Peptide - Based Immunotherapy for Food Allergy
2. Background Information
The gastrointestinal mucosa is constantly exposed to a multitude of innocuous food antigens that do not usually elicit an overt immune response. The reasons why certain individuals are more suscepti- ble to developing an allergic response to food anti-
2.2. Roles of T Lymphocytes in Food Allergy The contribution of T lymphocytes — and more par- ticularly, CD4 + T cells — is pivotal in facilitating the production of allergen - specifi c IgE, and in their role of effector cells secreting autocrine and paracrine factors, of which cytokines are the main players. As mentioned earlier, the cytokine environment encoun- tered by a naive T helper cell (Th0) plays a promi- nent role in determining whether Th0 will adopt a Th1 or a Th2 phenotype. While Th1 cells play a prominent role in cell - mediated immunity with the secretion of cytokines such as IFN - γ and TNF - β , which promote the development of cytotoxic T lym- phocytes (CTLs) and macrophages (Chehade and Mayer 2005 ), Th2 cells primarily promote humoral immune responses, with the production of cytokines such as IL - 4 and IL - 13 (IgE synthesis), but also IL - 5 (eosinophil proliferation) and IL - 9 (mast cell activa- tion). A key feature of the Th1/Th2 balance is that the two cell subsets are capable of counter - regulating one another (Mosmann and Coffman 1989 ; Mosmann et al. 1986 ). In recent years, although the paradigm of the Th1/Th2 balance is still recognized, more complex changes in lymphocyte responses were shown to arise in the course of an allergic response.
The Th1/Th2 model has, therefore, evolved to encompass the heterogeneous and complex popula- tions of regulatory T cells (Tregs). The revised model is schematically represented in Figure 8.2 . This has spurred interest in the prospective use of Tregs for the treatment of allergic conditions (Elkord 2006 ). Indeed regulatory T cells, previously known as suppressor T cells, are believed to play a critical role in the maintenance of immunological tolerance (Verhagen et al. 2006 ) at mucosal surfaces. They have been defi ned as a class of immune cells able to “ actively control or suppress the function of other immune and non - immune cells, generally in an inhibitory fashion ” (Prioult and Nagler - Anderson 2005 ). Within this conceptual framework, it was suggested that a disruption of Tregs activity may promote the development of a Th2 - skewed allergic response.
Mechanistically, regulatory T cells have been reported to exert their inhibitory activity on T helper the lumen to directly sample dietary antigens
(Chehade and Mayer 2005 ).
2.1.2. Allergic Response to Food Antigens: A Two - Phase Mechanism The etiology of IgE - mediated food allergy is classically described as a two - phase phenomenon: (1) the sensitization phase, without symptoms, during which the immune system is primed, resulting in the production of antibodies belonging to the immunoglobulin E class, and (2) the elicitation phase with specifi c clinical responses (i.e., allergic symptoms), which occurs after subse- quent exposure to the offending compound.
During the sensitization phase, it is believed that food antigens traverse the intestinal barrier and are captured by the underlying immune cells. They are processed and presented by a specialized class of immune cells known as antigen - presenting cells (APCs) for presentation to T lymphocytes. The cyto- kine environment encountered by a naive CD4 + T helper cell (Th0) plays a prominent role in determin- ing whether Th0 will exhibit a Th1 - or Th2 - cell phenotype. Allergic individuals preferentially develop an interleukin (IL) - 4 - rich microenvironment that drives the immune response toward a Th2 - biased response, which in turn promotes IgE production by B cells. Allergen specifi c - IgE then binds to high - affi nity receptors (Fc ε RI) present on the surface of mast cells and basophils. The infl uences responsible for an IL - 4 dominant environment are not completely understood, but innate immune cells such as natural killer (NK) and NK - T cells have been suggested as candidate sources of IL - 4 (Yoshimoto et al. 1995 ).
Upon subsequent ingestion of the food allergen (elicitation phase), allergenic fragments (epitopes) bind to receptor - bound IgE present on mast cells and basophils, triggering the aggregation of the recep- tors and the release of infl ammatory and vasoactive mediators such as leukotrienes, prostaglandins, and histamine. Simultaneously, allergen presentation by APCs leads to the rapid stimulation of sensitized Th2 cells, as well as the local recruitment and acti- vation of effector cells such as eosinophils and baso- phils (Figure 8.1 ). These events are responsible for the clinical reactions characteristics of an allergic response.
CD25, and FOXP3; and (2) inducible Tregs acti- vated in the periphery after antigen stimulation.
Inducible Tregs include T regulatory (Tr) - 1 cells, characterized by production of both IL - 10 and TGF - β ; Th3 cells, characterized by their prominent pro- duction of TGF - β (Taylor et al. 2006 ); and also a cells (both Th1 and Th2) and their downstream
effectors by either direct contact or by production of suppressive cytokines (for example, IL - 10 and TGF - β ) (Stock et al. 2006 ). Two major lineages of Tregs have been described: (1) natural thymus - derived Tregs, which constitutively express CD4,
Figure 8.1. Schematic representation of the sequence of events leading to type I hypersensitivity. Adapted from Prioult and Nagler - Anderson 2005 ; Kaza et al. 2007 . Note: A complex bidirectional interplay exists between mast cells/granulocytes and CD4+T cells (Ahern and Robinson 2005 ), but the phenomenon has not been illustrated in the fi gure.
include birds (e.g., chicken), crustaceans (e.g., crab), and red meat (e.g., beef), while plant groups include the apple family, grass family (e.g., wheat), legume family (e.g., peanut), and walnut family (Burks 2004a ). Proteins, lipids, and carbohydrates are the major constituents of foods; however, food allergens have been mainly characterized as glycoproteins having a molecular mass of 10 – 60 kDa (Bernstein et al. 2003 ) and soluble in water (i.e., albumins) or saline solutions (i.e., globulins) (Burks 2004b ).
They commonly own attributes that preserve them from the destructive effects of low pH, proteolysis, and thermal treatments (Bredehorst and David 2001 ; Huby et al. 2000 ; Breiteneder and Ebner 2000 ; Breiteneder and Mills 2005 ), but exceptions exist, especially for allergens found in fruits and vegeta- bles (Bannon 2004 ). Other properties, such as the presence of disulfi de bonds, N - glycosylation, struc- tural aggregation, rheomorphic or repetitive struc- tures, and lipid - binding capacity, are also believed to contribute to their inherent stability and, there- fore, to their allergenicity (Breiteneder and Mills 2005 ). Structurally, comparisons of primary amino acid sequences failed to reveal any typical pattern (Ivanciuc et al. 2003a, 2003b ). On the other hand, a recent study based on in silico approaches suggested that plant food allergens may be classifi ed into four structural superfamilies based on their conserved native surface features (Jenkins et al. 2005 ) and sup- ports the view that conserved spatial motifs of food proteins may be major determinants in their allerge- nicity (Sampson 2005 ). Features common to food allergens were gathered into a bioinfomatics data- bank and exploited in an attempt to create prediction tools for allergenicity (Kleter and Peijnenburg 2002 ; Ivanciuc et al. 2003a ; Stadler and Stadler 2003 ; Soeria - Atmadja et al. 2006 ); however, to date unique structural motifs or determinants responsible for food allergenicity have yet to be identifi ed.
2.4. Food Allergen B - and T - cell Epitopes 2.4.1. Defi nitions The immune system classi- cally recognizes antigens through two classes of molecules: (1) immunoglobulins, in the form of soluble or membrane - bound molecules produced by class of peripheral FOXP3 - expressing CD4 + CD25 + -
T cells. A major limitation in the study of Treg functions and cellular activities has been the lack of reliable and specifi c Treg molecular markers. This was further complicated by reports describing various types of Treg cells including innate immune cells such as natural killer T (NKT) cells, γ δ - T cells, CD8+ T cells, and B - cell subsets (Verhagen et al.
2006 ; Nagler - Anderson et al. 2004 ), as well as a newly reported subset of CD4+ T cells known as Th17 cells (Oboki et al. 2008 ).
There is undeniable evidence that T cells are essential in the pathogenesis and regulation of aller- gic reactions. However, it is important to note that allergic responses result from interactions involving many other players of the immune system (both cells and soluble mediators), contributing to the complexity of the allergic process and hindering the development of effi cient curative strategies.
2.3. Molecular Properties of Food Allergens Foods are typically derived from both animal and vegetable sources. Examples of animal groups
Figure 8.2. Currently accepted view on T - cell regulation.
An immune balance exists between the different subsets of CD4+ T cells involving Th1, Th2, and Treg cells. A disturbance in the balance can lead to a nonfavorable outcome for patients, such as an allergic response. Several populations of Treg have been reported, for example, IL - 10 producing inducible Tr1 cells, TGF - β - producing Th3 cells, naturally occurring and inducible CD4+CD25+FOXP3 Treg cells. TGF - β and IL - 10 could exert direct and/or indirect inhibitory effects on neighboring cells and are believed to play an important role in the induction of T - cell tolerance. Adapted from Jeurink and Savelkoul 2006 .
epitopes of food allergens to provide a better under- standing of their recognition by immune cells.
Knowledge of food allergen epitopes can provide important information on the pathogenesis of food allergy, and it can also be a guide for the develop- ment of pertinent experimental and therapeutic approaches for the prevention or the suppression of allergic disorders (Bohle 2006 ).
2.4.2. Allergen Epitope Identifi cation A detailed description of the methods available for the identi- fi cation of allergen epitopes (or epitope mapping) is beyond the scope of this chapter. A brief overview is presented in this section, but comprehensive information is available elsewhere (Morris 1996 ).
2.4.2.1. T - cell Epitope Mapping Activation and proliferation of CD4 + T lymphocytes is initiated upon contact with an APC presenting T - cell epit- opes complexed with MHC class II molecules.
This cognate recognition is critical to generation of the adequate immune response. Progress in the past decade allowed the identifi cation of T - cell epitopes in any allergen with a known primary sequence, and such investigation has been initiated for major food allergens. Experimental approaches toward B lymphocytes, or (2) T - cell receptors or TCRs,
present at the surface of T lymphocytes in the context of major histocompatibility complex presen- tation by APCs. The molecular regions specifi cally recognized by immunoglobulins and TCRs are com- monly known as B - cell and T - cell epitopes, respec- tively. B - cell epitopes can be either continuous (i.e., linear amino acid sequences) or discontinuous (i.e., residues forming the epitope arise from different positions brought together by folding). B - cell epit- opes do not have a defi ned length and can vary from 5 to 20 amino acids in length. The importance of linear vs. conformational epitopes has been the object of several studies, and it was proposed that the recognition of linear epitopes by certain patients was predictive of a persistent form of food allergy in these individuals (Mine and Yang 2008 ). On the other hand, the length of T - cell epitope peptides associated with MHC class II molecules is much more restricted and varies from 10 to 20 amino acids (Zeiler and Virtanen 2008 ). T - cell epitopes are strictly linear sequences as a result of the degra- dation of antigens through the cytosol of APCs (Figure 8.3 ).
In the past few years, a great deal of research has focused on the characterization of B - and T - cell
Figure 8.3. Schematic representation of antigen B - and T - cell epitopes or determinants.
minants are assayed by ELISA or by methods in which the antibody has been labeled with a fl uorochrome or other type of readily detectable reagent. The library of peptides can be provided on a solid support (e.g., SPOTS ® membranes or PEPSCAN on paper discs) or in the form of peptide libraries displayed by bacteriophages (Zegers et al.
1995 ).
On the other hand, methods to identify confor- mational B - cell epitopes are based on the use of x - ray crystallography often combined to site - directed mutagenesis. This latter method, however, requires a high degree of purity of the antigen and remains complex and time consuming. Alternatively, recent technology based on the use of chemical
“ scaffolds ” (i.e., Chemically Linked Peptides on Scaffolds, or CLIPS ™ ), onto which one or more peptides are attached to closely resemble the native structure of protein, may allow identifi cation of B - cell discontinuous epitopes yet to be tested for food allergens. Similar to T - cell epitopes, a multi- tude of predictive methods for identifi cation of B - cell epitopes have been documented based on physicochemical and structural propensity scales (e.g., hydrophilicity, fl exibility, exposure to the solvent, and occurrence of charged residues). Their limitations have been discussed in recent reviews (Ponomarenko and Bourne 2007 ; Roggen 2006 ).
2.5. Current Management of Food Allergy Current approaches toward the management of food - induced allergic conditions mainly consist of dietary avoidance and pharmacological interven- tions. Compliance with an avoidance regimen is often diffi cult because many prepackaged or pre- pared foods may contain minute amounts of milk, egg, or nut proteins, commonly referred to as “ hidden allergens, ” in a dose suffi cient to provoke allergic symptoms (Arshad 2001 ; Zeiger 2003 ). These hidden allergens may result from allergen contami- nation during food processing, misformulation, or erroneous labeling (Puglisi and Frieri 2007 ). More importantly, the elimination of essential foods for an extended period of time may not represent a viable identifi cation of T - cell epitopes rely traditionally on
the preparation of cell cultures. Cultures are pre- pared from peripheral blood mononuclear cells (PBMCs) in humans, and from lymph nodes or spleen when using murine models. Proliferation and cytokine assays are often conducted upon stimula- tion with libraries of overlapping sequences cover- ing the entire primary sequence of the protein of interest to allow localization of the immunogenic regions. In humans, establishment of allergen - specifi c T - cell lines or clones is often preferred due to the low frequency of specifi c T cells and the weak responses obtained with PBMC cultures. The prepa- ration of T - cell clones permits a detailed analysis of epitope specifi city and cell phenotype (Zeiler and Virtanen 2008 ), but can be labor intensive and tech- nically demanding. Alternative epitope mapping tools include the use of tetrameric MHC complexes and fl ow cytometry (Hoffmeister et al. 2003 ).
Computer - based programs such as TEPITOPE (Bian et al. 2003 ) or COREX/BEST (Melton and Landry 2008 ) have also been designed to predict MHC class II binding regions and were shown to be applicable to allergenic proteins. While such algo- rithms may be useful for predicting epitopes prior to conducting the actual mapping experiments, com- plete agreement with data generated by in vitro methods is not always observed.
2.4.2.2. B - cell Epitope Mapping Food allergens are a group of antigens classically defi ned by their ability to induce the production of specifi c IgE. The crosslinking of allergen specifi c IgE at the surface of mast cells or basophils is the event responsible for the clinical symptoms characteristic of an aller- gic response. As such, characterization of IgE - binding sites (or B - cell epitopes) is considered an important step toward the characterization of aller- gens. Identifi cation of antigenic determinants pri- marily requires (1) knowledge of the amino acid sequence of the protein and (2) the use of specifi c antibodies (monoclonal or polyclonal, obtained from animal or patient sera) for the antigen of inter- est. Indeed, conventional methods for identifi cation of B - cell linear epitopes rely on the use of overlap- ping linear peptide fragments where antigenic deter-
effects. For this reason, new approaches are being investigated. In line with the scope of this book, the utilization of peptides will be the main topic described in this section. The concept behind pep- tide - based immunotherapy is not new and stems from the development of peptide - based vaccines designed to elicit a protective immune response toward pathogens such as viruses, bacteria, and parasites. Such vaccines are designed to incorporate immunoreactive regions derived from the protein native structure and give rise to a specifi c immune response toward the intact protein. In the context of food allergy, the terminology associated with a good therapeutic prognosis remains controversial and is often referred to as “ desensitization ” or “ tolerance ” induction (Niggemann et al. 2006 ). A recent mono- graph emphasized that discrimination should be made between true “ tolerance, ” which refers to situ- ations where the food may be ingested without trig- gering any allergy symptoms even during elimination periods, and “ desensitization, ” which refers to cases where the allergen is ingested without symptoms during treatment but requires a maintenance regimen (Nowak - Wegrzyn and Sicherer 2008 ). Within this conceptual framework, a PIT - based approach aims at designing so - called “ tolerogenic ” peptides (Pecquet et al. 2000 ), that is, peptides capable of inducing a status of immune tolerance toward the allergen(s) of interest. Specifi c to food allergy, the rationale of PIT relies on the use of nonanaphylactic (synthetic or not) peptides, unable to crosslink IgE molecules (Ferreira et al. 2004 ). The reduced ability of peptides to react with IgE will prevent not only mediator release from mast cells and baso- phils (Figure 8.1 ) but also the uptake on IgE via Fc epsilon receptors of antigen presenting cells, which may contribute to an amplifi cation of the allergic response.
3.1.2. Strategies Potential PIT - based strategies for food allergy are presented in the following para- graphs based on the source of peptides (i.e., synthe- tisized vs. enzymatically obtained peptides) and on the nature of the immunogenic sequences contained in the preparation (i.e., T - cell epitopes vs. B - cell epitopes).
solution, particularly in young children, as it may lead to malnutrition (Sampson 1999 ). Additionally, recent studies have suggested that an elimination diet may not represent the safest alternative for aller- gic patients and may in fact have a deleterious impact by lowering the patient reactivity threshold (Morisset et al. 2007 ; Rolinck - Werninghaus et al.
2005 ). Current pharmacological treatments mainly induce symptomatic relief through the use of agents such as antihistamines, steroids, and, in severe cases, epinephrine (Thompson and Chandra 2002 ).
2.6. Overview of Novel Immunotherapeutic Strategies
The current lack of effi cient treatments for food allergy has prompted the development of novel pre- ventive and therapeutic approaches that can provide safer alternatives for combatting food allergies. This chapter focuses on the key fi ndings brought by pep- tide - based approaches. The interested reader is referred to a number of recent reviews focusing on the various immunotherapeutic strategies currently investigated for the treatment and prevention of IgE - mediated food allergy (Sicherer and Sampson 2008 ; Nowak - Wegrzyn and Sicherer 2008 ), as are listed in Table 8.1 . Immunotherapeutic approaches can be divided into specifi c and nonspecifi c approaches.
Specifi c immunotherapy (SIT) targets the individual allergens responsible for the patient ’ s disease, whereas nonspecifi c immunotherapy aims at modu- lating the immune system in an allergen - indepen- dent manner (Mine and Yang 2008 ). Specifi c immunotherapies can be further divided into those using native forms of food allergens and those using recombinant or altered forms of allergens.
3. Rationale, Strategies, and Potential