Engineering, Characterization and in vitro Efficacy of the Major Peanut Allergens for Use in Immunotherapy

Abstract

Background: Numerous strategies have been proposed for the treatment of peanut allergies, but despite the steady advancement in our understanding of atopic immune responses and the increasing number of deaths each year from peanut anaphylaxis, there is still no safe, effective, specific therapy for the peanut-sensitive individual. Immunotherapy would be safer and more effective if the allergens could be altered to reduce their ability to initiate an allergic reaction without altering their ability to desensitize the allergic patient. Methods: The cDNA clones for three major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, have been cloned and characterized. The IgE-binding epitopes of each of these allergens have been determined and amino acids critical to each epitope identified. Site-directed mutagenesis of the allergen cDNA clones, followed by recombinant production of the modified allergen, provided the reagents necessary to test our hypothesis that hypoallergenic proteins are effective immunotherapeutic reagents for treating peanut-sensitive patients. Modified peanut allergens were subjected to immunoblot analysis using peanut-positive patient sera IgE, T cell proliferation assays, and tested in a murine model of peanut anaphylaxis. Results: In general, the modified allergens were poor competitors for binding of peanut-specific IgE when compared to their wild-type counterpart. The modified allergens demonstrated a greatly reduced IgE-binding capacity when individual patient serum IgE was compared to the binding capacity of the wild-type allergens. In addition, while there was considerable variability between patients, the modified allergens retained the ability to stimulate T cell proliferation. Conclusions: These modified allergen genes and proteins should provide a safe immunotherapeutic agent for the treatment of peanut allergy.

Reduced Allergenic Potency of VR9-1, a Mutant of the Major Shrimp Allergen Pen a 1 (Tropomyosin)

“…To date, immunotherapy of food allergies is not the course of treatment, because potent allergenic foods, such as peanut, tend to induce severe adverse reactions (32,33). However, reducing or abolishing the IgE Ab binding capacity and preserving T cell reactivity are currently discussed as a promising new strategy for the treatment of allergies (34,35). Because Pen a 1 is responsible for at least 75% of the shrimp-specific IgE Abs (1), the IgE Ab-binding capacity of Pen a 1 has to be reduced substantially if it is to be used as a therapeutic reagent.…”

An Evolutionary Perspective on Human Cross-sensitivity to Tree Nut and Seed Allergens

“…Ewan (1996) tested 62 patients for cross-reactivity to six nuts: peanut, Brazil nut, almond, hazelnut, walnut, and cashew nut, but did not report the species involved in each treatment. Table based on references (Altenbach et al 1987;Arshad et al 1991;Fernandez et al 1995;Tariq et al 1996;Marinas et al 1998;Moneret-Vautrin et al 1998;Sutherland et al 1999;Teuber and Peterson 1999;Teuber et al 1999Teuber et al , 2003Diaz-Perales et al 2000;Bannon et al 2001;Poltronieri et al 2002;Wang et al 2002;de Leon et al 2003;Roux et al 2003;Asero et al 2004;Lerch et al 2005;Crespo et al 2006;Benito et al 2007;Willison et al 2008;Ahn et al 2009;Breiteneder 2009;Garino et al 2010;Allergen.org 2014). VOLUME 33 (2) Tree Nut Allergenscross-reactivity, though additional comparisons are needed to investigate this hypothesis.…”

A Phase 1 Study of Heat/Phenol Killed, E. coli-Encapsulated, Recombinant Modified Peanut Proteins Ara h 1, Ara h 2, and Ara h 3 (EMP-123) for the Treatment of Peanut Allergy

“…Specifically, three recombinant modified peanut proteins (Ara h 1, Ara h 2, and Ara h 3) were modified by amino acid substitutions to disrupt common IgE binding sites as previously described (20)(21)). These modified proteins are then separately expressed in E. coli strain BLR(DE3), and the E. coli are subsequently killed using heat and phenol.…”