Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen.

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.

Comparison of IgE-binding capacity, cross-reactivity and biological potency of allergenic non-specific lipid transfer proteins from peach, cherry and hazelnut.

BACKGROUND: Whether the observed clinical pattern of non-specific lipid transfer protein (nsLTP)-mediated food allergies is attributable to a primary sensitization by Pru p 3 from peach and subsequent cross-reactivity with Rosaceae- and non-Rosaceae-derived foods expressing homologous allergens is still unclear. OBJECTIVE: To investigate the allergenic properties of nsLTPs from Rosaceae and non-Rosaceae foods. METHODS: In peach-, cherry- or hazelnut-allergic patients, prevalence of sensitization, IgE-binding capacity, cross-reactivity and allergenic potency of Pru p 3 was compared with Pru av 3 (cherry) and Cor a 8 (hazelnut). RESULTS: Frequency of sensitization to corresponding nsLTPs was 88, 85, and 77% in peach-, hazelnut- and cherry-allergic patients, respectively. Concomitant allergic reactions to cherry and hazelnut were reported in 51 and 44% of peach-allergic patients, respectively. In contrast to cherry allergy, hazelnut allergy was not strictly associated to peach allergy. Sensitization to Cor a 8 or Pru av 3 was strongly correlated with IgE reactivity to Pru p 3, even when subjects tolerated peach. Specific IgE was highest for Rosaceae LTPs, and cross-inhibition experiments confirmed a stronger IgE-binding capacity of Pru p 3 than Cor a 8. The biological potency of Pru p 3 and Pru av 3 was similar but stronger for both nsLTPs than that of Cor a 8. CONCLUSION: Clinical cross-reactivity of food-allergic patients in the Mediterranean area is likely attributed to a primary sensitization to Pru p 3 and serological cross-reactivity with homologous food nsLTPs. In comparison to Cor a 8, Rosaceae nsLTPs showed a stronger IgE-binding capacity and allergenic potency indicating a different epitope pattern.

Identification and characterization of the major allergen of green bean (Phaseolus vulgaris) as a non-specific lipid transfer protein (Pha v 3).

BACKGROUND: Green bean (GB) has been reported to cause allergic reactions after ingestion, contact or inhalation of particles deriving from processing or cooking. Up-to-date no food allergens have been fully characterized in GB. OBJECTIVE: To characterize the GB major allergen(s) on a molecular level and to verify the involvement of non-specific lipid transfer proteins (nsLTPs) in GB allergy. METHODS: We recruited 10 Spanish patients reporting adverse reactions to GB. Skin prick tests, specific IgE detection and oral provocation were performed. Two nsLTP cDNAs were cloned from GB and over-expressed in Pichia pastoris. The recombinant LTPs (rLTPs) were characterized by circular dichroism spectroscopy and IgE-binding assays (immunoblotting and ELISA) with the patients' sera. Three natural LTPs (nLTPs) were further purified from GB fruit by chromatography. In vitro histamine release test was applied to compare the allergenic potency of rLTPs and nLTPs. RESULTS: Oral provocation test confirmed GB allergy. A 10kDa protein in GB extract was recognized by 80% of the sera and identified as nsLTP. The two rLTPs (named LTP1a and LTP1b), share 61.3% aa identity and present the typical nsLTP-like secondary structure. The IgE-binding and histamine release assays provided evidence that rLTPs and nLTPs possess different allergenic potency. CONCLUSIONS: nsLTP (Pha v 3) is the major allergen in GB and constitute a potential risk for patients affected by LTP-syndrome. GB encodes for several LTPs with different immune reactivity.