The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice

Abstract

Background: Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera.

Objectives: To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE.

Methods: An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood.

Results: Compared with mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (P < 0.05) and a smaller decrease in body temperature (P < 0.01). Results with the basophil histamine release assay corroborated these findings (P < 0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared with placebo (P < 0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges.

Conclusions and clinical relevance: Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy.

Figure 1. Removal of Ara h 2 and Ara h 6 from CPE

Removal by chromatography, A: Immunoblots of control CPE (1 μg, lanes 1 and 5), the 20 kD fraction (0.2 μg lane 2), fractionated CPE with proportional recombination of all fractions without (1 μg, CPE w/o 20 kD; lane 3) or the same material with inclusion of the fraction containing proteins of 20 kD (1 μg, CPE recombined; lane 4). Probes were rabbit anti-peptide antibodies to Ara h 1, 2, and 6. Removal by immunodepletion, B: Immunoblots of control CPE (lane 1), Ara h 2/6 immunodepleted CPE (lane 2). Control CPE was CPE passed over a column with affinity purified pre-immune IgG.

Figure 2

Characterization of the CPE recombined and CPE recombined w/o 20 kD. Fifty μg of CPE recombined (A) and 50 μg of CPE recombined w/o 20 kD (B) were minimally labeled with Cy3 (red) and Cy5 (green) CyDyes, respectively and run together. The superimposed images are shown (C) where identical spots appear in yellow. Approximate molecular weights (kD) are shown (C).

Figure 3. Specific IgE levels to various peanut preparations

Peanut-sensitized mouse sera were used in an ELISA assay for specific IgE binding to: CPE (black bar); control CPE and Ara h 2/6 immunodepleted preparations (gray bars); recombined and recombined w/o 20 kD CPE (white bars); Ara h 1 and 20 kD (cross-hatched bars). Bars represent means with standard deviation.

Figure 4. Allergic provocation challenges in peanut-sensitized mice with CPE recombined and CPE recombined w/o 20 kD

A: Symptom scores following challenge with CPE recombined or CPE recombined w/o 20 kD. B: Body temperatures at baseline, 30, and 45 minutes post-challenge. Closed circles represent individual mice; bars show means with standard deviation; * indicates p<0.05; ** indicates p<0.01. A second, independent experiment with 3 mice per group and several doses of these CPE preparations gave similar results.

Figure 5. Allergic provocation challenges in peanut-sensitized mice with Ara h 2/6 immunodepleted CPE

A: Symptom scores following challenge with control CPE and Ara h 2/6 immunodepleted CPE. B: Body temperatures at baseline, 30, 45, and 60 minutes post-challenge. C: MMCP-1 levels in sera of mice post-challenge. Closed circles represent individual mice; bars show means with standard deviation; ** indicates p<0.01. A second independent experiment with 5 mice per group gave similar results.

Figure 6. Effect of removing Ara h 2 and Ara h 6 in a basophil histamine release assay

Whole blood was stimulated with CPE (open circles) or CPE recombined w/o 20 kD (closed circles) and histamine release was measured. Subjects: A) D63, B) D19, C) D74, and D) D86. Each subject was studied once; each data point is the mean of duplicates. At the dose giving half-maximal for CPE, the CPE w/o 20 kD gave 16±11% (mean±SEM) of that response (n=4; p<0.01).

Figure 7. Allergic provocation challenge with 300 μg CPE in peanut-sensitized mice following immunotherpy with CPE, recombined w/o 20 kD, 20 kD fraction, or placebo

A: Symptom scores following CPE challenge in the four immunotherapy groups. B: Body temperatures at baseline, 30, and 45 minutes post-challenge. C: MMCP-1 levels in sera samples post-challenge. Five mice were in each treatment group. Closed circles represent individual mice; bars show means with standard deviation; * indicates p<0.05; ** indicates p<0.01.

Figure 8. Allergic provocation challenge with 450 μg CPE in peanut-sensitized mice following immunotherapy with CPE, recombined w/o 20 kD, or 20 kD fraction

A: Symptom scores following CPE challenge in mice given immunotherapy. B: Body temperatures at baseline, 30, and 45 minutes post-challenge. C: MMCP-1 levels in sera samples post-challenge. These are the same mice challenged in Fig. 7 except the mice desensitized with placebo were excluded from challenge. Closed circles represent individual mice; bars show means with standard deviation; ** indicates p<0.01. A second independent experiment measuring symptoms and temperature changes with 3-5 mice in each group gave similar results.