IgE-binding to vicilin-like antimicrobial peptides is associated with systemic reactions to macadamia nut

Background Macadamia nut can induce fatal allergic reactions and changes in dietary habits will raise their consumption in industrialised countries. Until now diagnosis of macadamia nut allergy by sIgE solely relies on the macadamia nut extract, but single components are lacking. Methods Macadamia nut proteins recognised by IgE from 2 macadamia nut extract positive sera were identified by mass spectrometry (vicilin-like antimicrobial peptides: VLAP). Sensitisation to macadamia nut extract and heterologously expressed isoform VLAP-2–3 was evaluated in 82 nut allergic (NA) and 27 tolerant (NT) patients (no tree nut allergy reported) comprehending 10 macadamia nut allergic (MA) and 18 explicitly reported macadamia nut tolerant patients (MT), using line blots. Co-sensitisation to additional VLAP isoforms and other vicilins was evaluated in 8 MA, 12 MT and 14 NA patients sensitised to VLAP-2–3. Functional properties were determined by indirect basophil activation. Results Even though proteins recognised by IgE were identified as VLAP-2–1, 2–2 and 2–3, only peptides specifically belonging to VLAP-2–3 were detected by mass spectrometry. The macadamia nut extract was recognised by 33% of NA patients (27/82) including 3 MA patients and 26% of NT patients (7/27, 3 MT). Similarly, 29% of NA (24/82) patients showed partly strong sIgE-binding to VLAP-2–3 including 3 MA patients with systemic reactions to macadamia nut. Contrary, VLAP-2–3 was recognised by only 2 NT (1 MT) patients (7%) with very low sIgE titres. Simultaneous recognition of the isoforms VLAP-2–1 and 2–2 was observed in all patients positive for VLAP-2–3 with partly reduced sIgE titres in 59% of these patients. Additionally, all three VLAP isoforms were able to repeatedly induce BAT reactivity upon sensitisation with a MA serum. Conclusion VLAP proteins are the first described macadamia nut components with serological and functional allergenic properties and they are associated with systemic reactions to macadamia nut.

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To the editor:
Macadamia nut belongs to the group of tree nuts being able to induce even fatal allergic reactions [1,2]. Even though macadamia nut is currently responsible for a small number of tree nut allergic reactions [3], it is hypothesized that the number will raise due to changes in dietary habits and increasing use in pastry and confectionery [4]. So far, specific IgE (sIgE) diagnostics of

Open Access
Clinical and Translational Allergy *Correspondence: a.m.ehlers@umcutrecht.nl macadamia nut allergy solely relies on crude macadamia nut extract and single components for stratification are lacking. In the present study, we identified vicilinlike antimicrobial peptides (VLAP) 2-1, 2-2 and 2-3 as novel allergens.
For identifying novel macadamia nut allergens, proteins of crude macadamia nut extract were separated by 2D gel electrophoresis and subsequently used for western blotting. Proteins recognised by sIgE from two commercially purchased sera were analysed by mass spectrometry (MS) and proteins with known amino acid sequences were heterologously expressed in E. coli [5]. For serological characterisation, tree nut allergic (NA, n = 82) and tree nut tolerant (NT) patients (n = 27) were retrospectively selected from patients visiting the University Medical Center (UMC) Utrecht between 2008 and 2018 based on food challenge or suggestive history by a trained physician. This selection included 10 NA patients with a diagnosed macadamia nut allergy and 18 NT patients with explicitly reported macadamia nut tolerance. Ethical approval (number 18-428) was acquired from the biobank committee of the UMC Utrecht. Obtained sera were applied onto a line blot in accordance with manufacturer's instructions (EUROLINE, EUROIMMUN AG, Germany) and sIgE levels were expressed as responsive units (RU). Binding to vicilins from other tree nuts, legumes and seeds was analysed in a subgroup of 8 MA, 12 macadamia nut tolerant (MT) and 14 NA patients sensitised to VLAP-2-3. The functionality of VLAP was assessed by indirect basophil activation test (i-BAT). Detailed description on patient selection and methods is given in Additional file 1 and information on the patient population is given in Table 1 and Additional file 2.
Particularly proteins with a molecular mass between 53 and 67 kDa and an isoelectric point (pI) between 6.3 and 8.7 were strongly bound by sIgE from 2 macadamia nut sensitised sera (Additional file 3: Figure S1; Serum 1: These proteins (spots 20-28, Additional file 3: Table S1) were identified as VLAP-2-1 (Q9SPL5), 2-2 (Q9SPL4) and 2-3 (Q9SPL3) whereof VLAP-2-3 showed the highest probability scores in MS analyses due to the detection of peptides solely specific for VLAP-2-3. Moreover, proteins with a molecular mass between 20 and 25 kDa and a pI between 6.5 and 7.9 (spots 42-44, 47, 48) were strongly bound by sIgE in western blot analyses. Despite high quality MS spectra with a great range of detected masses and high peak intensities, these proteins were not identified by aligning the spectra to the NCBI database (111 Macadamia integrifolia proteins).
Co-sensitisation to the VLAP isoforms 2-1 and 2-2 as well as to vicilins from seeds, tree nuts and legumes was studied in a subgroup of 8 MA, 12 MT and 14 additional NA patients sensitised to VLAP-2-3. Overall, patients recognizing VLAP-2-3 were mostly also positive for its isoforms but their sIgE-binding to VLAP-2-1 and 2-2 was reduced in 59% of the patients (Fig. 1b, Additional  file 4). Recognition of VLAP isoforms was accompanied by sIgE-binding to Ana o 1 (cashew nut) and Jug r 2 (walnut) in 59% of the patients sensitised to VLAP. These vicilins (Ana o 1 2/3 MA; Jug r 2 1/3 MA) were also corecognised in MA patients. Their functional ability to induce degranulation was evaluated by i-BAT (Fig. 1c). Patient MA-10 (NA-29) showed dose-dependent BAT reactivity upon stimulation with all three VLAP isoforms starting from 10 ng/ml and reaching a plateau at around 100 ng/ml for VLAP-2-2 and 2-3 and at 1000 ng/ml for VLAP-2-1. This dose-dependent basophil activation was confirmed by repetitive experiments on a second day with freshly obtained basophils. These basophils showed a more flat-angel reactivity curve and the plateau was reached at tenfold higher concentrations. For comparison, stimulation with native macadamia nut extract induced a stronger CD63 upregulation than stimulation with recombinant VLAPs. To ensure that no activation occurred by remaining donor sIgE, stripped basophils were stimulated under the same conditions leading to no CD63 upregulation (Fig. 1d). As internal positive control, basophils loaded with serum from a peanut allergic patients sensitised to Ara h 2 (Fig. 1e) showed dose-dependent CD63 upregulation upon stimulation with Ara h 2. Overall, VLAP 2-3 and its isoforms appeared functional, since they induced degranulation.
In the present study, macadamia nut proteins recognised by IgE were identified as VLAPs with serological recognition in 33% of NA and 30% of MA patients and the ability to induce basophil activation. While MA patients recognised VLAPs with highly increased sIgE titres (23 to 105 RU), MT patients recognised VLAPs with almost negligible sIgE titres, indicating the potential of VLAPs to discriminate between MA and MT patients positive for the macadamia nut extract. Moreover, MA patients sensitised to VLAPs experienced moderate to severe symptoms upon ingestion, highlighting the potential of VLAPs as potential markers for systemic reactions to macadamia nut. Accordingly, the recombinant vicilin from walnut, Jug r 2, has been described as a marker for severe allergic reactions in patients without pollen-related sensitisation [6,7]. Moreover, sensitisation to the vicilin from hazelnut, Cor a 11, was observed in children with severe hazelnut allergies while Cor a 11 was scarcely recognised by adults with oral allergy syndrome upon hazelnut ingestion [8].
In conclusion, VLAPs from macadamia nut appear to be supportive in identifying patients with systemic reactions to macadamia nut and should be incorporated in component-resolved diagnostics of macadamia nut allergy.