Indirect Network Effects and Trade Liberalization

Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins

Serum testing of genetically modified soybeans with special emphasis on potential allergenicity of the heterologous protein CP4 EPSPS

Roundup Ready soy contains the CP4-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein. Serum IgE from two distinct populations of soy-allergic patients were recruited to determine their IgE-binding specificity. One population consisted of 10 adult patients from Europe, whose primary diagnosis was soy food allergy with some also having mite allergy. In addition, 6 primarily mite-allergic, 6 food-allergic (celery, carrot, milk, shrimp, walnut, and apple), and 5 non-allergic patients were tested. Another population consisted of 13 children from Korea, whose primary diagnosis was atopic dermatitis and secondarily soy and egg sensitization. In addition, 11 non-allergic patients were tested. Each patient population was extensively characterized with respect to clinical symptoms, specific IgE (CAP) scores, and total IgE. Immunoblots and ELISA assays were developed using serum IgE from these patients and soy extracts, CP4 EPSPS, rice extract, ovalbumin, rubisco, purified major peanut allergen Ara h 2, the putative soy allergen Gly m Bd 30k and mite allergen Der f 2 proteins as the intended targets. Immunoblot results indicated that soy-allergic patients bound soy extracts but did not specifically bind rubisco or CP4 EPSPS. ELISA results were in general agreement with the immunoblot results except that rubisco bound significant quantities of serum IgE from some patients. These results indicate that the CP4 EPSPS protein does not bind significant quantities of IgE from two geographically distinct sensitive populations and there is no evidence for an increased allergenic potential of this biotech protein

Strategy for allergenicity assessment of "natural novel foods" : clinical and molecular investigation of exotic vegetables (water spinach, hyacinth bean and Ethiopian eggplant)

Background: Foods not commonly consumed in the European Union (EU) must be proven save before brought to market, including an assessment of allergenicity. We present a three-stepwise strategy for allergenicity assessment of natural novel foods using three novel vegetables as example (water spinach, hyacinth bean, Ethiopian eggplant). Methods: First, vegetable extracts were analysed for the presence of pan-allergens [Bet v 1 homologous proteins, profilins, non specific lipid transfer proteins (LTP)] by immunoblot analysis with specific animal antibodies. Second, the IgE-binding of the food extracts was investigated by EAST (Enzyme-allergosorbent test) and immunoblot analysis using sera with IgE-reactivity to known pan-allergens or to phylogenetically related foods from subjects 1) allergic to birch, grass and mugwort pollen, 2) with food allergy to soy, peanut, tomato, multiple pollen-related foods and 3) sensitised to LTP. Third, the clinical relevance of IgE-binding was assessed in vivo by skin prick testing (SPT) and open oral food challenges (OFC). Results: Profilin and LTP were detected by animal antibodies in all vegetables, a Bet v 1 homologue selectively in hyacinth bean. IgE-binding to LTP, profilin and a Bet v 1 homologue was proven by immunoblot analysis and EAST. Positive SPT and OFC results were observed for all vegetables in pollen-allergic patients. Conclusion: Our stepwise procedure confirmed the presence and IgE-binding capacity of novel vegetable proteins homologous to known allergens in endemic vegetable foods. In vivo testing proved the potential of the novel vegetables to elicit clinical allergy. Hence, our described algorithm seems to be applicable for allergenicity testing of natural novel foods

Amino acids critical forIgE and IgG cross-reactivity in a subgroup of Bet v 1-related proteins.

<p>(A) Binding of serial dilutions of pool serum IgE to surface-coated equimolar amounts of Bet v 1 and Bet v 1_4x (Bet v 1_{N43I/E45S/N47D/K55A}) in ELISA. (B) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of either Bet v 1 or Bet v 1_4x in ELISA. (C) Binding of serial dilutions of monoclonal BV16 to surface-coated equimolar amounts of Bet v 1 and Bet v 1_4x in ELISA. (D) Mediator release induced by recombinant NCS and Bet v 1 variants. Humanized RBL cells were sensitized with a pool of human birch-specific sera. Cross-linking of membrane-bound human IgE by IgE-protein interaction and subsequent release of β-hexosaminidase was determined with serial dilutions of the proteins.</p

Grafting of an epitope onto Δ29NCS.

<p>Left panel: 13 of the 16 amino acids comprising the Bet v 1 epitope (Bet v 1_{E42/N43/I44/E45/G46/N47/G48/G49/P50/G51/T52/R70/D72/H76/I86/K97}) of mouse monoclonal IgG antibody BV16 (yellow). Six amino acids of the epitope are labeled. Lys55 of Bet v 1 located adjacent to the epitope is highlighted (green). Middle panel: the corresponding 13 residues of the Bet v 1 epitope for BV16 and a lysine corresponding to K55 of Bet v 1 have been grafted onto Δ29NCS to generate Δ29NCS_4x (Δ29NCS_{N57/I58E/D60N/V63P}) (not shown) and Δ29NCS_5x (Δ29NCS_{N57/I58E/D60N/V63P/D68K}). Right panel: corresponding surface area of Δ29NCS. Protein models were based on the structures of NCS (pdb 2VNE) and Bet v 1a (pdb 1BV1), and modelled with a confidence of 100% and a sequence coverage of at least 85%.</p

Structural alignment of the Bet v 1 epitope forBV16 and amino acids critical for Ig cross-reactivity in PR-10 allergens.

<p>Upper panel: model structures of Δ29NCS_5x and PR-10 allergens showing IgE cross-reactivity with Δ29NCS_5x. The amino acids comprising the BV16-binding epitope and lysine 55 of Bet v 1 and the corresponding residues of Δ29NCS_5x, Gly m 4, and Cor a 1 are listed. Residues of the epitope and of position 55 of Bet v 1 that are critical for Ig cross-reaction are highlighted in green. Lower panel: model structures of Δ29NCS and allergens that do not show IgE cross-reactivity with Δ29NCS_5x. The amino acids corresponding to the epitope in the cross-reactive proteins are listed and highlighted in green.</p

Δ29NCS_5x presents a cross-reactive Ig epitope.

<p>(A) Inhibition of IgE binding to Δ29NCS_5x. Binding of serum IgE to Δ29NCS (lane 3) and to Δ29NCS_5x (lane 4) in the presence of increasing amounts of inhibitors Δ29NCS_5x (lanes 5–8) or Bet v 1 (lanes 9–12). No serum (lane 1) and negative serum pool (lane 2) served as control. (B) Dose-dependent inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of increasing concentrations of PR-10 allergens in ELISA. (C) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of both Bet v 1 and serial dilutions of Bet v 1-binding monoclonal mouse IgG antibody BV16 (–: no BV16; 10⁻⁵ to 10⁻³: dilutions of BV16) in ELISA. (D) Binding of mouse monoclonal IgG antibody BV16 to surface-coated Bet v 1-related proteins. 250 ng of protein (50 ng of Api g 1) was coated and incubated with dilution series of the monoclonal BV16 in ELISA.</p