Date of this Version
Allergy Asthma Proc 36:185–191, 2015; doi: 10.2500/aap.2015.36.3840.
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 Fce 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 ug/mL of peanut. B-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.