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.

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.

Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants.

RATIONALE: An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. METHODS: Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors. RESULTS: Following gel filtration, 75 +/- 7% of the applied protein and 76 +/- 16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 +/- 2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of approximately 20 kDa and a range of 13-25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 +/- 43% when measured with a pool of serum (n=2) and 66 +/- 7% when measured with individual sera (n=4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 +/- 2% (n=2) of the original extract when assayed with the serum pool and 10 +/- 4% (n=3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. CONCLUSIONS: Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.

Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation.

BACKGROUND: Ara h 2 is a potent peanut allergen but its contribution to the ability of a crude peanut extract (CPE) to cross-link IgE and activate mast cells has not been rigorously evaluated. OBJECTIVE: To measure the contribution that Ara h 2 makes to the effector function of a CPE. METHODS: Ara h 2 was specifically removed from a CPE as demonstrated by immunoblots, 2D gels, and an inhibitory ELISA. Functional assays of sham-treated and Ara h 2-depleted CPEs were performed with RBL SX-38 cells sensitized with IgE from highly peanut-allergic subjects and with naturally sensitized basophils. RESULTS: Depletion of approximately 99% of the Ara h 2 from the CPE led to an increase in the concentration of the CPE necessary to give 50% of maximal degranulation (EC50) of the SX-38 cells following sensitization with sera that contain anti-Ara h 2 IgE. Assays with a pool of 10 sera showed a small but significant increase in the EC50 following depletion of Ara h 2 (1.65+/-0.15-fold; P<0.05) and assays of seven individual sera showed a similar increase in the average EC50 (1.7+/-0.2-fold; P<0.02). The percent of the anti-peanut IgE that binds Ara h 2 correlated with an increase in the EC50 of the CPE following depletion of Ara h 2 (r=0.83; P<0.02). On the other hand, data from three of these patients studied with a basophil histamine release assay did not show a significant effect of depletion of Ara h 2. CONCLUSION: Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied.