Due to structural similarity and IgE cross-reactivity between Bet v 1 and the major apple allergen, Mal d 1 1, 2, a considerable number of birch pollen allergic patients exhibit oral allergy syndrome (OAS) when eating apples 3. Symptoms of OAS are usually mild but can also be severe depending on the sensitivity of the patients and the amount of consumed food. Recently we identified IgE-producing plasmablasts with pre-existing IgE epitope specificities distinct from the epitope specificities of Bet v 1-specific IgG as the major source for the increases in Bet v 1-specific IgE responses after seasonal birch pollen exposure 4. These increases in Bet v 1-specific IgE induced by Bet v 1 exposure re-establish Bet v 1-specific effector cell sensitivity. It has been reported earlier that seasonal birch pollen exposure may also boost clinical reactivity to apples 5 but the molecular basis for this clinical observation has not yet been investigated. Here, we hypothesized that the increase of Bet v 1-specific IgE due to seasonal birch pollen exposure can cause concomitant Mal d 1-specific IgE increases responsible for subsequent higher sensitivity to Mal d 1 and apple-induced OAS. Therefore, birch pollen allergic patients with apple OAS (group 1: n = 13) and without apple OAS (group 2: n = 12) were investigated before, during and after the birch pollen season (Figure 1A, Table S1). Testing for IgE reactivity to 112 micro-arrayed allergen molecules showed that Bet v 1-specific IgE levels were higher than those against any of the other Bet v 1-related PR-10 allergens identifying Bet v 1 as the culprit sensitizing allergen (Table S2). Group 1 patients reacted with more PR-10 allergens than group 2 patients (Figure S1, Table S2) and had higher Bet v 1-specific IgE levels than group 2 patients without OAS (Figure S2). Mal d 1-specific IgE levels were significantly higher in group 1 versus group 2 (Figure S2A, Table S3). ImmunoCAP measurements confirmed allergen microarray data (Figure S2B) and correlated highly (Figure S3). Skin sensitivity to apple was significantly higher in group 1 than in group 2 (Figure S3A, Table S3). Twelve out of 13 group 1 patients were positive by open oral provocation test (OOPT) whereas only one patient from group 2 showed a positive OOPT test at baseline (Table S3). Group 1 patients showed higher basophil sensitivity to Bet v 1 than group 2 and Mal d 1-specific basophil sensitivity was significantly greater in group 1 versus group 2 (Figure S3B, Table S4). After seasonal birch pollen exposure, group 1 and group 2 patients showed concomitant increases in Bet v 1 and Mal d 1-specific IgE levels (Figure 1B, Figure S4), suggesting that respiratory birch pollen exposure boosts Bet v 1-specific IgE cross-reacting with Mal d 1, which was confirmed by highly correlated Bet v 1- and Mal d 1-specific IgE levels at each of the time points (Figure S5). Also Bet v 1- and Mal d 1-specific basophil sensitivity increased after the birch pollen season although not all patients could be tested (Figure 2A, Figure S6A, and Table S4). In all patients, the median SPT responses to apple increased significantly at T2 and declined 4 months after the birch pollen season at T3 (Figure 2B, Figure S6B, and Table S3). Four out of 13 group 1 patients increased oral sensitivity to apple, eight remained equally sensitive after the birch pollen season, and one patient (i.e., patient 1.11) had a grade IV reaction at baseline and therefore was not tested at T2 and T3 for safety reasons. From 12 group 2 patients, three developed a positive oral reaction to apple after the birch pollen season, eight remained negative, and one stayed positive (Table S3). In summary, our study demonstrates that seasonal birch pollen exposure and, conceivably, respiratory exposure to Bet v 1, lead to a concomitant increase in Mal d 1-specific IgE, Mal d 1-specific basophil and skin sensitivity and increased sensitivity to apple. Exposure to Mal d 1 by ingestion does not seem to have such an effect because it has been reported that controlled exposure to Mal d 1 by apple ingestion rather induces tolerance to apples and eventually the induction of Mal d 1-specific IgG but not specific IgE 6. Our findings are of clinical importance because they demonstrate that sensitivity to apple is triggered and maintained by birch pollen exposure. Accordingly, this mechanism may be targeted by therapeutic strategies reducing Bet v 1-specific IgE levels, but also by preventing the binding of Bet v 1-specific IgE to effector cells or by targeting cells producing Bet v 1-specific IgE. Rudolf Valenta and Maria G. Byazrova were involved in data interpretation, study design, writing, reviewing, and revising the manuscript. Musa R. Khaitov and Alexander A. Filatov contributed to the study design and were involved in drafting, critically reviewing, writing, and revising the manuscript. Alla O. Litovkina, Evgenii V. Smolnikov, Alexandra A. Nikonova, Olga G. Elisyutina, Elena S. Fedenko, and Nataliya I. Ilina were involved in the clinical and experimental work, read, revised, and approved the manuscript. Oluwatoyin Akinfenwa and Raffaela Campana prepared and characterized recombinant allergens, read, revised, and approved the manuscript. All authors approved the final version of the manuscript. Open Access funding provided by Medizinische Universitat Wien/KEMö. This study was supported by a Megagrant of the Government of the Russian Federation, grant number 075-15-2021-632 (14.W03.31.0024), and in part by the Danube ARC grant of the country of Lower Austria and by grant P34472-B from the Austrian Science Foundation (FWF). Birch pollen allergic patients included in the study were recruited from the outpatient department of the NRC Institute of Immunology, FMBA of Russia. The study was approved by the Ethical Committee of the NRC Institute of Immunology, FMBA of Russia (Protocol code: #3, 16 February 2018), and written informed consent was obtained from all patients. The study was performed in accordance with the Declaration of Helsinki. Rudolf Valenta gave lectures for HVD Biotech, Vienna, Austria. The other authors have no conflicts of interest to declare. The authors with a Russian affiliation declare that they have prepared the article in their “personal capacity” and/or that they are employed at an academic/research institution where research or education is the primary function of the entity. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1: Supporting Information FIGURE S1: Amino acid sequence identities among PR-10 allergens. Amino acid sequences were aligned using the KAlign algorithm based on the Hirschberg method. The values represent pairwise sequence identities between PR-10 proteins. Color intensities reflect the degree of amino acid identities: white to red indicates > 50% identity, while blue indicates 50% or less. FIGURE S2: IgE levels specific for Bet v 1 (left panel) and Mal d 1 (right panel) (A), measured before the season using ImmunoCAP (y-axes: kUA/L) for the two groups (x-axes). (B) IgE levels specific for Bet v 1 (left panel) and Mal d 1 (right panel), measured before the season by ImmunoCAP ISAC (y-axes: ISU) for the two groups (x-axes). Shown are medians and ± IQR. Significant differences are indicated * p < 0.05. (C) Spearman's correlation between Bet v 1-specific (left panel) and Mal d 1-specific (right panel) IgE levels measured using ImmunoCAP (x-axes: kUA/L) and ISAC method (y-axes: ISU). Data are presented for individual subjects from group 1 (violet) and group 2 (green). Asterisks denote statistical significance, ****p < 0.0001. FIGURE S3: Allergen-specific sensitivity as determined by basophil activation test (BAT) and skin prick testing (SPT) before the birch pollen season. (A) Wheal areas determined by SPT before the birch pollen season (y-axis: mm2) for groups 1 and 2. (B) Allergen-specific basophil sensitivity (Bet v 1, Mal d 1) is expressed as median fluorescence intensity (MFI) corresponding to activated basophils, defined as CRTH2+CD203c+CD3− cells on the y-axes for the indicated allergen concentrations. Data are presented as median ± IQR. Statistical analyses were performed by Mann–Whitney test, *p < 0.05, **p < 0.01. FIGURE S4: Bet v 1- and Mal d 1-specific antibody levels in group 2 patients before, during and after the birch pollen season. IgE levels specific for Bet v 1 (A) and Mal d 1 (B), measured by ImmunoCAP (y-axes: kUA/L) at the different time points (x-axes). Shown are medians and ± IQR. FIGURE S5: Correlations of Bet v 1- (y-axes) and Mal d 1-specific IgE levels (x-axes) in group 1 (A) and group 2 patients (B) at the three different time points (T1, T2, T3). Spearman's correlation between Bet v 1-specific and Mal d 1-specific IgE levels measured by ImmunoCAP. For visualization purposes, values equal to 0 kUA/L were displayed as 0.01 kUA/L on panels B and B . FIGURE S6: Bet v 1- (left) and Mal d 1-specific basophil sensitivity (right) in group 2 patients (A) before, during and after the birch pollen season. Basophil activation is expressed as median fluorescence intensity (MFI) on the y-axes. All results are shown for an allergen concentration of 100 ng/mL. Data are presented as median ± IQR. Statistical analysis was performed using the Friedman test followed by Dunn's multiple comparisons test. No significant differences were found. (B) Skin sensitivity to apple before (T1), during (T2) and after (T3) the birch pollen season in group 2. Data are presented as median ± IQR of wheal areas (y-axes: mm2). Statistical analysis was performed using the Friedman test followed by Dunn's multiple comparisons test, *p < 0.05. TABLE S1: Clinical characteristics of birch pollen-allergic patients with (Group 1) or without (Group 2) oral allergy syndrome (OAS) to apple. Patients are identified according to the group (Group 1: 1.1–1.13; Group 2: 2.1–2.12). Shown are age, gender, birch pollen-related allergy symptoms and presence or absence of OAS. Abbreviations used: m: male; f: female; AD: Atopic dermatitis. TABLE S2: Molecular IgE reactivity profiles were determined by ImmunoCAP ISAC for all group 1 and group 2 patients. Bet v 1–cross-reactive PR-10 allergens are highlighted in green, followed by other allergen components listed alphabetically. Allergen-specific IgE levels are expressed in international standardized units (ISU), with corresponding color coding indicated in the legend. TABLE S3: Allergen-specific IgE levels, skin sensitivity and sensitivity by oral food provocation in birch pollen allergic patients at the three different time points. Bet v 1-specific and Mal d 1-specific IgE levels are shown as international standardized units (ISU) or kUA/L (Color codes indicated). Wheal areas for SPT with apple, histamine and saline are presented in mm2 and results of oral provocation test (OPT) with apple are displayed by grading (0: no symptoms; grades I-IV). TABLE S4: Basophil activation test results in response to Bet v 1 and Mal d 1 allergens at time points T1, T2, and T3 for group 1 and group 2 patients. Basophil sensitivities are shown as median fluorescence intensities (MFI) for different allergen concentrations, for the positive and the negative controls. ND: not done. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Litovkina et al. (Wed,) studied this question.