Cover Page. The handle holds various files of this Leiden University dissertation

Transcription

1 Cover Page The handle holds various files of this Leiden University dissertation Author: Voskamp, Astrid L. Title: Clinical allergy : basophils, T cells, and therapeutic design Issue Date:

2 Clinical Allergy: Basophils, T cells, and Therapeutic Design

3 Clinical Allergy: Basophils, T cells, and Therapeutic Design Proefschrift ISBN: ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof. mr. C.J.J.M Stolker, volgens besluit van het College voor Promoties ter verdediging op donderdag 16 juni 2016 klokke uur uur 2016 Astrid Voskamp All rights reserved. No part of this thesis may be reproduced in any form without permission of the author. The work presented in this thesis was performed at the Department of Immunology, Monash University, and the Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital, Australia. door The studies described in this thesis were financially supported by the Ilhan Food Allergy Foundation, the National Health and Medical Research Council of Australia, The Alfred Trusts and Novartis. Astrid Lida Voskamp geboren te Thurso (Schotland) in 1981 Printing: Printservice Ede

4 Promotors Prof. Dr. M. Yazdanbakhsh Prof. Dr. R.E. O Hehir (Monash University, Australia) Contents Chapter 1 General Introduction 9 Co-promotor Dr. S.R. Prickett (Monash University, Australia) Chapter 2 Clinically relevant IgE reactivity and basophil activation to goat s milk after cutaneous sensitization 43 Leden Promotiecommissie Prof. Dr. R.E.M. Toes Prof. Dr. R. Gerth van Wijk (Erasmus Medisch Centrum, Rotterdam) Prof Dr. R. van Ree (Academic Medical Center, Amsterdam) Prof. Dr. C. Taube Dr. H.H. Smits Chapter 3 Chapter 4 Chapter 5 MHC Class II expression in human basophils: induction and lack of functional significance Ara h 2 peptides comprising dominant CD4 + T-cell epitopes: candidates for a peanut allergy therapeutic Ara h 1 CD4 + T-cell epitope-based peptides: candidates for a peanut allergy therapeutic Chapter 6 Cysteine-to-serine substitution can alter susceptibility of therapeutic peptides to gastrointestinal enzyme digestion, affecting potential for oral delivery 125 Chapter 7 Clinical efficacy and immunological effects of omalizumab in allergic bronchopulmonary aspergillosis 139 Chapter 8 Summarizing Discussion 157 Appendix Nederlandse samenvatting Acknowledgements Curriculum Vitae List of publications

5 1 GENERAL INTRODUCTION

6 CHAPTER 1 GENERAL INTRODUCTION 1. The immune system The immune system is the body s defense mechanism to fight off pathogens and tumor growth. It comprises both innate and adaptive components. Innate cells are equipped to recognize conserved components shared by many pathogens, whereas the adaptive cellular response is developed to target specific antigens of different pathogens, and matures upon repeated exposure. Innate cells include monocytes, macrophages, dendritic cells (DC), natural killer cells, neutrophils, eosinophils, mast cells and basophils. These cells are equipped with receptors to detect conserved pathogen components. Many of these cells are also highly efficient at antigen uptake and processing for subsequent presentation to cells of the adaptive immune system, in order to initiate an adaptive response to the presented antigen(s) of the pathogen. Eosinophils, neutrophils and basophils, collectively known as granulocytes, respond primarily to extracellular parasites, and react accordingly. For parasites that are too large for single cells to ingest for either presentation to adaptive cells or intracellular destruction, granulocytes secrete specific compounds to attack the parasite directly and recruit more immune cells to the site. These cells are also capable of responding, via their immunoglobulin Fc receptors, to specific parasite antigens coated by antigen-specific immunoglobulin, produced by cells of the adaptive immune system. Adaptive immune cells include B and T lymphocytes. B cells can recognize whole antigens via surface-bound immunoglobulin molecules (B cell receptors). Antigen uptake, processing and presentation on MHC class II molecules on the surface of antigen presenting cells (APC) allows for T cell recognition of antigen fragments (peptides) via the T Cell Receptor (TCR). B cells, through interaction with T cells, produce more antigen-specific immunoglobulin with improved antigen specificity. Depending on the type of response required to deal with a pathogen, different T helper (Th) cells come into play 1-3. A Th1 response, characterized by the production of IFN-g and IL-2, is generally initiated towards intracellular pathogens including viruses, bacteria or fungi. Similarly, Th17 cells secrete IL-17 and IL-22, which stimulate neutrophils to combat extracellular bacteria or fungi. In contrast, a Th2 response, characterized by production of IL-4, IL-5 and IL- 13, is initiated upon infection with larger parasites, such as helminths. This response is required for recruitment of specific cells equipped to deal with the parasite, such as eosinophils and basophils. In addition, a regulatory T cell response (Treg), marked by Foxp3 expression or IL- 10 and TGF-β production, exists to dampen any excessive Th1 or Th2 responses that may be harmful to the body. Th1 responses can result in B cell production of antigen-specific IgG 1, IgG 2, or IgG 3, whereas Th2 responses typically induce IgG 1 and IgE production. Each of these antibody isotypes assists in the development of a targeted immune response, through either neutralizing the pathogen directly or marking it for attack by other cells. In a Treg environment (especially with IL-10), the production of IgG 4 is induced, an antibody isotype with many anti-inflammatory attributes 4. The adaptive cells of the immune system are primed to respond to specific antigens and, upon second exposure, can rapidly expand from a pool of memory cells in order to quickly and efficiently eradicate a recognized pathogen. This is the basis of developing immunity to a pathogen, and the fundamental mechanism involved in effective vaccination. Just as the development of the adaptive immune system is a milestone in our evolution, understanding and exploiting its mechanism of action is a milestone in the development of modern day medicine. 2. Allergy Although adaptive immunity is imperative to our survival, misdirection of this sophisticated mechanism can lead to a wide range of diseases. Central tolerance, originating in the bone marrow and thymus, prevents the immune system from recognizing self antigens, and thereby the development of T cell and/or B cell mediated autoimmune disorders. Peripheral tolerance occurs in the lymph nodes and is vital for restraining the adaptive immune system from attacking harmless environmental antigens. Failure to do so can result in allergic disease, characterized by an exaggerated Th2-skewed immune response to harmless components derived from food, pollens, dust mites, molds and other commonly encountered substances. Symptoms of allergic disease may range from mild itching to airway inflammation and even life-threatening anaphylaxis. An anaphylactic reaction is systemic and primarily observed in drug, insect venom and food allergies, although the mechanism behind the severity of symptoms associated with these particular allergies has yet to be determined. 2.1 Allergy prevalence Allergic disease can present in many different forms, including asthma, allergic rhinitis, atopic dermatitis, and food allergies. Progression from early-life atopic dermatitis to later allergic rhinitis, food allergy, and asthma has repeatedly been observed, and is now termed the allergic or atopic march 5. The actual prevalence of allergies is difficult to assess. Prevalence studies face challenges in several areas, including willingness to participate, which may bias recruitment to include those more likely to be affected; timing, as allergies may resolve or develop at different ages; and the method of diagnosis, from self reported to provocation challenges with the actual allergens. In addition, difficulties arise in the distinction between IgE and non-ige mediated reactions 6. Currently, allergic disease is estimated to affect approximately 20% of individuals in developed regions, with the prevalence having increased substantially in the past two decades 7. There is comparatively little information available on the prevalence of allergies in developing regions of the world. Although the prevalence of allergies and asthma is estimated to be lower than in westernized countries 8, increased prevalence has been reported in urban areas of developing countries, where a more westernized lifestyle has gradually been adopted Food allergies, most commonly directed towards cow s milk, egg, wheat, soy, peanut, tree nuts, fish, and shellfish 12 are now estimated to affect more than 1-2%, but no more than 10% of the population, based on a comprehensive review of the literature published between

7 CHAPTER 1 GENERAL INTRODUCTION and Cow s milk and egg allergies are primarily observed in young children, and often resolve by the age of five. Peanut, fish and shellfish allergy, in contrast, generally persist into adulthood 14. As reported for other allergic diseases the prevalence of food allergies is estimated to have more than doubled over the last two decades Launched in 2005, the EuroPrevall project was initiated with the aim to identify key risk factors for the development of food allergy and generate uniform European databases. The project was originally designed to assess European populations but has now extended to Russia, China and India, encompassing a wide range of lifestyles and socioeconomic status. The data resulting from these studies will provide the most comprehensive assessment of food allergy prevalence to date using gold standard methodology Allergic disease places a considerable burden on the health care system and society in general. In addition to the negative effects on the quality of life of those afflicted and their caregivers, significant economic consequences arise from both direct and indirect costs of managing this disease 7, 23, 24. Considering the high and increasing prevalence, slow resolution, negative effect on quality of life, and financial burdens of allergies, there is considerable interest in identifying risk factors and developing safe and effective therapeutics for this disease Risk factors for the development of allergies Genetic component The development of allergy is multi-factorial, however, many studies have conclusively shown that an underlying genetic component is involved 25, 26. This implies that specific environmental factors can trigger allergy development in those who are genetically susceptible. Variants in numerous genes have been associated with allergic disease many of which can be assigned to groups of genes involved in the immune response to environmental exposure, epithelial barrier integrity, Th1/Th2 response regulation or tissue responses to chronic inflammation 25. In addition to variation in DNA sequence, epigenetic modifications may mediate genetic susceptibility to allergy. Epigenetic modifications include DNA methylation and histone modifications, each altering the expression of a gene. For example, sequence variants in the HLA-DRB1 and HLA- DQB1 loci have been associated with peanut allergy in children of European descent 27, 28 and accounted for peanut allergy in 20% of the study population 28. The identified variants were linked to differential DNA methylation patterns in the HLA-DQB1 and HLA-DRB1 genes 28. Epigenetic modifications in these HLA class II loci may determine which peptides are presented to T cells by APC, thereby having a direct effect on the response mounted to an antigen. This avenue of research in allergy is in the early stages, and further research is needed to determine which, possibly manageable, environmental factors influence epigenetic modifications Hygiene Hypothesis Although genetic factors contribute to the development of allergic disease, they cannot account for the observed increase in prevalence during the past two decades. Allergy predominates in developed countries, which has led to the hypothesis that improved hygiene conditions and decreased exposure to infectious and microbial agents in childhood are key components in the development of the disease. This is termed the hygiene hypothesis and was introduced in 1989 by David P. Strachan 29. The hypothesis is based on the notion that reduced pathogenic microbial and viral exposure, which would induce a Th1 response, leads to a disturbed Th1/Th2 balance, in which the Th2 response dominates. More recently this hypothesis has been modified and expanded and now suggests that decreased exposure to common environmental microbes including bacteria, fungi and parasites, leads to a general excessive inflammatory state 30, 31. This old friends hypothesis, in contrast to the hygiene hypothesis, would explain the recent increase in both allergy and autoimmune disorders, as it does not imply a Th1/Th2 imbalance, but rather an overall hyper-inflammatory response 31. Furthermore, this theory would also support the reported correlation between helminth infection, which induces a strong Th2 type response, and decreased allergy prevalence, observed in helminth-endemic areas In essence, due to early, repeated exposure to diverse environmental microbes, including those that inhabit the skin, gut, and respiratory tract, an immune-regulatory network develops, suppressing hyperinflammatory and potentially chronic disorders. This hypothesis provides a general explanation for the differences in prevalence of allergy throughout various regions of the world, as well as other protective factors that have been identified, including natural as opposed to cesarean birth 35-37, siblings or large families 29, 38-40, communal childcare 40, 41, and farming lifestyle or animal exposure However, additional factors have been identified that may contribute to the development of allergy, and food allergy in particular Diet A recently identified protective factor for the development of allergy is high dietary fiber intake. Fiber and other undigested carbohydrates in the colon are fermented by specific strains of anaerobic bacteria, resulting in the production of short chain fatty acids (SCFA), predominantly acetate, butyrate and propionate 46. These SCFA affect immune cell gene expression, leading to differentiation of Foxp3 + or IL-10 + T regulatory cells 47-49, and bind to G-protein coupled receptors on APC, inducing a more tolerogenic cell phenotype. These effects suppress inflammation in the gut. The level of fiber intake also affects the composition of the gut microbiota by promoting the growth of certain strains of bacteria required for fiber fermentation, such as Clostridia. In addition, Clostridia induce IL-22 production by lymphocytes in the colon lamina propria 50, which increases intestinal epithelial barrier integrity, thereby reducing permeability to (potentially allergenic) dietary proteins 51, 52. Indeed, impaired intestinal permeability has been associated with food allergy or food hypersensitivity when compared to non-atopic controls. Furthermore, the degree of intestinal barrier permeability correlated with the severity of allergic symptoms experienced upon ingestion of the allergen 53. It is becoming clear that the typical Western

8 CHAPTER 1 GENERAL INTRODUCTION diet (high in fat and sugar and low in fiber) may negatively affect the composition of intestinal bacterial stains, resulting in decreased immune tolerance and increased epithelial barrier permeability, both contributing factors to the development of allergic disease Impaired epithelial barrier Another identified risk factor for the development of food allergy is impaired epidermal barrier function due to eczema 54 (atopic dermatitis) or filaggrin gene (FLG) loss of function mutations 55, 56. Filaggrin is an epidermal structural protein essential for skin integrity, and it is estimated that approximately 50% of moderate to severe cases of atopic dermatitis and about 15% of mild to moderate cases are associated with a FLG variant 57, 58. Disruptions to the epidermal barrier, as occurs with eczema, cause epithelial cells to release pro-inflammatory chemokines and cytokines, including those mediating Th2-cell polarization 59. Murine studies have shown that epicutaneous application of ovalbumin (OVA) and house dust mite (HDM) allergens to a disrupted epidermal barrier induces a local and systemic Th2 dominated response One study showed the development of peanut and OVA allergy in a mouse model after application of the allergens to adhesive tape-disrupted skin. The investigators reported IL-4 secretion from T cells in the draining lymph node and high levels of antigen-specific IgE and IgG 1. Furthermore, increased expression of MHC Class II and costimulatory molecules CD86 and CD40, along with CD54 and CD11c, were also observed on Langerhans cells in the disrupted epidermis; however, migration of these cells occurred only after the introduction of an antigen. In contrast, subcutaneous injection of the antigen into the dermal layer without prior disruption of the skin led to a predominantly Th1 response 65. Several other murine studies have shown that cutaneous exposure to an allergen, along with a danger signal through either barrier disruption or an adjuvant, results in allergen sensitization and an allergic response upon subsequent oral ingestion of the allergen 66, 67. This highlights the importance of barrier dysfunction in the development of a Th2-skewed response to an allergen. Furthermore, observational studies have suggested that the treatment of atopic skin with emollients containing food allergens manifests a higher risk of developing a corresponding food allergy This possible route of sensitization also brings into question the role of oral tolerance, and whether early introduction of allergen via the oral route may prevent the development of sensitization 71. This avenue of research in allergy requires further attention, as it not only identifies key risk factors for the development of allergy (in particular food allergy), but also elucidates the immunological mechanisms involved. 3. Mechanism of an Allergic Reaction The mechanism behind an allergic reaction consists of two phases (Figure 1). The first phase involves the initial sensitization, where an antigen is encountered by an APC and processed for presentation to naïve T cells. The antigen can be introduced through various routes, including the gastrointestinal tract (food allergens) and lung epithelial cells (aeroallergens) and, as mentioned previously, the skin. Accumulating evidence suggests that the state of the epithelial barrier (healthy versus damaged due to for example smoke or pollutant inhalation in the lung or eczema of the skin) at the antigen contact site can determine the type of response that is induced 72. Furthermore, many allergens contain proteolytic properties enabling penetration of the epithelial barrier 73. Barrier disruption and introduction of allergens accompanied by danger signals can lead to activation of epithelial cells with secretion of Th2 skewing cytokines IL-25, IL-33 and TSLP 59, In the next line of defense, immature DC respond to the encountered allergen both directly, through innate immune receptors such as Toll Like Receptors (TLR), Protease Activated Receptors (PAR) or C-type Lectin Receptors (CLR) and, indirectly, through cytokines produced by other cells, including epithelial cells 77. Upon activation and allergen uptake, maturation of the cells occurs with up-regulation of MHC Class II and costimulatory molecules CD80 or CD86, in preparation for subsequent antigen presentation to naive T cells in the lymph node 78. In order to present antigen to T cells, APC must internalize and process the antigen, then load the resulting peptides into the grooves of MHC Class II molecules for transport to the cell surface. Various components are required for successful peptide-mhc Class II complex formation and presentation. The MHC Class II peptide-binding groove is blocked by the invariant chain (CD74) during synthesis in the endoplasmic reticulum, to prevent binding of cellular or endogenous peptides before encountering peptides of the endocytosed digested proteins 79, 80. Upon fusion of the endosomes containing newly synthesized MHC class II with the late endosomes containing exogenous peptides, cathepsin proteases facilitate the removal of the invariant chain leaving only a small portion in the groove, termed CLIP 81, 82. Finally, CLIP is removed from the groove by HLA-DM 83, allowing for exogenous peptide loading and presentation of the newly formed MHC Class II-peptide complex on the cell surface. This complex interacts with the TCR of the naïve CD4 + T cells, providing the first of three signals required for T cell activation and differentiation. Costimulatory molecules expressed by the APC, including CD80 and CD86, interact with CD28 present on the T cell, providing the second signal 84, 85. The third signal determines the resulting T cell phenotype. In order to induce a Th1 phenotype, DC produce IL-12, which increases T cell IFN-g expression resulting in the induction of the master Th1 transcription regulator T-bet, resulting in further expression of IFN-g and suppression of IL The mechanism through which DC induce Th2 differentiation of naïve T cells, regulated by the master transcription factor GATA3 74,, is less clear. A number of different molecules expressed by DC have been associated with their ability to prime a Th2 response including, but not limited to, OX40 ligand 87, Notch ligand 88 and CD In addition, inhibition of IL-12p70 production by DC results in the suppression of Th1 skewing enabling the induction of a Th2 response 90, 91. Although not produced by DC, IL-4 is a potent inducer of Th2 immunity capable of up-regulating GATA3 expression through STAT6 signaling 92, 93. Various cells, including basophils, mast cells, and innate lymphoid type 2 cells (ILC2), do produce significant amounts of this cytokine 94. In some models of Th2-associated disease, including allergy, IL-4 is a necessity for the development of Th2 immunity; therefore accessory cells capable of IL-4 production may be required in the process of antigen presentation. Alternatively, it has been suggested that IL-4 producing cells, specifically

9 CHAPTER 1 GENERAL INTRODUCTION basophils, are able to function as APC inducing Th2 responses entirely independent of DC In the development of an allergic response, Th2 differentiated CD4 + T cells, capable of producing IL-4, IL-5 and IL-13, interact with B cells to induce isotype class switching resulting in antigen-specific IgE production. For this to occur, B cell receptor activation by the antigen, the presence of a Th2 cytokine environment, and signaling through CD40 is required. Although these signals are provided by T cells, T cell-independent class switching has also been described, where other cells such as basophils take on this role 98. The resulting antigen-specific IgE is a key player in the second phase of allergic inflammation, and therefore also a therapeutic target. Secreted locally in tissue and systemically in peripheral blood, IgE molecules are captured by IgE receptors on a variety of cells. Two types of IgE receptors exist. Low affinity receptors (CD23) are present on B cells, T cells, Langerhans cells, monocytes, macrophages, platelets, follicular DC and eosinophils 99, 100, and play an important role in IgE production regulation 101, 102. Binding of IgE or IgE-immune complexes induces a negative feedback signal preventing further IgE synthesis Figure 1. The mechanism of allergic reactions. During sensitization, immature dendritic cells (idc) encounter allergens at the epithelial barrier of the lung, skin or gut. Upon allergen uptake, DC mature and migrate to the lymph node to induce differential and clonal expansion of allergen-specific Th2 cells from naïve CD4 + T cells (nt). Th2 skewing can be facilitated by cytokines (TSLP) produced by disrupted epithelial cells, which induce OX40L upregulation on DC. IL-4 and IL-13 production by Th2 cells initiates immunoglobulin class switching in allergen-specific naïve B cells (nb), resulting in allergen-specific IgE producing plasma cells and IgE + memory B cells. Upon subsequent allergen encounter (challenge) mast cells and basophils are activated through FceRI bound allergen-specific IgE, producing inflammatory mediators responsible for the early phase allergic response. A late phase response may also be initiated upon infiltration of additional effector cells to the site of allergen encounter. The Th2 response to the allergen is further maintained and reinforced by stimulated allergen-specific Th2 cells. In contrast, soluble forms of CD23 up-regulate IgE production by B cells 104. High affinity IgE receptors (FcεRI) are present on basophils, mast cells, monocytes and DC, and are a key component of the immediate hypersensitivity that is observed in the second phase of allergic inflammation. The second phase of allergic inflammation is induced upon secondary encounter of the antigen (challenge). Conformational epitopes of the allergen will bind and cross-link allergen-specific IgE bound to FcεRI molecules on basophils and mast cells (effector cells). This induces cell activation through a cascade of intracellular protein phosphorylation and calcium influx, eventually resulting in granule release of pre-formed inflammatory mediators. These mediators include histamine, proteases (e.g. tryptase), proteoglycans (e.g. heparin) and, cytokines (IL-4 in basophils and TNF-α in mast cells). Activation of mast cells and basophils also results in 16 17

10 CHAPTER 1 GENERAL INTRODUCTION newly synthesized mediators including prostaglandin D2 (PGD2), leukotrienes, chemokines and cytokines. Each of these mediators have a specific effect on vascular permeability, vasodilation, bronchoconstriction and recruitment of effector cells to the site 105. Combined, these events manifest the immediate pathophysiology observed during an IgE-dependent allergic reaction. A delayed response, caused by the arrival of recruited effector cells to the site of allergen exposure, may also be observed The Basophil- Effector Cell and Valuable Tool Basophils and mast cells are key players in immediate hypersensitivity reactions through their ability to degranulate and release inflammatory mediators, including histamine, upon exposure to allergen. Basophils enter the circulation from the bone marrow as fully mature cells and have a relatively short lifespan of only several days 106, 107. Mast cells, in contrast, can survive for several months and reside in the tissue 108. Due to their unique attributes, basophils can serve as an excellent tool in allergy research and clinical diagnosis. They are easily accessible, only requiring a blood sample equivalent to that used to measure serum IgE, and their relatively short lifespan allows for a clear representation of the current serum IgE repertoire. Following identification of the degranulation marker CD63 in the early 1990s 109, the in vitro basophil activation test (BAT) was developed 110. CD63 is a tetraspanin protein located within the secretory granules that contain histamine. Upon activation and degranulation, this membrane along with the CD63 protein is transported to the cell surface to fuse with the outer membrane, allowing the CD63 molecules on live cells to become accessible to flow cytometry detection antibodies (Figure 2A,B). A range of allergen concentrations is included in the BAT to create a dose-response curve, from which both basophil reactivity (maximal percentage of CD63 + basophils detected) and basophil sensitivity (concentration of allergen required to induce 50% of maximal reactivity) can be measured 111 (Figure 2C). Within this test basophils can be detected with a number of different markers. Most commonly used markers include IgE ++, CD123 + /HLA-DR -, CCR3 + and CD203c +, often in combination with flow cytometry side scatter profile and CD3 to distinguish these cells from eosinophils and T cells, respectively 112. CD203c is a basophil lineage specific marker expressed at low levels on resting basophils, which is then upregulated upon activation. The marker is used both to identify basophils and as an activation marker, although its pathway of expression differs slightly from that of CD Positive controls of the BAT include anti-ige or anti-fceri antibodies that induce the IgE pathway of activation of the cells, and N-formylmethionine-leucyl-phenylalanine (fmlp), a bacterial peptide that induces basophil activation in an IgE-independent manner. These controls aid in confirming that the test procedure was followed correctly, and in identifying non-responder subjects. Despite the presence of cell surface IgE, the basophils of non-responders fail to activate upon IgE cross-linking, presumably due to a Syk protein deficiency in the IgE intracellular signaling pathway This occurs in approximately 10% of individuals 117, 118. Figure 2. Principle of the basophil activation test. Following membrane bound allergen-specific IgE crosslinking, basophil granules containing inflammatory mediators fuse with the outer membrane, releasing their contents and upregulating membrane CD63 (A). Basophil activation can be detected by flow cytometry (B) and the resulting dose-response curve is used to calculate commonly used parameters; basophil reactivity (the maximal basophil activation achieved) and basophil sensitivity (the concentration required to reach 50% of maximal activation) (C). Both CD63 and CD203c are robust basophil activation markers and have been shown to correlate well with histamine release and clinical symptoms. Basophil activation has proven to be sensitive and specific in the diagnosis of various IgE-mediated allergies, including hymenoptera venom , food and drug allergies 125, 126. The diagnosis of allergy currently relies on clinical history, skin prick testing and the detection of serum allergen-specific IgE, however, these methods can provide conflicting results 127. The gold standard in diagnosis is an allergen provocation test, which in some cases carries the risk of inducing a severe reaction. Furthermore, although serum allergen-specific IgE is a key component of IgE mediated allergy diagnosis, the presence of this molecule alone does not indicate its functional contribution to effector cell activation. Due to its close correlation with clinical symptoms and the gold standard allergen challenge 124, the BAT is applicable in not only allergy diagnosis but also in monitoring treatment efficacy and predicting the safety of novel allergy therapeutics 128. The BAT is currently applied as a research tool, however, future standardization and diagnostic laboratory implementation of this technique will greatly enhance its utility in difficult to diagnose allergies and allergies with high risk of anaphylaxis upon in vivo allergen challenge

11 CHAPTER 1 GENERAL INTRODUCTION 5. Therapeutics - Allergen Immunotherapy (AIT) Allergen Immunotherapy (AIT) has been used in clinical practice since the early 20 th century 129 and is currently the only therapy to deal with the underlying cause of allergy, by inducing immune desensitization or tolerance to the allergen with long term or permanent resolution of the disease and associated symptoms. Typically this is achieved by repeatedly administering increasing doses of the allergen to the patient. After the initial phase of increasing dose delivery, a maintenance phase follows, during which a consistent amount of allergen is administered on a regular basis for up to several years. Desensitization is achieved when an allergic subject can be exposed to or ingests an amount of allergen without the occurrence of an adverse reaction; however, the subject must encounter the allergen on a regular basis, as occurs during the maintenance phase of immunotherapy, in order to maintain desensitization. Tolerance or sustained unresponsiveness 130, on the other hand, is achieved when a subject who is no longer receiving an AIT maintenance dose, can be exposed to the allergen sporadically without adverse effects. Sustained unresponsiveness is the ultimate goal of AIT and the success in achieving this may be dependent on various factors, including the duration of AIT, severity of the allergy as suggested by clinical history, baseline skin prick test results, and serum antigen-specific IgE levels Routes of AIT delivery AIT is routinely delivered through either subcutaneous (SCIT) injection or sublingual (SLIT) drops or tablets, but may also be administered by intradermal injection or orally. Recently, novel routes of delivery have been investigated including epicutaneous application, whereby the allergen is applied to the skin, and intralymphatic injection, which involves injection of the allergen directly into the lymph node 132, 133. Both SCIT and SLIT have been shown to be effective in placebocontrolled trials 134, 135 although it is not yet clear whether effects are comparable between the two delivery methods There is some evidence that SCIT may be clinically more effective 136, 137, while SLIT can be self-administered at home and is associated with fewer adverse events , Oral immunotherapy has been used in clinical trials of immunotherapy for food allergies due to the severe side effects encountered after subcutaneous administration 151, 152. Although clinical efficacy has been achieved using this route of delivery, adverse events are still frequent and, depending on the allergen involved, in some cases severe 101, The efficacy of immunotherapy relies on adequate delivery of the allergen to APC, preferably without the encounter of highly vascularized areas or large numbers of mast cells to limit (systemic) adverse events. The skin contains several distinct subsets of DC; Langerhans cells (LC) in the epidermis 153 and CD1c + DC, CD14 + DC and CD141 + DC in the dermis (termed dermal DC) 154. Langerhans cells are positioned as the first line of defense, surveying the area for foreign antigen and migrating to the skin draining lymph nodes. They have been shown to induce a Th2 type response in vitro 155. CD141 + dermal DC, on the other hand, are major producers of IL-10 and capable of inducing a Treg response, thereby playing an important role in maintaining skin homeostasis 156. Subcutaneous delivery targets the connective tissue and adipose layer under the dermis, which also contains a dispersed population of DC. This route of delivery requires high doses of allergen to achieve tolerance. Intradermal delivery targets the relatively abundant population of dermal DC, with the added benefit of direct access to the lymphatic system. This may explain the relatively low dose of allergen required to achieve desensitization in intradermal delivery compared to subcutaneous delivery 157. Intralymphatic delivery deposits the allergen to the highly populated area of DC in the lymph node, where presentation of the allergen to T cells occurs. Although this route has recently been shown to be safe and effective for grass pollen and cat allergy 132, 133, further research is required to confirm its safety and applicability for other allergies, particularly those associated with anaphylaxis. The sublingual and oral routes involve APC within the oral cavity and intestinal mucosa, including tolerogenic LC capable of inducing a highly desirable Treg response to the allergen. This tolerogenic phenotype is a necessity in areas exposed to high loads of harmless foreign (food-derived) antigens and commensal bacteria encountered in the gut. As evidence for this, TLR4 ligation on oral LC results in up-regulation of coinhibitory molecules, down regulation of costimulatory molecules and increased IL-10 production. Subsequently, these cells are able to induce T cell production of regulatory and type 1 cytokines 158. Furthermore, allergen (Phl p 5) binding to oral LC not only induces IL-10 and TGF-b production, but also attenuates their maturation, which has been shown to induce tolerance 159. In addition to these tolerogenic features, the sublingual and oral routes of delivery can induce local allergen-specific IgA, providing an additional first level of protection from an allergic response to ingested food allergens 160, 161. Finally, the chosen route of delivery may also affect compliance of the patient, considering that a less invasive, home-administered therapeutic regimen may be easier to maintain than one that involves repeated hospital/clinic-administered injections. 5.2 Mechanism of conventional AIT The mechanism behind conventional immunotherapy for allergy consists of several immunemodulatory events. In the early phase, basophil reactivity, and thereby mediator release, is hampered by rapid up-regulation of the histamine receptor HR The following phase consists of the emergence of natural and/or induced allergen-specific regulatory T and B cells and the suppression of allergen-specific effector T cells. Induced regulatory T and B cells are characterized by production of the anti-inflammatory cytokines IL-10 (Tr1 and Br1 cells) or TGF-β (Tr3 and Br3 cells) 163, 164. TGF-β inhibits B cell IgE production while inducing anti-inflammatory mucosal allergen-specific IgA 165. Further effects of TGF-β include suppression of Th1 and Th2 lymphocyte differentiation 166, 167 and the induction of Treg development through the transcription factor Foxp IL-10 is a key component of the induction of tolerance and is initially produced by

12 CHAPTER 1 GENERAL INTRODUCTION Treg, however, in the following phases of AIT, monocytes and regulatory B cells also produce IL The suppressive effects of IL-10 result in further induction of regulatory cells and a decrease in allergen-induced proliferation and cytokine production by effector cells. Further suppression is achieved with the up-regulation of allergen-specific IgA, IgG 1 and IgG 4, although it has been shown that functional IgG 4, which competes directly with allergen-specific IgE molecules as opposed to merely immunoreactive IgG 4, is required for this effect 169. An additional effect of allergen-specific IgG is the prevention of IgE-facilitated antigen presentation through CD23-bound IgE on professional APC, which would otherwise boost the Th2 response in a positive-feedback loop Recently, it was shown that IL-10 producing B regulatory cells were responsible for bee venom allergen-specific IgG 4 production in tolerized beekeepers 173. Finally, upon nasal or cutaneous allergen challenge, a reduction in recruitment of eosinophils, mast cells and basophils and IgE-dependent mediator release occurs Allergen-specific IgE generally increases early after AIT induction followed by a gradual decrease The complete course of immunotherapy often lasts three to five years, however, the nadir of the allergen specific IgE levels may not be reached for several years after completion of the AIT. With successful AIT, a marked improvement in allergic symptoms is observed, with the additional advantage of a reduced risk of developing new allergic sensitizations 183. Although many aspects of the mechanism of AIT have been revealed, the precise mechanism behind the down-regulated Th2 cell response is not fully understood. There is evidence for a combination of Treg induction, T cell anergy and T cell deletion. T cell anergy describes the induction of non-responsiveness of T cells to their corresponding ligand. Anergy to any antigen can be induced in a number of different ways, primarily involving aspects of TCR and CD28 engagement with their respective ligand during antigen presentation. IL-10 can induce T cell anergy through inhibition of the CD28 signaling pathway. During low TCR triggering, CD28 engagement is necessary for T cell activation, a lack of which results in anergy 184. Furthermore, IL- 10 inhibits DC maturation leading to reduced MHC Class II and costimulatory ligand expression (CD80/CD86); antigen presentation by these immature DC also results in anergy of the T cells 185. In addition to the effects of IL-10, anergy can also be achieved in T cells through exposure to high concentrations of specific antigen , coupled with insufficient costimulation, or through altered peptide ligands , capable of only partial TCR binding. Another aspect of T cell anergy is that it can be reversed under specific circumstances. After bee venom immunotherapy, PBMC of treated patients displayed reduced antigen-specific T cell proliferation and Th1 (IL-2, IFN-g) and Th2 (IL-4, IL-5, IL-13) cytokine production. Through in vitro culture of the PBMC with antigen and IL-2, IL-15 or IL-4, proliferation and the production of Th1 and Th2 cytokines could be restored 192. This suggests that the cytokines present in the microenvironment of the cell can influence the phenotype of the restored responsiveness. Should the environment remain predominantly Th2, the restored T cells could potentially regain their Th2 phenotype, which may result in relapse in allergen sensitivity and render the AIT unsuccessful. Recently, evidence for allergen-specific T cell deletion in AIT has also emerged 193. Using a MHC Class II/peptide tetramer approach, Wambre et al. were able to track the fate of allergen- specific T cells during timothy grass AIT and found no change in allergen-specific Th1/Tr1 cell frequency, but rather a decrease in allergen-specific Th2 cell frequency. Along with the observed low level of the survival protein Bcl-2 in these Th2 cells compared to the Th1/Tr1 cells, targeted deletion of these cells seems the likely cause. This supports earlier studies showing induced apoptosis in antigen-specific IL-4+ T cells after HDM or grass pollen immunotherapy in atopic individuals, or through high dose allergen stimulation in vitro 187, 194, Development of conventional AIT for food allergies Although AIT is successful in treating allergic rhinitis, HDM allergy and animal dander allergy, its application is limited in treating food allergy, due to the risk of anaphylaxis during treatment. Subcutaneous administration of peanut immunotherapy has been performed in two clinical trials. Although signs of efficacy were observed in both, systemic adverse reaction rates were unacceptably high 151, with one trial ending prematurely due to fatal anaphylaxis in a placebo group patient who erroneously received an active dose intended for the treatment group 152. Since these early trials, subcutaneous administration of food allergens for immunotherapy has largely been abandoned, however, numerous clinical trials of oral immunotherapy (OIT) for milk , egg 130, 143, 144 and also peanut allergy have since been conducted and shown success in achieving allergen desensitization. Open studies, case series and controlled trials of OIT for peanut allergy have shown some evidence of allergen desensitization, through increased thresholds of allergen ingestion (several grams, the equivalent of 10 peanuts) without symptoms in oral food challenges In the first randomized placebo controlled trial of oral peanut immunotherapy in 19 children, investigators reported successful desensitization in 84% of the cohort after 1 year, with subjects passing a final oral challenge of 20 peanuts in the active group, compared to only 1 peanut in the placebo group 150. Desensitization is accompanied by immunological changes previously shown to correlate with AIT efficacy, including increased allergenspecific IgG 4 147, 149, 150, decreased Th2 cytokine production 147, 150 and increased Treg populations 149, 150. Unfortunately, adverse events including upper respiratory symptoms, nausea, abdominal pain and diarrhea during the initial escalation phase are frequent and, in some cases, severe 101, Importantly, anaphylaxis was reported in three separate studies and included patients with no prior history of anaphylaxis 148, 196, 197. In addition, subjects with a history of severe anaphylaxis are generally excluded from AIT, which is a significant limitation as these individuals have the most to gain from peanut immunotherapy and may therefore be more inclined to seek treatment. The results of these trials clearly indicate the potential for immunotherapy to treat food allergy, but also highlight the need for a safer alternative to conventional AIT

13 CHAPTER 1 GENERAL INTRODUCTION 6. Peptide immunotherapy (PIT) Several alternative approaches to AIT are under investigation in order to achieve desensitization and tolerance through allergen administration without inducing an IgE-mediated allergic reaction. One is the use of modified hypoallergenic recombinant proteins, which lack either key components required for protein folding or IgE-epitope sequences, eliminating the conformational epitope required for recognition by IgE. This has been demonstrated with Bet v 1, in which folding of the protein was disrupted by reduction and alkylation 198, 199. These non-allergenic proteins can also be developed through the introduction of point mutations to disrupt disulfide bonds of the protein, partial sequence deletion, or fusion with variant proteins 200. Many of these modifications have yet to be used in patient therapy, however, in a recent phase 1 clinical trial the safety of recombinant major allergens of peanut, modified by amino acid substitution at major IgE-binding epitopes, was assessed in peanut allergic patients. The vaccine, delivered rectally in an E. coli-encapsulated form, led to frequent and sometimes severe allergic reactions, indicating that despite best efforts, it may be challenging to remove the heterogeneous range of IgE-binding epitopes recognized by an allergic population 201. Another approach to reducing the allergenicity of the therapeutic for AIT is the use of short T cell targeted peptides derived from the allergen, which lack the ability to cause IgE crosslinking required for effector cell activation and an allergic response, as opposed to whole proteins. Although many aspects of the initiation of an allergic response are unclear, it is widely recognized that T cells play a fundamental role. Allergen-specific Th2 cells are necessary for the production of Th2 cytokines, which recruit effector cells, and initiate Ig class switching to IgE isotype in B cells. Antigen-specific Th2 cells are present in higher frequency in allergic subjects, whereas 193, 202, antigen-specific Th1 cells and Treg predominate in non-allergic or desensitized subjects 203. The existence of dominant T cell epitopes within major allergens, that are recognized across patient populations and over time, also supports the potential of a peptide-based therapeutic 204, 205. Finally, there is evidence of linked suppression in peptide immunotherapy, whereby the immune response is suppressed to not only the introduced peptide but also to other domains of the protein from which the peptide is derived 206. These factors combined constitute the rationale for T cell targeted therapy for allergic disease Efficacy of PIT This approach was first tested for safety and efficacy in clinical trial using immune-dominant T cell epitopes of major allergens involved in cat allergy (Fel d 1) and bee venom (PLA2) allergy. In the first study of bee venom PIT, five patients were treated with a mixture of three short (18 amino acids or less) PLA2-derived peptides 207. Clinical efficacy was observed in this study, with two patients experiencing only mild symptoms to bee sting challenge after PIT, and three being nonsymptomatic. Furthermore, no local or systemic reactions were observed during PIT. Concurrent immunological changes included suppression of T cell proliferative responses and both Th2 and Th1 cytokine production. Antigen-specific IgG 4 was induced upon challenge with the whole bee venom allergen after completion of PIT, but not during the treatment. Following on from this study, a mixture of three long synthetic overlapping peptides (between amino acids) of PLA2 were administered to patients in a placebo controlled trial. Immunological changes observed in this trial resembled those of conventional AIT, with decreased T cell responsiveness, increased IL-10 and IFN-g production, along with the emergence of allergen-specific IgG 4. During the course of PIT, mild late adverse reactions were observed in two patients 208. In the most recent study of PIT for bee venom allergy, twelve subjects received nine intradermal injections with a mixture of 18-mer PLA2 peptides. The treatment was received without any adverse events and, along with a reduction in the late phase response to bee venom challenge, reduced production of IL-13 and IFN-g and increased IL-10 from PLA2 stimulated PBMC was observed. A transient, but modest increase in PLA2-specific IgG 4 was also observed 209. Using a slightly different approach, a birch pollen specific immunotherapy was designed based on eight long (over 48 residues) contiguous overlapping peptides (COPs) spanning the entire sequence of the major allergen Bet v 1. Based on their inability to induce basophil activation or skin prick reactivity in allergic subjects 210, three of these peptides (collectively named AllerT) were tested for efficacy in a phase I/IIa clinical trial 211. Five injections of increasing doses of AllerT or placebo were administered to twenty randomized patients over two months. No immediate reactions or episodes of anaphylaxis occurred during treatment and the frequency and severity of local adverse events were comparable between the treatment and placebo groups. Two of the fifteen AllerT-treated patients experienced systemic adverse events several hours after injection, possibly caused by T cell cytokine production upon activation by the peptides. In vitro AllerT, Bet v 1 or birch pollen stimulated PBMC showed increased proliferative responses and increased production of IL-10, Th1 and Th2 cytokines in AllerT-treated but not placebo-treated patients. Although the nasal allergen challenge results were inconclusive, a trend towards improved quality of life was reported in the AllerT-treated group. Furthermore there was an increase in Bet v 1-specific IgG 4 level that was maintained for up to 3 years post treatment 211. The PIT designed to treat cat allergy, termed Cat-PAD, originated from studies performed by Larche and Kay in the late 1990s. It is the first in its class to reach phase III clinical trials, comprising seven short synthetic T cell epitope peptides derived from Fel d Earlier trials of the peptide immunotherapy Allervax CAT, demonstrated some clinical efficacy at high doses (750 mg) but also reported both immediate and late phase adverse events, some requiring adrenaline The Allervax CAT PIT consisted of two long peptides derived from Fel d 1, each 27 amino acids in length, and was administered subcutaneously. The length of the peptides, possibly allowing for formation of IgE reactive conformational epitopes, was thought to have contributed to the immediate adverse events. A subsequent Fel d 1-based PIT consisted of 12 short (16-17 residues) peptides. Clinical efficacy was assessed in several studies, revealing decreased airway hyper-responsiveness and a reduction in late phase cutaneous reactions and late asthmatic reactions to whole cat allergen, although not all patients responded to the

14 CHAPTER 1 GENERAL INTRODUCTION therapy Adverse reactions were minimal, consisting mainly of mild late asthmatic reactions that were readily reversed with inhaled bronchodilator. Decreased antigen-specific CD4 + T cell proliferation and cytokine Th2 production (IL-4, IL-5 or IL-13) were generally observed in PBMC cultures after PIT 218, 220, 221. Finally, Cat-PAD was designed comprising seven of the original twelve Fel d 1 peptides selected for their binding affinity to commonly expressed HLA-DR molecules, their ability to induce PBMC proliferation and cytokine production in vitro (IFN-g, IL-10 and IL-13), and their inability to cause histamine release from basophils 222. In addition, agents were included to prevent peptide dimer formation through disulfide bridging between cysteine residues. The selected peptides have since been tested clinically for efficacy and safety, with highly encouraging results 223, 224. Investigators reported a reduction in rhinoconjunctivitis symptoms upon challenge in an environmental exposure chamber (EEC), persisting for up to 2 years after a short course of treatment with 4 intradermal injections of 6 nmol Cat-PAD IT 223. Many details of the peptide characteristics, optimal dosing and schedule 224 as well as the route of delivery have been thoroughly investigated in the course of development of the current cat-pad therapy, providing valuable guidelines for the design of other allergy PIT in the future. Indeed, several clinical trials assessing the efficacy of similarly designed PIT for HDM 225, grass pollen 226, and ragweed pollen 227 allergy are currently underway, showing promising results. 6.2 Mechanism of PIT an earlier trial showed a significant increase in IL-10 production by allergen stimulated PBMC from allergic asthma patients after Fel d 1 PIT treatment compared to pre-treatment levels. It should be noted, however, that the change in IL-10 production did not differ significantly between the treatment and placebo groups 219. Further immunological analysis of participants in this trial revealed a role for linked epitope suppression. The patients were treated with 12 Fel d 1 derived peptides, but showed decreased PBMC proliferative and cytokine responses to another 4 peptides of Fel d 1 not included in the PIT mix 228. Further evidence of linked suppression was provided in this study using an HLA-DR1 transgenic mouse model sensitized to Fel d 1 to track allergen-specific T cells with Fel d 1 (29-45) DR1 tetramers after treatment with a low dose of the Fel d 1(29-45) peptide. Suppression of peptide (29-45)-specific T cell responses was observed, with linked suppression to other regions of the Fel d 1 protein. Furthermore, with this model the nature of the suppressive effects could be determined, which was based on immunoregulation through the production of IL-10 by peptide (29-45)-specific T cells as well as bystander T cells. There is also evidence that linked epitope suppression can be induced through cell-to-cell contact. Using a murine model of HDM allergy, it was determined that Delta 1 expression could be transiently upregulated on peripheral T cells after intranasal delivery of high dose Der p 1 ( ) peptide. T cell responses to both minor (e.g ) and dominant ( ) Der p 1 peptides were suppressed by Delta 1 transfected Der p 1 ( ) reactive T cells. In this case, linked epitope suppression may be induced through Delta-1/ Notch-1 interactions between T cells gathering at the cell membrane of APC, presenting both minor and dominant epitopes of the same protein Although clinical efficacy has been reported for peptide-based allergen immunotherapy, the underlying mechanisms of these effects have yet to be elucidated. Evidence derived from in vitro studies, mouse models, and current PIT trials, indicate that whilst some mechanisms overlap, others may differ to those thought to underlie conventional whole AIT (Figure 3) T cell regulation and linked epitope suppression Similarly to reports from conventional AIT, increased production of the immunoregulatory cytokine IL-10 has been observed in bee venom PIT trials, however, the role of IL-10 in immunesuppression by Fel d 1 derived PIT is less clear. There are, however, indications of a role for CD4 + T cells with a regulatory function. Allergen-dependent recruitment of CD4 + CD25 + cells to the skin was observed after Fel d 1 PIT (containing 12 peptides), but this was not related to IL-10 production 217. In a related study, increased Treg function, defined by increased suppression of allergen-specific proliferative responses of CD4 - cells by CD4 + cells after Fel d 1 PIT, was reported. Although no increase in CD4 + CD25 + cells was observed in PBMC, CD5 levels were increased on both CD4 + and CD8 + cells. CD5 is a negative regulator of TCR signaling, and elevated levels of this molecule are associated with decreased T cell responses to antigen 221. A subsequent placebo-controlled study of the same Fel d 1 PIT reported decreased allergen-specific CD4 + T cell responses, but no change in suppression by CD4 + CD25 + cells or IL-10 production 220. In contrast, T cell anergy In early in vitro studies, T cell anergy to HDM allergen was achieved through stimulation with supraoptimal levels of specific peptide, in which an initial increase in IL-4 production was followed by decreased T cell proliferation and IL-4 and IL-5, but not IFN-g cytokine production upon subsequent antigen exposure 230, 231. This anergic response has been shown to occur with both HLA-DR and HLA-DP restricted peptides, in the presence or absence of APC, and is associated with downregulation of TCR expression and upregulation of CD2 and CD Furthermore, significant CD28 downregulation at both the cytoplasmic mrna and protein level is involved in this process 234. Induction of anergy has also been shown in cloned human CD4 + bee venom (PLA)-specific Th2 cells upon incubation with PLA peptide and APC. An increase in T cell CD25 expression was observed, and although CD3 and CD28 levels remained unaltered, defective membrane TCR signaling through abrogation of p56lck or ZAP-70 tyrosine phosphorylation was detected 235. An additional aspect of PIT that may contribute to the induction of anergy is the introduction of the T cell epitope peptides in the absence of an accompanying danger signal. Danger signals can originate from various pathogenic components, but may also result from administration of whole allergen as used in conventional subcutaneous AIT. A murine study showed the induction of T cell tolerance to OVA peptide when delivered alone, but not OVA peptide combined with LPS. The mechanism behind this is thought to be dependent on peptide 26 27

15 CHAPTER 1 GENERAL INTRODUCTION presentation to T cells by DC in the absence of TLR activation, which would otherwise induce DC costimulatory molecule upregulation and T cell activation 236. Murine studies also provide in vivo evidence of T cell anergy following transient T cell activation after high dose PIT 237, 238. In line with the previously mentioned in vitro studies, the anergic state of the allergen-specific T cells correlated with decreased TCR expression 237. Furthermore, the induction of anergy was shown to be reversible but long-lasting, with anergic T cells surviving for up to several months in the lymphoid tissue T cell deletion To date, direct evidence of T cell deletion during PIT is limited, however, it has been noted that the dose of peptide administered may influence the mechanism of tolerance in PIT, with high doses associated with deletion of peptide-specific T cells. In transgenic mice expressing TCR specific for a peptide derived from the influenza virus hemagglutinin, intravenous injection of high dose (750 mg), but not low dose (75 mg), of the peptide resulted in both thymic and peripheral T cell apoptosis, in addition to T cell anergy 239. Evidence of T cell deletion following conventional AIT has been reported 193 and by applying the knowledge of the epitopes involved, PIT could enable a targeted approach to inducing specific T cell deletion. It is clear that PIT can be clinically effective and associated with important immunological changes, consisting of decreased allergen-specific T cell responsiveness at both the proliferative and cytokine production level. The specific mechanism involved is not entirely clear, but may depend on both the peptide dose and the dosing regimen employed. Induction of allergenspecific IgG 4 production, although a prominent component of conventional AIT, so far has not proven to play a comparable role in PIT containing short peptides 207, 209. Figure 3. Mechanisms involved in T-cell epitope peptide mediated tolerance. Based on murine and human studies suppression of the allergic response by PIT can be achieved through induced anergy of allergen-specific naïve CD4 + T cells (nt) and Th2 cells, deletion of allergen-specific Th2 cells, and the emergence of allergen-specific Treg cells, capable of inhibiting of Th1, Th2 and effector cell function. In contrast to conventional allergen immunotherapy, the role of allergen-specific IgG 4 in PIT is unclear. [adapted from Prickett, Clin & Exp Allergy 2015] 7. Biologicals Based on increased knowledge of the mechanisms behind an allergic reaction, several targeted therapeutics have been developed. Many of these consist of humanized antibodies specific for various components of the Th2 pathway, including TSLP, IL-4, IL-5, IL-13, CD23 or IgE Omalizumab, targeting human IgE, was designed to capture free IgE before it attaches to its corresponding high affinity receptor (FcεRI) on effector cells, including basophils, mast cells and DC. The antibody specifically targets the CH3 region of IgE that is necessary for binding to the alpha-chain of FcεRI. Therefore, it not only prevents free IgE from binding to empty receptors but also lacks the ability to bind to and crosslink FcεRI bound IgE, which would otherwise result in effector cell activation, as occurs in an allergic reaction 243. In addition, due to the high turnover of FcεRI molecules, newly produced FcεRI molecules will not encounter IgE upon administration of omalizumab, and remain empty. Empty FcεRI molecules are rapidly internalized, and the 28 29

16 CHAPTER 1 GENERAL INTRODUCTION overall expression of FcεRI is then down regulated in response to the decreased levels of IgE in the environment of the cell. It is known that FcεRI expression levels are directly related to the level of IgE encountered by the cell 244, 245. Omalizumab was developed by Roche/Genentech and Novartis, and has been available for clinical use since The drug is currently indicated for severe allergic asthma, and has been shown to be effective in reducing exacerbation frequency 247, 248. More recent results have even shown that this treatment can lead to an improvement in lung function 249 and reduction in the use of systemic corticosteroids 250, however, it is becoming clear that its efficacy may vary, depending on subtle differences in the disease endotype. Although originally intended for allergic asthma, omalizumab may also be effective in treating other (IgE-related) diseases 251 including mastocytosis 252, allergic rhinitis 253, chronic urticaria , and allergic bronchopulmonary aspergillosis (ABPA) This has been indicated in various case studies and case series, however, for conclusive evidence of efficacy in these disorders, placebo-controlled clinical trials are essential. 8. Scope of this Thesis The first aim of this thesis is to investigate basic components of the initiation of an allergic response, including the route of sensitization and the cells responsible for the initiation of Th2 skewing in allergic subjects. More specifically, in Chapter 2 both clinical and immunological evidence of food allergy development through cutaneous sensitization are presented in the form of a case study. In Chapter 3 the ability of human basophils to initiate a Th2 response through direct antigen presentation to T cells is investigated. The second aim of this thesis is to investigate the use of current therapies and the development of new therapies for difficult-to-treat allergic diseases. In Chapters 4 and 5 dominant T cell epitopes of major peanut allergen proteins are identified and characterized providing the first step in the design of a safe and effective peptide based T cell targeted immunotherapy to treat peanut allergy. The identified dominant T cell epitopes are then further investigated in Chapter 6, assessing their stability and resistance to enzymatic digestion for potential oral administration. Finally in Chapter 7, the immunological and clinical efficacy of omalizumab for the treatment of allergic bronchopulmonary aspergillosis is assessed, for the first time, in a randomized placebo controlled clinical trial. In Chapter 8 the findings of this thesis are summarized and discussed, focusing on the contribution they have made to the discipline of allergy and future directions in the field. Throughout these studies, basophils play an important role as either the object of investigation or as a tool in diagnosis, allowing determination of the efficacy of existing therapeutics, or assessment of the safety of novel therapies. References 1. Miossec P, Korn T, et al. Interleukin-17 and type 17 helper T cells. N Engl J Med. 2009;361: Vignali DA, Collison LW, et al. How regulatory T cells work. Nat Rev Immunol. 2008;8: Zhu J, Yamane H, et al. Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol. 2010;28: Vidarsson G, Dekkers G, et al. IgG subclasses and allotypes: from structure to effector functions. Front Immunol. 2014;5: Spergel JM, Paller AS. Atopic dermatitis and the atopic march. J Allergy Clin Immunol. 2003;112:S Sicherer SH. Epidemiology of food allergy. J Allergy Clin Immunol. 2011;127: Pawankar R, Canonica GW, et al. The WAO White Book on Allergy Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet. 1998;351: Hamid F, Wiria AE, et al. Risk Factors Associated with the Development of Atopic Sensitization in Indonesia. PLoS One. 2013;8:e Nicolaou N, Siddique N, et al. Allergic disease in urban and rural populations: increasing prevalence with increasing urbanization. Allergy. 2005;60: Weinmayr G, Weiland SK, et al. Atopic sensitization and the international variation of asthma symptom prevalence in children. Am J Respir Crit Care Med. 2007;176: Diseases NIoAaI. Food allergy an overview. NIH, services USDoHaH; Chafen JJ, Newberry SJ, et al. Diagnosing and managing common food allergies: a systematic review. JAMA. 2010;303: Prescott SL, Pawankar R, et al. A global survey of changing patterns of food allergy burden in children. World Allergy Organ J. 2013;6: Turner PJ, Allen KJ, et al. Knowledge, practice, and views on precautionary allergen labeling for the management of patients with IgEmediated food allergy-a survey of Australasian and UK health care professionals. J Allergy Clin Immunol Pract Dyer AA, Lau CH, et al. Pediatric emergency department visits and hospitalizations due to food-induced anaphylaxis in Illinois. Ann Allergy Asthma Immunol. 2015;115: Liew WK, Williamson E, et al. Anaphylaxis fatalities and admissions in Australia. J Allergy Clin Immunol. 2009;123: Mullins RJ, Dear KB, et al. Time trends in Australian hospital anaphylaxis admissions in to J Allergy Clin Immunol. 2015;136: Rudders SA, Arias SA, et al. Trends in hospitalizations for food-induced anaphylaxis in US children, J Allergy Clin Immunol. 2014;134:960-2 e Wong GW, Mahesh PA, et al. The EuroPrevall- INCO surveys on the prevalence of food allergies in children from China, India and Russia: the study methodology. Allergy. 2010;65: Keil T, McBride D, et al. The multinational birth cohort of EuroPrevall: background, aims and methods. Allergy. 2010;65: de Blok BM, Vlieg-Boerstra BJ, et al. A framework for measuring the social impact of food allergy across Europe: a EuroPrevall state of the art paper. Allergy. 2007;62: Gupta R, Holdford D, et al. The economic impact of childhood food allergy in the United States. JAMA Pediatr. 2013;167: Zuberbier T, Lotvall J, et al. Economic burden of inadequate management of allergic diseases in the European Union: a GA(2) LEN review. Allergy. 2014;69: Holloway JW, Yang IA, et al. Genetics of allergic disease. J Allergy Clin Immunol. 2010;125:S Portelli MA, Hodge E, et al. Genetic risk factors for the development of allergic disease identified by genome-wide association. Clin Exp Allergy. 2015;45: Madore AM, Vaillancourt VT, et al. HLA- DQB1*02 and DQB1*06:03P are associated with peanut allergy. Eur J Hum Genet. 2013;21: Hong X, Hao K, et al. Genome-wide association study identifies peanut allergyspecific loci and evidence of epigenetic mediation in US children. Nat Commun. 2015;6:

17 CHAPTER 1 GENERAL INTRODUCTION 29. Strachan DP. Hay fever, hygiene, and household size. BMJ. 1989;299: Rook GA, Martinelli R, et al. Innate immune responses to mycobacteria and the downregulation of atopic responses. Curr Opin Allergy Clin Immunol. 2003;3: Rook GA, Adams V, et al. Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders. Springer Semin Immunopathol. 2004;25: van den Biggelaar AH, van Ree R, et al. Decreased atopy in children infected with Schist os om a haematobium: a role for parasite-induced interleukin-10. Lancet. 2000;356: Yazdanbakhsh M, Kremsner PG, et al. Allergy, parasites, and the hygiene hypothesis. Science. 2002;296: Smits HH, Everts B, et al. Chronic helminth infections protect against allergic diseases by active regulatory processes. Curr Allergy Asthma Rep. 2010;10: Eggesbo M, Botten G, et al. Is delivery by cesarean section a risk factor for food allergy? J Allergy Clin Immunol. 2003;112: Kolokotroni O, Middleton N, et al. Asthma and atopy in children born by caesarean section: effect modification by family history of allergies - a population based cross-sectional study. BMC Pediatr. 2012;12: Renz-Polster H, David MR, et al. Caesarean section delivery and the risk of allergic disorders in childhood. Clin Exp Allergy. 2005;35: Strachan DP, Harkins LS, et al. Childhood antecedents of allergic sensitization in young British adults. J Allergy Clin Immunol. 1997;99: Svanes C, Jarvis D, et al. Childhood environment and adult atopy: results from the European Community Respiratory Health Survey. J Allergy Clin Immunol. 1999;103: Svanes C, Jarvis D, et al. Early exposure to children in family and day care as related to adult asthma and hay fever: results from the European Community Respiratory Health Survey. Thorax. 2002;57: Kramer U, Heinrich J, et al. Age of entry to day nursery and allergy in later childhood. Lancet. 1999;353: Ege MJ, Mayer M, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med. 2011;364: Kilpelainen M, Terho EO, et al. Childhood farm environment and asthma and sensitization in young adulthood. Allergy. 2002;57: Riedler J, Braun-Fahrlander C, et al. Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey. Lancet. 2001;358: Wickens K, Lane JM, et al. Farm residence and exposures and the risk of allergic diseases in New Zealand children. Allergy. 2002;57: Tan J, McKenzie C, et al. The role of shortchain fatty acids in health and disease. Adv Immunol. 2014;121: Arpaia N, Campbell C, et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature. 2013;504: Park J, Kim M, et al. Short-chain fatty acids induce both effector and regulatory T cells by suppression of histone deacetylases and regulation of the mtor-s6k pathway. Mucosal Immunol. 2015;8: Smith PM, Howitt MR, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science. 2013;341: Stefka AT, Feehley T, et al. Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci U S A. 2014;111: Cao S, Feehley TJ, et al. The role of commensal bacteria in the regulation of sensitization to food allergens. FEBS Lett. 2014;588: Sonnenberg GF, Fouser LA, et al. Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22. Nat Immunol. 2011;12: Ventura MT, Polimeno L, et al. Intestinal permeability in patients with adverse reactions to food. Dig Liver Dis. 2006;38: Martin PE, Eckert JK, et al. Which infants with eczema are at risk of food allergy? Results from a population-based cohort. Clin Exp Allergy. 2015;45: Brown SJ, Asai Y, et al. Loss-of-function variants in the filaggrin gene are a significant risk factor for peanut allergy. J Allergy Clin Immunol. 2011;127: Ogrodowczyk A, Markiewicz L, et al. Mutations in the filaggrin gene and food allergy. Prz Gastroenterol. 2014;9: Brown SJ, McLean WH. Eczema genetics: current state of knowledge and future goals. J Invest Dermatol. 2009;129: Palmer CN, Irvine AD, et al. Common loss-offunction variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet. 2006;38: Soumelis V, Reche PA, et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol. 2002;3: Herrick CA, MacLeod H, et al. Th2 responses induced by epicutaneous or inhalational protein exposure are differentially dependent on IL-4. J Clin Invest. 2000;105: Bartnikas LM, Gurish MF, et al. Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and foodinduced anaphylaxis. J Allergy Clin Immunol. 2013;131: e Hsieh KY, Tsai CC, et al. Epicutaneous exposure to protein antigen and food allergy. Clin Exp Allergy. 2003;33: Kondo H, Ichikawa Y, et al. Percutaneous sensitization with allergens through barrierdisrupted skin elicits a Th2-dominant cytokine response. Eur J Immunol. 1998;28: Oyoshi MK, Murphy GF, et al. Filaggrindeficient mice exhibit TH17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen. J Allergy Clin Immunol. 2009;124:485-93, 93 e Strid J, Hourihane J, et al. Disruption of the stratum corneum allows potent epicutaneous immunization with protein antigens resulting in a dominant systemic Th2 response. Eur J Immunol. 2004;34: Dunkin D, Berin MC, et al. Allergic sensitization can be induced via multiple physiologic routes in an adjuvant-dependent manner. J Allergy Clin Immunol. 2011;128: e Strid J, Hourihane J, et al. Epicutaneous exposure to peanut protein prevents oral tolerance and enhances allergic sensitization. Clin Exp Allergy. 2005;35: Boussault P, Leaute-Labreze C, et al. Oat sensitization in children with atopic dermatitis: prevalence, risks and associated factors. Allergy. 2007;62: Guillet G, Guillet MH. [Percutaneous sensitization to almond oil in infancy and study of ointments in 27 children with food allergy]. Allerg Immunol (Paris). 2000;32: Lack G, Fox D, et al. Factors associated with the development of peanut allergy in childhood. N Engl J Med. 2003;348: Du Toit G, Roberts G, et al. Randomized trial of peanut consumption in infants at risk for peanut allergy. N Engl J Med. 2015;372: Lambrecht BN, Hammad H. The airway epithelium in asthma. Nat Med. 2012;18: Liao YW, Wu XM, et al. Proteolytic antigens interfere with endosome/lysosome fusion in epithelial cells. Biochem Cell Biol. 2013;91: Wang YH, Liu YJ. Thymic stromal lymphopoietin, OX40-ligand, and interleukin-25 in allergic responses. Clin Exp Allergy. 2009;39: Ohno T, Morita H, et al. Interleukin-33 in allergy. Allergy. 2012;67: Ziegler SF. Thymic stromal lymphopoietin and allergic disease. J Allergy Clin Immunol. 2012;130: Salazar F, Ghaemmaghami AM. Allergen recognition by innate immune cells: critical role of dendritic and epithelial cells. Front Immunol. 2013;4: Banchereau J, Briere F, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18: Cresswell P. Assembly, transport, and function of MHC class II molecules. Annu Rev Immunol. 1994;12: Peters PJ, Neefjes JJ, et al. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature. 1991;349: Blum JS, Cresswell P. Role for intracellular proteases in the processing and transport of class II HLA antigens. Proc Natl Acad Sci U S A. 1988;85: Romagnoli P, Germain RN. The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding, transport, and peptide occupancy. J Exp Med. 1994;180: Denzin LK, Cresswell P. HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers

18 CHAPTER 1 GENERAL INTRODUCTION and facilitates peptide loading. Cell. 1995;82: Seder RA, Germain RN, et al. CD28-mediated costimulation of interleukin 2 (IL-2) production plays a critical role in T cell priming for IL-4 and interferon gamma production. J Exp Med. 1994;179: Webb LM, Feldmann M. Critical role of CD28/ B7 costimulation in the development of human Th2 cytokine-producing cells. Blood. 1995;86: Szabo SJ, Kim ST, et al. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell. 2000;100: Ito T, Wang YH, et al. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J Exp Med. 2005;202: Amsen D, Blander JM, et al. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell. 2004;117: MacDonald AS, Straw AD, et al. Cutting edge: Th2 response induction by dendritic cells: a role for CD40. J Immunol. 2002;168: Ghaemmaghami AM, Gough L, et al. The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level. Clin Exp Allergy. 2002;32: Traidl-Hoffmann C, Mariani V, et al. Pollenassociated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization. J Exp Med. 2005;201: Kurata H, Lee HJ, et al. Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells. Immunity. 1999;11: Zhu J, Guo L, et al. Stat6 is necessary and sufficient for IL-4 s role in Th2 differentiation and cell expansion. J Immunol. 2001;166: Mjosberg J, Bernink J, et al. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. Immunity. 2012;37: Perrigoue JG, Saenz SA, et al. MHC class II-dependent basophil-cd4+ T cell interactions promote T(H)2 cytokine- dependent immunity. Nat Immunol. 2009;10: Sokol CL, Chu NQ, et al. Basophils function as antigen-presenting cells for an allergeninduced T helper type 2 response. Nat Immunol. 2009;10: Yoshimoto T, Yasuda K, et al. Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide- MHC class II complexes to CD4+ T cells. Nat Immunol. 2009;10: Yanagihara Y, Kajiwara K, et al. Cultured basophils but not cultured mast cells induce human IgE synthesis in B cells after immunologic stimulation. Clin Exp Immunol. 1998;111: Delespesse G, Suter U, et al. Expression, structure, and function of the CD23 antigen. Adv Immunol. 1991;49: Yokota A, Kikutani H, et al. Two species of human Fc epsilon receptor II (Fc epsilon RII/ CD23): tissue-specific and IL-4-specific regulation of gene expression. Cell. 1988;55: Cho SW, Kilmon MA, et al. B cell activation and Ig, especially IgE, production is inhibited by high CD23 levels in vivo and in vitro. Cell Immunol. 1997;180: Yu P, Kosco-Vilbois M, et al. Negative feedback regulation of IgE synthesis by murine CD23. Nature. 1994;369: Sherr E, Macy E, et al. Binding the low affinity Fc epsilon R on B cells suppresses ongoing human IgE synthesis. J Immunol. 1989;142: Cooper AM, Hobson PS, et al. Soluble CD23 controls IgE synthesis and homeostasis in human B cells. J Immunol. 2012;188: MacGlashan DW, Jr. Biochemical events in basophil/mast cell activation and mediator release. In: Adkinson N, Bochner B, Busse W, Holgate S, Lemanske R, Simons F, editors. Middleton s Allergy: Principles and Practice 7th ed: Mosby Elsevier; Ohnmacht C, Voehringer D. Basophil effector function and homeostasis during helminth infection. Blood. 2009;113: Yamaguchi M, Hirai K, et al. Hemopoietic growth factors regulate the survival of human basophils in vitro. Int Arch Allergy Immunol. 1992;97: Padawer J. Mast cells: extended lifespan and lack of granule turnover under normal in vivo conditions. Exp Mol Pathol. 1974;20: Knol EF, Mul FP, et al. Monitoring human basophil activation via CD63 monoclonal antibody 435. J Allergy Clin Immunol. 1991;88: Sainte-Laudy J, Vallon C, et al. [Analysis of membrane expression of the CD63 human basophil activation marker. Applications to allergologic diagnosis]. Allerg Immunol (Paris). 1994;26: Nopp A, Johansson SG, et al. Basophil allergen threshold sensitivity: a useful approach to anti-ige treatment efficacy evaluation. Allergy. 2006;61: Ebo DG, Bridts CH, et al. Basophil activation test by flow cytometry: present and future applications in allergology. Cytometry B Clin Cytom. 2008;74: MacGlashan D, Jr. Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes. Clin Exp Allergy. 2010;40: Kepley CL, Youssef L, et al. Syk deficiency in nonreleaser basophils. J Allergy Clin Immunol. 1999;104: Kepley CL, Youssef L, et al. Multiple defects in Fc epsilon RI signaling in Sykdeficient nonreleaser basophils and IL-3- induced recovery of Syk expression and secretion. J Immunol. 2000;165: Yamaguchi M, Hirai K, et al. Nonreleasing basophils convert to releasing basophils by culturing with IL-3. J Allergy Clin Immunol. 1996;97: Knol EF, Mul FP, et al. Intracellular events in anti-ige nonreleasing human basophils. J Allergy Clin Immunol. 1992;90: Nguyen KL, Gillis S, et al. A comparative study of releasing and nonreleasing human basophils: nonreleasing basophils lack an early component of the signal transduction pathway that follows IgE cross-linking. J Allergy Clin Immunol. 1990;85: Balzer L, Pennino D, et al. Basophil activation test using recombinant allergens: highly specific diagnostic method complementing routine tests in wasp venom allergy. PLoS One. 2014;9:e Erdmann SM, Sachs B, et al. The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring specific immunotherapy. Allergy. 2004;59: Korosec P, Silar M, et al. Clinical routine utility of basophil activation testing for diagnosis of hymenoptera-allergic patients with emphasis on individuals with negative venom-specific IgE antibodies. Int Arch Allergy Immunol. 2013;161: Santos AF, Douiri A, et al. Basophil activation test discriminates between allergy and tolerance in peanut-sensitized children. J Allergy Clin Immunol. 2014;134: Santos AF, Du Toit G, et al. Distinct parameters of the basophil activation test reflect the severity and threshold of allergic reactions to peanut. J Allergy Clin Immunol. 2015;135: Song Y, Wang J, et al. Correlations between basophil activation, allergen-specific IgE with outcome and severity of oral food challenges. Ann Allergy Asthma Immunol. 2015;114: Hoffmann HJ, Santos AF, et al. The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease. Allergy. 2015;70: Sanz ML, Gamboa PM, et al. Basophil activation tests in the evaluation of immediate drug hypersensitivity. Curr Opin Allergy Clin Immunol. 2009;9: Cox L, Williams B, et al. Pearls and pitfalls of allergy diagnostic testing: report from the American College of Allergy, Asthma and Immunology/American Academy of Allergy, Asthma and Immunology Specific IgE Test Task Force. Ann Allergy Asthma Immunol. 2008;101: McGowan EC, Saini S. Update on the performance and application of basophil activation tests. Curr Allergy Asthma Rep. 2013;13: Noon L. Prophylactic inoculation against hayfever. Lancet. 1911;1: Burks AW, Jones SM, et al. Oral immunotherapy for treatment of egg allergy in children. N Engl J Med. 2012;367: Vickery BP, Scurlock AM, et al. Sustained unresponsiveness to peanut in subjects who have completed peanut oral immunotherapy. J Allergy Clin Immunol. 2014;133: Senti G, Crameri R, et al. Intralymphatic immunotherapy for cat allergy induces tolerance after only 3 injections. J Allergy Clin Immunol. 2012;129: Senti G, Prinz Vavricka BM, et al

19 CHAPTER 1 GENERAL INTRODUCTION Intralymphatic allergen administration renders specific immunotherapy faster and safer: a randomized controlled trial. Proc Natl Acad Sci U S A. 2008;105: Canonica GW, Bousquet J, et al. Sub-lingual immunotherapy: World Allergy Organization Position Paper Allergy. 2009;64 Suppl 91: Cox L, Calderon M, et al. Subcutaneous allergen immunotherapy for allergic disease: examining efficacy, safety and costeffectiveness of current and novel formulations. Immunotherapy. 2012;4: Chelladurai Y, Suarez-Cuervo C, et al. Effectiveness of subcutaneous versus sublingual immunotherapy for the treatment of allergic rhinoconjunctivitis and asthma: a systematic review. J Allergy Clin Immunol Pract. 2013;1: Di Bona D, Plaia A, et al. Efficacy of subcutaneous and sublingual immunotherapy with grass allergens for seasonal allergic rhinitis: a meta-analysis-based comparison. J Allergy Clin Immunol. 2012;130: e Nelson H, Cartier S, et al. Network metaanalysis shows commercialized subcutaneous and sublingual grass products have comparable efficacy. J Allergy Clin Immunol Pract. 2015;3: e Cox LS, Larenas Linnemann D, et al. Sublingual immunotherapy: a comprehensive review. J Allergy Clin Immunol. 2006;117: Morisset M, Moneret-Vautrin DA, et al. Oral desensitization in children with milk and egg allergies obtains recovery in a significant proportion of cases. A randomized study in 60 children with cow s milk allergy and 90 children with egg allergy. Eur Ann Allergy Clin Immunol. 2007;39: Pajno GB, Caminiti L, et al. Oral immunotherapy for cow s milk allergy with a weekly up-dosing regimen: a randomized single-blind controlled study. Ann Allergy Asthma Immunol. 2010;105: Skripak JM, Nash SD, et al. A randomized, double-blind, placebo-controlled study of milk oral immunotherapy for cow s milk allergy. J Allergy Clin Immunol. 2008;122: Caminiti L, Pajno GB, et al. Oral Immunotherapy for Egg Allergy: A Double-Blind Placebo-Controlled Study, with Postdesensitization Follow-Up. J Allergy Clin Immunol Pract. 2015;3: Meglio P, Giampietro PG, et al. Oral food desensitization in children with IgE-mediated hen s egg allergy: a new protocol with raw hen s egg. Pediatr Allergy Immunol. 2013;24: Anagnostou K, Clark A, et al. Efficacy and safety of high-dose peanut oral immunotherapy with factors predicting outcome. Clin Exp Allergy. 2011;41: Anagnostou K, Islam S, et al. Assessing the efficacy of oral immunotherapy for the desensitisation of peanut allergy in children (STOP II): a phase 2 randomised controlled trial. Lancet. 2014;383: Blumchen K, Ulbricht H, et al. Oral peanut immunotherapy in children with peanut anaphylaxis. J Allergy Clin Immunol. 2010;126:83-91 e Clark AT, Islam S, et al. Successful oral tolerance induction in severe peanut allergy. Allergy. 2009;64: Jones SM, Pons L, et al. Clinical efficacy and immune regulation with peanut oral immunotherapy. J Allergy Clin Immunol. 2009;124: , e Varshney P, Jones SM, et al. A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response. J Allergy Clin Immunol. 2011;127: Nelson HS, Lahr J, et al. Treatment of anaphylactic sensitivity to peanuts by immunotherapy with injections of aqueous peanut extract. J Allergy Clin Immunol. 1997;99: Oppenheimer JJ, Nelson HS, et al. Treatment of peanut allergy with rush immunotherapy. J Allergy Clin Immunol. 1992;90: Merad M, Ginhoux F, et al. Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells. Nat Rev Immunol. 2008;8: Haniffa M, Gunawan M, et al. Human skin dendritic cells in health and disease. J Dermatol Sci. 2015;77: Segura E, Valladeau-Guilemond J, et al. Characterization of resident and migratory dendritic cells in human lymph nodes. J Exp Med. 2012;209: Chu CC, Ali N, et al. Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation. J Exp Med. 2012;209: Rotiroti G, Shamji M, et al. Repeated lowdose intradermal allergen injection suppresses allergen-induced cutaneous late responses. J Allergy Clin Immunol. 2012;130: e Allam JP, Peng WM, et al. Toll-like receptor 4 ligation enforces tolerogenic properties of oral mucosal Langerhans cells. J Allergy Clin Immunol. 2008;121: e Allam JP, Wurtzen PA, et al. Phl p 5 resorption in human oral mucosa leads to dosedependent and time-dependent allergen binding by oral mucosal Langerhans cells, attenuates their maturation, and enhances their migratory and TGF-beta1 and IL-10- producing properties. J Allergy Clin Immunol. 2010;126: e Kulis M, Saba K, et al. Increased peanutspecific IgA levels in saliva correlate with food challenge outcomes after peanut sublingual immunotherapy. J Allergy Clin Immunol. 2012;129: Queiros MG, Silva DA, et al. Modulation of mucosal/systemic antibody response after sublingual immunotherapy in mite-allergic children. Pediatr Allergy Immunol. 2013;24: Novak N, Mete N, et al. Early suppression of basophil activation during allergen-specific immunotherapy by histamine receptor 2. J Allergy Clin Immunol. 2012;130: e Akdis CA, Blesken T, et al. Role of interleukin 10 in specific immunotherapy. J Clin Invest. 1998;102: Palomares O, Martin-Fontecha M, et al. Regulatory T cells and immune regulation of allergic diseases: roles of IL-10 and TGF-beta. Genes Immun. 2014;15: Pilette C, Nouri-Aria KT, et al. Grass pollen immunotherapy induces an allergen-specific IgA2 antibody response associated with mucosal TGF-beta expression. J Immunol. 2007;178: Gorelik L, Constant S, et al. Mechanism of transforming growth factor beta-induced inhibition of T helper type 1 differentiation. J Exp Med. 2002;195: Gorelik L, Fields PE, et al. Cutting edge: TGF-beta inhibits Th type 2 development through inhibition of GATA-3 expression. J Immunol. 2000;165: Chen W, Jin W, et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003;198: Shamji MH, Ljorring C, et al. Functional rather than immunoreactive levels of IgG4 correlate closely with clinical response to grass pollen immunotherapy. Allergy. 2012;67: van Neerven RJ, Knol EF, et al. IgE-mediated allergen presentation and blocking antibodies: regulation of T-cell activation in allergy. Int Arch Allergy Immunol. 2006;141: van Neerven RJ, Wikborg T, et al. Blocking antibodies induced by specific allergy vaccination prevent the activation of CD4+ T cells by inhibiting serum-ige-facilitated allergen presentation. J Immunol. 1999;163: Wachholz PA, Soni NK, et al. Inhibition of allergen-ige binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol. 2003;112: van de Veen W, Stanic B, et al. IgG4 production is confined to human IL-10- producing regulatory B cells that suppress antigen-specific immune responses. J Allergy Clin Immunol. 2013;131: Durham SR, Ying S, et al. Grass pollen immunotherapy inhibits allergen-induced infiltration of CD4+ T lymphocytes and eosinophils in the nasal mucosa and increases the number of cells expressing messenger RNA for interferon-gamma. J Allergy Clin Immunol. 1996;97: Nouri-Aria KT, Pilette C, et al. IL-9 and c-kit+ mast cells in allergic rhinitis during seasonal allergen exposure: effect of immunotherapy. J Allergy Clin Immunol. 2005;116: Otsuka H, Mezawa A, et al. Changes in nasal metachromatic cells during allergen immunotherapy. Clin Exp Allergy. 1991;21: Varney VA, Hamid QA, et al. Influence of grass pollen immunotherapy on cellular infiltration and cytokine mrna expression during allergen-induced late-phase cutaneous responses. J Clin Invest. 1993;92: Wilson DR, Irani AM, et al. Grass pollen

20 CHAPTER 1 GENERAL INTRODUCTION immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium. Clin Exp Allergy. 2001;31: Creticos PS, Van Metre TE, et al. Dose response of IgE and IgG antibodies during ragweed immunotherapy. J Allergy Clin Immunol. 1984;73: Delaunois L, Salamon E, et al. Influence of hyposensitization with Dermatophagoides pteronyssinus extract on clinical score, total and specific IgE levels, and skin test in asthmatic patients. Ann Allergy. 1985;55: Gleich GJ, Zimmermann EM, et al. Effect of immunotherapy on immunoglobulin E and immunoglobulin G antibodies to ragweed antigens: a six-year prospective study. J Allergy Clin Immunol. 1982;70: Van Ree R, Van Leeuwen WA, et al. Measurement of IgE antibodies against purified grass pollen allergens (Lol p 1, 2, 3 and 5) during immunotherapy. Clin Exp Allergy. 1997;27: Pajno GB, Barberio G, et al. Prevention of new sensitizations in asthmatic children monosensitized to house dust mite by specific immunotherapy. A six-year follow-up study. Clin Exp Allergy. 2001;31: Joss A, Akdis M, et al. IL-10 directly acts on T cells by specifically altering the CD28 costimulation pathway. Eur J Immunol. 2000;30: Steinbrink K, Wolfl M, et al. Induction of tolerance by IL-10-treated dendritic cells. J Immunol. 1997;159: Friedman A, Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci U S A. 1994;91: Gardner LM, O Hehir RE, et al. High dose allergen stimulation of T cells from house dust mite-allergic subjects induces expansion of IFN-gamma+ T Cells, apoptosis of CD4+IL-4+ T cells and T cell anergy. Int Arch Allergy Immunol. 2004;133: Taams LS, van Eden W, et al. Dose-dependent induction of distinct anergic phenotypes: multiple levels of T cell anergy. J Immunol. 1999;162: Sloan-Lancaster J, Evavold BD, et al. Induction of T-cell anergy by altered T-cellreceptor ligand on live antigen-presenting cells. Nature. 1993;363: Sloan-Lancaster J, Evavold BD, et al. Th2 cell clonal anergy as a consequence of partial activation. J Exp Med. 1994;180: Tsitoura DC, Holter W, et al. Induction of anergy in human T helper 0 cells by stimulation with altered T cell antigen receptor ligands. J Immunol. 1996;156: Akdis CA, Akdis M, et al. Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro. J Clin Invest. 1996;98: Wambre E, DeLong JH, et al. Specific immunotherapy modifies allergen-specific CD4(+) T-cell responses in an epitopedependent manner. J Allergy Clin Immunol. 2014;133:872-9 e Guerra F, Carracedo J, et al. TH2 lymphocytes from atopic patients treated with immunotherapy undergo rapid apoptosis after culture with specific allergens. J Allergy Clin Immunol. 2001;107: Tsai YG, Chien JW, et al. Induced apoptosis of TH2 lymphocytes in asthmatic children treated with Dermatophagoides pteronyssinus immunotherapy. Pediatr Allergy Immunol. 2005;16: Hofmann AM, Scurlock AM, et al. Safety of a peanut oral immunotherapy protocol in children with peanut allergy. J Allergy Clin Immunol. 2009;124:286-91, 91 e Fleischer DM, Burks AW, et al. Sublingual immunotherapy for peanut allergy: a randomized, double-blind, placebocontrolled multicenter trial. J Allergy Clin Immunol. 2013;131: e Kahlert H, Suck R, et al. Characterization of a hypoallergenic recombinant Bet v 1 variant as a candidate for allergen-specific immunotherapy. Int Arch Allergy Immunol. 2008;145: Meyer W, Narkus A, et al. Double-blind, placebo-controlled, dose-ranging study of new recombinant hypoallergenic Bet v 1 in an environmental exposure chamber. Allergy. 2013;68: Valenta R, Ferreira F, et al. From allergen genes to allergy vaccines. Annu Rev Immunol. 2010;28: Wood RA, Sicherer SH, et al. A phase 1 study of heat/phenol-killed, E. coliencapsulated, recombinant modified peanut proteins Ara h 1, Ara h 2, and Ara h 3 (EMP- 123) for the treatment of peanut allergy. Allergy. 2013;68: Wambre E, DeLong JH, et al. Differentiation stage determines pathologic and protective allergen-specific CD4+ T-cell outcomes during specific immunotherapy. J Allergy Clin Immunol. 2012;129:544-51, 51 e Akdis M, Verhagen J, et al. Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med. 2004;199: Jarman ER, Hawrylowicz CM, et al. Inhibition of human T-cell responses to house dust mite allergens by a T-cell receptor peptide. J Allergy Clin Immunol. 1994;94: Wedderburn LR, O Hehir RE, et al. In vivo clonal dominance and limited T-cell receptor usage in human CD4+ T-cell recognition of house dust mite allergens. Proc Natl Acad Sci U S A. 1993;90: Hoyne GF, Callow MG, et al. Characterization of T-cell responses to the house dust mite allergen Der p II in mice. Evidence for major and cryptic epitopes. Immunology. 1993;78: Muller U, Akdis CA, et al. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol. 1998;101: Fellrath JM, Kettner A, et al. Allergen-specific T-cell tolerance induction with allergenderived long synthetic peptides: results of a phase I trial. J Allergy Clin Immunol. 2003;111: Tarzi M, Klunker S, et al. Induction of interleukin-10 and suppressor of cytokine signalling-3 gene expression following peptide immunotherapy. Clin Exp Allergy. 2006;36: Pellaton C, Perrin Y, et al. Novel birch pollen specific immunotherapy formulation based on contiguous overlapping peptides. Clin Transl Allergy. 2013;3: Spertini F, Perrin Y, et al. Safety and immunogenicity of immunotherapy with Bet v 1-derived contiguous overlapping peptides. J Allergy Clin Immunol. 2014;134: e [cited 2016 Jan]. Available from: clinicaltrials.gov/ct2/show/study/nct Maguire P, Nicodemus C, et al. The safety and efficacy of ALLERVAX CAT in cat allergic patients. Clin Immunol. 1999;93: Norman PS, Nicodemus CF, et al. Clinical and immunologic effects of component peptides in Allervax Cat. Int Arch Allergy Immunol. 1997;113: Norman PS, Ohman JL, Jr., et al. Treatment of cat allergy with T-cell reactive peptides. Am J Respir Crit Care Med. 1996;154: Alexander C, Tarzi M, et al. The effect of Fel d 1-derived T-cell peptides on upper and lower airway outcome measurements in cat-allergic subjects. Allergy. 2005;60: Alexander C, Ying S, et al. Fel d 1-derived T cell peptide therapy induces recruitment of CD4+ CD25+; CD4+ interferon-gamma+ T helper type 1 cells to sites of allergen-induced late-phase skin reactions in cat-allergic subjects. Clin Exp Allergy. 2005;35: Oldfield WL, Kay AB, et al. Allergenderived T cell peptide-induced late asthmatic reactions precede the induction of antigen-specific hyporesponsiveness in atopic allergic asthmatic subjects. J Immunol. 2001;167: Oldfield WL, Larche M, et al. Effect of T-cell peptides derived from Fel d 1 on allergic reactions and cytokine production in patients sensitive to cats: a randomised controlled trial. Lancet. 2002;360: Smith TR, Alexander C, et al. Cat allergen peptide immunotherapy reduces CD4(+) T cell responses to cat allergen but does not alter suppression by CD4(+) CD25(+) T cells: a double-blind placebo-controlled study. Allergy. 2004;59: Verhoef A, Alexander C, et al. T cell epitope immunotherapy induces a CD4+ T cell population with regulatory activity. PLoS Med. 2005;2:e Worm M, Lee HH, et al. Development and preliminary clinical evaluation of a peptide immunotherapy vaccine for cat allergy. J Allergy Clin Immunol. 2011;127:89-97, e Couroux P, Patel D, et al. Fel d 1-derived synthetic peptide immuno-regulatory epitopes show a long-term treatment effect in cat allergic subjects. Clin Exp Allergy. 2015;45: Patel D, Couroux P, et al. Fel d 1-derived peptide antigen desensitization shows a persistent treatment effect 1 year after the

21 CHAPTER 1 GENERAL INTRODUCTION start of dosing: a randomized, placebocontrolled study. J Allergy Clin Immunol. 2013;131:103-9 e Hafner RP, Couroux P, et al. Four doses of Der p derived synthetic peptide immunoregulatory epitopes over 3 months results in a persistent treatment effect at 1 year on symptoms of House Dust Mite allergy. Allergy. 2014;69: Ellis AK, Frankish CW, et al. A short course of synthetic peptide immuno-regulatory epitopes derived from grass allergens leads to a reduction in grass allergy symptoms. Allergy. 2014;69: Hafner R, Salapatek A, et al. Validation of peptide immunotherapy as a new approach in the treatment of allergic rhinoconjunctivitis: the clinical benefits of treatment with Amb a 1- derived T cell epitopes. J Allergy Clin Immunol. 2012; Campbell JD, Buckland KF, et al. Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression. J Exp Med. 2009;206: Hoyne GF, Dallman MJ, et al. Linked suppression in peripheral T cell tolerance to the house dust mite derived allergen Der p 1. Int Arch Allergy Immunol. 1999;118: O Hehir RE, Lake RA, et al. Regulation of cytokine and chemokine transcription in a human TH2 type T-cell clone during the induction phase of anergy. Clin Exp Allergy. 1996;26: O Hehir RE, Yssel H, et al. Clonal analysis of differential lymphokine production in peptide and superantigen induced T cell anergy. Int Immunol. 1991;3: Higgins JA, Lamb JR, et al. Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite). J Allergy Clin Immunol. 1992;90: Higgins JA, Thorpe CJ, et al. Overlapping T-cell epitopes in the group I allergen of Dermatophagoides species restricted by HLA- DP and HLA-DR class II molecules. J Allergy Clin Immunol. 1994;93: Lake RA, O Hehir RE, et al. CD28 mrna rapidly decays when activated T cells are functionally anergized with specific peptide. Int Immunol. 1993;5: Faith A, Akdis CA, et al. Defective TCR stimulation in anergized type 2 T helper cells correlates with abrogated p56(lck) and ZAP-70 tyrosine kinase activities. J Immunol. 1997;159: Hochweller K, Anderton SM. Kinetics of costimulatory molecule expression by T cells and dendritic cells during the induction of tolerance versus immunity in vivo. Eur J Immunol. 2005;35: Hoyne GF, Askonas BA, et al. Regulation of house dust mite responses by intranasally administered peptide: transient activation of CD4+ T cells precedes the development of tolerance in vivo. Int Immunol. 1996;8: Pape KA, Khoruts A, et al. Antigen-specific CD4+ T cells that survive after the induction of peripheral tolerance possess an intrinsic lymphokine production defect. Novartis Found Symp. 1998;215:103-13; discussion 13-9, Liblau RS, Tisch R, et al. Intravenous injection of soluble antigen induces thymic and peripheral T-cells apoptosis. Proc Natl Acad Sci U S A. 1996;93: Rosenwasser LJ, Meng J. Anti-CD23. Clin Rev Allergy Immunol. 2005;29: Gauvreau GM, O Byrne PM, et al. Effects of an anti-tslp antibody on allergen-induced asthmatic responses. N Engl J Med. 2014;370: Chung KF. Targeting the interleukin pathway in the treatment of asthma. Lancet. 2015;386: Presta LG, Lahr SJ, et al. Humanization of an antibody directed against IgE. J Immunol. 1993;151: MacGlashan DW, Jr., Bochner BS, et al. Serum IgE level drives basophil and mast cell IgE receptor display. Int Arch Allergy Immunol. 1997;113: MacGlashan DW, Jr., Bochner BS, et al. Down-regulation of Fc(epsilon)RI expression on human basophils during in vivo treatment of atopic patients with anti-ige antibody. J Immunol. 1997;158: Available from: committee-meeting-info/adec-221stmeeting-resolutions-4-5-april Bousquet J, Cabrera P, et al. The effect of treatment with omalizumab, an anti-ige antibody, on asthma exacerbations and emergency medical visits in patients with severe persistent asthma. Allergy. 2005;60: Busse W, Corren J, et al. Omalizumab, anti- IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma. J Allergy Clin Immunol. 2001;108: Busse WW, Massanari M, et al. Effect of omalizumab on the need for rescue systemic corticosteroid treatment in patients with moderate-to-severe persistent IgE-mediated allergic asthma: a pooled analysis. Curr Med Res Opin. 2007;23: Saralaya D, Menzies-Gow A, et al. Lung function response to omalizumab in severe allergic asthma patients in UK clinical practice: APEX II study. Eur Respir J. 2014; El-Qutob D. Off-Label Uses of Omalizumab. Clin Rev Allergy Immunol Douglass JA, Carroll K, et al. Omalizumab is effective in treating systemic mastocytosis in a nonatopic patient. Allergy. 2010;65: Kopp MV, Stenglein S, et al. Omalizumab (Xolair) in children with seasonal allergic rhinitis: leukotriene release as a potential in vitro parameter to monitor therapeutic effects. Pediatr Allergy Immunol. 2007;18: Spector SL, Tan RA. Effect of omalizumab on patients with chronic urticaria. Ann Allergy Asthma Immunol. 2007;99: Kaplan AP, Joseph K, et al. Treatment of chronic autoimmune urticaria with omalizumab. J Allergy Clin Immunol. 2008;122: Boyce JA. Successful treatment of coldinduced urticaria/anaphylaxis with anti-ige. J Allergy Clin Immunol. 2006;117: Tillie-Leblond I, Germaud P, et al. Allergic bronchopulmonary aspergillosis and omalizumab. Allergy. 2011;66: Perez-de-Llano LA, Vennera MC, et al. Effects of omalizumab in Aspergillus-associated airway disease. Thorax. 2011;66: Moss RB. Treatment options in severe fungal asthma and allergic bronchopulmonary aspergillosis. Eur Respir J. 2014;43:

22 2 Clinically relevant IgE reactivity and basophil activation to goat s milk after cutaneous sensitization adapted from Goat s cheese anaphylaxis following cutaneous sensitization by moisturizer containing goat s milk Voskamp AL a,b, Zubrinich CM a, Abramovitch JB a,b, Rolland JM a,b, O Hehir RE a,b a Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Australia b Department of Immunology, Monash University, Melbourne, Australia Journal of Allergy and Clinical Immunology: In Practice 2014;2:629-30

23 CHAPTER 2 ANAPHYLAXIS FOLLOWING CUTANEOUS SENSITIZATION Capsule summary We report anaphylaxis to goat s cheese after cutaneous sensitization with a moisturizer containing goat s milk applied to eczematous skin confirmed by specific in vitro basophil activation and inhibition serum IgE immunoblotting. To the Editor: but no activation was found with cow s skimmed milk (Figure 1). Basophils from a non-allergic donor were also assessed, and failed to respond to any of the antigens (data not shown). Positive controls included the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fmlp) and anti-ige antibody induced activation. We also performed serum IgE immunoblotting to the moisturizer and goat s milk. The concentration of goat s milk within the moisturizing product was calculated to be approximately 0.44 mg/ml based on the equivalent levels of basophil activation achieved with a known concentration of goat s milk (Figure 1). 2 The current treatment for atopic eczema includes topical corticosteroid application during exacerbations, and regular use of emollients and moisturizers to optimally hydrate affected areas. Many creams to treat dry skin and eczema are advertised as natural products, but they may contain potential food allergens. For example, ingredients can include goat s milk, cow s milk, coconut milk or oil, oats and nut oil. Application of such food allergens to barriercompromised skin could cause sensitization to the allergen, leading to severe reactions when the food is subsequently ingested. A 55 year old female medical research administrator developed a generalized allergic reaction, characterized by urticaria and rapidly evolving oral and upper airway angioedema, immediately after eating two mouthfuls of a salad at a restaurant. This resulted in Emergency Department attendance and administration of intramuscular adrenaline. The salad contained zucchini, goat s cheese, broad beans, mustard, vinegar, olive oil, basil and lemon zest. Her medical history was unremarkable, except for lifelong extensive atopic eczema and seasonal asthma. Direct questioning revealed that four months prior to the anaphylactic episode, for several weeks, she had been applying a skin moisturizer that contained goat s milk. After an application resulted in acute erythema and itch, she subsequently ceased applying the product. Investigational serum specific-ige was strongly positive for goat s milk (65.7 ku/l; ImmunoCAP, Phadia, Uppsala, Sweden) and negative for mustard. The remaining salad ingredients have been subsequently eaten without reaction, and she regularly consumes cow s milk products. The patient confirmed ingestion of goat s cheese, without any adverse effects, prior to the use of the moisturizer. We hypothesized that IgE mediated sensitization occurred during application of the moisturizer that contained goat s milk to inflamed, eczematous skin, which resulted in the generalized allergic reaction when the foodstuff was subsequently encountered orally. The basophil activation test (BAT) is an in vitro test for functional IgE reactivity. The test assesses IgE-mediated basophil activation in response to stimulation with allergen by detection of CD63 on the basophil surface by flow cytometry 1. Results from this test have previously shown high correlation with clinical symptoms 2. We performed a BAT using whole blood of the patient stimulated with both goat s milk (Greer Laboratories, NC, USA) and her moisturizing product. The moisturizer was diluted at a 1:2 ratio in phosphate buffered saline (PBS; Life Technologies, NY, USA) before use. As a control, we also tested cow s skimmed milk protein. Dose-dependent basophil activation to both the goat s milk extract and the moisturizing product were confirmed, Figure 1. Basophil activation test. Dose-dependent basophil activation induced by goat s milk and moisturizing product used by the patient. ( ) Goat s milk (µg/ml), ( ) cow s milk (µg/ml), ( ) moisturizing product (µl), ( ) anti-ige, ( ) fmlp, (m) no antigen. Antigen preparations (3µg/lane) were resolved by 4% to 12% gradient SDS-PAGE under reducing conditions and then transferred to nitrocellulose and probed with the patient s serum at a 1:20 dilution. IgE immunoblotting revealed serum IgE binding to components in both the moisturizing product and goat s milk (Figure 2A, lane 2 and Figure 2B, respectively), consistent with the ImmunoCAP and basophil activation test results. To verify our hypothesis that the initial sensitization developed through use of the moisturizing product we performed IgE inhibition immunoblotting 3. For this, prior to probing the nitrocellulose, the patient s serum was incubated with increasing concentrations of goat s milk, moisturizing product or control protein keyhole limpet hemocyanin (KLH) for 1 hour. IgE binding to the moisturizing product (lane 2) is shown in Figure 2, which is completely inhibited by pre- incubation with goat s milk (lanes 3-6) as well as the moisturizing product (lanes 7-10), which confirms that the IgE target protein within the moisturizing product contains B-cell epitopes present in goat s milk. Controls for the IgE inhibition immunoblotting included KLH (lane 11-13), which did not cause any nonspecific inhibition of IgE binding to the moisturizing product, and a ryegrass (Greer Laboratories) immunoblot with serum from a ryegrass allergic donor in which no nonspecific inhibition of IgE binding occurred with 44 45

24 CHAPTER 2 ANAPHYLAXIS FOLLOWING CUTANEOUS SENSITIZATION goat s milk (Figure 2, C). Together, these results support the conclusion that an allergic response was induced by the moisturizing product, and this response could be attributed to the goat s milk proteins within the product. The immunoblot results show strong IgE reactivity to proteins with a molecular weight between 20 and 28 kda within the moisturizing product, which may correspond with goat b-casein (approximately 27 kda) and g-casein (22 kda) and which differs from typical cow s milk allergy, in which a-casein and whey proteins (eg, b-lactoglobulin) are the dominant allergens 4, which likely explains the lack of cross-reactivity with cow s milk in this case. References 1. Drew AC, Eusebius NP, et al. Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity. J Immunol 2004; 173: McGowan EC, Saini S. Update on the performance and application of basophil activation tests. Curr Allergy Asthma Rep 2013; 13: Abramovitch JB, Kamath S, et al. IgE Reactivity of Blue Swimmer Crab () Tropomyosin, Por p 1, and Other Allergens; Cross-Reactivity with Black Tiger Prawn and Effects of Heating. PLoS One 2013; 8:e Umpierrez A, Quirce S, et al. Allergy to goat and sheep cheese with good tolerance to cow cheese. Clin Exp Allergy 1999; 29: Chinuki Y, Morita E. Wheat-dependent exercise-induced anaphylaxis sensitized with hydrolyzed wheat protein in soap. Allergol Int 2012; 61: Boussault P, Leaute-Labreze C, et al. Oat sensitization in children with atopic dermatitis: prevalence, risks and associated factors. Allergy 2007; 62: Lack G, Fox D, et al. Factors associated with the development of peanut allergy in childhood. N Engl J Med 2003; 348: Mullins RJ. Allergy to topical and oral goat products. Med J Aust 2012; 197: Dunkin D, Berin MC, et al. Allergic sensitization can be induced via multiple physiologic routes in an adjuvant-dependent manner. J Allergy Clin Immunol 2011; 128: e2. 2 Figure 2. Inhibition IgE immunoblotting. A, Inhibition of patient serum IgE binding to moisturizing product with 10-fold dilutions of inhibiting extract. Lane 1, non-allergic control serum; lane 2, no inhibitor; lanes 3-6, µg/ml goat s milk.; lanes 7-10, µg/ml moisturizing product.; lanes 11-13, µg/ml KLH. B, Patient serum IgE reactivity to goat s milk. C, Inhibition of ryegrass allergic patient serum IgE reactivity to ryegrass. Lane 1, no serum; lane 2, no inhibitor.; lanes 3 and 4, 10,100 µg/ml ryegrass; lanes 5-8, µg/ml goat s milk. When combined, the clinical history and in vitro immunological data clearly support the route of sensitization as being through topical application of the allergen to inflamed and compromised skin with subsequent anaphylaxis to the ingested allergen. This route of sensitization has been suggested in clinical studies of the development of wheat, oat, peanut and goat s milk allergy 5-8 with speculation in several of these studies that the suspected sensitizing product consisted of soaps and oils used to alleviate the symptoms of eczema. Murine studies demonstrate systemic sensitization after cutaneous allergen exposure 9. Our data provide, to our knowledge, the first direct evidence of an immunological response in a patient to the suspected causative agent involved in the development of the allergy. We remind clinicians and patients that skin care ought to be bland, and we advocate avoidance of agents capable of sensitization, especially foods

25 3 MHC Class II expression in human basophils: induction and lack of functional significance Voskamp AL a,b, Prickett SR a,b Mackay F b, Rolland JM a,b, O Hehir RE a,b a Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Australia b Department of Immunology, Monash University, Melbourne, Australia PLoS One 2013;8:e81777

26 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS Abstract Introduction The antigen-presenting abilities of basophils and their role in initiating a Th2 phenotype is a topic of current controversy. We aimed to determine whether human basophils can be induced to express MHC Class II and act as antigen presenting cells for T cell stimulation. Isolated human basophils were exposed to a panel of cytokines and TLR-ligands and assessed for MHC Class II expression. MHC Class II was expressed in up to 17% of isolated basophils following incubation with a combination of IL-3, IFN-γ and GM-CSF for 72 hours. Costimulatory molecules (CD80 and CD86) were expressed at very low levels after stimulation. Gene expression analysis of MHC Class II-positive basophils confirmed up-regulation of HLA-DR, HLA-DM, CD74 and Cathepsin S. However, MHC Class II expressing basophils were incapable of inducing antigenspecific T cell activation or proliferation. This is the first report of significant cytokine-induced MHC Class II up-regulation, at both RNA and protein level, in isolated human basophils. By testing stimulation with relevant T cell epitope peptide as well as whole antigen, the failure of MHC Class II expressing basophils to induce T cell response was shown not to be solely due to inefficient antigen uptake and/or processing. Basophils are circulating effector cells involved in protection against helminth infection, by producing histamine and other inflammatory mediators upon activation by helminth-derived antigens 1-3. By a similar mechanism, they also play a key role in eliciting allergic reactions to normally innocuous environmental antigens. In addition, basophils are significant producers of IL-4 4, 5 and IL-13 6, 7 and can induce T cell-independent IgE class switching in B cells 8, 9. In 2009, there were several reports from murine studies that basophils play a direct role in the initiation of a Th2 type response to particular antigens through antigen presentation This suggested a new paradigm for induction of Th2 response and the potential importance of basophils in initiating the development of allergic disorders. However, in 2010, these findings were contradicted, implicating a discrepancy in the basophil population depletion methods of the previous studies 13, 14. At this time a transgenic mouse model with constitutive and selective deletion of basophils was developed. Using this model, basophils were shown to be important for protective immunity against helminths, for the development of chronic allergic inflammation, but dispensable for the initiation of a primary Th2 response to antigens 15. However, a more recent study using basophil conditionally-depleted transgenic mice showed that basophils can induce Th2 skewing to haptens and peptide antigens, but not protein antigens in vivo. In this model, basophils were shown to express MHC Class II and costimulatory molecules and could present exogenous peptide, but they were unable to take up and process whole antigen 16 Therefore, the full extent of the role of the basophil in antigen presentation and Th2 development is as yet unclear. Basophils comprise less than 1% of circulating leukocytes in humans and are generally considered to be HLA-DR negative. Therefore this marker is often used to distinguish basophils from circulating CD123 + plasmacytoid dendritic cells (pdc). Freshly isolated basophils from PBMC of healthy individuals have consistently been found to lack MHC Class II molecules regardless of the isolation method used Short-term stimulation (24 hours or less) of isolated basophils with allergens, anti-ige or the TLR 2 ligand peptidoglycan did not induce HLA-DR expression 19 and only marginal levels were detected after stimulation with IL-3 and IFN-γ 17, 18. Accordingly, stimulated basophils were unable to induce T cell activation through presentation of whole antigen in these studies. Whether there are conditions under which these properties can be induced in human basophils has not yet been reported, nor has the ability of human basophils to present peptide to T cells, as recently shown in mice 16 Evidence of HLA-DR expressing basophils in humans has been found in relation to disease. One study reported increased expression of HLA-DR on circulating basophils and basophil migration to lymph nodes and spleen of patients suffering from Systemic Lupus Erythematosus, but not healthy controls 20. Basophils have also been detected in mucosal and other tissues and during inflammation in Th2 21, 22 and Th17 23 associated diseases, revealing their ability to migrate from the circulation to areas where they may be exposed to MHC Class II inducing factors. It is therefore feasible that mature, circulating human basophils could be induced to express

27 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS significant levels of MHC Class II under certain conditions, which may have relevance to other specific physiological conditions or disease states. Although basophils share many similar cellular features with mast cells, human basophils may have a closer lineage relationship with eosinophils 24, 25. Previous in vitro studies showed MHC Class II expression and antigen presenting functions in eosinophils upon stimulation with specific cytokines. Maximal MHC Class II expression levels occurred at two to four days of stimulation 26-31, durations longer than examined in prior basophil studies. In order to ascertain the potential for mature human basophils to express MHC Class II and participate in antigen presentation, we chose to follow the methods used in these studies, including stimulation with IL-3, IFN-γ, GM-CSF and TLR ligands for up to 72 hours. In this report we assess the induction of MHC Class II and costimulatory molecule expression in mature human basophils and their ability to present antigen to induce specific T cell proliferation. As antigen, we chose a clinically relevant house dust mite (HDM) allergen extract and we compared T cell responses of Th2-predisposed HDM-allergic subjects with non-atopic subjects. As a tool to dissect antigen processing and presenting capability of activated basophils, we also tested peptide presentation to peptide-specific T cells. Materials and methods Subjects Four non-atopic and five atopic, HDM-allergic Caucasian adults were recruited through The Alfred Allergy Clinic, Melbourne, Australia. Allergic subjects had clinical symptoms of HDM allergy, HDM-specific IgE CAP score 2 (Pharmacia Diagnostics, Uppsala, Sweden) and skin prick test wheals > 4mm diameter to HDM extract. Non-atopic subjects had no history of clinical symptoms of allergy and were skin prick test or IgE CAP score negative to a range of common allergens including HDM. The study was approved by The Alfred Hospital Ethics Committee (Project number 509/11) and the Monash University Human Research Ethics Committee (CF12/0702), and informed written consent was obtained from all subjects. Antigens and other stimulants Lyophilised HDM extract was obtained from Greer Laboratories (NC, USA), reconstituted with PBS ph 7.2 and filter sterilized (0.2 µm) before use. The extract was confirmed to be neither mitogenic nor toxic as described 32 and the endotoxin level was 179 EU/mg (Endpoint Chromogenic LAL assay; Lonza, Walkersville, USA). Lyophilized Arachis hypogaea 1 (Ara h 1) peptide (ALMLPHFN) was synthesized by Mimotopes (Victoria, Australia) and reconstituted in 10% dimethyl sulfoxide/ PBS. Lyophilized cytokines rhil-3 (R&D Systems, Minneapolis, USA), rhifn-γ, rhgm-csf and rhtnf-α (MiltenyiBiotech, BergischGladbach, Germany) and TLR 4, 2 and 9 ligands LPS (Sigma- Aldrich, St Louis, MO), PGN and CpG (Invivogen, San Diego, USA) respectively were reconstituted in 0.1% BSA/ PBS ph7.2 according to manufacturer s recommendations. Basophil isolation, purity and culture Basophils were isolated from ficoll-purified PBMC with a Human Basophil Enrichment kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer s protocol (removing cells expressing CD2, CD3, CD14, CD15, CD19, CD24, CD34, CD36, CD45RA, CD56 and glycophorin A). Basophil purity was assessed with a combination of anti-ige-fitc (Invitrogen, Carlsbad, USA), anti-cd123-pe (BD Pharmingen, San Diego, USA), anti-cd203c-apc (Miltenyi Biotech, Germany), anti-fcεri-pe, anti-cd19-ef450, anti-cd4-apc ef780 (ebioscience, San Diego, USA) and, when sufficient cells were available, anti-lineage-1-fitc (containing antibodies specific for CD3, CD19, CD14 and CD56; BD Pharmingen). Following isolation, basophils were stimulated with cytokines, TLR ligands or HDM in 96-U-well plates at 2x10 5 cells/ml in 10% FCS RPMI consisting of RPMI-1640 containing 2 mm L-glutamine, 100 IU/mL penicillin-streptomycin and 10% FCS (Sigma-Aldrich, St Louis, USA) for 72 hours. Annexin V-PE (ebioscience) and 7AAD (BD Pharmingen) were used according to the manufacturers protocols to determine basophil (identified with FcεRI-APC antibody, ebioscience) viability after culture. MHC Class II and costimulatory molecule expression MHC Class II expression was detected on freshly isolated and stimulated basophils with combined FITC conjugated HLA-DR, -DP and -DQ antibodies and the corresponding IgG2a isotype-control antibody (BD Pharmingen). Costimulatory molecules were detected with anti- CD80-PE, anti-cd40-fitc, and anti-cd86-fitc (BD Pharmingen) and corresponding isotype control antibodies (IgG1, BD). Basophils were blocked with 5% human AB serum in PBS (FACS buffer) prior to antibody staining to inhibit non-specific Fc-IgG2a binding on the cell surface. Subsequent antibody and isotype control incubations were also performed in FACS buffer on ice for 20 minutes. The basophil population was identified as IgE hi or FcεRI hi and CD203c +, CD4, CD19 after strict exclusion of doublets and non-viable, 7AAD + cells. RT-PCR was performed to confirm corresponding gene expression as described below. Gene expression analysis Freshly isolated and stimulated basophils (FcεRI hi, CD203c +, 7AAD, CD4, CD19 cells) were sorted (FACS Aria, BD) according to positive or negative MHC Class II expression directly into a 96-well skirted PCR-plate (Eppendorf, Hamburg, Germany) containing RT reaction mix. Flow cytometry sorted CD19 + B cells and CD123 + IgE low pdc from freshly isolated PBMC served as positive controls for MHC Class II related gene expression. The RT reaction mix consisted of 25 µg/ml Oligo dt18 (Geneworks, Thebarton, Australia), 1% Triton X (Sigma-Aldrich), 0.8 mm dntp, 23 U RNase OUT Ribonuclease Inhibitor, 75 U SuperScript III Reverse Trascriptase, 8 mm DTT, 1x First Strand buffer and ultrapure distilled H 2 O (Invitrogen). The plate was then heated at 50ºC for 50 minutes followed by 70ºC for 15 minutes in a PCR machine to transcribe the mrna to cdna. PCR of the obtained cdna was performed with the platinum pfx polymerase kit (Invitrogen) according to the manufacturer s protocol. Primers (Table 1) were designed to cross exon-exon boundaries using UCSC In-Silico PCR software and synthesized by GeneWorks

28 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS (Thebarton, Australia). PCR was performed on Mastercycler (Eppendorf) as follows: 94 ºC for 5 minutes followed by 36 cycles of 94 ºC for 30 seconds, 58 ºC for 30 seconds and 68 ºC for 30 seconds. The PCR products were then visualized by ultraviolet illumination after electrophoresis in 1.5% agarose gels (Invitrogen) containing 0.5 µg/ml ethidium bromide (Sigma-Aldrich). CD80 primers were tested on cdna of CD19 + cells sorted from 1 µg/ml CpG-stimulated PBMC as a positive control prior to use. Reverse Transcriptase negative controls of the CpG-stimulated CD19 + cell population were included to rule out amplification of genomic regions. Table 1: Primer sequences used for RT-PCR ALMLPHFN, 10 µg/ml) or crpmi alone for a further 72 to 120 hours. Basophil-T cell co-cultures were then washed and stained with anti-fcεri-apc, anti-cd3-ef450 (ebiosciences), anti-cd4- APC ef780, anti-cd25-pe (or the corresponding isotype control IgG1-PE) (BD Pharmingen) and 7AAD in FACS buffer for 20 minutes on ice. Proliferation was assessed by loss of CFSE fluorescence and increased CD25 expression and data presented as percentage of CD3/CD4 positive T cells proliferating. Gates were set according to isotype control and unstimulated T cell controls. As a positive control for antigen presentation, autologous CD14 + monocytes separated from PBMC by flow cytometry using anti-cd14-apc (BD Pharmingen) were irradiated (5000 rad) and incubated at T cell: monocyte ratios ranging from 1:1 to 50:1. 3 Gene Forward primer (5-3 ) Reverse primer (5-3 ) B2M GAGGCTATCCAGCGTACTCCAAAG GCTGCTTACATGTCTCGATCCCAC CD40 TGCCAGCCAGGACAGAAACTG CCAGGTCTTTGGTCTCACAGCTTG CD80 GGG AAC ACC TGG CTG AAG TGA C CAT CTT GGG GCA AAG CAG TAG G CD86 CACAGGAATGATTCGCTCCAC GGCTGAGGGTCCTCAAGCTCTATAG HLA-DRα TGGGACCATCTTCATCATCAAGG GGGCATTCCAT AGCAGAGACAGAC HLA-DMα AGTTGGGGAAGCTGGGTTGG CTGAGCCCAGTCAGCAAATTCG CD74 GAAGCAGGAGCTGTCGGGAA GGAAGCTTCATGCGCAGGTTC Cathepsin S GTGGTTGGCTATGGTGAT CTTAATG AACTCCTGGCCTCAAGTTGATATGC CD123 GAGCTTCAGATACAAAAGAGAATGCAGC GGGTCTTTCATGTGAGGGATGC Generation of T cell lines HDM-specific T cell lines were generated from freshly isolated PBMC of two atopic HDMallergic donors and one non-atopic donor using 5,6-CFSE according to our previously described protocol 33. Ara h 1 peptide (ALMLPHFN)-specific T cells were generated in a previous study according to the same protocol 34. Antigen-specific responses of all T cell lines were shown to be HLA-DR restricted as described 33. T cell proliferation assays Basophils were isolated and incubated at 37 C with IFN-γ (100 ng/ml), GM-CSF (100 ng/ml) and IL-3 (10 ng/ml) in 10% FCS RPMI for 72 hours to induce MHC Class II expression. For the HDMspecific T cell assays, HDM extract (10 µg/ml) or medium alone was also added to the basophil stimulation culture. Basophils were then washed and plated into a 96-well plate at 2x10 5 cells/ml in complete medium (crpmi) consisting of 2 mm L-glutamine, 100 IU/mL penicillinstreptomycin and 5% human AB serum in RPMI-1640 (Sigma-Aldrich). Previously generated autologous antigen-specific T cells were CFSE stained (0.1 μm) and added to each well at 2x10 5 cells/ml crpmi. Cells were incubated at 37 C with antigen (HDM extract or Ara h 1 peptide Results Highly purified basophils require IL-3 for survival in culture Freshly isolated basophils had high levels of surface bound IgE and expressed CD123 and FcεRI together with intermediate levels of the basophil-specific marker CD203c. They were CD4 and CD19 negative and, when included, lineage marker Lin-1 negative (Figure 1A, B, C). Purity levels of over 95% were consistently achieved as determined by expression of CD123 and high levels of surface bound IgE (Figure 1A). An additional pdc population expressing high levels of CD123, FcεRI, intermediate levels of CD4, but not CD203c was identified within freshly isolated PBMC. This population was excluded during the basophil isolation process. The viability of isolated basophils was assessed in two separate experiments with the early apoptotic marker Annexin V and cell death marker 7AAD after 72 hours of culture with a panel of cytokines and TLR ligands. Less than half of the isolated basophil population were viable after culture for 72 hours in medium alone. Of the panel of stimuli tested, IL-3 was found to be most efficient at maintaining basophil viability for 72 hours of culture (Figure 1D, E, F). Therefore, 10 ng/ml of IL-3 was added to all cultures containing isolated basophils. Using IL-3 supplementation, viabilities of over 95% were obtained for isolated basophil preparations after 72 hours of culture throughout this study, as assessed by 7AAD. Basophils can be induced to express MHC Class II molecules We tested the induction of MHC Class II expression on circulating human basophils from 3 atopic HDM-allergic and 3 non-atopic donors with a panel of cytokines and TLR ligands selected on the basis of on known basophil receptors and previous eosinophil studies 26-30, 35. We found that freshly isolated basophils did not express MHC Class II (Figure 2A, E), however a small but significant (p<0.05) increase in expression was observed when cells were cultured with IL-3 for 72 hours (Figure 2E). The addition of individual cytokines (IFN-γ, GM-CSF or TNF-α at 10 ng/ml), TLR ligands (PGN, LPS and CpG) or HDM allergen did not significantly increase this expression, but the addition of IFN-γ and GM-CSF (both at 100 ng/ml) combined, induced a higher percentage of MHC Class II positive basophils as compared to IL-3 alone (p<0.01) 54 55

29 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS (Figure 2E). Two additional donors (1 atopic HDM-allergic and 1 non-atopic) were assessed for basophil MHC Class II expression upon stimulation with IL-3 (10 ng/ml), IFN-γ and GM-CSF (100 ng/ml) resulting in levels of 12.8 and 14.3% respectively, and in subsequent experiments the percentage of MHC Class II positive basophils in such culture conditions reached up to 17% (Figure 3D). The addition of HDM extract (10 µg/ml) to these cultures did not affect the percentage of MHC class II positive basophils (Figure 5i). The atopy status of subjects had no impact on basophil MHC Class II expression (Figure 2E). compared to that for MHC Class II expressing cells. The addition of HDM extract (10 µg/ml) to these cultures did not increase costimulatory molecule expression (data not shown). 3 Figure 1: Isolated basophil purity and viability. Representative dot plots showing proportion of basophils in PBMC and after isolation assessed by antibodies specific for (A) IgE and CD123, (B) FcεRI and CD203c, (C) FcεRI and Lineage-1. Example of basophil viability after culture for 72 hours with (D) IL-3 (10 ng/ml) or (E) medium alone. (F) Percentage of viable basophils (7AAD, Annexin V ) after 72 hours of culture with a panel of cytokines and TLR ligands. Representative of 2 separate experiments. Figure 2: MHC Class II expression on stimulated basophils. Representative dot plots showing MHC Class II expression on basophils freshly isolated (A), and after 72 hours culture with medium alone (B) or IL-3 (10 ng/ml), IFN-γ and GM-CSF (100 ng/ml) (C). Corresponding isotype control (D). Percentage of MHC Class II positive basophils for individual atopic HDM-allergic (open shapes) and non-atopic (closed shapes) donors after 72 hours culture with various stimuli (E). Differences between groups were calculated with One-way ANOVA and Dunett s Multiple comparison post-test with IL-3 as the control group for complete data sets of the 6 donors, * p < 0.05, ** p < Costimulatory molecule expression on basophils remained minimal upon stimulation Up-regulation of costimulatory molecules CD40, CD80 and CD86 was assessed by flow cytometry of basophils stimulated with a combination of IFN-γ, GM-CSF (100 ng/ml) and IL-3 (10 ng/ml) for 3 donors (2 atopic HDM-allergic, 1 non-atopic) (Figure 3). Freshly isolated basophils did not express these costimulatory molecules. A small percentage of stimulated basophils (< 5%) expressed CD80 and CD86 for both atopic donors however, this percentage was minimal 56 57

30 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS HLA-DR, CD74 and Cathepsin S transcripts were also detected (at decreased intensity levels) in stimulated basophils that were MHC Class II negative. Freshly isolated basophils did not contain any detectable MHC Class II related transcripts. Transcripts for CD40, CD80 or CD86 could not be detected in freshly isolated or stimulated basophils. Freshly isolated B cells and pdc expressed all transcripts of the MHC Class II related proteins as well as CD40 and CD86. CD80 transcripts were detected in CpG stimulated B cells. 3 Figure 3: Costimulatory molecule expression on stimulated basophils. CD40 (A), CD80 (B), CD86 (C) and MHC Class II (D) expression on IL-3, IFN-γ and GM-CSF stimulated basophils of 2 atopic HDM-allergic (i,ii) and 1 non-atopic (iii) donor. CpG-stimulated B cells served as a positive control for costimulatory molecule detection (iv). Gates were determined using corresponding isotype controls. Stimulated basophils contain RNA transcripts of MHC Class II components required for assembly and surface expression Stimulated basophils contain RNA transcripts of MHC Class II components required for assembly and surface expression In order to confirm the flow cytometric detection of MHC Class II expression on isolated basophils after culture with IL-3, IFN-γ and GM-CSF and HDM extract, analysis of corresponding gene transcripts in sorted MHC Class II positive and negative basophils was performed with RT-PCR (representative data shown in Figure 4). The proteins HLA-DR, CD74, HLA-DM and Cathepsin S each play a role in achieving functional peptide-mhc Class II complex assembly on the cell surface. Expression levels of these genes were assessed with B2M used as the control housekeeping gene. HLA-DR, CD74 and Cathepsin S transcripts were detected at varying levels in MHC Class II positive basophils of all donors tested (3 atopic HDM-allergic, 2 non-atopic). HLA-DM transcripts were detected in MHC Class II positive basophils from 3 of the 5 donors. Figure 4: Gene expression of MHC Class II components and costimulatory molecules. RT-PCR of RNA from freshly isolated basophils (1), MHC Class II negative (2) and MHC Class II positive basophils (3) after 72 hours of culture with IL-3, IFN-γ, GM-CSF and HDM extract, compared with CD123 + IgE low pdc (4), CD19 + B cells from freshly isolated PBMC (5) and CD19 + B cells from CpG stimulated PBMC (6) as positive controls, and a RT negative control (7). Data for a non-atopic donor are shown, representative of 5 experiments. Basophils do not induce antigen-specific T cell proliferation We next assessed the ability of stimulated human basophils to present antigen to established antigen-specific T cell lines and induce T cell proliferation. Isolated basophils were stimulated for 72 hours with IL-3, IFN-γ and GM-CSF (to induce MHC Class II expression) together with 10 µg/ml HDM or medium alone. Autologous CFSE-stained HDM-specific T cells were then added (1:1 ratio) and the cells cultured in the presence of 10 µg/ml HDM or medium alone for an additional 72 hours. CD3 + CD4 + 7AAD T cells were assessed by flow cytometry for activation and proliferation by up-regulation of CD25 expression and loss of CFSE fluorescence respectively. HDM-specific T cell lines from 1 non-atopic donor and 2 atopic HDM-allergic donors were tested (Figure 5A). No antigen-specific T cell proliferation was induced by stimulated basophils for any donor, indicating that although up to 17% of these cells expressed MHC Class II, they were incapable of antigen processing and/or presentation. As a positive control, flow cytometry-sorted monocytes were used to compare antigen-presenting abilities of basophils with professional antigen presenting cells. This revealed that even at a 1:20 monocyte to T 58 59

31 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS cell ratio, these highly efficient antigen-presenting cells were capable of inducing substantial antigen-specific T cell activation and proliferation, whereas basophils at a 1:1 ratio with T cells were incapable of these tasks. Basophils do not present exogenous peptide to peptide-specific T cells To investigate the possibility that the lack of T cell stimulatory activity was due to an inability to ingest or process antigen, we tested basophils for presentation of an exogenous peptide to a T cell line specific for that peptide. Basophils were stimulated with IL-3, IFN-γ and GM-CSF for 72 hours to induce MHC Class II expression, then washed and added to autologous CFSE-stained Ara h 1 peptide-specific T cells with 10 µg/ml peptide or medium alone (Figure 5B). Again, in contrast to monocytes, MHC Class II expressing basophils were incapable of inducing peptidespecific T cell activation/proliferation through peptide presentation, even after 5 days of culture. Furthermore, an increase in basophil to T cell ratio, from 1:1 to 2:1, did not improve their ability to induce proliferation. 3 Discussion The potential for basophils to present antigen to T cells has been of great interest in recent years. As basophils are capable of producing high levels of Th2 type cytokines and can be directly activated by cysteine proteases derived from both parasites and allergens 36, it was hypothesized that these cells could provide the answer to the initiation and development of a Th2 type T cell response. However, recent reports examining circulating human basophils have found these cells to be incapable of antigen presentation, showing lack of antigen uptake and insufficient MHC Class II and costimulatory molecule expression. We sought to induce these qualities in isolated human basophils through stimulation with a panel of cytokines, TLR ligands and HDM allergen for 72 hours. In this report we demonstrate for the first time that expression of essential components of MHC Class II can be induced in isolated human basophils with an appropriate cytokine mix, but expression of conventional costimulatory molecules including CD40, CD80 and CD86 is minimal. Despite the presence of surface MHC Class II, basophils were incapable of processing and presenting whole HDM allergen to HDM-specific T cells. Furthermore, basophils were unable to present exogenously added Ara h 1 peptide to induce proliferation of peptidespecific T cells. Although some have previously reported a small percentage of basophils within freshly isolated PBMC to be MHC Class II positive 37, 38, our findings agree with others that show highly purified basophils enriched from freshly isolated PBMC are MHC Class II negative. Discrepancies between studies regarding the presence of MHC Class II molecules on unstimulated basophils may be due to the basophil isolation process or the detection technique used within whole PBMC. Our focus was on the ability to induce expression on mature human Figure 5: T cell proliferation assay. (i) Percentage of MHC Class II positive basophils after 72 hours culture with IL-3, IFN-γ and GM-CSF prior to co-culture with T cells. (ii) Percentage of CD25 + CFSE low proliferating CD3 + CD4 + T cells after culture of T cells alone or with autologous basophils or monocytes in the presence of 10 μg/ml HDM (A, non-atopic donor; similar results for 2 HDM-allergic donors) for 72 hours or 10 μg/ml peptide (B) for 120 hours. T cell: APC ratio indicated below. (C) Representative dot plot showing gating strategy for identification of proliferating CD3 + CD4 + T cells. basophils, and we found up to 17% of basophils expressed MHC Class II on the cell surface following culture for 72 hours with IL-3, IFN-γ and GM-CSF. To our knowledge, this is the highest percentage of isolated mature human basophils induced to express MHC Class II reported to date. Furthermore, we are the first to show that the presence of surface bound MHC Class II was accompanied by expression of RNA transcripts of components required for assembly and surface expression of the complex, including HLA-DR, CD74, Cathepsin S and HLA-DM. Interestingly, MHC Class II-related transcripts were also found in surface MHC Class II negative basophils from the stimulated cultures indicating the potential for an even larger percentage of basophils to express the complex under appropriate conditions or upon prolonged stimulation. In order to obtain a pure basophil population, we used a basophil enrichment kit based on negative selection that does not contain an HLA-DR-specific antibody. Furthermore, we applied strict exclusion of any doublets, FcεRI and CD203c negative cells, and CD19 and CD4 positive cells during FACS sorting for gene expression analysis. Since pdc of healthy individuals as well as basophils express CD123, we sorted CD123 + IgE low pdc and confirmed that, like the purified basophils, they expressed CD123 gene transcripts, but they were clearly distinguished 60 61

32 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS from basophils by their high gene expression levels of all MHC Class II components as well as CD86 (Figure 4). Although measurable up-regulation of MHC Class II was detected in basophils after stimulation, similar studies involving eosinophils showed a much larger increase in the percentage of MHC Class II positive cells often reaching values of between 50 and 90% 26, 29. In addition, expression of the costimulatory molecule CD86 has been detected on stimulated eosinophils 27, 28. Expression of conventional costimulatory molecules remained minimal in basophils of our donors upon stimulation as detected by both flow cytometry and RT-PCR. Our T cell proliferation assays revealed that, despite the presence of MHC Class II molecules on the surface of stimulated basophils, these cells were incapable of processing and/or presenting T cell epitopes from whole antigen (HDM) to established T cell lines. In order to investigate this further, and remove the requirement for antigen processing, we also assessed the ability of stimulated basophils to present exogenous peptide to established T cell lines. Again, basophils were incapable of inducing peptide-specific T cell activation/proliferation, indicating additional functional deficiencies in antigen presentation distinct from an inability to process whole antigen. The inability of basophils to induce peptide-specific T cell activation/proliferation may be due to insufficient costimulatory molecule expression, conventional or otherwise. Costimulatory molecules including ICAM-1 and LFA-3 39 have been shown to assist in antigen presentation by eosinophils lacking conventional costimulatory molecules 31. There are two reports supporting LFA-3 expression by human basophils 40, 41, but the results from our peptide assay suggest that this costimulatory molecule expression (if present) together with MHC Class II is not sufficient to induce T cell activation/proliferation to peptide. Although only 11-17% of basophils were found to express MHC Class II on their cell surface prior to T cell co-culture, by testing both a 1:1 and 1:2 T cell to basophil ratio in our assays, the actual ratio of T cells to MHC Class II positive basophils was within the range found to result in strong T cell response when stimulated by monocytes. In fact, monocytes could still induce high levels of T cell proliferation at much higher T cell to monocyte ratios than were employed for the basophils. In our T cell assays, we chose to include the entire basophil population to minimize manipulation of the cells. The flow cytometry-based method of assessing antigen presentation allows for highly sensitive detection of both early T cell activation (CD25 expression) and proliferation (CFSE loss), therefore even very low levels of T cell activation would have been detected. In conclusion, we demonstrate that mature human basophils can be induced to express MHC Class II, however this expression is insufficient to induce antigen-specific T-cell proliferation with either whole antigen or exogenous peptide. Acknowledgements The authors thank Geza Paukovic, Michael Thomson and Jeanne Lemasurier for flow cytometry assistance, research nurses Karen Symons, Kirsten Deckert and Anita Hazard for patient sample collection and Professor Maria Yazdanbakhsh for helpful intellectual discussions

33 CHAPTER 3 MHC CLASS II EXPRESSION IN HUMAN BASOPHILS References 1. Ohnmacht C, Voehringer D. Basophil effector function and homeostasis during helminth infection. Blood. 2009;113: Valent P, Bettelheim P. The human basophil. Crit Rev Oncol Hematol. 1990;10: Valent P, Bettelheim P. Cell surface structures on human basophils and mast cells: biochemical and functional characterization. Adv Immunol. 1992;52: MacGlashan D, Jr., White JM, et al. Secretion of IL-4 from human basophils. The relationship between IL-4 mrna and protein in resting and stimulated basophils. J Immunol. 1994;152: Schroeder JT, MacGlashan DW, Jr., et al. IgE-dependent IL-4 secretion by human basophils. The relationship between cytokine production and histamine release in mixed leukocyte cultures. J Immunol. 1994;153: Li H, Sim TC, et al. IL-13 released by and localized in human basophils. J Immunol. 1996;156: Ochensberger B, Daepp GC, et al. Human blood basophils produce interleukin-13 in response to IgE-receptor-dependent and -independent activation. Blood. 1996;88: Gauchat JF, Henchoz S, et al. Induction of human IgE synthesis in B cells by mast cells and basophils. Nature. 1993;365: Yanagihara Y, Kajiwara K, et al. Cultured basophils but not cultured mast cells induce human IgE synthesis in B cells after immunologic stimulation. Clin Exp Immunol. 1998;111: Perrigoue JG, Saenz SA, et al. MHC class II-dependent basophil-cd4+ T cell interactions promote T(H)2 cytokinedependent immunity. Nat Immunol. 2009;10: Sokol CL, Chu NQ, et al. Basophils function as antigen-presenting cells for an allergeninduced T helper type 2 response. Nat Immunol. 2009;10: Yoshimoto T, Yasuda K, et al. Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide- MHC class II complexes to CD4+ T cells. Nat Immunol. 2009;10: Hammad H, Plantinga M, et al. Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen. J Exp Med. 2010;207: Kim S, Prout M, et al. Cutting edge: basophils are transiently recruited into the draining lymph nodes during helminth infection via IL-3, but infection-induced Th2 immunity can develop without basophil lymph node recruitment or IL-3. J Immunol. 2010;184: Ohnmacht C, Schwartz C, et al. Basophils orchestrate chronic allergic dermatitis and protective immunity against helminths. Immunity. 2010;33: Otsuka A, Nakajima S, et al. Basophils are required for the induction of Th2 immunity to haptens and peptide antigens. Nat Commun. 2013;4: Eckl-Dorna J, Ellinger A, et al. Basophils are not the key antigen-presenting cells in allergic patients. Allergy. 2012;67: Kitzmuller C, Nagl B, et al. Human blood basophils do not act as antigen-presenting cells for the major birch pollen allergen Bet v 1. Allergy. 2012;67: Sharma M, Hegde P, et al. Circulating human basophils lack the features of professional antigen presenting cells. Sci Rep. 2013;3: Charles N, Hardwick D, et al. Basophils and the T helper 2 environment can promote the development of lupus nephritis. Nat Med. 2010;16: Guo CB, Liu MC, et al. Identification of IgEbearing cells in the late-phase response to antigen in the lung as basophils. Am J Respir Cell Mol Biol. 1994;10: Ito Y, Satoh T, et al. Basophil recruitment and activation in inflammatory skin diseases. Allergy. 2011;66: Wakahara K, Baba N, et al. Human basophils interact with memory T cells to augment Th17 responses. Blood. 2012;120: Arock M, Schneider E, et al. Differentiation of human basophils: an overview of recent advances and pending questions. J Leukoc Biol. 2002;71: Grundstrom J, Reimer JM, et al. Human cord blood derived immature basophils show dual characteristics, expressing both basophil and eosinophil associated proteins. PLoS One. 2012;7:e Lucey DR, Nicholson-Weller A, et al. Mature human eosinophils have the capacity to express HLA-DR. Proc Natl Acad Sci U S A. 1989;86: Jung YJ, Woo SY, et al. Human eosinophils show chemotaxis to lymphoid chemokines and exhibit antigen-presenting-cell-like properties upon stimulation with IFN-gamma, IL-3 and GM-CSF. Int Arch Allergy Immunol. 2008;146: Celestin J, Rotschke O, et al. IL-3 induces B7.2 (CD86) expression and costimulatory activity in human eosinophils. J Immunol. 2001;167: Weller PF, Rand TH, et al. Accessory cell function of human eosinophils. HLA- DR-dependent, MHC-restricted antigenpresentation and IL-1 alpha expression. J Immunol. 1993;150: Mawhorter SD, Kazura JW, et al. Human eosinophils as antigen-presenting cells: relative efficiency for superantigen- and antigen-induced CD4+ T-cell proliferation. Immunology. 1994;81: Hansel TT, De Vries IJ, et al. Induction and function of eosinophil intercellular adhesion molecule-1 and HLA-DR. J Immunol. 1992;149: Eusebius NP, Papalia L, et al. Oligoclonal analysis of the atopic T cell response to the group 1 allergen of Cynodon dactylon (bermuda grass) pollen: pre- and postallergen-specific immunotherapy. Int Arch Allergy Immunol. 2002;127: Prickett SR, Voskamp AL, et al. Ara h 2 peptides containing dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic. J Allergy Clin Immunol. 2011;127: e Prickett SR, Voskamp AL, et al. Ara h 1 CD4+ T-cell epitope-based peptides: candidates for a peanut allergy therapeutic. Clin Exp Allergy. 2013;43: Komiya A, Nagase H, et al. Expression and function of toll-like receptors in human basophils. Int Arch Allergy Immunol. 2006;140 Suppl 1: Phillips C, Coward WR, et al. Basophils express a type 2 cytokine profile on exposure to proteases from helminths and house dust mites. J Leukoc Biol. 2003;73: Poulsen BC, Poulsen LK, et al. Detection of MHC class II expression on human basophils is dependent on antibody specificity but independent of atopic disposition. J Immunol Methods. 2012;381: Siegmund R, Vogelsang H, et al. Surface membrane antigen alteration on blood basophils in patients with Hymenoptera venom allergy under immunotherapy. J Allergy Clin Immunol. 2000;106: Damle NK, Klussman K, et al. Differential costimulatory effects of adhesion molecules B7, ICAM-1, LFA-3, and VCAM-1 on resting and antigen-primed CD4+ T lymphocytes. J Immunol. 1992;148: Fureder W, Agis H, et al. The surface membrane antigen phenotype of human blood basophils. Allergy. 1994;49: Munitz A, Bachelet I, et al. 2B4 (CD244) is expressed and functional on human eosinophils. J Immunol. 2005;174:

34 4 Ara h 2 peptides comprising dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic Prickett SR a,b, Voskamp AL a,b, Dacumos-Hill A a,b, Symons K a, Rolland JM a,b, and O Hehir RE a,b a Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Australia b Department of Immunology, Monash University, Melbourne, Australia Journal of Allergy Clinical Immunology 2011;127: e1-5

35 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Abstract Introduction Background: Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy. Objective: This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4 + T cells but do not bind IgE, as candidates for safe peanut-specific immunotherapy. Methods: Multiple CD4 + T-cell lines specific for the major peanut allergen Ara h 2 were generated from peripheral blood mononuclear cells of 16 HLA-diverse subjects with peanut allergy using CFSE-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA-restriction of core epitope recognition was investigated using anti-hla blocking antibodies and HLA-genotyping. Serum-IgE peptide-binding was assessed by dot-blot. Results: Five dominant CD4 + T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, -DP and -DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants encompassing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE and could directly target T H 2-type T-cells in peripheral blood of allergic subjects. Conclusions: Short CD4 + T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell targeted peanut-specific immunotherapy for an HLA-diverse population. Peanut allergy is a major health care problem affecting 1 to 2% of the population 1-3, with clinical symptoms ranging from mild oropharyngeal irritation and vomiting to life-threatening systemic anaphylaxis. It is the leading cause of food-induced anaphylactic fatalities worldwide 2, 4. Unlike egg and milk food allergy that presents in infancy and typically resolves by school age, peanut allergy persists through adulthood in 80% of cases, significantly impairing the quality of life of afflicted individuals and their families 4-7. There is currently no cure for peanut allergy, leaving avoidance as the only preventative option and adrenaline for anaphylactic crises. Even with diligent precautions, most subjects with allergy have accidental exposures that can result in severe and sometimes fatal reactions 4, 8. Research trials of conventional allergen desensitization, or more recently oral immunotherapy using whole peanut extract, provide encouragement on feasibility, but the potential for adverse reactions highlights major safety concerns This risk is especially pertinent with peanut allergens since they may induce anaphylaxis at minute doses, even in individuals who previously experienced only mild symptoms 13, 16. Consequently, there is an urgent need to develop a safe, disease-modifying therapeutic for individuals with peanut allergy. Mechanistic studies on clinically effective immunotherapy with other allergens demonstrate immune deviation from a predominantly T H 2 to T H 1 immune response, and induction of regulatory T cells (Treg) that down-regulate T H 1 and T H 2 responses. Specific immunotherapy is also accompanied by decreased specific IgE, increased protective specific IgG 4 antibody levels, and reduced number and activation of mast cells, eosinophils and basophils Stimulation of appropriate T-cell responses during allergen-specific immunotherapy is necessary for effective desensitization 22. Conventional immunotherapy administers whole allergen extracts, but peptides containing T-cell epitopes of major allergens provide an effective, safer (non- IgE reactive) alternative 23, 24. In proof-of-concept studies, T-cell epitope-based peptides from 25, 26 the major allergens phospholipase A 2 and Fel d 1 have been used to treat bee venom and cat allergy respectively, without causing IgE-mediated reactions. Importantly, there is evidence for linked suppression using peptide immunotherapy. In in vitro and murine studies, the dominant T-cell epitope peptide of the house dust mite major allergen Der p 1 induced tolerance not only to this peptide but to whole Der p 1 and house dust mite extract 32, 33. Clinical administration of selected Fel d 1 peptides altered T-cell responses to those peptides, other nonrelated Fel d 1 peptides, and whole cat allergen extract 29. We aim to develop a T-cell targeted peptide immunotherapy as a safe treatment option for individuals with peanut allergy. However, data on CD4 + T-cell epitopes of peanut allergens are limited. Only a few reports document CD4 + T-cell responses to individual peptides spanning whole peanut allergen(s). These include analyses of CD4 + T-cell proliferation to unsequenced digestion products of the major peanut allergens Ara h 1 and Ara h 2 34, 35, and two reports, including our own pilot study, of T-cell reactivity to overlapping 15- or 20-mer peptides spanning Ara h 2 36, 37. Whilst these latter studies implicated broad regions of CD4 + T-cell reactivity within

36 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Ara h 2, core epitopes were not determined, population sizes were small and analyses were limited. Other studies identifying peanut allergen peptides with therapeutic value focused on identifying short peptides that bound IgE from individuals with peanut allergy, and subsequently developing non-ige-binding variants that retained T-cell reactivity 36, Given the pivotal role of CD4 + T cells in driving the immune response to allergen, we took the more direct strategy of identifying the full repertoire of dominant CD4 + T-cell epitopes of a major peanut allergen recognized by proliferative T cells from an HLA-diverse peanut-allergic cohort and subsequently determining their therapeutic potential. Here we present definitive data identifying and characterizing 5 dominant CD4 + T-cell epitopes of Ara h 2, the most widely recognized and potent allergen for individuals with peanut allergy We design peptides containing the identified epitopes and demonstrate key properties that make them prime candidates for a specific immunotherapy for subjects with peanut allergy. Methods Subjects Sixteen Caucasian adult subjects with peanut allergy and 3 healthy, nonatopic control (negative skin-prick testing to common allergens) Caucasian adult subjects were recruited from The Alfred Allergy Clinic, Melbourne, Australia (Table S1). Subjects with peanut allergy had clinical symptoms of IgE-mediated peanut allergy and peanut-specific IgE CAP score 2 ( 1.16 ku A /l; Pharmacia CAP System TM, Pharmacia Diagnostics, Uppsala, Sweden) and were genotyped (HLA- DRB1, -DQB1 and -DPB1, exon 2) by the Victorian Transplantation and Immunogenetics Service. The study was approved by The Alfred and Monash University Ethics Committees and informed written consent obtained from each subject. Antigens Crude peanut extract (CPE) was prepared from commercial unsalted, dry-roasted peanuts as described 46, dialyzed against phosphate-buffered saline (PBS) and filter-sterilized (0.2 μm). Natural Ara h 2 (nara h 2) was enriched from CPE based on published methodology 47. Briefly, CPE was buffer exchanged into 20 mmol/l TRIS-bis-propane (TBP), ph 7.2, using Vivaspin columns (Sartorius Stedim Biotech S.A., Aubagne, France) and applied onto a 5 ml Mono-Q 10/10 column (Pharmacia FPLC System, St Albans, UK) equilibrated with TBP. After washing with TBP, a linear gradient of 30 ml 0 to 1 mol/l NaCl/TBP was applied to elute bound proteins (1 ml/min). Fractions, 0.5 ml, were analyzed by SDS-PAGE and those containing nara h 2 with minimal other proteins pooled and dialyzed against PBS. Endotoxin contents were 1.7 and 78 EU/mg for CPE and nara h 2 respectively (Endpoint Chromogenic LAL assay, Lonza, Walkersville, USA). Ara h 2 peptides (Mimotopes, Clayton, Victoria, Australia; Table S2) were reconstituted at 2 mg/ml 10% dimethyl sulfoxide/pbs. All antigens were confirmed to be neither mitogenic nor toxic. Generation of Ara h 2-specific CD4 + T-cell lines (TCL) Ara h 2-specific oligoclonal TCL were generated from peripheral blood mononuclear cells (PBMC) of subjects with peanut allergy using 5,6-carboxyfluorescein diacetate succinimidylester (CFSE)- based methodology 48. Briefly, culturing was performed in RPMI-1640 containing 2 mmol/l L-glutamine, 100 IU/mL penicillin-streptomycin and 5% human AB serum (crpmi; Sigma-Aldrich, St Louis, USA) (crpmi). PBMC were labeled with 0.1 mmol/l CFSE (Molecular Probes, Eugene, USA) and cultured ( /ml) with crpmi alone, CPE (100 µg/ml), nara h 2 (10 µg/ml), Ara h 2 20-mer-peptide pools (Table S3; 10 μg/ml/peptide) or as a control, tetanus toxoid (TT; 10 LfU/mL; Statens Serum Institute, Copenhagen, Denmark) for 7 days at 37 C. After staining with CD4-PE and 7AAD (BD Pharmingen, San Diego, USA), CD4 + CFSE dim 7AAD - cells were sorted (10 cells/well) into 96-U-well plates containing irradiated allogeneic feeder-cells, anti-cd3 (OKT-3), ril-2 (Cetus, Emeryville, USA) and Fungizone (Invitrogen, Carlsbad, USA) as described 48. Cells were fed with ril-2 as required and after 10 to 14 days, transferred to 48-well plates and tested for proliferation to nara h 2 (10 μg/ml). Ara h 2-positive TCL were expanded with anti-cd3 and ril-2 48 in T25 culture flasks (BD, Franklin Lakes, USA) for 10 to 12 days then tested for specificity (proliferation) to overlapping 20-mer peptides spanning Ara h 2 (10 μg/ml). Proliferation assays Proliferation assays were performed on 72-hour duplicate or triplicate cultures in 96-U-well plates containing T cells/well, irradiated (5000 rad) autologous EBV-transformed PBMCs (EBV-B cells) as antigen-presenting cells and antigens as specified. Negative control was crpmi alone. Cells were pulsed with 3 H-thymidine (0.5 μci/well) for the last 16 hours and uptake recorded as mean counts per minute (cpm) of replicate cultures. A stimulation index (SI; cpm antigen-stimulated T cells/cpm unstimulated T cells) 2.5 was considered positive and all positive responses confirmed in 2 assays. HLA class II blocking assays T cells and irradiated EBV-B cells ( of each) were incubated with 0.1 to 10 μg/ml blocking monoclonal antibody (mab) against HLA-DR (L243, BD Pharmingen), HLA-DQ (SVP-L3) or HLA- DP (B7/21) (gifts from S. Mannering, St Vincent s Research Institute, Melbourne, Australia) or isotype-control antibodies (IgG 2a : BD Pharmingen; IgG 1 : BioLegend, San Diego, USA) for 1 hour at 37 C before addition of peptides (2-10 μg/ml) or CPE (100 μg/ml) and testing proliferative response as discussed. Cytokine ELISPOT assays MAIP ELISPOT plates (Millipore, Billerica, USA) were coated overnight at 4 C with 10 μg/ml IL- 4, IFN-γ or IL-5 antibodies (ebioscience, San Diego, USA) in PBS. Wells were blocked (crpmi, 1 hour, 37 C) then PBMC ( ) or T cells and irradiated EBV-B cells ( of each) added in duplicate 100 μl cultures with CPE (100 μg/ml), nara h 2 (10 μg/ml) or peptides (10 μg/ml)

37 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Controls were crpmi alone, TT (10 lfu/ml) and phytohaemagglutinin (1 μg/ml; Sigma-Aldrich). After 48 hours culture at 37 C, plates were incubated with biotinylated IL-4, IL-5 or IFN-γ antibodies (ebioscience; 1 μg/ml PBS, 2 hours) followed by ExtrAvidin -alkaline phosphatase (Sigma-Aldrich) (1/3,000 PBS, 2 hours) before developing with alkaline phosphatase substrate (Bio-Rad). When spots appeared in positive-control wells, plates were washed, air dried and read (AID ELISPOT 4.0 h reader, Autoimmun Diagnostika, Strassberg, Germany). T-cell epitopes. Dot-blot IgE-binding assay Nitrocellulose membrane (0.2μm pore size) was dotted with 10 μg CPE or 20 μg peptide, blocked, incubated with individual subject sera (diluted 1:10) and washed as described 49. Membranes were then incubated with rabbit polyclonal anti-human IgE antibody (1:500; DAKO, Glostrup, Denmark) followed by horseradish peroxidase-labelled goat anti-rabbit IgG (1:1000; Promega, Madison, USA) for 1 hour each. IgE binding was detected using substrate 4-chloro-1-naphthol (Sigma-Aldrich) and hydrogen peroxide Results Selection of Ara h 2 20-mer peptides containing dominant CD4 + T-cell epitopes recognized by subjects with peanut-allergy A total of 69 Ara h 2-specific TCL were generated from PBMC of 16 subjects with peanut allergy by isolating and expanding antigen-specific (proliferated) CD4 + CFSE dim T cells from 7-day CFSE-labeled PBMC cultures stimulated with CPE, nara h 2 or, for six subjects, pools of Ara h 2 20-mer peptides. The 20-mer peptides recognized (SI 2.5) by each TCL are shown in Table S3 and data summarized in Figure 1. As reported for other allergens, 50 patterns of T-cell peptide recognition were more restricted for some subjects (e.g. 8 and 9) than others (e.g. 1 and 10). Nonetheless, certain 20-mers were consistently recognized by multiple subjects. Ara h 2(91-110), ( ), (28-47) and (37-56) had the most responders, being recognized by 9 (56%), 8 (50%), 7 (44%) and 7 (44%) of the 16 subjects with peanut allergy respectively (Figure 1A). At least 1 TCL from each of the 16 subjects (in total, 80% of all TCL) recognized one or more of these four 20-mers, with reactive TCL typically showing strong proliferative responses (SI>8). Furthermore, each of these 20-mers was recognized by multiple TCL from many responders reflecting a prevalence of T cells specific for these peptides among the subjects T-cell repertoires. Indeed, 13 of 14 (93%) TCL generated from PBMC stimulated with Ara h 2 peptide pools were specific for one or more of these 20-mers, further demonstrating that these 20-mers could directly target Ara h 2-specific T cells among PBMC of individuals with peanut allergy and preferentially over other Ara h 2 20-mers. Because Ara h 2(28-47), (37-56), (91-110) and ( ) collectively showed the highest donor and TCL responder frequencies, strongest T-cell responses and preferential ability to target PBMC T cells, these 4 peptides were identified as containing dominant Ara h 2 Figure 1: Responder frequency profiles for Ara h 2 20-mer peptides. Aggregate donor responder frequencies (A) and total numbers of specific TCL (B) are shown for each Ara h 2 20-mer peptide for all 16 subjects with peanut allergy. Mapping core T-cell epitopes of dominant Ara h 2 20-mer peptides Keeping immunotherapeutic peptides as short as possible minimizes risk of cross-linking cellbound IgE on inflammatory cells and facilitates therapeutic production. The minimum T cell stimulatory sequence (core epitope) within each dominant 20-mer was determined by testing proliferation of reactive TCL to peptide sets truncated from the N- or C-terminus of the 20- mer (Table S2). Truncations of Ara h 2(37-56) were not required because most TCL which recognized this peptide also responded with comparable magnitude to Ara h 2(28-47) (Table S3) and mapping using Ara h 2(28-47) truncations revealed that the core epitope recognized by these lines lay within the overlap between these 2 peptides. In total, 5 core CD4 + T-cell epitopes were identified (Figure 2). Ara h 2(28-47) and (91-110) each contained 2 distinct but overlapping T-cell epitopes: (32-44) and (37-47) (Figure 2A, upper and lower panels, respectively), and (91-102) and (95-107) (Figure 2B, upper and lower panels, respectively). No single TCL responded to both epitopes of an overlapping pair

38 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES For Ara h 2(32-44)-specific TCL, the lack of response to epitope (37-47) is demonstrated in Figure 2A (truncation 37-47, upper panel). Similarly, for Ara h 2(95-107)-specific TCL, the lack of response to truncation (91-102) is evident in Figure 2B (lower panel). Only a single epitope, ( ), was found within Ara h 2( ) (Figure 2C). For each epitope, the number of residues required to induce maximal TCL proliferation varied from 9 to 15 aa between subjects (Figure 2, bolded sequences), but there was a minimum core sequence of 9 to 11 aa required for recognition by every reactive TCL (Figure 2, underlined sequences). In selecting epitope sequences for further analyses, residues required for maximal stimulation of all TCL tested were incorporated to ensure broadest possible recognition. Figure 2: Mapping core T-cell epitopes within dominant Ara h 2 20-mer peptides. Dominant 20-mer-specific TCL proliferation to truncated peptide sets. Representative data show each epitope identified (mean cpm replicate wells +SD). Sequences required for recognition by all tested subjects boldface; conserved cores underlined. A) Ara h 2(28-47); upper panel: n = 3 (5 TCL); lower panel: n = 4 (7 TCL); B) Ara h 2(91-110); upper panel: n = 3 (6 TCL); lower panel n = 3 (5 TCL); C) Ara h 2( ); n = 5 (6 TCL). 3 H-TdR, 3 H-thymidine; Ag, antigen Determining HLA class II restriction specificity of dominant Ara h 2 T-cell epitopes There is no confirmed HLA class II (HLA-II) association with peanut allergy, 51 therefore peptides selected for therapy must bind diverse HLA-II molecules for wide applicability. To determine the HLA-II type presenting each epitope, anti-hla-dr, -DP or -DQ mabs were used to block epitope presentation to T cells. For each TCL tested, epitope recognition was prevented by 1 or more HLA-mAb in a dosedependent manner (Figure 3). Isotype control antibodies (10 μg/ml) had no effect (data not shown). Each HLA-mAb prevented recognition of one or more epitopes, collectively indicating presentation by HLA-DR, -DP and -DQ. In each case, the same mab blocked recognition of CPE (data not shown), demonstrating consistency for presentation of naturally processed and synthetic epitope forms. In all subjects tested, T-cell recognition of epitope (32-44) was consistently blocked by anti-hla-dp (Figure 3A), epitope (95-107) by anti-hla-dq (Figure 3E), and epitopes (91-102) and ( ) by anti-hla-dr (Figure 3D and 3F). In contrast, recognition of epitope (37-47) was blocked by anti-hla-dr in subjects 8 and 13 (Figure 3B) and anti-hla-dq in subjects 9 and 11 (Figure 3C). Each epitope of an overlapping pair was blocked by a different HLA-mAb, further demonstrating their distinction. The ability to block recognition of a given epitope by the same HLA-mAb in multiple subjects indicated preferential presentation by a specific HLA type. To assess potential for degeneracy of epitope-presentation by allelic variants of the respective HLA type, HLA alleles of subjects recognizing a given epitope were compared (Table S1). The absence of a shared HLA-DQB1 allele between all subjects for whom recognition of epitope (95-107) was blocked by anti-hla- DQ indicated that this epitope must be presented by multiple HLA-DQB1 molecules. Similarly, the diversity in HLA-DRB1 alleles between subjects for whom recognition of epitopes ( ) or (37-47) was blocked by anti-hla-dr indicated binding-degeneracy of both epitopes for multiple HLA-DRB1 molecules. Epitope (37-47) was also presented by HLA-DQB1*06:09 as both subjects (9 and 11) who recognized this epitope in the context of HLA-DQ had this allele, and for subject 9 it was the only DQB1 allele present. Because DPB1*04:01 or DRB1*15:01 alleles were present in all subjects recognizing epitopes (32-44) (blocked by anti-hla-dp) or (95-107) (blocked by anti-hla-dr) respectively, degeneracy of these epitopes could not be determined. However, because DPB1*0401 and DRB1*1501 are prevalent in populations worldwide 52, epitopes presented by these HLA-molecules would still be broadly recognized

39 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES 4 Figure 4: T-cell recognition of peptides containing cysteine-to-serine substituted epitopes. TCL proliferation in response to parent or cysteine-substituted Ara h 2 peptides. Graphs show representative TCL for each epitope (mean cpm replicate wells +SD). A) Ara h 2(32-44), n = 2 (3 TCL); B) Ara h 2(37-47) n = 3 (5 TCL); C) Ara h 2(91-102), n = 3 (3 TCL); D) Ara h 2(95-107), n = 2 (2 TCL); E) Ara h 2( ), n = 3 (3 TCL). 3 H-TdR, 3 H-thymidine; Ag, antigen Figure 3: HLA class II restriction specificity of T-cell recognition of dominant epitopes. Proliferation of specific TCL to each epitope in the presence of HLA-DR (circles), -DQ (squares) or -DP (triangles) mabs. Graphs show representative TCL (mean cpm replicate wells +SD). A) Ara h 2(32-44), n = 2 (4 TCL); B) Ara h 2(37-47), n = 2 (4 TCL); C) Ara h 2(37-47), n = 3 (4 TCL); D) Ara h 2(91-102), n = 2 (3 TCL); E) Ara h 2(95-107), n = 3 (4 TCL); F) Ara h 2( ), n = 3 (3 TCL). 3 H-TdR, 3 H-thymidine; Ag, antigen Testing T cell reactivity of cysteine-substituted Ara h 2 T-cell epitope peptides In order to minimize the number of peptides for a therapeutic and avoid unnecessary sequence duplication, each overlapping epitope pair was combined into a single peptide, i.e. Ara h 2(32-47) and Ara h 2(91-107). The resultant peptides were less than 18 aa and efficiently stimulated T cells specific for either epitope (Figure 4). Together with Ara h 2( ), this provided a panel of 3 HLAdegenerate peptides. However, each peptide contained 1 or more cysteine residues, which are prone to oxidation and disulphide bridge formation that can hinder synthesis, stability and bioavailability of therapeutic peptides. To avoid these problems, cysteines were replaced with structurally conserved but less reactive serines (Table S2). The cysteine-substituted peptides stimulated comparable T-cell proliferation (Figure 4) and cytokine production (Figure S1) to the parent peptides. Sequence analysis and synthesis of these modified peptides confirmed water solubility and ease of generation at high purity (Mimotopes). Testing candidate peptides for serum-ige binding To provide a safe alternative to whole allergens, peptides must not bind and cross-link cellbound IgE. Using methodology optimized for Ara h 2 peptides 49, serum from each subject with peanut allergy was tested for IgE-binding to cysteine-substituted (candidate) peptides (Figure 5). Although all subjects with peanut allergy showed clear IgE-reactivity to CPE, no IgE-binding was detected to any candidate peptide (Figure 5A). Serum from a latex-allergic subject with known IgE-reactivity to peptides of major latex allergen Hev b 5 provided a positive control for peptide-ige-binding (Figure 5B)

40 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES 4 Figure 5. IgE-reactivity to T-cell epitope-derived peptides. Dot-blots showing binding of CPE or candidate peptides by IgE from peanut-allergic sera (A) (representative blots for 4 of 16 subjects). Positive control blot for peptide-binding (B) using serum of latex-allergic subject with known IgE-reactivity to Hev b 5 peptides. Validation of candidate peptides for targeting peanut-specific T cells in PBMC of subjects with peanut allergy Successful allergen desensitization mandates a decreased T H 2-response to allergen. To demonstrate that candidate peptides could target pathogenic T H 2-type T cells directly in whole blood from individuals with peanut allergy, ELISPOT cytokine assays were performed on PBMC from a panel of subjects with peanut allergy (donors 6, 12 and 15). Because the hallmark of allergen-specific T cells from allergic subjects is elevated production of T H 2-type cytokines in response to specific allergen compared to subjects without peanut allergy, PBMC from subjects without peanut allergy were analyzed as controls (Figure 6). Figure 6: PBMC cytokine secretion in response to T-cell epitope-derived peptides. PBMC from 3 nonatopic control (A) and 3 subjects with peanut allergy (B) subjects stimulated with CPE, nara h 2, candidate peptides or TT (control). IL-4, IL-5 and IFN-g secretion determined by ELISPOT. Mean spots of replicate wells (+SD) shown for each subject. Ag, antigen. In response to the control antigen TT, IL-4, IL-5 and IFN-g were produced by PBMC from both subject groups (Figure 6). In contrast, only PBMC from donors with peanut allergy secreted cytokines in response to peanut allergens or candidate peptides, with prominent IL-4 and IL-5 production and a detectible but limited IFN-γ response induced by one or more peptides for each subject with peanut allergy tested. Responses to parent and cysteine-substituted candidate peptides were comparable and reflected those of specific TCL (Figure S1). These data further confirmed that TCL reflected the peripheral blood repertoire of pathogenic T cells in individuals with peanut allergy, and that candidate peptides were recognized by, and could target, pathogenic T H 2-type T cells in PBMC of these subjects. Discussion An appropriately selected T-cell-targeted peptide immunotherapeutic will provide a safe treatment option for individuals with peanut allergy. Candidate peptides must (1) comprise dominant CD4 + T-cell epitopes of major peanut allergens recognized by multiple individuals with peanut allergy, (2) be presented on different HLA class II molecules to target T cells in genetically diverse individuals, (3) not bind and cross-link cell-bound IgE, and (4) be scalable and amenable to therapeutic production. This comprehensive study is the first to report core 78 79

41 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES sequences of dominant CD4 + T-cell epitopes of a major peanut allergen. We have identified and characterized five, HLA-diverse CD4 + T-cell epitopes of Ara h 2, the most potent and widely recognized peanut allergen among individuals with peanut allergy. Using these sequences we have designed candidate peptides that meet all the above criteria for therapeutic development. To commence this study we selected four dominant 20-mers of Ara h 2 based on responses of multiple TCL from each of 16 HLA-diverse subjects with peanut allergy. Two of these 20-mers differed to those we reported previously 37. Importantly however, when data from both studies were pooled, this panel of four 20-mers remained the most frequently recognized among TCL from the combined cohort of 24 subjects with peanut allergy, further validating our final 20- mer selection for core-epitope mapping. Although our approach may have excluded analysis of nonproliferative but functional T cells, proof-of-concept studies indicate epitopes recognized by proliferative T H 2 cells are sufficient for effective therapy 53 Despite varied patterns of peptide recognition by TCL between subjects (influenced by many factors including subject genetics and patterns of allergen encounter 50 ), 1 or more of the 5 epitopes identified within these 20-mers was recognized by each of the 16 subjects with peanut allergy in this study. Together with their collective presentation by HLA-DR, -DQ and -DP molecules (commonly observed for allergen T-cell epitopes ) this emphasized their broad applicability, particularly as the HLA-allele profile of our cohort is typical of Caucasian populations 52 in countries with the highest incidence of peanut allergy 59. Each candidate peptide contains at least 1 HLA-degenerate epitope for presentation by multiple HLA-molecules, with Ara h 2(32-47) alone containing epitopes presented on all 3 HLA-types and at least 4 different HLA-molecules. Inclusion of HLA-DQ and -DP-restricted epitopes in a therapeutic is particularly advantageous as these alleles are less variable and thus more prevalent in mixed populations than HLA-DR alleles 60. The main rationale for developing a peptide immunotherapy for peanut allergy is to identify an effective allergen preparation that does not invoke the adverse effects seen with whole peanut extract 10-14, 17, 61. Consistent with the absence of reported linear IgE epitopes 36, 38, 49, 62 within our candidate peptides, we were unable to detect peptide-binding by IgE from peanut-allergic sera, despite detection of CPE-reactive IgE in all samples. This emphasizes the potential for these peptides to provide a safe alternative to whole allergen extract for immunotherapy. In summary we report the novel identification of 5 dominant CD4 + T-cell epitopes of Ara h 2 that collectively show diverse HLA class II-restriction. We incorporated cysteine-free variants of these epitopes into a combinatorial panel of 3 short ( 17 aa), HLA-degenerate peptides that can target pathogenic T cells in HLA-diverse allergic individuals without binding peanutreactive serum IgE. Because immunological suppression to whole allergen can be induced by administration of isolated T-cell epitopes from the allergen sequence, 29 these peptides are prime candidates for a therapeutic for peanut allergy. Acknowledgements We thank Dr Stuart Mannering for advice with CFSE-T-cell cloning protocols and provision of cell lines and reagents, Dr Nicole Mifsud for helpful intellectual discussions and Neeru Varese for technical assistance

42 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES References 1. Mullins RJ, Dear KB, et al. Characteristics of childhood peanut allergy in the Australian Capital Territory, 1995 to J Allergy Clin Immunol. 2009;123: Burks AW. Peanut allergy. Lancet. 2008;371: Hourihane JO, Aiken R, et al. The impact of government advice to pregnant mothers regarding peanut avoidance on the prevalence of peanut allergy in United Kingdom children at school entry. J Allergy Clin Immunol. 2007;119: Bock SA, Munoz-Furlong A, et al. Further fatalities caused by anaphylactic reactions to food, Journal of Allergy & Clinical Immunology. 2007;119: Kemp AS, Hu W. Food allergy and anaphylaxis - dealing with uncertainty. Med J Aust. 2008;188: Yun J, Katelaris CH. Food allergy in adolescents and adults. Intern Med J. 2009;39: O Hehir RE, Douglass JA. Risk-minimisation strategies for peanut allergy. Lancet. 2007;370: Sampson MA, Munoz-Furlong A, et al. Risk-taking and coping strategies of adolescents and young adults with food allergy. J Allergy Clin Immunol. 2006;117: Thyagarajan A, Varshney P, et al. Peanut oral immunotherapy is not ready for clinical use. J Allergy Clin Immunol. 2010;126: Oppenheimer JJ, Nelson HS, et al. Treatment of peanut allergy with rush immunotherapy. [see comment]. Journal of Allergy & Clinical Immunology. 1992;90: Nelson HS, Lahr J, et al. Treatment of anaphylactic sensitivity to peanuts by immunotherapy with injections of aqueous peanut extract. Journal of Allergy & Clinical Immunology. 1997;99: Jones SM, Pons L, et al. Clinical efficacy and immune regulation with peanut oral immunotherapy. Journal of Allergy & Clinical Immunology. 2009;24: Hofmann AM, Scurlock AM, et al. Safety of a peanut oral immunotherapy protocol in children with peanut allergy. Journal of Allergy & Clinical Immunology. 2009;124: Varshney P, Steele PH, et al. Adverse reactions during peanut oral immunotherapy home dosing. J Allergy Clin Immunol. 2009;124: Blumchen K, Ulbricht H, et al. Oral peanut immunotherapy in children with peanut anaphylaxis. J Allergy Clin Immunol. 2010;126:83-91 e Pumphrey R. Anaphylaxis: can we tell who is at risk of a fatal reaction? Current Opinion in Allergy & Clinical Immunology. 2004;4: Rolland JM, Gardner LM, et al. Allergenrelated approaches to immunotherapy. Pharmacology & Therapeutics. 2009;121: O Hehir RE, Sandrini A, et al. Sublingual allergen immunotherapy: immunological mechanisms and prospects for refined vaccine preparation. Current Medicinal Chemistry. 2007;14: Scadding G, Durham S. Mechanisms of sublingual immunotherapy. Journal of Asthma. 2009;46: O Hehir RE, Gardner LM, et al. House dust mite sublingual immunotherapy: the role for transforming growth factor-beta and functional regulatory T cells. Am J Respir Crit Care Med. 2009;180: Akdis M. Immune tolerance in allergy. Curr Opin Immunol Akdis M, Akdis CA. Mechanisms of allergenspecific immunotherapy. J Allergy Clin Immunol. 2007;119: Larche M. Update on the current status of peptide immunotherapy. Journal of Allergy & Clinical Immunology. 2007;119: Larche M. Peptide immunotherapy for allergic diseases. Allergy. 2007;62: Muller U, Akdis CA, et al. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. Journal of Allergy & Clinical Immunology. 1998;101: Kammerer R, Chvatchko Y, et al. Modulation of T-cell response to phospholipase A2 and phospholipase A2-derived peptides by conventional bee venom immunotherapy. Journal of Allergy & Clinical Immunology. 1997;100: Alexander C, Ying S, et al. Fel d 1-derived T cell peptide therapy induces recruitment of CD4+ CD25+; CD4+ interferon-gamma+ T helper type 1 cells to sites of allergen-induced late-phase skin reactions in cat-allergic subjects. Clinical & Experimental Allergy. 2005;35: Alexander C, Tarzi M, et al. The effect of Fel d 1-derived T-cell peptides on upper and lower airway outcome measurements in cat-allergic subjects. Allergy. 2005;60: Campbell JD, Buckland KF, et al. Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression. Journal of Experimental Medicine. 2009;206: Kay AB, Larche M. Allergen immunotherapy with cat allergen peptides. Springer Seminars in Immunopathology. 2004;25: Oldfield WL, Larche M, et al. Effect of T-cell peptides derived from Fel d 1 on allergic reactions and cytokine production in patients sensitive to cats: a randomised controlled trial. Lancet. 2002;360: Higgins JA, Lamb JR, et al. Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite). Journal of Allergy & Clinical Immunology. 1992;90: Hoyne GF, O Hehir RE, et al. Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice. Journal of Experimental Medicine. 1993;178: Hong SJ, Michael JG, et al. Pepsin-digested peanut contains T-cell epitopes but no IgE epitopes. Journal of Allergy & Clinical Immunology. 1999;104: Eiwegger T, Rigby N, et al. Gastro-duodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potential. Clinical & Experimental Allergy. 2006;36: King N, Helm R, et al. Allergenic characteristics of a modified peanut allergen. Molecular Nutrition & Food Research. 2005;49: Glaspole IN, de Leon MP, et al. Characterization of the T-cell epitopes of a major peanut allergen, Ara h 2. Allergy. 2005;60: Stanley JS, King N, et al. Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. Archives of Biochemistry & Biophysics. 1997;342: Burks AW, Shin D, et al. Mapping and mutational analysis of the IgE-binding epitopes on Ara h 1, a legume vicilin protein and a major allergen in peanut hypersensitivity. Eur J Biochem. 1997;245: Rabjohn P, West CM, et al. Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation. International Archives of Allergy & Immunology. 2002;128: Palmer GW, Dibbern DA, Jr., et al. Comparative potency of Ara h 1 and Ara h 2 in immunochemical and functional assays of allergenicity. Clinical Immunology. 2005;115: Koppelman SJ, Wensing M, et al. Relevance of Ara h1, Ara h2 and Ara h3 in peanutallergic patients, as determined by immunoglobulin E Western blotting, basophilhistamine release and intracutaneous testing: Ara h2 is the most important peanut allergen. Clinical & Experimental Allergy. 2004;34: McDermott RA, Porterfield HS, et al. Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation. Clinical & Experimental Allergy. 2007;37: Flinterman AE, van Hoffen E, et al. Children with peanut allergy recognize predominantly Ara h2 and Ara h6, which remains stable over time. Clinical & Experimental Allergy. 2007;37: Blanc F, Adel-Patient K, et al. Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors. Clin Exp Allergy. 2009;39: de Leon MP, Glaspole IN, et al. Immunological analysis of allergenic cross-reactivity between peanut and tree nuts. Clinical & Experimental Allergy. 2003;33: de Jong EC, Van Zijverden M, et al. Identification and partial characterization of multiple major allergens in peanut proteins. Clinical & Experimental Allergy. 1998;28: Mannering SI, Dromey JA, et al. An efficient method for cloning human autoantigenspecific T cells. Journal of Immunological Methods. 2005;298: Albrecht M, Kuhne Y, et al. Relevance of IgE binding to short peptides for the allergenic activity of food allergens. J Allergy

43 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Clin Immunol. 2009;124:328-36, 36 e Woodfolk JA. T-cell responses to allergens. J Allergy Clin Immunol. 2007;119:280-94; quiz Shreffler WG, Charlop-Powers Z, et al. Lack of association of HLA class II alleles with peanut allergy.[see comment]. Annals of Allergy, Asthma, & Immunology. 2006;96: Middleton D, Menchaca L, et al. New allele frequency database: Tissue Antigens. 2003;61: Tanabe S. Epitope peptides and immunotherapy. Curr Protein Pept Sci. 2007;8: Bateman EA, Ardern-Jones MR, et al. Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes.[see comment]. Clinical & Experimental Allergy. 2008;38: Verhoef A, Higgins JA, et al. Clonal analysis of the atopic immune response to the group 2 allergen of Dermatophagoides spp.: identification of HLA-DR and -DQ restricted T cell epitopes. International Immunology. 1993;5: Higgins JA, Thorpe CJ, et al. Overlapping T-cell epitopes in the group I allergen of Dermatophagoides species restricted by HLA- DP and HLA-DR class II molecules. Journal of Allergy & Clinical Immunology. 1994;93: Ruiter B, Rozemuller EH, et al. Role of human leucocyte antigen DQ in the presentation of T cell epitopes in the major cow s milk allergen alphas1-casein. Int Arch Allergy Immunol. 2007;143: van Neerven RJ, van de Pol MM, et al. Characterization of cat dander-specific T lymphocytes from atopic patients. J Immunol. 1994;152: Shek LP, Cabrera-Morales EA, et al. A population-based questionnaire survey on the prevalence of peanut, tree nut, and shellfish allergy in 2 Asian populations. J Allergy Clin Immunol. 2010;126: e Larche M. Of cats and men: immunodominance and the role of HLA-DP/ DQ.[comment]. Clinical & Experimental Allergy. 2008;38: de Leon MP, Rolland JM, et al. The peanut allergy epidemic: allergen molecular characterisation and prospects for specific therapy. Expert Reviews in Molecular Medicine. 2007;9: Ramos ML, Huntley JJ, et al. Identification and characterization of a hypoallergenic ortholog of Ara h Plant Molecular Biology. 2009;69:

44 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES SUPPLEMENTARY TABLES Table S1. Demographics and genotype of subjects with peanut allergy. HLA-genotypes Peanut CAP DRB1 DQB1 DPB1 ku A /L (score) Donor Sex Age Atopic Asthma Anaph-ylaxis PA 1 F 32 yes yes yes (3) 07:01 15:01 02:01G 06:02 02:01 04:01 PA 2 F 53 yes yes yes (5) 03:01 08:01 02:01G 04:02 03:01/104:01 04:01 PA 3 M 30 yes no yes (4) 04:01 04:04 03:02 04:02 13:01/107:01 04:01 PA 4 F 22 yes no yes 4.72 (3) 11:01 15:01 03:01G 06:02 03:01/104:01 04:01 PA 5 F 25 yes no yes 2.12 (2) 11:04 15:01 03:01G 06:02 02:01 14:01 PA 6 M 37 yes yes yes (4) 11:01 15:01 03:01G 06:02 04:01 PA 7 M 36 yes yes yes (5) 01:03 04:01 03:02 05:01 03:01/104:01 02:01 PA 8 M 30 yes no yes >100 (6) 12:01G 15:01 03:01G 06:02 13:01/107:01 04:01 PA 9 M 30 yes yes yes (4) 13:02 06:09 05:01 04:02/105:01 PA 10 F 23 yes yes yes >100 (6) 08:01 10:01 04:02 05:01 03:01/104:01 04:01 PA 11 F 26 yes yes yes 2.82 (2) 03:01 13:02 02:01G 06:09 01:01 04:01 PA 12 M 35 yes yes no 1.23 (2) 04:05 15:01 03:02 06:02 03:01/104:01 04:01 PA 13 M 30 yes yes yes (4) 09:01 13:01 03:03 06:03 03:01/104:01 04:02/105:01 PA 14 F 19 yes no yes (5) 11:01 15:01 03:01G 06:02 04:01 PA 15 F 36 yes no yes 6.94 (3) 04:01 04:04 03:01G 03:02 02:01 04: (2) 04:04 13:01 03:02 06:03 02:01 04:01 PA 16 F 20 yes yes yes NA 1 F 32 no no no 0.00 (0) NA 2 F 24 no no no 0.03 (0) NA 3 M 24 no no no 0.00 (0) F, Female; M, male; PA, peanut allergy; NA, nonatopic control; All HLA abbreviations comply with recent changes to allele nomenclature ( and Alleles followed by a G represent groups of alleles that share common sequences in exon 2 (

45 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Table S2. Ara h 2 peptides a. Residues Sequence Residues Sequence Residues Sequence NLPQQCGLRAPQRCD LPQQCGLRAPQRCD PQQCGLRAPQRCD QQCGLRAPQRCD QCGLRAPQRCD CGLRAPQRCD GLRAPQRCD GLRAPQRCD KRELRNLPQQCGLRAPQRC KRELRNLPQQCGLRAPQR KRELRNLPQQCGLRAPQ KRELRNLPQQCGLRAP KRELRNLPQQCGLRA KRELRNLPQQCGLR KRELRNLPQQCGL KRELRNLPQQCG KRELRNLPQQC KRELRNLPQQ KRELRNLPQ RRCQSQLERANLRP RRCQSQLERANLR RRCQSQLERANL RRCQSQLERAN RRCQSQLERA RRCQSQLER N- and C-terminal truncations of peptide (91-110) LNEFENNQRCMCEALQQIM NEFENNQRCMCEALQQIM EFENNQRCMCEALQQIM FENNQRCMCEALQQIM ENNQRCMCEALQQIM NNQRCMCEALQQIM NQRCMCEALQQIM QRCMCEALQQIM RCMCEALQQIM 20-mer peptides spanning Ara h 2 with 11 aa overlap 1-20 LTILVALALFLLAAHASARQ FLLAAHASARQQWELQGDRR RQQWELQGDRRCQSQLERAN RRCQSQLERANLRPCEQHLM ANLRPCEQHLMQKIQRDEDS LMQKIQRDEDSYERDPYSPS DSYERDPYSPSQDPYSPSPY PSQDPYSPSPYDRRGAGSSQ PYDRRGAGSSQHQERCCNEL SQHQERCCNELNEFENNQRC ELNEFENNQRCMCEALQQIM IMENQSDRLQGRQQEQQFKR KRELRNLPQQCGLRAPQRCD QCGLRAPQRCDLDVESGGRD CDLDVESGGRDRY Core epitope peptides SQLERANLRPCEQ ANLRPCEQHLM ELNEFENNQRCM FENNQRCMCEALQ RELRNLPQQCGLRA Peptides comprising overlapping epitope pairs SQLERANLRPCEQHLM ELNEFENNQRCMCEALQ Cysteine-to-serine substituted therapeutic candidate peptides C42S SQLERANLRPSEQHLM ELNEFENNQRSMSEALQ C[ ]S C137S RELRNLPQQSGLRA CMCEALQQIM MCEALQQIM ELNEFENNQRCMCEALQQI ELNEFENNQRCMCEALQQ ELNEFENNQRCMCEALQ ELNEFENNQRCMCEAL ELNEFENNQRCMCEA ELNEFENNQRCMCE ELNEFENNQRCMC ELNEFENNQRCM ELNEFENNQRC ELNEFENNQR ELNEFENNQ N- and C-terminal truncations of peptide ( ) RELRNLPQQCGLRAPQRCD ELRNLPQQCGLRAPQRCD LRNLPQQCGLRAPQRCD RNLPQQCGLRAPQRCD N- and C-terminal truncations of peptide (28-47) RCQSQLERANLRPCEQHLM CQSQLERANLRPCEQHLM QSQLERANLRPCEQHLM SQLERANLRPCEQHLM QLERANLRPCEQHLM LERANLRPCEQHLM ERANLRPCEQHLM RANLRPCEQHLM ANLRPCEQHLM NLRPCEQHLM LRPCEQHLM RRCQSQLERANLRPCEQHL RRCQSQLERANLRPCEQH RRCQSQLERANLRPCEQ RRCQSQLERANLRPCE RRCQSQLERANLRPC a Peptide sequences based on Ara h 2 sequence published by Stanley JS, King N, et al. Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. Arch Biochem Biophys 1997;342:

46 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES 4 Table S3. Proliferative responses of T-cell lines of subjects with peanut allergy to Ara h 2 20-mer peptides. Ara h 2 20-mer peptides Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 Pool cpm of unstimulated TCL (mean ± SD) Subject Driving Antigen TCL A CPE ± CPE ± CPE ± CPE ± CPE ± CPE ± CPE ± CPE 1 99 ± CPE ± CPE ± Ah ± Ah ± Ah ± Ah ± Ah ± Ah ± Ah ± Ah ± CPE ± CPE ± Ah ± Ah ± Ah ± Ah ± Ah ± Ah ± Ah ± CPE ± Ah ± CPE ± CPE ± CPE ± CPE ± CPE ± Ah ± CPE ± CPE ± CPE ± CPE ± CPE ± CPE ± Ah ± Ah ± Ah ± Ah ± Ah ±

47 CHAPTER 4 ARA H 2 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES SUPPLEMETARY FIGURE 4 Ah ± CPE ± CPE ± CPE ± CPE ± Ah ± Ah ± Ah ± Ah ± Pool ± Pool ± B Pool ± Pool ± Pool ± Pool ± Pool ± Pool ± Pool ± Pool ± Pool ± Pool ± Pool ± Pool ± Only positive simulation indices ( 2.5) are shown: Grey, ; Black, >5.0. A, Allergen-driven TCL; B, peptide-driven TCL. Figure S1: T-cell recognition of peptides containing cysteine-to-serine substituted epitopes. TCL cytokine secretion in response to parent or cysteine-substituted Ara h 2 peptides determined by ELISPOT. Graphs show representative TCL specific for each epitope (mean spots of replicate wells +SD). IL-4, black bars; IL-5, hatched bars; IFN-g, white bars; A) Ara h 2(32-44), n = 2 (3 TCL); B) Ara h 2(37-47) n = 3 (5 TCL); C) Ara h 2(91-102), n = 3 (3 TCL); D) Ara h 2(95-107), n = 2 (2 TCL); E) Ara h 2( ), n = 3 (3 TCL). Ag, Antigen

48 5 Ara h 1 peptides comprising dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic Prickett SR a,b, Voskamp AL a,b, Phan T a,b, Dacumos-Hill A a,b, Mannering SI c, Rolland JM a,b, and O Hehir RE a,b a Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Australia b Department of Immunology, Monash University, Melbourne, Australia c Immunology and Diabetes, St Vincent s Institute of Medical Research, Melbourne, Australia Clinical and Experimental Allergy. 2013;43:684-97

49 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Abstract Introduction Background: Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions and even fatalities in peanut allergy. Objective: To identify short, HLAdegenerate CD4 + T-cell epitope-based peptides of the major peanut allergen Ara h 1 that target allergen-specific T cells without causing IgE-mediated inflammatory cell activation, as candidates for safe peanut-specific immunotherapy. Methods: Ara h 1-specific CD4 + T-cell lines (TCL) were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using CFSE-based methodology. Dominant T-cell epitopes were identified using CFSE and thymidine-based proliferation assays. Epitope HLA-restriction was investigated using blocking antibodies, HLA-genotyping and epitope prediction algorithms. Functional peanut-specific IgE reactivity to peptides was assessed by basophil activation assay. Results: 145 Ara h 1-specific TCL were generated from 18 HLA-diverse peanut-allergic subjects. The TCL recognized 20-mer peptides throughout Ara h 1. Nine 20-mers were selected as containing dominant epitopes and their recognition confirmed in 18 additional peanut-allergic subjects. Ten core epitopes were mapped within these 20-mers. These were HLA-DQ and/or HLA DR restricted, with each presented on at least two different HLA-molecules. Seven short ( 20 aa) non-basophil-reactive peptides encompassing all core epitopes were designed and validated in peanut-allergic donor PBMC T-cell assays. Conclusions and Clinical Relevance: Short CD4 + T-cell epitope-based Ara h 1 peptides were identified as novel candidates for a safe, T-cell targeted peanut-specific immunotherapy for HLA-diverse populations. Peanut allergy is the leading cause of food-induced anaphylactic fatalities world-wide 1, 2. It is a major health care problem affecting 1-2% of the population 2-4, with clinical symptoms ranging from mild oropharyngeal irritation to life-threatening anaphylaxis. Unlike egg and milk food allergy that is present in infants and typically resolve by school age, peanut allergy is life-long in 80% of cases. This significantly impairs the quality of life of afflicted individuals and their families 5-7, with further impact on the wider community through efforts to manage this severe condition 2, 8. There is currently no cure for peanut allergy. Avoidance is the only means of control, with epinephrine as emergency treatment for anaphylaxis. Even with diligent precautions, most peanut-allergic subjects have accidental exposures which can have severe or even fatal consequences 1, 4, 5, 9. Whole allergen extracts are currently used for specific immunotherapy for respiratory and insect venom allergies, but are unavailable in clinical practice for treatment of food allergy due to risks of severe side effects or even death in the case of peanut allergy. The limited studies on specific immunotherapy for peanut allergy provide encouragement that desensitization is feasible, but the observed adverse reactions highlight major safety concerns These risks are especially pertinent for peanut allergy since peanut allergens may induce anaphylaxis at minute doses with little correlation between previous severity of reactions and a person s first anaphylactic episode 11, 19. Consequently, there is an urgent need to develop a safe, diseasemodifying therapeutic for peanut-allergic individuals. Anaphylaxis results from the release of inflammatory cell mediators triggered by binding and cross-linking of cell-bound allergen-specific IgE by the relevant allergen. During specific immunotherapy, stimulation of appropriate T-cell responses is considered essential for successful desensitization and the subsequent reduction and/or inhibition of allergen-specific IgE Although conventional immunotherapy administers whole allergen extracts, studies on cat allergy and bee venom allergy 28, 29 clearly demonstrate that short T-cell epitope-based peptides of major allergens are sufficient for effective desensitization without causing adverse IgE-mediated reactions. Importantly, targeting T cells specific for immunodominant epitopes of major allergens can alter responses to whole allergen extracts (linked suppression). Many studies reporting successful peptide immunotherapy in murine models of allergy demonstrated that administration of immunodominant T-cell epitope peptides of major allergens induced tolerance not only to those peptides, but also to purified allergen and whole allergen extracts More recently, clinical administration of Fel d 1 T-cell epitope peptides in humans altered T-cell responses to those peptides, other non-related Fel d 1 peptides, and whole cat allergen extract 25. We aimed to design peptides based on most reliably recognized CD4 + T-cell epitopes of major peanut allergens for a T-cell-targeted immunotherapy for peanut allergy as a safe (non- IgE reactive) and effective alternative to whole allergens. Of eleven peanut allergens identified (Ara h 1-11) 36, Ara h 1 and Ara h 2 are the two designated major allergens whose recognition is most consistently reported in >50% of cohorts tested 4, 37, 38. Although a number of studies have

50 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES indicated Ara h 2 to be the more potent of these two allergens 39-41, Ara h 1 also plays a major role in the pathogenesis of peanut allergy, with numerous studies reporting strong correlations between symptom severity and IgE reactivity to both Ara h 1 and Ara h Ara h 1 is the most abundant major allergen in peanut, accounting for 12-16% of total peanut protein 48. This is an important consideration for driving linked epitope suppression in allergen immunotherapy, since inducing T-cell suppressor activity against abundant major allergens will undoubtedly facilitate reduced responses to whole allergen extracts. We recently designed a panel of T-cell epitopebased Ara h 2 peptides for inclusion in a peptide therapeutic 49. In a single report of sequences of T-cell-reactive peptides from Ara h 1 using predictive tetramer-based epitope mapping 50, core epitopes were not determined and only ten HLA-DR tetramers were used, preventing detection of epitopes presented on other HLA-types. Here we provide a comprehensive report of precise core T-cell epitopes of Ara h 1 based on analysis of full T-cell repertoires from a large cohort of HLA-diverse peanut-allergic subjects. We reveal novel HLA-DQ-restricted epitopes as well as epitopes within previously reported T-cell reactive Ara h 1 20-mers 50. We also demonstrate presentation of the latter epitopes on additional HLA-molecules to those previously reported 50. Using these sequences we designed a panel of HLA-degenerate, T-cell reactive Ara h 1 peptides to combine with those we identified previously from Ara h 2 49, providing a broadly acting therapeutic to take forward for pre-clinical and clinical testing to treat HLA-diverse peanutallergic populations. Methods Subjects Peanut-allergic adult subjects were recruited from The Alfred Allergy Clinic, Melbourne, Australia (Table S1). All subjects had clinical symptoms of IgE-mediated peanut allergy and peanut-specific IgE CAP score 1 ( 0.49 ku A /l; Pharmacia CAP System TM, Pharmacia Diagnostics, Uppsala, Sweden). Subjects used for T-cell line (TCL) generation were genotyped (HLA-DRB1, -DQB1 and -DPB1, exon 2) by the Victorian Transplantation and Immunogenetics Service (Table S2). The study was approved by The Alfred and Monash University Ethics Committees and informed written consent obtained from each subject. Antigens Crude peanut extract (CPE) was prepared from commercial unsalted, dry-roasted peanuts as described 49, 51. Ara h 1 and Ara h 2 were enriched from CPE by liquid chromatography as described 49. Endotoxin contents were 1.7, 4.0 and 78.0 EU/mg for CPE, Ara h 1 and Ara h 2 respectively (Endpoint Chromogenic LAL assay, Lonza, Walkersville, USA). Ara h 1 peptides (Mimotopes, Victoria, Australia and GenScript USA Inc, New Jersey, USA; Table S3) were reconstituted at 2 mg/ml in 10% dimethyl sulfoxide/pbs (20-mers and truncated peptide sets) or PBS alone (custom-synthesized core epitope peptides). All antigens were confirmed to be neither mitogenic nor toxic as described 52. Generation of Ara h 1-specific CD4 + T-cell lines (TCL) Ara h 1-specific oligoclonal TCL were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using 5,6-carboxyfluorescein diacetate succinimidylester (CFSE)-based methodology 53 as described 49, with CPE (100 µg/ml), Ara h 1 (10 µg/ml) or 20- mer peptides spanning the Ara h 1 sequence (11 amino acid (aa) overlap (17 aa overlap for the last peptide); Table S3; 10 μg/ml/peptide) as the driving antigens. All TCL were tested for specificity (proliferation) to individual Ara h 1 20-mers (10 μg/ml) as well as CPE (100 μg/ml) and/or Ara h 1 (10 μg/ml). Core epitope sequences were mapped within selected 20-mers using peptide sets truncated from the N- or C-terminus of the 20-mer as described 49. T-cell assays All culturing was performed in RPMI-1640 containing 2 mm L-glutamine, 100 IU/mL penicillinstreptomycin and 5% heat-inactivated human AB serum (crpmi; Sigma-Aldrich, St Louis, USA). Antigen-induced TCL proliferation was assessed by 3 H-thymidine ( 3 H-TdR) uptake assays as described 49. A stimulation index (SI; cpm antigen-stimulated T cells/cpm unstimulated T cells) 2.5 was considered positive and all positive responses confirmed in 2 assays. HLA-restriction of epitope recognition by TCL was assessed using monoclonal antibodies (mab) against HLA-DR (L243), HLA-DQ (SVP-L3) or HLA-DP (B7/21) to block epitope presentation as described 49. To allow detection of peptide-induced CD4 + T-cell proliferation within whole PBMC, 7-day cultures of CFSE-labelled PBMC were set up as described for TCL generation 49. At least 10,000 CD4 + T cells were analyzed per sample and SI calculated as percentage of CD4 + CFSE lo (proliferated) cells with antigen/percentage of CD4 + CFSE lo cells without antigen (background). The detection threshold for a specific response in this assay was assessed by expanding peptide-specific TCL from proliferated CD4 + cells over a range of SI values for three subjects. Specific TCL could be generated from divided T cells with SI as low as 1.1 in all three subjects (data not shown) allowing designation of an SI 1.1 as positive. Basophil activation test Basophil activation was assessed by CD63 upregulation detected by flow cytometry as described 54. Positive controls were rabbit anti-human IgE antibody (7.5 µg/ml; DAKO Corporation, CA, USA), N-formyl-methionine-leucine-phenylalanine (fmlp) (0.4 µg/ml; Sigma) and CPE. CPE, Ara h 1 and peptides were tested over a 3-log concentration range (5, 0.5 and 0.05 µg/ml)

51 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Results Selection of Ara h 1 20-mer peptides containing dominant CD4 + T-cell epitopes most reliably recognized by peanut-allergic subjects A total of 145 Ara h 1-specific T-cell lines (TCL) were generated from PBMC of 18 HLA-diverse peanut-allergic subjects (Table S1 and S2) by isolating and expanding antigen-specific (proliferated) CD4 + CFSE lo T cells from 7-day CFSE-labelled PBMC cultures stimulated with CPE, Ara h 1 or pools of Ara h 1 20-mer peptides collectively spanning the Ara h 1 sequence (Table S3). The 20-mer peptide(s) recognized (SI 2.5) by each subject are shown in Table 1 and data summarized in Figure 1. For some subjects, CPE or Ara h 1 stimulation generated most TCL whilst for others it was the peptide pools. Where TCL were generated from a given subject using different antigen preparations (CPE, Ara h or peptide pools), TCL 20-mer specificities were comparable. Overall, there was no bias in the TCL 20-mer specificity generated depending on antigen preparation. Table 1. Proliferative responses (thymidine uptake) of T-cell lines to Ara h 1 20-mer peptides. Subject No. TCL/Subject Ara h 1 20-mer peptide residues 5 TCL, T-cell line. Only positive stimulation indices (SI 2.5) are shown. For subjects with multiple TCL specific for a given 20-mer, the highest SI is shown. SIs above 10 have been rounded to the nearest whole number. Dark grey, SI 2.5<5.0; Black, SI

52 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Figure 1: Donor responder frequency profile for Ara h 1 20-mer peptides. Donor responder frequencies for TCL recognition of Ara h 1 20-mer peptides (n= 18 peanut-allergic subjects). The 145 TCL collectively recognized epitopes throughout the entire Ara h 1 sequence, with only four of the sixty-nine 20-mers failing to stimulate any TCL. Fourteen 20-mers (23, 24, 26, 38, 40, and 57) were each recognized by four (22%) or more subjects, with peptides 50 and 51 having the most responders (six subjects; 33%; Figure 1). Although dominant allergen epitopes are most simply defined as being those peptides/regions most frequently recognized within the respective allergen sequence 55-59, we considered a number of factors in addition to TCL responder frequencies to further define and refine our selection of epitopes for inclusion in a therapeutic. These included the magnitude of TCL response, number of specific TCL per subject, reproducibility of specific TCL response and ability to target specific T cells in PBMC. Based on these parameters, nine of the fourteen 20-mers (peptides 23, 24, 40, 46, 47, 49, 50, 51 and 57) were selected for subsequent analyses. These nine 20-mers were collectively recognized by 16 of the 18 subjects (89%) in this cohort, and typically induced strong and consistent proliferative responses in specific TCL, with the majority of SI over five and many considerably higher (Table 1). Furthermore, each of these 20-mers was recognized by multiple TCL from many responders reflecting a prevalence of T cells specific for these peptides among the subjects T-cell repertoires. To assess recognition in a wider cohort, PBMC from an additional 20 peanut-allergic subjects were screened by CFSE assay for CD4 + T-cell proliferation in whole PBMC following seven days stimulation with each peptide (Table 2, upper panel and Figure S1). This assay provided a sensitive and accurate screen for detecting the full repertoire of peptidespecific CD4 + T cell proliferative responses within whole PBMC. All 20 subjects showed PBMC T-cell proliferation to CPE or a combination of enriched Ara h 1 and Ara h 2. The 20-mers were collectively recognized by 18 (90%) of these subjects, with 40-79% responding to each 20-mer. Analysis of four subjects from the original cohort used for TCL generation confirmed they also had T cells specific for other 20-mers in addition to those recognized by their TCL (Table 2, lower panel). Table 2. CFSE-based detection of peanut-allergic donor CD4+ T-cell proliferation in response to Subject No Antigen* selected Ara h 1 20-mers. Stimulation Indices (SI) +ve 20-mers Ara h 1 20-mers SI>1.1 SI>1.5 CPE No. % No. % nt nt nt /6 67 4/ nt nt nt /6 50 1/ nt nt nt /6 17 1/ nt nt nt /6 17 1/ nt nt nt /6 33 1/ nt nt nt /6 33 2/ /9 56 4/ /9 78 6/ /9 33 1/ / / /9 89 6/ /9 56 4/ / / /9 78 4/ /9 33 2/ ^ /9 66 6/ ^ /9 44 3/ ^ /9 89 7/ /9 0 0/ /9 0 0/9 0 Responders w SI>1.1 Responders w SI>1.5 # 20/20 11/20 10/20 8/20 7/14 10/14 11/14 8/20 11/20 11/20 % # 17/20 10/20 7/20 4/20 6/14 7/14 9/14 7/20 8/20 10/20 % ^ /9 78 5/ /9 89 8/ nt nt nt / / nt nt nt / /6 100 Responders w SI>1.1 Responders w SI>1.5 # 24/24 15/24 14/24 12/24 9/16 11/16 13/16 11/24 14/24 15/24 % # 21/24 14/24 11/24 8/24 7/16 8/16 11/16 10/24 11/24 13/24 % Upper panel shows new peanut-allergic donor cohort; lower panel shows four subjects from peanutallergic donor cohort used for TCL generation with combined totals from upper and lower panels. CPE, crude peanut extract; +ve, positive; nt, not tested (peptide stocks not available at time of testing); Grey, stimulation indices 1.1<2.5; Black, stimulation indices 2.5; * Background proliferation with no antigen, % CD4 + CFSE lo T cells of total CD4 + T cells ^A combination of enriched Ara h 1 and Ara h 2 (10 µg/ml of each) was used instead of CPE for these subjects

53 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Combined totals for all 24 subjects tested with the CFSE assay showed 46-81% responded to each 20-mer. If a higher SI of 1.5 was used as a positive cut off, the frequency of responders per 20-mer was only slightly reduced to 33-69%. Overall, T-cell recognition of one or more of the selected panel of nine 20-mers was confirmed in 35 (92%) of 38 subjects analyzed using either cut-off. Mapping core T-cell epitopes within selected Ara h 1 20-mer peptides Minimal length peptides decrease risk of cross-linking cell-bound IgE on inflammatory cells during clinical administration and facilitate therapeutic production. The minimum T-cell stimulatory sequence (core epitope) within each selected 20-mer was determined by testing proliferation of reactive TCL from different subjects to truncated peptide sets (e.g. Figure 2 and Table 3). The number of residues required to induce maximal T-cell proliferation varied from 6-19 aa between different TCL and/or subjects (Table 3), consistent with previous reports for CD4 + T-cell epitopes 60, 61. Due to variation in the number of flanking-residues required for optimal epitope recognition 61, TCL were considered to recognize the same epitope if peptides containing a common core sequence induced recognition. Based on this criterion, ten distinct CD4 + T-cell epitopes were identified ( consolidated epitopes, Table 3), with common cores varying from 5-12 aa (underlined sequences, Table 3). Consolidated epitope sequences were selected to encompass residues required for maximal stimulation of all specific TCL tested to ensure broadest possible recognition. At least one epitope was found within each of the nine 20-mers, with 20-mers 50 and 51 each containing two distinct but overlapping T-cell epitopes: one unique to each 20-mer (( ) and ( )), and the other within the overlap sequence (( ), Table 3 and Figure 2). No single TCL responded to both epitopes within either 20-mer, further confirming the distinction of these epitopes (data not shown). HLA-epitope prediction algorithms 62, 63 also highlighted one or more strong HLA class II (HLA-II) binding motifs within each of our minimalstimulatory sequences. Data are shown for the Propred 62 HLA-DR binding algorithm in Table S4. This algorithm did not predict HLA-DR epitopes within peptide 40, but algorithms of the Immune Epitope Database (IEDB) and Analysis Resource 63 predicted epitopes within this peptide to bind most strongly to HLA-DP and/or -DQ molecules. Finally, to avoid unnecessary sequence duplication and to minimize peptide numbers for a therapeutic, six of the consolidated epitopes (comprising three overlapping epitope pairs) were combined into three single peptides of 20 aa or less (( ), ( ) and ( ); grey shading, Table 3). The combined epitope peptides efficiently stimulated TCL specific for either epitope (data not shown) and together with the remaining four consolidated epitopes (( ), ( ), ( ) and ( )), provided a panel of seven candidate peptides for further characterization (see asterisks, Table 3). CFSE-based screening of nine subjects from our cohorts confirmed that these peptides could each directly target detectable numbers of Ara h 1-specific T cells among whole PBMC of peanut-allergic subjects (Table 4). In a few cases the T cell response of a given subject to the original 20-mer and Figure 2: Mapping core T-cell epitopes within Ara h 1 20-mer peptides 50 and mer-specific TCL proliferation to truncated peptide sets. Representative TCL shown for peptides 50 (A) and 51 (B) (mean cpm replicate wells +SD). Upper panels indicate the epitope in overlap between the 20- mers (n = 2; 3 TCL). Lower panels indicate epitopes unique to each 20-mer; A) n = 3; 6 TCL. B) n = 4; 7 TCL. Epitope sequences recognized by represented TCL are bolded and consolidated epitopes recognized by all specific TCL are underlined. the corresponding candidate peptide differed. Where responses to candidates were reduced as compared to the 20-mer, flanking residue(s) required for optimal T cell recognition or HLAbinding may have been removed. It is well recognized that flanking residues can stabilize peptide binding to HLA class II molecules. In contrast, improved responses could reflect generation of additional new epitopes, improved epitope purity (cores were synthesized at high purity (>95%) whilst 20-mers were produced as peptide sets with a minimum estimated purity of 70%) or alteration of epitopes (or flanking residues) to enable better interaction with HLA and/or T cell receptor (TCR) molecules. Indeed, this was considered to be the case where responses to candidate peptide ( ) were much stronger than to 20-mer 50. Since this region contains multiple adjacent hydrophobic residues, even single residue changes could significantly alter the charge and structure of this peptide, thus affecting its biochemical properties and interactions with HLA and/or TCR molecules. Nonetheless, most responses to candidates were comparable or improved compared to responses to the original 20-mers

54 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Table 3. Core T-cell epitope sequences mapped within selected Ara h 1 20-mers. 20-mer peptide Minimum sequence required for T-cell recognition Consolidated epitope (common core underlined) Confirmed Responders # Residues Residues Sequence Residues/ aa Sequence TCL Subjects 23 ( ) ( ) ( ) FQNLQNHR FQNLQNHRIV ( ) 10 aa 24 ( ) ( ) RIVQIEAKPN ( ) RIVQIEAKPNTLV ( ) ( ) IVQIEA 13 aa FQNLQNHRIV 6 3 RIVQIEAKPNTLV 6 3 Determining HLA class II restriction specificity of Ara h 1 T-cell epitopes There is no identified HLA-II association with peanut allergy 64, therefore peptides selected for therapy must bind diverse HLA-II molecules for wide applicability. To determine the HLA-II type presenting each epitope, anti-hla-dr, -DP or -DQ mabs were used to block individual epitope presentation to T cells. For each TCL tested, epitope recognition was prevented by one or more HLA-mAb in a dose-dependent manner (e.g. Figure S2, Supporting information) and the same mab blocked recognition of CPE (data not shown), demonstrating consistency for presentation of naturally processed and synthetic epitope forms. At least two subjects and/or TCL were tested per epitope (Table 5). Consistent with predictions of the HLA-II algorithms described above 62, 63, anti-hla-dr blocked recognition of all but one epitope, ( ), which was blocked by Overlapping epitopes combined ( ) 20 aa 40 ( ) ( ) WSTRSSENNEGVIVKVSKE ( ) ENNEGVIVKVSKE ( ) NEGVIVKVSK ( ) 19 aa FQNLQNHRIVQIEAKPNTLV* 12 6 WSTRSSENNEGVIVKVSKE* 3 3 anti-hla-dq in both subjects tested. For epitopes ( ) and ( ), recognition was blocked by anti-hla-dr for some TCL but by anti-hla-dq for others, confirming HLA-binding degeneracy for these epitopes ( ) ( ) NNFGKLFEVK ( ) NNFGKLFEVKPDKKNPQ ( ) FGKLFEVK ( ) 17 aa 47 ( ) ( ) EVKPDKKNPQLQ ( ) 12 aa NNFGKLFEVKPDKKNPQ 3 2 EVKPDKKNPQLQ 2 1 Table 4. CFSE-based detection of peanut-allergic donor CD4+ T-cell proliferation in response to selected Ara h 1 candidate peptides. Overlapping epitopes combined ( ) 19 aa 49 ( ) ( ) VEIKEGALML ( ) VEIKEGALMLPHFN ( ) ( ) EGALMLPHFNSKA 17 aa 50 ( ) ( ) ( ) ALMLPHFNSKAMVIVVV LMLPHFNSKAMVIVV ( ) PHFNSKAMVIV ( ) KAMVIVVVN ( ) AMVIVVVNKG ( ) IVVVNKG 51 ( ) ( ) AMVIVVVNKGTGNLEL ( ) AMVIVVVNKGTGNLELV ( ) VVNKGTGNLELVA ( ) VVNKGTGNLELVAV ( ) 17 aa ( ) 11 aa ( ) 19 aa Overlapping epitopes combined ( ) 20 aa 57 ( ) ( ) GDVFIMPAAHPVAINASS ( ) VFIMPAAHPVAINASS ( ) FIMPAAHPVAIN ( ) IMPAAHP ( ) IMPAAHPVAIN ( ) 18 aa NNFGKLFEVKPDKKNPQLQ* 3 2 VEIKEGALMLPHFNSKA* 5 2 ALMLPHFNSKAMVIVVV* 6 3 KAMVIVVVNKG 3 2 AMVIVVVNKGTGNLELVAV 7 4 KAMVIVVVNKGTGNLELVAV* 10 6 GDVFIMPAAHPVAINASS* 12 4 Grey shading indicates overlapping consolidated epitope pairs combined into single peptides for further analyses as outlined in the text. *The seven candidate peptides proposed for a therapeutic Subject No Ag* CPE Stimulation Indices (SI) +ve peptides Candidate peptides SI > 1.1 SI > ^ /7 71 4/ /7 57 3/ /7 86 4/ /7 71 5/ /7 14 0/ ^ / / ^ /7 71 4/ ^ / /7 71 Responders No. 8/8 6/8 5/8 8/8 6/8 6/8 4/8 5/8 w SI>1.1 % Responders w SI>1.5 No. 8/8 5/8 4/8 6/8 5/8 6/8 2/8 3/8 % CPE, crude peanut extract; +ve, positive; Grey, stimulation indices 1.1<2.5; Black, stimulation indices 2.5. * Background proliferation with no antigen, % CD4 + CFSE lo T cells of total CD4 + T cells ^A combination of enriched Ara h 1 and Ara h 2 (10 µg/ml of each) was used instead of CPE for these subjects No. % No. %

55 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Table 5. HLA class II restriction of core epitope peptides. the threshold of positive activation 65 and was negligible compared to the activation induced by Ara h 1 (80-90%) or CPE (74-76%) in this subject. 20-mer Epitope Subject HLA-restriction Corresponding HLA-allele(s) 23 ( ) 18 3 HLA-DR HLA-DR DRB1 04:05 DRB1 03:01 DRB1 15:01 DRB1 08:01 24 ( ) HLA-DR HLA-DR DRB1 08:01 DRB1 11:01 DRB1 10:01 DRB1 15:01 40 ( ) HLA-DQ HLA-DQ nt DQB1 03:01 DQB1 03:01 DQB1 06:09 DQB1 06:02 DQB1 06:02 46 ( ) 47 ( ) HLA-DR nt HLA-DR nt DRB1 04:04 DRB1 03:01P DRB1 04:04 DRB1 03:01P DRB1 13:01 DRB1 04:01 DRB1 13:01 DRB1 04: ( ) ( ) 17 9 HLA-DQ HLA-DR HLA-DR HLA-DR DQB1 03:02 DRB1 04:05 DRB1 11:04 DRB1 09:01 DQB1 06:02 DRB1 15:01 DRB1 15:01 DRB1 13: ( ) 12 6 HLA-DR HLA-DR DRB1 08:01 DRB1 04:01 DRB1 10:01 DRB1 04:04 51 ( ) HLA-DR nt DRB1 11:01 DRB1 13:02 DRB1 15:01 57 ( ) HLA-DR HLA-DQ DRB1 11:04 DQB1 03:01 DRB1 15:01 DQB1 06:02 nt = not tested (TCL not available); Grey shading indicates overlapping epitope pairs combined into single peptides for further analyses as outlined in the text. To assess HLA-binding degeneracy of epitopes whose recognition was blocked by a single HLAmAb, the respective HLA-alleles of at least two subjects with TCL specific for that epitope were compared (Table S2 and Table 5). The absence of shared HLA-DRB1 or HLA-DQB1 alleles between subjects recognizing HLA-DR- or HLA-DQ-restricted epitopes respectively confirmed that each epitope was presented on at least two different HLA-molecules. The HLA-binding algorithms further supported these data, with each epitope containing motifs predicted to bind multiple HLA-molecules 62, 63 (e.g. Table S4). Figure 3: Basophil activation in response to candidate Ara h 1 peptides. Box-and-whiskers plot showing percentage of activated (CD63 hi ) basophils (IgE hi ) in response to Ara h 1 or candidate peptides for seven peanut-allergic subjects. Negative control was no antigen (unstimulated) and positive controls were anti-ige, fmlp and CPE. Whiskers show minimum to maximum values. Testing candidate peptides for basophil activation To provide a safe alternative to whole allergens, peptides must not bind and cross-link cellbound IgE. Basophil reactivity to peptides was assessed in fresh blood from seven of the peanut-allergic subjects recruited for this study (Figure 3). All seven subjects showed high levels of basophil activation to CPE over a concentration range. Whilst responses to Ara h 1 varied between subjects at the lowest dose, the highest concentration induced high activation in all subjects. However, none of the candidate peptides induced activation at any concentration tested. One subject showed a very low response (8%) to peptide ( ), but this was below

56 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Discussion An appropriately selected T-cell-targeted peptide immunotherapeutic will provide a safe treatment option for peanut-allergic individuals. Candidate peptides must comprise HLAdegenerate CD4 + T-cell epitopes of the major peanut allergens recognized by HLA-diverse peanut-allergic individuals, without cross-linking cell-bound IgE and activating inflammatory cells. In order to maximize the population coverage and efficacy of a therapeutic, we designed a peptide set containing T-cell epitopes from the two major allergens, Ara h 1 and Ara h 2. Following on from our previous study of Ara h 2 49, we now provide the first report of core sequences of CD4 + T-cell epitopes of the most abundant major peanut allergen, Ara h 1. We identified and characterized ten, HLA-diverse CD4 + T-cell epitopes of Ara h 1, and used these sequences to design candidate Ara h 1 peptides to combine with our candidate Ara h 2 peptides for therapeutic development. To commence this study we selected nine 20-mers of Ara h 1 containing most frequently recognized epitopes based on responses of 145 TCL from 18 HLA-diverse peanut-allergic subjects. The cohort HLA profile was typical of Caucasian populations 66 in countries where peanut allergy is prevalent 67. We further validated our 20-mer selection by demonstrating their collective recognition by PBMC T cells directly ex vivo from an additional 18 peanut-allergic subjects (Table 2), resulting in a total responder frequency of 92% for the 38 subjects analyzed. Although we could not confirm T-cell recognition of these 20-mers in four subjects, it is possible that specific T cells went undetected for two of these subjects (5 and 7), as data were only obtained from three and four TCL respectively (Table 1). The minimum T-cell stimulatory sequences identified within our selected 20-mers varied from 6 to 19 aa (Table 3), consistent with reports of different peptides processed for HLA-II presentation and/or required for HLA- and/or TCR-binding both within and between subjects 60, 61. As we used oligoclonal TCL, it is possible that the longer sequences contained more than one epitope. Indeed, algorithms 62, 63 predicted up to three HLA-binding motifs within some of our consolidated epitopes (Table S4). Seven of our epitopes showed overlap with T-cell-reactive Ara h 1 20-mers recently identified using HLA-DR tetramers 50, providing further support for recognition of these peptides in larger peanut-allergic populations. In addition, we confirmed presentation of five of these peptides on additional HLA-molecules to those used for the tetramer mapping. 50 However, epitopes ( ), ( ) and ( ) were unique to our study. Consistent with this observation, we showed these epitopes were either presented on HLA-DQ molecules (commonly observed for allergen T-cell epitopes ), or HLA-DR types for which no tetramer-specific T cells were detected. 50 Inclusion of HLA-DQ-restricted epitopes is particularly advantageous for therapeutics as these alleles are less variable and thus more prevalent in mixed populations than HLA-DR alleles 73. However, we confirmed HLA-binding degeneracy for all epitopes identified (both HLA-DR and -DQ-restricted) (Table 5), with further degeneracy predicted by algorithms 62, 63 (e.g. Table S4), emphasizing their collective suitability for targeting HLA-diverse peanut-allergic populations. The main rationale for developing a peptide immunotherapy for peanut allergy is to identify an effective allergen preparation that does not invoke the adverse effects seen with whole peanut extract 10, 12-14, 19, 37, 74. Mapping the core sequences of T-cell epitopes enables refined peptide design for a therapeutic, but selecting optimal peptide combinations is a balance between peptide length and number. Longer peptides will increase population coverage by encompassing more T-cell epitopes, and being fewer in number, will reduce the complexity of therapeutic standardization compared to using a greater number of shorter peptides. However, the main concern with longer peptides is the increased potential for IgE binding and crosslinking, resulting in adverse reactions. We opted to combine overlapping epitopes into peptides up to 20 aa in length. Of over 23 linear IgE epitopes reported for Ara h , only two minor epitopes ( and ) fell within our candidate peptides 75. Most importantly, none of these peptides caused activation of peanut-reactive basophils in all of the seven peanutallergic subjects tested (Figure 3) emphasizing the potential for these peptides to provide a safe alternative to whole allergen extract for immunotherapy. In summary we report the novel identification of ten dominant CD4 + T-cell epitopes of Ara h 1 that collectively show diverse HLA class II-restriction. We incorporated these epitopes into a panel of seven short ( 20 aa), HLA-degenerate peptides that can target T cells within PBMC of HLA-diverse allergic individuals without causing activation of peanut-allergic donor basophils. The combination of these Ara h 1 peptides with our three T-cell epitope-based Ara h 2 peptides 49 provides strong candidates for a broad acting and safe peptide-therapeutic to treat peanut allergy. Acknowledgements We thank Dr Nicole Mifsud for helpful intellectual discussions, Neeru Varese for technical assistance and Karen Symons and Kirsten Deckert for patient recruitment and blood collection. This project was supported by grants from the Ilhan Food Allergy Foundation and the National Health and Medical Research Council of Australia

57 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES References 1. Bock SA, Munoz-Furlong A, et al. Further fatalities caused by anaphylactic reactions to food, Journal of Allergy & Clinical Immunology. 2007;119: Burks AW. Peanut allergy. Lancet. 2008;371: Sicherer SH, Munoz-Furlong A, et al. US prevalence of self-reported peanut, tree nut, and sesame allergy: 11-year follow-up. J Allergy Clin Immunol. 2010;125: Husain Z, Schwartz RA. Peanut allergy: an increasingly common life-threatening disorder. J Am Acad Dermatol. 2012;66: Kemp AS, Hu W. Food allergy and anaphylaxis - dealing with uncertainty. Med J Aust. 2008;188: Busse PJ, Nowak-Wegrzyn AH, et al. Recurrent peanut allergy. New England Journal of Medicine. 2002;347: Yun J, Katelaris CH. Food allergy in adolescents and adults. Intern Med J. 2009;39: Avery NJ, King RM, et al. Assessment of quality of life in children with peanut allergy. Pediatric Allergy & Immunology. 2003;14: Sampson MA, Munoz-Furlong A, et al. Risk-taking and coping strategies of adolescents and young adults with food allergy. J Allergy Clin Immunol. 2006;117: Oppenheimer JJ, Nelson HS, et al. Treatment of peanut allergy with rush immunotherapy. [see comment]. Journal of Allergy & Clinical Immunology. 1992;90: Pumphrey R. Anaphylaxis: can we tell who is at risk of a fatal reaction? Current Opinion in Allergy & Clinical Immunology. 2004;4: Varshney P, Steele PH, et al. Adverse reactions during peanut oral immunotherapy home dosing. J Allergy Clin Immunol. 2009;124: Nelson HS, Lahr J, et al. Treatment of anaphylactic sensitivity to peanuts by immunotherapy with injections of aqueous peanut extract. Journal of Allergy & Clinical Immunology. 1997;99: Jones SM, Pons L, et al. Clinical efficacy and immune regulation with peanut oral immunotherapy. Journal of Allergy & Clinical Immunology. 2009;24: Varshney P, Jones SM, et al. A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response. J Allergy Clin Immunol. 2011;127: Anagnostou K, Clark A, et al. Efficacy and safety of high-dose peanut oral immunotherapy with factors predicting outcome. Clin Exp Allergy. 2011;41: Allen KJ, O Hehir RE. The evolution of oral immunotherapy for the treatment of peanut allergy. Clin Exp Allergy. 2011;41: Thyagarajan A, Varshney P, et al. Peanut oral immunotherapy is not ready for clinical use. J Allergy Clin Immunol. 2010;126: Hofmann AM, Scurlock AM, et al. Safety of a peanut oral immunotherapy protocol in children with peanut allergy. Journal of Allergy & Clinical Immunology. 2009;124: Sabatos-Peyton CA, Verhagen J, et al. Antigen-specific immunotherapy of autoimmune and allergic diseases. Curr Opin Immunol. 2010;22: Rolland JM, Gardner LM, et al. Functional regulatory T cells and allergen immunotherapy. Curr Opin Allergy Clin Immunol. 2010;10: Akdis CA, Akdis M. Mechanisms of allergenspecific immunotherapy. J Allergy Clin Immunol. 2011;127:18-27; quiz Alexander C, Ying S, et al. Fel d 1-derived T cell peptide therapy induces recruitment of CD4+ CD25+; CD4+ interferon-gamma+ T helper type 1 cells to sites of allergen-induced late-phase skin reactions in cat-allergic subjects. Clinical & Experimental Allergy. 2005;35: Alexander C, Tarzi M, et al. The effect of Fel d 1-derived T-cell peptides on upper and lower airway outcome measurements in cat-allergic subjects. Allergy. 2005;60: Campbell JD, Buckland KF, et al. Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression. Journal of Experimental Medicine. 2009;206: Kay AB, Larche M. Allergen immunotherapy with cat allergen peptides. Springer Seminars in Immunopathology. 2004;25: Oldfield WL, Larche M, et al. Effect of T-cell peptides derived from Fel d 1 on allergic reactions and cytokine production in patients sensitive to cats: a randomised controlled trial. Lancet. 2002;360: Muller U, Akdis CA, et al. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. Journal of Allergy & Clinical Immunology. 1998;101: Kammerer R, Chvatchko Y, et al. Modulation of T-cell response to phospholipase A2 and phospholipase A2-derived peptides by conventional bee venom immunotherapy. Journal of Allergy & Clinical Immunology. 1997;100: Yang M, Yang C, et al. Multiple T cell epitope peptides suppress allergic responses in an egg allergy mouse model by the elicitation of forkhead box transcription factor 3- and transforming growth factor-betaassociated mechanisms. Clin Exp Allergy. 2010;40: Yoshitomi T, Nakagami Y, et al. Intraoral administration of a T-cell epitope peptide induces immunological tolerance in Cry j 2-sensitized mice. J Pept Sci. 2007;13: Marazuela EG, Rodriguez R, et al. Intranasal immunization with a dominant T-cell epitope peptide of a major allergen of olive pollen prevents mice from sensitization to the whole allergen. Mol Immunol. 2008;45: Rupa P, Mine Y. Oral immunotherapy with immunodominant T-cell epitope peptides alleviates allergic reactions in a Balb/c mouse model of egg allergy. Allergy. 2012;67: Hoyne GF, O Hehir RE, et al. Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice. Journal of Experimental Medicine. 1993;178: Hall G, Houghton CG, et al. Suppression of allergen reactive Th2 mediated responses and pulmonary eosinophilia by intranasal administration of an immunodominant peptide is linked to IL-10 production. Vaccine. 2003;21: Allergen Nomenclature, International Union of Immunological Societies [Internet]. [cited 2015]. Available from: de Leon MP, Rolland JM, et al. The peanut allergy epidemic: allergen molecular characterisation and prospects for specific therapy. Expert Reviews in Molecular Medicine. 2007;9: Palmer K, Burks W. Current developments in peanut allergy. Curr Opin Allergy Clin Immunol. 2006;6: Blanc F, Adel-Patient K, et al. Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors. Clin Exp Allergy. 2009;39: Koppelman SJ, Wensing M, et al. Relevance of Ara h1, Ara h2 and Ara h3 in peanutallergic patients, as determined by immunoglobulin E Western blotting, basophilhistamine release and intracutaneous testing: Ara h2 is the most important peanut allergen. Clinical & Experimental Allergy. 2004;34: Palmer GW, Dibbern DA, Jr., et al. Comparative potency of Ara h 1 and Ara h 2 in immunochemical and functional assays of allergenicity. Clinical Immunology. 2005;115: Glaumann S, Nopp A, et al. Basophil allergen threshold sensitivity, CD-sens, IgEsensitization and DBPCFC in peanutsensitized children. Allergy. 2012;67: Chiang WC, Pons L, et al. Serological and clinical characteristics of children with peanut sensitization in an Asian community. Pediatr Allergy Immunol. 2009;21:e Asarnoj A, Moverare R, et al. IgE to peanut allergen components: relation to peanut symptoms and pollen sensitization in 8-yearolds. Allergy. 2010;65: Moverare R, Ahlstedt S, et al. Evaluation of IgE antibodies to recombinant peanut allergens in patients with reported reactions to peanut. Int Arch Allergy Immunol. 2011;156: Lin YT, Charles Wu CT, et al. Patterns of sensitization to peanut allergen components in Taiwanese Preschool children. J Microbiol Immunol Infect. 2012;45: Peeters KA, Koppelman SJ, et al. Does skin prick test reactivity to purified allergens correlate with clinical severity of peanut allergy? Clin Exp Allergy. 2007;37: Koppelman SJ, Vlooswijk RA, et al. Quantification of major peanut allergens Ara h 1 and Ara h 2 in the peanut varieties Runner, Spanish, Virginia, and Valencia, bred in different parts of the world. Allergy

58 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES 2001;56: Prickett SR, Voskamp AL, et al. Ara h 2 peptides containing dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic. J Allergy Clin Immunol. 2011;127: e DeLong JH, Simpson KH, et al. Ara h 1-reactive T cells in individuals with peanut allergy. J Allergy Clin Immunol. 2011;127: e de Leon MP, Glaspole IN, et al. Immunological analysis of allergenic cross-reactivity between peanut and tree nuts. Clinical & Experimental Allergy. 2003;33: Eusebius NP, Papalia L, et al. Oligoclonal analysis of the atopic T cell response to the group 1 allergen of Cynodon dactylon (bermuda grass) pollen: pre- and post-allergen-specific immunotherapy. International Archives of Allergy & Immunology. 2002;127: Mannering SI, Dromey JA, et al. An efficient method for cloning human autoantigenspecific T cells. Journal of Immunological Methods. 2005;298: Drew AC, Eusebius NP, et al. Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity. Journal of Immunology. 2004;173: Etto T, de Boer C, et al. Unique and crossreactive T cell epitope peptides of the major Bahia grass pollen allergen, Pas n 1. International Archives of Allergy and Immunology. 2012;159: Pascal M, Konstantinou GN, et al. In silico prediction of Ara h 2 T cell epitopes in peanut-allergic children. Clinical and Experimental Allergy. 2013;43: Cardaba B, Del Pozo V, et al. Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule. Clinical and Experimental Allergy. 1998;28: de Silva HD, Gardner LM, et al. The hevein domain of the major latex-glove allergen Hev b 6.01 contains dominant T cell reactive sites. Clinical and Experimental Allergy. 2004;34: Oseroff C, Sidney J, et al. Molecular determinants of T cell epitope recognition to the common Timothy grass allergen. Journal of Immunology. 2010;185: Hemmer B, Kondo T, et al. Minimal peptide length requirements for CD4(+) T cell clones--implications for molecular mimicry and T cell survival. Int Immunol. 2000;12: Suri A, Lovitch SB, et al. The wide diversity and complexity of peptides bound to class II MHC molecules. Curr Opin Immunol. 2006;18: Singh H, Raghava GP. ProPred: prediction of HLA-DR binding sites. Bioinformatics. 2001;17: Vita R, Zarebski L, et al. The immune epitope database 2.0. Nucleic Acids Res. 2010;38:D Shreffler WG, Charlop-Powers Z, et al. Lack of association of HLA class II alleles with peanut allergy.[see comment]. Annals of Allergy, Asthma, & Immunology. 2006;96: Boumiza R, Debard AL, et al. The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives. Clin Mol Allergy. 2005;3: Middleton D, Menchaca L, et al. New allele frequency database: Tissue Antigens. 2003;61: Shek LP, Cabrera-Morales EA, et al. A population-based questionnaire survey on the prevalence of peanut, tree nut, and shellfish allergy in 2 Asian populations. J Allergy Clin Immunol. 2010;126: e Bateman EA, Ardern-Jones MR, et al. Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes.[see comment]. Clinical & Experimental Allergy. 2008;38: Verhoef A, Higgins JA, et al. Clonal analysis of the atopic immune response to the group 2 allergen of Dermatophagoides spp.: identification of HLA-DR and -DQ restricted T cell epitopes. International Immunology. 1993;5: Higgins JA, Thorpe CJ, et al. Overlapping T-cell epitopes in the group I allergen of Dermatophagoides species restricted by HLA- DP and HLA-DR class II molecules. Journal of Allergy & Clinical Immunology. 1994;93: Ruiter B, Rozemuller EH, et al. Role of human leucocyte antigen DQ in the presentation of T cell epitopes in the major cow s milk allergen alphas1-casein. Int Arch Allergy Immunol. 2007;143: van Neerven RJ, van de Pol MM, et al. Characterization of cat dander-specific T lymphocytes from atopic patients. J Immunol. 1994;152: Larche M. Of cats and men: immunodominance and the role of HLA-DP/ DQ.[comment]. Clinical & Experimental Allergy. 2008;38: Rolland JM, Gardner LM, et al. Allergenrelated approaches to immunotherapy. Pharmacology & Therapeutics. 2009;121: Burks AW, Shin D, et al. Mapping and mutational analysis of the IgE-binding epitopes on Ara h 1, a legume vicilin protein and a major allergen in peanut hypersensitivity. Eur J Biochem. 1997;245: Shreffler WG, Beyer K, et al. Microarray immunoassay: association of clinical history, in vitro IgE function, and heterogeneity of allergenic peanut epitopes. J Allergy Clin Immunol. 2004;113: van Boxtel EL, Koppelman SJ, et al. Determination of pepsin-susceptible and pepsin-resistant epitopes in native and heattreated peanut allergen Ara h 1. J Agric Food Chem. 2008;56:

59 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES SUPPLEMENTARY TABLES Table S1. Subject demographics Subject Sex Age Atopic* Asthma Peanut CAP ku A /l (score) Anaphylaxis Use of patient samples TCL 20-mer CFSE Core CFSE BAT 1 M 39 Yes No 2.18 (2) Yes X X X 2 M 34 Yes Yes 0.78 (2) Yes X X 3 F 53 Yes as a child (5) Yes X 4 F 19 Yes No (5) Yes X X 5 F 22 Yes No 4.72 (3) Yes X 6 M 30 Yes No (4) Yes X 7 M 42 No No (3) Yes X 8 M 36 Yes Yes (5) Yes X 9 M 30 Yes Yes (4) Yes X 10 M 37 Yes Yes (4) Yes X X X 11 F 26 Yes Yes 2.82 (2) Yes X 12 F 23 Yes Yes >100 (6) Yes X 13 M 30 Yes No >100 (6) Yes X 14 M 30 Yes Yes (4) Yes X X 15 F 31 Yes No (5) No X 16 F 20 Yes Yes 1.16 (2) Yes X 17 F 25 Yes No 2.12 (2) Yes X X 18 M 35 Yes Yes 1.23 (2) No X 19 M 27 Yes Yes 6.19 (3) Yes X 20 F 25 Yes Yes 87.2 (5) Yes X 21 F 53 Yes No 1.43 (2) No X 22 F 28 Yes Yes 9.53 (3) na X 23 F 37 Yes No 6.94 (3) Yes X 24 M 38 Yes Yes 2.42 (2) Yes X 25 M 28 Yes Yes >100 (6) Yes X 26 F 70 No No 2.18 (2) Yes X X 27 F 26 Yes No 1.37 (2) No X 28 F 35 Yes No SPT 14mm Yes X 29 F 23 na No 2.37 (2) na X 30 F 28 Yes Yes 9.2 (3) No X X 31 F 30 Yes Yes (3) Yes X X 32 M 53 Yes No 2.01 (2) Yes X X 33 M 26 Yes Yes 12.00(3) Yes X X 34 M 43 Yes Yes 1.63 (2) No X X X 35 F 33 Yes na 0.49 (1) No X X 36 M 28 Yes na 0.72 (2) no X X 37 F 21 Yes Yes 1.51 (2) Yes X 38 M 28 Yes Yes 1.43(2) Yes X 39 M 29 Yes No (4) Yes X 40 F 52 Yes Yes 7.23 (3) Yes X * Atopic is defined by specific IgE to one or more of a panel of common aeroallergens either by RAST or skin prick test. TCL, T cell line; 20-mer CFSE, screen for T cell reactivity to selected Ara h 1 20-mers; Core CFSE, screen for T cell reactivity to candidate Ara h 1 peptides; BAT, basophil activation test; na, data not available; SPT, skin-prick test (RAST not available for this subject). Table S2. HLA genotyping for subjects used for T-cell line generation Subject HLA-genotypes DRB1 DQB1 DPB1 1 07:01 15:01 02:01 06:02 04: :01 03:01 05:01 06:02 04:01 04: :01 08:01 02:01P 04:02 03:01P 04: :01 15:01 03:01P 06:02 04: :01 15:01 03:01P 06:02 03:01P 04: :01 04:04 03:02 04:02 13:01P 04: :01 08:01 03:03 04:02 04:01 06: :03 04:01 03:02 05:01 03:01P 02: :01 13:01 03:03 06:03 03:01P 04:02P 10 11:01 15:01 03:01P 06:02 04: :01 13:02 02:01P 06:09 01:01 04: :01 10:01 04:02 05:01 03:01P 04: :01P 15:01 03:01 06:02 13:01P 04: :02 06:09 05:01 04:02P 15 03:01P 04:01 04:01P 02:01P 03:01P 16 04:04 13:01 03:02 06:03 02:01 04: :04 15:01 03:01P 06:02 02:01 14: :05 15:01 03:02 06:02 03:01P 04:01 All HLA abbreviations comply with recent changes to allele nomenclature ( announcement.html and Alleles followed by a P represent groups of alleles that share common sequences in exon 2 (

60 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Table S3. Ara h 1 20-mer peptides Pool No. Residues Sequence Pool No. Residues Sequence MRGRVSPLMLLLGILVLASV FSRNTLEAAFNAEFNEIRRV LLLGILVLASVSATHAKSSP FNAEFNEIRRVLLEENAGGE SVSATHAKSSPYQKKTENPC RVLLEENAGGEQEERGQRRW SPYQKKTENPCAQRCLQSCQ GEQEERGQRRWSTRSSENNE PCAQRCLQSCQQEPDDLKQK RWSTRSSENNEGVIVKVSKE CQQEPDDLKQKACESRCTKL NEGVIVKVSKEHVEELTKHA QKACESRCTKLEYDPRCVYD KEHVEELTKHAKSVSKKGSE KLEYDPRCVYDPRGHTGTTN HAKSVSKKGSEEEGDITNPI YDPRGHTGTTNQRSPPGERT SEEEGDITNPINLREGEPDL TNQRSPPGERTRGRQPGDYD PINLREGEPDLSNNFGKLFE RTRGRQPGDYDDDRRQPRRE DLSNNFGKLFEVKPDKKNPQ YDDDRRQPRREEGGRWGPAG FEVKPDKKNPQLQDLDMMLT REEGGRWGPAGPREREREED PQLQDLDMMLTCVEIKEGAL AGPREREREEDWRQPREDWR LTCVEIKEGALMLPHFNSKA EDWRQPREDWRRPSHQQPRK ALMLPHFNSKAMVIVVVNKG WRRPSHQQPRKIRPEGREGE KAMVIVVVNKGTGNLELVAV RKIRPEGREGEQEWGTPGSH KGTGNLELVAVRKEQQQRGR GEQEWGTPGSHVREETSRNN AVRKEQQQRGRREEEEDEDE SHVREETSRNNPFYFPSRRF GRREEEEDEDEEEEGSNREV NNPFYFPSRRFSTRYGNQNG DEEEEGSNREVRRYTARLKE RFSTRYGNQNGRIRVLQRFD EVRRYTARLKEGDVFIMPAA NGRIRVLQRFDQRSRQFQNL FDQRSRQFQNLQNHRIVQIE AAHPVAINASSELHLLGFGI NLQNHRIVQIEAKPNTLVLP SSELHLLGFGINAENNHRIF IEAKPNTLVLPKHADADNIL GINAENNHRIFLAGDKDNVI LPKHADADNILVIQQGQATV IFLAGDKDNVIDQIEKQAKD ILVIQQGQATVTVANGNNRK VIDQIEKQAKDLAFPGSGEQ TVTVANGNNRKSFNLDEGHA KDLAFPGSGEQVEKLIKNQK RKSFNLDEGHALRIPSGFIS EQVEKLIKNQKESHFVSARP HALRIPSGFISYILNRHDNQ QKESHFVSARPQSQSQSPSS ISYILNRHDNQNLRVAKISM RPQSQSQSPSSPEKESPEKE NQNLRVAKISMPVNTPGQFE SSPEKESPEKEDQEEENQGG SMPVNTPGQFEDFFPASSRD KEDQEEENQGGKGPLLSILK FEDFFPASSRDQSSYLQGFS QEEENQGGKGPLLSILKAFN RDQSSYLQGFSRNTLEAAFN

61 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES Table S4. Predicted HLA-DR binding binding motifs motifs in selected in selected Ara Ara h 1 20-mers h 1 20-mers Ara h 1 20-mer peptide 23 ( ) 24 ( ) 46 ( ) 47 ( ) 49 ( ) 50 ( ) 51 ( ) 57 ( ) HLA molecule DRB1_0101 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0102 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0301 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0305 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0306 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0307 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0308 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0309 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0311 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0401 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0402 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0404 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0405 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0408 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0410 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0421 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0423 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0426 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0701 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0703 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0801 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0802 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0804 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0806 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0813 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_0817 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1101 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1102 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1104 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1106 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1107 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1114 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1120 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1121 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1128 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1301 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1302 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1304 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1305 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1307 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1311 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1321 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1322 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1323 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1327 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1328 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1501 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1502 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1506 FDQRSRQFQNLQNHRIVQIE LQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1502 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB1_1506 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB5_0101 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV DRB5_0105 FDQRSRQFQNLQNHRIVQIE NLQNHRIVQIEAKPNTLVLP DLSNNFGKLFEVKPDKKNPQ FEVKPDKKNPQLQDLDMMLT LTCVEIKEGALMLPHFNSKA ALMLPHFNSKAMVIVVVNKG KAMVIVVVNKGTGNLELVAV HLA-DR binding motifs (grey shading) were predicted using the ProPred algorithm published by Singh H, Raghava GP, et al. ProPred: prediction of HLA-DR binding sites. Bioinformatics. 2001;17: , ( accessed 30 th January 2012). Predicted primary anchor residues are bolded and underlined. Peptide 40 ( ) is not shown as no HLA-DR binding motifs were predicted for this peptide by this algorithm

62 CHAPTER 5 ARA H 1 PEPTIDES COMPRISING DOMINANT CD4+ T-CELL EPITOPES SUPPLEMENTARY FIGURES 5 Figure S2. Representative HLA class II restriction specificity of T-cell epitope recognition. Proliferation of specific TCL to selected epitopes in the presence of HLA-DR (circles), -DQ (squares) or -DP (triangles) mabs (Ai and Bi) or isotype control antibodies (10 μg/ml) (Aii and Bii), (mean cpm replicate wells +SD). Graphs show sample data for an HLA-DR-restricted epitope ( ) (A) and an HLA-DQ restricted epitope ( ) (B). Figure S1. Representative CFSE-based assay for detecting CD4+ T-cell proliferation in PBMC. Proliferation of CFSE-labelled PBMC from peanut-allergic subject 26 following 7 days stimulation with selected Ara h 1 20-mer peptides. Medium alone (No Antigen) or crude peanut extract (CPE) provided negative and positive controls respectively. At least 10,000 live CD4 + T cells were analyzed per sample. Gates indicate percentage CD4 + CFSE lo (proliferating) T cells of total CD4 + T cells with stimulation indices (SI) in parentheses

63 6 Cysteine-to-serine substitution can alter susceptibility of therapeutic peptides to gastrointestinal enzyme digestion, affecting potential for oral delivery Voskamp AL a,b, Phan T a,b, Rolland JM a,b, O Hehir RE a,b, Prickett SR a,b a Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Australia b Department of Immunology, Monash University, Melbourne, Australia Manuscript in preparation

64 CHAPTER 6 THERAPEUTIC PEPTIDE SUSCEPTIBILITY TO ENZYMATIC DIGESTION ABSTRACT Introduction We recently designed three peptides based on dominant CD4 + T cell epitopes of the major peanut allergen Ara h 2 for inclusion in a peptide-based therapeutic for peanut allergy. In this study, we evaluated and compared the stability of native (cysteinecontaining) and therapeutic (serine-containing) forms of these peptides in the presence of different gastrointestinal digestive enzymes, to assess potential for oral administration of these peptides. Two of the three peptides tested were resistant to enzyme digestion at physiological concentrations and therefore show potential for oral delivery. Importantly, significant changes (both beneficial and detrimental) occurred in the resistance of these peptides to high concentrations of enzyme following cysteine to serine substitutions. Our data suggest that such changes could both create and remove enzymatic access to digestion sites, highlighting the complexity of peptide conformation and stability, with implications for therapeutic design. The development of peptide-based therapeutics for a wide range of conditions has escalated in the last decade, with close to 100 products currently on the market, representing 1.5% of global drug sales. In addition, an estimated 850 peptide-products are now in preclinical or clinical development heralding an even greater expansion of peptide therapeutics in coming years 1-4. Peptides hold many advantages over larger molecules such as proteins or antibodies; they are relatively easy to synthesize cost effectively and are amenable to up scaling and standardization 5. In addition, peptides can be rapidly cleared from the body without accumulating in specific organs, thus minimizing toxic side effects. An important advantage of peptides is their amenability to modifications that optimise therapeutic applicability. Considerations for modification include ease of peptide synthesis, solubility, risk of aggregation and stability, and, importantly, their biological reactivity. Cysteine residues are particularly problematic in therapeutic peptides due to susceptibility to oxidation and disulphide bridge formation, which impedes synthesis, solubility, stability and biological reactivity. A frequent approach to avoid these issues is to substitute cysteine residues with structurally conserved, but less chemically reactive, serine residues. Peptides are being trialled as a new class of therapeutic for allergic diseases, termed SPIRE (Synthetic Peptide Immuno-Regulatory Epitope) 6. Current phase II and III clinical trials show promising results for rapid and long-lasting treatment of allergies to cat, grass pollen and house-dust mites SPIRE therapy consists of carefully selected short ( 20 amino acids) T cell epitope-based peptides of major allergens, which can ameliorate allergic responses via interactions with T cells, but are too short to cross-link inflammatory cell-bound IgE. This allows for an effective immunotherapy, without risk of the patient experiencing allergic reactions during treatment. These properties make SPIRE therapy an attractive alternative to the current practice of using whole allergen extracts for allergenspecific immunotherapy, particularly for subjects who are unable to use existing products due to the risk of severe reactions during treatment. Furthermore, SPIRE therapy may be the only suitable treatment option for more severe allergies such as peanut allergy, for which no product has yet been approved for specific immunotherapy. The established route of SPIRE delivery is via intradermal injection, with early trials demonstrating enhanced efficacy compared to subcutaneous administration 11. Mechanistically, it is thought that intradermal administration enables preferential targeting of peptides to tolerogenic antigen presenting cells (APC), known to be abundant in the dermis 12. The formation of peptide-hla complexes on the surface of these cells in the absence of inflammatory signals is thought to facilitate induction of tolerance, deletion and/or regulatory activity in allergen-specific T cells, leading to the observed reduction in allergic response 6. For this reason, oral delivery could potentially offer an attractive

65 CHAPTER 6 THERAPEUTIC PEPTIDE SUSCEPTIBILITY TO ENZYMATIC DIGESTION alternative route of administration. As in the dermis, APC in the mucosa of the oral cavity are thought to promote tolerance induction and efficacy of immunotherapy 13. These tolerogenic APC are not only present in the oral cavity but can also be found throughout the intestinal mucosa 14, 15. Finally, in addition to the contribution of tolerogenic immune cells, antigen-specific IgA has been detected in saliva after successful sublingual immunotherapy, and may provide an additional barrier to an allergic response upon subsequent exposure to the allergen 16. This type of protection is highly desirable in food allergies. From a practical point of view, oral delivery is less invasive, likely resulting in further improved safety and higher patient compliance. However, for oral immunotherapy to be successful, peptides must first be resistant to degradation by enzymes encountered in the oral cavity and, to some extent, along the digestive tract. We recently designed peptides based on dominant CD4 + T cell epitopes of the major peanut allergen Ara h 2 for inclusion in a safe SPIRE therapeutic for peanut allergy 17. We replaced native cysteine residues with serine residues to facilitate production, and demonstrated retained T cell reactivity of the variant peptides 17. Cysteine-to-serine substitutions serve the additional purpose of preventing formation of tertiary structures that could induce inflammatory cell-bound IgE cross-linking and unwanted allergic sideeffects. Here we evaluate and compare the stability of native (cysteine-containing) and therapeutic (serine-containing) forms of these peptides in the presence of different gastrointestinal digestive enzymes, to assess potential for oral administration of these peptides in a future treatment for peanut allergy. Australia) and peptide 3 (Genscript, NJ, USA) were synthesized. Enzymatic digestion of these peptides and crude peanut extract (CPE) was carried out in reaction mixes containing α-amylase (purified from human saliva), pepsin (from porcine gastric mucosa), trypsin or chymotrypsin (from bovine gastric mucosa) (Sigma-Aldrich, Deisenhofen, Germany) in relevant buffers with ph adjusted as required (Table 1). Mitogenicity and toxicity of the peptides and buffers were tested, as described previously 17. A low and high enzyme:protein ratio were used to mimic biological digestion and to observe the maximum effect of the enzyme, respectively. Equal volumes of enzyme mix were added to protein solution and incubated at 37 o C in a water bath. Aliquots were collected at specified time points and heated at 100 o C for 5 minutes to stop the digestion reaction. For time point zero, the enzyme was heated prior to addition to the protein solution. Enzymatic activity was evaluated by digestion of CPE following the same protocol and visualization of the resulting fragments with SDS-PAGE. Before use, α-amylase activity was confirmed using an iodine-starch based assay 20. All enzyme treated samples were aliquoted and stored at -80 o C until further use. Table 1. Enzyme digest reaction conditions Enzyme Time points (minutes) Ratio ph Buffer (enzyme:protein) α-amylase :50, 1: H 2 O 6 Pepsin :5, 1: Saline (0.15M) Methods Peptide specific T cell line generation Trypsin :50, 1: M Tris-HCl 20nM CaCl 2 Chymotrypsin :50, 1: M Tris-HCl Ara h 2-specific oligoclonal T cell lines (TCL) were expanded from peripheral blood mononuclear cells (PBMC) of peanut allergic subjects using 5,6-carboxyfluorescein diacetatesuccinimidylester (CFSE)-based methodology 18, following a 7 day stimulation with CPE (100mg/mL), Ara h 2 (10mg/mL) or 20-mer peptides (10mg/mL) spanning the Ara h 2 sequence as reported 17, 19. Briefly, TCL specificity was verified by proliferative response to individual Ara h 2 20-mers (10mg/mL) as well as CPE (100mg/mL) and/or Ara h 2 (10mg/ ml). Core epitope sequences within the 20-mer peptides were mapped using sets of peptides truncated from the N- or C-terminus of the 20-mer as described 17. Enzyme digestion of protein and peptide Based on the previously identified dominant epitopes of Ara h 2 17, three peptides, designated peptide 1 (Think Peptides, Clayton, Australia), peptide 2 (Mimotopes, Clayton, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Digested and whole CPE, 15µg/lane, were separated by electrophoresis under reducing conditions using 4% to 12% Bis-Tris SDS-PAGE gels (NuPage, Carlsbad, CA). The gel was stained with Coomassie brilliant blue. Pre-stained standard (1 SeeBlue Plus2, Invitrogen, Carlsbad, CA) was used as the molecular weight marker. T cell proliferation assays T cell proliferation assays were performed in duplicate with 10,000 irradiated (5,000 rads) Epstein Barr-transformed B cells and 10,000 peptide-specific TCL incubated with Ara h 2 peptides (10mg/mL) or CPE (100mg/mL) exposed to the digestive enzymes. The

66 CHAPTER 6 THERAPEUTIC PEPTIDE SUSCEPTIBILITY TO ENZYMATIC DIGESTION enzymes in the corresponding buffers alone served as negative controls, with untreated peptides in the same buffers as positive controls. These control samples were also heated at 100 o C for 5 minutes, in keeping with the protocol applied to the test samples. After 48-hour incubation, 3 H-thymidine ( 3 H-TdR; 0.5μCi/well) was added for the last 16 hours of culture. Thymidine uptake was measured as mean counts per minute (cpm) of replicate cultures. A stimulation index (SI; cpm antigen-stimulated T cells/cpm unstimulated T cells) 2.5 was considered positive, indicating adequate peptide presentation for specific T cell proliferation 17. Clonality testing of TCL Clonality of each of the reactive TCL was assessed to exclude the possibility of multiple epitopes being recognized within CPE. TCL were stained with proliferation dye efluor 670 (ebioscience, CA, USA) according to manufacturer s instructions and stimulated with peptide or CPE as described in the T cell proliferation assay section. The T cell receptor Vb repertoire of the reactive TCL was determined using the IOTest BetaMark TCR Vb Repertoire Kit, as per manufacturer s instructions (Beckman Coulter, Krefeld, Germany), with the addition of anti-cd4-apc ef750 antibody (ebioscience) for CD4 + T cell identification and 7AAD (BD Pharmingen, Hamburg, Germany) for the exclusion of dead cells. Results and Discussion We previously identified three short peptides (designated peptides 1, 2 and 3) as candidates for T cell targeted immunotherapy, containing a total of five dominant T cell epitopes of the major peanut allergen Ara h 2 (Figure 1) 17. Here we evaluated the proliferative response of T cells to three of these epitopes within the candidate peptides, before and after exposure to several concentrations and durations of enzymatic digestion with a-amylase, pepsin, chymotrypsin and trypsin to represent the main enzymes encountered following oral delivery. Although a-amylase hydrolyses alpha bonds of large polysaccharides and does not digest proteins, it was included to account for any unforseen effects the enzyme may have on the peptide, interfering with subsequent T cell recognition. 6 Enzyme cutting predictors Predicted sites of digestion by gastrointestinal enzymes were obtained from ExPASy PeptideCutter algorithms ( Predicted digestion sites did not differ between cysteine and serine versions of the peptides. Figure1. Dominant CD4 + T cell epitopes of Ara h 2. Five previously identified dominant CD4 + T cell epitopes of the major peanut allergen Ara h 2 are indicated in bold or underlined (when overlapping) within the entire Ara h 2 protein sequence (gray). Peptide 1: Ara h 2(32-47), Peptide 2: Ara h 2(91-108), Peptide 3: Ara h 2( ). Retained T cell proliferative reactivity of each peptide following enzymatic digestion demonstrated that both native and therapeutic forms of all three peptides were resistant to a-amylase (at all concentrations) and pepsin digestion (at physiological concentrations) (e.g. Figure 2a for native peptide 3). In addition, both forms of peptides 1 and 2 were also resistant to chymotrypsin and trypsin digestion at physiological concentrations, indicating that these two peptides, in therapeutic form, are potential candidates for sublingual/ oral administration. However, the T cell proliferative reactivity of the native form of peptide 3 was lost within minutes of digestion with low (physiological) concentrations of chymotrypsin or trypsin, demonstrating that peptide 3 was highly susceptible to digestion by these enzymes (Figure 2b). Similar results were obtained for digestion of the therapeutic version of peptide 3 with chymotrypsin (data not shown)

67 CHAPTER 6 THERAPEUTIC PEPTIDE SUSCEPTIBILITY TO ENZYMATIC DIGESTION 6 Figure 2. Digestion of peptide 3 by amylase, pepsin, chymotrypsin and trypsin Proliferative responses of TCL 5 to enzyme alone (enzyme), peptide 3 containing cysteine residues before (peptide) and after 5 time points (t=0 t=4) of digestion with A. a-amylase (1:300 enzyme:protein ratio, black bars) and pepsin (1:50 enzyme:protein ratio, grey bars) and B. chymotrypsin (1:100 enzyme:protein ratio, black bars) and trypsin (1:500 enzyme:protein ratio, grey bars); C. Peptide 3 sequence with predicted digestion sites from the PeptideCutter program, indicated by vertical lines for pepsin (black dash), chymotrypsin (grey dash) and trypsin (grey solid). Precise TCL epitope indicated below sequence. Cysteine residues indicated in bold were replaced with serine in the therapeutic version of the peptide. n.d: not determined. The T cell proliferative reactivity to both native and therapeutic forms of peptides 1 and 2 was maintained after enzymatic digestion with trypsin at all concentrations and times tested (data not shown). Upon digestion of the native form of peptide 1 with high concentrations of pepsin or chymotrypsin, T cell proliferative reactivity to the NLRPCEQHL epitope was maintained (TCL 1, Figure 3a). However, T cells specific for a slightly longer version of the epitope (NLRPCEQHLM) no longer responded (TCL 2 and 3, Figure 3a). This indicates a site of digestion between the L and M amino acid near the C-terminus of the peptide, consistent with predicted digestion sites for both pepsin and chymotrypsin using peptide cutter programs (Figure 3b). When the same peptide- specific T cells (TCL 2 and 3) were stimulated with CPE after digestion with the same enzymes, proliferation was either entirely (TLC 2) or partially (TCL 3) maintained (Figure 3c). Figure 3. Digestion of peptide 1 by pepsin and chymotrypsin A. Proliferative responses of TCL 1, 2 and 3 to enzyme alone (enzyme), peptide 1 containing cysteine or serine residues before (peptide), and after five time points (t=0 t=4) of digestion with pepsin (1:5 enzyme:protein ratio, black bars) or chymotrypsin (1:100 enzyme:protein ratio, grey bars). B. Peptide 1 sequence, with predicted digestion sites from the PeptideCutter program indicated by dotted lines for both pepsin (black line) and chymotrypsin (grey line). Precise TCL epitopes indicated below sequence. Cysteine residues indicated in bold were replaced with serine in the therapeutic version of the peptide. C. Proliferative responses of TCL 2 and 3 to enzyme alone (enzyme), CPE before (CPE) and after five time points (t=0 t=4) of digestion with pepsin (1:5 enzyme:protein ratio, black bars) and chymotrypsin (1:100 enzyme:protein ratio, grey bars). Although the TCR Vb specificity of TCL 2 could not be determined with the IOTest Beta Mark TCR Vb Repertoire Kit (which covers 70% of the human TCR Vb repertoire), 85% of T cells proliferated in response to peptide 1. Furthermore, clonality of reactive T cells within TCL 3 was confirmed, therefore the possibility of peptides other than peptide 1 being recognized within CPE was minimal (data not shown). These findings demonstrate that the epitope was protected, to some degree, from enzymatic digestion when located within intact allergen. This is in agreement with previous studies evaluating the stability of major peanut proteins, in which Ara h 2 was found to be more stable and resistant to enzymatic digestion with pepsin, chymotrypsin and trypsin than Ara h 1 and 3. The resistance was attributed to the presence of disulfide bonds within the protein, as reduction with dithiothreitol (DTT) prior to digestion resulted in increased fragmentation of the protein Interestingly, the serine-substituted version of peptide 1 showed a

68 CHAPTER 6 THERAPEUTIC PEPTIDE SUSCEPTIBILITY TO ENZYMATIC DIGESTION higher resistance to enzymatic digestion by pepsin and chymotrypsin, with no or little reduction in proliferation of TCL 2 and 3. The increased resistance was not predicted by peptide cutter programs suggesting the sequence change could be conferring resistance through changes other than direct alteration of the digestion site. Assessment of T cell responses to peptide 2 revealed the opposite effect, with retained T cell proliferation to the native cysteine-containing peptide following pepsin and chymotrypsin digestion, but loss of proliferation following digestion of the serinesubstituted version by both enzymes (Figure 4a). In this instance, the serine substitution directly affected a predicted chymotrypsin digestion site, but not the predicted pepsin sites (Figure 4b). In contrast to peptides 1 and 2, peptide 3 was resistant to high concentrations of pepsin (Figure 2a) in both the native and therapeutic forms (serine data not shown). Previous reports on gastrointestinal enzyme digestion of peanut allergens have shown a strong resistance of native Ara h 2 to digestion with trypsin, resulting in a stable ~10 kda fragment Sen et al. indicated that this fragment was the result of protective disulphide bridge formation, and that reduction of these bonds prior to digestion inhibited the production of any significant enzyme-resistant fragments. Our results show that epitopes within peptides 1 and 2, both present in the 10 kda digestion resistant fragment, were still recognized by T cells after trypsin digestion. In contrast, peptide 3 was no longer recognized, indicating its susceptibility to digestion. Sensitization to peanut allergens through oral ingestion would require persistence of digestion-resistant fragments long enough to elicit a response, as is seen following digestion of Ara h 2 and 6. However, IgE epitopes and dominant T cell epitopes can still be found in fragments of other major peanut allergens such as Ara h The demonstration that peptide 1 was protected from digestion with chymotrypsin and peptide when present within CPE, but not as an isolated peptide (Figure 3c), also suggests that allergens such as Ara h 1 may be better protected when ingested within whole peanut than when directly exposed to enzymes as a purified protein in vitro. Alternatively, it could be that sensitization has occurred to these allergens through alternate routes of exposure such as the skin, where the allergens are more likely to remain intact. In summary, we have demonstrated that peptides derived from dominant T cell epitopes of the major peanut allergen Ara h 2 can resist enzymatic digestion (even at very high concentrations) and therefore have potential to be administered via the sublingual/ oral delivery route for treatment of peanut allergy. Importantly, however, we also report significant changes in the resistance of these peptides to pepsin digestion following cysteine to serine substitutions. Enzyme digestion site predictions suggest that changes in enzyme resistance may not only result from removal of digestion sites, but could also be caused by other conformational changes to the peptide. Given the tendency of cysteines to form disulphide bridges, it is quite likely that substitution with serine residues results in significant conformational changes, which directly affect enzyme access to restriction sites. Our data suggest that such changes could both create and remove enzymatic access to digestion sites. This finding highlights the complexity of peptide conformation and stability and emphasizes the need for careful consideration of multiple factors in the modification of peptides for therapeutic use. The data also highlight a need for further research into the implications of using different peptide delivery routes. 6 Figure 4. Digestion of peptide 2 by pepsin and chymotrypsin A. Proliferative responses of TCL 4 to enzyme alone (enzyme), peptide 2 containing cysteine or serine residues before (peptide) and after five time points (t=0 t=4) of digestion with pepsin (1:5 enzyme:protein ratio, black bars) or chymotrypsin (1:100 enzyme:protein ratio, grey bars). B. Peptide 2 sequence with predicted digestion sites from the PeptideCutter program, indicated by dotted lines for both pepsin (black lines) and chymotrypsin (grey lines). Precise TCL epitope indicated below sequence. Cysteine residues indicated in bold were replaced with serine in the therapeutic version of the peptide