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Keywords:

  • birch pollen allergy;
  • blocking antibodies;
  • IgE;
  • IgG4;
  • specific immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information

Background

IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes.

Methods

IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen–allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm.

Results

BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap.

Conclusion

Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition.

Abbreviations
aa

aamino acid

Ab

antibody

BAT

basophil activation test

BP

birch pollen

FAB

facilitated antigen binding

OAS

oral allergy syndrome

SIT

allergen-specific immunotherapy

TCL

T-cell line

Allergen-specific immunotherapy (SIT) is an effective treatment of IgE-mediated hypersensitivity to birch pollen [1]. Successful BP-SIT has been associated with the induction of Bet v 1-specific IgG4 Abs that are capable of inhibiting IgE binding to the major birch pollen allergen [2]. Consequently, IgE-mediated activation of effector cells and Bet v 1-specific T lymphocytes is reduced [3-5].

Bet v 1, the major birch pollen allergen, has been well characterized regarding its structure [6] and B- and T-cell epitopes [7-10]. Bet v 1 shares IgE epitopes with structurally related food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple [9, 11]. These Bet v 1 homologs show 67.3% and 55.7% amino acid (aa) sequence identity with Bet v 1, respectively, and a very similar three-dimensional structure [12]. So far, only very few studies have assessed whether BP-SIT-induced IgG4 Abs recognize Bet v 1-related food allergens and exert blocking activity [13-15]. This tempted us to study the kinetics of production, cross-reactivity, and IgE-blocking capacity of BP-SIT-induced Bet v 1-specific IgG4 Abs. We quantified IgE and IgG4 levels specific for Bet v 1, Cor a 1, and Mal d 1 in sera from 42 patients before and at different time points during therapy. IgE-blocking activity was assessed by IgE-facilitated allergen-binding (FAB) assays [16], basophil activation tests [17], and IgE-facilitated antigen presentation to allergen-specific T cells [5].

We found Bet v 1-specific IgG4 Abs that cross-reacted with Cor a 1 and Mal d 1 and exerted IgE-blocking activity. Currently, it is not known whether this blocking is due to competition of IgG4 for IgE epitopes or sterical hindrance after binding of IgG4 to sites different from IgE epitopes. To address this aspect, we performed competitive immunoscreening of phage-displayed peptides [9] and predicted IgE and IgG4 epitopes on Bet v 1, Mal d 1, and Cor a 1 using the algorithm EpiSearch [18].

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information

Allergens

Recombinant Bet v 1.0101 (termed ‘Bet v 1’) and recombinant Mal d 1.0801 (termed ‘Mal d 1’) were purchased from Biomay, Vienna, Austria. Recombinant Cor a 1.0401 (Cor a 1) was kindly provided by S. Scheurer, Paul-Ehrlich Institut, Langen, Germany.

Patients

Birch pollen–allergic patients (age range 10–57 years, median 35 years) who received their first SIT were included. P1-P9 received ALK-depot SQ108 (ALK-ABELLO, Hørsholm, Denmark) [3], and blood samples were collected as follows: months 0, 1, 3, 12, 24, and 36 out of the birch pollen season; months 6, 18, and 30 during the birch pollen season. P10-P42 received ALK-ALUTARD SQ197 (ALK-ABELLO), and all blood samples were collected out of the birch pollen season. Both vaccines contain Al(OH)3 as adjuvant. The cumulative dose of Bet v 1 was around 150 μg for 1 year and around 500 μg for 3 years of SIT.

Assessment of allergen-specific antibodies

Allergen-specific IgE and IgG4 Abs were quantified by ImmunoCAP (Thermo Fisher Scientific, Uppsala, Sweden).

IgE-FAB assay

For all tests, the same indicator serum was used. Indicator serum (20 μl) containing >100 kU/l of birch pollen-specific IgE and test sera (15 μl) or an equal volume of medium was pre-incubated with titrated concentrations (1, 0.1, 0.01 μg/ml) of allergens (5 μl) for 1 h at 37°C. Then, EBV-transformed B cells (1 × 105/5 μl) were added and incubated for 1 h at 4°C. After washing and saturation with 20% human AB serum for 20 min at 4°C, cells were stained with FITC-labeled anti-human IgE (KPL, Gaithersburg, MD, USA). After washing, IgE-positive B cells were detected by flow cytometry using a FACS Canto and Diva software (BD Biosciences, San Jose, CA, USA). All assays were performed in duplicate, and 5000 viable cells per sample were acquired.

Basophil activation tests (BAT)

Heparinized blood from untreated birch pollen–allergic patients was incubated for 30 min at 37°C with different concentrations of Bet v 1 or Cor a 1 (100, 10, 1, 0.1, 0.01 ng/ml) in RPMI supplemented with human IL-3 (5 ng/ml; PeproTech, Rocky Hill, NJ, USA). Medium alone served as negative and anti-human IgE (1 μg/ml; Nordic Immunological Laboratories, Maidenhead, UK) as positive control. After adding PBS containing 20 mM EDTA, cells were stained with CD63-PE, CD123-FITC (both from Biolegend, San Diego, CA, USA), CD203c-APC, CD45-PerCP (both from Miltenyi Biotec, Bergisch Gladbach, Germany), or the respective isotype controls (all from BD Biosciences). After lysis of erythrocytes, the percentage of CD63+ cells in the CD45+ CD123+ population was assessed by flow cytometry. All analyses were performed in duplicate. For inhibition experiments, allergens (1 ng/ml) were pre-incubated with sera from SIT-treated patients for 1 h at 37°C prior to the addition to whole blood.

Bet v 1-specific T-cell lines (TCL)

Bet v 1-specific TCL were generated from non-SIT-treated birch pollen–allergic individuals by stimulating PBMC (1.5 × 106) with Bet v 1 (10 μg/ml) as described in the study by Mutschlechner et al. [19]. Bet v 1-specific TCL cross-reactive with Mal d 1 and/or Cor a 1 were expanded to cell numbers necessary for inhibition experiments. After incubation of allergens (each at 0.01 μg/ml in 5 μl) with indicator serum (20 μl) plus test sera (20 μl) or an equal volume of medium, EBV-transformed B cells (5 × 104/5 μl) were added. After washing twice, cells were incubated for 2 h at 37°C, irradiated (60 Gy), and added to Bet v 1-specific TCL (5 × 104 cells/well). All experiments were performed in duplicate. After 48 h, proliferation was assessed by uptake of 3[H] thymidine (0.5 μCi/well) and expressed as mean counts per minute (cpm).

Competitive immunoscreening of phage-displayed peptides

Competitive immunoscreening of a Ph.D.-12 Phage Display Peptide Library (New England BioLabs, Beverly, MA, USA) was performed as described with slight modifications [9]. Briefly, 2.5 mg of Dynabead M-280 tosylactivated beads (Invitrogen Dynal AS, Oslo, Norway) was coupled with 50 μg mouse anti-human IgE or IgG4 Abs (Southern Biotech, Birmingham, AL, USA) at 37°C overnight, blocked with 0.5% BSA in PBS at RT for 2 h, and incubated with patients' sera (500 μl) at 4°C overnight. After extensive washing, beads were resuspended in 200 μl of PBS containing 0.1% BSA. Then, 100 μl was used for the first round of selection, while the remaining 100 μl was kept for the second round of selection.

First round of panning

Beads (100 μl) were incubated with 211 phages for 3 h at RT. After extensive washing, bound phages were amplified by direct infection of ER2738 Escherichia coli cells at 37°C for 4.5 h. Phages were purified by PEG precipitation and tittered.

Second round of panning

Beads were incubated with 211 phages selected from the first round. After extensive washing, beads were resuspended in 300 μl of PBS containing 0.1% BSA. For specific elution, beads (100 μl) were incubated with 400 μl of PBS, 0.1% BSA containing 25 μg of either Bet v 1, Mal d 1, or Cor a 1 for 1 h at RT. Eluted phages were amplified, purified, and tittered. Single plaques were picked and amplified, and amino acid (aa) sequences of the selected phage-displayed peptides were determined by DNA sequencing using M13 phage-specific primers.

Mapping of peptides onto the allergen surface

Epitopes were predicted using the default settings for 12-mer peptides of ‘EpiSearch’ (http://curie.utmb.edu/episearch.html) [18]. This algorithm matches the sequences of antibody-binding peptides with surface-exposed aa in patches of similar physicochemical nature as the input peptides on the 3D structure of the allergen. Only peptides with highest matching scores >1.0 were considered as putative epitopes. The following three-dimensional experimentally solved or modeled structures of the allergens were used for epitope prediction: Bet v 1 (PDB code: 1BV1) [6], Mal d 1, and Cor a 1 [20].

Defining overlapping IgE and IgG4 epitopes

Available crystal structures of antibody–antigen interactions suggest that a typical epitope covers ∼600–900 Å2 on the surface of an antigen [21, 22]. We calculated the solvent-accessible surface areas (ASAs) of predicted IgE and IgG4 epitopes using GETAREA (http://curie.utmb.edu/getarea.html) [23]. The ASAs of IgE and IgG4 epitopes were compared. Two epitopes were considered as distinct, if there was >600 Å2 of contiguous area outside the intersection of the two predicted surface patches. Representative examples of overlapping and distinct IgE/IgG4 epitopes are depicted in Fig. 4.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information

Allergen-specific IgE and IgG4 responses to BP-SIT

In sera from 42 birch pollen–allergic patients collected before, at 12 and 36 months of BP-SIT, Bet v 1-, Mal d 1-, and Cor a 1-specific IgE and IgG4 levels were quantified by ImmunoCAP (Fig. 1). Bet v 1-specific IgE levels continuously decreased during the course of BP-SIT (Fig. 1A, m0/m12, P = 0.034; m0/m36, P = 0.001; and m12/m36, P < 0.001). Mal d 1- and Cor a 1-reactive IgE levels were significantly reduced at 36 months of BP-SIT (Mal d 1: m0/m36, P = 0.004, and m12/m36, P = 0.001; Cor a 1: m0/m36, P = 0.005, and m12/m36, P = 0.001). At each time point, Bet v 1-specific IgE levels were significantly higher than food allergen-reactive IgE levels (m0: Bet v 1/Mal d 1, P < 0.001; Bet v 1/Cor a 1, P < 0.001; m12: Bet v 1/Mal d 1, P < 0.001; Bet v 1/Cor a 1, P < 0.001; and m36: Bet v 1/Mal d 1, P < 0.001; Bet v 1/Cor a 1, P = 0.002). During BP-SIT, a continuous and significant increase in allergen-specific IgG4 levels occurred (Fig. 1A, Bet v 1: m0/m12, P < 0.001; m0/m36, P < 0.001; and m12/m36, P = 0.016; Mal d 1: m0/m12, P < 0.001; m0/m36, P < 0.001; m12/m36, P = 0.252; Cor a 1: m0/m12, P < 0.001; m0/m36, P < 0.001; and m12/m36, P = 0.014). After 12 and 36 months, respectively, Bet v 1-specific IgG4 levels were significantly higher than food allergen-reactive IgG4 levels (m12: Bet v 1/Mal d 1, P = 0.001; Bet v 1/Cor a 1, P < 0.001; and m36: Bet v 1/Mal d 1, P < 0.001; Bet v 1/Cor a 1, P < 0.001).

image

Figure 1. Allergen-specific IgE and IgG4 levels during the course of BP-SIT. (A) Ab levels and IgG4/IgE ratios specific for Bet v 1 (white box plots), Mal d 1 (light gray), and Cor a 1 (dark gray) in 42 sera before (0) and after 12 and 36 months of BP-SIT, respectively. Wilcoxon signed-rank test, *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Time kinetics of IgG4 specific for Bet v 1 (full line), Mal d 1 (dotted line), and Cor a 1 (dashed line) in patients P1–P9.

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Similar production kinetics of Bet v 1 and food allergen-reactive IgG4 Abs during birch pollen SIT

We monitored the course of allergen-specific IgG4 levels in sera from nine birch pollen–allergic individuals collected before and at months 1, 3, 6, 12, 24, 30, and 36 of BP-SIT (P1-P9 in Table 1, obtained from the clinical trials performed by Mobs et al. [2, 24]. In general, Bet v 1-specific IgG4 Abs started to rise after 3–6 months of BP-SIT (Fig. 1B). In seven of nine patients, food allergen-reactive IgG4 levels increased at the same time point, whereas two individuals (P1 and P9) showed a delayed rise of food allergen-reactive IgG4 levels. Of note, only one of nine individuals (P4) showed a significant increase in both Mal d 1- and Cor a 1-reactive IgG4 Abs. The remaining eight individuals developed remarkable levels of Cor a 1-reactive IgG4 Abs and rather weak levels of Mal d 1-reactive IgG4 Abs, although all individuals showed IgE reactivity to both food allergens.

Table 1. Allergen-specific IgG4 responses before (0) and after 36 months of BP-SIT in patients sensitized to Bet v 1, Mal d 1, and Cor a 1
Patient noIgE (kU/l)IgG4 (mg/l)
Bet v 1Mal d 1Cor a 1Bet v 1Mal d 1Cor a 1
036036036036036036
  1. Gray boxes highlight patients who show no increase in IgG4 Ab.

P121.9658.823.729.1410.0624.000.2826.800.092.070.066.69
P259.5127.7430.2015.7228.4015.920.555.290.070.200.332.14
P371.8249.2519.4010.4483.6061.001.222.410.900.891.421.86
P423.870.350.740.350.880.350.019.460.025.530.037.22
P520.8623.879.0810.7820.4023.200.002.490.000.040.011.68
P69.514.751.461.204.122.980.031.100.020.110.041.88
P71.782.121.640.862.541.460.022.050.000.370.021.80
P814.2011.944.004.0411.6610.580.000.560.000.050.000.20
P969.7773.8722.6019.7657.0056.600.000.150.000.040.000.07
P1013.728.044.003.629.5010.000.626.500.343.200.484.82
P114.042.341.121.141.861.820.093.420.041.330.053.04
P1230.0042.0012.4222.8020.4032.200.162.420.060.970.081.03
P1359.2024.8020.208.1029.8012.940.398.750.710.360.691.65
P1451.4013.285.741.1022.804.920.031.920.000.450.031.12
P156.737.064.862.2010.384.800.534.200.361.200.523.62
P1639.600.3520.404.9828.408.060.193.360.330.700.411.06
P1764.0053.008.605.8634.0024.000.581.630.181.350.250.57
P1818.925.062.300.848.602.680.222.330.000.170.000.93
P1960.0020.2022.004.0836.808.920.020.640.130.420.010.60
P2096.600.705.628.664.2023.600.074.540.000.290.000.72
P2134.6017.6411.903.7624.4010.680.253.150.190.280.261.43
P2241.2019.2015.1210.5028.8017.705.2414.202.947.206.4914.60
P2363.2025.8010.605.5622.609.020.144.060.371.200.121.06
P2437.4016.009.543.4411.265.080.591.270.720.380.320.48
P2514.027.023.081.6210.804.660.052.340.000.120.040.87
P2622.605.325.260.9017.182.820.070.310.031.270.060.23
P2750.6034.889.634.9442.6223.800.241.010.120.840.060.71
P2872.4035.6013.6011.7058.6229.000.341.960.263.760.421.34
P2924.2018.749.807.0414.9412.400.010.120.000.350.000.03
P30116.8096.6025.6020.8060.2046.200.130.450.310.960.020.05
P3123.4011.703.322.4011.006.181.142.241.433.301.472.50
P3263.6089.2025.2038.6061.6088.001.091.941.023.580.801.16
P3372.0039.3220.4013.3646.2025.000.330.570.400.900.130.16
P3431.6017.787.884.1222.6011.800.130.220.150.630.120.15
P3525.4012.143.240.8411.606.600.032.200.011.170.020.16

Birch pollen SIT does not induce food allergen-reactive IgG4 Abs in all food-sensitized patients

To substantiate the divergent food allergen-reactive IgG4 responses observed in the nine patients shown in Fig. 1B, allergen-specific IgG4 levels before and at 36 months of BP-SIT were compared in additional 26 patients sensitized to both Mal d 1 and Cor a 1 (Table 1). A difference of >0.05 mg/l, which was the standard deviation of the assay determined by fivefold repeated analyses of three sera, was considered as positive. Accordingly, 6 of 35 (17%) patients showed no increase in Mal d 1-reactive IgG4, and 4 of 35 (11%) did not develop Cor a 1-reactive IgG4. Some individuals developed Mal d 1-reactive Abs, but no Cor a 1-reactive Abs and vice versa. Thus, not all patients who showed IgE cross-reactivity with Mal d 1 and Cor a 1 developed cross-reactive IgG4 Abs during BP-SIT.

Post-birch pollen SIT sera containing food allergen-reactive IgG4 Abs show blocking activity

Next, the blocking activity of sera with ample volumes at each time point (before and at months 1, 3, 6, 12, 24, 30, and 36 of BP-SIT) from four patients who had developed Cor a 1-reactive IgG4 was tested in FAB assays. In addition, the sera were compared for their capacity to inhibit Bet v 1- and Cor a 1-induced basophil activation by evaluating CD63 expression. The presence of post-BP-SIT sera containing allergen-specific IgG4 clearly reduced allergen IgE binding to B cells (Fig. 2A) and basophil activation by both Bet v 1 and Cor a 1 (Fig. 2B). The production kinetics of allergen-specific IgG4 are shown in Fig. 2C. Sera containing Bet v 1-IgG4 and no Cor a 1-IgG4 Abs served as controls and inhibited Bet v 1-IgE complex formation, but not Cor a 1-IgE complex formation (data not shown). To elucidate whether food allergen-reactive IgG4 Abs showed equivalent blocking activity for Bet v 1 and Cor a 1, sera from eight additional patients randomly selected from (P10-P17 (Table 1) were tested in FAB assays. All sera contained lower levels of Cor a 1- than Bet v 1-specific IgG4 (Table 1). Accordingly, the reduction in Cor a 1-IgE immune complex binding to B cells was significantly lower than inhibition of Bet v 1-IgE complex binding (P = 0.025, Fig. 2D). However, after adjusting Bet v 1- and Cor a 1-specific IgG4 levels, a similar inhibitory activity was observed for both allergens (Fig. 2D).

image

Figure 2. IgE-blocking activity of post-BP-SIT sera. Bet v 1 (full line) and Cor a 1 (dashed line) were pre-incubated with sera from four patients obtained before (0) and during BP-SIT. Pre-BP-SIT sera were normalized to 100%. The percentage of inhibition of (A) basophil activation and (B) IgE allergen binding to B cells is shown. (C) IgG4 levels specific for Bet v 1 (full line) and Cor a 1 (dashed line) in the respective sera at indicated time points. (D) FAB assays of eight patients who developed Bet v 1- and Cor a 1-reactive IgG4. Sera containing higher levels of Bet v 1- (a) than Cor a 1-reactive (b) IgG4 levels show significantly stronger inhibition activity which is reduced after adjusting Bet v 1- to Cor a 1-reactive IgG4 levels (c). Horizontal lines indicate median values. Wilcoxon signed-rank test, *P < 0.05.

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Post-birch pollen SIT sera containing food allergen-reactive IgG4 Abs inhibit IgE-mediated T-cell activation

Bet v 1-specific TCL cross-reactive with the food allergens were stimulated with either Bet v 1, Mal d 1, or Cor a 1 and pre-incubated with sera containing high levels of allergen-specific IgE in the presence of pre- and post-BP-SIT sera from four patients. Sera from three patients contained Bet v 1- and food allergen-reactive IgG4 Abs, and the fourth serum contained Bet v 1-reactive IgG4 Abs, but not food allergen-reactive IgG4 Abs (Fig. 3). Sera inhibited IgE-mediated proliferation of allergen-specific T-cell lines in response to Bet v 1 and its food homologs (Fig. 3).

image

Figure 3. Post-BP-SIT sera inhibit IgE-mediated allergen-induced T-cell proliferation. (A) T-cell proliferation to Bet v 1 (white bars), Mal d 1 (light gray bars), and Cor a 1 (dark gray bars) pre-incubated with sera from four patients collected before (0) and during BP-SIT. n.t., not tested, because TCL were not cross-reactive with Cor a 1. (B) IgG4 levels specific for Bet v 1 (full line), Mal d 1 (dotted line), and Cor a 1 (dashed line) in the respective sera at indicated time points.

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Predicted IgE and IgG4 epitopes show a high degree of overlap

For each serum and allergen, individual sets of phage-displayed 12-mer peptides were competitively eluted from IgE- or IgG4-loaded beads with the recombinant allergens, respectively (Table 2). Several peptides were detected multiple times with the same serum and allergen or with different allergens. Moreover, identical peptides were selected with sera from different patients. These findings point to the cross-reactivity of Bet v 1, Cor a 1, and Mal d 1. Of note, nine identical mimotopes bound to both IgE- and IgG4-loaded beads, suggesting that IgE and IgG4 may recognize the same epitopes. All peptides were mapped on the surface of the eluting allergen, and conformational epitopes were predicted by EpiSearch [18] and checked for overlap by GETAREA as described in 'Materials and methods' (Fig. 4). Table S1 shows all mimotope sequences and aa residues of predicted IgE- and IgG4-epitopes on Bet v 1, Mal d 1 and Cor a 1 for five patients. Table 2 summarizes the total numbers of these IgE- and IgG4-epitopes. For each individual and allergen, the overlap of predicted IgG4 with IgE epitopes ranged from 33 to 100% (mean for Bet v 1: 71.4%, Mal d 1: 74.8%, Cor a 1: 86.4%) (Table 2).

Table 2. Summary of mimotopes, predicted IgE/IgG4 epitopes and their overlap
 Bet v 1-IgEBet v 1-IgG4Mal d 1-IgEMal d 1-IgG4Cor a 1-IgECor a 1-IgG4
  1. Peptides binding both IgE and IgG4 and/or detected multiple times with different sera are underlined.

P1
Mimotopes

TCAKATSTPPLS

DPQIAYERANTS

KPPSLTPIHLTA

DVVPASTRDHTD

GTTTLNHNYSAK

QMSLQYRSHYTS

SIPRYYHTENSP

SSPSAAQWLNIG

LPGRAHDPWKVP

HPATSNTKYWIN

FTTESGGLNPHA

VEAHNQWTGLVA

YHPNGMNPYTKA

APTIAYNTFMPM

DRLPGTQAVRLV

YHPNGMNPYTKA

SNLSWPANMKHP

TYNTHPDFTYSS

VEAHNQWTGLVA

EQVPASSAQWSR

MDRANLVKPSTL

KFNPGNSEWQRT

SATNGSLTRPVH

GYSPTKDRMESA

GTTTLNHNYSAK

YHPNGMNPYTKA

YHPNGMNPYTKA

STNSPPASMRTN

RSDALEYMSKLR

KFNPGNSEWQRT

LWAYPVIYTGKW

TDPYLSTRLSLH

TSPDVVAPQLTK

APKSTVDSAQNL

TCAKATSTPPLS

No of predicted epitopes333241
No of IgG4 epitopes overlapping with IgE (%) 2 (66%) 2 (100%) 1 (100%)
P2
Mimotopes

HHFEKRWGAETL

YHKSPNACDLSP

YHPNGMNPYTKA

NHSFDKSTLSTG

TAKMQVNNMKPS

ESIHASGKMGRH

ANHLSGNNYGIS

GTTTLNHNYSAK

YHPNGMNPYTKA

NDNYPATIKHSN

SVDMPHPYGQRP

QLYPGTPAAERL

MHNRPTTYAHGD

SQPFKTWQSHDL

NQAKDHHNLQRY

TVEPNKLKFTQT

HGIPKHNKDMRT

YKPMADRSSAVF

ACQHKYCRADFV

VLIPSNFSPDKS

HLHTYQRPVQFY

GNNASKDVSATR

SSLHSSHHEMNK

MDTTRSAEKIKW

VQHNTKYSVVIR

SEASWTKVSQLK

TSYPSYLHRTMP

QNGNITALHSYV

VNLEKSSSDTRH

FEDHRGWAVDRM

QQSWPSHLSPTK

No of predicted epitopes424423
No of IgG4 epitopes overlapping with IgE (%) 1 (50%) 3 (75%) 2 (66%)
P3
Mimotopes

QHMGVSPAAAAA

IFAALDYNLGRH

DSHSTHKALMAH

GTTTSNHNYSAK

YHPNGMNPYTKA

SNLSWPANMKHP

GTTTSNHNYSAK

HMGMTKINYSAL

HMGMTKINYSAL

HMGMTKINYSAL

LSSHKATSTRAD

SLPFGPNTTIRP

LESHYTQASYTQ

SNLSWPANMKHP

YNDMSCCMAVHH

AVEQEAARHYNW

YHPNGMNPYTKA

WSEYDIPTPQIP

EVEMKGRHLAQM

YYNPSPPNPRTQ

TGSWATPKGVRM

HSHELQSSAKIA

TAYTLTNPETGY

YHPNGMNPYTKA

MADHSRHPNSSM

LNAKVPLPGHKS

YSINAIKNARHF

NRPDSAQFWLHH

KLHISKDHIYPT

No of predicted epitopes544323
No of IgG4 epitopes overlapping with IgE (%) 3 (75%) 2 (66%) 2 (66%)
P6
Mimotopes

VQHNTKYSEVIR

KPVAYYPWTGSQ

IHNPQQNQQTST

EQVPASSAQWSR

STNSPPASMRTN

GYTSSEPRLHTL

YTDIHIQNTEAA

YHPNGMNPYTKA

IHNPQQNQQTST

VPQARWHDMPRP

VTQEYRGNNNIS

DSRPWMESWKTP

FDFPHGKLDKRD

SAVEKEGYPPLR

AQENIYRIMAPA

APCPKNMCSSTV

SLAGATATAALR

IFAALDYNLGRH

NTALTEDNSFQR

QVFAVRDWHHVP

AKAADLPMYGQN

KFNPGNSEWQRT

EQHNTKYSVVIR

TPPKQMINTFIN

TQPHQAVDVLHS

SASIFSHGHARV

MTFDTVSNIYKM

GEAKTRLLPSQH

YHPNGMNPYTKA

KFNPGNSEWQRT

DPVYVKRQDRIV

IQTSRGDWTDMA

GKMPSNPGLKSG

No of predicted epitopes332352
No of IgG4 epitopes overlapping with IgE (%) 2 (66%) 1 (33%) 2 (100%)
P17
Mimotopes

TSVPLDGTLLQH

AWNTRVDSLSDK

TWTNDQQRSLQR

SEASWTKVSQLK

GDSAGGTRLYVV

EHYMASTYFSPK

AHRDAHASLPMH

GVKDVNAMDRVT

SHEPVLMIQKFK

QLYPGTPAAERL

TSNNTLTFTKFQ

TLETTIKHNYWL

DLAKNGVMTKGF

SVTVNATSLGTT

KALDNRWSRTQL

THLGLYQRNTMN

FDFPHGKLDKRD

LPGRAHDPWKVP

IFAALDYNLGRH

HANSYNPTKDHK

YHPNGMNPYTKA

HNDGQSRPMASH

VLDNGTQKHTHK

NLNGSQHTVSPI

DVSMVPVPPPNR

ESTMNNHSYITL

GEQAKTPVGSFE

GTTTLNHNYSAK

AGLHFMKPSERN

SFMNNSVRYNNM

SELNGQTPTMTA

NSIDKTLVIPPT

KFNPGNSEWQRT

FMPILSSPSTYT

FTTESGGLNPHA

TSRELPVLMASN

TTITTHKLPVFR

No of predicted epitopes333242
No of IgG4 epitopes overlapping with IgE (%) 3 (100%) 2 (100%) 2 (100%)
image

Figure 4. Distinct and overlapping epitopes on Bet v 1. Representative examples for (A) distinct epitopes: mimotope GTTTLNHNYSAK predicting epitope Bet v 1N4, Y5, N82, Y83, L95, K97, S99, K115, S117, N118, K119, H121, K134, A135, S136, K137, G140 (ASA = 902.2 Å2, red) and mimotope YHPNGMNPYTKA predicting epitope Bet v 1K55, P59, G61, P63, K65, Y66, G88, G89, P90, G92, A135, M139 (ASA = 498.7 Å2, green). The intersection of the two epitopes (A135) is 56.8 Å2 (yellow), leaving 707.9 Å2 (>600 Å2) of contiguous surface area (Bet v 1N4, Y5, N82, Y83, L95, K97, S99, K115, S117, N118, K119, H121, K134) for potential independent immunoglobulin binding. (B) overlapping epitopes: mimotope DVVPASTRDHTD predicting epitope Bet v 1D25, D27, P31, V33, P35, S39, S40, V41, S57, P59, D69, T142, R145, A146 (ASA = 670.2 Å2, red) and mimotope APTIAYNTFMPM predicting epitope Bet v 1F22, I23, N28, F30, P31, P35, I56, F58, P59, M139, T142, A146, Y150 (ASA = 498.7 Å2, green). The intersection of the two epitopes (P31, P35, P59, T142, T146) has 230.2 Å2 ASA (yellow), leaving a largest contiguous surface area (Bet v 1S39, S40, V41, S57) of 134.16 Å2 (<600 Å2). Protein models (pdb 1BV1) were displayed using Polyview3D [36].

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information

This study evaluated the production kinetics, cross-reactivity, IgE-blocking activity and specificity of Bet v 1-specific IgG4 Abs induced by BP-SIT performed for up to 36 months. Monitoring Bet v 1-, Mal d 1-, and Cor a 1-reactive Ab levels in 42 individuals revealed an overall significant increase in allergen-specific IgG4 and a decrease in IgE during the course of treatment. Notably, Bet v 1-specific IgE levels were significantly reduced already after 12 months of BP-SIT, whereas a significant reduction in food allergen-reactive IgE started at later time points. Bet v 1- and food allergen-reactive IgG4 appeared simultaneously after 3 months of BP-SIT. To study whether food allergen-reactive IgG4 Abs blocked IgE allergen binding, we employed different experimental approaches, namely FAB assays, BAT, and IgE-mediated allergen presentation to specific T cells. Concordantly, all results showed that post-SIT sera containing Bet v 1- and food allergen-reactive IgG4 Ab inhibited IgE binding to the major birch pollen allergen and its food homologs. Previous studies have attributed most of the inhibitory capacity in post-SIT sera to IgG4 Ab [25]. Accordingly, the blocking activity of individual sera correlated with allergen-specific IgG4 levels in our experimental setups, indicating that BP-SIT can induce IgG4 Abs able to inhibit IgE binding to Bet v 1, Mal d 1, and Cor a 1. However, at early time points of SIT, we noticed blocking activity despite still low allergen-specific IgG4 levels. It has been suggested that IgE shows a higher tendency toward cross-reactivity than IgG4 [26]. Accordingly, we calculated the individual ratios of Mal d 1/Bet v 1 and Cor a 1/Bet v 1 for IgE at m0 and IgG4 at m12 and m36, respectively, and found preliminary evidence that Mal d 1-IgE has a lower tendency to cross-react than Mal d 1-IgG4, whereas Cor a 1-IgE has a higher tendency to cross-react than Cor a 1-IgG4. However, experimental proof for this hypothesis is still needed.

Vaccination with genetically modified non-IgE-binding Bet v 1-variants was reported to induce IgG Abs that inhibit IgE recognition of Bet v 1- and Bet v 1-related food allergens due to sterical hindrance after binding to new sequential epitopes not recognized by IgE [27, 28]. To investigate whether Bet v 1-specific IgG4 Abs induced by administration of birch pollen extract recognized IgE epitopes, we combined competitive immunoscreening of phage-displayed 12-mer peptides with EpiSearch. This algorithm has been demonstrated to predict conformational epitopes as determined by X-ray structures of antigen–Ab complexes [18]. EpiSearch has also predicted conformational epitopes on the major cockroach allergen Bla g 2 in good accordance with the results from co-crystal structural analysis [29]. All our predicted IgE epitopes on the surface of Bet v 1 matched those reported by Mittag et al. [9] who employed a random phage-displayed 7-mer peptide library as well as those identified by other experimental approaches, for example by determination of the X-ray structure of an antigen–Ab complex or by employment of peptide-specific monoclonal Ab and human monoclonal IgE [7, 30-33]. Hence, we proceeded to predict IgG4 epitopes on Bet v 1, Mal d 1, and Cor a 1, respectively, and calculated their potential overlap with IgE epitopes (Fig. 4). Comparing predicted binding sites of IgG4 and IgE Abs from five different individuals on all three allergens revealed that on average, 77.5% IgG4 epitopes overlapped with IgE epitopes. The high degree of overlap suggests that the majority of BP-SIT-induced IgG4 Abs recognize IgE binding areas. This hypothesis is further supported by the elution of several identical mimotopes from IgE- and IgG4-loaded beads.

In summary, our data provide evidence that not all but most patients receiving BP-SIT develop Bet v 1-specific IgG4 Abs that cross-react with homologous food allergens and block IgE allergen binding by direct epitope competition. However, our data also indicate that SIT-induced IgG4 Abs are not simply IgE epitope identical responses as more than 35% of predicted IgE epitopes were not recognized by IgG4 Abs (mean for Bet v 1: 36%, Mal d 1: 38%, Cor a 1: 37%). Thus, SIT-induced IgG4 Abs may not cover all IgE specificities. On the other hand, around 20% of IgG4 epitopes did not overlap with IgE epitopes (mean for Bet v 1: 28%, Mal d 1: 25%, Cor a 1: 13%). The induction of de novo IgG4-specificities has been reported for SIT with grass pollen and recently also for oral immunotherapy with peanut [34, 35]. Based on our results, we conclude that the majority of SIT-induced IgG4 Abs recognize IgE epitopes, and a minority represent de novo specificities.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information

The work was supported by the Austrian Science Fund/Medical University of Vienna doctoral program (DK W1212) ‘Inflammation and Immunity’, FWF-SFB-F4610-B19, the Christian Doppler Research Association and Biomay AG, Vienna, Austria. B.S. received a Research Fellowship 2011 from the European Academy of Allergy and Clinical Immunology.

Author contributions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information

B.S. and B.B. designed the experiments; B.S. and D.S. performed the experiments and analyzed the data; C.M., N.J., C.E., N.R., W.P., and R.G.W. provided patients' sera, D.B., J.L., and S.V. performed ImmunoCAP; B.S. and B.B. wrote the manuscript.

Conflicts of interest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information

S. Vieths has received consultancy fees from the Food Allergy Resource and Research Program, Lincoln, NE, USA, the Institute for Product Quality, Berlin, Germany, and Fresenius Academy, Dortmund, Germany; has received an honorarium for evaluation of a PhD thesis from the Medical University of Vienna, Austria; has received lecture fees from the American Academy of Asthma, Allergy and Immunology, the Deutsche Dermatologische Gesellschaft, the Spanish Society of Allergy and Clinical Immunology, the Westdeutsche Arbeitsgemeinschaft für pädiatrische Pneumologie und Allergologie e.V., Köln, Germany, the Gesellschaft für pädiatrische Allergologie und Umweltmedizin, and the Ärzteverband Deutscher Allergologen; and has received royalties from Schattauer Allergologie Handbuch and Elsevier Nahrungsmittelallergien und Intoleranzen. His institution has received grants from Monsanto Company and Pioneer Hi-Bred International, Inc.; and has received travel/accommodations/meeting expenses from the German Research Foundation, the Federal Institute for Risk Assessment, the Austrian Society for Allergology and Immunology, the French Society of Allergology, the European Directorate for the Quality of Medicines and Health Care, the European Academy of Allergy and Clinical Immunology, the World Allergy Organization, the Technical University of Munich, the Deutscher Allergie- und Asthmabund, the Association Monégasque pour le Perfectionnement des Connaissances des Médicins, the Federal Office of Consumer Protection and Food Safety, the German Chemical Society (GDCh), the Austrian Society for Dermatology and Venerology and AKM Allergiekongress. [Correction added on 8 October 2013, after first online publication: conflict of interest statement added.]

References

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflicts of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
all12236-sup-0001-TableS1.docWord document564KTable S1. Amino acid sequences of mimotopes and predicted IgE and IgG4 epitopes on Bet v 1, Mal d 1 and Cor a 1.

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