Food, drug, insect sting allergy, and anaphylaxis
Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen

https://doi.org/10.1016/j.jaci.2011.04.020Get rights and content

Background

Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy.

Objective

We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties.

Methods

A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice.

Results

Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced.

Conclusion

Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy.

Section snippets

Allergen preparation

Native Pru p 3 (nPru p 3) was purified from a freshly prepared peach peel extract, as previously described.30, 31 Purified nPru p 3 was reduced and alkylated (R/A) and subsequently dialyzed against PBS buffer to disrupt the tertiary structure of Pru p 3.32 A protocol for the reduction and alkylation is described in the Methods section of this article’s Online Repository at www.jacionline.org.

The protocols for circular dichroism spectroscopy and protease digestion are also described in the

Unfolded Pru p 3 reduces resistance to gastrointestinal digestion

A Pru p 3 folding variant was generated by means of disruption of disulfide bonds on reduction and alkylation. R/A Pru p 3 showed a reduced apparent molecular mass of 6 kDa in contrast to nPru p 3 (9 kDa) on SDS-PAGE (Fig 1, A). Both samples were investigated for structural integrity and stability against gastrointestinal digestion. The circular dichroism spectrum of R/A Pru p 3 revealed a typically unfolded protein in contrast to nPru p 3, showing 2 minima at 208 nm and 222 nm and indicating

Discussion

nsLTPs are important allergens not only in Rosaceae fruits but also in other plant foods, such as vegetables, cereals, and nuts, as well as in different kinds of pollen.23, 24, 36 The protein structure of lipid transfer proteins, an all α-type compact structure with 4 α-helices, is stabilized by 4 intramolecular disulfide bonds. Lipid transfer proteins retain most of their immunogenic and allergenic capacity after passing through the gastrointestinal tract and are capable of eliciting severe

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    Supported in part by Paul-Ehrlich-Institut and the Austrian Science Fund, projects SFBF1807-B13 and W1212.

    Disclosure of potential conflict of interest: B. Bohle receives research support from the Austrian Science Fund and the Christian Doppler Research Society. S. Vieths is an associate for the Institute for Product Quality; received an honorarium as an expert reviewer from the Food Allergy Resource and Research Program; receives honorarium for an expert seminar from Fresenius Academy; receives research support from the European Union, the German Research Foundation, the Research Fund of the German Food Industry, Monsanto Company, Pioneer Hi-Bred International, the Food Allergy Research and Resource Program, the European Directorate for the Quality of Medicines and Health Care, and the German Ministry for Education and Research; is a member of the executive committee for the European Academy of Allergy and Clinical Immunology; has served as chairman and as secretary of the Allergen Standardization Subcommittee for the International Union of Immunological Societies; is a registered expert for the European Agency for the Evaluation of Medicinal Products and the European Pharmacopoeia Commission; is chairman of Technical Committee 275 Working Group 12 Food Allergens for CEN; and is a member of the Food Allergy Working Group for the German Society for Allergy and Clinical Immunology. The rest of the authors have declared that they have no conflict of interest.

    These authors contributed equally to this work.

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