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.

Pichia pastoris is superior to E. coli for the production of recombinant allergenic non-specific lipid-transfer proteins.

Non-specific lipid-transfer proteins (nsLTP) from food and pollen are clinically important allergens, especially in patients recruited from the Mediterranean area. For the use of recombinant nsLTPs in allergy diagnosis and preclinical allergy studies the preparation of nsLTPs in a properly folded and biologically active form is required. Using hazelnut nsLTP (Cor a 8) as a model allergen, heterologous over-expression in Escherichia coli and Pichia pastoris was compared. Recombinant Cor a 8 derived from E. coli and P. pastoris was purified by IMAC and SEC or ammonium sulphate precipitation followed by IEC and SEC, respectively. The recombinant proteins were characterized with regard to IgE-binding by immunoblotting and ELISA, structure by N-terminal sequencing, CD-spectroscopy and LS and to their biological activity using an in vitro basophil histamine release assay. Purification of hazelnut nsLTP from bacterial lysate under native conditions resulted in a low yield of Cor a 8. In addition, the preparation contained non-IgE-reactive aggregations besides the IgE-reactive monomer. In contrast, the yield of rCor a 8 produced in P. pastoris was approximately 270-fold higher and impurities with oligomers have not been detected. Purified monomeric Cor a 8 from bacteria and yeast showed similar IgE-antibody reactivity and secondary structures, and both were capable of inducing histamine release from basophils. In summary, P. pastoris is superior to E. coli as expression system for the production of large quantities of soluble, properly folded, and biologically active rCor a 8.

Molecular characterisation of Lac s 1, the major allergen from lettuce (Lactuca sativa).

BACKGROUND: IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce (Lactuca sativa), but has not yet been investigated on the molecular basis. OBJECTIVE: Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3. METHODS: Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay. RESULTS: Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identity (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays. CONCLUSION: Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.

Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression.

Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.

Molecular basis of pollen-related food allergy: identification of a second cross-reactive IgE epitope on Pru av 1, the major cherry (Prunus avium) allergen.

Birch (Betula verrucosa) pollen-associated food allergy is a well-characterized syndrome, which is due to the cross-reactivity of IgE antibodies to homologous allergens in various foods. One crossreacting area on the major birch pollen allergen Bet v 1 and its homologue in cherry (Prunus avium) Pru av 1 has already been identified. This is the so-called 'P-loop' region, which encompasses amino acid residues around position 45 and is found on the two virtually identical tertiary protein structures. We tried to determine an additional IgE cross-reacting patch on Pru av 1 and Bet v 1. The putative IgE-binding region on Pru av 1 was localized with a mAb (monoclonal antibody) that was generated against Bet v 1, and cross-reacts with several Bet v 1 homologues in food and inhibits the binding of patients' IgE to Pru av 1. mAb reactivity pattern was analysed and amino acid positions 28 and 108 of Pru av 1 were selected and mutated by site-directed mutagenesis. The Pru av 1 mutants were produced as recombinant proteins and characterized for their folding, mAb- and IgE-binding capacity and allergenic potency with a cellular assay using the humanized rat basophilic leukaemia cell line RBL-25/30. Amino acid position 28 is involved in a second major IgE-binding region on Pru av 1 and probably on Bet v 1. The identification of this second major IgE-binding region is an essential prerequisite to understand the phenomenon of cross-reactivity and its clinical consequences, and to produce hypoallergenic proteins for an improved immunotherapy of type I allergy.

Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.

Immune reactivity of candidate reference materials.

Immune reactivity is a key issue in the evaluation of the quality of recombinant allergens as potential reference materials. Within the frame of the CREATE project, the immune reactivity of the natural and recombinant versions of the major allergens of birch pollen (Bet v 1), grass pollen (Phl p 1 and 5), olive pollen (Ole e 1), and house dust mite (Der p 1 and 2, and Der f 1 and 2) was analysed. The IgE binding capacity of the allergens was studied by direct RAST and RAST inhibition, and their biological activity by basophil histamine release, using sera of allergic patients selected across Europe. For birch pollen, rBet v 1 is an excellent mimic of the natural allergen. For grass pollen, rPhl p 1 showed a significant lower IgE reactivity and was not considered a suitable candidate, whereas rPhl p 5a exhibited an immune reactivity closer to that of its natural counterpart. For olive, rOle e 1 had a lower IgE binding capacity in RAST but a higher biological activity in histamine release. For house dust mite, recombinant group 1 allergens were significantly less potent than their natural counterparts, but recombinant group 2 allergens were close mimics of their natural homologues.

Skin prick tests reveal stable and heritable reduction of allergenic potency of gene-silenced tomato fruits.

BACKGROUND: Today, for patients with food allergy, the only possibility to prevent allergic reactions is avoidance of the allergenic food. Genetic engineering of hypoallergenic plants by means of RNA interference (RNAi) could be an approach to improve the quality of life of subjects with food allergy. OBJECTIVES: We sought to achieve stable inhibition of expression of the allergenic nonspecific lipid transfer protein Lyc e 3 in tomato and to analyze the reduction of allergenicity in vitro by using histamine release assays and in vivo by using skin prick tests with transgenic tomato fruits. METHODS: Gene silencing was performed by means of RNAi and monitored by using Western blotting with nonspecific lipid transfer protein-specific antibodies and sera from patients with tomato allergy. Dose-dependent basophil histamine release assays, prick-to-prick skin testing, and determination of endogenous histamine content were performed with fruits harvested from plants of the first and second generation to assess the allergenic potency compared with that of wild-type fruits. RESULTS: We demonstrated that silencing of Lyc e 3 by means of RNAi contributes to reduced skin reactivity and is passed on to the next generation of fruits. A significant reduction of allergenic potency was determined in vitro and confirmed by using skin prick tests. CONCLUSION: Taken together, these results indicate that RNAi technology is an effective tool to generate foods with reduced allergenicity. CLINICAL IMPLICATIONS: Allergen-reduced plant foods might allow reduction of dietary restrictions for patients allergic to panallergen families.

Orange-induced skin lesions in patients with atopic eczema: evidence for a non-IgE-mediated mechanism.

Oranges are suspected of inducing adverse skin reactions in patients with atopic eczema. We studied 21 adult patients with atopic eczema and a history of adverse reactions to oranges and 10 patients without. A dietary history, skin tests, serum IgE and oral provocation tests with oranges were obtained. Severity of eczema was monitored by SCORAD, and serum tryptase, eosinophil cationic protein and urinary methylhistamine were measured. No allergic reactions were found to orange in skin prick or patch tests. However, 23 patients (74%) had specific serum IgE to orange. Oral provocation testing resulted in pruritic eczematous or maculopapular skin lesions predominantly at the predilection sites in 16 patients (52%). The SCORAD increased significantly in patients positive to the oral provocation test (p <0.05). Specific IgE to orange did not correlate with the clinical outcome of the oral provocation test. No significant changes were found in serum mast cell tryptase, eosinophil cationic protein or in urinary methylhistamine excretion. The negative results in the skin tests and a lack of correlation between specific IgE and oral provocation tests indicate that non-IgE-mediated mechanisms are involved in cutaneous adverse reactions to oranges in patients with atopic eczema.