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

Abstract

The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90–98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use.

Cloning, Expression, and Mapping of Allergenic Determinants of αS1-Casein, a Major Cow’s Milk Allergen

“…␣S1-casein is considered as a true food allergen, also classified as class I food allergen, which is comparable to the major fish allergen parvalbumin (31), the major peanut allergens (Ara h 1, Ara h 2, Ara h 3) (32), and the major shrimp allergen (33), and it can induce severe and life-threatening anaphylactic reactions. Interestingly, we found that Ͼ50% of patients with gastrointestinal symptoms, skin and respiratory symptoms, or severe systemic reactions, but fewer patients with only skin symptoms, showed IgE reactivity to r␣S1-casein (Fig.…”

Expression of a Biologically Active GFP-αS1-Casein Fusion Protein in Lactococcus lactis

“…Of these four proteins, α S1 -casein thought to be the most potent for inducing a specific IgE response [1]. α S1 -casein is considered as a true food allergen, also classified as class I food allergen, which is comparable to the major fish allergen parvalbumin [21], the major peanut allergens [13], and the major shrimp allergen [15], and it can induce severe and life-threatening anaphylactic reactions. α S1 -casein, which is a single-chain linear phosphoprotein of 199 amino acid residues, has only a small amount of secondary structure and lacks disulfide bonds, which results in a reduction of tertiary interactions [8].…”

From Molecule Studies of Allergens to Development of Immunotherapy of Allergies

“…Reese et al [4] constructed a hypo-allergen VR9-1 by converting 12 critical amino acids located in the five IgE epitope regions in the shrimp tropomyosin Pen a 1 previously mapped [14] into homologous sequences of the non-allergenic vertebrate tropomyosin. Results showed that VR9-1 exhibited reduced allergenic potency up to 90-98% in a humanized rat basophil leukemia cells (RBL) assay.…”

“…T cell epitopes refer to the short peptide fragments activating Th2 via MHC class II molecules while B cell epitopes, or IgE-binding epitopes, refer to the regions recognized by IgE on the allergen. With the identification of B cell epitopes at the molecular level, hypo-allergens possessing reduced allergenicity can be constructed through the introduction of point mutations on these IgE-binding regions [3,4]. The lack of IgE reactivity reduces the risk of these hypo-allergens to form cross-links with IgE and thus prevents the development of allergic side effects during treatment [5].…”