Effect of chemical modifications on allergenic potency of peanut proteins

Abstract

Background: Modification of native peanut extracts could reduce adverse effects of peanut immunotherapy.

Objective: We sought to compare native and chemically modified crude peanut extract (CPE) and major peanut allergens Ara h 2 and Ara h 6 in a mediator-release assay based on the rat basophilic leukemia (RBL) cell line transfected with human Fcε receptor.

Methods: Native Ara h 2/6 was reduced and alkylated (RA), with or without additional glutaraldehyde treatment (RAGA). CPE was reduced and alkylated. Sera of subjects with peanut allergy (16 males; median age 7 years) were used for overnight RBL-passive sensitization. Cells were stimulated with 0.1 pg/mL to 10 μg/mL of peanut. β-N-acetylhexosaminidase release (NHR) was used as a marker of RBL degranulation, expressed as a percentage of total degranulation caused by Triton X.

Results: Median peanut-specific immunoglobulin E was 233 kUA/L. Nineteen subjects were responders, NHR ≥ 10% in the mediator release assay. Responders had reduced NHR by RA and RAGA compared with the native Ara h 2/6. Modification resulted in a later onset of activation by 10- to 100-fold in concentration and a lowering of the maximum release. Modified RA-Ara h 2/6 and RAGA-Ara h 2/6 caused significantly lower maximum mediator release than native Ara h 2/6, at protein concentrations 0.1, 1, and 10 ng/mL (p < 0.001, < 0.001, and < 0.001, respectively, for RA; and < 0.001, 0.026, and 0.041, respectively, for RAGA). RA-CPE caused significantly lower maximum NHR than native CPE, at protein concentration 1 ng/mL (p < 0.001) and 10 ng/mL (p < 0.002). Responders had high rAra h 2 immunoglobulin E (mean, 61.1 kUA/L; p < 0.001) and higher NHR in mediator release assay to native Ara h 2/6 than CPE, which indicates that Ara h 2/6 were the most relevant peanut allergens in these responders.

Conclusions: Chemical modification of purified native Ara h 2 and Ara h 6 reduced mediator release in an in vitro assay ∼100-fold, which indicates decreased allergenicity for further development of the alternative candidate for safe peanut immunotherapy.

Figure 1.

(A and B) Allergenic potency and dose-response curve of NHR (%) between native Ara h 2/6 and its modified form, RA-Arah 2/6, and (C and D) CPE and its modified form. The dilution that gives the ED₅₀ was calculated. The reciprocal value of ED₅₀ (1/ED) was defined as the allergenic potency of the extract (1.E) and compared between native extract and its modified form on a logarithmic scale. Lines connect the symbols that represent the allergenic potency of native and RA-Ara h 2/6 extracts from the sera of the same individual subject. The overlapping number of subjects is shown. One responder (subject no. 12) was excluded because no NHR was induced by the RA-Ara h 2/6 extract. Allergenic potency of RAGA-Ara h 2/6 extract was not shown due to the unreliability of the allergenic potency from very low NHR (%). (A) Allergenic potency of native Ara h 2/6 and its modified form, RA-Ara h 2/6 in 19 responders. (B) Dose-response curve of NHR induced by native Ara h 2/6 and its modified forms, RA-and RAGA-Ara h 2/6 in four representative responders. The curves represent NHR (%) from the lowest peanut protein concentration (0.1 pg/mL, left) to the highest protein concentration (10 μg/mL, right). (C) Allergenic potency of CPE and its modified form, RA-CPE in 19 responders. Lines connect the symbols that represent the allergenic potency of CPE and RA-CPE extracts from the sera of the same individual subject. The overlapping number of subjects was shown. One responder (subject no. 12) was excluded due to no NHR induced by the RA-CPE extract. (D) A dose-response curves of NHR induced by CPE and its modified form, RA-CPE in four representative responders; the curves represent NHR (%) from the lowest peanut protein concentration (0.1 pg/mL, left) to the highest protein concentration (10 μg/mL, right).