverse food reactions can be broadly divided into those that are mediated by IgE antibodies (type I hypersensitivity) and those that are not. The development of IgE mediated allergy is a multistep process, and the mechanisms leading to sensitization, production of IgE antibodies, and manifestation of an allergic response is complex and not yet fully deciphered. The antigen fragments are processed by antigen presenting cells (APCs) and displayed on their surface in association with MHC class II molecules that can be recognized by a specific T cell receptor on T cells. The allergic immune response is ultimately dependent on the cytokine profile that develops is response to an allergen.

In the presence of IL-4, naive antigen-specific TH2 cells get activated into effector cells. TH2 cells produce cytokines like IL-4, IL-5, IL-9, and IL-13 that induce class switching in B cells to develop into IgE producing plasma cells.13 The secreted antigen-specific IgE antibodies are distributed systemically and bind to the high affinity receptor FcεRI on mast cells and basophils. Upon re-exposure, the causal allergenic protein(s) interacts with the receptor bound specific IgE on the effector cells (mast cell, basophil), promoting cross-linking of adjacent IgE antibodies and aggregation of FcεRI receptors, and triggering degranulation and the release of inflammatory mediators (histamine, serotonin, prostaglandins, leukotrienes, cytokines), causing the symptoms of allergy. 13;15;28;41;44 These anaphylactic reactions occur within minutes to hours after ingestion of the offending food and are potentially life-threatening. The non-IgE mediated reactions to food can mimic food allergy but their patho-physiology are distinctly different and are not as clearly defined.11 The development and pathophysiology of an allergic response is known to be influenced by several factors, including diet and culture, genetic susceptibility of an individual, allergen dose, route of exposure, processing, cooking, digestion, and also the intrinsic structural and 12 functional characteristics of an allergen. A skewing towards a TH2 response has been reported in allergies, with elevated IL-4, IL-5, and IL-13 levels that are responsible for class switching to the ε heavy chain for IgE production, whereas non-allergic individuals have higher levels of the 13 TH1 cytokines IFN-γ and TNF-α, and the regulatory cytokine IL-10. Thus a dysregulated immune response appears to be an important pathogenesis mediating factor in allergy.

In the U.S., food allergy is the leading cause of life-threatening anaphylactic reactions and accounts for ~ 1500 deaths/year.1;2;4 It is hypothesized that the increase in food allergy is tied to a lack of microbial challenge in early development (which is believed to shift the immune 29 system profile to TH2) due to better hygiene in developed countries.

FOOD ALLERGY The food allergic reactions typically manifest at mucosal sites, however primary sensitization could occur via various routes of exposure. In addition to the complexity of the gastrointestinal tract (GI), and the characteristics of allergens and also the sequence of events culminating an allergic response, it is difficult to pin point the contributing factor(s) responsible for the allergic reaction. Thus a multiplicity of factors is required in development of food allergy. These comprise the genetic predisposition of the host, the biochemical properties of the allergen(s), the processing of the food, and the environment. Other factors include age at which food antigen is introduced, formula versus breast feeding, dietary and gut flora composition, and gastrointestinal infection status.14 In the recent years, several food allergens have been identified and characterized, thus contributing to our understanding of how these proteins induce IgE mediated immune reactions. They are classified as class 1 and class 2 food allergens. Class 1 food allergens are also referred as true or complete allergens that induce allergic sensitization in the GI tract.15 The symptoms of class 1 food allergy range from mild rashes to life-threatening systemic anaphylaxis and are of four main types: dermatological (hives, local swelling, dermatitis, and eczema), gastrointestinal (nausea, vomiting, diarrhea, and abdominal pain), respiratory (runny nose, asthma, and tightening of the throat), and systemic (anaphylactic shock, organ failure, cardiac arrhythmia, and death).9 Class 2 allergens are also referred as incomplete allergens that are highly homologous with proteins in pollen (e.g. Mal d 1 in apple and Bet v 1 in birch pollen).16;18 Sensitization occurs in the respiratory tract as a consequence of sensitization to the cross-reactive pollen allergens thereby leading to oral allergy syndrome (OAS).16 The symptoms of class 2 induced OAS are generally mild compared to class 1 food allergens. OAS symptoms include oropharyngeal itch, lip or tongue swelling and tightness in the throat pharyngitis, laryngeal edema.9 OAS has been frequently used to describe associations between allergy to pollen and the concomitant hypersensitive reactions to certain kinds of fruits, vegetables, and spices.16;17

There is evidence that Bet v 1 homologs, the pan allergens profilin and LTPs, as well as high MW allergens and/or glycoallergens are involved in pollen allergy with concomitant food hypersensitivity.18;19 Class 1 food allergens are typically heat and acid stable, water soluble glycoproteins ranging in molecular size from 10 to 70 kDa.20 In contrast, class 2 food allergens are heat-labile and susceptible to digestion. According to the stability of class 2 allergens during the digestive process, they can cause symptoms ranging from mild oral reactions (typical for the birch-fruit syndrome) to anaphylactic shock, which is not rare within the celery-mugwort-spice syndrome.16;17 Cooking can reduce the allergenicity of some allergens by destroying the conformational epitopes; but in other cases it can increase the allergenicity of proteins through modifications that provide increased stability during digestion.21 While fruits mainly cause oral symptoms, legumes, seeds and tree nuts are likely to provoke acute generalized symptoms and even severe anaphylactic reactions.

DIGESTION AND ABSORPTION OF FOOD The cells (enterocytes) lining the GI tract perform three main functions digestion, secretion and absorption. Antigen handling in the gut is normally associated with the generation of OT. However, in the immature gut, because of immature absorptive functions, antigen exposure may result in priming for immune responses instead of OT.11 Animal studies show that intestinal uptake of macromolecules is at a maximum during the neonatal period and decreases overtime with maturation of the intestinal barrier.11;22;23 Any alterations in maturation of the gut barrier can lead to increased exposure to orally ingested antigens and cause a primed immune response.22 Evidence supporting this hypothesis comes from the presence of circulating antibodies to common food antigens during the first 3 months of life.6;22 Increased uptake of intact food antigens in the immature gut has been explained by increased binding of antigens to the microvillus membrane.23 Moreover, incomplete degradation may result in the generation of sensitization rather than tolerance.14

The gut epithelial barrier The single layer of intestinal epithelial cells in the small intestine, interfaces tissue compartments and luminal contents, and represents a primary cellular barrier of the gut and has

an extensively large surface area (~ 400 m2), due to highly convoluted projections called villi and microvilli.23 The tight junction seals the space between adjacent epithelial cells and, in intact gastrointestinal epithelia, tight junction permeability is the rate limiting step that defines the overall epithelial permeability.14 The intestinal lumen is replete with digestive enzymes that facilitate the digestion of ingested foods and consequently followed by absorption by enterocytes. The intercellular tight junctions restrict the passage of very small (2 kDa) molecules.34;40 Tight junctions function as a semipermeable gate that regulates passive movement of luminal fluid and solutes through the paracellular pathway. The intestinal epithelium also boasts a number of specialized protective adaptations including mucus, antimicrobial peptides (defensins), secretory IgA (sIgA), mucins, and trefoil peptides.11;14;34;40;42;43 However, the gut epithelial barrier does not completely prevent luminal antigens from entering the tissues/systemic circulation. A small amount of residual, non-degraded, dietary proteins can, however, potentially trigger the gut associated immune system.23;40

Macromolecular transport through the enterocyte Macromolecules can be transported through the enterocyte (transcellular transport) or between adjacent epithelial cells (paracellular transport).23 The transcellular pathway allows molecules to enter the cell from the luminal side and exit on the serosal side and is also important in the regulation of intestinal permeability.40 After uptake, enzymes present in the lysosome or in the endocytic vesicle destroy most of the macromolecules. However, some of them escape degradation and are released into the interstitial space, after which they enter the systemic circulation with or without prior absorption into the lymphatic vessels. The paracellular pathway is mainly controlled by tight junctions between epithelial cells and is a key regulator of intestinal permeability to macromolecules. The open tight junction has a pore radius of 5 nm and allows the passage of small macromolecules (4000–5500 Da).14;40 However, the tight junctions could be altered rapidly by osmotic load or certain disease conditions e.g. inflammatory bowel disease.23;40

MUCOSAL IMMUNE SYSTEM The gut is constantly exposed to infectious agents and harmless food substances. While an immune response is required against the former, tolerance – immunosuppression – is of vital

necessity against the latter. The integrity of the GI tract along with the delicate balance between the necessity to mount an immune response and establish a non-immunoresponsive state is maintained via both innate and adaptive mucosal immune mechanisms. In the gut, the mucosal immune responses are primarily induced in gut associated lymphoid tissue (GALT) consisting of both organized lymphoid tissues, such as mesenteric lymph nodes (MLN) and Peyers patches (PP), and more diffusely scattered lymphocytes in the intestinal lamina propria (LP) and epithelium including large numbers of IgA plasmablasts.42 PP are organized aggregates of lymphoid tissue numbering ~200 in the small intestine of an average adult.11 PPs are overlaid by specialized epithelial cells termed as M cells whose function is to transport luminal antigens.

M-Cells The follicle-associated epithelium (FAE) of PPs is composed of cuboidal absorptive enterocytes interrupted by delicate membranous “M” cells, which have luminal microfolds.22;23 The M cells are highly specialized antigen sampling cells, and play a key role in mucosal immunity through their capacity to sample and transport macromolecules from the gut lumen either via phagocytosis, transepithelial vesicular endocytosis or fluid phase pinocytosis.23;40 A typical feature of M cells is that, the basolateral surface is deeply invaginated to form intraepithelial pockets folded around underlying lymphocytes and APCs, which take up the transported material released from the M cells and process it for antigen presentation. Thus, M cell pockets provide the first opportunity for contact between antigens penetrating the intestinal epithelial barrier and the underlying lymphoid system.23 The antigen is in turn delivered into the subepithelial dome region of the PPs, an area rich in DCs, that in turn ingests and processes the antigens and presents it to the underlying B cell follicles.11 These events stimulate sIgA production under the influence of TGF-β secreted by T cells, consequently contributing to OT.22;23;42

Dendritic cells These are potent APCs and are present in different compartments of the gut, including LP, PPs, and MLN.11 Besides the above-mentioned macromolecular transport pathways, a new possibility for antigen delivery to the mucosal immune system has recently been described. This novel possible mechanism implies that DC protrusions extend through the tight junctions of

epithelial cells into the intestinal lumen, to sample luminal material without disrupting epithelial integrity.23;42 The phenomenon of DC luminal antigen uptake, however, is mainly aimed at collecting luminal bacteria by picking them up directly within the intestinal lumen, especially in inflammatory conditions.46 It is less clear whether food antigens can also be captured directly in the lumen by this process. DCs have a pivotal role in directing the delicate balance between tolerance and active immunity in the intestine and is dependent on the cytokine microenvironment and expression of costimulatory molecules by these cells.23;42 These DCs minimally express CD80/CD86 indicating an immature state. Harmless food antigens normally fail to fully activate DCs and therefore induce antigen-specific tolerance. Upon exposure to inflammatory stimuli, DCs mature and are activated and shown to upregulate CD80/CD86 molecules and elicit a potent stimulatory response.23

Intestinal epithelial cells The intestinal epithelial cells constitutively express MHC class II molecules on their basolateral membranes and are capable of antigen presentation to primed T and B cells and might also act as nonprofessional APCs.24 In contrast to professional APCs, intestinal epithelial cells normally selectively activate CD8+ suppressor T cells thereby involved in local immunosuppression.24 Another feature is the capacity of enterocytes to secrete membrane vesicles (exosomes), enriched in MHC class II/peptide complexes and presenting high immunogenic capacity.43 These vesicles could play a role as messengers, transmitting peptidic information from the intestinal lumen to the underlying immune system, via enterocytes, by diffusing through pores in the basal membrane, in order to interact with non-adjacent immune cells.34;40;43

Basophils and Mast cells Basophils are major effector cells of IgE mediated hypersensitivity, and are usually found in the blood in very low numbers (0.2-1% of total leukocytes).25;47 Upon stimulation they migrate through the endothelium into tissues.25;27;28;41;44;47 Although basophils are the least circulating leukocytes, in atopic individuals they represent the largest population of antigen- specific cells in the circulation.25;41

Basophils contain cytoplasmic granules that stain with basic dyes and were first described in 1879 by Paul Ehrlich, who 1 year earlier had found a morphologically similar cell type present in tissues that he termed Mastzellen (mast cells). Human mast cells are a heterogeneous group of multifunctional tissue-dwelling cells with roles in parasitic infections, allergy, inflammation, angiogenesis, and tissue remodeling.25;28 They are found in tissues throughout the body and are particularly associated with blood vessels, nerves and surfaces underneath the skin.28 Mast cells and basophils have several features in common--they are the two principal reservoirs of the biogenic amine, histamine (110 Da), which is formed within each cell type from histidine by histidine decarboxylase.25;28;41 Basophils and mast cells contain numerous cytoplasmic granules containing vasoactive inflammatory mediators.22;41;47 These secretory granules exhibit characteristic crystalline ultrastructures and are usually not released until the cell becomes activated. There are two categories of inflammatory mediators in mast cells and basophils - preformed and newly synthesized mediators.41 Preformed mediators stored in secretory granules and secreted upon cell activation, include histamine, heparin, chondroitin sulphates, and a spectrum of neutral proteases. Newly synthesized mediators are typically produced during IgE-mediated activation, and consist of arachidonic acid metabolites, principally leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) and cytokines like TNF-α, IL-4, IL-5, and IL-13.37;41;47 The phenotypic hallmark of these cells is expression of high affinity IgE receptors (FcεRI) on their cell surface.41 In addition, these cells express various adhesion receptors, virus- and complement-binding sites, surface membrane enzymes, and cytokine receptors. These surface structures are associated with distinct functional properties including proliferation, adhesion, or mediator secretion.26

FcεRI, the high affinity IgE receptor FcεRI is abundant in mast cell and basophil cell membranes (~200,000 molecules/cell) and is also expressed at much lower numbers in eosinophils, platelets, APCs like monocytes, DC and Langerhans cells.41;47 The concentration of FcεRI on mast cells and basophils is 3-10 times greater in atopic individuals in comparison to non-atopic individuals and its expression is directly associated to the titer of serum IgE.47 The FcεRI belongs to the immunoglobulin superfamily of multichain immune recognition class of receptors (MIRRs).27. It is expressed as a

tetrameric surface protein complex (αβγ2), consisting of the IgE binding α-chain, a signal amplifying membrane-tetraspanning β-chain, and a disulfide-linked homodimer of the γ- chains.27;28;41;47 27;47 In humans FcεRI is expressed as both trimeric (αγ2) and tetrameric (αβγ2) forms. The tetrameric form is expressed exclusively on mast cells and basophils and the trimeric form is found on eosinophils, APCs mentioned above, and platelets.27 Cross-linking of the FcεRI on mast cells and basophils triggers a network of processes that culminates in secretion of granule- stored mediators and the de novo synthesis and secretion of arachidonic acid metabolites, prostaglandins, and several cytokines.27;28;47 The binding of IgE to FcεRI occurs via its Fc domain in a 1:l ratio with very high affinity (Ka = 1010 - 1012 M-1), this interaction has a slow rate of dissociation thus, greatly prolonging the in vivo half-life of IgE.29 The Cε3 domain of the IgE-Fc region contains the principal binding site with the receptor.27;30 The α-chain of FcεRI belongs to the immunoglobulin (Ig) superfamily and comprises two extracellular Ig-related domains that bind a single IgE molecule, a transmembrane domain, and a short cytoplasmic tail.27 The crystallization studies revealed that the α-chain is made up of two heavily glycosylated extracellular domains (D1 and D2) containing the IgE Fc binding site.31 The two Ig domains (D1 and D2) of FcεRIα are positioned at an acute angle to one another, forming a convex surface at the top of the molecule and a marked cleft enclosed by the two globular domains.27 Four grouped tryptophan residues at the top surface (contributed by D1, D2 and the interface region) form a hydrophobic patch that is a putative contact point for the binding of IgE Fc.27;28;41;47 The FcεRI - and -chains have no role in ligand binding.27 The -chain has four transmembrane domains and both the amino and carboxy terminal have cytoplasmic tails. The homodimeric subunits consist of a short extracellular region, a transmembrane region and cytoplasmic tail.47 The and chains of FcεRI contain immunoreceptor tyrosine based activation motifs (ITAMs), which are a conserved feature of antigen and some Fc receptors and essential in the amplification and signaling competence of these receptor chains.27;37 The tyrosine residues in ITAMs serve as phosphoacceptor sites for the action of receptor-associated protein tyrosine kinases (PTKs) and are critical for signal transduction.27; 37 In addition to cell signaling, the β chains acts as an amplifier of FcεRI cell-surface expression on human

basophils/mast cells by promoting maturation and trafficking of associated FcεRIα and possibly by stabilizing the cell surface-expressed FcεRI complex. The -chains are the main and indispensable FcεRI signaling units and co-expression with the FcεRI α-chains is a requirement for reaching the surface and ultimately for the FcεRI complex to function.27;32;34 The cell-surface expression of the FcεRI complex is regulated through the availability of specific receptor components and by the amount of IgE.32;33 Moreover, the IgE-mediated upregulation of FcεRI expression leads to increased effector-cell functions, such as mast cell/ basophil mediator release and IgE-dependent antigen-presenting functions of DCs.32;34 The binding of monomeric IgE to the FcεRI does not initiate mast cell/basophil activation and degranulation.47 Cross-linking of the FcεRI-bound IgE by a multivalent antigen, followed by receptor aggregation, is required, and this cross-linking induces phosphorylation of β and γ chain ITAMs within 5-15 secs.47 In addition it has been reported that exposure of mast cells and basophils to IgE results in a striking (~35-fold) upregulation of surface expression of FcεRI, both in vitro and in vivo.32;34 This enhanced expression is caused due to the increased half-life of the surface-resident FcεRI rather than increased synthesis and/or transport to the plasma membrane.35 Additionally, binding of IgE to FcεRI enhances the expression of cytokines including IL -4, IL-6, IL-13 and TNF-α36 and histidine decarboxylase, the rate limiting enzyme of histamine synthesis. IgE induced increases in FcεRI expression have been associated with increased sensitivity to antigen and increased degranulation and cytokine production in response to antigen challenge.32

Activation of Basophils/Mast cells in an allergic reaction - intracellular signaling cascades The Type I IgE-mediated immediate hypersensitivity reaction is initiated by the cross- linking of FcεRI-bound IgE with multivalent allergen on the surface of the mast cells or basophils.27 IgE cross-linking by the respective allergen initiates the activation of mast cells or basophils by promoting aggregation of FcεRI and stimulates a series of membrane and cytoplasmic events, culminating in the activation and degranulation of basophils/mast cells. The diversity of mediators released as an event of degranulation is accomplished by a highly complex and inter-related network of secondary messengers.41;47 The FcεRI-IgE dependent mast cell/basophil activation process results in downstream events leading to the secretion of three classes of inflammatory mediators within minutes of allergenic challenge. Tyrosine

phosphorylation drives intracellular signal transduction cascades, leading to phosphorylation and recruitment of numerous adaptor proteins like LAT, Vav, SLP-76, and members of Gab2 family.27;41;47 The major target of antigen-receptor signaling is the activation of enzymes, such as phospholipase C1 and 2 (PLC1 and 2), PI-3 kinase, protein kinase C (PKC) to generate two secondary messengers--inositol triphosphate (IP3) and diaylglycerol (DAG). The resulting signaling events regulate intracellular calcium release, wherein IP3 binds to the IP3 receptors on the endoplasmic reticulum surface, leading to opening of calcium channel and influx and elevation of cytoplasmic levels of calcium.27 The intracellular calcium influx and DAG activates PKC and MAP kinases and culminate in the activation of the small GTPases such as Rac, Ras, and Rho.41,47 These pathways induce cytoskeletal changes, such as microtubule and actin- myosin filament networks, promoting granule vesicle translocation and fusion of secretory granules to the plasma membrane resulting in exocytosis.27;28;41 Thus tyrosine phosphorylation of ITAMs is a major regulatory mechanism involved in mast cell/basophil activation, signaling, regulating protein-protein interactions and subcellular location of several proteins. These intracellular events lead to degranulation, resulting in subsequent release of histamine and other performed inflammatory mediators, as well as synthesis of lipid mediators and cytokines by the effector cells.27;37;38;44;47 Basophils have been proposed to participate in modulating immune responses because of their ability to rapidly generate two archetypal cytokines involved in TH2 responses, namely IL-4 and IL-13. These effector cells play a key role in allergy by directly inducing the switch to the IgE isotype in B cells independently of T cells.47 The FcεRI expression on APCs function as an allergen focusing molecule on these cells.37 Studies have shown that allergens are more efficiently taken up, processed, and presented to T cells following targeting by APCs via FcεRI in comparison with allergen binding to APC in the conventional manner in vivo.37 In addition, engagement of FcεRI on APCs has been shown to induce major signal transduction events, such as activation of PTKs and increased activation of transcription factors, eventually resulting in secretion of several cytokines (TNF-α, IL-8, MCP-1).37;38

FcεRII/CD23 The other IgE receptor identified is FcεRII (CD23), and is a 45 kDa type II membrane protein that is expressed on B cells, activated macrophages, eosinophils, follicular DC, and

platelets.39 Binding studies revealed this receptor to have a 100-1000 fold lower affinity for IgE (Ka = 106–107 M-1), than the high affinity FcεRI on mast cells and basophils. Consequently, FcεRII /CD23 is termed as the low affinity IgE receptor.29 Aside from binding IgE, CD23 is known to share very little in common with FcεRI and does not participate directly in Type I hypersensitivity reactions.39 FcεRII/CD23 has multiple functions; it is both a positive and negative regulator of IgE synthesis.40 In humans, two isoforms of CD23 that differ by only seven amino acids in the short N-terminal cytoplasmic domain are observed. The two forms of isoforms of CD23 are designated as: CD23a and CD23b. CD23a is unconditionally present on B cells, while CD23b requires IL-4 to be expressed on T-cells, monocytes, Langerhans cells, eosinophils and macrophages.41;42 The soluble CD23 promotes the differentiation of germinal center B-cells into plasma cells,43 stimulates IgE synthesis in B-cells, and induces the secretion of proinflammatory cytokines in monocytes.195 The form that is constitutively expressed on B cells is CD23a and it is thought to play a role in endocytosis of IgE-coated particles. In the case of macrophages, it has been shown that IgE-mediated endocytosis is observed only in FcεRIIα-expressing cells, whereas IgE-dependent phagocytosis is observed only in FcεRIIβ-expressing cells.41 The IgE-dependent functions of FcεRII/CD23 vary according to the cell type on which it is expressed. On macrophages, eosinophils and platelets FcεRII/CD23 can mediate IgE-dependent cytotoxicity and, in the case of macrophages, can promote phagocytosis of IgE-coated particles, whereas binding of IgE to CD23 on monocytes induces the release of IL- 1 and TNF-α. CD23 protein appears to be expressed constitutively on human intestinal 285 epithelial cells. A predominance of TH2 cytokines may be the cause for increased CD23 expression observed on bronchial epithelial cells in asthma, upregulation of intestinal CD23 in cow’s milk protein intolerant adult patients,285 and intestinal CD23 expression in infants with IgE-mediated and non-IgE mediated cow’s milk protein adverse reactions.286 Cross-linking of CD23 on the surface of B cells has been shown to suppress IgE production in both human and animal studies.287

T cells Allergic diseases are inflammatory disorders in which aberrant immune regulation occurs, and susceptible individuals mount allergen specific TH2 responses, which drive disease 12

pathology. The archetypal cytokines of allergic diseases are IL-4 and IL-13, which instigate IgE production, and IL-5 that promotes eosinophil infiltration and inflammation.15;29;44;47 For establishing tolerance, and maintaining gut immune homeostasis, immunosuppression is critical. Among others, several types of regulatory T cells (Tregs), γδ T cells, TGF-β producing Th3 cells, and IL-10-producing Tr1 cells, appear to be involved in modulating the immune response to harmless food antigens.6;14 In the recent years, naturally occurring CD4+CD25+ Tregs, have received attention as a possible mucosal immune regulator.44;288 The naturally occurring CD4+CD25+ Tregs develop in the thymus and represent 5-10% of CD4+ T cells in the periphery, and are a central component of active immune suppression.288 The most specific marker that distinguishes regulatory from conventional T cells is FOXP3, an X-chromosome-linked transcription factor belonging to the forkhead family.288;289 Indeed, FOXP3 expression is now considered the gold standard property of nTreg and serves as a master switch in the development, differentiation, and function of Treg cells. The other subsets of Tregs (Th3 and Tr1) are inducible (adaptive) in the peripheral lymphoid tissue in response to allergens or foreign antigens. They produce both IL-10 and TGF-β and are known to suppress inflammation in both normal and atopic subjects.43;288 Several studies have elucidated the enormous potential of these Tregs to prevent pathological immune responses in autoimmune diseases, transplantation, graft-vs-host diseases, and allergy.288 Indeed, Treg-deficiency is associated with multiple sclerosis, Type-1 diabetes, rheumatoid arthritis as well as several allergic responses.290;291 The first indication that Tregs might play a role in allergic disorders in human subjects came from the observation that FOXP3- deficient patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked autoimmune syndrome (IPEX) have atopic disease.290 The IPEX syndrome is characterized by elevated IgE levels, eosinophilia, and food allergies. 290;291;292 The best evidence in humans that Tregs participate beneficially to control allergy comes from results of SIT trials and observations in persons who have resolved their allergic disease.292 Beekeepers who are repeatedly stung by bees no longer suffer bee allergy and have high numbers of antigen driven IL-10 producing CD4+ cells.45;293 Likewise, in the birch pollen- allergic patients, the Tregs displayed an impaired ability to suppress birch pollen-stimulated effector cells as compared with non-allergic controls. 45;293 In addition, children who outgrow cow’s milk allergy have increased numbers of circulating Tregs compared with children with

persistent active allergy.43 Thus the generation of a population of allergen-specific CD4+CD25+ Tregs and increased production of their suppressive cytokines are essential for effective SIT.45

ORAL TOLERANCE One of the key functions of GALT is to distinguish innocuous antigens from pathogenic micro-organisms and to elicit an appropriate response.22;34 Normal tissue homeostasis in the small intestine is maintained by exquisite interplay between the contents of the gut lumen, the surface epithelium and the immune cells in the lamina propria. Despite the large extent of dietary antigenic exposure, only a small percentage of individuals have food allergy. This is due to development of OT to dietary proteins, and refers to a state of systemic immunologic non- responsiveness to an antigen by means of prior exposure to that antigen through the oral route.34;42 The journey for a dietary protein antigen involves multiple steps before B and T cells can respond to it and cause either tolerance or food hypersensitivity. After undergoing modification in the lumen, the antigen is in contact with specific APCs with distinct activation requirements, which then help to activate Treg cells, resulting in the net suppression of an immune response. It is postulated that a breakdown in OT mechanisms or a failure of induction of OT results in food hypersensitivity. 34;42 In genetically susceptible individuals, allergic reactions to food typically occur, presumably when OT fails.11 These patients are referred as “atopic”, and it has been observed that a family history of allergies enhances the chances of developing atopic disorders. Genetic profile studies of the atopic individuals reveal a number of defects or abnormalities that could probably augment the predisposition towards development of allergy.42;43 The mechanism(s) responsible for OT is dependent on the nature and dose of the oral antigen. Soluble proteins induce tolerance, whereas particulate or globular antigens fail to do so.46 Higher doses are considered to cause clonal anergy or deletion of T cells, and multiple lower doses stimulate Tregs of the Th3 or Tr1 subset that induce immunosuppression.11;42;43 It has been reported that Treg cells produce anti-inflammatory cytokines like IL-10 and TGF-β that play an important role in the modulation of intestinal mucosal immune responses.13;42,43;45 In the absence of Tregs, food antigens and gut commensal bacteria act as a trigger for the development of allergy and irritable bowel syndrome (IBD).47 Oral administration of antigens has been shown to be effective in the treatment of autoimmune diseases in experimental animal models as well as

in human clinical trials.43 The induction and activation of Tregs and the production of immunosuppressive cytokines play a pivotal role in maintaining OT. In addition, DCs are also crucial in the differentiation and expansion of Tregs. DCs in the periphery are predominantly immature and are characterized by low level expression of surface MHC class II and costimulatory molecules (CD80, CD86, and CD40).27;42;43 Upon antigen uptake and migration to secondary lymphoid organs, immature DCs acquire a mature phenotype and present antigens to naive T cells. Depending upon the cytokine mileu and the inherent allergenicity of food proteins, the naive TH cells differentiate into TH1 or TH2 cells. TH2 polarization results in allergic disorders in atopic individuals. The type of antigen presenting DC, its maturation and the level and form of costimulation in turn influences OT.43 There is increasing evidence that induction of OT to protein antigens can be affected significantly by the intestinal microflora.34 Colonization of newborn’s intestinal flora begins at birth and the maternal microflora can be a source of bacteria. This is also affected by environmental factors and by infant feeding patterns. Breast-feeding is found to encourage the growth of bifidobacteria, whereas formula-fed infants have a more complex flora made up of bifidobacteria, bacteroides, clostridia and streptococci. The microbial community generally stabilizes by adulthood, and is composed of both permanent members and transient colonizers, which are briefly introduced from an exogenous source. Both prokaryotic and eukaryotic microbes are present although bacterial species predominate. The majority of the bacterial species are strict anaerobes (97%), while only 3% are aerobic (facultative anaerobes). The most common anaerobic genera in terms of concentration within the adult GI tract are Bacteroides, Bifidobacterium, Eubacterium, Fusobacterium, Clostridium and Lactobacillus. Among the facultative anaerobes are the Gram-negative enteric bacteria (Escherichia coli and Salmonella spp.), the Gram-positive cocci (Enterococcus, Staphylococcus and Streptococcus) and yeast (predominantly Candida albicans). Antibiotics and diet can dramatically affect the stability of the microbiota populations.34;42;43 Probiotics are live lactobacilli and bifidobacteria strains that are ingested to promote beneficial effects on health by altering indigenous microflora. The beneficial effects of probiotics are achieved through a variety of mechanisms including regulation of cytokine production, enhancement of IgA secretion, production of antibacterial substances and enhanced tight junction of the intestinal barrier to protect against intercellular bacterial invasion, and

competition with pathogenic micro-organisms for enterocyte adherence. Non-enteropathogenic E.coli or Lactobacillus GG reduce infection and protect against the development of food-induced atopic dermatitis.14 The inflammatory cytokines disrupt tight junctions of epithelial cells, leading to increased permeability and paracellular transport of luminal antigens.14;22;23;34 Commensal bacteria modulate key epithelial cell functions that help to maintain intestinal epithelial barrier integrity against injury and enhance specific sIgA responses.14 It is reported that delivery of sIgA and other molecules such as TGF- and IL-10 through breast milk play a very impottant role in OT.34 sIgA interacts with antigens that have reached the LP and the resultant immune complexes are either taken up by phagocytosis or transcytosed back to the lumen via binding to the polymeric Ig receptor. This process leads to immune exclusion and thus prevents inflammatory processes. In humans and mice, low levels of sIgA have been associated with food allergy and IgA-deficient individuals are predisposed towards developing food allergy.14 It is intriguing that only a limited number of the numerous proteins in a given allergenic food act as allergens, and it remains to be elucidated why even in atopic individuals only a small proportion of food proteins are associated with allergy. In addition, many questions are still unanswered regarding the underlying cellular mechanisms as well as site(s) of OT induction.

HYGIENE HYPOTHESIS The development of food allergy is influenced by both genetic and environmental risk factors. Several gene polymorphisms have been identified that show an association to one or more manifestations of allergic disorders.11;14;22;43;46;48 Environmental changes and subsequently complex gene - environment interactions are thought to be responsible for these changes. In addition, factors such as differences in diet between industrialized vs developing countries have been noted. 43;46;48 The hygiene hypothesis was initially formulated by David Strachan in 198948 to explain the steep increase in the prevalence of allergies in Western societies observed over the past few decades. It proposed that reduced exposure to infections and bacteria, viral, or fungal antigens in early childhood owing to a combination of factors including diminishing family size, modern health care, improved living standards and higher personal hygiene might result in increased risk of developing allergic disease.48 Epidemiological studies validate the significant increase in

allergic diseases over the years, which is paralleled by a tremendous decrease in incidence and prevalence of bacterial and viral diseases.48 Improved vaccination strategies, use of antibiotics as well as higher hygienic living conditions contribute to reduced incidence of infectious diseases. 43 Consequently, reduced exposure to infections in early childhood, leads to an imbalance in the immune system of predisposed individuals and triggers allergic responses.43;46;48 The strong protective effect of traditional farming environments against allergic sensitization and asthma is one of the strongest arguments in favor of this hypothesis and consistently points to bacterial products such as endotoxin, muramic acid, and hypomethylated CpG motifs.294 It is evident from epidemiological studies that strong associations exist between certain TLR gene polymorphisms and increased prevalence of general atopy or specific allergic 294; 295 diseases. It is well established that allergies demonstrate skewed TH2 responses. Hygienic environments typical of western lifestyles deprive the immune system of stimuli required to 294; 295 boost TH1 responses, thus leading to a surge in the prevalence of TH2 mediated allergies.

GUT BARRIER DYSFUNCTION & FOOD ALLERGY: CAUSE OR CONSEQUENCE The major antigenic challenge facing the gut derives from the ingested food antigens. There is evidence that small quantities of intact proteins do cross the gastrointestinal in animals and humans, and that this is a physiologically normal process required for antigen sampling by the gut immune system.43 Under normal circumstances, oral administration of protein antigens induces systemic unresponsiveness (OT).34;43 However it appears that a small number of people absorbing these normal amounts react idiosyncratically; also some individuals may absorb excessive amounts of dietary antigens leading to clinically significant allergic reactions. Gut barrier dysfunction is a common feature of digestive and cutaneous manifestations of food allergy.14 There are at least two pre-requisites for the development of food allergy. First, intra- luminal antigens must penetrate the intestine's mucosal barrier. Second, the absorbed antigens must cause harmful immune responses. Increased intestinal permeability and enhanced antigen transfer has been reported in children with cow's milk allergy caused by the hypersensitivity reaction.34 Particularly in infants with atopic eczema and cow's milk allergy, more extensive barrier dysfunction can ensue, refractory to an elimination diet. Resistance to digestion in the gastrointestinal tract is considered to be one of the crucial factors that may contribute to the allergenic potential of food proteins by permitting sufficient

intact (or large fragment(s) or peptides) proteins to be taken up by the gut and consequently sensitize the mucosal immune system. The capacity of a given food antigen to resist enzymatic hydrolysis is highly variable and often determines its allergenicity.20 The mucosal and systemic immune responses are preferentially stimulated by particulate antigens, taken up through M cells of the FAE, while soluble dietary antigens transported by enterocytes are likely to induce tolerance.22 Abnormalities in intestinal permeability are not the hallmarks of food allergy, since they are reported in many other inflammatory diseases (celiac disease, cystic fibrosis, Crohn's disease, acute and chronic diarrhea).23 Although allergic inflammation leads to epithelial dysfunction with altered antigen handling, most studies have concluded that constitutive modification of epithelial permeability is not involved in the development of food allergy.14 Indeed, increased epithelial permeability could be more the consequence, rather than the cause, of food sensitization, although it participates in a self-perpetuating cycle that maintains allergic inflammation. Although constitutive abnormalities in intestinal permeability are not recorded in atopic patients, environmental events such as infection or stress may have consequences on the susceptibility to allergic diseases.

DIGESTIBILITY – A CRITERION FOR ALLERGENICITY In order for a food protein to retain its allergenicity, the epitopes on proteins to which the human IgE antibody is directed (i.e., the structural and linear epitopes) must survive in vivo digestion. A number of food allergens are known to be stable toward the proteolytic enzymes encountered in the GI tract in vivo49 and also to in vitro proteolysis when tested under simulated gastric fluid (SGF) conditions.50 Stability to digestion and processing has thus been considered as one of the properties shared by food allergens.49-51 It has been suggested that linear epitopes in comparison to conformational epitopes may play a more crucial in persistent food allergy. This is evident in cow’s milk allergic children with persistent symptoms; significantly higher ratio of IgE antibodies observed to linear than to conformational epitopes.21 However the in vitro digestive stability of most allergenic proteins has not been determined and less is known about the relative stability in comparison with non-allergenic proteins. Yet such information is crucial to validate the use of digestive stability as a predictive tool for protein allergenicity assessment. In an in vitro study SGF, in general the allergens and

lectins were resistant to pepsin digestion (stable in SGF for the full 60 min of reaction) whereas the other non-allergenic proteins were more rapidly and completely digested (within 30s).50 Allergenic proteins from cow's milk as well as chicken egg white ovomucoid (OVM), presented different susceptibility to the sequential hydrolysis by pepsin and trypsin.296 In a more recent study, immunoblotting profiles of SGF-digested pecan proteins, displayed several low molecular mass (16-20 and 28 kDa) antigenic peptides that persisted throughout the 4 hours of digestion.52 In this study pecan allergic IgE antibodies from patient sera could detect antigenic peptides even after 4 h of digestion, thereby suggesting that such stable peptides that resist gastric digestion may therefore be able to trigger allergenic response. Certain groups of proteins, such as storage proteins or structural proteins are inherently more stable to proteolysis in cellular environments than other types of proteins, such as enzymes. It is not clear whether the higher stability reported among those food allergens occurred because of this inherent differential stability. However, the stability to digestion as a significant and valid parameter that distinguishes food allergens from non-allergens has been under scrutiny. Several studies examining the stability of a variety of proteins in pepsin digestion assays report that distinctions between allergens and non-allergenic proteins were less clear.51;54;55 The significance of simulated digestion studies has therefore been under skepticism due to the observation that resistance to digestion is not a defining characteristic of food allergens. Some of the perceived limitations of in vitro digestion studies include use of extreme pepsin/protein ratios (such as 12.5:1 w/w), which may not accurately represent the in vivo ratio in the human GI tract; use of an insufficient range of conditions to accurately represent the possible in vivo digestion conditions. In addition, the purity of pepsin, target protein and structural conformation and changes in pH used in an assay may affect the relative digestibility measured. Furthermore, failure to probe the digested proteins to reveal the presence or absence of relevant IgE-binding epitopes in the protein digests. Despite such drawbacks, in vitro proteolysis of a targeted protein can furnish useful preliminary information in the assessment of stability potential of food allergens. Pepsin- resistant fragments of ovomucoid bind IgE and correlates well with the pattern of persistent allergy to egg white proteins. Other studies have also tested the IgE binding capacity of the residual fragments following pepsin digestion of allergens and found that they bind IgE, or that

they contain major IgE binding domains or epitopes.52;57;58 The fact that pepsin-resistant proteins or fragments may have the potential to bind IgE, suggests that linear epitopes may play a more important role.

PLANT FOOD ALLERGENS Considering the diversity of the human diet, only few foods account for the majority of food allergies.2 It is worth noting that plant food allergens also fall into rather few protein families.19 Several studies are focused on answering the crucial question -- what establishes a protein as an allergen. Analysis of a variety of allergenic foods has resulted in the identification of certain physicochemical properties shared by many but not all food allergens that enhance stability and promote allergenicity.12;19;20;59 In addition, the levels, route of exposure, and certain structural and functional properties of dietary proteins confer them the ability to resist proteolytic digestion and increase gut mucosal uptake, thus affecting the GI tract immune milieu.6;20;60 The vast majority of plant food allergens that sensitize via the GI belong to three major groups of plant proteins: the prolamins (2S albumins, nonspecific lipid transfer proteins (nsLTPs), α-amylase/trypsin inhibitors), cupins (7S and 11S seed storage proteins) and cysteine proteases.19

MOLECULAR PROPERTIES OF PROTEINS CONTRIBUTING TO ALLERGENICITY

Abundance Seed storage proteins account for about 10-50% of the total protein in mature cereal grains, legumes and nuts, forming a major source of dietary protein, and are involved in allergic reactions.39 However, the enzyme ribulose-1, 5-bisphosphate carboxylase that is present in all plants in very large quantities accounts for 30-40% of total leaf protein, has never been reported as allergenic. In contrast, nsLTPs are potent allergens but not very abundant. Thus, the amount of protein alone does not explain its allergenicity. While abundance is an important factor, protein stability might be more crucial in conferring allergenic property.

Protein stability

The ability of a protein to resist pH conditions and proteolytic degradation by the GI tract enzymes is termed as stability. There are some indications that thermostable proteins might have a greater propensity to be allergenic. One of the structural features clearly related to stability is the presence of disulfide bonds. Both the intrachain and interchain disulfide bonds constrain the 3-D scaffold, as a result perturbation of this structure by heat or chemical means is limited and frequently reversible. There are some notable food allergens that are highly disulfide bonded, including members of the prolamin superfamily - nsLTP, 2S albumin, and trypsin and α-amylase inhibitors. The 2S albumins are compact molecules because of the presence of 4 conserved disulfide bonds which exhibit high thermostability and resistance to extreme pH and enzymatic proteolysis, as indicated by the allergenic 2S albumin from mustard seeds 61 and Brazil nut.62 Similarly, the tightly packed three-dimensional structure accounts for the extreme resistance of nsLTP to both thermal denaturation and proteolysis.63-66 The allergenic corn nsLTP maintained its IgE-binding capacity after cooking at 100oC.297 Similarly, heating resistance has been demonstrated for other LTPs in foods, such as the LTP in barley, which survives malting and brewing processes, and LTP from peach nectar.67 The structural features, such as extensive disulfide bonding, that increase protein stability, such as compactness with few mobile loops, will also render them resistance to proteolytic digestion by pepsin.19 However, absence of disulfide bonds does not indicate a lack of stability because the cupin barrel, for example, is highly stable and possesses just one disulfide bond.68

Aggregation, Glycosylation and Glycation The globulin seed storage proteins have a propensity to form large structures in the form of trimers and hexamers, which are held together by noncovalent interactions. Both 11S and 7S globulins, as with other members of the cupin superfamily, are thermostable.69 Peanuts and tree nuts are often subjected to thermal processing at low water levels, such as roasting. This affects protein stability because protein denaturation requires the presence of water, with proteins becoming more thermostable in low-water systems.69 There is evidence that N-glycosylation increases the stability of 7S globulin of pea and peanut to resistance to enzymatic and thermal denaturation.70 Germin like proteins have been described as allergens in pepper, orange, and tangerine, apparently owing much of their IgE-binding capacity to their glycosylation.298

Additionally, glycation reactions might be responsible for the apparent increase in allergenic activity of peanuts after processes such as curing and roasting.71;72;72;73 It has been proposed that thermal treatments might result in disulfide exchange and modifications of other amino acids, further cross-linking the proteins. The presence of large aggregated protein structures in the consumed food might potentiate their allergenic properties by containing repeating units and multiple IgE epitopes, and might trigger histamine release more effectively than soluble monomeric forms.73 The result of these modifications causes stabilizing effect on protein structure and influences the biologic properties of allergens.19;298

Ligand binding A number of food allergens are able to bind ligands, ranging from metal ions to lipids.298 Ligand binding can have the overall effect of reducing mobility on the polypeptide backbone, increasing both thermal stability and resistance to proteolysis. Proteins such as the lipocalins and nsLTPs, which possess a lipid-binding pocket, show increased stability when the pocket is occupied.19;74 Thus the thermostability of α- and β-lactoglobulin increases on lipid binding.19;75 The nsLTPs have a been identified as allergens from fruits, nuts, seeds, and vegetables, e.g., peach Pru p 3, apple Mal d 3 and apricot Pru ar 3, sweet cherry Pru av 3, plum Pru d 3, hazelnut Cor a 8, corn Zea m 14, asparagus Aspa o 1, and grape Vit v 1, wheat LTP. Their common structural features are designated the basis of their allergenic clinical cross-reactivity.76;77;298

Enzymatic properties It has been demonstrated that certain biochemical functions, such as enzymatic activities may influence a protein to become allergenic.12 The families of proteases reported to contain allergenic proteins are, the papain-like cysteine proteases, and the alkaline serine proteases.94 The allergenic papain-like plant enzymes include papain from papaya, ficin from fig, bromelain from pineapple, and actinidin from kiwi.18 A major allergen from soybean seed storage vacuoles, designated as P34/Gly m Bd 30K, shows sequence similarity to papain-like proteases.78 Cucumisin (Cuc m 1) from melon, bound IgE from more than half of the patients with melon allergy and is the sole allergenic member of the subtilisin family of serine proteases described so far.94;298

Proteins of the plant defense system pathogenesis-related (PR) proteins Plants protect themselves against pathogen attack as well as against phytophagous insects by different strategies. Besides, establishing a physical barrier by strengthening their cell walls, plants produce antibiotic compounds called phytoalexins and accelerate cell death to suppress the spread of infectious pathogens. Furthermore, a number of proteins are induced by various types of pathogens (viruses, bacteria and fungi) or adverse environmental factors and are designated PR proteins.79 The common biochemical features of PR proteins are - low molecular weight size, stability at low pH, and resistance to proteases.18 They represent a collection of 14 unrelated protein families that function as part of the plant defense system.79;80 Plant chitinases (PR-3) are endochitinases that hydrolyze chitin. Chitin is a major structural component of the exoskeleton of insects and the cell walls of most fungi, and chitinases therefore can function as part of the plant’s defense system. Allergenic class I chitinases have been identified in latex, avocado, banana, and chestnut.81;94 The family of PR-5 proteins comprises unique proteins with diverse functions including antifungal activity. Members of this family identified as major allergens include Mal d 2, an important allergenic TLP of apple, and cherry Pru av 2.18 Bet v 1 is a member of the PR-10 family, and its homologs form a ubiquitous group of proteins involved in the oral allergy syndrome.19;94,298 The majority of Bet v 1–homologous food allergens have been found in the fruits of Rosaceae species (apple, pear, cherry, apricot) or vegetables of Apiaceae species (celery, carrot, parsley).19;201 LTPs belonging to the PR-14 family of proteins are involved in plant defense, having potent antifungal and antibacterial activities. They are the most important allergens of the Prunoideae, such as peach, apricot, plum, and cherry.82 In fruits and vegetables with high water content, such as cherries, papayas, bananas, or avocados, that are prone to fungal attack, the chitinases, proteases, and antifungal proteins are the predominant allergens. The allergens in hard and dry seeds, legumes, and nuts fall into two classes: the seed storage proteins and the enzyme inhibitors. They prevent the endosperm from being digested by insects or fungi. Thus the molecular characteristics of plant allergens may indicate that the allergenicity of at least some of these allergens depends on their biochemical functions.

CHAPTER 2

CLONING AND CHARACTERIZATION OF PROFILIN (PRU DU 4), A CROSS- REACTIVE ALMOND (PRUNUS DULCIS) ALLERGEN

Introduction

Tree nuts are one of the 'big 8' allergenic foods299 and include cashew nut, almond, pistachio, walnut, hazelnut, and Brazil nut.83 Allergic reactions to tree nuts range from mild oral symptoms, often seen in the pollen-food syndrome, to life-threatening anaphylaxis.84 The prevalence of allergy to tree nuts in the US is estimated at 0.2% in young children and 0.5% in adults.2 A variety of allergens from cashew nut, walnut, hazelnut, and Brazil nut have been isolated, cloned, and characterized.83 Thus far, several native almond allergens have been identified and characterized to varying degrees, including members of the legumin85-87, 86;88vicilin89, and 2S albumin89 families, but none have been cloned and tested for reactivity with almond-allergic sera. Two well-studied low molecular weight tree nut allergens are 2S albumin and lipid transfer protein80;83;90 which have molecular weights in the range of 12 to 14 kDa and 9-10 kDa, respectively. Profilin is another small (12-16 kDa), ubiquitous eukaryotic protein that is recognized as allergenic in pollens, vegetables, and fruits. Profilin can also be allergenic in seeds, nuts, and latex, often as a consequence of cross-reactivity with IgE directed to pollen profilin.18;80;90 Profilins are best known for binding to monomeric actin (G-actin) and regulating the polymerization of actin into filaments.91 Sequence homology is high (70-85%) among plants, and, in some plants, multiple isoforms have been identified.92 The emerging evidence suggests that there is some degree of tissue specificity to the expression of profilin isoforms, which is reflected in their pattern of sequence homology.91;91-93;93;94;94 Thus the fruit and seed profilin sequences cluster together whereas the pollen isoforms comprise several disparate clusters.94 Of the various plant food allergenic protein families, profilins rank third behind the prolamin and Bet v 1 families with respect to the number of allergens thus far identified.95 Many plant profilins exhibit significant cross-reactivity, apparently due to conserved amino acid sequences and shared IgE-reactive epitopes prompting the designation of profilins as panallergens.96-100 Evidence for both linear and conformational epitopes has been reported.101-103

102;103 The co-sensitization of patients to hazelnut pollen and nut profilins was first verified with the aid of cross-reactive rabbit anti-celery profilin antibodies.96 Thus it is not surprising that profilin from one plant source can cross-sensitize an individual to the tissues of multiple plant species and pollen profilins can sensitize individuals to food profilins.94;104 94 Here we purify native almond profilin, express a recombinant form of the profilin, demonstrate their reactivity with serum IgE from almond-allergic patients, and show cross- reactivity with ryegrass pollen profilin (RGPP). These studies have been published in JACI in 2006 (Tawde 2006).105

Methods

Human Sera Blood samples were drawn, after informed consent, from patients with self-reported allergic reactions to almonds. The reactions were not confirmed by oral challenges. Eleven of the 18 patients reported potentially life-threatening reactions to almonds with symptoms of bronchospasm, hypotension, or throat swelling/laryngoedema. Seven patients reported more mild reactions, particularly oral angioedema or pruritus to raw, but not cooked almonds, and three of the seven reported reactions to multiple fresh fruits. The latter seven patients were included, even though food challenges were not performed, because of the known association between IgE anti-profilin and the pollen-food syndrome.106 The three patients with self-reported allergy to multiple fresh fruits also reported pollinosis, as did 15 of the 18 overall. The sera were stored at -70°C. The study was approved by the institutional review board of the University of California, Davis. The presence of almond-reactive IgE was confirmed by Phadia CAP FEIA assay (Phadia, Inc., Uppsala, Sweden), modified RAST, or immunoblot. The patient clinical characteristics are shown in Table 2.1.

Table 2.1. Clinical characteristics of almond-allergic subjects

Serum Sex/Age Age at Other Reaction Other food In vitro Positive Positive No. (years) onset atopy* to allergy IgE to dot blot dot blot of tree almond** almond*** to to nut kIU/L almond profilin allergy 2 F/55 53 AD, U Peanut, 1.84 yes yes AR, walnut As (severe allergy, unlike to almond) 5 F/53 1 AD, AE, walnuts, 4.72 yes no AR, oral/throat pecans, As pruritus cashews 7 F/25 child AD, Wheezing, Peanuts, Class 2 yes no AR AE Walnut, other tree nuts 8 F/38 1 AD, Wheeze, Peanuts, Class 5 yes yes AR, V, AE walnut, As cashew 10 F/31 2 AR, Wheeze, peanut, Class 2 yes no As stridor walnut 11 M/50 1 AR, AE, throat Multiple tree 0.64 yes yes As swelling, V nuts, mustard 12 F/26 3 AR, Wheeze, Pistachio, <0.35 yes no As AE, throat cashew, swelling, hazelnut V/D, U 18 F/62 1 AR Wheeze, Peanut, 1.15 yes yes U, oral AE walnut, cashew 20 F/48 4 AD, Wheeze, Peanut, <0.35 yes no AR, AE, throat walnut, kIU/L, As swelling, pecan, Class 4 V/D, U, hazelnut, modified LOC cashew RAST 27 M/38 2 AD, Stridor, Peanut, other Class 2 yes no AR, AE, U tree nuts As 29 F47 3 AD, Wheeze, Sesame, <0.35 yes no AR, stridor, walnut As V/D, U, AE 32 M/38 1 As Wheeze, Walnut, <0.35 yes No AE, throat pecan, swelling, cashew V, U 41 F/45 1 AD Oral AE, Walnut, <0.35 yes yes oral pecan, pruritus sesame

Table 2.1. - continued Subjects Sex/Age Age at Other Reaction Other food In vitro Positive Positive with a (years) onset atopy* to allergy IgE to dot blot dot blot fruit of tree almond** almond*** to to allergy nut kIU/L almond profilin allergy 34 M/33 child AR Oral Multiple <0.35 yes yes pruritus, D fresh fruits, to raw cooked are almond OK only, cooked is OK 37 M/57 1 AD, V/D, U Peanut, <0.35 yes yes AR walnut, avocado 39 F/52 1 AD, AE, throat Peanut, other 0.49 yes no As swelling tree nuts, raw carrots, avocado, banana 42 F/16 10 AR Oral Walnut, <0.35 yes yes pruritus to pecan, raw melon, almond banana only, cooked is OK 43 F/48 30 AR Oral AE, Raw <0.35 , yes no oral walnuts, positive pruritus to pecans, prick/prick raw pistachios, to raw almond cherries, foods only, peaches, cooked is apricots, OK avocado

* AD = atopic dermatitis, AR = allergic rhinitis (pollinosis), As = asthma ** AE = angioedema, V = vomiting, D = diarrhea, U = urticaria *** Test performed was the Pharmacia ImmunoCAP FEIA, but was reported in Classes that were related to percent of the Pharmacia reference value prior to the hospital’s switch to the IU reporting system. Class 0: <60% of reference, Class 1: 70-110% of the reference, Class 2: 110- 220% of the reference, Class 3: 220-600% of the reference, Class 4: 600-2000% of the reference, Class 5: 2000-6000% of the reference.

Almond extract and native profilin Defatted Nonpareil almond flour, and an aqueous protein extract thereof, were prepared as previously described.107 Almond profilin was purified by affinity chromatography on poly-

(L-proline)-Sepharose 4B (PLP-Sepharose).108-110 Briefly, 100 mg of poly(L-proline) of mol. wt. 1000-10,000 (Sigma-Aldrich., St. Louis, MO) was coupled to 15 mL CNBr-activated Sepharose 4B.28-30 The PLP-Sepharose 4B was packed into a glass column (1.6 x 7.5 cm), and almond extract (from 20 g defatted almond flour) was passed through the column at 4°C. The column was first eluted using extraction buffer containing 3 M urea to remove profilactin (profilin-actin) complexes, followed by elution of profilin with extraction buffer containing 7 M urea. The 7 M urea fraction was dialyzed against distilled water at 4°C, and concentrated using Centricon-3 (mol. wt. cutoff, 3 kDa) concentrators. Protein purity was verified by SDS-PAGE (data not shown). Rye grass pollen profilin was similarly prepared from 4.0 g of rye grass pollen.

Almond Nut cDNA Expression Library Construction Almond cDNA library construction was performed by Dr. Fang Wang in our lab, using mRNA derived from immature almond kernels as previously described in detail for cashew cDNA library generation.111;112 Briefly, nuts were chopped, frozen in liquid nitrogen, and ground with a mortar and pestle. Total RNA was extracted in TRIzol (GIBCO BRL Life Technologies Inc., Rockville, MD) as previously described 111;112 and mRNA was isolated using a PolyATtract kit (Promega Co., Madison, WI) according to the manufacturer’s instructions. Synthesis and cloning of cDNA was performed using the Uni-ZAP XR Gigapack Cloning Kit (Stratagene Inc., Cedar Creek, TX) following the manufacturer’s instructions. The double- stranded cDNAs with EcoRI (using a 5’ end adapter) and XhoI (using a 3’ end PCR primer) cohesive ends were cloned into the lambda Uni-ZAP XR expression vector. The library was amplified on E. coli strain XL1-Blue.

Expression Library Screening and IgE Immunopositive Clone(s) Identification After amplification the almond cDNA library was screened as previously described.111;112 Briefly, the transfected XL1-Blue cells were incubated with E. coli (strain Y1090) for 15 minutes (min) at 37° C and then plated in a layer of Luria-Bertani (LB) soft agar for approximately 5 hours (hr) at 42° C at a density of approximately 20,000 plaques per plate. A nitrocellulose filter soaked in 10 mmol/L isopropyl thiogalactose (IPTG) was dried, placed on the agar plate, and further incubated 10 to 14 hr at 37° C. The filter was placed in blocking solution (4% nonfat milk in phosphate-buffered saline, pH 7.4) for 1 hr, and primary pooled sera from patients that

displayed IgE reactivity to multiple bands on whole almond extract immunoblots were diluted 1:5 in 4% nonfat milk and incubates at 4° C overnight with the filter. 125I labeled anti-human IgE (Hycor Biomedical, Garden Grove, CA) was added after washing and allowed to incubate overnight. The filter was exposed for 96 hr to Kodak XRT film and developed. The immunopositive clones were picked, plaque-purified, and stored in SM buffer supplemented with 2% chloroform at 4oC.

Sequencing, Amplifying, and Analysis of Selected Genes Inserts from the selected phage clones were amplified with M13 forward and reverse primers by polymerase chain reaction (PCR) and ; both strands of the PCR products were then sequenced on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) using capillary electrophoresis and Version 2 Big Dye Terminators as described by the manufacturer.111;112 Similarity searches and alignments of deduced amino acid sequences were performed on Genetics Computer Group software using the BLAST 2.0 program (www.ncbi.nlm.nih.gov/BLAST/). Leader peptide prediction was performed using the SignalP V1.1 World Wide Web Prediction Server (www.cbs.dtu.dk/services/SignalP/).

Recombinant almond profilin production105 As described in detail (for cashew nut),111 the cDNA coding sequences were ligated into a maltose-binding protein (MBP) fusion expression vector pMAL-c2 (New England BioLabs Inc., Beverly, MA), into which a thrombin cleavage site had been engineered. The cDNA insert was produced by PCR amplification of the portion of the sequence extending from the presumed first codon (following the leader peptide) through to the last codon prior to the stop codon using the primers: 5’gtcctctagaatgtcgtggcagcagtacg3’ (forward) and 5’gcttctgcagttacagaccctgctcgataag3’ (reverse). Cut products were then purified using a QIAquick Gel Extraction Kit (Qiagen) and ligated with T4 DNA Ligase H.C. (Invitrogen) to their respective sites of the RE cut maltose binding protein (MBP) fusion expression vector pMAL-c2 (New England BioLabs Inc., Beverly, MA), according to the manufacturers instructions. The pMAL-c2 expression vector (pMAL-c2-His) used for all experiments was previously modified to contain a His-tag, thrombin cleavage site, and modified restriction sites. For expression of the cDNA/pMAL-c2 plasmids, competent E. coli DH5-α cells were

transformed with complete ligation mixtures. Positive clones were selected via PCR using gene specific primers (Table 2.1) and used to innoculate 5 ml LB overnight cultures containing 100 μg/ml ampicillin. Plasmid preps were performed using 3 ml of the overnight culture and the QIAprep Spin Miniprep Kit (Qiagen) following the manufacturers instructions. Insert positive plasmids were subsequently used to transform competent BL21 (DE3) cells (Novagen Inc, Madison, WI). Transformed cells were plated overnight and positive clones were identified via

PCR as described above. Single colonies were grown at 37°C to an OD600 of 0.5-0.8, at which point protein synthesis was induced with the addition of isopropyl-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM for 3-4 hr. The cells were harvested by centrifugation at 4000g for 20 min, resuspended in amylose resin column buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.4) supplemented with 10 mM β-mercaptoethanol (β−ME) and 1 mM EDTA and lysed using a microfluidizer. The whole lysate was centrifuged as described above and the fusion protein containing supernatant diluted 1:4 in column buffer passed over an amylose affinity column, (New England Biolabs) and eluted with column buffer containing 10 mM maltose. Pure recombinant fusion proteins were dialyzed overnight vs. borate saline buffer (BSB; 100 mM boric acid, 25 mM sodium borate, 75 mM NaCl, pH 8.2), while changing the dialysis buffer every 4 hr, concentrated using Centricon Centrifugal Filter Devices (YM-30, Millipore Corporations, Bedford, MA) and either stored (briefly) at 4°C until use or frozen at - 80°C. The fusion protein was cleaved with thrombin (Sigma-Aldrich) at 1 mg of fusion protein per unit of thrombin. Cleaved and purified rPru du 4 was used in the dot-blot and ELISA assays.

One-dimensional electrophoresis (1-DE) (SDS-PAGE) and immunoblotting Aqueous whole extract samples (10-15 μg/4 mm well width) were boiled for 5 min in reducing sample buffer (60 mM Tris-HCl, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 100 mM dithiothreitol (DTT), pH 6.8) and loaded onto Tris-HCl mini-gels (7X10 mm), 12% monomer acrylamide concentration resolving gel and 4% stacking gel) made according to Laemmli and electrophoresis carried out at 125 V using a Mini Protean 3 electrophoresis unit (Bio-Rad).105;111 The gels were either stained with colloidal Coomassie stain (Sigma), or transferred to transferred onto 0.2 m Immobilon-PSQ nitrocellulose (NC) transfer membrane (Millipore Corp. Billerica, MA) using a Mini Trans-Blot Transfer Cell (BioRad Laboratories, Inc., Hercules, CA).105 for immunoblotting assays as described below.

Direct and Inhibition Immunoblotting

Nitrocellulose membranes containing blotted almond extract proteins were cut into 3-4 mm wide strips and blocked for 1 hr at room temperature in 20 mM phosphate buffered saline (PBS; pH 7.3) containing 5% nonfat dry milk and 0.2% Triton X-100 (TX-100). Diluted human almond allergic sera (1:5 in the blocking buffer or 1:20 for highly reactive sera) were added to the strips and incubated overnight at 4°C. Additional strips were incubated with non-allergic control sera in blocking buffer to check for non-specific IgE or secondary antibody binding. The strips were then washed for 20 min three times in PBS containing 0.01% TX-100 and incubated overnight at 4°C with horseradish peroxidase (HRP) conjugated mouse anti-human IgE ((Invitrogen/Zymed Laboratories Inc, Carlsbad, CA), diluted 1:15,000 in blocking buffer. Washing was repeated as above and the IgE-reactive bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology Inc. Rockford, IL) and subsequent exposure to Kodak X-OMAT X-ray film for 10 sec to 2 min. To identify the molecular weight of the native allergenic proteins in whole nut extracts, inhibition experiments were carried out in which 40 μg of purified native almond profilin were pre-incubated with three human almond allergic serum (pt#11, #41, and #34) for 3 hr at room temperature prior to probing whole almond extract blotted strips, as described above.

Two dimensional gel electrophoresis (2-DE), immunoblotting, and N-terminal sequencing

Two-dimensional gel electrophoresis was performed using ZOOM IPGRunner System (Invitrogen Life Technologies, Carlsbad, CA). The almond extract samples (300 μg in 10 μl) were diluted in 155 μl of sample rehydration buffer (8M urea, 2% CHAPS, 0.5% (v/v) ZOOM Carrier Ampholytes (pH 3-10) non-linear, 0.002% bromophenol blue, 20 mM DTT) and were then applied to immobilized pH gradient gel strip (pH 3-10 NL, Invitrogen Life Technologies) incubated o/n at RT. Isoelectric focusing (IEF) was performed using the ZOOM IPGRunner Mini-cell (Invitrogen Life Technologies) as described by the manufacturer. The second dimension electrophoresis was performed using Novex 4 – 20%Tris-Glycine gels (Invitrogen) in an Xcell SureLock Mini-Cell (Invitrogen Life Technologies) as described by the manufacturer. The 2-DE gel was then transferred to 0.2 μM Immobilon-PSQ membrane (Millipore Inc.) and the profilin isoforms detected using rabbit anti-maize profilin antiserum (anti-ZM Pro5, generously provided by Dr. Chris J. Staiger, Purdue University, W. Lafayette,

IN), using the Enhanced Chemiluminescence (ECL) Plus kit (GE Healthcare, Piscataway, NJ) and subsequent exposure to Biomax X-ray film for 10 sec to 2 min (Kodak, Rochester, NY).105 Additionally the gel was either stained with colloidal Coomassie stain (Sigma-Aldrich) or transferred to ProBlot PVDF membrane (Applied Biosystems, Foster City, CA) for N-terminal sequence analysis using a ABI 477A Protein Sequencing System (Applied Biosystems).

Dot-Blot Analysis Defined quantities of almond extract, and purified native and recombinant profilin were applied to NC membrane using a 96-well Bio-Dot microfiltration apparatus (Bio-Rad Laboratories). Bovine serum albumin (BSA) served as a negative control. The membranes were then cut into strips, each of which contained the desired sets of protein dots, and probed as described above. For inhibition dot blots the inhibitor (native Pru du 4 or RGPP) concentration used was 10 μg/ml.

ELISA Almond extract (40 μg/ml), rPru du 4 (20 μg/ml), and native almond profilin (20 μg/ml) diluted in coating buffer (0.1 M carbonate–bicarbonate buffer, pH 9.6) were coated (50 μl/well) onto the wells of 96-microwell round bottom polyvinyl microtiter ELISA plates (Seracluster “U” Vinyl, no. 2797, Costar, Cambridge, MA) at 4oC overnight. Plates were washed three times between steps with PBS/0.1% Tween-20 (PBS–T). Blocking was carried out using 5% nonfat dry milk in PBS–T at 37°C for 1 h. Sera from almond-allergic patients (listed in Table 2.1) (diluted 1:10, 50 μL/well) were added and incubated o/n at 4°C. After washing, bound IgE was reacted with HRP-conjugated mouse anti-human IgE (Zymed Laboratories, Inc.) at a dilution of 1:1000 and incubated for 1 h at 37°C. IgE reactivity was detected by colorimetric reaction using o-phenylenediamine (OPD, Zymed Laboratories Inc.) and H2O2 as substrate. Optical density (OD) was measured in a KC4 v2.5 ELISA reader (Bio-Tek Instruments Inc, Winooski, VT) at 495 nm.

Results cDNA library screening and gene characterization

A screening of the cDNA library with a pool of human almond-allergic sera (patient # 7, 8, 34) yielded two clones (a 924 bp fragment, clone 1 (AY081850), and a 742 bp fragment, clone 2 (AY081852)) which, upon translation, had identical 131 amino acid coding sequences but differed by two silent mutations (Α Τ and A G at positions 310 and 322, respectively (Figure 2.1). When analyzed by a GenBank BLAST search, the sequence was homologous with plant profilins. Following the guidelines of the International Union of Immunological Societies’ Allergen Nomenclature Subcommittee 113 the allergen was designated Pru du 4. The almond profilin polypeptide had a calculated molecular mass of 14,061 Da, a pI of 4.67, and and A280nm at 10 mg/mL of 11.8.

1 CTCCATCCATCTTCGCACCTTCCATTTTCGTTTCAGCTCTAGAACCTTCTC 52 CTCTCTCCCCAAAAAGCACTCACCGCCAGAGCAGAAGATCAGCCAAAGCAA 103 GCAAGAAGAAGAAGAAATCAGAAGCAACGAAACGATGTCGTGGCAGCAGTA 1 M S W Q Q Y 154 CGTCGATGACCACTTGATGTGCGATATTGACGGCAACCGCCTCACCGCGGC 7 V D D H L M C D I D G N R L T A A 205 GGCAATCCTCGGCCAAGACGGCAGCGTTTGGTCTCAGAGCGCCACCTTTCC 24 A I L G Q D G S V W S Q S A T F P 256 TGCGTTTAAGCCTGAAGAGATAGCTGCCATTCTGAAAGATTTCGACCAACC 41 A F K P E E I A A I L K D F D Q P 307 CGGAACGCTTGCCCCAACTGGGTTATTCCTTGGTGGGACAAAGTATATGGT 58 G T L A P T G L F L G G T K Y M V 358 GATCCAAGGTGAAGCCGGTGCTGTCATTCGAGGAAAGAAGGGCTCTGGTGG 75 I Q G E A G A V I R G K K G S G G 409 CATTACTGTTAAGAAGACCAATCAGGCCCTCATCATTGGTATATATGATGA 92 I T V K K T N Q A L I I G I Y D E 460 GCCTTTGACTCCAGGGCAGTGCAACATGATTGTCGAAAGGCTTGGCGATTA 109 P L T P G Q C N M I V E R L G D Y 511 CCTTATCGAGCAGGGTCTGTAATTGCTCCGAGTGACTATTTTATTTGAAGT 126 L I E Q G L * 562 TATGGTGAACTTGGTTTGAAAACAAAGTTGTTGCTTGCTGCTCGGGATGTG 613 ATGGGTATATGTATGAACCATTTATCTTTGCATTCTGGGACCTCTGGATAT 664 GGATTGTCTTTGTGAATGATAGTGGTTTAATTTTACAATTATTTTTGCTTA 715 AGTTATTTTCGTCCTTTTGCTTGGAAAAGCGTATGTAATTGGAGTTGTCAT 766 CGCTGGAGTGCCATGAATGAAACGGGTTATGTACTAAGTAGTTTTGTTCTA 817 TCAGAAAACGAGTTGGGTGAAAGACATTAAACATTTTTGCTTACCTTCCCT 868 TTCATTGAGAAAGCTTGTGGATTTCCGCAATTTAACTGAAAAA

Figure 2.1. Nucleotide and deduced amino acid sequence (bold) of rPru du 4, clone 1 (AY081850), showing start (yellow) and stop (red) codons. Also indicated are 2 codons (underlined) that differ (A310/T and A322/G, variant nucleotides highlighted in purple) in rPru du 4, clone 2 (AY081852, not shown). The amino acids encoded by the codons with these silent mutations are highlighted in green.105

Protein sequence homology comparisons Table 2.2 in the lists the proteins having the highest degrees of identity and similarity to almond profilin and represent products from a diversity of tree and plant species. Those in the same family, such as peach and sweet cherry, have very high degrees of similarity and identity (98-99%). Even those more distantly related species, such as muskmelon and orange, displayed identity and similarity of ≥ 97 and 98%, respectively, to almond profilin. Many other plant profilins displayed ≥ 80% similarity and ≥ 90% identity. Almost all of the plant profilins listed in Table 2.2) have been previously identified as allergens.

Table 2.2. Profilins displaying sequence homology to almond profilin105 Organism (common name) Accession No. Identity Similarity Allergen % % designation Prunus persica (peach) CAD37201 99 99 Pru p 4.01 Prunus avium (sweet cherry) AAD29411 98 98 Pru av 4 Cucumis melo (muskmelon) CAD 92666 97 98 Cuc m 2 Citrus sinensis (orange) CAI23765 96 97 Cit s 2 Malus x domestica (apple) AAD29414 93 98 Mal d 4 Capsicum annuum (bell pepper) CAD10376 87 94 Cap a 2 Lycopersicon esculentum (tomato) AAL 29690 87 93 Lyc e 1 Pyrus communis (pear) AAD29410 85 93 Pru c 4 Hevea brasilienis (rubber tree) AAF34341 85 94 Hev b 8.0201 Cynodon dactylon (Bermuda grass) CAA69670 80 93 Cyn d 12 Glycine max (soybean) CAA11756 80 90 Gly m 3 Corylus avellana (hazelnut) AAK01236 79 91 Cor a 2 Olea europaea (olive) CAA73039 79 90 Ole e 2 Betula pendula (birch) AAA16522 75 88 Bet v 2

Production of recombinant profilin For immunological characterization, we cloned and expressed clone 1 almond profilin in E. coli. The resulting ~55 kDa almond profilin fusion protein was affinity purified and digested with thrombin to yield a polypeptide of ~14 kDa (i.e., the expected molecular mass of profilin) as well as the 41-kDa MBP polypeptide.

Purification of native profilin Although profilin is ubiquitous, it is present in minute amounts in plant tissues. Nevertheless, we attempted purification, based on the natural affinity of profilin for polyproline,

for comparative studies with the recombinant almond profilin. Approximately 25 µg of profilin was obtained per g of defatted almond flour (excluding profilactin complexes). In contrast, a yield of ~2.5 - 3 mg protein of cleaved and purified recombinant profilin was obtained per L of culture medium. The yield of ryegrass pollen profilin was ~ 40 µg per g of pollen.

Immunological analysis of almond profilin Because of the well-known property of cross-reactivity among plant profilins, we attempted to identify the almond profilin using rabbit antibody (Ab) to maize profilin (ZMPro5). Both cleaved and uncleaved recombinant molecules and total almond extract were reactive (Fig 2.1A). To identify the corresponding peptide in immunoblot, three almond-allergic patients’ sera (pts # 11, 41, and 34) were reacted with electrophoresed and blotted almond extract and compared to similar blots inhibited by nPru du 4 (Fig 2.1B). The inhibition of antisera with nPru du 4 resulted in the loss of a protein band at 14 kDa as expected.

Figure 2.2. Immunoreactivity of rabbit anti-maize profilin polyclonal antibody (ZMPro5)105 (A) with total almond extract (lane 1), rPru du 4–MBP fusion protein (lane 2), and cleaved and purified rPru du 4 (lane 3), and inhibition immunoblot of almond extract (B) probed with patient sera (pateints #11, 41, 34) and with (1) and without (–) added nPru du 4 inhibitor. Arrows indicate positions of inhibited bands. MW, Molecular weight; Pt, patient.

SDS-based immunoblotting is known to substantially denature proteins and diminish or abolish the reactivity of some constituent epitopes, and IgE-binding epitopes of some profilins are known to be largely conformational.114;115 We confirmed that SDS treatment and/or boiling of almond profilin substantially reduced or eliminated binding to some patients’ IgE (data not shown). Consequently, we screened almond-allergic patients sera for reactivity to recombinant and native profilin by dot blot under non-denaturing conditions as shown in Fig 2.3 Of the 18 tested sera from patients with histories of mild to life threatening reactions to almond, 8 (44%) demonstrated IgE reactivity to the nPru du 4. The qualitative IgE reactivity to nPru du 4 was strong with six (33%) and moderate with 2 sera (11%) (Fig 2.3). On the other hand, sera from 6 of 18 (33%) patients demonstrated IgE reactivity to the rPru du 4. Of these 6 reactive sera, 2 (11%) showed strong IgE reactivity (from pts # 34 and 41) and 4 sera (from pts # 2, 8, 18, and 37) exhibited weak IgE reactivity.

Figure 2.3. Dot-blot analysis of nPru du 4, rPru du 4, and almond extract (AE) with sera from patients with almond allergy.105 BSA was used as negative control. NC, Nonatopic control serum; Pt, patient.

Antigen used in ELISA is even less likely to be denatured than in dot blotting. ELISA with rPru du 4 (as a fusion protein) using 18 almond-allergic sera indicated that 8 of 18 (44%) were positive (≥ 0.6 absorbance) (Fig 2.4). Four of the 8 sera were strongly positive (pts # 2, 8, 18, and 41), and 4 were moderately positive (pts # 5, 7, 11, and 34). Similarly, ELISA with native profilin showed 9 of 18 (50%) almond-allergic sera were positive, 5 strongly positive (pts

# 8, 11, 18, 34, and 41), and 4 moderately positive (pts # 2, 5, 7, and 37). Overall, IgE reactivity of almond allergic sera to nPru du 4 gave stronger signals as compared to those of rPru du 4. It is possible that these differences in IgE binding reflect variations in antigen coating efficiency or epitope accessibility under the assay conditions rather than the innate differences in epitope expression.

Figure 2.4. ELISA analysis of specific IgE reactivity of sera from patients with almond allergy to almond extract (AE) proteins and native (nPd4) and recombinant almond profilin (rPd4).105 NC, Nonatopic control serum; Pt, patient.

There was general concordance between positive IgE binding to the dot blot and ELISA in most patients (pts # 2, 8, 18, 34, and 41) though some unexpected patterns were noted. For example, IgE from pt # 7 demonstrated reactivity to both nPru du 4 and rPru du 4 in ELISA, but no detectable reactivity to either protein in the dot-blot assay. Pt # 11 displayed high reactivity with nPru du 4 in both assays but failed to react with rPru du 4 in dot blot even though the dot blot signal with nPru du 4 was strong. Pts #37 and 42 also demonstrated some discordance in their reactivity profiles between the dot blot assays and ELISAs. To better assess relative reactivity of the native and recombinant Pru du 4, an inhibition dot-blot was performed using pt # 34 serum (Fig 2.5), the serum most reactive with nPru du 4 by

ELISA. Pre-adsorption with nPru du 4 nearly abolished IgE reactivity to both nPru du 4 and rPru du 4 on the dot-blot. On the other hand, pre-adsorption with rPru du 4 inhibited IgE binding to rPru du 4, but, at best, only partially inhibited reactivity to nPru du 4 (Fig 2.5).

Figure 2.5. Inhibition dot-blot with patient #34 serum (diluted 1:5).105 Immunoreactivity of patient #34 serum with rPru du 4 (rPd4), nPru du 4 (nPd4), and almond extract (AE) showing inhibition by rPru du 4 and nPru du 4 (sera diluted 1:5 and pre-incubated with nPru du d4 and rPru u d4). BSA was used as a negative control.

The presence of more than one isoform of almond profilin was detected by 2-DE immunoblot of almond extract probed with rabbit anti-maize profilin (ZMPro5) polyclonal antibodies (Fig 2.6). The N-terminal amino acid sequence of reactive spots “Y” and “Z” revealed an identical sequence (MSWQQYVDDHLM) for both, matching that of rPru du 4.

Figure 2.6. Detection of almond profilin isoforms with rabbit anti-maize profilin polyclonal antibody (anti-ZMPro5) on 2-DE immunoblot of almond extract.105 N-terminal sequences were obtained for immunoreactive spots Y and Z. MW, Molecular weight.

The IgE cross-reactivity between almond and grass pollen profilin was demonstrated by inhibition dot-blot (Fig. 2.7) using sera from three almond allergic patients (pts#11, 41, and 34). nPru du 4 and nRGPP demonstrated complete reciprocal inhibition with pt#11 and 41 sera, whereas the binding of serum IgE to nRGPP from pt#34 showed only partial inhibition with nPru du 4.

Figure 2.7. Inhibition dot blot demonstrating cross-reactivity between nPru du 4 and nRGPP.105 IgE cross-reactivity demonstrated by inhibiting reactivity of serum IgE (from patients #11, 41, and 34) to nRGPP and nPru du 4 with soluble nPru du 4 and nRGPP.(+) with inhibitor; (–) no inhibitor. Pt, Patient.

Discussion

The most common tree nut allergies in the US are to walnut, cashew, and almond.7 In a double-blind, placebo-controlled food challenge (DBPCFC) evaluation of 163 French food- allergic asthmatic children, 1.5% showed sensitivity to almond.116 Almond vicilins, legumins and 2S albumins have been identified as allergens, primarily through immunoblotting and N- terminal amino acid sequencing.85;87;87;89 In an effort to better characterize almond allergens, we screened an almond cDNA library with human almond-allergic sera and identified profilin as an allergen. Almond profilin has ≥90% sequence identity with profilins from a variety of plant sources including apple, cherry, peach, orange, and melon.117 Among the tree nuts, only almond belongs to the Rosaceae (synonym: Prunaceae) family. However, the Rosaceae family comprises a large number of commonly consumed fruits such as strawberry, raspberry, plum, apricot, cherry, peach, apple, and pear. Apple Mal d 4 was the first profilin allergen to be cloned from this plant food family. Other cloned Rosaceae fruit profilins include cherry (Pru av 4), pear (Pyr c 4), and peach.117;118 Banana, pineapple, lychee, tomato, and bell pepper profilins have also been cloned.119 As can be seen in Table 2.2, almond profilin shows a high degree of identity and similarity to profilins from other members of Rosaceae. Slightly lower, but still substantial, homology is seen with other plant profilins, which is a characteristic of this highly conserved ubiquitous protein.96;98;99;118 Profilin has been found to be a minor but important allergen in a variety of plant foods with reactivity ranging between 8 and 57%.18;58;100 Our results are in line with these and approach, at least in some assays, the rather arbitrary 50% threshold indicative of a major allergen. When the immunoreactivity of the recombinant almond profilin was compared to that of its native counterpart, we found that 44% of sera from almond-allergic subjects exhibited reactivity with recombinant profilin and 50% with native profilin. Inhibition studies also showed rPru du 4 to be less inhibitory of IgE-nPru du 4 interactions than nPru du 4. The reduced reactivity of the rPru du 4 is likely due to the presence of multiple native profilin isoforms, each displaying a somewhat differing array of epitopes as compared to the single recombinant isoform tested. In support of this interpretation, we identified two strongly staining, and several weakly staining, Pru du 4 spots on the 2-DE blots of almond extract suggesting multiple isoforms.

Others have also reported that recombinant profilins from various plant sources display a subset of epitopes as compared to native counterparts.120-123 Even though the degree to which recombinant profilin displays the full panoply of native epitopes has been questioned,104 the analysis of cherry profilin, for example, reveals that the use of rPru av 4 in skin prick tests strongly correlates with patient symptoms124 indicating that the cloning of all isoforms in a given plant need not be a prerequisite for their useful application. Two related manifestations of the conserved sequence of the various profilins are the observation that profilins from a wide variety of sources are allergenic and that the various profilins are cross-reactive in their interaction with patient IgE. We took advantage of this cross- reactivity by using rabbit anti maize profilin antisera to identify the profilin band in 1-DE and 2- DE immunoblots of almond extract. The IgE cross-reactivity extends to sensitization of patients by pollen profilin causing reactivity to food profilins. For example, of 29 peach allergic patients, the 15-patient subset that demonstrated reactivity to birch pollen profilin (Bet v 2) also reacted with recombinant peach profilin (Pru p 4.01).117 In contrast, none of the 14 patients allergic to peach but not to Bet v 2 reacted with peach profilin, Pru p 4.01. A strong correlation between the reactivity to birch pollen profilin (Bet v 2) and sweet cherry (Pru av 4, 92%) and pear profilins (Pyr c 4, 88%) in patients allergic to the respective fruits has also been noted.18 Bet v 2 has been shown to completely inhibit the binding of patient IgE to a variety of fruit and vegetable profilins in vitro.99 Clinical reactivity to melons, citrus, tomato and banana have all been strongly correlated with profilin hypersensitivity and also to Bet v 2 cross-reactivity.64 In contrast, a study on German patients noted that only 16% of cherry/birch pollen allergic patients were reactive with profilin demonstrating that this association is not universal.118 Others have found that reactivity to grass profilin is more strongly correlated with fruit allergy than is birch profilin sensitivity.94;118;125;126 Our demonstration of IgE cross-reactivity between almond and grass pollen profilins in some of our reactive patients is in line with these studies and, since most of our patients reported pollinosis, suggests the possibility that the route of initial sensitization to Pru du 4 may have been via prior pollen exposure. Despite the strong evidence of cross-reactivity between various profilins, in several studies there appears to be only a fair to poor correlation between profilin reactivity in in vitro tests and clinical food allergy in patients sensitive to both pollens and food.104;127 Some of this discordance may be traced to the observation that degranulating mast cells release chymase, an

enzyme which lyses profilins and their IgE-reactive epitopes, thus potentially limiting the triggering effect of profilin-specific IgE.128 Unlike many other food allergens, profilins display only moderate molecular stablity.90;129;129 For example, roasting has been shown to significantly decrease allergenicity of native hazelnut Cor a 2.130-132 Similarly, melon profilin allergen is readily digested by simulated gastric fluid.117 This degree of sensitivity to denaturation and digestion may explain the observation that profilin-sensitive peanut allergic patients typically suffer milder symptoms (oral allergy syndrome) than patients allergic to other constituent peanut allergens.133 Our finding that higher percentages of patients react with Pru du 4 in those immunoassays in which the allergens are less subject to denaturing conditions (i.e., dot blot and ELISA) is consistent with this observation and suggests that labile conformational epitopes may be primarily targeted by IgE in some patients. A primary role for conformational epitopes has previously been suggested for patient reactivity to other profilins.115 The somewhat labile nature of the allergens coupled with the low levels found in almond nuts may explain why this allergen was not previously detected in immunoblot screens.89;134;135 Overall from this study, we can conclude that almond profilin is an IgE-binding protein in 4 of 11 patients with a self-reported history of severe almond allergy, and in 5 of 7 of those with milder allergy (not confirmed by DBPCFC). The key history in common between these patients may be that 8 of the 9 with IgE reactive with profilin had a history of pollinosis. Indeed, preincubation of sera with ryegrass pollen extract was able to inhibit IgE-binding to Pru du 4. Whether there is a clinical contribution to the food allergy by almond profilin, either mild or severe in nature, cannot be determined unless food challenges with purified profilin are done, or unless profilin is the only IgE-binding protein detected in almond extract in patients whose mild allergy is proven by DBPCFC. Our study did not include such patients.

CHAPTER 3

ALMOND AND WALNUT 60S RIBOSOMAL PROTEIN P2: A NEW CLASS OF IGE- BINDING FOOD PROTEIN WITH FUNGAL AEROALLERGEN CROSS-REACTIVITY

Introduction

Tree nuts are energy-rich foods with recognized health benefits.136;137 However, for people with tree nut allergy, consumption can lead to allergic reactions ranging from mild to lethal.84 Several native (n) and recombinant (r) nut allergens have been identified and characterized thus far.83 Some allergens, such as profilins and members of the Bet v 1-related family, represent minor constituents in tree nuts, but are frequently cross-reactive with other food and pollen homologues in vitro. Other allergens such as lipid transfer proteins and the major seed storage proteins, the legumins, vicilins, and 2S albumins, are major allergens in tree nuts.83 Almond ranks first among tree nuts in per capita consumption in the USA138 and almond allergy ranks third (15% reactive) behind walnut (34% reactive) and cashew nut (20% reactive) among U.S. patients reporting allergy to tree nuts.7 Almonds belong to the Rosaceae family, which also includes apples, pears, peaches, plums, and raspberries. Several almond proteins bind patient serum IgE including a 14S (375-475 kDa) globulin (legumin) known as almond major protein (AMP, or amandin)87 with homology to prunin 1 and 2,86 a 7S vicilin–like protein,139 a 12 kDa 2S albumin,89 and profilin (Pru du 4).105 All but profilin are recognized by greater than 50% of almond-allergic patients.86;87;89;105;139 In contrast to almond, the walnut antigens are better defined antigenically. The commonly consumed English walnut (Juglans regia) is a member of the Juglandaceae family, which also includes black walnut, pecan, and butternut. Cloned English and black walnut allergens include 2S albumin (Jug r 1 and Jug n 1, respectively),140;141 vicilin (7S globulin, Jug r 2 and Jug n 2),141;142 nsLTP (Jug r 3),143 and legumin (11S globulin, Jug r 4)84;144 all of which are major allergens. Immunological characterization of protein allergens is essential, not only to better understand the pathogenesis of atopic diseases, but also to develop more efficacious and specific diagnostic and therapeutic modalities. In this report, we employed molecular cloning and

expression to identify the 60S acidic ribosomal protein P2 (60S RP) as IgE-reactive proteins in almonds and walnuts. 60S RP represent a protein class previously recognized as a highly cross- reactive aeroallergen in molds145-150 but not previously identified as an allergen in plant or animal tissues. The acidic ribosomal P proteins (P0, P1, and P2) consist of highly conserved small- phosphorylated ribosomal proteins with acidic pIs that have functional roles in protein synthesis.151;152 Phylogenetically conserved and homologous 60S RPs are recognized as minor allergens in four fungi; Alternaria alternata (Alt a 5),147 Aspergillus fumigatus (Asp f 8),149;150 Cladosporium herbarum (Cla h 5),147;148 and Fusarium culmorum (Fus c 1).145;146 Interestingly, in certain autoimmune diseases, e.g., systemic lupus erythematosus (SLE), human 60S RPs are reported to be auto-antigens.153;154 We observed that all tree nut-allergic patients with serum IgE reactive with almond and walnut 60S RP had IgE that cross-reacts with a fungal homologue (Fus c 1) from F. culmorum. Our data represent the first examples of an IgE-reactive 60S RP protein in plant tissues. The manuscript with the data presented below is submitted to Clinical and Experimental Allergy and in currently under revision (Tawde 2008).305

Methods

Tree nut extracts Almond and walnut extracts were prepared from defatted flours as previously described.107

Human Sera This study was approved by the institutional review board of the University of California, Davis. Blood samples were drawn, after informed consent, from patients with self-reported allergic reactions to almonds and/or walnuts. The reactions were not confirmed by oral challenges. Twenty-four of 27 patients reported potentially life-threatening reactions to one or both of the nuts including bronchospasm, throat swelling/laryngoedema, and/or, rarely, hypotension. Three patients reported only milder reactions consistent with pollen-food syndrome. Patients were asked about histories of asthma, allergic rhinitis, childhood atopic

dermatitis, and current atopic dermatitis, but not about reactions to specific aeroallergens nor were they queried about autoimmune disorders. The sera were stored at -70°C. The presence of almond/walnut-reactive IgE was confirmed by Phadia CAP FEIA assay (Phadia, Inc., Uppsala, Sweden), modified RAST, or dot blot. Patient characteristics are shown in Table 3.1.

Table 3.1. Clinical characteristics of almond and/ or walnut allergic subjects.

Serum Sex/ Age at Other Reaction Other food In vitro Positive Positive No. Age onset of atopy* to allergy IgE to dot blot to dot blot (year tree nut almond** almond almond to s) allergy *** rPru du 5 walnut rJug r 5 1 M/23 3 As Tolerates Walnut throat <0.35 no yes almonds swelling, kIU/L wheezing, U, N/V) (7.20 kIU/L) 2 F/55 53 AD, U Peanut, 1.84 no no AR, walnut (0.86 kIU/L As kIU/L) (severe systemic reaction, unlike to almond) 3 F/25 1 AD, Wheeze, Walnut (27.0 Class 2 no no AR, AE, N/V kIU/L), As cashew 4 M/26 child As AE, throat Brazil nut, 1.35 no no swelling, walnut, kIU/L wheezing cashew 5 F/53 1 As AE, throat Walnuts, 4.72 no no swelling, pecans, kIU/L oral/throat cashews pruritus 7 F/25 child AD, AE, throat Peanuts, Class 2 no no AR swelling, Walnut, other wheezing tree nuts 9 F/34 1 AD, Wheeze, Walnut (1.17 Class 3 no no AR, AE, N/V kIU/L), As cashew, pecan 10 F/31 2 AR, Wheeze, Peanuts, <0.35 no no As stridor walnut (2.02 kIU/L, kIU/L), peas Class 2 11 M/50 1 AD, AE, throat Walnut (12.0 0.64 yes yes AR, swelling, V kIU/L), other kIU/L As tree nuts

Table 3.1. – continued Serum Sex/ Age at Other Reaction Other food In vitro Positive Positive No. Age onset of atopy* to allergy IgE to dot blot to dot blot (year tree nut almond** almond almond to s) allergy *** rPru du 5 walnut rJug r 5 13 F/39 1 AD as Throat Peas, walnut <0.35 yes yes child, swelling, (8.98 kIU/L), kIU/L As, N/V, pistachio, AR wheezing, peanut, Brazil U nut 14 F/38 5 As as itchy Cashew (near 1.9 no no child mouth, fatal kIU/L N/V reaction), pistachio, brazil nut, walnut (0.43 kIU/L) 18 F/62 1 AR Wheeze, U, Peanut, 1.15 no no oral AE walnut (0.45 kIU/L kIU/L), cashew 20 F/48 4 AD, Wheeze, Peanut, <0.35 no no AR, AE, throat walnut (4.27 kIU/L, As swelling, kIU/L), Class 4 V/D, U, pecan, modifie LOC hazelnut, d RAST cashew 23 F/22 6 AD, no reaction Walnut Class 3 no no AR, (throat As swelling, U, AE)(Class 3), macadamia 27 M/38 2 AD, Stridor, Peanut, Class 2 yes yes AR, AE, U walnut (Class As 3), other tree nuts 32 M/38 1 As Wheeze, Walnut (3.94 <0.35 no no AE, throat kIU/L), kIU/L swelling, pecan, V, U cashew 34**** M/33 child AR Oral Multiple <0.35 yes yes pruritus, D fresh fruits, kIU/L to raw cooked are almond OK only, cooked is OK 35 F/49 1 AD, Tolerates Walnut (AE, <0.35 no no As as almonds wheezing, kIU/L child N/V, U, throat swelling) (10.2 kIU/L) 36 M/43 child AD, Wheeze, U, Walnut (5.28 Class 3 no no AR, AE kIU/L)

Table 3.1. - continued Serum Sex/ Age at Other Reaction Other food In vitro Positive Positive No. Age onset of atopy* to allergy IgE to dot blot to dot blot (year tree nut almond** almond almond to s) allergy *** rPru du 5 walnut rJug r 5 39 F/32 1 AD, Oral AE, Walnut (AE, 0.49 yes yes As oral throat kIU/L pruritus swelling, wheezing) (1.42 kIU/L), pecan, sesame 41 F/45 1 AD, Oral Walnut, <0.35 yes yes As pruritus to pecan, melon, kIU/L raw almond banana only, cooked is OK 42**** F/16 10 AR Oral AE, Raw walnuts, <0.35 yes yes oral pecans, kIU/L pruritus to pistachios, raw almond cherries, peaches, apricots, avocado 43**** F/48 30 AR Wheezing, Never eaten <0.35 no no throat other nuts kIU/L, swelling, positive AE, N/V prick/pr ick to raw almond 44 M/30 child AD as U, N/V/D, Walnut (2.60 2.88 no no child, wheezing kIU/L), kIU/L AR, cashew, As pistachio, macadamia, kiwi, mango, avocado, papaya, buckwheat, melons 45 F/37 5 AD, <0.35 yes yes AR, kIU/L As

* AD = atopic dermatitis, AR = allergic rhinitis, As = asthma ** AE = angioedema, V = vomiting, D = diarrhea, U = urticaria, N = nausea *** Reported in Classes by modified RAST prior to the hospital’s switch to Pharmacia ImmunoCAP FEIA and the IU reporting system ****Mild oral symptoms suggestive of pollen-food syndrome

Production and screening of the almond cDNA library and recombinant protein expression Almond seeds in late maturation were chopped, frozen, and ground. RNA extraction and cDNA cloning were as previously described.105;111;112 As described in detail for cashew nut,111 the cDNA coding sequences were ligated into the maltose-binding protein (MBP) fusion expression vector, pMAL-c2 (New England BioLabs Inc., Beverly, MA), into which a thrombin cleavage site had been engineered. The cDNA insert was produced by PCR amplification of the sequence extending from aa residue 21 (following the 20 aa leader peptide) through to the codon preceding the stop codon using 60S forward (5’ ACGCGTCGACGAAGATCTCAAGGACATCCTTG 3’) and reverse (5’ CCCAAGCTTGCAACCACTCTAGTCAAAGAG 3’) primers. Recombinant fusion protein was produced in E. coli and cleaved using one unit of thrombin (Sigma-Aldrich, St. Louis, MO) per mg of fusion protein and purified as previously described.105 Cleaved and purified rPru du 5 was used in the dot-blot and ELISA assays.

PCR amplification, cloning, expression, and purification of walnut 60S ribosomal gene PCR primers (forward 5’ ACGCGTCGACACCCCTTGCACTGATGATTTG 3’, reverse 5’ CCCAAGCTTTTAGTCAAATAGGCTAAAACCC 3’) were designed based on a previously published (GenBank number AJ278460) walnut homologue of the 60S RP cDNA sequence and used to amplify the genetic sequence from a walnut embryo cDNA library (SU2 genotype). The products were cloned, expressed, and the protein purified as described above.105;305

Recombinant Fus c 1 production Recombinant Fus c 1 was produced in E. coli as previously described.145

Anti-rPru du 5 rabbit polyclonal antiserum Polyclonal antiserum to purified rPru du 5 was produced in a New Zealand White rabbit (Oryctulagus cuniculus) as previously described.87

Dot-blot analysis The IgE-binding properties of almond, walnut, and F. culmorum r60S RPs were analyzed in vitro by dot-blot immunoassay using a panel of sera from 27 almond and/or walnut sensitive

patients. Briefly, different amounts (1, 5, and 10 μg) of almond extract (positive control), the r60S RPs, and BSA (negative control) were applied to 0.2 μm Immobilon-PSQ NC membrane (Millipore Corp. Billerica, MA) using a 96-well Bio-Dot microfiltration apparatus (BioRad Laboratories). The membranes were cut into strips and probed with human almond/walnut allergic serum and rabbit anti-rPru du 5 antisera as described previously.105;111 For inhibition dot blots, the inhibitor (rPru du 5, rJug r 5, or rFus c 1) concentration used was 10 μg/mL. For inhibition dot blots with native almond and walnut extracts, the inhibitor (almond extract, AE and walnut extract, WE) concentration range used was 10-1,000 μg/mL.

ELISA Tree nut-allergic patient IgE reactivity to rPru du 5, rJug r 5 and rFus c 1 was assayed by ELISA as previously described.105 For IgE inhibition ELISA, solid phase rPru du 5 was assayed with pooled (pts# 13, 27, 39, 41, 42) rPru du 5- and rJug r 5-reactive sera pre-incubated with soluble inhibitors (rPru du 5, rJug r 5, rFus c 1, AE, or WE) at concentrations from 0.001 – 100 μg/mL. The resultant IgE binding was measured as described above and the percentage of inhibition was calculated as follows: (S+, serum with inhibitor; S-, serum without inhibitor; NSB, non-specific baseline binding; OD, optical density.)

Inhibition (%) = OD(S-) - OD(S+) x 100 OD(S-) - OD(NSB)

Multiple sequence alignment and phylogenetic analysis The program, ClustalW, available at http://www.ebi.ac.uk/clustalw/, was used to perform multiple sequence alignment and to produce the phylogenetic tree.

Results

Cloning of almond and walnut 60S RP This study was initially undertaken to identify almond allergens by screening an almond cDNA E. coli expression library with pooled almond allergic patient sera. An initial screening with a pool of sera from 8 almond-allergic individuals identified two strongly IgE-reactive

clones. The plaques did not react with normal non-allergic human sera. Translation of the 604 bp inserts (Fig 3.1A) revealed identical sequences encoding a 93-aa protein (Fig 3.1B) with a calculated molecular mass of 9,456 Da (accession numbers AY08185.1 and DQ836316.1).

A Nucleotide sequence of Pru du 5: ATGAAGGTTGTTGCAGCATACTTGTTGGCTGTGTTGGGGGGCAATACCACCCCTTCTGCCGAAGATCTCAAGGACAT CCTTGGCTCTGTTGGAGCTGAGACTGATGATGATAGGATTCAGCTTCTTCTTTCGGAAGTGAAGGGTAAGGACATTA CAGACTTATTGCTTCTGGAAGGGAGAAGTTGGCCTCTGTACCATCTGGTGGTGGTGCTGTTGCAGTGGCTGCACCTG GTGCTGGTGCTGGTGCCGCTGCTCCTGCTGCAGCTGAGCCAAAGAAAGAAGAGAAGGTTGAGGAGAAAGAGGATACC GATGATGATATGGGCTTCAGTCTCTTTGACTAG

B Amino acid sequence of Pru du 5: M K V V A A Y L L A V L G G N T T P S A E D L K D I L G S V G A E T D D D R I Q L L L S E V K G K D I T E L I A S G R E K L A S V P S G G G A V A V A A P G A G A G A A A P A A A E P K K E E K V E E K E D T D D D M G F S L F D Stop

Figure 3.1. rPru du 5 nucleotide and amino acid sequence.305 (A) Coding nucleotides (Genbank accession code: AY081851) and (B) derived amino acid sequences of the expressed almond r60S RP cDNA clone. The deduced amino acid sequence begins at the first potential in-frame initiation codon (ATG) and ends with the TAG stop codon. The boxed 20 amino acid sequences represent the presumptive signal sequences.

Upon discovering that almond 60S RP was IgE-reactive, we searched Genbank and noted that its walnut homologue had previously been described but not tested for allergenicity. Consequently, we PCR amplified, cloned, expressed, and analyzed the walnut 60S RP for IgE reactivity with sera from walnut- and almond-allergic patients. Because 60S RP had been reported to be a minor aeroallergen in molds145-148 and the aa sequence of this protein is highly conserved, we included the homologous F. culmorum allergen, Fus c 1146 in our assays to test for potential cross-reactivity.

Sequence comparisons BLAST analysis revealed the almond cDNA clone to be 84% identical to the walnut homologue (Fig 3.2 and Table 3.2) and share considerable homology with 60S RPs from other

plants (68-84% identity and 82-91% similarity). The encoded polypeptides from almond and walnut were designated as rPru du 5 and rJug r 5, respectively, in accordance with the naming convention set by the International Union of Immunological Societies (I.U.I.S) Allergen Nomenclature Subcommittee, http://www.allergen.org/Editoral. Two isoforms of walnut 60S RP (AJ27846 and EF043535), displaying 98% aa sequence identity, were identified and designated as rJug r 5.0101 and rJug r 5.0102 (Fig 3.2).

Pru du 5 MKVVAAYLLAVLGGNTTPSAEDLKDILGSVGAETDDDRIQLLLSEVKGKDITELIASGRE 60 + + + + + Jug r 5(1) MKVVASYLLAVLGGNTTPCTDDLKHILGSVGAEADDDKIELLLSEVKGKDITELIAAGRE 60

Pru du 5 KLASVPSGGGAVAVAAPGAGAGAAAPAAAEPKKEEKVEEKEDTDDDMGFSLFD 113 + ++ Jug r 5(1) KLASVPSGGGGIAVAAAAGGAPAAAAAAAEPKKEEKVEEKEESDDDMGFSLFD 113 Jug r 5(2) K

Figure 3.2. BLAST alignment of rPru du 5 and the two isoforms of rJug r 5.305 Identical amino acids are indicated with a vertical line (|), while similar residues are highlighted in yellow and depicted as (+). The amino acids in rPru du 5 and rJug r 5 that differ are highlighted in blue and the residue that differs in the two isoforms of rJug r 5 are in red type.

Homology to fungal 60S RPs, while still significant (56-61% identity), was considerably lower (Table 3.2) demonstrating that the almond tissue-derived sequence was not that of a contaminating fungus.

Table 3.2. Homology comparisons between almond, walnut, fungal, and human 60S acidic ribosomal P2 proteins.305

Organism Accession Allerg en % identity with % similarity with No. designation Pru du 5 Pru du 5 Juglans regia AJ278460 Jug r 5 84 91 Fusarium culmorum AAL79930 Fus c 1 61 74 Cladosporium herbarum CAA54470 Alt a 5 59 71 Alternaria alternata CAA55066 Asp f 8 56 73 Aspergillus fumigatus CAB64688 Cla h 3 59 72 Homo sapiens AAA36472 - 61 77

A more detailed phylogenetic cross- comparison analyses between allergenic 60S RPs is shown in figure 3.3.

r 5 du 5 P2 f 8 c 1 a 5 h 3

60S RP Pru du 5 Jug r 5 Fus c 1 Alt a 5 Cla h 3 Asp f 8 Human P 2 Pru du 5 100% 84%(I); 91%(S) 61%(I); 74%(S) 59%(I); 71%(S) 59%(I); 72%(S) 56%(I); 73%(S) 61%(I); 77%(S) Jug r 5 84%(I); 91%(S) 100% 61%(I); 76%(S) 62%(I); 73%(S) 55%(I); 72%(S) 55%(I); 73%(S) 60%(I); 72%(S) Fus c 1 61%(I); 74%(S) 61%(I); 76%(S) 100% 85%(I); 89%(S) 73%(I); 86%(S) 77%(I); 87%(S) 61%(I); 78%(S) Alt a 5 59%(I); 71%(S) 62%(I); 73%(S) 85%(I); 89%(S) 100% 76%(I); 88%(S) 73%(I);82%(S) 61%(I); 77%(S) Cla h 3 59%(I); 72%(S) 55%(I); 72%(S) 73%(I); 86%(S) 76%(I); 88%(S) 100% 71%(I); 81%(S) 58%(I); 76%(S) Asp f 8 56%(I); 73%(S) 55%(I); 73%(S) 77%(I); 87%(S) 73%(I);82%(S) 71%(I); 81%(S) 100% 59%(I); 82%(S)

Figure 3.3. (A) Phylogenetic analysis of 60S RPs and (B) cross-comparison of allergenic 60S RPs with human 60S ribosomal protein P2.305 I = identity; S = similarity.

Recombinant Pru du 5 and Jug r 5 expression Both almond and walnut 60S RP-encoding clones were subcloned into the pMAL c 2 expression system, verified by sequencing, and expressed as MBP fusion proteins. Elution from an amylose affinity column with maltose yielded 10-15 mg of almond and walnut 60S RP protein/liter of E. coli culture. The purified rPru du 5 and rJug r 5.0101 isoform proteins were used for further analysis.

Allergic Patients’ reactivity to the 60S RPs To test for IgE reactivity to recombinant almond and walnut 60S RPs, we screened 27 self-reported tree nut-allergic patients’ sera by dot-blot and ELISA. Of these patients, 23 reported clinical histories of allergy to both nuts, 3 (pts# 1, 23, and 35) were clinically allergic to walnut only and 1 (pt# 44) was allergic to almond and had never eaten walnut. The dot-blot and ELISA results were fully concordant identifying 10 (37%) reactive patients’ sera. Two of these

sera (pts# 34 and 42) were from patients reporting only very mild clinical allergy to almond or walnut consistent with pollen-food syndrome. Thus, 8 of 24 sera (33%) from patients with potentially severe tree nut allergy showed IgE binding (Table 3.3). Interestingly, 9 of the 27 serum samples were reactive to both allergens, with one (pt# 1) reacting only with walnut rJug r 5. This particular patient is allergic to walnut but tolerates almond (Table 3.1).

Table 3.3. IgE reactivity of individual almond and/or walnut allergic sera with rPru du 5, rJug r 5, and rFus c 1 analyzed by non-reducing dot-blot and ELISA.305

Subject Dot-blot ELISA no. Pru du 5 Jug r 5 Fus c 1 Pru du 5 Jug r 5 Fus c 1 1 -a ++ ++ - +++ +++ 2------3------4------5------7------9------10 ------11 ++ ++ ++ +++ +++ +++ 12 + + + ++ ++ ++ 13 ++ ++ ++ + + + 14 ------18 ------20 ------23 ------27 +++ +++ +++ +++ +++ +++ 32 ------34 + + + ++ ++ ++ 35 ------36 ------38 ------39 ++ ++ ++ +++ +++ +++ 41 ++ ++ ++ + + + 42 ++ ++ ++ ++ ++ ++ 43 ------44 ------45 +++ +++ +++ +++ +++ +++

a(+++) strongly positive, (++) moderately positive, (+) weakly positive, (-) negative

Our results raise the possibility of cross-reactivity between the two nut allergens for the 9 patients, a property that was confirmed by demonstrating complete inhibition of IgE reactivity to each nut homologue by the alternative cloned protein by dot-blot (Fig 3.4).

Figure 3.4. Inhibition dot-blot demonstrating cross-reactivity between rPru du 5, rJug r 5, and rFus c 1.305 IgE cross-reactivity demonstrated by inhibition of IgE reactivity (pooled almond and walnut allergic patients’, pt#27, and pt#13, sera) to rPru du 5, rJug r 5, and rFus c 1 with soluble rPru du 5, rJug r 5, and rFus c 1. (+), inhibitor added; Pt, patient.

This pattern was further confirmed by demonstrating complete cross-reactivity between rPru du 5 and rJug r 5 in inhibition ELISA with the three tested 60S RP-reactive patients’ sera (Fig 3.5).

100%

80%

60% Pru du 5 Jug r 5 40% Fus c 1 Inhibition (%)

20%

0% 0.001 0.01 0.1 1 10 100 Inhibitor (ug/ml)

Figure 3.5. IgE inhibition ELISA. Solid phase rPru du 5 assayed with pooled rPru du 5- and rJug r 5-reactive patients’ sera pre-incubated with rPru du 5, rJug r 5, or rFus c 1 as soluble inhibitors.305

Because 60S RP has been reported to be an aeroallergen in several common molds,146;147 we included the previously described F. culmorum allergen homologue, Fus c 1, in our assays to test for potential cross-reactivity and the possibility that molds could be the primary sensitizers.

The data show that each of the 10 patients’ sera positive for almond and/or walnut 60S RP, but not other sera, tested positive for Fus c 1. For 9 of the 10 patients’ sera, the relative intensity of binding was similar for each of the three tested 60S RPs, a result consistent with both cross- reactivity and the possibility that sensitization could have occurred through fungal exposure. Multiple sequence alignment of rPru du 5, rJug r 5, and rFus c 1 reveals regions of high homology (Fig 3.6), which could account for these results.

N-terminal part Flexible hinge C-terminal part

Jug r 5 MKVVASYLLAVLGGNTTPCTDDLKHILGSVGAEADDDKIELLLSEVKGKDITELIAAGREKLASVPSGGGGIAVAAAAGGAPAAAAAAAEPKKEEKVEEKEESDDDMGFSLFD Pru du 5 MKVVAAYLLAVLGGNTTPSAEDLKDILGSVGAETDDDRIQLLLSEVKGKDITELIASGREKLASVPSGGGAVAVAAPGAGAGAAAPAAAEPKKEEKVEEKEDTDDDMGFSLFD Fus c 1 MKHLAAYLLLGLGGNTSPSAADVKAVLTSVGIDADEDRLNKLISELEGKDIQQLIAEGSEKLASVPSGG----AGGASGGAAAAGGAAEEAKEEEKEEEKEESDEDMGFGLFD ** :*:*** *****:*.: *:* :* *** ::*:*::: *:**::**** :*** * ********** ...... ** **. ** *.*:*** ****::*:****.***

Figure 3.6. Multiple amino acid sequence alignment of almond (rPru du 5), walnut (rJug r 5), and fungus (rFus c 1) 60S RPs.305 The N-terminal portion, indicated by the red bar, is responsible for protein-protein interactions. The conserved acidic C-terminal region is depicted by the blue bar, and the highly conserved (identical) C-terminal 11 amino acid sequence is shown in blue type. Other identical residues are in green. (*) = identity, (:) = strongly conservative substitution; (.) = weakly conserved substitution; (-) = gap inserted for alignment.

Expression and stability of 60S RPs in nut tissues To verify the presence of 60S RP in the nut extracts and determine the molecular mass of the native protein, we performed inhibition Western blots consisting of total almond extract probed with pooled rPru du 5-reactive patients’ sera that had been adsorbed with rPru du 5. Surprisingly, no evidence of band inhibition was detected (data not shown). Possible interpretations include, levels of native Pru du 5 (nPru du 5) in the extract below the level of detection by human sera, labile epitopes disrupted by Western blot conditions, co-migration of nPru du 5 with another stronger allergen thereby masking inhibition, or that the patients’ IgE that originally served to detect the reactive almond cDNA clone was actually raised against a cross- reactive homologue from another source (e.g., Alternaria spores), and that mature nut tissues do not contain the allergen in detectable levels. To address these issues, we generated rabbit antisera by immunizing with almond rPru du 5 under the assumption that targeted immunization

of rabbits would yield stronger monospecific antisera than that found in naturally sensitized patients’ sera.

Figure 3.7. Dot-blot analysis of almond, walnut and fungal r60S RP in complex with rabbit anti-rPru du 5.305 (A) Binding of antibody to recombinant (top 3 dots) and native (bottom 2 dots) 60S RPs (AE, almond extract; WE, walnut extract). (B) Effect of reducing and non-reducing conditions upon reactivity of native almond (AE) and walnut (WE) 60S RP extracts with anti-Pru du 5. TBS, Tris buffered saline; NRB, non-reducing buffer; RB, reducing buffer; BSA, bovine serum albumin (negative control).

Dot-blots performed under non-reducing conditions, demonstrated strong reactivity between the rabbit antisera and both purified rPru du 5, as anticipated, and with rJug r 5 indicating cross-reactivity. Considerably lower levels of reactivity were observed with crude aqueous almond and walnut extracts, even when dotted at high protein concentration, suggesting low levels of nPru du 5 and nJug r 5 in the respective nut extracts (Fig 3.7A).

Figure 3.8. Inhibition dot-blot demonstrating the presence of native 60S RPs in almond and walnut extracts.305 Pooled almond and walnut allergic patients’ sera was preincubated with

almond/walnut extracts and then probed with nitrocellulose containing rPru du 5, rJug r 5, and rFus c 1 as solid phase. (+), inhibitor added; Pt, patient, NC-Negative control used was non- allergic serum.

It was estimated from these results that the concentration of the native 60S RP in these nut extracts were ~ 0.1-0.2 μg/mL. Loss of reactivity of anti-rPru du 5 with total almond and walnut extracts upon reduction in dot-blot assays was also demonstrated (Fig 3.7B), explaining our inability to detect reactivity with almond and walnut extract in (reducing) Western blots with rabbit anti-rPru du 5. Similar results were noted when probing recombinant proteins with patients’ sera (data not shown).

A 100%

80%

60% % n Pru du 5 io it Almond Ext ib h 40% In

20%

0% 0.001 0.01 0.1 1 10 100 Inhibitor ฀g/mL

B 100%

80%

60% % n Jug r 5 io it Walnut Ext ib 40% h In

20%

0% 0.001 0.01 0.1 1 10 100 Inhibitor ฀g/mL

Figure 3.9. IgE inhibition ELISA.305 (A) Solid phase rPru du 5 was assayed with pooled rPru du 5- and rJug r 5-reactive patients’ sera pre-incubated with soluble inhibitors (rPru du 5, AE) in the concentration range of 0.001-100 μg/mL, and (B) Solid phase rJug r 5 was assayed with

pooled rPru du 5- and rJug r 5-reactive patients’ sera pre-incubated with soluble inhibitors (rJug r 5, WE) in the concentration range of 0.001-100 μg/mL.

Both dot blot (Fig 3.8) and ELISA inhibition assays (Fig 3.9) confirmed low levels of native 60S RP in both the nut extracts. Together, these data suggest the presence of low levels of a somewhat labile native 60S RP in nut extracts. No detectable IgG reactivity to both the nut extracts was observed with pre-immune rabbit sera.

Discussion

In this report, we describe the molecular cloning and immunological characterization of 60S RP, in almond (rPru du 5) and walnut (rJug r 5) kernels. Although 60S RPs are described as minor aeroallergens in some molds,145;147-150 our findings represent the first report of an IgE- reactive 60S RP in plants where they may serve as food allergens. These IgE-reactive proteins were initially identified by screening a developing almond nut cDNA library with pooled almond-allergic sera, and subsequently by PCR amplification of the homologue from a walnut embryo cDNA library. The two IgE-reactive proteins are 84% identical and 91% similar in aa sequence. About a third of the almond and walnut allergic patients in the tested population are IgE reactive to both Pru du 5 and Jug r 5, making 60S RP a potential minor food allergen in these nut-allergic patients (i.e., <50 % of patients were reactive). Certain ribosomal proteins, such as 60S RP P1, are degraded within a few hours of expression, and thus are not present in mature seed tissue.155 However, 60S RP P2, the subject of this report, has a longer half-life 31 and has previously been detected in mature seed tissue such as maize kernels.156 Our attempts to detect 60S RP P2 in extracts of mature almond and walnut kernels by dot blot were successful but we could not detect reactive bands on Western blots. A series of assays using both – the allergic patients’ sera and a mono-specific rabbit anti-almond rPru du 5 anti-serum indicates low concentrations of native Pru du 5 and Jug r 5 in the nut extracts and a predominance of labile epitopes. These findings contrast with the binding of patient IgE to the two original cDNA

expression library plaques, which was quite strong. It is therefore highly unlikely that 60S RP would have been recognized as a potential tree nut allergen based on identification of reactive bands on a typical Western blot. Could 60S RP be a significant contributor to symptoms in allergic individuals? Certainly other important allergens present in trace amounts, such as profilin and pathogenesis-related proteins, are allergenic in some foods.18 In our patients with almond or walnut allergy, serum IgE has been demonstrated against other more abundant almond or walnut allergens in most of the 60S RP-positive sera (data not shown), thus a contribution to clinical reactivity by IgE binding to trace quantities of 60S RP would be difficult to prove. However, there is a precedent that suggests 60S RP can be allergenic in food. A patient demonstrating pronounced symptoms of food allergy after ingesting Quorn, a food product derived from the mold, F. venenatum, displayed IgE reactivity to r60S RP (Fus c 1) from the closely related F. culmorum.145 The data indicated that Fus c 1 was the dominant allergen recognized by this patient. Fus c 1 is also recognized by IgE in 35% of F. culmorum-sensitized patients.145 In fact, it is this sensitization to the aeroallergen that is the probable basis for the (pre)sensitization to Quorn in the above- described patient.145 An important question is how sensitization to 60S RP occurred in our patient population. It is certainly possible our almond/walnut- reactive patients were initially sensitized by a fungal aeroallergen (Quorn is not commonly consumed in the US); all patients with IgE reactive to 60S RP from one or both tree nuts and also displayed similar degrees of reactivity with rFus c 1 (Fig 3.4, Table 3.3) and no Fus c 1 reactivity was detected among 4 atopic control patients (Table 3.3). Perhaps subsequent oral ingestion of cross-reactive nut proteins can serve to enhance allergic reactivity. On the other hand, the 2 nut and the fungal homologues were mutually and completely inhibitory in IgE assays (Fig 3.4, 3.5) supporting no particular IgE-binding protein as the stimulating allergen. Fus c 1 in Quorn was classified as an “incomplete” food allergen, capable of eliciting allergic symptoms in patients with a pre-existing respiratory sensitization to molds but not able to the induce de novo IgE responses upon ingestion.145 Pru du 5 and Jug r 5 similarly may be “incomplete” food allergens. It would be interesting to test sera from patients known to have clinical allergies to fungi and IgE demonstrable to fungal 60S RP, but not displaying allergy to tree nuts, for reactivity to tree nut 60S RP in similar immunoassays, and such studies are

planned. Sensitization to molds is seen in about 25% of patients with allergic rhinitis or asthma in the region of California from which the current patient pool was drawn (about 24% to Alternaria, 4% to Aspergillus, 7% to Cladosporium, and 8% to Penicillium) (unpublished data). No patients in the current study had known histories of allergic bronchopulmonary aspergillosis (ABPA) or allergic fungal sinusitis, but all had other atopic disorders: 6 of 10 had atopic dermatitis, 7 of 10 had asthma, and 7 of 10 had allergic rhinitis. Consistent with the cross-reactivity scenario is the relatively high degree of identity (56- 61%) and similarity (71-74%) observed between nut and fungal 60S RPs (Table 3.2). The cross- comparison (Fig 3.3B) and phylogenetic analysis (Fig 3.3A) further illustrate the homology between IgE-reactive 60S RPs. In fact, one 9 aa segment (residues 61-69) displays 100% identity and another 12 aa segment (residues 98-109), 75-92% identity and 100% similarity between the 2 nut and 4 fungal homologues (Fig 3.6). Such regions could well serve as the basis for the observed cross-reactivity. With this in mind, it would be interesting to compare the (cross-)reactive epitope profiles of the 8 patients that recognized all three 60S RPs to the one patient that recognized rJug r 5 and rFus c 1 but not rPru du 5. 60S RP sequence conservation and cross-reactivity may also extend to auto- reactivity. Significant IgE-mediated cross-reactivity, including strong type 1 skin reactions to human 60S RP, has been documented in individuals sensitized to fungal 60S RP of A. fumigatus.149;150 Patients with SLE154;157 and those infected with pathogenic intracellular parasites including Trypanosoma cruzi and several Leishmania species154 can express high titers of antibodies against the C-terminal end of human ribosomal P 2 protein.158 Our findings allow us to report 60S RPs from almond and walnut as IgE-reactive proteins and potential minor food allergens, possibly on the basis of newly demonstrated IgE cross-reactivity with fungal Fus c 1, a homologous aeroallergen from F. culmorum. Definitive testing of the degree of clinical relevance of the tree nut 60S RPs must await further studies with direct clinical challenge.305

CHAPTER 4

IDENTIFICATION OF PISTACHIO 7S AND 11S GLOBULIN AND DETECTION OF ALLERGENIC CROSS-REACTIVITY BETWEEN HOMOLOGOUS CASHEW AND PISTACHIO ALLERGENS

Introduction

Food hypersensitivity is reported to affect more than 4 million people in the United States, resulting in about 29,000 anaphylactic reactions each year with a minimum of 150 deaths.9 More than 90% of these reactions are caused by eight foods: crustacea, eggs, fish, milk, peanuts, soybeans, tree nuts and wheat.6 Approximately 0.5% of the US population is considered to be allergic to tree nuts.7 Tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence.3;7;300 The consumption of tree nuts is steadily increasing due to the general perception of their health benefits.159 According to the USDA in 2005, the per capita tree nut consumption was 2.7 pounds, with cashew nut being the most commonly consumed imported nut.138 Over the recent years, the consumption of pistachio has been steadily rising, which can be attributed to their incorporation into baked goods, ice cream, candies, and other food dishes. A random digit dialed phone survey revealed that, of 82 tree nut-allergic individuals, 44% reported allergy to cashew and 22% to pistachio.7 It has been estimated that 30% of patients who are allergic to at least one food in the nut group are allergic to several tree nuts.160 The prevalence of allergenic cross-reactivity between various tree nuts may be due to the presence of cross-reactive IgE targeted against epitopes present in different tree nuts.161;162 Considerable research has been conducted in recent years in an attempt to characterize the tree nut allergens that are most responsible for allergy sensitization and triggering. The major allergens identified so far in the tree nuts include seed storage proteins.83 To date three major cashew allergens, Ana o 1 (7S vicillin), Ana o 2 (11S globulin), and Ana o 3 (2S albumin), have been identified which are characterized as seed storage proteins.112;163;164 Ana o 1, a 7S vicilin, is a homotrimer of 45 kDa subunits, recognized by 10 of the 20 cashew-allergic patients’ sera and identified as a major allergen (i.e., ≥50% reactive).163 Vicilins are typically homotrimeric proteins with a molecular mass of 150 to 190 kDa, composed of protomers of 40 to

80 kDa, and belong to the cupin superfamily which contains structurally related proteins built from the β-barrel cupin motif.18 Vicilins have previously been identified as allergens in walnut (Jug r 2)142, hazelnut (Cor a 11)301, peanut (Ara h 1)165, lentil (Len c 1)166, pea (Pis s 1)167, sesame seed (Ses i 3).168 Several vicilins have been shown to be resistant to digestion and food processing, a common characteristic of allergenic proteins.169 The other major cashew allergen Ana o 2 belongs to the 11S globulin or legumin family.112 The 11S globulins are hexameric heteroligomeric proteins of 300-450 kDa MW, with each subunit comprising an acidic polypeptide 30-40 kDa in size that is disulfide-linked to a 20- 25 kDa basic polypeptide.68 In addition to cashew, several 11S globulins have been identified as major plant food allergens, including peanut Ara h 359;170 and Ara h 4134, soybean G1 and G2 glycinins171;172, walnut Jug r 4144, hazelnut Cor a 9173, almond 11S globulin174, Brazil nut Ber e 2175, buckwheat Fag e 1176;177, sesame seed Ses i 6168;178;302, yellow mustard seeds Sin a 2179, and coconut 11S globulin.180 It has been estimated that 30% of patients who are allergic to at least one food in the nut group are allergic to several tree nuts. 160 Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. The major seed storage protein constituents of tree nuts are legumins, vicilins, and 2S albumins, each of which can be allergenic. Other allergens such as lipid transfer proteins, profilins, and members of the Bet v 1-related family represent minor constituents in tree nuts. These latter groups of allergens are frequently cross-reactive with other food and pollen homologues, and are thus considered panallergens.83;162 There have been several reports of serologic as well as clinical allergenic cross-reactivity between pistachio and cashew nut proteins, which is not surprising since both are members of the Anacardiaceae family.83;181-187 Adverse reactions to other members of the Anacardiaceae family, e.g., mango fruit, have also been reported.184;188;189;304 In a study reported previously, two pistachio-allergic individuals who had never eaten cashews exhibited IgE specific to both cashew and pistachio nuts using skin prick tests, immunoblotting, and radioallergosorbent tests (RAST).183 Western immunoblot assays demonstrated IgE binding to pistachio proteins ranging from 14 to 70 kDa and cashew proteins from 20 to 67 kDa.183 In another study, ImmunoCAP- inhibition assays were used to demonstrate cross-reactivity between pistachio and cashew using three patients; one allergic to only pistachio and two allergic to pistachio but had never eaten

cashew.182 It was reported that pre-incubation of patients’ sera with cashew could significantly inhibit IgE binding to pistachio nut.182 IgE binding to 34, 41, 52, and 60 kDa bands in pistachio nut extract was detected by immunoblotting and, in agreement with Fernandez et al., the 34 kDa band exhibited the strongest IgE binding signal.182 Cross-reactivity between cashew and pistachio proteins using rabbit anti-pistachio anti-sera was demonstrated in a double immune- diffusion assay.187 Pistachio allergy is however not extensively investigated, resulting in limited information on pistachio allergens. Additionally, none of the previous reports identify or characterize the specific proteins responsible for the observed cross-reactivity beyond the estimates of their molecular masses.182;183;187 It is evident that we need careful biochemical and immunological studies to evaluate IgE-binding proteins in both nut species, with a goal of identifying homologous proteins that can potentially elicit serious allergic cross-reactions. Our aim was to identity these cross-reactive allergens. In this study we (1) report the identification and immunological characterization of a cloned pistachio allergen, a vicilin designated Pis v 3, (2) perform cloning and characterization of pistachio 11S globulin designated as Pis v 2, and (3) demonstrate allergenic and antigenic cross-reactivity between cashew and pistachio (vicilin and legumin homologues respectively) by assaying with serum IgE from tree nut allergic individuals, anti-cashew rabbit polyclonal antisera, and with mouse anti-cashew vicilin and legumin monoclonal antibodies (mAbs) (4) compare amino acid sequences of homologous vicilin and legumin from both the nuts for deducing regions that could potentially confer cross-reactivity. The cashew cDNA library construction was performed by Fang Wang.111 The cloning and characterization studies with cashew Ana o 1 was performed by Fang Wang, Jason Robotham, and myself and is published in JACI in 2002.111 The cloning of pistachio Pis v 3 was performed in collaboration with Jason Robotham and Richard Penney. The immunological characterization of Pis v 3 was performed by Leanna Willison and me, and the manuscript has been accepted for publication in Clinical and Exp. Allergy, 2008.306 The experimental studies with cashew Ana o 2 is published in International archives of Allergy in 2003.112 The epitope mapping of both cashew Ana o 1 and Ana o 2 were performed by Jason Robotham and the data are published in the above mentioned journals.111;112

Methods

Tree nut Allergic Human Sera Blood samples were drawn after informed consent from patients with life-threatening systemic reactions to cashew and pistachio nut. Sera were frozen at -70°C until use. The presence of pistachio- and cashew-reactive IgE was confirmed by means of Pharmacia ImmunoCAP assay or Western immunoblotting. Clinical characteristics of the subjects are shown in Table 1. Control sera were obtained from patients with histories of pollinosis to weeds, trees, and/or grasses but who were not food-allergic.

Table 4.1. Clinical characteristics of pistachio and/or cashew-allergic subjects.

Pt Sex/ Age at Pistachio Cashew Other Other 2ImmunoCap, RAST, or No. Age onset of allergic allergic Atopy Food positive IgE Immunoblot years tree nut history Allergy * allergy 1 M/25 3 Yes Yes Asthma walnut, Pistachio 5.65 kIU/L pecan, Cashew 6.95 kIU/L hazelnut 3 F/26 2 Yes Yes AD, peanut, Pistachio Class 5 AR, walnut Cashew 9.51kIU/L Asthma 5 F/54 10 Yes Yes AR, walnut, Pistachio 7.24 kIU/L Asthma pecans, Cashew 1.62 kIU/L hazelnut 7 F/30 10 NE1 Yes AD, AR Peanut, Pistachio 2.80 kIU/L walnut, Cashew 4.04 kIU/L hazelnut 9 F/35 2 NE Yes AD, Walnut, Pistachio Class 5 AR, pecans, Cashew 35.1 kIU/L Asthma almond 11 M/50 1 Yes Yes AD, Multiple Pistachio 4.60 kIU/L AR, tree nuts Cashew 5.19 kIU/L Asthma 12 F/26 3 NE Yes AR, Multiple Pistachio 2.22 kIU/L Asthma tree nuts Cashew 2.41 kIU/L 13 F/39 1 Yes NE AD, Peanut, Pistachio 12.5 kIU/L AR, walnut, Cashew 9.53 kIU/L Asthma hazelnut, pine nut, Brazil nut 14 F/39 5 Yes Yes Asthma Tree nuts Pistachio 57.4 kIU/L Cashew 94.7 kIU/L 20 F/48 1 NE Yes AD, Peanut, Pistachio 0.56 kIU/L AR, walnut, Cashew +blot Asthma hazelnut 29 F/49 3 Yes Yes AD, Peanut, Pistachio 0.38 kIU/L

Table 4.1. - continued Pt Sex/ Age at Pistachio Cashew Other Other 2ImmunoCap, RAST, or No. Age onset of allergic allergic Atopy Food positive IgE Immunoblot years tree nut history Allergy * allergy 32 M/38 1 Yes Yes AD, Walnut, Pistachio 1.17 kIU/L Asthma pecan, Cashew 1.64 kIU/L hazelnut 33 F/63 53 NE Yes AD, Peanut, Pistachio 66.9 kIU/L AR, almond, Cashew 81.3 kIU/L Asthma fish, eggs 35 F/54 2 Yes Yes AD, AR Walnut, Pistachio Class 2 Asthma hazelnut, Cashew 0.85 kIU/L pecan, Brazil nut 46 M/39 18 Yes No AR, Sunflower Pistachio 2.29 kIU/L Asthma seed, Cashew <0.35 kIU/L mango, fruit 47 F/65 child Yes Yes AR, Banana, Pistachio 38.2 kIU/L Asthma avocado, Cashew 52.9 kIU/L mango, melon 48 M/59 4 Yes Yes AD, Peanut, Pistachio 3.53 kIU/L Asthma walnut, Cashew 7.82 kIU/L almond, pecan, hazelnut, Brazil nut, pine nut 49 M/43 2 NE NE AD, Walnut, Pistachio Class 2 AR, almond Cashew <0.35 kIU/L Asthma 50 M/35 6 Yes Yes AR, Walnut, Cashew +blot Asthma pecan, Pistachio +blot hazelnut, Brazil nut

1 NE= never eaten, AD = Atopic dermatitis; AR = allergic rhinitis 2ImmunoCAP results are shown as kIU/L, RAST as Class * Reported in Classes by modified RAST prior to the hospital’s switch to Pharmacia ImmunoCAP FEIA and the IU reporting system

Cashew and Pistachio Protein Extract Cashew and pistachio protein extracts were obtained from defatted cashew or pistachio flour as previously described.190

Production of Rabbit Polyclonal Antiserum A rabbit was immunized with 5 mg of cashew extract in Freund’s complete adjuvant and boosted 4 weeks later with 5 mg of cashew extract in incomplete Freund’s adjuvant. The rabbit was subsequently bled, and the serum was stored at –20°C.163

cDNA Production, PCR Amplification, and DNA Sequencing Total RNA from cashew nuts was extracted in TRIzol (GIBCO BRL Life Technologies Inc, Rockville, MD), and mRNA was isolated with a Poly-ATtract kit (Promega, Madison, WI), as previously described.163 The construction of the cashew cDNA library was performed with the Uni-ZAP XR Gigapack Cloning Kit (Stratagene Inc, Cedar Creek, TX), according to the manufacturer’s instructions. The double-stranded cDNAs with EcoRI (using a 5′ end adapter) and XhoI (using a 3′ end PCR primer) cohesive ends were cloned into the lambda Uni ZAP XR expression vector. The amplified library was initially screened with rabbit anti-cashew serum at 1:5000 dilution. Bound IgG was detected with HRP–conjugated goat anti-rabbit IgG antibody (Sigma, St. Louis, MO) at 1:50,000 dilution and developed with the ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ). The immunopositive clones were picked, plaque purified, and stored in SM buffer supplemented with 2% chloroform at 4°C. Similarly, mature pistachio nuts were frozen in liquid nitrogen and ground with a mortar and pestle. Total RNA was extracted as described earlier163 using TRIzol (Gibco BRL, New York, NY). mRNA was isolated using a PolyATract® mRNA Isolation kit (Promega, Madison, WI) as described by the manufacturer. Both 5'- and 3'-RACE were used to generate pistachio cDNA as described in the SMART RACE cDNA Amplification Kit user manual (BD Biosciences Clonetech, Palo Alto, CA). Degenerate primers were designed based upon conserved homologous sequences found in 7S globulins from cashew, hazelnut, sesame, soybean, and fava bean. The degenerate primer 5’-IGIKATYTTYGTTGCMIKCGAGTTGTA-3’ and a universal primer based on the 5' linker sequence on the 5'-RACE cDNA were used. Sequencing of the PCR products lead to the identification of the pistachio 7S globulin. Gene specific primers (forward: 5’- TGCTCTAGAAAGACAGACCCAGAGCTGAAAC-3’, reverse: 5’- AAACTGCAGTCATTCATCAGCACGCCCTTG-3’) were then designed and used to amplify

full length pistachio 7S globulin cDNA which was then TA cloned (TOPO TA Cloning Kit, Invitrogen, Carlsbad, CA) and sequenced on an ABI 3100 Genetic Analyzer (Foster City, CA).

Cloning, Expression and Purification of cDNA-encoded Proteins from Cashew cDNA Library The cashew nut and pistachio cDNA coding sequences of respective IgE reactive clones were ligated into a modified version of the maltose-binding protein (MBP) fusion expression vector pMAL-c2 (New England BioLabs Inc, Beverly, MA).163 The cloning, expression, and purification of rAna o 1 and rPis v 3 fusion proteins was performed as previously described for rAna o 1.163 Briefly, cDNA/pMAL-c2-His plasmids encoding rAna o 1 or rPis v 3 were used to transform competent E. coli BL21 (DE3) cells (Novagen Inc, Madison, WI). Bacterial colonies o were grown at 37 C with shaking to an OD600 nm of 0.5, followed by incubation with 0.3 M IPTG. The cells were harvested, resuspended in amylose resin buffer lysed with mild sonication, centrifuged at 10,000 g, and the supernatant passed over amylose affinity column. The fusion protein was eluted with column buffer containing 10 mmol/L maltose and stored at 4°C until use or frozen at -80°C.

SDS-Polyacrylamide Gel Electrophoresis (1-DE) Recombinant proteins (0.5 μg per 4 mm well width) or aqueous total cashew/pistachio extracts (12 to 14 μg per 4 mm well width) were subjected to SDS-PAGE (12%) followed by either staining with Coomassie Brilliant Blue R (Sigma-Aldrich, St. Louis, MO) or transferred to NC membranes as previously described.163;191

IgE Immunoblotting and Inhibition NC strips (4 mm wide) containing 12 to 14 μg of nut protein extract or 0.5 μg of recombinant/native Pis v 3 protein per strip were used for immunoblotting as described earlier.105;163 For inhibition immunoblots and dot-blots, human sera at 1:5 or 1:50 dilution in were pre-incubated with 100 μg/mL of rAna o 1 or rPis v 3 inhibitor (both with associated MBP) o/n at 4°C or at 37°C for 1 h and used as described previously.105;163 Controls included strips/dots exposed to IgE without inhibitor and strips/dots exposed to serum from an atopic individual without a history of tree nut allergies.

Dot-blot Analysis and Inhibition Recombinant Pis v 3 and rAna o 1 were applied to NC membranes using a 96-well Bio- Dot Microfiltration Apparatus (Bio-Rad Laboratories) as previously described.105 Briefly, recombinant proteins (0.5 μg per 2 mm dot) were spotted onto a NC membrane and strips containing dotted proteins were excised and probed as described below. For inhibition dot-blots, rPis v 3 and rAna o 1 were used as inhibitors at 100 μg/mL and pre-incubated with patient’s sera at 1:50 (v:v) dilution o/n at 4°C prior to incubation with the dotted protein. The membranes were developed following the same procedure described above.

Monoclonal Antibody Production and Ana o 1 and Ana o 2 Immunoreactivity Ana o 1 and Ana o 2-reactive monoclonal antibodies (mAbs) were generated in the Hybridoma Core Facility at Florida State University according to standard procedures.192 Those mAbs that were found to be reactive with whole cashew extract in this initial screening were then checked for reactivity to rAna o 1/rAna o 2 in subsequent ELISAs using plates coated with 5 μg/ml rAna o 1/rAna o 2. Reactive clones of interest were subcloned until a total of 20 monospecific Ana 2-binding mAbs were obtained.

Dot Blot Screening of Proteins with Ana o 1-specific mAbs rAna o 1 and rPis v 3 were dotted onto 0.4 μm NC using the Bio-Rad Dot Blot apparatus (Bio-Rad) as described previously.105 The dotted membranes were probed with the rAna o 1- specific MAbs at 1:400 or 1:500 dilutions in TBS-T at RT for 1 h. Reactive dots were identified after 5-min incubation in ECL+ kit solution (Amersham GE Healthcare, Piscataway, NJ), prepared following manufacturer’s instructions, and subsequent exposure to Kodak X-XAR x- ray film as described previously.105

Solid-Phase Peptide (SPOTs) Synthesis and Binding to IgE On the basis of the derived amino acid sequence of the 540-amino-acid Ana o 1 protein, 66 overlapping 15-amino-acid peptides, each offset by 8 amino acids, were synthesized.163 Similarly, for Ana o 2, overlapping peptides were synthesized as described for Ana o 1.112 The Ana o 1 and Ana o 2 SPOTs membranes probed with pooled sera diluted 1:5 (v/v) in Genosys blocking buffer, followed by washing, incubation with 125I-labeled anti-human IgE (Hycor

Biomedical Inc., Garden Grove, CA), and 48 hours’ exposure at –70°C to Kodak Biomax x-ray film, as described above.163

Results

Gene Characterization of Cashew Vicilin Ana o1 The screening of cashew cDNA library included separate probings with cashew allergic human serum (IgE immunodetection) and rabbit anti-cashew antisera (IgG immunodetection). Clones that gave positive signals with both screenings were selected.

Ao1.1 GCCATAATGGGTCCGCCTACAAAGTTTTCTTTTTCTCTTTTTCTCGTTTCTGTTTTGGTCCTGTGTTTAGGTTTTGCT Ao1.2 ------******************************************************************

Ao1.1,2 GCTAAAATAGACCCGGAGCTGAAACAGTGCAAGCACCAGTGCAAAGTCCAGAGGCAGTATGACGAGCAACAGAAGGAG Ao1.1,2 CAGTGTGTGAAAGAGTGTGAAAAGTACTACAAAGAGAAGAAAGGACGGGAACGAGAGCATGAGGAGGAAGAAGAAGAA Ao1.1,2 TGGGGAACTGGTGGCGTTGATGAACCCAGCACTCATGAACCAGCTGAAAAGCATCTCAGTCAGTGCATGAGGCAGTGC Ao1.1,2 GAGAGACA AGAAGGAGGACAACAAAAGCAACTATGCCGCTTTAGGTGTCAGGAGAGGTATAAGAAAGAGAGAGGACA Ao1.1,2 ACATAATTACAAGAGAGAAGACGATGAAGACGAAGACGAAGACGAAGCCGAGGAAGAAGATGAGAATCCCTATGTATT Ao1.1,2 CGAAGACGAAGATTTCACCACCAAAGTCAAGACTGAGCAAGGAAAAGTTGTTCTTCTTCCCAAGTTCACTCAAAAATC Ao1.1,2 GAAGCTTCTTCATGCCCTGGAGAAATACCGTCTAGCCGTTCTCGTTGCGAATCCTCAGGCTTTTGTAGTTCCAAGCCA Ao1.1,2 CATGGATGCTGACAGTATTTTCTTCGTTTCTTGGGGACGAGGAACGATCACCAAGATCCTTGAGAACAAACGAGAGAG Ao1.1,2 CATTAATGTCAGACAGGGAGACATCGTCAGCATTAGTTCTGGTACTCCTTTTTATATCGCCAATAACGACGAAAACGA Ao1.1,2 GAAGCTTTACCTCGTCCAATTCCTCCGACCAGTCAATCTTCCAGGGCATTTCGAAGTGTTTCATGGACCAGGCGGTGA Ao1.1,2 AAATCCAGAGTCTTTCTACAGAGCTTTCAGCTGGGAAATACTAGAAGCCGCACTGAAGACCTCAAAGGACACACTTGA Ao1.1,2 GAAACTTTTCGAGAAACAGGACCAAGGAACTATCATGAAAGCCTCCAAAGAACAAATTCGGGCTATGAGCCGGAGAGG Ao1.2 *******************************************************G**********************

Ao1.1,2 CGAAGGCCCTAAAATTTGGCCATTTACAGAGGAATCAACGGGATCATTCAAACTTTTCAAAAAGGATCCCTCTCAATC Ao1.1,2 CAATAAATACGGCCAACTCTTTGAAGCTGAACGTATAGATTATCCGCCGCTTGAAAAGCTTGACATGGTTGTCTCCTA Ao1.1,2 CGCGAACATCACCAAGGGAGGAATGTCTGTTCCATTCTACAACTCACGGGCAACGAAAATAGCCATTGTTGTTTCAGG Ao1.1,2 AGAAGGATGCGTTGAAATAGCGTGTCCTCATCTATCCTCTTCGAAAAGCTCACACCCAAGTTACAAGAAATTGAGGGC Ao1.1,2 ACGGATAAGAAAGGACACAGTGTTCATTGTCCCGGCGGGTCACCCTTTCGCGACTGTTGCTTCGGGAAATGAAAACTT Ao1.1,2 GGAAATCGTGTGCTTTGAAGTAAACGCAGAAGGCAACATAAGGTACACACTTGCGGGGAAGAAGAACATTATAAAGGT Ao1.1,2 CATGGAGAAGGAAGCGAAAGAGTTGGCATTCAAAATGGAAGGAGAAGAAGTGGACAAAGTGTTTGGAAAACAAGATGA Ao1.1 GAGAAGAAATGGGAAGGTTGTTTGGGGTCTGAGAAAGGCTGAGCTACTGACTAGTGAACGTTATATATGGATAACGTA Ao1.2 ***************************************************------

Ao1.1 TATATGTATGTAAATGTGAGCAGCGGACATCATCTTCCCAACTGCATTAAGCAAAACTAAATAAAAAGAAAAGGCTTA Ao1.2 ------

Ao1.1 AGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Ao1.2 ------

Figure 4.1. Nucleotide sequence of the Ana o 1 cDNA clones Ana o 1.0101 (Ao1.1) and Ana o 1.0102 (Ao1.2). The presumed allelic difference between the 2 genes is indicated in bold type and highlighted in yellow. The presumed start and stop codons are underlined and highlighted in green and red respectively. Asterisks denote identity and dashes denote gaps. The GenBank accession numbers for Ana o 1.01.01 is AF395893 and Ana o 1.01.02 is AF395894.

Two positive clones from cashew cDNA library were identified and designated as Ana o1.0101 and Ana o1.0102 (Figure 4.1). Ana o1.0101 (AF395894) represents the longer version of the cDNAs, and Ana o1.0102 (GenBank Acc. No. AF395893) the truncated version.163 Comparison of the nucleotide sequences revealed that these differed in the length of their 3′ ends. In addition, a single nucleotide substitution (A for G) at residue 994 distinguishes Ao1.1 and Ao1.2 in their region of common overlap, suggesting that this represents an allelic difference. Analysis of the deduced amino acid sequence revealed a 540-amino-acid open-reading frame, a possible start codon at position 3, and possible leader peptide from 3 to 28 (Figure 4.2). A search of Genbank revealed that Ao1.1 and Ao1.2 encode members of the 7S (vicilin) superfamily of proteins.

Ao1.1 AIMGPPTKFSFSLFLVSVLVLCLGFALAKIDPELKQCKHQCKVQRQYDEQQKEQCVKECEKYYKEKKGRER Ao1.2 ----*******************************************************************

Ao1.1 EHEEEEEEWGTGGVDEPSTHEPAEKHLSQCMRQCERQEGGQQKQLCRFRCQERYKKERGQHNYKREDDEDE Ao1.2 ***********************************************************************

Ao1.1 DEDEAEEEDENPYVFEDEDFTTKVKTEQGKVVLLPKFTQKSKLLHALEKYRLAVLVANPQAFVVPSHMDAD Ao1.2 ***********************************************************************

Ao1.1 DSIFFVSWGRGTITKILENKRESINVRQGDIVSISSGTPFYIANNDENEKLYLVQFLRPVNLPGHFEVFHG Ao1.2 ***********************************************************************

Ao1.1 PGGENPESFYRAFSWEILEAALKTSKDTLEKLFEKQDQGTIMKASKEQIRAMSRRGEGPKIWPFTEESTGS Ao1.2 ************************************************V**********************

Ao1.1 FKLFKKDPSQSNKYGQLFEAERIDYPPLEKLDMVVSYANITKGGMSVPFYNSRATKIAIVVSGEGCVEIAC Ao1.2 ***********************************************************************

Ao1.1 PHLSSSKSSHPSYKKLRARIRKDTVFIVPAGHPFATVASGNENLEIVCFEVNAEGNIRYTLAGKKNIIKVM Ao1.2 ***********************************************************************

Ao1.1 EKEAKELAFKMEGEEVDKVFGKQDEEFFFQGPEWRKEKEGRADE Ao1.2 ********************************************

Figure 4.2. Amino acid sequence of the Ana o 1 cDNA clones Ana o 1.0101 (Ao1.1) and Ana o 1.0102 (Ao1.2). Asterisks denote identity and dashes denote gaps. The differences between the 2 aa sequences is indicated in bold type and highlighted in yellow. The predicted signal peptide is indicated in red.

Reactivity of the Recombinant Ana o 1 Protein with Human IgE and Rabbit IgG For immunologic characterization, we cloned a long version of the Ao1.1 cDNA (designated Ao1.1a) beginning (at K29) after the presumptive leader peptide (Fig. 4.2). The DNA segment was ligated into an expression vector designed to allow for purification of the recombinant molecules by way of a maltose-binding protein fusion domain in conjunction with an amylose affinity column and a thrombin-specific cleavage site. The resulting approximately 93 kDa fusion protein of Ana o 1 were affinity purified and digested with thrombin to yield approximately 55 kDa Ana o 1, as well as the 43 kDa MBP. Both cleaved and uncleaved peptides were reactive with specific human IgE and rabbit IgG. IgE from 10 of 20 sera from patients with a history of life-threatening reactions to cashews bound the rAna o 1 by means of Western immunoblotting. In 3 cases the intensity of the signal was strong, but it was weak in 7 cases, implying, although not proving, variable titers of antibody directed to this protein. In contrast, 2 of 8 sera from patients tolerant of cashew but clinically with life-threatening reactions to other tree nuts reacted with the rAna o 1. One of these (pt #22) showed weak binding (the patient self-reported mild throat scratchiness with cashew), whereas the other (pt #21) showed strong binding, yet the patient reconfirmed no symptoms on cashew ingestion but has experienced 4 emergency department visits after accidental walnut or pecan ingestion and recent strong wheal-and-flare reactions to walnut and cashew on skin prick testing.

Detection of Antigenic and Allergenic Cross-reactivity between Cashew and Pistachio Cashew and pistachio nuts belong to Anacardiaceae family and in order to investigate antigenic and allergenic cross-reactivity between cashew and pistachio, SDS-PAGE, 2-DE and immunoblotting of total pistachio extract with rabbit anti-cashew polyclonal antisera and pooled cashew and tree nut allergic patient sera was performed. Comparison of cashew and pistachio extract by SDS-PAGE followed by Coomassie Brilliant Blue staining is illustrated in Fig. 4.3. Protein bands at 6-9, 25-30, 35, 45, 55, and 66 kDa MW are seen in both the nut extracts (Fig. 4.3, lanes C and P). This pattern is consistent with the close family relationship between the two nuts as previously reported.183

MW C P 170 100 72 55 40

Figure 4.3. SDS-PAGE and Coomassie Blue stain of cashew (C) and pistachio (P) protein extract. MW= molecular weight marker.

The immunoreactivity of cashew (Fig. 4.4 A, lane 3) and pistachio (Fig. 4.4B, lane 3) proteins analyzed with rabbit anti-cashew globulin antisera demonstrated antigenic cross- reactivity between cashew and pistachio protein bands at, 20-22, 31-35 and 55 kDa MW.

A B

Vicilin-like protein

11S globulin Legumin acidic subunit 11S globulin Legumin basic subunit 2S albumin

Figure 4.4. Comparative SDS-PAGE and immunoblotting profile of (A) Cashew, and (B) Pistachio extracts. Lane 1 –SDS-PAGE, Lane 2- Immunoblot with cashew and tree nut allergic human sera, and Lane 3 – Immunoblot with rabbit polyclonal anti-cashew antiserum.

The human cashew and tree nut allergic serum demonstrated IgE reactivity to cashew (Fig. 4.4A, lane 2) and pistachio (Fig. 4.4B, lane 2) proteins respectively at bands: 9-12, 25, 31- 35, and 55 kDa MW. No reactivity was detected by the rabbit sera to the basic subunit of 11S globulins (22-27 kDa MW) in either tree nuts (Fig. 4.4 A and B, lane 3). The human IgE reactivity pattern with pistachio (Fig. 4.4B, lane 2) was similar to that of cashew (Fig. 4.4A, lane 2). Patient IgE reactivity to low MW bands at 6.5-12 kDa was detectable in both the nuts extracts, probably belonging to the 2S albumin family of seed storage proteins (Fig. 4.4A and B, lane 2).

Two-dimensional Electrophoresis (2-DE) Immunoblot of Cashew and Pistachio Proteins 2-DE, immunoblotting, and subsequent N-terminal sequencing enabled us to separate, identify, and characterize IgE-binding proteins in cashew and pistachio. Several distinct spots at ~35 and 55 kDa with pI of 5-6, 25 kDa with pI of 8-9, and ~ 6-9 kDa with pI of 3-5 were observed in both the cashew (Fig. 4.5) and pistachio (Fig. 4.6) extract protein preparations. Several immunoreactive spots having the same molecular weight but expressing different pIs were detected, thereby suggesting the existence of isoforms for these storage proteins.

MW (kDa) 200 4% 116 C N-terminal amino acid sequence 97 S 66 D Spot A – GIEETICTMRLK S 45 - Spot B – SRQEWQQQDECQI 34 P A Spot C – KIDPELKQCKHQ G E B Spot D – RQESFRQCCQ 22 E

D Spot E – RQESLRECCQQ 6.5 A 20% 3 10 pI Figure 4.5. 2-DE immunoblot of cashew extract probed with human cashew allergic sera. Proteins in the spots A, B, C, D, and E were identified based upon N-terminal amino acid sequencing of 10-12 residues and compared with known proteins in NCBI databases.

The N-terminal sequences of spots A and B (Fig. 4.3) appeared to be identical to the earlier published sequences of basic and acidic subunits of Ana o 2, the cashew 11S globulin.163 Additionally, the sequence of spot C corresponds to previously published sequence of Ana o1, the cashew 7S globulin163, and spots D and E correspond to the sequences of Ana o 3, cashew 2S albumin respectively.164 All the three allergens have been cloned from a cashew cDNA library and been identified earlier as major allergens in cashew.112 The pistachio 2-DE immunoblot (Fig. 4.6) probed with cashew and tree nut allergic patients sera is demonstrated similar reactivity patterns as observed in cashew 2-DE immunoblot (Fig. 4.5). The N-terminal amino acid sequences of IgE reactive spots from cashew and pistachio share high homology, and provided preliminary data indicative of a shared structural motif and potential cross-reactivity between these two nuts. Several spots in the acidic pI range of 4-6 and MW of 30-40, 50-55 kDa, and spots in the basic pI range of 8-9 with MW of 20-27 kDa, show IgE immunoreactivity (Fig. 4.6).

MW (kDa) 4% 200 N-terminal amino acid sequence 116 S Spot A – GLEETIXTMK 97 D Spot B – SRQQEQQQNE S F 66 B - C Spot C – GLEETFCTMTLK P G A 45 Spot D – GLEETFCTMTLKLN G 34 A E Spot E – RQESFRQCCQELQE 22 E Spot F – KTDQELKQQCK Spot G – KTDPEEKQQQ 6.5 D 20% 3 10 pI

Figure 4.6. 2-DE immunoblot of pistachio extract probed with human cashew and pistachio allergic sera. Proteins in the spots A, B, C, D, E, F, and G were identified based upon N-terminal amino acid sequencing of 10-12 residues and compared with known proteins in NCBI databases.

The acidic (spot B) and basic (spots A, C, and D) spots were identified as acidic and basic subunits of 11S globulins and revealed significant sequence similarity with the 11S

globulins from several nuts, seeds, and legumes including cashew Ana o 2, sesame seed, soybean glycinin, and almond prunin. The N-terminal aa sequence of spot E (Fig. 4.6) revealed remarkably high homology (87-94%) to 2S albumins from cashew (Ana o 3), walnut, sunflower and sesame seed. Additionally, the N-terminal aa of spots F and G demonstrated high homology to 7S globulins from cashew (Ana o 1). These studies provided information suggesting the presence of homologous cross- reactive allergens in both the nuts. Additional studies involving cloning and characterization of pistachio allergens were thus performed.

Gene Characterization of Pistachio Vicilin Pis v 3306 The 7S globulin gene was amplified from the pistachio 5` RACE cDNA by means of PCR with a degenerate forward and universal lock dock reverse primer. Subsequently, gene specific primers were used to clone the full length gene.

A 1 atgggttcgc gaacaaagtt ttgtttaact ctttttctcg tttctgtttt gattctgtgt 61 gccggtttag ctttggctaa gacagaccca gagctgaaac aatgcaagca ccagtgcaaa 121 gtccagaggc agtatgacga ggaacagaag gagcagtgtg cgaaaggatg tgaaaagtac 181 tacaaagaga agaaaggacg cgagcaagag gaagaggaag aagaggaatg gggaagcggt 241 cgcggtcggg gtgatgaatt cagcacgcat gaacccggtg aaaagcgttt gagccagtgc 301 atgaagcagt gcgagagaca agacggaggg cagcagaagc agctgtgccg cttcaggtgt 361 caggagaagt ataagaaaga gagaagagaa catagttaca gtagagacga agaagaggaa 421 gaggaaggcg atgaggaaca agaggaagaa gatgagaatc cttacgtatt tgaagacgaa 481 catttcacca ccagagtcaa gaccgaacaa ggaaaagttg ttgttcttcc caagttcact 541 aaacgatcaa agcttctccg tggcctggag aaataccgtc tggcctttct tgtcgctaat 601 cctcaagctt ttgtagttcc aaaccacatg gatgctgaca gtattttctt tgtttcctgg 661 ggacgaggaa caatcaccaa gattcgtgag aataagagag agagcatgaa cgtcaaacag 721 ggagatataa ttaggattcg tgctggtact cctttttata tcgtcaatac cgatgaaaat 781 gagaagcttt acattgtcaa actccttcaa cccgtcaatc ttcctggcca ttacgaagta 841 tttcatggac caggaggtga aaacccagag tcgttctaca gagctttcag cagggaagta 901 ctcgaagccg ctctgaagac tccaagggac aaactggaga aattgttcga gaaacaggac 961 gagggagcca tcgtaaaagc ctccaaagaa caaattcggg ctatgagccg gaggggtgaa 1021 ggtcctagca tttggccatt tacagggaaa tcaacgggta cattcaatct cttcaaaaag 1081 gatccctctc aatccaataa ctatggccaa ctc tttgaaa gcgaattcaa agattatccg 1141 ccactccaag agctcgacat tatggtctct tatgtcaaca tcaccaaggg aggaatgtca 1201 ggtccattct acaactcaag ggcaacgaag atagccattg ttgtttcagg agagggacgc 1261 cttgaaatag cctgccctca cctctcctct tccaaaaact caggccagga aaaaagtggc 1321 ccgagttaca agaaattaag ctcgagtatc agaaccgatt cagtgttcgt tgtcccggcg 1381 ggtcaccctt ttgtcaccgt tgcttctgga aaccaaaact tggaaatcct ctgttttgaa 1441 gttaatgcag aaggaaatat caggtatact cttgctggga agaagaacat tatagaggtg 1501 atggagaagg aagcgaaaga attggcattt aaaacgaaag gagagga ggt ggacaaagtg 1561 tttggaaaac aagatgaaga gttcttcttc caggggccga aatggcgaca acatcaacaa 1621 gggcgtgctg atgaatga

B 1 MGSRTKFCLTLFLVSVLILCAGLALAKTDPELKQCKHQCKVQRQYDEEQKEQCAKGCEKY 61 YKEKKGREQEEEEEEEWGSGRGRGDEFSTHEPGEKRLSQCMKQCERQDGGQQKQLCRFRC 121 QEKYKKERREHSYSRDEEEEEEGDEEQEEEDENPYVFEDEHFTTRVKTEQGKVVVLPKFT 181 KRSKLLRGLEKYRLAFLVANPQAFVVPNHMDADSIFFVSWGRGTITKIRENKRESMNVKQ 241 GDIIRIRAGTPFYIVNTDENEKLYIVKLLQPVNLPGHYEVFHGPGGENPESFYRAFSREV 301 LEAALKTPRDKLEKLFEKQDEGAIVKASKEQIRAMSRRGEGPSIWPFTGKSTGTFNLFKK 361 DPSQSNNYGQLFESEFKDYPPLQELDIMVSYVNITKGGMSGPFYNSRATKIAIVVSGEGR 421 LEIACPHLSSSKNSGQEKSGPSYKKLSSSIRTDSVFVVPAGHPFVTVASGNQNLEILCFE 481 VNAEGNIRYTLAGKKNIIEVMEKEAKELAFKTKGEEVDKVFGKQDEEFFFQGPKWRQHQQ 541 GRADE

Figure 4.7. Nucleotide and derived amino acid sequence of Pis v 3 cDNA. (A) Nucleotide sequence (GenBank accession no. EF116865), and (B) amino acid sequence of the Pis v 3 coding region (GenBank accession no. ABO36677). The predicted signal peptide is indicated in red.

The resulting 1560-bp PCR product (GenBank Acc. No. EF116865) shown in Figure 4.7A encodes a 519-aa protein designated Pis v 3 (Fig. 4.7B). The SignalP program (www.expasy.org, Swiss Institute of Bioinformatics, 4056 Basel, Switzerland) was used to identify a 26 aa presumptive signal sequence (in red, Fig. 4.7B). Comparison of the aa sequence with the NCBI database using BLAST analysis identified homology with other members of the 7S globulin family of seed storage proteins, several of which are known food allergens (Table 4.2). In line with the familial relationship between pistachio and cashew, their respective vicilins are 90% aa sequence similar and 80% identical. In contrast, the aa sequence comparisons with the 10 nut and seed proteins listed in Table 4.2 revealed only 51-72% similarity and 31-55% identity to pistachio vicilin.

Table 4.2. Vicilins demonstrating the greatest homology to Pis v 3.306

Protein Organism Accession % % Ref. Allergen Description No. Identity Similarity Designation Vicilin-like Anacardium AAM73730 80 90 163 Ana o 1 protein occidentale (cashew) 7S globulin Sesamum AAK15089 47 65 168 Ses i 3 indicum (sesame seed) 48-kDa Corylus AAL86739 55 72 66 Cor a 11 glycoprotein avellana precursor (hazelnut)

Sucrose Glycine max AAF05723 46 65 binding (soy bean)

protein homolog S-64 Sucrose- Glycine max AAO48716 46 65 binding (soy bean) protein 2

Vicilin Macadamia AAD54244 39 60 193;194 precursor integrifolia

Table 4.2. – continued Protein Organism Accession % % Ref. Allergen Description No. Identity Similarity Designation Vicilin seed Juglans AAM54366 40 63 195 Jug n 2 storage nigra protein (black walnut) Vicilin-like Juglans AAF18269 41 65 142 Jug r 2 protein regia precursor (English walnut) 7S seed Arachis AAL27476 31 51 165 Ara h 1 storage hypogaea protein (peanut) (vicilin)

Epitope mapping of Ana o 1 with pooled cashew allergic human sera Overlapping solid-phase synthetic peptides spanning the full length of Ana o 1 aa sequence were probed with rAna o 1–reactive sera from 12 patients randomly assigned to 3 pools.

Table 4.3. IgE- reactive linear epitopes identified on Ana o 1.111

Epitope Amino acid sequence* Position Pool reactivity † No. Pool 1 Pool 2 Pool 3 1 AIMGPPTKFSFSLFL 1-15 ++ ++ + 2 CKVQRQYDEQQKEQC 41-55 + - - 3 EQQKEQCVKECEKYY 49-53 + - - 4 KECEKYYKEKKGRER 57-71 +++ +++ ++ 5 EKKGREREHEEEEEE 65-79 - ++ - 6 DEAEEEDENPYVFED 145-159 +++ - - 7 RRGEGPKIWPFTEES 337-351 ++ - - 8 NITKGGMSVPFYNSR 393-407 + - - 9 TKIAIVVSGEGCVEI 409-423 + - - 10 SSHPSYKKLRARIRK 433-447 + - - 11 EEFFFQGPEWRKEKE 521-535 + +++ + + = reactivity, - = no reactivity

Collectively, the 3 pools reacted with 11 linear IgE-binding epitopes, which were distributed throughout the entire length of the protein (Table 4.3).111 Three of the identified epitopes (highlighted in bold) were bound by patient sera from all 3 pools and are thus referred as immunodominant epitopes.

Comparison of the Pis v 3 Protein Sequence to that of Ana o 1 and its IgE-reactive Peptides A sequence alignment of the pistachio vicilin, Pis v 3 and the cashew homologue, Ana o 1, was used to evaluate homology. In addition, compare the aa sequence of the 10 known cashew IgE binding peptides111 with the corresponding aa sequence on pistachio (Fig. 4.8). As described above, the sequence comparison reveals 80% overall aa identity and 90% similarity. Of the two peptides previously identified to contain immunodominant epitopes in rAna o 1, peptide #3 had 13 of 15 identical residues and one similar residue, and peptide #10 had 8 of 15 identical residues and 4 similar residues.

4 3 2 1 A o 1 1 KIDPELKQCKHQCKVQRQYDEQQKEQCVKECEKYYKEKKGREREHEEEEEEWGTGGV--D 58 P v 3 1 *T*******************|*****A*G************|**********|*RGRG* 59

A o 1 59 EPSTHEPAEKHLSQCMRQCERQEGGQQKQLCRFRCQERYKKERGQHNYKREDDEDEDEDE 118 P v 3 60 *F*****G********|*****|**************|*****R|*|*S*|||*|*|G**5 119 A o 1 119 A-EEEDENPYVFEDEDFTTKVKTEQGKVVLLPKFTQKSKLLHALEKYRLAVLVANPQAFV 177 P v 3 120 EQ*************H***|*********|*****||****RG*******F********* 179

A o 1 238 VQFLRPVNLPGHFEVFHGPGGENPESFYRAFSWEILEAALKTSKDTLEKLFEKQDQGTIM 297 P v 3 240 |*L*|*******|*******************R*|*******P|*K*********|*A*|6 299 A o 1 298 KASKEQIRAMSRRGEGPKIWPFTEESTGSFKLFKKDPSQSNKYGQLFEAERIDYPPLEKL 357 P v 3 300 *****************S*****G|***|*N**********N******|*FK*****||* 359 7 8 9 A o 1 358 DMVVSYANITKGGMSVPFYNSRATKIAIVVSGEGCVEIACPHLSSSKS-----SHPSYKK 412 P v 3 360 *||***V********G******************R|***********|SGQEK*G***** 419

A o 1 413 LRARIRKDTVFIVPAGHPFATVASGNENLEIVCFEVNAEGNIRYTLAGKKNIIKVMEKEA 472 P v 3 420 *S|S**T*|**|*******V******|****|*********************|****** 479 10

A o 1 473 KELAFKMEGEEVDKVFGKQDEEFFFQGPEWRKEKEGRADE 512 P v 3 480 ******T|********************|WR|H||***** 519

Figure 4.8. Sequence alignment of rAna o 1 and rPis v 3. The boxed sequences indicate linear epitope-bearing peptides previously identified on Ana o 1.163 Peptides 3 and 10 (highlighted in red) contain immunodominant epitopes. “*” = identical amino acid, “|” = similar amino acid.

All of the variant amino acids were clustered at the C-terminal end of peptide #10 leaving the N-terminal end, which is 100% identical, as a potential source of cross-reactivity. Sequence comparisons of the other epitope sites show similar degrees of homology with the exception of peptide #9 where minimal homology is evident. The high degree of sequence homology between the two allergens suggests the likelihood of considerable allergenic cross-reactivity and prompted additional empirical studies.

Immunoreactivity of Recombinant Proteins with Human IgE IgE reactivity to rAna o 1 and rPis v 3 was detected using 20 patients’ sera: 12 cashew- and pistachio-allergic, five cashew-allergic but who had never eaten pistachios, one pistachio allergic but had never eaten cashew, one only pistachio-allergic, and one who had never eaten pistachio or cashew (Table 4.1). Of the 14 pistachio-allergic patients (#’s 1, 3, 5, 11, 13, 14, 29, 30, 32, 35, 46, 47, 48, and 50), five (36%) showed IgE reactivity to rPis v 3 by dot-blot (Fig. 4.9). Interestingly, of the five cashew allergic patients (#’s 7, 9, 12, 20, and 33) who report that they had never eaten pistachio, two (50%) showed IgE reactivity to rPis v 3 by dot-blot. Each of the seven rPis v 3-reactive sera (patient #’s 3, 9, 13, 14, 33, 35, and 48) also reacted positively to rAna o 1. Only patient #47 was reactive to the cashew rAna o 1 alone and not to the pistachio homolog (Fig. 4.9).

Patient # 33 # 14 # 9 # 35 # 3 # 11

rPis v 3

rAna o 1

Figure 4.9. IgE reactivity of cashew-allergic patients’ sera to dot-blot containing rAna o 1 and rPis v 3 (sera showing no signals not shown).306

To investigate further the potential IgE cross-reactivity relationship, inhibition dot blot was performed, wherein soluble rAna o 1 or rPis v 3 served as inhibitors to inhibit the reaction

between patients’ sera and solid phase bound recombinant cashew and pistachio 7S globulins (Fig. 4.10).

35 35 35 9 9 9 14 14 14 (-) (+Pv3) (+Ao1) (-) (+Pv3) (+Ao1) (-)(+Pv3)(+Ao1) NC rPis v 3

rAna o 1

Figure 4.10. Inhibition dot-blot with recombinant allergens in which patients’ sera #35, #9, and #14 were pre-incubated with either rPis v 3 or rAna o 1.306

The IgE reactivity of patient #35, 9, and 14 demonstrated significant cross-reactivity between the homologous vicilin proteins (Fig. 4.10). In order to identify the native vicilin-like protein band(s) in pistachio protein nut extract, an inhibition immunoblot was performed using rPis v3 and rAna o 1 as inhibitors in conjunction with a pool of patient sera (# 3, 9, and 14).

MW kDa + + Un rPv3 rAo1 NC 170 135 100

72 55

40 33

Figure 4.11. Inhibition immunoblot of pistachio protein extract probed with a pool of patient sera (# 3, 9, and 14) either unabsorbed (U), pre-absorbed with rPis v 3 (+rPv3), or with rAna o 1 (+rAo1). NC = negative control. Putative native Pis v 3 band indicated by an arrow.306

Pre-incubation of sera with either inhibitor showed inhibition of IgE binding to a 45 kDa band in the pistachio nut extract indicating that this band represents the native vicilin-like protein (Fig. 4.11).

Reactivity of Anti-Cashew rAna o 1 mAbs with rAna o 1 and rPis v 3 The above described sequence alignment and specific IgE binding data revealed a high degree of homology between the cashew and pistachio vicilin. To further assess the nature of the cross-reactivity between cashew and pistachio vicilin proteins, a panel of mouse IgG mAbs, previously generated against cashew rAna o 1, was assayed. Of the nine anti-rAna o 1 mAbs tested, six (67%) also recognized rPis v 3 to varying degrees on dot-blots (Fig. 4.12) indicating considerable epitope homology between rPis v 3 and rAna o 1.

Figure 4.12. Dot-blot analysis of anti-cashew rAna o 1 mAbs probed with rPis v 3 and rAna o 1. NC - negative control - no primary antibody.306

Linear epitope mapping of Ana o 1 reactive mAbs identified two linear epitopes for mAbs 1A7 and 2G7. The linear mAb 1A7 recognizes CKVQRQYDEQQKEQC and 2G7 recognizes HEEEEEEWGTGGVDE peptides, respectively. The other tested mAbs mentioned above are probably targeted against discontinuous or conformation dependent epitopes.

Gene Characterization of Cashew 11S globulin Ana o 2112 The cashew cDNA library was screened for reactivity with both cashew extract-specific rabbit IgG and cashew-allergic human IgE. An immunoreactive clone was selected for

sequencing and identified to encode for 11S globulin and is designated as Ana o 2. The nucleotide and derived amino acid sequence analysis of Ana o 2 cDNA clone is illustrated in Figure 4.13. Analysis of the nucleotide (Fig. 4.10A) and deduced amino acid sequence (Fig. 4.13B) revealed a 457-amino acid open reading frame with a predicted leader peptide from 1 to 15.112

Of our previously reported N-terminal and tryptic peptide sequences for several native IgE- reactive cashew proteins, four were found to have similarity to the aa of Ana o 2.60;112 Considerable aa sequence and structural homology is observed among the 11S globulin family of seed storage proteins and are implicated as major allergens in several nuts, seeds and legumes.112

A 1 TCTTTCTGTTTGCTTTTTAATTCTCTTTCATGGTTGCCTAGCTTCTCGCCAGGAATGGCAACAACAAGATGAGTGCCAAA 81 TCGATAGGCTGGATGCCCTTGAACCCGATAACCGAGTTGAGTATGAAGCCGGTACGGTGGAAGCCTGGGATCCTAACCAT 161 GAGCAATTCCGATGCGCTGGTGTTGCCTTGGTTAGGCATACCATCCAACCTAATGGCCTTCTCTTGCCTCAATATTCTAA 241 TGCTCCTCAACTTATTTACGTTGTCCAGGGTGAGGGTATGACAGGAATATCATATCCAGGATGCCCAGAAACTTACCAAG 321 CGCCCCAACAGGGACGACAACAGGGACAGAGTGGTAGGTTCCAGGACCGGCATCAAAAGATTCGACGCTTCCGTCGAGGC 401 GATATCATCGCAATCCCCGCCGGAGTAGCACACTGGTGCTACAACGAGGGCAATTCCCCGGTCGTCACTGTTACTCTTCT 481 AGACGTCTCAAACAGTCAAAATCAGCTTGATAGGACCCCACGAAAATTCCATCTGGCTGGTAACCCAAAAGATGTGTTCC 561 AGCAGCAGCAACAACACCAATCTCGCGGGCGTAACCTTTTTTCTGGCTTCGATACAGAGTTATTGGCTGAGGCTTTCCAA 641 GTGGACGAACGTCTCATAAAGCAGCTCAAAAGCGAGGACAACAGGGGTGGCATTGTTAAGGTGAAGGATGACGAACTTCG 721 GGTGATCCGCCCATCAAGGAGTCAGAGCGAGCGTGGAAGTGAGAGTGAAGAGGAAAGTGAGGATGAAAAACGCCGATGGG 801 GACAGCGTGACAATGGGATTGAAGAGACCATTTGCACTATGAGACTCAAAGAGAATATCAATGATCCTGCTCGCGCTGAC 881 ATTTACACCCCAGAAGTCGGTCGTCTTACCACACTCAACAGCCTCAACCTCCCAATCCTCAAATGGCTTCAACTCAGTGT 961 TGAAAAGGGTGTGCTATACAAAAATGCTCTAGTGCTGCCACACTGGAACCTCAACTCGCACAGCATAATATACGGGTGCA 1041 AGGGTAAAGGCCAGGTTCAAGTAGTAGACAACTTCGGCAACAGAGTGTTCGACGGCGAAGTCCGCGAGGGACAGATGTTG 1121 GTGGTGCCACAAAACTTTGCAGTAGTGAAACGTGCAAGAGAGGAAAGATTCGAATGGATTTCTTTCAAGACCAATGATCG 1201 GGCCATGACGAGTCCTCTCGCTGGACGCACCTCGGTGCTTGGTGGCATGCCAGAGGAAGTGTTAGCCAATGCGTTCCAGA 1281 TCTCAAGAGAAGATGCTAGGAAGATCAAGTTCAACAATCAGCAGACAACTTTGACAAGTGGAGAGTCAAGCCACCACATG 1361 AGGGATGATGCTTAAATTTTAAGTAATTTGAGCTGAGCTAGTGGTGATTTAAAGCCGAATGCATGTGGTGTACGTACTAT 1440 GTTTTTTGTTTTGCTTTGTAAGGGGGATAGGTAATGAATAATAAAGGAGAGCTTGGATAGTCTCTGCTGTGAGAGGGGAG 1521 AAGAAAGCAGGGAGCAGAGAGCAGAGAGCTTGTATGTAGTTAAGTTAATATTACTACTACTACTACTACGATGTGAATGA 1601 ACTCTTGATGAGTTCTGTCCAATAAAAAACTACTTTTCCTACTCAAAAAAAAAAAAAAAAAAAAAAAAAAA

B 1 LSVCFLILFH GCLASRQEWQ QQDECQIDRL DALEPDNRVE YEAGTVEAWD PNHEQFRCAG 61 VALVRHTIQP NGLLLPQYSN APQLIYVVQG EGMTGISYPG CPETYQAPQQ GRQQGQSGRF 121 QDRHQKIRRF RRGDIIAIPA GVAHWCYNEG NSPVVTVTLL DVSNSQNQLD RTPRKFHLAG 181 NPKDVFQQQQ QHQSRGRNLF SGFDTELLAE AFQVDERLIK QLKSEDNRGG IVKVKDDELR 241 VIRPSRSQSE RGSESEEESE DEKRRWGQRD NGIEETICTM RLKENINDPA RADIYTPEVG 301 RLTTLNSLNL PILKWLQLSV EKGVLYKNAL VLPHWNLNSH SIIYGCKGKG QVQVVDNFGN 361 RVFDGEVREG QMLVVPQNFA VVKRAREERF EWISFKTNDR AMTSPLAGRT SVLGGMPEEV 421 LANAFQISRE DARKIKFNNQ QTTLTSGESS HHMRDDA

Figure 4.13. Nucleotide and derived amino acid sequence of Ana o 2 cDNA. (A) Nucleotide sequence (GenBank accession no. AF453947), and (B) amino acid sequence of the Ana o 2

coding region (GenBank accession no. AAN76862). The predicted signal peptide is indicated in red.

Comparison of the aa sequence with the NCBI database using BLAST analysis identified homology with other members of the 11S globulin family of seed storage proteins, several of which are known food allergens (Table 4.4) and includes proteins described as legumin-like, legumin precursor, legumin A precursor, 11S globulin and 11S globulin β-subunit precursor, representing a diversity of tree and plant species.

Table 4.4. Sequences demonstrating the greatest homology to Ana o 2.

Protein Organism Accession AA1 % % Publication/ No. Overlap2 Report Identity Similarity Ricinus communis Sherarer Legumin-like protein (castor bean) AAF73007 1-446 58 74 (unpubl. results) Juglans regia 11S globulin (English walnut) AAW29810 4-445 57 73 144 Corylus avellana 11S globulin (hazelnut) AF449424 2-444 55 71 173 Quercus robur Legumin precursor (English Oak) CAA67879 2-445 55 72 Fischer (unpubl. results) Sesamum indicum 196;302 11S globulin (sesame) ABB60054 20-451 51 69 Amaranthus 11S globulin hypochondriacus S49422 1-454 48 68 Barba de la Rose 1986 (grain amaranth) (unpubl. results) Bertholletia excelsa 11S globulin (Brazil nut) AAO38859 1-457 48 65 Beyer (unpubl. results) Glycine max Glycinin G1 subunit (soybean) AAB23209 2-439 47 66 197 Prunus dulcis Pru2 Prunin (almond) CAA55010 4-436 47 66 86 Coffea arabica 11S Globulin (coffee) AAC61881 1-457 46 64 198 Vicia faba Legumin A precursor (tick bean) CAA38758 1-439 45 65 197 Lupinus albus Legumin-like protein (white lupine) CAI83773 11-439 43 61 307 Arachis hypogaea Glycinin Ara h 3 (peanut) AF093541 21-439 43 59 59 Arachis hypogaea Glycinin Ara h 4 (peanut) AF086821 4-439 42 58 134

AA1= Amino acid, Overlap2 = Ana o 2 residue numbers

For immunological characterization, we cloned and expressed Ana o 2 from nucleotide 47 (after the presumptive leader peptide) through to nucleotide 1375. The DNA segment was ligated into modified pMAL-c2 expression vector (New England BioLabs Inc., Beverly, MA, USA), designed to allow for purification of the recombinant molecule via an MBP fusion domain in conjunction with an amylose affinity column and a thrombin-specific cleavage site. The resulting approximately 93 kDa Ana o 2 fusion protein was affinity purified as previously described112;163

Identification of rAna o 2 as an Allergen Twenty one cashew allergic sera were used to study the IgE immunoreactivity to the rAna o 2 by western immunoblotting. IgE from 13 of these 21 sera (62%) reacted positively with the recombinant cashew protein.112 The intensity of the signal was strong in six cases, but with the remaining seven it was weak. Of the control sera used that were tolerant to cashews but were allergic to other tree nuts, none reacted with rAna o 2.112

Table 4.5. Linear IgE reactive epitopes of Ana o 2 identified by SPOTs technology.112

Each pool consisted of sera from 4 patients and included sera from at least 1 patient that reacted strongly to the rAna o 2 in our Western blots and from 3 other patients with varying degrees of reactivity. Collectively, the three pools reacted weakly (phosphoimaging score from 2.0 - 3.9 x 10–3) with 12, moderately (4.0 - 6.9 x 10–3) with 3, and strongly (>7.0 x 10–3) with 7 linear IgE-binding epitopes. The 22 IgE reactive epitopes were distributed throughout the entire length of the Ana o 2 protein (Table 4.5).

Gene characterization of pistachio 11S globulin Pis v 2 The 11S globulin gene was amplified from the pistachio 5` RACE cDNA by means of PCR with a degenerate forward and universal lock dock reverse primer. Subsequently, gene specific primers were used to clone the full length gene. The resulting 1419 bp PCR product (GenBank Acc. No. EF562454) shown in Figure 4.14A encodes a 472 aa protein designated Pis v 2 (Fig. 4.14B). The SignalP program (www.expasy.org, Swiss Institute of Bioinformatics, 4056 Basel, Switzerland) was used to identify a 22 aa presumptive signal sequence (in red, Fig. 4.14B). In line with the familial relationship between pistachio and cashew, their respective 11S globulins are 66% similar and 50% identical at the aa sequence level. In addition, the pistachio 11S globulin is remarkably homologous with other members of the legumin family.

A 1 atgggttact cttctttgct ctcttttagt cttggctttc ttcttctctt 51 tcattgcagt tttgctcaga tagaacaggt tgtaaattca cagcaacgac 101 aacagcagca acgcttccag actcaatgcc aaatccagaa cctcaatgct 151 cttgaaccca agaggaggat tgaatctgag gccggtgtta ccgagttctg 201 ggaccagaat gaggaacaac tacaatgcgc taatgtcgct gtgtttcgcc 251 acactatcca gagcagagga ctcttagtcc cttcatacaa caatgctcct 301 gagctagtct acgttgttca aggtagtggc attcatggag ctgtattccc 351 aggttgtcct gagacattcc aagaagaatc acagtcacag tcacggtcac 401 agcactcaag atcagaaagg agtcagcaat caggagaaca gcaccaaaag 451 gtcaggcata tccgggaggg tgatatcatt gcattgcctg caggagtagc 501 tcactggatt tataacaatg gccagtccaa gcttgttttg gtcgcacttg 551 cggacgttgg caattccgag aaccagctcg accagtacct taggaaattc 601 gtccttggtg gaagcccgca acaagaaatt caaggcggtg gtcagagctg 651 gagccaaagc cgaagcagca gaaaaggcca gcaaagcaac aatattttga 701 gcgcctttga cgaggagata ctcgcacaat ctttaaacat tgacactcag 751 ctagtcaaaa aattgcagag agaggagaag cagaggggca ttattgtcag 801 agtcaaggag gatcttcagg tactatcacc acagagacaa gaaaaagaat 851 actccgataa tgggttagag gaaaccttct gcacaatgac gctgaagctc 901 aacatcaatg acccatcacg cgctgacgtc tataacccac gtggtggacg 951 cgttaccagc atcaatgcct taaaccttcc catcctcaga ttcctccagc 1001 ttagcgttga gaaaggcgtg ctttaccaga atgctatcat ggcaccacac 1051 tggaacatga atgcccacag catagtgtac atcacaagag gcaacggcag 1101 gatgcaaatt gtctcggaga atggagaatc cgtattcgac gaggaaattc 1151 gtgagggtca gctggtggtt gttccacaaa attttgcagt ggtgaagaga 1201 gcaagcagcg atggattcga atgggtatca ttcaagacca acggacttgc 1251 caagattagc cagctggctg gacgtatctc agtgatgaga ggactaccac 1301 tggatgtgat acagaactcg tttgatattt ccagggagga tgcctggaac 1351 ttgaaggaga gtaggtctga gatgacaatt tttgctccag gttcaagatc 1401 acagaggcag agaaactaa B 1 MGYSSLLSFS LGFLLLFHCS FAQIEQVVNS QQRQQQQRFQ TQCQIQNLNA LEPKRRIESE 61 AGVTEFWDQN EEQLQCANVA VFRHTIQSRG LLVPSYNNAP ELVYVVQGSG IHGAVFPGCP 121 ETFQEESQSQ SRSQHSRSER SQQSGEQHQK VRHIREGDII ALPAGVAHWI YNNGQSKLVL 181 VALADVGNSE NQLDQYLRKF VLGGSPQQEI QGGGQSWSQS RSSRKGQQSN NILSAFDEEI 241 LAQSLNIDTQ LVKKLQREEK QRGIIVRVKE DLQVLSPQRQ EKEYSDNGLE ETFCTMTLKL 301 NINDPSRADV YNPRGGRVTS INALNLPILR FLQLSVEKGV LYQNAIMAPH WNMNAHSIVY 361 ITRGNGRMQI VSENGESVFD EEIREGQLVV VPQNFAVVKR ASSDGFEWVS FKTNGLAKIS 421 QLAGRISVMR GLPLDVIQNS FDISREDAWN LKESRSEMTI FAPGSRSQRQ RN

Figure 4.14. Nucleotide and derived amino acid sequence of Pis v 2 cDNA. (A) Nucleotide sequence (GenBank accession no. EF562454), and (B) amino acid sequence of the Ana o 2 coding region (GenBank accession no. ABU42022). The predicted signal peptide is indicated in red.

Comparison of the Pis v 2 aa sequence to that of Ana o 2 and its IgE-reactive linear peptides A sequence alignment of the pistachio 11S globulin, Pis v 2 and the cashew homologue, Ana o 2, was used to evaluate homology. In addition, the aa sequence of the 22 IgE binding

peptides identified on Ana o 2112 was compared with the corresponding aa sequence on pistachio Pis v 2 (Fig. 4.15).

4 3 2 1 Ana o 2 SVCFLILFHGCLA------SRQEWQQQ---DECQIDRLDALEPDNRVEYEAGTVEAWDPN 52 Pis v 2 *|G**|***CSF*QIEQVVN*|*RQ***RFQT|***QN*|****KR*|*S***VT*F**Q* 62 5 6 Ana o 2 HEQFRCAGVALVRHTIQPNGLLLPQYSNAPQLIYVVQGEGMTGISYPGCPETYQAPQQGRQ 113 Pis v 2 E**L|**N**|F*****SR***|*S*|***|*|*****S*|H*AV|******|*EES*S|S 124 8 7 9 Ana o 2 QGQSGRFQDR------HQKIRRFRRGDIIAIPAGVAHWCYNEGNSPVVTVTLLDVSNSQNQ 168 Pis v 2 |S*HS*S|RSQQSGEQ***|*HI*E*****|*******I**N*Q*K|*L*A*A**G**|** 184 10 11 12 Ana o 2 LDRTPRKFHLAGNPKDVFQQQQQHQSRGR------NLFSGFDTELLAEAFQVDERLIK 220 Pis v 2 **|YL***V*G*|*|QEI*GGGQSW*QS|SRRKGQQSN*|L*A**E*|**||LN|*T|*|* 245 14 12 13 15 Ana o 2 QLKSEDN-RGGIVKVKDDELRVIRPSRSQSERGSESEEESEDEKRRWGQRDNGIEETICTM 280 Pis v 2 |*|R*|KQ**I**|**|*-*|*|S*Q*Q|K*YS------***|***F*** 288 18 16 17 Ana o 2 RLKENINDPARADIYTPEVGRLTTLNSLNLPILKWLQLSVEKGVLYKNALVLPHWNLNSHS 341 Pis v 2 I**L*****|***|*N*RG**|*||*|******||***********|**||A****|*|** 349 20 18 19 Ana o 2 IIYGCKGKGQVQVVDNFGNRVFDGEVREGQMLVVPQNFAVVKRAREERFEWISFKTNDRAM 402 Pis v 2 *|*IT|*N*||*|*SEN*ES***E*|****||************SS|G***|*****GL*K 410 22 21 Ana o 2 TSPLAGRTSVLGGMPEEVLANAFQISREDARKIKFNNQQTTL-TSGESSHHMRD 455 Pis v 2 I*Q****I**|R*|*L|*|Q*|*D******WN|*E|RS|MT|FAP*SR*QRQ*| 464

Figure 4.15. Sequence alignment of rAna o 2 and rPis v 2. The boxed sequences indicate linear epitope-bearing peptides previously identified on Ana o 2.112 Peptides #3, #6, #11, #13, #14, #15, and #22 (highlighted in red) contain immunodominant epitopes. “*” = identical amino acid, “|” = similar amino acid, “---” = gap.

As described above, the sequence comparison reveals 50% overall aa identity and 66% similarity. Of the seven peptides composed of 98 aa, previously identified to contain immunodominant epitopes in rAna o 2, 34 aa residues were identical, 14 aa were similar to residues in Pis v 2. Marked differences were observed with Ana o 2 peptides and Pis v 2, especially peptide # 11 and #15. With peptide #11, a gap was observed and was encompassed

with 9 aa in Pis v 2. Similarly a similar gap was seen in Pis v 2 in comparison with Ana o 2 peptide # 15, demonstrating total absence of this immunodominat epitope in Pis v 2.

Monoclonal Antibody Production and Ana o 2 Reactivity BALB/c mice were immunized with whole cashew extract to generate mAbs. These antibodies were tested for reactivity against the recombinant proteins Ana o 1 and Ana o 2. Based on this information, we identified about 8 clones that were reactive to rAna o 2 and did not cross react to Ana o 1. We chose to use six of the strongly reactive mAbs for future experiments; 1F5, 2B5, 4C3, 4F5, 5G10, and 4H9. These six aforementioned mAbs were used for determining the corresponding linear epitopes by SPOTs assay consisting of 58 overlapping 15-mer peptides offset by 8 amino acids. Two peptides (#6 and #33) were found to bind to the six pooled rAna o 2 reactive mAbs. The sequences of the 2 reactive peptides are as follows: Peptide #6 --YEAGTVEAWDPNHEQ; Peptide #33-- EESEDEKRRWGQRDN. In order to determine which mAbs within the pool were actually recognizing the 2 reactive peptides, strips containing the peptide #6 and #33 were synthesized and each of the 6 mAbs were tested individually. Peptide #6 was recognized by the mAb 4H9 and peptide #33 by mAb 1F5. Peptide #33 was found to be an immunodominant epitope -- bound strongly by all 3 pools of Ana o 2 allergenic patient sera. Peptide #33 was thus selected for alanine scanning mutagenesis on the sequence in order to define the amino acids critical for antibody reactivity. Furthermore, this approach was used to determine whether the amino acids that we found to be critical for human IgE binding were also those that are involved in mouse IgG (mAb 1F5) binding. The critical amino acids for both the human IgE and mAb 1F5 were comparable (with some additional aa), thus demonstrating conserved binding reactivity. Since mAbs 4F5, 2B5, 5G10, and 4C3 were part of the pool that did not recognize these peptides individually, it was determined that they do not bind linear epitopes. Additionally, these 4 mAbs did not show reactivity to rAna o 2 and cashew extract on Western blot performed under reducing conditions, suggesting that they must be directed towards some conformational dependent or discontinuous binding epitopes.

Dot Blot reactivity of anti-cashew rAna o 2 mAbs with rAna o 2 and rPis v 2 The six above mentioned cashew Ana o 2-specific mAbs were tested for reactivity with homologous pistachio rPis v 2. The dot blot results are shown in Figure 4.16. Of the 6 tested Ana o 2 reactive mAbs 4H9 and 5G10 demonstrated reactivity to both Ana o 2 and Pis v 2 proteins.

4H91F5 2B5 4C3 5G10 4F5

Pis v 2

Ana o 2

Figure 4.16. Dot-blot immunoblotting of rAna o 2 and rPis v 2 with Ana o 2 specific mAbs.

As mentioned above, the mAb 4H9 recognizes a linear epitope (peptide #6) on Ana o 2 and mAb 5G10 a conformational epitope (unidentified as yet). There is 60% identity between the aa sequence of peptide #6 on Ana o 2 and corresponding aa sequence on Pis v 2. The mAb 1F5 reactive against a linear epitope (peptide #33) on Ana o 2 did not show reactivity with rPis v 2. Comparison of the Pis v 2 aa sequence demonstrated the absence of this analogous sequence, thus explaining the lack of 1F5 reactivity. Additionally, mAb 2B5 reactive against a conformational epitope in Ana o 2, recognizes rAna o 2 and not rPis v2. Interestingly, 2B5 is reactive with total pistachio extract (data not shown) suggesting the presence of other 11S globulin isoforms.

Discussion It has been estimated that 30% of patients who are allergic to at least one food in the nut group are allergic to several tree nuts.160 A pattern that is frequent in food-allergic individuals can be as a result of several independent sensitization events or due to allergenic cross-reactivity. Deciphering the patterns of allergic cross-reactivities are important because they may reflect (a)

the pattern of clinical sensitivities, (b) regulation of allergic sensitization via B- and T-cell epitope cross-reactivity, and (c) the need for screening of novel foods for potential allergenicity. Considerable research has been conducted in recent years in an attempt to characterize the allergens that are responsible for allergy sensitization and triggering. This information has not only helped to define the proteins that are directly responsible for food allergies, but has also revealed structural relationships between allergens including, in some cases, the structural basis for allergen cross-reactivity.18;19;74;146;162;199 A large proportion of patients with pollen allergies show hypersensitivity after eating fruits or vegetables.16 In accordance, the allergenic cross- reactivity of the major allergens of birch pollen (Bet v 1), sweet cherry (Pru a 1), apple (Mal d 1), pear (Pyr c 1), celery tuber (Api g 1), and carrot (Dau c 1) were traced to high structural and amino acid sequence similarities.200 Likewise, high sequence homology is related to pan- allergenicity as observed with lipid transfer proteins 63, and profilins.105;201 Cross-reactivity between allergens occurs when IgE originally primed against one allergen recognizes and binds to a structurally similar protein from a different source even in the absence of prior exposure to the cross-reacting agent.74;199 The FAO/WHO guidelines for the assessment of potential cross-reactive allergenicity of food proteins include sequence comparison and homology search against protein databases and multiple sequence analysis.299 Accordingly, cross-reactivity has to be considered if there is >35% homology with any known allergen across any 80-amino acid window of a query protein, or if there is an identity of six or more contiguous amino acids. The greater use of tree nuts in the food industry raises concerns for tree nut allergy and its impact on people with known or latent allergies to peanuts and other tree nuts. Patients allergic to cashew often report allergy to pistachio as well, which is likely as a result of cross-reactivity between the two closely related tree nuts, belonging to the Anacardiaceae family.83;181-187 Adverse reactions to other members of the Anacardiaceae family, e.g., mango fruit, have also been reported.184;188;189 Significant serologic cross-reactivity among cashew and pistachio has been reported in earlier studies. However pistachio allergy is not extensively investigated, resulting in relatively limited information regarding the identity of the relevant pistachio allergens. It is evident that we need careful biochemical and immunological studies to evaluate IgE- binding proteins in both nut species, with a goal of identifying homologous proteins that can

potentially elicit serious allergic cross-reactions. However none of the previous reports identify the cross-reactive allergens at the protein level. Our aim was to identity these cross-reactive allergens. Over the past few years, both native and recombinant cashew allergens have been identified and characterized in our lab and, the IgE-reactive epitopes described. The major seed storage protein constituents and allergens of the tree nuts are legumins, vicilins, and 2S albumins.83 In this study, sera from cashew and tree allergic patients, containing IgE reactive with both cashew and pistachio proteins were used for the detection, identification and immunological characterization of the cross-reactive proteins in these two nuts. The antigenic and allergenic profile of cashew and pistachio was analyzed by 1-DE/2-DE followed by immunoblotting, and subsequently by N-terminal amino acid sequencing. The comparative SDS-PAGE analysis of cashew and pistachio extracts shows similar profiles (Fig. 4.4A and B). Our extraction technique was expected to solubilize both albumins and globulins, and indeed our 1-DE and 2-DE gels show that the albumins and globulins are represented. The reactivity pattern of both cashew and pistachio extracts with rabbit anti-cashew globulin polyclonal antiserum was demonstrated to be identical and detected proteins at 30-35 and 45-50 kDa MW (Fig. 4.4A, Lane 3 and Fig. 4.4B, Lane 3). However no reactivity was observed with the 22 kDa protein from pistachio as opposed to that observed in cashew extract (Fig. 4.4A, Lane 2). Additionally, the cashew and pistachio immunoblots probed with sera from cashew and tree nut allergic patients show a similar IgE reactivity pattern, recognizing proteins at MW of 6-9, 22-25, 30-35, and 45- 50 kDa (Fig. 4.4A and B, Lane 2). In a study reported earlier, the immunoblot screening of 15 cashew allergic patients with cashew extract resulted in dominant IgE reactivity to the 20-27 and 31-35 kDa MW polypeptides.60 The N-terminal and partial amino acid sequencing characterized these proteins as the basic subunit and acidic subunit of the 11S globulins, respectively.60;190 Seed storage proteins belong to families of proteins that are known to exist as isoforms.202 The 2-DE immunoblotting and N-terminal sequencing of allergens have contributed to the identification of putative allergens and their multiple isoforms.168;307;308 The high resolution of 2-DE detected several distinct spots in both the cashew (Fig. 4.5) and pistachio (Fig. 4.6) extracts. N-terminal aa sequence analysis lead to the identification of 7S globulin, acidic and basic subunits of 11S globulin, and 2S albumin from both the cashew and pistachio 2- DE immunoblot probed with pooled sera from cashew and tree nut allergic patients (Fig. 4.5 and 4.6). Protein spots having the same molecular weight but slightly different pIs, possibly due to

posttranslational modifications and multi-gene complexity, suggests the presence of isoforms, which is a characteristic of the seed storage proteins and also a common feature of several seed, legume, and tree nut allergens 83;166;203. The cashew 7S, 11S, and 2S N-terminal aa sequences were identified to be identical to the cloned Ana o 1, Ana o 2, and Ana o 3 aa sequences respectively.112;163;164 The detection of homologous IgE reactive proteins in pistachio by 1-DE and 2-DE prompted us to clone these proteins from a pistachio cDNA library for further studies. The pistachio 7S vicilin-like protein, designated as Pis v 3 was identified as an allergen (35% of the tested patients’ serum were reactive). The N-terminal aa sequences of the 45-50 kDa band in 1- DE and similar molecular size spots in 2-DE pistachio immunoblots correspond to the cloned Pis v 3 aa sequence. A sequence alignment of Pis v 3 with cashew Ana o 1, revealed a high degree of homology (80% identity and 90% similarity) between the two proteins (Fig. 4.8). Epitope mapping preformed on cashew Ana o 1 and Ana o 2 using synthetic overlapping peptides identified eleven and 22 linear epitopes, respectively that bind IgE from cashew allergic individuals.163 Moreover, the presence of considerable similarity between them in the regions corresponding to the previously identified linear epitopes of cashew Ana o 1163 could explain the observed IgE mediated cross-reactivity. All but one of the tested pistachio and/or cashew allergic patients’ sera that recognized the vicilin from one nut also reacted with the other in an IgE dot-blot assay (Fig. 4.9). Inhibition studies by dot-blots (Fig. 4.10) and Western immunoblots (Fig. 4.11) further validate IgE cross-reactivity between these homologous proteins. Furthermore, antigenic cross-reactivity is not limited to patient IgE as six of nine randomly selected anti-rAna o 1 mAbs also react positively to Pis v 3 (Fig. 4.12). Together, these data suggest that antibody recognition of these proteins, whether by patient IgE in a natural allergenic situation or in an artificial murine immunization/hybridoma situation, is targeted towards the most conserved regions of the proteins. In addition to vicilin, 11S globulins were identified as IgE reactive in pistachio extract by both 1-DE and 2-DE immunoblotting (Fig.4.6). Thus we designed degenerate and subsequently gene specific primers to clone 11S globulin from pistachio. The pistachio 11S globulin designated as Pis v 2, demonstrated significant homology with cashew Ana o 2. However, it was observed that Pis v 2 in comparison to Ana o 2 demonstrated much reduced IgE reactivity.

The globular 7S and 11S storage proteins are very stable proteins classified as two- domain cupins that share a common architecture, which has been described as 'double-stranded β-helix' or 'jelly-roll' barrel-like structure.204 It is speculated that some aspects of protein structure are relevant for allergenicity, such as solubility, compactness, stability, and possibly an ability to interact with lipids in the membrane. In this regard the structures of 7S and 11S globulins are highly compact, thereby conferring stability to thermal denaturation and to digestion by proteolytic enzymes. Such properties, coupled with the abundance in the cashew and pistachio nuts, may contribute to these proteins being able to act as potent allergens. Primary and structural similarity observed among vicilins and legumins from different species suggests a strong link between sequence homology and clinical crossreactivity.205;309;310 The major vicilin allergens Ara h 1 of peanut, Jug r 2 of walnut, Cor a 11 of hazelnut, and Ana o 1 of cashew nut share very similar amino acid sequences, especially at their C-termini where most of the minor IgE-binding epitopes are located.205;310 Furthermore, three-dimensional models of these vicilin allergens built by homology modeling demonstrated the occurrence of surface-exposed epitopes of similar conformation that could account for some of the IgE-binding cross-reactivity.205;310 In comparison to rAna o 2, fewer number of cashew/tree allergic patients demonstrate IgE reactivity with rPis v 2. Allergen isoforms with diverging IgE binding properties have been reported for Bet v 1 and Pru av 1.311;312 Seven distinct genes encode a diverse array of naturally occurring Bet v 1 isoforms with varying IgE reactivity ranging from high, moderate to low.313 The isoform Bet v 1.0101 demonstrates the highest IgE-reactivity and the isoforms Bet v 1.0401 and Bet v 1.1001 showed very low IgE reactivity.162 In addition, IgE induced by Bet v 1.0101 only partly cross-reacts with the 2 other isoforms.312 These Bet v 1 isoforms share sequence identities between 87.4% and 96.3% with the Bet v 1.0101 isoform.314 The differences in allergenicity as well as immunogenicity are speculated to be attributed to the small structural differences between the isoforms at secondary and tertiary level as well. When two relatively homologous or similar proteins from phylogenetically related or unrelated food sources are capable of inducing a clinical relevant reaction (affecting one or more organ systems) in a food allergic individual the allergen is considered a clinically relevant cross- reactive allergen. Cross-reactivity is largely contingent upon high sequence and structural homology, aspects that have been recognized by several previous studies.18;199 Likewise, a

single amino acid exchange may drastically influence the extent of IgE reactivity and allergenicity.24 As a consequence of minor sequence alterations demonstrated by the exchange of 1 amino acid residue in the dust mite allergen Lep d 2, it resulted in the complete loss of the ability to bind IgE.315 The data presented in our study suggests that analysis of allergen isoforms could facilitate identification and cloning of naturally occurring hypoallergenic isoforms that could aid in designing efficient allergen specific immunotherapeutic candidates. The importance of these studies is evident in accurately characterizing the allergen(s) responsible for the sensitization and in determining the risk of reaction(s) to related foods in previously sensitized individuals. The application of molecular biology techniques to the analysis of allergens can help to improve the knowledge of their structures, immunologic properties, and functions and aid in predicting the potential allergenicity of new or existing cross-reactive protein(s) and identify the candidate’s usefulness in allergen therapy, vaccine development, and food engineering.

CHAPTER 5

HYPOALLERGENIC MUTANTS OF ANA O 2, A MAJOR CASHEW ALLERGEN

Introduction

The type I IgE-mediated allergic reactions represent a significant health problem in industrialized countries affecting almost 500 million people worldwide.1;2;3 Allergic patients are characterized by the increased production of immunoglobulin E (IgE) antibodies against per se harmless antigens from different sources (e.g. pollen, mites, animal dander and food. A considerable increase in the prevalence of allergic diseases has been recorded in the past few decades, thus posing an increasingly important clinical problem. A skin test survey conducted by the American Academy of Allergy, Asthma and Immunology (AAAAI), estimated that allergies affect as many as 40-50 million people in the United States, making it the sixth most common cause of chronic illness.316 Recent epidemiological studies suggest that nearly 4% of adults and 6-8% of children are afflicted with IgE-mediated food allergies in US, prevalence much higher than appreciated in the past.1;2;3;6 The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) proposed that fish, shellfish, soybean, wheat, egg, milk, peanuts, and tree nuts cause the majority of all allergic reactions to foods.299 Peanut and tree nuts allergies cause over 90% of fatalities in the US and are characterized by high frequency of life-threatening anaphylactic reactions.1;2 Allergies to milk, eggs, wheat and soy in children are usually outgrown by age three, however allergies to fish, shellfish, peanuts and tree nuts often persist throughout life.1;3;6;7 Food allergies display a range of disorders encompassing acute, potentially chronic and fatal reactions mainly affecting the skin and gastrointestinal (GI) tract. The dietary proteins that are sampled by the GI immune system generally evoke a state of specific immunological unresponsiveness termed oral tolerance.11 However, when this immune tolerance is abrogated, adverse reactions to food proteins arise.14 The development of allergy is a multistep process, and the mechanisms leading to sensitization, production of IgE antibodies, and manifestation of an allergic response is complex and not yet fully deciphered.22;34;41;477 As a result of allergen cross- linking of at least two adjacent IgE antibodies attached to FcεRI receptors on the cell surface of

the effector cells, degranulation occurs with the release of inflammatory mediators (e.g. histamine, serotonin, prostaglandins, and leukotrienes).25;27 The pathophysiology of an allergic response is known to be influenced by several factors, including genetic susceptibility of an individual, allergen dose, route of exposure, and also the structural and functional characteristics of an allergen. A skewing towards a TH2 response has been reported in allergies, with elevated IL-4, IL-5, and IL-13 levels that are responsible for class switching to the ε heavy chain for IgE production, whereas non-allergic individuals have higher levels of the TH1 cytokines IFN-γ and TNF-α, and the regulatory cytokine IL-10.15 Thus a dysregulated immune response appears to be an important pathogenesis factor in allergy.25;27;28 Allergic reactions to tree nuts are typically severe, fatal and long lasting, additionally there is also a potential for frequent accidental dietary contamination.1;2 It has been reported recently that as many as 20% of children outgrow peanut allergy and a subset of children with tree nut allergy outgrow their allergy over time, although the resolution rate appears to be less as that seen in peanut allergy.10 Data from a voluntary registry of peanut-and tree nut-allergic US patients records that 62% of those reporting allergy to tree nuts list sensitivity to walnut and 44% report sensitivity to cashew.7 Based on the clinical symptoms and the underlying immunological mechanisms, tree nut allergy belongs to class I food allergies, where the primary sensitization occurs via the GI tract.1;20 Treatment of food allergic patients is primarily based on avoidance of the offending food, thereby having a considerable social impact and leading to nutritional deficiencies. The various pharmaco-therapeutic approaches that are employed for the treatment of allergic disorders are aimed primarily at neutralizing the inflammatory chemical mediators or at inhibiting/suppressing the function of inflammatory effector cells.206 These therapies provide relief from the allergic symptoms, however they remain unsuccessful in altering the underlying inflammatory cascade leading to allergic disorders. Additionally, discontinuation of the therapy invariably results in redevelopment of allergic symptoms on subsequent exposure to offending allergens. Moreover, certain groups of patients respond unfavorably to the conventional pharmacotherapy.207 The allergen specific immunotherapy is one of the few causative and reliable treatment approaches for type I allergies as it modifies the causal cascade of adverse immune responses in allergic reactions in an antigen-specific manner, leading to long-term relief of allergic symptoms and life-long immunological tolerance.45 The premise of SIT is based on the administration of

increasing doses of allergenic molecules to allergic individuals resulting in the induction of allergic-specific non-responsiveness also referred as desensitization.45;206;208 A number of hypotheses have been proposed regarding the mechanisms underlying immunotherapy, these include the modulation of the allergen-specific immune response from TH2 cells in favor of TH1 13;38;45;214 cells , and induction of allergen-specific blocking IgG4 and IgA antibodies that antagonize IgE-mediated effects.209;216;219;226 Additionally, SIT is found to be associated with reduction in the number of effector cells and IL-4/IL-5 production at the site of allergic inflammation, and a shift towards increased IFN-γ production by TH1 cells and induction of regulatory T cells producing anti-inflammatory cytokines like IL-10 and TGF-β.13;45 Several studies have shown the efficacy of SIT in the treatment for pollinosis, animal, mite and insect venom allergy.208;210;211 In contrast, SIT for food allergy has been hindered by severe side effects in the past, due to the anaphylactic potential associated with the high IgE- binding capacity of purified food allergens. Although immunotherapy for food allergy was initially described in 1930212, it was not until 1992 that a controlled study with peanut-allergic patients was performed.213 The major problem with SIT is the use of crude or relatively undefined allergen extracts comprising of variable proportions of allergenic components that lead to the risk of local and in some cases severe anaphylactic side effects and may also contribute inadvertently to a broadening of the patients sensitization spectrum.38;214 Vaccination with natural non-standardized allergen extracts therefore yields highly heterogeneous immune responses, with sometimes poor or no protective immunity.215 The latter may be attributed to the under representation of certain allergens in the therapeutic vaccines and/or their poor immunogenicity.13 The availability of well-defined purified recombinant allergens and techniques for gene manipulation offers the potential for designing novel therapeutic approaches for improved, standardized and safer means of allergen immunotherapy. In the last few years, much research is therefore geared into making immunotherapy safer and more efficacious by altering the various components: antigens, adjuvants, and delivery systems.13;45 The availability of well-defined purified recombinant allergens and techniques for gene manipulation offers the potential for designing novel therapeutic approaches for improved, standardized and safer means of allergen immunotherapy.226 Recombinant allergens mimicking the immunological and structural properties of natural allergens could be used for immunotherapy, offering the advantage that defined molecules in defined quantities can be

formulated as vaccine.211;216 Such a treatment would be tailored to the sensitization profile of the patient, which would ultimately minimize the possibility of therapy-induced de novo sensitizations. Recombinant allergens have been used in animal models to induce blocking IgG antibodies (e.g., major fish allergen, grass pollen and Parietaria) and induce tolerance (latex and venom allergens).217 However, the retained allergenic activity of recombinant allergens may be a disadvantage leading to allergenic side effects during treatment. Moreover, due to purity of the recombinant allergen preparations, the anaphylaxis inducing potential is expected to be very high, even at very low doses.216;218 Therefore it is essential to explore strategies designed to reduce the ability of allergens to elicit unwarranted dangerous allergic responses, yet retain their ability to interact with T cells and modulate the immune system to a tolerant state. With the advancement in the characterization of the immunological and structural features of recombinant and natural allergens it has become possible to modify allergens in ways that simultaneously reduce IgE binding and preserve relevant T-cell epitopes and structures that are necessary for the induction of protective blocking IgG4 antibody responses resulting in reduced allergenic activity. Chemical modifications of allergens (allergoids) to abrogate IgE binding have been used in clinical practice.219 The experience with these hypoallergenic allergoids paved the way for designing hypoallergenic vaccines based on recombinant proteins or synthetic peptides. Developments in molecular cloning and the availability of aa sequences and structures of several allergens have made it possible to design novel therapeutic approaches with reduced allergenic activity and thus improved and safer forms of allergen immunotherapy. A very promising approach involving genetic engineering and site-directed mutagenesis of recombinant allergens offers an exciting possibility for developing new allergen variants for use in SIT displaying low IgE reactivity and thus reduced allergenic activity.206;216 One major advantage of recombinant hypoallergenic derivatives is that they can be engineered to induce protective antibody responses and increased induction of TH1 type cytokines. This seems to be important because there is evidence for the necessity of both T cell and B cell-mediated effects for successful immunotherapy. Based on these advantages, the use of recombinant hypoallergenic allergen-derivatives has been suggested not only for treatment of established allergy but also for prophylactic vaccination.38 Safer and more efficacious vaccines would greatly increase patient compliance and ultimately lead to more widespread use of immunotherapy.

Several strategies to construct hypoallergenic derivatives which have so far been successfully applied include: production of recombinant allergen fragments to destroy the three- dimensional structure of the allergen and thus conformational IgE epitopes220-222; introduction of point mutations to alter the IgE epitopes and retaining T cell epitopes and immunogenicity206;223- 229; selection of natural hypoallergenic isoforms230;312; deletion of allergen portions222;231;232; production of defined allergen oligomers or hybrids228;233;233, restructured317, and misfolded allergen proteins234;318; DNA shuffling of allergens319; and conjugation to immunostimulatory 320 sequences (ISS-ODN) to induce TH1-biased immune responses. By site-directed mutagenesis, several food allergens have been transformed into hypoallergens, including major peanut allergens235-239, major peach allergen Pru p 3240, major apple allergen Mal d 1241, major beef allergen242, major shrimp allergen Pen a 1243, egg white ovomucoid allergen244, and major celery allergen Api g 1234, demonstrating a decrease or complete loss of IgE binding to the modified allergens. For the past few years our lab along with Teuber and Sathe labs have extensively collaborated on investigating biochemical, immunochemical and clinical studies of tree nut allergens. The allergenic proteins associated with most tree nut allergies thus far include the seed storage, structural, and several pathogenesis-related proteins.18;83 Much of the recent work on tree nut allergens is derived from the study of recombinant proteins by virtue of the availability of cDNA libraries of walnut, hazelnut, cashew and almond.83;105;111;142;173 The cashew allergens identified to date include seed storage proteins: Ana o 1, a 7S globulin (vicilin)111, Ana o 2, an 11S globulin (legumin)112, and Ana o 3, a 2S albumin.164 This dissertation study has been focused on Ana o 2, as it is a major allergen and protein in cashew. The selection criteria included the frequency of sensitization, the clinical relevance, the magnitude of IgE responses, and the extent to which IgE epitopes are represented in this allergen. Ana o 2, is recognized by serum IgE from 13 of 21 (62%) tested sera from patients with a history of life-threatening reactions to cashews.112 The 11S globulins represent a family of most abundant seed storage proteins, and have been shown to be the most immunogenic (i.e., react with high percentage of patients sera) of the seed storage proteins (allergenic in cashew, pistachio, almond, walnut, Brazil nut, and hazelnut). Therefore, Ana o 2 allergen has been our first target to design hypoallergenic derivatives for allergen-specific immunotherapy.

Linear IgE-reactive epitopes on the primary sequence of Ana o 2 were located by overlapping synthetic peptide analysis using cashew allergic sera.112 Additionally, alanine scanning mutagenesis of the immunodominant IgE reactive linear epitopes resulted in the identification of critical amino acid residues important for IgE binding, and would serve as targets for further studies. Our aim was to create hypoallergenic variants of Ana o 2, by introducing point mutations of the critical amino acids using site-directed mutagenesis and unaltering the overall structural fold of the molecule. Thus, by reducing the anaphylactic potential of Ana o 2 while preserving T cell epitopes, we could potentially use these hypoallergens for SIT. This strategy has been successfully applied to a number of important allergens for the development of hypoallergenic molecules for immunotherapy.24;206;223-229 The goal of these studies was to compare the IgE reactivity of Ana o 2 wt and mutants by in vitro and ex vivo basophil assays. The steady increase in the incidence of allergic disease has intensified the need for successful therapeutic approaches, and the modified allergens are expected to make safer allergen-specific immunotherapy more widely used in the future for the treatment of food- allergic patients. The experimental studies involving cloning and immunological characterization with cashew Ana o 2 has been performed by myself in collaboration with Fang Wang and Jason Robotham and is partly published in International archives of Allergy in 2003.112 The epitope peptide mapping and alanine mutagenesis of cashew Ana o 2 was performed by Jason Robotham and the data are partly published.112 The site-directed mutagenesis of cashew Ana o 2 cDNA, the empirical data demonstrating the in vitro reactivity of the Ana o 2 mutants, and ex vivo assays demonstrating the hypoallergenic potential of the Ana o 2 mutants in comparison with Ana o 2 wt has been performed by myself. The homology models of Ana o 2 have been built in collaboration with Greg Hoffman at FSU. The manuscript for these data is under construction.

Methods

Recombinant Protein Production cDNA insert coding Ana o 2 amino sequence was ligated to maltose-binding protein (MBP) fusion expression vector pMAL-c2 (New England BioLabs Inc., Beverly, Mass., USA),

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into which a thrombin cleavage site had been engineered.112 Competent E. coli BL21 (DE3) cells (Novagen Inc, Madison, WI) were transformed with Ana o 2 cDNA/pMAL-c2 plasmids,

and single colonies were grown at 37°C to an OD600 nm of 0.5, followed by induction with isopropyl-D-thiogalactopyranoside. The cells were harvested, resuspended in amylose resin column buffer (20 mM/LTris-HCl, pH 7.4, 200 mM/L NaCl, 10 mM/L -mercaptoethanol, and 1 mM/L EDTA), lysed with mild sonication, centrifuged at 12,000g, and passed over an amylose affinity column. The fusion protein was eluted with column buffer containing 10 mM/L maltose. A yield of 15–20 mg of soluble fusion protein per liter of cultured cells was routinely recovered after column or batch purification. Various hypoallergenic mutants of Ana o 2 were also expressed using competent E. coli BL21 (DE3) using the procedure described above. These recombinant proteins were concentrated and either stored (briefly) at 4°C until use or frozen at - 80°C.

Human Sera Blood samples were drawn after informed consent from patients with life-threatening systemic reactions to walnut or cashew nut and the sera frozen at -70°C until use. The study was approved by the Institutional Review Board of the University of California at Davis, and Florida State University, Tallahassee. The presence of walnut and cashew-reactive IgE was confirmed by Pharmacia ImmunoCAP assays (Pharmacia Diagnostics, Uppsala, Sweden) or by Western immunoblotting (described below).

Linear Epitope Mapping of Ana o 2 Based on the derived aa sequence of the 457-amino acid Ana o 2 protein, 57 overlapping 15-amino acid peptides, each offset by 8 amino acids, were synthesized as shown in figure 5.2A.112 Peptides were synthesized on derivatized cellulose sheets using 9-fluorenylmethoxy carbonyl-derived amino acids (Genosys Biotechnologies Inc., TX, USA) and probed with pooled serum from cashew allergic patients diluted 1:5 (v/v) in Genosys blocking buffer by using the immunoblotting protocol described below.111;112 The serum IgE reactivity to the spotted peptides was detected with 125I-labeled anti-human IgE (Hycor Biomedical Inc., Garden Grove, CA), and after a 1-week exposure at –70°C to X-ray film or a 17-hour incubation with a Molecular Dynamics phosphor-imaging screen from Kodak.112 Phosphoimaging data were quantified using

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a Storm 860 scanner (Molecular Dynamics Inc., Sunnyvale, Calif., USA) and its accompanying software.

Alanine Scanning Mutagenesis Three immunodominant IgE-reactive epitopes of Ana o 2 (#3, #6, and #15) were selected for alanine scanning mutagenesis in order to identify the critical aa residues important for IgE binding. Synthetic peptides represented as spots were manually synthesized on a SPOTs membrane. SPOTs layout and IgE reactivity as detected to epitope #3 (SRQEWQQQDECQIDR), epitope #6 (YQAPQQGRQQGQSGR), epitope #15 (EESEDEKRRWGQRDN) is depicted in figure 5. For each of the three epitopes analyzed, the wild-type unmutated peptide was used as control. In addition, an unrelated peptide was used as a negative control. Alanine substitutions are shown in red (Fig. 5.3). The membrane was probed with pooled cashew and tree nut-sensitive sera and bound IgE was detected with 125I-labeled anti-human IgE (Hycor Biomedical Inc., Garden Grove, CA), and after a 1-week exposure at – 70°C to X-ray film or a 17-hour incubation with a Molecular Dynamics phosphor-imaging screen from Kodak.112

Site-Directed Mutagenesis Specific point mutations to alanine were introduced into the cDNA of Ana o 2 in pMAL c-2 by using the Quick-Change Site-directed Mutagenesis kit from Stratagene (La Jolla, CA), according to the manufacturer’s instructions. The primers used for introducing the specific point mutation in cDNA of Ana o 2 are as shown below: Q20A For 5'- CGCCAG GAATGGGCGCAACAAGATGAGTGC - 3` C25A For 5`- GCAACAACAAGATGAGGCGCAAATCGATAGG - 3` Q110A For 5`- CCAAGCGCCCCAAGCGGGACGACAACAGG - 3` R112A For 5`- GCCCCAACAGGGAGCGCAACAGGGACAG - 3` D261A For 5`- GAGGAAAGTGAGGCGGAAAAACGCCGATGG - 3` W266A For 5`- GGATGAAAAACGCCGAGCGGGACAGCGTGAC - 3` G267A For 5`- CGCCGATGGGCGCAGCGTGACAATGG - 3`

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DNA sequencing of each plasmid construct confirmed the presence of the point mutations. After mutagenesis, each clone was transformed into competent BL21 cells and recombinant protein production was carried out using the protocol described above.

Immunoblotting Purified recombinant proteins wt Ana o 2 and mutants were tested for IgE reactivity by immunoblotting (i.e., SDS-PAGE using a Bio-Rad Mini-Protean II electrophoresis unit followed by blotting onto NC and probing with cashew allergic patients' sera). The purified recombinant proteins 0.5 μg/4 mm well width is typically used for immunoblotting. Blots are blocked with 5% nonfat dry milk in TBS-T buffer containing 0.2% Tween 20. Diluted sera (1:5 v:v in the blocking buffer) added to the strips and incubated overnight at 4oC. The strips are then washed with TBS-T and incubated overnight (o/n) at 4oC with polyclonal 125I-anti-human IgE (Hycor Biomedical Inc., Garden Grove, CA) diluted 1:5 (v:v) in the non-fat milk buffer in PBS-T, washed and exposed to X-ray film (Kodak X-OMAT). The density of the bands was analyzed with Gel DOC 2000 imaging system (Quantity One software; Bio-Rad). The background density was subtracted from the values obtained from Ana o 2 wt and mutants for each of the patient sera tested.

Dot-Blot Analysis Defined quantities of purified recombinant proteins Ana o 2 wt and mutants were applied to NC membrane using a 96-well Bio-Dot microfiltration apparatus (Bio-Rad Laboratories). Bovine serum albumin (BSA) served as a negative control. The membranes were then cut into strips, each of which contained the desired sets of protein dots, and probed as described above. The density of the bands was analyzed with Gel DOC 2000 imaging system (Quantity One software; Bio-Rad). The background density was subtracted from the values obtained from Ana o 2 wt and mutants for each of the patient sera tested.

ELISA ELISAs were preformed as previously described.105 Briefly, 96 well micro-titer plates were coated with 50 μl/well of protein solution (Ana o 2 wt and mutants) at 20 μg/ml in coating buffer (0.1 M carbonate–bicarbonate buffer, pH 9.6). Sera from cashew-allergic patients (diluted

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1:10 v:v, 50 μL/well) were added and incubated o/n at 4°C. After washing, bound IgE was reacted with HRP-conjugated mouse anti-human IgE (Zymed Laboratories, Inc.) at a dilution of 1:1000 and incubated for 1 h at 37°C. IgE reactivity was detected by colorimetric reaction using

o-phenylenediamine (OPD, Zymed Laboratories Inc.) and H2O2 as substrate. Optical density (OD) was measured in a KC4 v2.5 ELISA reader (Bio-Tek Instruments Inc, Winooski, VT) at 495 nm.

Inhibition ELISA For IgE inhibition ELISA, solid phase wt Ana o 2 was assayed with pooled (6 patients) cashew allergic Ana o 2 reactive sera pre-incubated with soluble inhibitors (wt Ana o 2 and Ana o 2 mutants) at concentrations from 5 – 20 μg/mL. The resultant IgE binding was measured as described above and the percentage of inhibition was calculated as follows:

Inhibition was calculated for each dilution using the formula: % inhibition = (A1-NSB)-(A2-NSB) x 100 (A1-NSB) where A1 is the average OD of the positive control wells without inhibitor and A2 is the average OD of the well with an inhibitor. NSB represents the average OD of the negative control wells for nonspecific binding (sample without serum).

Monoclonal Antibodies to Ana o 2 Ana o 2-reactive mAbs were generated in the Hybridoma Core Facility at Florida State University according to standard procedures as described briefly below.192 Briefly, BALB/c mice were immunized with 25 μg of whole cashew extract in RIBI adjuvant (Corixa Inc., Hamilton, MT) according to the manufacturer’s instructions. The initial immunization was given ½ subcutaneously and ½ intraperitonealy. Three weeks after the immunization, these mice were given a primary boost with 20-25 μg of respective antigen, again using the RIBI adjuvant. Two weeks following the boost, mice were test bled for and their Ab titer against whole cashew extract or Ana o 1 were determined via ELISA as previously described.87 When the titer approached 1:10,000, the mice were given a final boost with 25 μg antigen in saline, ½ intravenously in the tail vein and ½ intraperitonealy. Three days after the final boost, the mice

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were sacrificed and their spleens were removed. Spleen cells were dispersed, washed, and red blood cells removed with ammonium chloride prior to fusion with NS-2 myeloma cells (ATCC, Manassas, VA) which were mixed at a ratio of 1:5 (myeloma cells:spleen cells). Fused cells, achieved using PEG, were plated in HAT media and allowed to grow for two weeks. Surviving hybridoma cells were screened for reactivity to whole cashew extract via ELISA. Those mAbs that were found to be reactive with whole cashew extract in this initial screening were then checked for reactivity to rAna o 2 in subsequent ELISAs using plates coated with 5 μg/ml rAna o 2. Reactive clones of interest were sub-cloned until mono-specific Ana o 2-binding mAbs were obtained. The Ana o 2 mutants were tested with these mAbs and the IgG reactivity comparison with wt Ana o 2 was performed with ELISA.

Circular Dichroism Circular Dichroism (CD) spectrometer (Model 202, Aviv Instruments, Inc.) was used to assess the secondary structure of the wt and mutants of Ana o 2. CD measurements was performed in the far-ultraviolet region (200-260 nm). The secondary structure parameters will be calculated using CDPro: http://lamar.colostate.edu/~sreeram/CDPro/index.html.

Modified Basophil Activation Test Basophil collection and enrichment - Basophils were enriched from whole blood collected from non-food-allergic human donors in a single step using a Percoll gradient. Approximately 25 mL of venous blood collected in sodium-heparin Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) was layered on top of the gradient and centrifuged at 700 x g for 15 minutes at 20oC without brakes. The band (basophils) at the interface of the 1.070 and 1.079 gm/mL gradients was aspirated. Antibody stripping and passive Sensitization - Pelleted basophils were re-suspended in an ice-cold pH 3.9 lactic acid buffer (stripping buffer) on ice for 1 minute before being spun at 700 x g for 5 minutes. The pellet was washed in PBS and finally resuspended in 1 mL of a solution containing 20% serum from a food allergic donor, 4 mM EDTA, and 10 µg/mL heparin, then placed in a heated water bath for 90 minutes at 37oC. Allergen challenge and antibody staining - Prior to challenging basophils with allergens, the basophil solutions were washed in 20 mM HEPES buffer (pH 7.4) and 0.5% human serum

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albumin. After washing, cells were incubated with allergenic and hypoallergenic variants of Ana o 2. The triggering reaction was stopped after 15 minutes and cells were pelleted and resuspended in a staining buffer. The fluorescent stains phycoerythrin (PE)-conjugated anti-human CD203c (Immunotech, Marseille, France), and fluorescein isothiocyanate (FITC)-conjugated CD63 (Serotec, Raleigh, NC) were used according to the manufacturers’ recommendations. Data acquisition was completed using a Becton Dickinson FACScan (San Jose, CA) and analyzed using FlowJo software (Treestar Inc., Ashland, OR). Each mBAT included challenges with a relevant allergen (positive control), formyl-methionyl-leucyl (fMLP, Sigma, St. Louis, MO), an irrelevant allergen (negative control), and PBS. Data analysis - Cells were gated for 7-AAD - CD203c+ to identify viable basophils. To determine basophil reactivity, the percentage of 7-AAD - CD203c+ CD63+ cells werecompared to total 7-AAD-CD203+ cells and converted to an activation index (AI). The AI is the ratio of the percentage of 7-AAD - CD203c+ CD63+ basophils in challenged samples compared to unchallenged basophils. An AI > 0.25 was used as a cut-off value based on preliminary experiments (Dr. S Teuber personal communication) with positive and negative controls to optimize the assay.

Homology Modeling and Surface Mapping of Ana o 2 Homology based 3D modeling was performed for Ana o 2 based on the solved X-ray crystal structure of soybean 11S G2 proglycinin A1aB1b homotrimer as template (PDB 1FXZ)245. The spatial arrangement of the identified linear IgE epitopes and the critical mutated aa residues in the three immunodominant linear IgE reactive epitopes are highlighted in color. Models were constructed using SWISS MODEL246 (Deep-Viewer http://www.expasy.ch/spdbv /mainpage.html and ViewerLite: - http://www.accelrys.com/dstudio/ds_viewer/viewerlite/). The target sequence and the quaternary structure template were loaded into SwissPDB Viewer (SPDBV)247 and the target sequence was threaded onto the template. This PDB file was submitted to the Swiss Model automated protein homology modeling server and the resulting structure file was subjected to successive rounds of steepest descent energy minimization using GROMOS 96 (http://www.igc.ethz.ch/gromos/)

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Results

Amino Acid Sequence of Ana o 2 The cashew cDNA library was screened for reactivity with both cashew extract-specific rabbit IgG and cashew-allergic human IgE. An immunoreactive clone was selected for sequencing and identified to encode for 11S globulin and is designated as Ana o 2. The derived amino acid sequence analysis of Ana o 2 cDNA clone is illustrated in Figure 5.1.112 Analysis of the deduced amino acid sequence revealed a 457-amino acid open reading frame with a predicted leader peptide from 1 to 15 (Fig. 5.1)

LSVCFLILFHGCLASRQEWQQQDECQIDRLDALEPDNRVEYEAGTVEAWDPNHEQFRCAGVALVRH TIQPNGLLLPQYSNAPQLIYVVQGEGMTGISYPGCPETYQAPQQGRQQGQSGRFQDRHQKIRRFRR GDIIAIPAGVAHWCYNEGNSPVVTVTLLDVSNSQNQLDRTPRKFHLAGNPKDVFQQQQQHQSRGRN LFSGFDTELLAEAFQVDERLIKQLKSEDNRGGIVKVKDDELRVIRPSRSQSERGSESEEESEDEKR RWGQRDNGIEETICTMRLKENINDPARADIYTPEVGRLTTLNSLNLPILKWLQLSVEKGVLYKNAL VLPHWNLNSHSIIYGCKGKGQVQVVDNFGNRVFDGEVREGQMLVVPQNFAVVKRAREERFEWISFK TNDRAMTSPLAGRTSVLGGMPEEVLANAFQISREDARKIKFNNQQTTLTSGESSHHMRDDA

Figure 5.1. Amino acid sequence of the Ana o 2 coding region (GenBank accession no. AAN76862). The predicted signal peptide is shown in grey, the acidic subunit in red and the basic subunit in blue, respectively.112

For immunological characterization, we cloned and expressed Ana o 2 after the presumptive leader peptide. The DNA segment was ligated into modified pMAL-c2 expression vector (New England BioLabs Inc., Beverly, MA, USA), designed to allow for purification of the recombinant molecule via an MBP fusion domain in conjunction with an amylose affinity column and a thrombin-specific cleavage site.112 The resulting ~ 93 kDa Ana o 2 fusion protein was affinity purified as previously described111;112 and digested with thrombin to yield a peptide of approximately 52 kDa as well as the 43 kDa MBP.

Identification of rAna o 2 as an Allergen

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Twenty one cashew allergic sera were used to study the IgE immunoreactivity to the rAna o 2 by western immunoblotting. IgE from 13 of these 21 sera (62%) reacted positively with the recombinant cashew protein.112 The intensity of the signal was strong in six cases, but with the remaining seven it was weak. Of the control sera used that were tolerant to cashews but were allergic to other tree nuts, none reacted with rAna o 2.112

Identification of IgE-reactive linear epitopes on Ana o 2 The full length amino acid sequence of Ana o 2 was studied by probing 58 overlapping solid-phase synthetic peptides with sera from 12 patients assigned to three pools.112 Each pool consisted of sera from 4 patients and included sera from at least 1 patient that reacted strongly to the rAna o 2 in our Western blots and from 3 other patients with varying degrees of reactivity.

A 111 103 LSVCFLILFHGCLASRQEWQQQDECQIDRLDALEPDNRVEYEAGTVEAWDPNHEQFRCAGVALVRHTIQPNGLLLPQYSNAPQLIYVVQGEGMTGISYPGCPE 1 2 3 4 5 6 7 8 9 10 11 12 04 20 6 TYQAPQQGRQQGQSGRFQDRHQKIRRFRRGDIIAIPAGVAHWCYNEGNSPVVTVTLLDVSNSQNQLDRTPRKFHLAGNPKDVFQQQQQHQSRGRNLFSGFDTE 13 14 15 16 17 18 19 20 21 22 23 24 B

Figure 5.2. Linear epitope analysis of Ana o 2.112 (A) Schematic representation of solid phase overlapping peptide synthesis of Ana o 2. (B) IgE binding analysis of the Ana o 2 peptide spots. SPOTs array depicting all 58 -overlapping 15-aa Ana o 2 peptides probed with pooled sera from 12 cashew-allergic patients.

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Collectively, the three pools reacted weakly (phosphoimaging score from 2.0 - 3.9 x 10–3) with 12, moderately (4.0 - 6.9 x 10–3) with 3, and strongly (>7.0 x 10–3) with 7 linear IgE-binding epitopes (Fig. 5.2). Twenty two IgE reactive linear epitopes were distributed throughout the entire length of the Ana o 2 protein (Table 5.1). Interestingly, fifteen of the 22 (68%) epitopes reside on the acidic chain region of the Ana o 2 protein. Additionally, of the seven immunodominant epitopes identified, six were on the Ana o 2 acidic chain.

Table 5.1. Linear IgE reactive epitopes of Ana o 2 identified by overlapping synthetic peptide technology.112

Epitope AA position AA sequence Phosphoimaging dataa Pool 1 Pool 2 Pool 3 1 1-15 LSVCFLILFHGCLAS + 2 9-23 FHGCLASRQEWQQQD 2.4 31 15-29 SRQEWQQQDECQIDR +++ + + 4 17-31 QEWQQQDECQIDRLD + 5 89-103 QGEGMTGISYPGCPE + 62 105-119 YQAPQQGRQQGQSGR +++ ++ +++ 7 113-127 QQGQSGRFQDRHQKI + + 8 121-135 QDRHQKIRRFRRGDI + 9 137-151 AIPAGVAHWCYNEGN ++ 10 169-183 LDRTPRKFHLAGNPK ++ 11 185-199 VFQQQQQHQSRGRNL +++ 12 217-231 RLIKQLKSEDNRGGI ++ + 13 233-247 KVKDDELRVIRPSRS + +++ 14 241-255 VIRPSRSQSERGSES +++ 15 257-271 EESEDEKRRWGQRDN +++ +++ 16 313-327 LKWLQLSVEKGVLYK + 17 329-343 ALVLPHWNLNSHSII + 18 337-351 LNSHSIIYGCKGKGQ + 19 377-391 QNFAVVKRAREERFE + + 20 385-399 AREERFEWISFKTND + 21 417-431 PEEVLANAFQISRED + + 22 425-439 FQISREDARKIKFNN +++

1Bold print denotes strongly reacting epitopes. 2Underlining denotes those epitopes bound by sera from all three patient pools

Alanine scanning mutagenesis of synthetic peptides of Ana o 2 Of the 22 linear IgE epitopes identified on Ana o 2112, three immunodominant epitopes were selected for further analysis via alanine scanning mutagenesis of synthetic peptides in order to identify the individual aa residues that were critical for epitope specific recognition by IgE antibodies (Fig. 5.3). The substitution of each aa residue to alanine in each of the 3 epitopes was 109

accomplished by synthesizing peptides to determine the critical amino acids involved in IgE binding (Fig. 5.3B). The effects of these alanine substitutions were measured by evaluating the IgE reactivity patterns of pooled cashew allergic sera exhibited by the mutations. Key amino acid residues were identified in these epitopes, showing reduced or loss of IgE reactivity in the spots analysis (Fig. 5. 3A). Probing of the mutated peptides with pooled patient sera demonstrated that, for epitope #3, aa residues W19, Q20, D23, E24, C25, Q26, R29 would reduce patient-IgE reactivity by more than 50% if mutated to alanine (Fig. 5.3).

A 1234567 8 9101112 18 19 20 21 22 23 24 34 35 36 37 38

13 14 15 16 17 25 26 27 28 29 30 31 32 33 34 39 40 41 42 43 44 45 46 47 48 49 50

B SPOT Epitope # 6 Mutation SPOT Epitope # 15 Mutation SPOT Epitope # 3 Mutation 1 YQAPQQGRQQGQSGR (+) 18 EESEDEKRRWGQRDN (+) 34 SRQEWQQQDECQIDR (+) 2 AQAPQQGRQQGQSGR Y105A 19 AESEDEKRRWGQRDN E257A 35 ARQEWQQQDECQIDR S15A 3 YAAPQQGRQQGQSGR Q106A 20 EASEDEKRRWGQRDN E258A 36 SAQEWQQQDECQIDR R16A 4 YQAPQQGRQQGQSGR A107A 21 EEAEDEKRRWGQRDN S259A 37 SRAEWQQQDECQIDR Q17A 5 YQAAQQGRQQGQSGR P108A 22 EESADEKRRWGQRDN E260A 38 SRQAWQQQDECQIDR E18A 6 YQAPAQGRQQGQSGR Q109A 23 EESEAEKRRWGQRDN D261A 39 SRQEAQQQDECQIDR W19A 7 YQAPQAGRQQGQSGR Q110A 24 EESEDAKRRWGQRDN E262A 40 SRQEWAQQDECQIDR Q20A 8 YQAPQQARQQGQSGR G111A 25 EESEDEARRWGQRDN K263A 41 SRQEWQAQDECQIDR Q21A 9 YQAPQQGAQQGQSGR R112A 26 EESEDEKARWGQRDN R264A 42 SRQEWQQADECQIDR Q22A 10 YQAPQQGRAQGQSGR Q113A 27 EESEDEKRAWGQRDN R265A 43 SRQEWQQQAECQIDR D23A 11 YQAPQQGRQAGQSGR Q114A 28 EESEDEKRRAGQRDN W266A 44 SRQEWQQQDACQIDR E24A 12 YQAPQQGRQQAQSGR G115A 29 EESEDEKRRWAQRDN G267A 45 SRQEWQQQDEAQIDR C25A 13 YQAPQQGRQQGASGR Q116A 30 EESEDEKRRWGARDN Q268A 46 SRQEWQQQDECAIDR Q26A 14 YQAPQQGRQQGQAGR S117A 31 EESEDEKRRWGQADN R269A 47 SRQEWQQQDECQADR I27A 15 YQAPQQGRQQGQSAR G118A 32 EESEDEKRRWGQRAN D270A 48 SRQEWQQQDECQIAR D28A 16 YQAPQQGRQQGQSGA R119A 33 EESEDEKRRWGQRDA N271A 49 SRQEWQQQDECQIDA R29A 17 RFRRGDIIAIPAGVA (-) 34 RFRRGDIIAIPAGVA (-) 50 SRQEWQQQDECQIDR (-)

Figure 5.3. Alanine scanning mutagenesis of immunodominant Ana o 2 linear epitopes. The sequential mutation of aa residue to alanine is shown in red. The critical aa for IgE reactivity identified are shown in red (A) and boxed in red (B).

Similarly, IgE binding to epitope #6 was reduced >50% when aa Q110, G111, and R112 were mutated to alanine and completely eliminated when aa Q113 or Q114 were mutated (Fig. 5.3). Epitope #15 demonstrated highest IgE reactivity with two pools of cashew allergic sera as seen in the phosphoimaging data in Table 5.2 and was selected for alanine scanning mutagenesis.

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In epitope #15, the mutation of aa residue D261 to alanine, resulted in >50% reduction in IgE reactivity and total abrogation following mutation of W266, G267 to alanine (Fig. 5.3).

Site-directed mutagenesis of the Ana o 2 cDNA The information generated by alanine scanning mutagenesis to develop hypoallergenic mutants of the Ana o 2 protein by site-directed mutagenesis of the Ana o 2 cDNA. Specific point mutations to alanine were introduced into the cDNA of Ana o 2 in pMAL c-2 by using the QuickChange Site-directed Mutagenesis kit from Stratagene (La Jolla, CA). The point mutations in the cDNA of Ana o 2 include Q20A, C25A, R29A, Q110A, R112A, Q113A, Q114A, D261A, MBP in E. coli and purified by affinity chromatography using amylose coupled to agarose as described earlier. The yield of the purified proteins was around 30-35 mg/L of E. coli culture. Purified Ana o 2 mutants expressed as fusion protein with MBP showed as a ~ 97 kDa band as determined by Coomassie Blue stained SDS-PAGE (Fig. 5.4).

kDa M C25A Q20A Q110A D261A W266AG267A R112A WT Ao2 200

116 97 66

Figure 5.4. Expression of Ana o 2 mutants as observed by SDS-PAGE stained by Coomassie Blue. (M-Molecular weight marker)

IgE reactivity of Ana o 2 wt and single point and multiple epitope mutants In order to assess the IgE binding capacity of the mutated Ana o 2 proteins ELISA, immunoblot and inhibition ELISA assays were performed using individual serum or pooled sera IgE from documented cashew allergic patients. Figure 5.4 depicts IgE reactivity of the Ana o 2

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wt and mutants analyzed by ELISA with six individual Ana o 2 reactive sera from cashew allergic patients. In comparison with wt Ana o 2, the mutants demonstrated reduction in IgE binding capacity by 75-25% depending on the individual serum samples tested (Fig. 5.5). All the point mutations of the critical aa residues in IgE reactive epitopes of Ana o 2 demonstrated substantial decrease in IgE binding.

1.6

1.4 wtAna o 2 1.2 Q20A 1 C25A Q110A 0.8 R112A 0.6 D261A

0.4 W266A G267A 0.2

IgE to Ana o 2 and mutants(O.D) 0 Pt#1 Pt#2 Pt#3 Pt#4 Pt#5 Pt#6

Figure 5.5. IgE-binding capacity of the Ana o 2 mutants in comparison with the wild-type protein using six cashew allergic sera by ELISA.

In the immunoblot assay, equal amounts of the wt Ana o 2 and mutant proteins were electrophoresed on 12% SDS-PAGE gels, transferred to NC, and then probed with six individual sera from cashew sensitive patients. As evident in Fig. 5.6A, as a result of mutation in epitope #6 of glutamine to alanine at position 110 (Q110A), significant reduction in IgE reactivity, is detected in comparison with wt Ana o 2 with all six cashew allergic sera tested. With the aid of densitometric analysis it was recorded that the intensity of IgE to the Ana o 2 mutant Q110A was reduced in the range of 23-84% in comparison with Ana o 2 wt. (Fig. 5.6B)

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Pt # 1 Pt #2 Pt #3 Pt #4 Pt #5 Pt #6 A WT MUT WT MUT WT MUT WT MUT WT MUT WT MUT

Pt#1 Pt#2 Pt#3 Pt#4 Pt#5 Pt#6 B 36% 23% 52% 46% 62% 84%

Figure 5.6. IgE immunoblot analysis (A) of unmutated wt Ana o 2 and mutant Q110A with six cashew allergic patient sera. (B) Densitometric analysis demonstrating the percentage reduction in IgE reactivity to mutant Q110A in comparison with Ana o 2 wt.

The immunoblot technique is performed under reducing conditions inducing denaturation and misfolding and thus we conducted IgE inhibition ELISA assays in solution with pooled sera from the above six cashew allergic patients to further analyze the ability of the Ana o 2 mutants to inhibit IgE binding to the wt Ana o 2. The soluble Ana o 2 mutants and wt proteins were used as inhibitors at different concentrations and incubated in solution with the pooled Ana o 2 reactive sera prior to adding the mixture to a micro-titer plate coated with wt Ana o 2. An HRP coupled anti-human IgE antibody was used to determine the amount of IgE binding to the solid phase wt Ana o 2. The results of the IgE inhibition ELISA are in good accordance with the immunoblot experiments. The soluble wt Ana o 2 was able to completely inhibit IgE binding to the solid phase wt Ana o 2 in a dose response manner (shown in red, Fig. 5.7). However, all the Ana o 2 mutants tested exhibited a clear reduction of inhibition capacity of IgE to solid phase wt Ana o 2 when compared to the level of inhibition obtained using wt Ana o 2 (Fig. 5.7).

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100%

90% 80% 70% wt rAna o2 60% W266A 50% C25A G267A 40% Q110A %Inhibition 30% 20%

10%

0% 0 5 10 15 20

Protein concentration of inhibitors (μg/mL)

Figure 5.7. IgE inhibition ELISA with pooled cashew allergic sera and Ana o 2 wt and four mutants at concentration ranging from 1-20 μg/mL.

In addition to the single point mutations, six combinations of point mutations of Ana o 2 were produced in E. coli as explained for point mutants. The six combination mutants are shown in figure 5.8. The mutant combinations were designed so as to disturb IgE binding of more than one immunodominant epitope. Dot-blot analysis of these mutants with six cashew allergic sera (reactive to Ana o 2), demonstrated total loss of IgE binding to three Ana o 2 mutants in 5 of the 6 sera as illustrated in figure 5.8. Interestingly, each of the individual serum tested demonstrated a dramatic and unique reduction or total loss of IgE binding patterns to the six combinatory mutants of Ana o 2. Total loss of IgE binding to all the Ana o 2 mutants tested was observed with Pt#4 sera (Fig. 5.8). The IgE reactivity of Pt#1 was intense with wt Ana o 2, and although total loss of IgE was not observed to any of the mutants, the intensity was nonetheless drastically reduced in comparison.

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A Pt #1 Pt #2 Pt #3 Pt #4 Pt #5 Pt #6 wtAna o 2

Epitope #3 and #6 C25A+Q110A

Epitope #15 D261A+W266A

Epitope #3 and #6 C25A+Q110A +R112A

Epitope #3, #6, C25A+Q110A+ and #15 D266A+W266A

Epitope #3, #6, C25A+Q110A+ and #15 R112A+D261A +W266A

Epitope #3, #6, Q20A+R112A+ and #15 D261A+W266A

B C25A+ D261A+ C25A+Q110A C25A+Q110A C25A+Q110A Q20A+R112A Q110A W266A +R112A +D261A +R112A+D261A +D261A +W266A +W266A + W266A Pt#1 91% 86% 90% 92.6% 92% 79% Pt#2 98.4% 81% 100% 100% 100% 100% Pt#3 89% 0% 100% 100% 100% 100% Pt#4 100% 100% 100% 100% 100% 100% Pt#5 100% 83% 100% 100% 100% 80% Pt#6 76% 100% 100% 100% 100% 100%

Figure 5.8. Dot-blot analysis (A) of six combinatory Ana o 2 mutants and sera from six cashew allergic Ana o 2 reactive patients. (B) Percentage decrease in IgE reactivity in comparison with Ana o 2 wt as determined by densitometry.

Although the reactivity of the mutants in vitro varied considerably with either the patients or mutants, the most consistent and strong reduction in IgE binding was obtained for the combination mutants (Fig. 5.8). The results of the ELISA as well as inhibition ELISA

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demonstrated that disruption of the three immunodominant IgE-reactive epitopes of Ana o 2 considerably decreased the IgE-binding capacity of the allergen.

Reactivity of Ana o 2 specific mAbs with Ana o 2 wt and mutants Twenty mAbs reactive with whole cashew-reactive were identified via ELISA. Of these 20, only eight demonstrated IgG binding to rAna o 2 in subsequent screenings with ELISA. Six of the eight strongly reactive mAbs were selected for future experiments: 1F5, 2B5, 4C3, 4F5, 5G10, and 4H9. These six Ana o 2-specific mAbs were tested for reactivity to Ana o 2 wt and mutant proteins by ELISA. With aid of SPOTs assay linear epitopes for two Ana o 2 specific mAbs 4H9 and 1F5 were determined. The sequences of the 2 reactive peptides are as follows: Peptide #6 -- YEAGTVEAWDPNHEQ; Peptide #33-- EESEDEKRRWGQRDN. Peptide #6 was recognized by the mAb 4H9 and peptide #33 by mAb 1F5. Similar studies with 3 pools of human cashew allergic sera demonstrated the immunodominant reactivity of peptide #33. Peptide #33 was thus selected for alanine scanning mutagenesis in order to define the amino acids critical for mAb 1F5 reactivity. Furthermore, this approach was used to determine whether the amino acids that we found to be critical for human IgE binding were also those that are involved in mouse IgG (mAb 1F5) binding. The critical amino acids for both the human IgE and mAb 1F5 were comparable (with some additional aa), thus demonstrating conserved binding reactivity. Since mAbs 4F5, 2B5, 5G10, and 4C3 were part of the pool that did not recognize these peptides individually, it was determined that they do not bind linear epitopes. Additionally, these 4 mAbs did not show reactivity to rAna o 2 and cashew extract on Western blot performed under reducing conditions, suggesting that they must be directed towards some conformational dependent or discontinuous binding epitopes. Using an indirect ELISA, we examined the ability of the six above mentioned Ana o 2 specific mAbs to react with solid-phase Ana o 2 mutant proteins (Fig. 5.9). Antibodies were used at dilutions that gave approximately 1-1.5 OD in the indirect ELISA when 20 μg/mL of wt Ana o 2 was adsorbed to the plate 1/500 dilution for 2B5 and 5G10, 1/1000 for 1F5 and 4F5, 1/4000 for 4C3, 1/2000 for 4H9. None of the mutations had any effect on antibody binding of mAb 4H9 in the ELISA.

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Figure 5.9. Ana o 2 specific mAb reactivity to Ana o 2 wt and mutant proteins by indirect ELISA. O.D = Optical density

Mutation of C25A reduced antibody binding by 36% for 2B5, 77% for 4C3, and 17% for 1F5, 42% for 5G10. Mutation of D261A had a major reduced binding (81%) of mAb 5G10; R112A reduced mAb 5G10 reactivity by 88%. Mutation of W226A demonstrated reduced antibody reactivity by 71% for 1F5, 72% for 5G10, and 55% for 2B5. Double mutant C25A+Q110A reduced antibody binding by 80% for 4C3, 44% for 2B5, and 57% for 5G10 (Fig. 5.8). The reactivities of mAb 1F5, 4F5, and 4H9 were maintained inspite of these mutations. The combination point mutants in three immunodominant epitopes had a considerable impact on the binding of all the tested mAbs except 4H9 as listed in Table 5.4. The marked decrease of mAb reactivity for mutations made in three immunodominant epitopes demonstrates the importance of these residues.

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Table 5.2. Reactivity of Ana o 2 specific mAbs with Ana o 2 wt and mutants.

Ana o 2 proteins 1F5 2B5 4C3 4F5 4H9 5G10

Ana o 2 wt 1.2461 1.008 1.273 1.459 1.536 1.367

C25A 1.025 (83%)2 0.647 (64%) 0.289 (23%) 1.374 (94%) 1.374 0.797 (58%)

D261A 0.547 (44%) 0.984 (98%) 0.988 (78%) 1.267 (87%) 1.59 0.264 (19.3%)

Q110A 1.278 1.116 1.134 (89%) 1.413 (98%) 1.426 1.126 (83%)

R112A 1.267 0.962 (95%) 1.212 (95%) 0.839 (58%) 1.573 0.165 (12%)

W266A 0.363 (29%) 0.453 (45%) 0.919 (72%) 1.428 (98.4%) 1.397 0.379 (28%)

C25A+Q110A 1.114 (89.4%) 0.569 (56%) 0.252 (20%) 1.204 (84%) 1.345 0.593 (43%) C25A+Q110A +D261A+W266A 0.346 (28%) 0.327 (32%) 0.202 (16%) 1.144 (79%) 1.312 0.057 (4%) C25A+Q110A+ R112A+D261A+W266A 0.141(11.3%) 0.302 (30%) 0.164 (13%) 0.786 (54%) 1.239 0.072 (5%) Q20A+R112A +D261A+W266A 0.327 (26%) 0.287 (28.4%) 1.002 (78%) 0.677 (46%) 1.426 0.243 (16%)

1Data presented as adjusted O.D values (O.D of tested sample-background reading). 2These values denote % of Ana o 2 wt binding.

Allergen-specific basophil activation demonstrate reduction of allergenicity for Ana o 2 mutants The allergenic activity of six Ana o 2 mutants was further investigated by evaluating the activation of human basophils using the ex vivo mBAT. The mBAT represents a functional assay, and in the absence of the gold standard of food challenges with these purified rAna o 2 wt and mutants, provides evidence that modifying the immunodominant IgE reactive epitopes leads to considerable decreased cellular activation. Basophils from healthy non-atopic donors were isolated using density gradient centrifugation. The inherent IgE from the isolated basophils were stripped using lactic acid buffer and the cells were subsequently loaded with IgE from three individual cashew allergic sera (Ana o 2 reactive). These three sera (Pt#1, #4, and #6) were selected from the six Ana o 2 reactive cashew allergic patients, based on availability of an adequate amount of sera. The basophils loaded with the above mentioned patient IgE, were challenged with wt rAna o 2 or the mutants. The cells were then analyzed by two-color flow cytometry in a BD FACSCanto™ flow cytometer (Becton Dickinson, San Jose, CA). The resultant activation of basophils, as a result of

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allergen-mediated IgE cross-linking was analyzed by detecting CD203c cells demonstrating upregulation of CD63 marker.

rAna o 2 wt Q110A

C25A+Q110A+R112A C25A+Q110A +D61A+W266A +D261A+W266A

fMLP – Positive BSA – Negative control control

Figure 5.10. Representative dot-plot using pt#1 serum IgE and reactivity of Ana o 2 wt, Ana o 2 mutants (Q110A, C25A+Q110A+D261A+W266A, C25A+Q110A+R112A+D61A+W266A), positive (fMLP), and negative (BSA) control. Cells were initially based on forward and side scatter characteristics, and then the viable cells identified by CD203c+ 7AAD-. Positive activation was considered when the cells showed CD63+CD203c+ 7AAD- staining profile.

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rAna o 2 wt Q110A

C25A+Q110A+R112A C25A+Q110A +D61A+W266A +D261A+W266A

fMLP – Positive BSA – Negative control control

Figure 5.11. Dot-plot using pt#4 serum IgE and reactivity of Ana o 2 wt, Ana o 2 mutants (Q110A, C25A+Q110A+D261A+W266A, C25A+Q110A+R112A+D61A+W266A), positive (fMLP), and negative (BSA) control. Cells were initially based on forward and side scatter characteristics, and then the viable cells identified by CD203c+ 7AAD-. Positive activation was considered when the cells showed CD63+CD203c+ 7AAD- staining profile.

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The basophil marker CD203c was used to set a basophil gate including only CD203c positive cells. Two hundred events were collected within the gate and were analyzed for CD63 positivity (marker for basophil activation and granule release). The extent of basophil activation was calculated as the percentage of CD63 positive events among the 200 events in the basophil gate (CD63/CD203c). Basophils from non-atopic individuals loaded with serum IgE from Pt#1, #4 and #6 respectively, and stimulated with wt rAna o 2, resulted in the activation of basophils with all the three Ana o 2-reactive cashew allergic sera tested. Data from a representative experiment(s) shows that the rAna o 2 wt protein can activate basophils loaded with IgE from food allergic individuals (Figs. 5.10, 5.11, and 5.12). On the other hand, the Ana o 2 mutants tested showed a decrease in the intensity of basophil activation as observed in the reduction of CD63 expression in comparison with wt Ana o 2 (Figs. 5.10, 5.11, and 5.12). The allergen concentrations used in this study were standardized for comparative purposes and did not necessarily reflect the minimum concentration required for activation. The ability to induce degranulation/activation of basophils was decreased with an increased number of mutations within the identified immunodominant epitope regions. Accordingly, the triple mutant-- C25A+Q110A+R112A+D261A+W266A, showed the lowest capacity to activate basophils (Figs. 5.10, 5.11, and 5.12). The positive control used for activating basophils was fMLP and demonstrated upregulation of CD63 as shown in figures 5.10, 5.11, and 5.12. The negative control antigen (BSA) did not activate basophils with either cashew allergic patient sera. Furthermore, stimulation with sera from a non-atopic individual with rAna o 2 did not result in basophil activation (data not shown).

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rAna o 2 wt Q110A

C25A+Q110A C25A+Q110A+R112A +D261A+W266A +D61A+W266A

fMLP – Positive BSA – Negative control control

Figure 5.12. Dot-plot using pt#6 serum IgE and reactivity of Ana o 2 wt, Ana o 2 mutants (Q110A, C25A+Q110A+D261A+W266A, C25A+Q110A+R112A+D61A+W266A), positive (fMLP), and negative (BSA) control. Cells were initially based on forward and side scatter characteristics, and then the viable cells identified by CD203c+ 7AAD-. Positive activation was considered when the cells showed CD63+CD203c+ 7AAD- staining profile.

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The AI recorded for Ana o 2 was considered to be 100% and extrapolated to compare the relative activation of the four Ana o 2 mutants (Fig. 5.13)

100 90 80 % Decrease 70 in CD63 in comparison 60 with 50 Q110A Ana o 2 wt 40 C25A+Q110A+D261A 30 +W266A 20 C25A+Q110A+R112A 10 +D261A+W266A 0 Pt#1 Pt#4 Pt#6 Ana o 2 reactive cashew allergic sera

Figure 5.13. The percentage decrease in CD63 expression observed with Ana o 2 mutants. The AI with Ana o 2 wt with three individual cashew allergic sera tested was considered as 100%.

All the three Ana o 2 mutants tested using mBAT demonstrated significant reduction in basophil activation as reflected by lower levels of CD63 expression in comparison with unmutated wt Ana o 2. The decrease in the basophil activation was recorded to be in the range with of 23-91% with respect to Ana o 2 wt (Fig. 5.13).

Secondary structure comparison of Ana o 2 wt and mutants Optically clear solutions in phosphate buffer were used to record CD spectra (200–260 nm) in 1 mm quartz cuvette (Fisher Scientific, Atlanta, GA) with AVIV CD spectrometer (Proterion Corp., Santa Barbara, CA). For each of the samples tested three CD spectra were recorded and the averaged values were used for analysis. The results of the far-UV CD analysis

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are expressed as mean residue molar ellipticity. Estimation of secondary structure from the recorded CD spectra of rAna o 2 wt and mutants indicated a combination of β-strands and α- helices (Fig. 5.14) The differences in the Ana o 2 wt and single point mutants are minor in comparison to the combination point mutants targeting three immunodominant epitopes (Fig. 5.14).

20 Ana o 2 wt 15 m Q110A d 10 m 5 C25A d Mean 0 residue 210 220 230 240 250 260 D261A+W266A ellipticityp -5 C25A+Q110A+D261A -10 u +W266A d -15 C25A+Q110A+R112A n +D261A+W266A -20 M -25

-30 Wavelength (nm)

Figure 5.14. The CD spectra of Ana o 2 wt and mutants. Spectra were obtained with an Aviv m spectrometer and the mean residue ellipticity is in degrees times square centimeters divided by decimoles.

Homology Modeling and Surface Mapping of Ana o 2 Homology-based models of the tertiary structure of Ana o 2 were generated to predict the 3D location of epitopes on the molecules. Interestingly, the identified linear IgE epitopes on Ana o 2 allergens were located throughout the exterior of the molecule when modeled as a trimer (Fig. 5.15). Almost all of those epitopes recognized strongly by one (red), all (green) or at least weakly by all patient sera pools (yellow), were located on what could be considered protruding regions of the molecule.

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IA Face IE Face

Figure 5.15. Surface epitope mapping of the Ana o 2 trimer model. Epitopes are colored as previously described and labeled on the molecules IA (left) and IE (right) faces as described by Adachi et al.248

The three immunodominant linear IgE-reactive epitopes identified by synthetic peptide approach were targeted for site-directed mutagenesis.112 The three epitopes are shown in different colors as seen in figure 5.16 (epitope #3 in teal, eptipoe#6 in blue, and epitope#15 in orange color).

A A B

Figure 5.16. The three immunodinant IgE reactive epitopes of Ana o 2 were targeted for mutational analysis. A and B are faces of Ana o 2.

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A B

Figure 5.17. The three immunodinant IgE reactive epitopes of Ana o 2 were targeted for B mutational analysis. The point mutations in the three epitopes are colored in red. A and B are faces of Ana o 2.

The mutations in the critical aa residues in the three immunodominant epitopes are depicted in red color as shown in figure 5.17. As can seen the immunodominant IgE reactive epitopes are flexible loops and perturbing them causes reduced and in some cases total loss of IgE reactivity. In addition, it can be speculated that these linear epitopes could be part of conformational epitopes or they could also cause conformational change that might explain the observed in vitro and ex vivo decreased IgE reactivity.

Discussion IgE-mediated allergies affect 20% of the population in western countries.1;2,6 Allergenicity of an allergen especially dietary can be attributed to a range of factors, including intrinsic physical and structural characteristics, thereby conferring stability and resistance to processing, pH, temperature, as well as enzymatic digestion.18 Tree nut allergic patients demonstrate increased specific IgE levels in the sera, and Ana o 2 has been identified to be a

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major allergen in our tested cashew allergic patients.60;112 IgE plays an important role in Type I allergies, culminated as an event of cross-linking, thus activating the basophils and mast cells and subsequently leading to the release of pro-inflammatory mediators.27;28;41;44 Determination of allergen-specific IgE binding epitopes appears to be of great importance, given the critical role allergen-specific IgE plays in the etiology of allergic reaction, and for enhancing our understanding of the allergenic nature of foods and for possible therapeutic intervention. Two categories of IgE binding epitopes, linear and conformational have been identified to occur in food allergens. Linear epitopes may be defined as short contiguous stretches of amino acids that are reactive with antibody, whereas conformational epitopes, are constituted by several sequentially discontinuous peptide segments that are brought together by the folding of the antigen into its native structure. While there is a tendency to categorize epitopes, it should be noted that epitopes could have both conformational and linear characteristics. Conformational IgE epitopes are more prevalent and important in allergies mediated by aeroallergens,321 while linear epitopes are important to food allergens mainly because the immune system will encounter them only after they have been partially denatured and digested by the human gastrointestinal tract.19;20 It has also been suggested that a differential responsiveness to linear vs conformational epitopes in allergic patients is of considerable clinical and diagnostic significance. For example, children who outgrow allergies to ovomucoid (a major allergen in eggs),249;322 and casein in cow’s milk21;250 may develop IgE to mostly conformational epitopes, whereas those that remain allergic into adulthood typically target linear epitopes. Moreover as tree nut and peanut allergies are persistent and life-long in nature, the linear IgE epitopes of these food allergens have garnered more attention than conformational epitopes, at least partly due to the difficulty in mapping conformational epitopes. Twenty-two linear IgE-binding epitopes were identified in Ana o 2 using the overlapping synthetic peptide approach. These epitopes are distributed throughout out the length of the Ana o 2 protein (Table 5.2).112 Interestingly, fifteen of the 22 (68%) epitopes reside on the acidic chain region of the Ana o 2 protein. Additionally, of the seven immunodominant epitopes identified, six were on the Ana o 2 acidic chain. Likewise, epitope mapping of several identified allergenic 11S globulins revealed higher incidence of IgE epitopes on the acidic chain as compared to the basic chain.171;238;251

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Homology-based models of the tertiary structures of Ana o 2 were generated to predict the 3D location of the identified linear IgE epitopes. It is evident that they are most likely surface exposed and thus would be involved in the non-covalent interactions with Fabs of IgE antibodies (Fig. 5.15 and 5.16). It has been observed that the overall aa composition between the epitope and non-epitope surfaces differs significantly.323 The epitope surfaces are generally enriched with tyrosine, tryptophan, charged, and polar aa residues, and are underrepresented by the aliphatic hydrophobic residues. The propensity of these aa in epitope surfaces is also reflected in protein-protein interfaces due to their capability to form a multitude of interactions. Additionally, it has also been suggested that aa that are proximal to the epitope act cooperatively, thus enhancing the binding interaction.324 A characteristic feature of an epitope is accessibility of aa residues via their exposed side chains thus rendering them chemically active and facilitating contact and interaction with the antibody(ies). Analyses of the secondary structures of epitopes revealed that they are predominantly loops and are significantly devoid of helices and strands compared to non-epitope antigen surfaces.323 These observations suggest that epitopes are flexible, as loops tend to be more flexible in comparison to other secondary structure elements. This flexibility in turn may aid the epitope surfaces to undergo conformational adjustments upon antibody binding. It is alleged that aa modifications in surface loops typically do not perturb the 3-D structure of the protein since surface loops are flexible.325 The structural epitope of a protein usually covers a 5–10 nm2 (500–1000 Å2) surface area involving 14–20 amino acid side chains. Only few of these side chains are responsible for the majority of the binding affinity, and the functional epitope may only involve 4–14 aa side chains. Testing the binding efficiency of antibodies to antigens with amino acid substitutions is one way to determine the functional epitope, from which the position of the structural epitope may be considered. Site-directed mutagenesis often leads to less radical changes to the overall protein structure although its effect on the biological function can be substantial.252 Site-directed substitutions are therefore used to locate active sites of protein as well as to identify functional epitopes and to reduce IgE binding of an allergen molecule.232;235 229;239;242;244 Of the 22 linear IgE epitopes identified on Ana o 2112, three immunodominant epitopes were selected for further analysis via alanine scanning mutagenesis of synthetic peptides. The substitution of a single amino acid was accomplished by synthesizing peptides with a single amino acid mutation to alanine in each of the 3 epitopes to determine the critical amino acids

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involved in IgE binding (Fig. 5.3). The effects of these alanine substitutions were measured by evaluating the IgE reactivity patterns of pooled cashew allergic sera exhibited by the mutations. Single-amino-acid substitutions in the major IgE-binding epitopes of Ana o 2 resulted in a significant reduction or loss of IgE binding as evident from the reactivity patterns (Fig. 5.3A). In this study, the information generated by alanine scanning mutagenesis was applied to develop hypoallergenic mutants of the Ana o 2 protein by site-directed mutagenesis of the Ana o 2 cDNA by introducing specific point mutations to alanine. The purpose is to generate hypoallergens of Ana o 2 that exhibit properties favorable for allergen specific immunotherapy. These properties comprise reduced binding of IgE from individual cashew allergic patients sera to the various mutated Ana o 2 proteins in immunoblotting as well as ELISA; and the need for larger amounts of the modified allergen in an ELISA-based inhibition assay for serum IgE from a pool of cashew allergic patients. In comparison with wt Ana o 2, the mutants demonstrated reduction in IgE binding capacity by 75-25% depending on the individual serum samples tested (Fig. 5.5). The critical aa residues are hence thought to be indispensable in binding to IgE. Furthermore mAbs raised against cashew Ana o 2, highlighted the impact of the aa substitutions, and demonstrated that some of them were clearly antigenically significant, because the mAbs reduced its binding with the mutant(s), which carried this substitution. These anti-Ana o 2 mabs are reactive to either linear or conformational epitopes, and thus served to observe the relative effect of these mutations on the respective epitopes. The mAb 4C3, 2B5, and 5G10 recognized aa residue substitutions in two or more distantly located antigenic sites on Ana o 2 and thus could probably identify critical residues on the respective conformational epitope(s) on Ana o 2. Some of the tested Ana o 2 mAbs have been demonstrated to mimic the reactivity of human IgE by competitive ELISAs and immunoblots (data not shown), suggesting that the IgG and IgE epitopes at least partially overlap, or are separate, but closely spaced causing steric hindrance or conformational change. These empirical data provide evidence of reduction and complete loss of IgE and mAb binding capacity of the Ana o 2 point mutants and combinatory mutants in vitro, and provide encouraging results to evaluate the potential of the Ana o 2 mutants as hypoallergenic engineered molecules in vivo. It has been reported that the results of the in vitro immunochemical analyses often do not correlate with data obtained from skin testing as well as clinical histories of the allergic individuals and certainly do not provide any information on the ability of a protein to

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induce type I allergic reactions. It has been reported that a decrease in IgE-binding potency (2– 10 times) as evaluated by immunoblot or ELISA translates into a much stronger inhibition of biological activity (10-200 times).241 This implies that assessment of hypoallergenicity by measuring IgE-binding activity is not adequate and biological potency tests like basophil mediator release assay or basophil activation test and/or SPT are essentially crucial for evaluating the allergen mutants. Thus evaluating the basophil-activating potential of the Ana o 2 mutants was an important aspect and was the final focus of this study to determine the efficacy of modified proteins for specific immunotherapy. Intradermal tests, however, cannot be used in quality control for ethical reasons and in vivo SPT using recombinant purified allergens would require the production of these materials under good manufacturing practice (GMP) conditions, which limits its use.253 A positive SPT strongly suggests that the tested allergen is capable of activating basophils and tissue mast cells, however, the positive prediction of a reactive SPT is only about 50%.254 In combination with SPTs, in vitro estimates of serum IgE facilitate the accuracy of diagnosing food allergy.254 Most importantly skin tests may induce fatal anaphylaxis, and oral challenge tests, considered as the “gold standard” for allergy diagnosis, cannot be used in routine testing for obvious ethical reasons.255 For this purpose, considerable efforts have been directed towards developing in vitro methods based on the activation and mediator release of effector cells involved in an allergic reaction. In recent years, some studies have shown strong correlations between basophil activation tests and clinical reactivity.256-258 These biological tests involving mediator release by basophils include histamine, serotonin, tryptase, β-hexosaminidase, prostaglandin D2, cysteinyl leukotrienes, and eosinophil cationic protein.259-261 However they may not always correlate well with the skin prick test and the clinical symptoms of the allergic patients, thus the clinical benefit of basophil mediator release test has remained controversial.261;262 In addition to the release of several soluble mediators, basophil activation as a consequence of allergen challenge leads to characteristic changes in the expression of cell surface membrane bound activation markers viz. CD45, CD63, and CD203c.25;263;264 In the early 1990s, it was reported that the basophil activation marker CD63 is expressed with high density on activated basophils versus only weakly (<5%) on resting cells.263 This discovery promoted the development of a flow cytometric technique to analyze allergen-specific ex vivo activation of

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basophils.25;256;265-267 In resting basophils, CD63 is anchored in basophilic intracytoplasmic granules and is only weakly expressed on the cell surface.268 As a consequence of basophil activation and degranulation, CD63 expression is upregulated with high density on the cell surface due to fusion between granules and plasma membranes, and mirrors mediators release following stimulation with allergens, anti-IgE, or N-formyl-methionyl-leucyl-phenylalanine (fMLP).256;263;266;269 Thus, CD63 expression has been proposed as a reliable tool to monitor basophil activation. The flow cytometric basophil activation test (BAT), based on the detection of allergen induced CD63 expression, is a relatively new technique , and has proven to be effective in the diagnosis of IgE mediated allergies to various allergens such as natural rubber latex, foods, hymenoptera venoms, and aeroallergens (pollen, dust mites).256;258;264;266;267;270-274 In addition to CD63, another marker CD203c has been proposed as a valuable tool for the detection of both non-activated and activated basophils by flow cytometry.275 Due to its restricted expression pattern on the basophil/mast cell lineage, CD203c is considered to be a specific marker for monitoring allergen-induced activation of basophils in flow cytometric basophil activation tests.275 Allergen challenge leads to a rapid up-regulation of both CD203c and CD63 activation markers.26;276;277 278 CD203c and CD63 upregulation appear to be regulated by different pathways and follows different kinetics as CD203c upregulation of cell surface expression on activated basophils reaches a maximum after 5 min, and precedes CD63 upregulation which requires 15 min of antigen stimulation.278 The combined use of CD63 and CD203c for the analysis of allergen reactivity is recommended as it increases the sensitivity of the assay.258 The ex vivo BATs are becoming increasing common assay systems for predicting clinical reactivity. In the absence of the gold standard of food challenges with native or recombinant protein, these assays represent functional assays providing evidence that an allergen can result in IgE-crosslinking and cellular activation. A modified BAT (mBAT) has recently been shown by our collaborator Dr. Teuber, to be successful in evaluating allergenic cross-reactivity between sesame seed and walnut.303 In this study we have used a modified BAT (mBAT) to examine the reactivity patterns and potential clinical relevance of Ana o 2 wt and mutants. The percentage of CD63 membrane expression on CD203c positive peripheral blood basophils, after activation with specific Ana o 2 wt allergen and mutants, was analyzed. The Ana o 2 mutants tested show significantly reduced

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basophil activation profile with basophils obtained from non-atopic individuals that were loaded with IgE from two cashew Ana o 2-allergic patients. These observations indicate that perturbations of the immunodominant IgE epitopes have reduced the relative affinities of the specific IgE antibodies for Ana o 2. As a result of introducing mutations in Ana o 2, the local changes on the Ana o2 topography may explain why the IgE and mAb binding ability of these mutants was dramatically reduced in both in vitro and ex vivo based assays. A decrease and or loss in IgE binding potential minimizes the risk for anaphylaxis and increases the likelihood of development of proteins or peptides suitable for use in allergen-specific immunotherapy. Even though the traditional skin prick test remains the gold standard for IgE antibody detection, the cell-based ex vivo methods offer numerous advantages like speed, precise quantitation, and absolute safety. This study demonstrated the successful use of the flow cytometry assay system, as tools to determine the hypoallergenicity of Ana o 2. In combination with current assays (immunoblotting, ELISA, ImmunoCAP, and skin prick testing, etc.), the mBAT may be a useful supplement for predicting clinical reactivity. The mBAT using non- atopic human basophil donors and passive sensitization may be a simpler, faster, and more biologically relevant assay compared with traditional BATs. Passive sensitization is a promising tool for analyzing the reactivity of sera because the antibodies from sera are more stable and can be frozen, as opposed to working directly with patient basophils. In conclusion, these studies are encouraging in the development of new generations of immunological reagents for allergy testing and specific immunotherapy. Moreover, detailed information concerning IgE-binding epitopes, T-cell epitopes, and 3D structure can help in the design of hypoallergenic candidate vaccines. The advantages of using the hypoallergenic approach are that the modified allergens can be precisely defined and the design features validated in respect to the specific immuno-therapeutic applications. A decrease and or loss in IgE binding potential minimizes the risk for anaphylaxis and increases the likelihood of development of proteins or peptides suitable for use in allergen-specific immunotherapy. In addition, the advantages of genetically modified allergens include intact T cell epitopes that induce protective antibody responses upon immunization that can antagonize IgE-mediated effects.

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CONCLUSIONS

Allergies represent a health problem of increasing importance in the industrialized world. This type I allergy is based on the formation of IgE antibodies against, in principle, harmless proteins, allergens. The application of molecular biology techniques to the analysis of allergens can help to improve the knowledge of their structures, immunologic properties, and functions. Our studies have been instrumental in identifying and characterizing various tree nut allergens. The major tree nut allergens have been the seed storage proteins – 7S and 11S globulins. In addition to these seed storage proteins other relevant allergens like profilin and 60S ribosomal P2 protein have been identified in almond. In comparison to aeroallergens that apparently sensitize allergic patients as intact, structurally folded proteins via the respiratory mucosa, many food allergens such as milk, egg, peanut and tree nut allergens become digested in the gastrointestinal tract and may sensitize in the form of unfolded polypeptides. IgE antibodies of food allergic patients are therefore frequently directed against continuous and sequential epitopes. Continuous epitopes can be mapped with overlapping peptides synthesized according to the primary aa sequence or by fragmentation. In contrast, the determination of conformational epitopes represents a difficult task. Linear epitope mapping of cashew Ana o 1 and Ana o 2, have identified linear IgE reactive epitopes. The elucidation of allergenic cross-reactivity was demonstrated between cashew and pistachio seed storage proteins. Deciphering the patterns of allergic cross-reactivities are important because they may reflect (a) the pattern of clinical sensitivities, (b) regulation of allergic sensitization via B- and T-cell epitope cross-reactivity, and (c) the need for screening of novel foods for allergenicity. Identification of common structural motifs is likely to improve the quality of assessment of cross-reactivity and allergenicity. Allergen specific immunotherapy remains the only truly disease-modifying treatment for allergic diseases. Conventional allergen extracts used in immunotherapy are prepared from natural sources and have numerous disadvantages, such as the presence of undefined material, huge variability in sample composition, and contamination of allergens from other sources.209 These problems can be overcome with recombinant allergens and modified hypoallergen proteins.24

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Our current research focuses on the design of hypoallergenic Ana o 2 proteins. We have generated a panel of single point and multiple point Ana o 2 mutants, primarily targeting the various critical residues for IgE reactivity identified by alanine scanning mutagenesis. These mutants were evaluated by in vitro and ex vivo analysis. Our objective was to use a rational approach to design hypoallergenic candidates of the major cashew allergen Ana o 2 for potential application in SIT. By using the whole modified allergen, the IgE epitopes are perturbed and the T cell epitopes are maintained, which has been shown to be important to modify the immune response.45 The modified hypoallergenic proteins that have intact T cell epitopes have the capacity to induce an IgG antibody response that serve as blocking antibodies and are suggested to play a central mechanistic role in SIT.24 Since IgE cross-linking by allergen is an obligatory step in the initiation of reactions, therapeutic strategies aimed at inhibiting IgE+allergen interactions could provide a mode of treatment for these diseases. In conclusion, detailed information concerning IgE-binding epitopes, T-cell epitopes, and 3D structure can help in the design of hypoallergenic candidate vaccines. The advantages of using the hypoallergenic approach are that the modified allergens can be precisely defined and the design features validated in respect to the specific immuno-therapeutic applications.

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FUTURE DIRECTIONS

For future studies, the T cell responses to these modified allergens needs to be explored

and may uncover opportunities for modulation of the TH2 response to TH1. It is crucial to identify dominant T-cell epitopes of Ana o 2, as this information is critical to the rational design of allergen vaccines. Retention of the allergen’s ability to interact with T cells allows the shift

from an allergen-specific TH2 response to a desired immuno-protective allergen-specific TH1 response.45 In addition, animal studies need to be considered in order to further confirm the allergenicity of allergens and would provide valuable information regarding the in vivo sensitizing capacity of the hypoallergenic candidates. Another innovative SIT option suggested for food allergy immunotherapy includes the use of DNA vaccination of allergens and conjugation with CpG motifs.279 Preliminary phase I and II trials in ragweed allergic patients have shown that allergen DNA-CpG conjugates are well tolerated, less allergenic, and induce IgG anti-allergen antibodies.280 It would be worth investigating DNA vaccination with Ana o 2 (mutants) as well as conjugates of Ana o 2 mutants (DNA)-CpG motifs in animal models to study the efficacy of these candidates. An additional interesting approach reported, involves making hybrid molecules of different allergens.24;210 An engineered hybrid vaccine composed of three different bee venom allergens reported markedly reduced IgE binding activity and skin test reactivity in bee allergic patients.210 Since, three major cashew allergens have been identified so far (Ana o 1, Ana o 2, and Ana o 3), it seems likely that it would be possible to produce hypoallergenic forms of all three allergens, and use as a cocktail or hybrid immunotherapeutic vaccine for cashew allergy. It has been reported that co-aggregation of FcεRI with FcRIIb can block FcεRI-mediated reactivity.281;282 An approach using a chimera fusion protein of human Fc:cat allergen (Fel d 1) protein, demonstrated successful inhibition of allergen-driven IgE-mediated mediator release in vitro from human basophils and cord blood-derived mast cells.39;283 Such chimera fusion protein with human Fc:Ana o 2 (mutated/wt) would be worth investigating and possibly could serve as a promising vaccine candidate given the success reported with the cat allergen Fel d 1. Another novel approach recently reported, targets enhancement of allergen presentation through the MHC class II pathway via engineered modular antigen translocating (MAT) vaccines, to achieve a high dose effect, without actually increasing the allergen dose, to make

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284 SIT more efficient. MAT-allergen vaccines induce a strong secretion of TH1 type cytokines 284 and IL-10 paralleled by a decreased secretion of TH2 type cytokines. It would certainly be interesting to produce MAT-allergen vaccines with mutated Ana o 2 and study the therapeutic potential of these constructs in cashew allergy.

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APPENDIX A

HUMAN SUBJECTS COMMITTEE APPROVAL MEMORANDUM

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APPENDIX B

INFORMED CONSENT FORM

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322. Jarvinen KM, Beyer K, Vila L, Bardina L, Mishoe M, Sampson HA. Specificity of IgE antibodies to sequential epitopes of hen's egg ovomucoid as a marker for persistence of egg allergy. Allergy 2007;62:758-65.

323. Rubinstein ND, Mayrose I, Halperin D, Yekutieli D, Gershoni JM, Pupko T. Computational characterization of B-cell epitopes. Mol. Immunol. 2007 Nov 17(epub.)

324. Neuvirth H, Raz R, Schreiber G. ProMate: a structure based prediction program to identify the location of protein-protein binding sites. J. Mol. Biol. 2004;338:181-99.

325. Saunders, C.T., Baker, D. Evaluation of structural and evolutionary contributions to deleterious mutation prediction. J. Mol. Biol. 2002;322:891-901.

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BIOGRAPHICAL SKETCH

Personal

Pallavi Dattatray Tawde Born in Mumbai (Bombay), India.

Education

April, 2008 Ph.D. in Biological Science Florida State University, Tallahassee, FL

Dec, 2004 M.S. in Biological Science Florida State University, Tallahassee, FL

June, 1993 M.Sc. in Microbiology University of Bombay, India

June, 1990 B.Sc. in Microbiology University of Bombay, India

Professional Experience

2001- 2008 Graduate Research Assistant Department of Biological Science, Florida State University

2001- 2008 Graduate Teaching Assistant Department of Biological Science, Florida State University

PUBLICATIONS:

Tawde P, Robotham JM, Hoffman GG, Teuber SS, Sathe SK, Roux KH. Allergy vaccine therapy: Mutational epitope analysis of Ana o 2, a major cashew allergen. (Manuscript in preparation)

Robotham JM, Tawde P, Hoffman GG, Teuber SS, Sathe SK, Roux KH. Identification of a human-IgE binding conformational epitope on the cashew nut (Anacardium occidentale) important allergen, Ana o 2. (manuscript in preparation)

Tawde P, Abolhassani M, Seamon V, Wang F, Teuber SS, Uratsu S. Dandekar AM, Vieths S, Sathe SK, and Roux KH. IgE-reactive 60S ribosomal protein P2 in almond (Prunus dulcis) and walnut (Juglans regia), a new class of food allergen cross-reactive with fungal aeroallergens. Clinical Exp Allergy (manuscript under revision).

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Comstock SS, Robotham JM, Tawde P, Kshirsagar H, Roux KH, Sathe SK, Teuber SS. IgE- Reactive Proteins in Cashew Apple Juice Concentrate. Clinical Exp Allergy (manuscript under revision)

Willison LN, Tawde P, Robotham JM, Penny R, Teuber SS, Sathe SK, Roux KH. Pistachio vicilin, Pis v 1, is allergenic and cross-reactive with the homologous cashew allergen, Ana o 1. Clin. Exp. Allergy (manuscript accepted, will be published in 2008)

Baker VS, Imade G, Molta N, Tawde P, Pam S, Obadofin M, Sagay S, Egah D, Iya D, Afolabi B, Baker M, Ford K, Ford R, Roux KH, Keller TCS III. Cytokine-associated neutrophil extracellular traps and antinuclear antibodies in Plasmodium falciparum infected children under six years of age. Malar J. 2008;29;7(1):41.

Tawde P, Yeldur PV, Wang F, Teuber SS, Sathe SK, Roux KH. Cloning and characterization of profilin (Pru du 4), a cross-reactive almond allergen. J. Allergy Clin. Immunol. 2006; 118(4): 915-22.

Wang F, Robotham JM, Teuber SS, Tawde P, Sathe SK, Roux KH. Ana o 1, a cashew (Anacardium occidentale) allergen of the vicilin seed storage protein family. J. Allergy Clin.Immunol. 2002;110: 160-6.

ABSTRACTS & PRESENTATIONS:

"IgE-reactive 60S ribosomal protein P2 in almond (Prunus dulcis) and walnut (Juglans regia), a new class of food allergen." 7th Annual Conference of FOCIS, San Diego, CA, 2007.

"Pistachio vicilin, Pis v 1, is allergenic and cross-reactive with the homologous cashew allergen, Ana o 1." 7th Annual Conference of FOCIS, San Diego, CA, 2007.

"Identification of Profilin as an Almond Nut Allergen." Annual Meeting of AAAAI 2005, San Antonio, TX. 2005.

"Identification of Allergenic Cross-Reactive Proteins in Cashew and Pistachio Nut." Annual Meeting of AAAAI 2005, San Antonio, TX. 2005.

"Linear IgE Epitope Mapping of Various Tree Nut and Legume 11S globulins." 12th International Congress of Immunology and 4th Annual Conference of FOCIS, Montreal, Canada. 2004.

"Hypoallergenic Variants of the Legumin, Ana o 2, a Major Cashew Allergen." 12th International Congress of Immunology and 4th Annual Conference of FOCIS, Montreal, Canada. 2004.

"Mutational Analysis of the Cashew Vicilin Allergen, Ana o 2," American Academy of Allergy, Asthma and Immunology, 60th Annual Meeting, San Francisco, CA. 2004.

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"Ana o 1 and Ana o 2, major cashew allergens of the vicilin and legumin families," Collegium Internationale Allergologicium Conference. 2002.

Awards and Honors

2004-2006 Nominated three times – Outstanding Teaching Assistant

2004 Nominated for Brenda Weems Bennison Memorial Scholarship for Graduate Studies

1990-1994 The Foundation for Medical Research Fellowship, India

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