Allergenicity and allergen dominance: structural requirements for IgE-dependent recognition by B cells & effector cells

The incidence of allergic disease has increased alarmingly in the UK and is now among the highest in the world. One in six children in the UK suffer from asthma, and life-threatening anaphylactic reactions to common food allergens such as peanuts, once rare, are now increasingly common; peanut allergy in children has doubled in ten years. Allergic reactions occur when apparently innocuous substances are recognised by the immune system and treated as dangerous. The antibodies produced by the immune system in response to these allergenic substances are of a type called IgE, with different properties to the more commonly produced protective (IgG) antibodies with which the body defends itself again bacterial, viral and other ?foreign? invaders. While much is known about IgE antibodies and their mechanism of action, a fundamental question in allergy remains unanswered, namely, why are only certain substances allergenic and others not? Our research has brought us to the point at which we can address this question experimentally, and we propose to do this using peanut allergy as our example. This is a particularly good example not only because of its medical importance, but also because the individual allergenic proteins within the peanut have been identified, and particular allergens have been shown to be ?dominant?, i.e. people with antibodies to these dominant allergens are clinically reactive, while those with antibodies to ?minor? peanut allergens do not react. We aim to understand these differences, and have developed methods to isolate IgE antibodies from the cells of patients with known allergic reactivity. Using a range of techniques that include determining the detailed 3D structures of the complexes formed between the IgE antibodies and the allergens, and studying whether or not the different allergens (individually and in various combinations) can trigger either the cells (called B cells) that produce the IgE antibodies or the cells (mast cells and basophils) that cause immediate hypersensitivity (anaphylactic) reactions. Our hypothesis is that the 3D structure of the allergenic protein, together with the unique structure of the IgE antibody molecule, determines whether a protein is recognized as an allergen or not. We shall also investigate whether bacterial proteins known as ?superantigens?, and a particular (and perhaps unstable) form of IgE, play a role in allergic disease. Our results will underpin immunotherapeutic methods to treat allergic disease, and lead to the development of methods to profile and predict patient?s potential for allergic reactivity.

IgE is the antibody isotype involved in mediating allergic reactions. Our structural and functional studies on B-cell switching to IgE expression, regulation of IgE synthesis, and the mechanism of IgE/receptor interactions (which has spawned an anti-IgE small-molecule drug discovery programme), together with the development of a novel cloning/expression system to produce IgE molecules from human B-cells and plasma cells from allergic subjects, places us uniquely to investigate a fundamental question in allergy, i.e. what makes a protein allergenic? We shall test our hypothesis that the disposition of epitopes on the allergen structure, and their IgE-binding affinity, determine whether they are recognized by the IgE-B-cell receptor (to cause B-cell proliferation and IgE synthesis) and by receptor-bound IgE on mast cells and basophils (to cause immediate hypersensitivity reactions); antigens that are not allergens may fail at one or both of these stages. We further propose that the bent structure of IgE places isotype-specific constraints upon these recognition steps. We shall study peanut allergy as a paradigm, not only for its medical importance, but also because certain peanut allergens are known to be dominant - IgE antibodies to these correlate with clinical reactivity - while others are not. Specifically we shall: 1) Clone and express peanut allergen-specific IgEs from single B-cells and plasma cells from allergic subjects; these will have naturally ?matched? H- and L-chains, which we believe is critical for understanding pathogenicity in allergic disease. 2) Determine the structures of allergen/IgE/receptor complexes to understand the constraints of the Fab regions in IgE. 3) Employ TIRF microscopy with lipid bilayers to study interactions of allergens (and combinations of allergens) with membrane-bound and receptor-bound IgE using the peanut allergen-specific antibodies. 4) Investigate the bias in VH gene usage in the peanut allergen-specific IgEs, explore the role of known B-cell superantigens (SAg) with SAg-specific IgEs from human tissue, and discover whether dominant allergens behave as ?superallergens?. 5) Determine the structural basis of ?cytokinergic? IgE and its pathogenic potential in allergic disease by comparing IgEs with matched and unmatched VH/VL domains from allergic patients. 6) Correlate the structural studies with functional assays of IgE-BCR signalling and mast cell/basophil activation, using peanut allergens with combinations of the allergen-specific IgEs. Answers to the fundamental questions addressed here in peanut allergy will underpin immunotherapeutic and potentially disease-modifying ?cures? for other allergies, and may lead to the development of clinically relevant diagnostic tools to profile patient reactivity to dangerous allergens.