A hypoallergenic peptide mix containing T cell epitopes of the clinically relevant house dust mite allergens

Abstract Background In the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects. Methods IgE reactivity of HDM‐allergic and non‐HDM‐sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty‐three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively. Results House dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM‐allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen‐specific IgG levels were found in HDM allergics. PBMC from HDM‐allergics produced higher levels of IL‐5 whereas non‐HDM‐sensitized individuals mounted higher levels of IFN‐gamma, IL‐17, pro‐inflammatory cytokines, and IL‐10. Conclusion IgG antibodies in HDM‐allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen‐specific IgG antibodies do not protect against IgE‐mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.


| INTRODUC TI ON
House dust mites (HDMs) are one of the most important elicitors of immunoglobulin E (IgE)-associated allergies worldwide. 1 HDM-allergic patients suffer from severe and chronic respiratory symptoms such as rhinitis and asthma as well as from skin symptoms, mainly atopic dermatitis. [2][3][4] Dermatophagoides pteronyssinus (Der p) and D.
farinae (Der f) are the most important mite species and contain more than 30 different allergen molecules. [5][6][7] Der p and Der f allergens show extensive cross-reactivity and several allergens seem to have intrinsic properties promoting their ability to induce allergic sensitization. 8 Assessment of IgE to individual Der p allergens in crosssectional population studies as well as in longitudinal birth cohort studies have shown that Der p 1, Der p 2, Der p 4, Der p 5, Der p 7, Der p 21, and Der p 23 are the most frequently recognized allergens. [9][10][11][12][13] Among those, Der p 1, 2, 5, 7, 21, and 23 seem to be clinically most relevant, as they were shown to comprise the majority of IgE epitopes of the HDM proteome. 14 At present, several molecular approaches for allergen-specific immunotherapy (AIT) and prevention are under development. [15][16][17] They include recombinant hypoallergens targeting B cell as well as T cell responses, B cell epitope-targeting vaccines based on fusion proteins, which consist of allergen-derived peptides fused to a nonallergenic carrier protein [18][19][20] and, nonallergenic peptides comprising the T cell epitope repertoire for the induction of therapeutic as well as preventive immune tolerance. 16,21 Approaches that target allergen-specific T cells with synthetic peptides are mainly based on small peptides of less than 20 amino acids to avoid IgE sensitization, IgE recognition, and associated side effects. 21,22 With such an approach it is difficult, or even impossible, to cover the majority of T cell epitopes of several allergens with a reasonable number of synthetic peptides. Therefore, we have synthesized a panel of 33 peptides ranging from 27 to 43 amino acids, which allowed us to cover the sequences of the most important HDM allergens, that is Der p 1, 2, 5, 7, 21, and 23. These peptides and the complete allergen molecules were used to study allergen/peptide-specific IgE and IgG responses, as well as T cell and cytokine responses to the allergens and peptides in HDM-allergic patients and subjects without HDM sensitization. Our study identified a cocktail of 31 defined synthetic hypoallergenic peptides containing T cell epitopes of the six important HDM allergens. This peptide mix might be suitable for T cell-directed approaches to induce immune tolerance for therapy and prevention of HDM allergy. It also revealed interesting differences regarding allergen-specific antibody responses in allergic and nonallergic subjects that explain why natural allergen-specific IgG antibodies do not protect against HDM allergy.  Table S1 . Peptides were purified by HPLC (Dionex UltiMate 3000; Thermo Fisher Scientific) and their molecular weights confirmed by MALDI-TOF (Microflex, Bruker). Purified peptides (>85%) with correct molecular weight were dissolved in endotoxin-free water and stored at −20°C.

| HDM-allergic patients and control subjects
Twenty-seven HDM-allergic patients, five non-HDM-sensitized allergic patients, and five nonallergic individuals were analyzed.
The subjects were recruited by advertisement with approval of the Ethics Committee of the Medical University of Vienna (EK 1641/2014). Symptoms of allergy were recorded using ISAACbased questionnaires. 24 Blood samples (serum, heparinized blood) were obtained from the subjects after written informed consent was obtained. The demographic and clinical characteristics of the subjects are summarized in Table S2 . The 27 HDM-allergic patients had a positive case history and perennial airway symptoms indicative for HDM allergy (ie, rhinitis, conjunctivitis, asthma, and/ or atopic dermatitis; Table S2 ). Patients ' treatments included measures for allergen avoidance, systemic or topical antihistamines and/ or topical steroids but none of the patients had received AIT during the last 5 years or had systemic intake of steroids. The presence of specific IgE antibodies to D. pteronyssinus was determined by ImmunoCAP (d1 ImmunoCAP, Thermo Fisher Scientific/Phadia, Uppsala, Sweden) and was ≥0.7 kUA/L. All HDM-allergic patients showed IgE reactivity to at least one of the 13 D. pteronyssinus allergens which were present on the MeDALL allergen chip 13,25 as determined by ImmunoCAP ISAC technology (Thermo Fisher Scientific/Phadia). The five non-HDM-sensitized subjects and five nonallergic subjects were negative to HDM allergen molecules in ImmunoCAP ISAC (Table S3 ). The five non-HDM-sensitized and five nonallergic subjects were combined to obtain a group of 10 non-HDM-sensitized subjects. Table S3 provides the complete IgE   sensitization profiles to 163 microarrayed allergens of all patients and control subjects analyzed in this study.

| ELISA and ImmunoCAP assays
Microplates (MaxiSorp; Thermo Fisher Scientific) were coated with 0.5 μg/well (in 100 μL PBS) of the individual HDM allergens or peptides overnight at 4°C. Microplates were washed three times with phosphate-buffered saline with Tween-20 (PBST) and nonspecific binding sites were blocked for 3 hours at RT with PBST containing 1% BSA (Carl Roth). Sera and antibodies were diluted with PBST F I G U R E 1 Overview of peptides spanning the sequences of Der p 1, 2, 5, 7, 21, and 23. Allergens are shown as black bars from the N (left) to the C terminus (right) in the same scale indicating the first, the last and every 50 th amino acid. Peptides are indicated as grey bars and are numbered for each allergen Detection of IgE binding was performed as described 25 (Table S4 ). A monoclonal anti-IgE antibody (1 μg/mL) was used as a positive control. Upregulation of CD203c expression on basophils was measured by flow cytometry as reported. 27 The stimulation index (SI) was calculated as a ratio of mean fluorescence intensity of allergen or peptide mixturetreated basophils to mean fluorescence intensity of unstimulated basophils.

| Cytokine measurements
The Assay; Bio-Rad) according to the manufacturer ' s instructions as described. 29 The Bio-Plex ® 200 System (Bio-Rad) was used for analysis.

| Statistical analysis
Mann-Whitney U test was performed to analyze the differences between HDM-allergic patients and non-HDM-sensitized individuals. A P value of less than 0.05 was considered as statistically significant. Figure 2 shows the comparison of the six important HDM allergens with the allergen-derived peptides regarding IgE reactivity with sera from HDM-allergic patients (Table S2 ). According to ELISA and ImmunoCAP ISAC testing (Table S3 ), we found the F I G U R E 2 IgE reactivity of Der p allergens and allergen-derived peptides. Allergens (Der p 1, Der p 2, Der p 5, Der p 7, Der p 21, and Der p 23) and allergen-derived peptides ( x -axes) were tested by ELISA for IgE reactivity ( y -axes: OD values correspond to specific IgE levels) with sera from individuals sensitized to the respective allergen (red) and individuals without HDM sensitization (black). Statistically significant differences (**** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05) are indicated None of the non-HDM-sensitized allergic patients and none of the nonallergic individuals showed IgE reactivity to any of the six HDM allergens in the ELISA and micro-array (Table S3 ). Thus patients mounted IgE reactivity to the complete allergen molecules but only two of the 33 allergen-derived peptides (ie, peptide 4 from Der p 5, peptide 3 from Der p 23) showed IgE reactivity ( Figure 2 ). Accordingly, 31 peptides comprising almost the complete sequences of Der p 1, 2, 5, 7, 21, and 23 lacked IgE reactivity (Table S1 ). The lack of IgE reactivity of the mix of these 31 allergen-derived peptides was confirmed by quantitative IgE measure-

| The peptide mix is hypoallergenic as determined by basophil activation testing
The allergenic activity of the HDM allergens and the peptide mix In contrast to IgE antibodies (Figures 2 and 3 ), IgG antibodies of allergic patients were directed to sequential peptide epitopes ( Figure 5 , Table S5 ). Also, non-HDM-sensitized subjects showed IgG reactivity to the allergen-derived peptides ( Figure 5 , Table S5 ).
F I G U R E 3 Quantification of allergen-and peptide-specific IgE. IgE levels (y-axis: kUA/L) specific for six Der p allergens (Der p 1, 2, 5, 7, 21, and 23) or allergen-derived peptides ( x -axis) were measured by ImmunoCAP in sera from HDM-allergic patients (red) and non-HDM-sensitized individuals (black). Statistically significant differences (**** P < 0.0001) are indicated  sensitized sensitized sensitized and PA23) but these patients also showed no detectable T cell response to the complete allergens ( Figure 6 ).

| PBMC from HDM-allergic patients produce higher levels of Th2 cytokines whereas non-HDMsensitized individuals produce higher levels of IFNgamma, pro-inflammatory cytokines and IL-10 in response to allergens
In parallel to the assessment of T cell proliferation, multiple cytokine responses to the allergens and peptides were determined in cultured PBMCs from HDM-allergic patients and non-HDM-sensitized individuals. The results are presented in Figure 7 A-E and   The levels of the other cytokines determined by the Luminex technology were either very low (eg, IL-2, IL-4, IL-7 and IL-12 (p70)) or above the detection limit (IL-8; data not shown).

| DISCUSSION
Der p 1, 2, 5, 7, 21, and 23 have been identified as the most frequently recognized HDM allergens comprising the majority of HDM-specific IgE epitopes. 14 Therefore, they should be considered as components included in a therapeutic or prophylactic vaccine for HDM allergy. A major goal of our study was to identify a panel of synthetic hypoallergenic allergen-derived peptides containing T cell epitopes of these allergens for therapeutic and preventive strategies which target T cells. Studies on T cell epitopes have so far only been conducted for Der p 1, 2, and 23 but not for the other clinically relevant HDM allergens, Der p 5, 7, and 21. [30][31][32] This is the first study to compare the T cell responses to the panel of clinically relevant HDM allergens (Der p 1, 2, 5, 7, 21, and 23). In order to reduce the number of peptides needed for a tolerogenic peptide cocktail, we synthesized peptides which were longer (ie, 27-43 aa) than the peptides used in previous T cell epitope-based approaches (<20 aa). 21,22 Peptides were designed to span hydrophilic regions and the exact populations with different antigen specificity are engaged in the production of allergen-specific IgE and IgG antibodies. Whether class switch recombination to IgE production occurs in a sequential mode by a switching from IgM via IgG to IgE or by direct and independent switching from IgM either to IgG or IgE production is a matter of considerable debate, 33 but our finding that IgE and IgG antibodies recognize different epitopes speaks for the latter scenario. HDM-allergic patients with IgE sensitization to a certain allergen always had higher levels of IgG to this allergen than non-HDM-sensitized subjects. This finding is in agreement with an earlier study which has performed a similar comparison in HDM-allergic children. 11 This study also found that allergen-specific IgG levels were higher in children who were sensitized to the same allergen than in children who were not sensitized, especially for Der p 1, Der p 2, and Der p 23. A similar result was obtained also in another study which showed that nonhospitalized, nonallergic individuals had low allergen-specific IgG levels when compared to allergic subjects. 34 We suggest that this could be due to a genetic restriction of allergen presentation to T cells which provide help to IgE as well as IgG production. HLA-restricted allergen presentation has been suggested already earlier to be associated with IgE recognition of allergens. 35 the tolerogenic cytokine IL-10. 43 A limitation of our study was that we could measure the cytokine levels only at one time point, which may have been not optimal for all of the cytokines due to different kinetics of production and there may be also consumption of cytokines in the cultures. The latter may also explain differences to previous studies which have reported that levels of IFN-gamma were higher or equal in PBMC from allergic compared to nonallergic subjects when stimulated with purified allergen molecules. [44][45][46] There are at least two possibilities for how the hypoallergenic cocktail of synthetic peptides containing T cell epitopes of the six important HDM allergens may be used for allergy treatment. First, the peptides may be applied in a therapeutic setting as has been proposed for T cell peptide approaches earlier, for example for the treatment of cat allergy. 21 However, different results were obtained for the therapeutic Fel d 1 peptide vaccination studies. Initial clinical trials were promising 47 but the large phase III field study ( https :// www.circa ssia.com/media/ press-relea ses/circa ssia-annou nces-topline-resul ts-from-cat-aller gy-phase-iii-study/ ) did not reveal significant differences regarding clinical improvement between active and placebo-treated patients. 48 Therefore, we suggest to use the hypoallergenic peptide mix rather in a preventive setting by applying the peptides systemically either via the oral route 16 or by injection, 49 with the goal to prevent allergic sensitization early in childhood.
The production of a cocktail of more than 30 peptides may be difficult, but it might be possible to narrow the peptide set for clinical applications. Since some peptides (eg, peptide 4 and peptide 5 from Der p 23) showed weak IgE reactivity and some T cell epitopes may be lacking because not all peptides were overlapping, the exact location and length of some peptides need to be adapted. Modifications of certain peptide sequences will be necessary to improve solubility and stability as well as to prevent aggregation. 50 For oral treatment, the peptides may need to be protected from degradation in the gastrointestinal tract. 51 Additionally, one may consider to reduce the number of peptides by identifying dominant T cell epitopes. 50 Nevertheless, our study may be considered as a first important step toward such an ambitious goal as it provides a set of hypoallergenic peptides containing T cell epitopes of the HDM allergens recognized by humans.

AUTH O R CO NTR I B UTI O N S
HJH contributed to design of the study, performed experiments,