Isotype-specific binding patterns of serum antibodies to multiple conformational epitopes of Bet v 1.

BACKGROUND: Birch pollen is an important elicitor of respiratory allergy. The major allergen, Bet v 1, binds IgE exclusively via conformational epitopes. OBJECTIVE: We identified Bet v 1-specific epitope repertoires of IgE and IgG from birch pollen-allergic and nonallergic subjects. METHODS: Chimeric proteins were created by grafting individual epitope-sized, contiguous surface patches of Bet v 1 onto a nonallergenic structural homolog and expressed in Escherichia coli. Binding of IgE, IgG1, and IgG4 from sera of 30 birch pollen-allergic and 11 nonallergic subjects to Bet v 1, 13 chimeric proteins, and 4 bacterial Bet v 1 homologs were measured by ELISA. The proportion of epitope-specific in-total Bet v 1-specific IgE and the cross-reactivity of Bet v 1-specific IgE with bacterial homologs were determined by competitive ELISA. RESULTS: Thirteen soluble, correctly folded chimeric proteins were produced. IgE from 27 of 30 birch pollen-allergic patients bound to 1 to 12 chimeric proteins (median, 4.0), with patient-specific patterns evident. Three chimeras binding IgE from the majority of sera were identified, the grafted patches of which overlapped with previously published epitopes. Patterns of IgG1 and IgG4 binding to the chimeric proteins did not correspond to the binding patterns of IgE. Sera of 19 of 30 birch pollen-allergic patients contained low amounts of IgE to bacterial homologs. Bacterial proteins were able to partially inhibit IgE binding to Bet v 1. CONCLUSION: Epitopes recognized by Bet v 1-specific antibodies from birch pollen-allergic patients are specific to each patient and differ between IgE, IgG1, and IgG4.

Pru du 1, the Bet v 1-homologue from almond, is a major allergen in patients with birch pollen associated almond allergy.

Background: Almond allergy is common and can manifest in two different forms. Primary almond allergy has been reported to be associated with sensitization to almond legumin Pru du 6. In birchendemic regions, there is a link between birch-pollinosis which is likely based on a cross-reactive Bet v 1 homologue, a yet unidentified allergen in almond. Therefore, we sought to identify and characterize a Bet v 1-homologue in almond. Methods: The expression of a Bet v 1 homologue in almond kernels was confirmed by mass spectrometry. The recombinant protein was produced in Escherichia coli and its cross-reactivity and allergenic potency was analyzed by IgE quantitative and competitive ELISA, immunoblotting and basophil histamine release using sera from 17 almond allergic patients. Results: The identified Bet v 1 homologue received the designation Pru du 1.0101. Pru du 1.0101 bound IgE from 82 % of almond allergic patients. Bet v 1 was able to inhibit IgE-binding to rPru du 1 by 100%, while rPru du 1 inhibited IgE binding to rBet v 1 by 48%. Pru du 1.0101 activated basophils, though 100- to 1000-fold higher concentrations were required for maximum activation in comparison to rBet v 1. Conclusion: Considering the strong inhibition capacity and higher allergenic potency of Bet v 1, the results provide compelling evidence for primary sensitization to Bet v 1 in case of birch pollen associated almond allergy. Combining Pru du 6 and Pru du 1 in diagnostic approaches may help to discriminate between primary and birch-pollen associated almond allergy.

Adjuvants and Vaccines Used in Allergen-Specific Immunotherapy Induce Neutrophil Extracellular Traps.

Aluminum hydroxide (alum) and monophosphoryl-lipid A (MPLA) are conventional adjuvants in vaccines for allergen-specific immunotherapy (AIT). Alum triggers the release of neutrophil extracellular traps (NETs) by neutrophils. NETs contain expelled decondensed chromatin associated with granular material and may act as danger-associated molecular patterns and activate antigen-presenting cells. We investigated whether adjuvant-induced NETs contribute to innate responses to AIT-vaccines. Human neutrophils were incubated with alum, MPLA and adjuvant-containing AIT-vaccine preparations. NETs were verified by time-lapse and confocal fluorescence microscopy and quantitatively assessed by DNA and elastase release and ROS production. In contrast to MPLA, alum represented a potent trigger for NET release. Vaccine formulations containing alum resulted in less NET release than alum alone, whereas the vaccine containing MPLA induced stronger NET responses than MPLA alone. NETs and alum alone and synergistically increased the expression of molecules involved in antigen presentation, i.e., CD80, CD86 and CD83, by peripheral blood monocytes. Monocyte priming with NETs resulted in individually differing IL-1ß- and IL-6-responses. Thus, NETs induced by adjuvants in AIT-vaccines can provide autonomous and cooperative effects on early innate responses. The high diversity of individual innate responses to adjuvants and AIT-vaccines may affect their therapeutic efficacy.