Bet v 1-independent sensitization to major allergens in Fagales pollen: Evidence at the T-cell level.

BACKGROUND: In birch-dominated areas, allergies to pollen from trees of the order Fagales are considered to be initiated by the major birch pollen allergen Bet v 1. However, the sensitizing activity of Bet v 1-homologs in Fagales pollen might be underestimated. Allergen-specific T-cells are crucial in the sensitization process. The T-cell response to major allergens from alder, hazel, oak, hornbeam, chestnut, beech, and chestnut pollen has not yet been analyzed. Here, we characterized the cellular cross-reactivity of major allergens in Fagales pollen with Bet v 1. METHODS: T-cell-lines (TCL) were established from allergic individuals with Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1, and Que a 1, and tested for reactivity with Bet v 1 and synthetic overlapping 12-mer peptides representing its primary sequence. Aln g 1-specific TCL was additionally tested with Aln g 1-derived peptides and all allergens. IgE-competition experiments with Aln g 1 and Bet v 1 were performed. RESULTS: T-cell-lines initiated with Fagales pollen allergens varied strongly in their reactivity with Bet v 1 and by the majority responded stronger to the original stimulus. Cross-reactivity was mostly restricted to the epitope Bet v 1142-153 . No distinct cross-reactivity of Aln g 1-specific T-cells with Bet v 1 was detected. Among 22 T-cell epitopes, Aln g 1 contained two immunodominant epitopes. Bet v 1 inhibited IgE-binding to Aln g 1 less potently than Aln g 1 itself. CONCLUSION: The cellular cross-reactivity of major Fagales pollen allergens with Bet v 1 was unincisive, particularly for Aln g 1, most akin to Bet v 1. Our results indicate that humoral and cellular responses to these allergens are not predominantly based on cross-reactivity with the major birch pollen allergen but suggest a Bet v 1-independent sensitization in individuals from birch tree-dominated areas.

Identification of apple cultivars hypoallergenic for birch pollen-allergic individuals by a multidisciplinary in vitro and in vivo approach.

BACKGROUND: Birch pollen-related apple allergy is the most frequent IgE-mediated food allergy in Central-Northern Europe with Mal d 1 as major allergen. Its concentration in apples varies with the cultivar and storage time. Year-round appealing, hypoallergenic cultivars still are needed to satisfy the nutritional needs of affected individuals. We characterized three promising cultivars by multidisciplinary in vitro assays including long-term storage and by clinical challenges of allergic individuals before and after the birch pollen season. METHODS: Proteins were extracted from fruits of 'Santana', 'Golden Delicious' (GD), and three genuine cultivars in November 2018 and April 2019. Mal d 1-levels were analysed by mass spectrometry, SDS-PAGE, immunoblotting, competitive ELISA, and basophil activation tests. Twenty-eight allergic individuals underwent single-blinded open food challenges and skin testing with the cultivars and birch pollen in November 2018 and May 2019. Allergen-specific IgE-levels were determined. RESULTS: After storage all cultivars except 'Santana' were of appealing appearance and taste. Their Mal d 1 content had increased, also reflected by significantly amplified basophil activation and stronger reactions in clinical challenges. Besides, individuals showed boosted reactivity after pollen exposure indicated by enhanced allergen-specific IgE-levels and skin reactions to birch pollen. Still, all cultivars remained significantly less allergenic than GD and comparable to Santana in November 2018 in all assessments except for skin testing. CONCLUSIONS: Combined expertise in pomology and allergology identified promising new cultivars for allergic consumers. The evaluation of hypoallergenic apples should incorporate long-term storage and birch pollen exposure. Basophil activation tests may be suitable in the selection of promising cultivars for oral challenges.

IgE-cross-blocking antibodies to Fagales following sublingual immunotherapy with recombinant Bet v 1.

BACKGROUND: Evidence has accumulated that birch pollen immunotherapy reduces rhinoconjunctivitis to pollen of birch homologous trees. Therapeutic efficacy has been associated with IgE-blocking IgG antibodies. We have recently shown that sera collected after 16 weeks of sublingual immunotherapy with recombinant Bet v 1 (rBet v 1-SLIT) display strong IgE-blocking bioactivity for Bet v 1. Here, we assessed whether rBet v 1-SLIT-induced IgG antibodies display cross-blocking activity to related allergens in Fagales pollen. METHODS: IgE, IgG1 and IgG4 reactivity to recombinant Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1 and Que a 1 were assessed in pre- and post-SLIT samples of 17 individuals by ELISA. A basophil inhibition assay using stripped basophils re-sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to allergens after incubation with pre-SLIT or post-SLIT samples. IgG1 and IgG4 were depleted from post-SLIT samples to assess its contribution to IgE-cross-blocking. RESULTS: Sublingual immunotherapy with recombinant Bet v 1 boosted cross-reactive IgE antibodies and induced IgG1 and IgG4 antibodies with inter- and intra-individually differing reactivity to the homologs. Highly variable cross-blocking activities of post-SLIT samples to the different allergens were found. IgG1 and IgG4 antibodies displayed cross-blocking activity with individual variance. CONCLUSIONS: Our mechanistic approach suggested that immunotherapy with the reference allergen Bet v 1 induces individual repertoires of cross-reactive IgG1 and IgG4 antibodies. The cross-blocking bioactivity of these antibodies was also highly variable and neither predictable from protein homology nor IgE-cross-reactivity.