A Recombinant Hypoallergenic Parvalbumin Mutant for Immunotherapy of IgE-Mediated Fish Allergy

Abstract

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients’ IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

Allergenicity attributes of different peanut market types

“…Table 3 shows the IC 50 values which ranged from 0.20 to 0.41 mg/mL. Attempts to make allergen extracts or allergens hypo-allergenic, for example by chemical modification or biotechnological modification, aim to reduce the IgE-binding at least 100-fold, preferably more (Apostolovic et al, 2013;Ibarrola et al, 2004;Swoboda et al, 2007;van Bilsen et al, 2013;Versteeg et al, 2011). The narrow range of IC 50 values reported here (only a 2-fold difference) indicates that the peanut samples have a highly comparable IgE-binding potency.…”

Mapping of Conformational IgE Epitopes with Peptide-Specific Monoclonal Antibodies Reveals Simultaneous Binding of Different IgE Antibodies to a Surface Patch on the Major Birch Pollen Allergen, Bet v 1

“…Sequential IgE epitopes are rare and have been described mainly for food allergens, which may be due to the fact that true food allergens undergo proteolysis in the gastrointestinal tract before they induce an IgE sensitization. However, it has been shown that true food allergens also contain conformational epitopes (9)(10)(11). Several lines of evidence support the concept that IgE epitopes represent mainly conformational epitopes.…”

Hypoallergenic molecules for subcutaneous immunotherapy

“…Carp parvalbumin (Cyp c 1) contains most cross-reactive IgE epitopes present in the family of homologous fish parvalbumin. [23] In 2007, a mutant (m) Cyp c 1, [24] was created with the two functional Ca 2+ -binding sites mutated, rendering the molecule hypoallergenic. As in many similar papers, the tentative conclusion: "this molecule might be an interesting candidate for future immunotherapy" was proposed.…”