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.

Keywords: T cell epitope; allergen; house dust mite allergy; recombinant allergen; synthetic peptide.

Figure 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

Figure 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

Figure 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

Figure 4

Comparison of the allergenic activity of Der p allergens and allergen‐derived peptides. Blood samples from seven HDM‐allergic patients (PA 6, 7, 10, 11, 13, 19, and 23) and one nonallergic individual (NA4) were incubated with anti‐IgE (1 µg/mL), medium buffer alone (control), different concentrations c1‐c5 (x‐axes: c1: 0.06 ng/mL, c2: 0.6 ng/mL, c3: 6 ng/mL, c4: 60 ng/mL, c5: 600 ng/mL) of mixtures containing the six Der p allergens (Der p 1, 2, 5, 7, 21, 23) (black) or approximately equimolar mixtures of the allergen‐derived peptides (c1: 0.076 ng/mL, c2: 0.76 ng/mL, c3: 7.6 ng/mL, c4: 76 ng/mL, c5: 760 ng/mL) (gray). Up‐regulations of CD203c expression on basophils are displayed as mean stimulation indices (SIs) ± SD (y‐axes)

Figure 5

IgG 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 for IgG reactivity (y‐axes: OD values correspond to specific IgG levels) with sera from individuals sensitized to the respective allergen (red) and individuals without HDM sensitization (black). The buffer control was subtracted from the data and cutoffs were represented by dashed lines. Statistically significant differences (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05) are indicated

Figure 6

T cell reactivity of Der p allergens and allergen‐derived peptides for each subject. Shown are percentages of proliferated CD4+ T cell in responses to the allergens and allergen‐derived peptides (A, Der p 1, Der p 2, Der p 5, B, Der p 7, Der p 21, Der p 23; Means of triplicates; color codes for different percentages of proliferated cells are shown with a cut off of 2%‐white) in PBMC cultures from each allergen‐sensitized patient (PA), non‐HDM‐sensitized allergic individuals (NDP) and nonallergic individuals (NA). Median percentages are shown for each allergen and peptide for sensitized and nonsensitized individuals at the bottom of each Table

Figure 7

Cytokine and chemokine responses to Der p allergens and allergen‐derived peptides. Cultured PBMCs from individuals sensitized to the respective allergen (red) and individuals without HDM sensitization (black: n = 10) were incubated with allergens (Der p 1, Der p 2, Der p 5, Der p 7, Der p 21, Der p 23) and the corresponding allergen‐derived peptides (x‐axes) and cytokine and chemokine levels were measured in the supernatants (y‐axis: pg/mL). A, IL‐5, B, IL‐13, C, IFN‐gamma, D, IL‐10, E, IL‐17. Shown are box plots representing the first and third quartile. Horizontal bars denote medians and outliers are presented as circles. Statistically significant differences (**P < 0.01; *P < 0.05) are indicated