Epitope analysis of Ara h 2 and Ara h 6: characteristic patterns of IgE-binding fingerprints among individuals with similar clinical histories

Abstract

Background: Ara h 2 and Ara h 6 are moderately homologous and highly potent peanut allergens.

Objective: To identify IgE-binding linear epitopes of Ara h 6, compare them to those of Ara h 2, and to stratify binding based on clinical histories.

Methods: Thirty highly peanut-allergic subjects were stratified by clinical history. Sera were diluted to contain the same amount of anti-peanut IgE. IgE binding to overlapping 20-mer peptides of Ara h 2 and Ara h 6 was assessed using microarrays.

Results: Each subject had a unique IgE-binding fingerprint to peptides; these data were coalesced into epitope binding. IgE from subjects with a history of more severe reactions (n = 19) had a smaller frequency of binding events (BEs) for both Ara h 2 (52 BEs of 152 (19X8epitopes) possible BEs and Ara h 6 (13 BEs of 133 (19X7 epitopes) possible BEs) compared to IgE from those with milder histories (n = 11) (Ara h 2: 47 BEs of 88 (11X8 epitopes) possible BEs, P < 0.01; Ara h 6: 25 BEs of 77 (11X7 epitopes) possible BEs, P < 0.001). Using an unsupervised hierarchal cluster analysis, subjects with similar histories tended to cluster. We have tentatively identified a high-risk pattern of binding to peptides of Ara h 2 and Ara h 6, predominantly in subjects with a history of more severe reactions (OR = 12.6; 95% CI: 2.0-79.5; P < 0.01).

Conclusions and clinical relevance: IgE from patients with more severe clinical histories recognize fewer linear epitopes of Ara h 2 and Ara h 6 than do subjects with milder reactions and bind these epitopes in characteristic patterns. Close examination of IgE binding to epitopes of Ara h 2 and Ara h 6 may have prognostic value.

Keywords: Ara h 2; Ara h 6; IgE; allergens; microarrays; peanut; peptides.

Fig. 1

Microarray analysis of IgE binding to overlapping 20-mer peptides of Ara h 2, left, and Ara h 6, right. Sera are from 4 of 30 representative patients with varying histories regarding the severity of reactivity. From top to bottom: D61, grade I (less severe); D71, grade II; D74 grade III; and D60, grade IV (most severe). Linear peptide regions that bind peanut-specific IgE are colour-coded (see Fig. 2). The Y axis is a calculated z-score (see Methods). The established cut-off score of z = 3 is shown as a horizontal line.

Fig. 2

Amino acid alignment is shown for Ara h 2 and Ara h 6 (a). Different colours denote epitopes for Ara h 2 and Ara h 6; these are listed (b). The asterisks (*) in A, denote linear sequences for Ara h 2 that have been previously identified [38, 46]. A colour-coded 3-D image is shown (c).

Fig. 3

Correlation of IgE binding to linear epitopes. Spearman correlation analysis was performed with z-scores for each homologous pair of epitopes of Ara h 2 and Ara h 6 (horizontal and vertical axes, respectively): epitope 1 of Ara h 2 and epitope A of Ara h 6 (a, r = 0.37, P = 0.04, p_c = ns); epitope 2 of Ara h 2 and epitope B of Ara h 6 (b, r = 0.55, p_c = 0.01); epitope 3 of Ara h 2 and epitope C of Ara h 6 (c, r = 0.21, p_c = ns); epitope 4 of Ara h 2 and epitope D of Ara h 6 (d, r = 0.64, p_c < 0.01); epitope 5 of Ara h 2 and epitope E of Ara h 6 (e, r = 0.65, p_c < 0.01); epitope 7 of Ara h 2 and epitope F of Ara h 6 (f, r = 0.82, p_c < 0.001); and epitope 8 of Ara h 2 and epitope G of Ara h 6 (g, r = 0.90, p_c < 0.001).

Fig. 4

Heat maps of binary (yes or no; z ≤ 3/z > 3) binding of IgE to epitopes for Ara h 2 (a) and Ara h 6 (b) for all 30 sera sorted according to grades of reaction based on clinical histories, ‘yes’ is denoted by yellow and ‘no’ is denoted by blue.

Fig. 5

Unsupervised cluster analysis of the data from Fig. 4 for Ara h 2 (a) and Ara h 6 (b). ‘yes’ is denoted by yellow; ‘no’ is denoted in blue. Sera from subjects with a history of grade I or II reactivity are in black. Those with a history of grade III or IV reactivity are in red.

Fig. 6

Contingency plots of key data from the unsupervised cluster analysis shown in Fig. 5. Totals for each horizontal and vertical set of numbers are shown. For all plots, n = 30. (a) Frequency of subjects with a history of grades I or II severity cluster in either node 1 or 2A of Ara h 2 (P = 0.03); (b) frequency of subjects with a history of grades III or IV severity cluster in node 2B2B1 of Ara h 6 (P = 0.02); (c) frequency of subjects who cluster in node 2B2B1 of Ara h 6 and do not cluster in either node 1 or 2A of Ara h 2 (P < 0.01); and (d) frequency of subjects having the high-risk pattern [h 6 (2B2B1), yes and h 2 (1 + 2A), no] (P < 0.01).