title: 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.
authors.affiliation: Division of Pediatric Allergy and Immunology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Ramon
authors.initials: R
authors.lastname: Bencharitiwong
authors.forename: Hanneke P M
authors.initials: HP
authors.lastname: van der Kleij
authors.forename: Stef J
authors.initials: SJ
authors.lastname: Koppelman
authors.forename: Anna
authors.initials: A
authors.lastname: Nowak-Węgrzyn
mesh_terms: D055516:2S Albumins, Plant / Q000276:immunology; D000293:Adolescent; D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000818:Animals; D052179:Antigens, Plant / Q000276:immunology; D010367:Arachis / Q000009:adverse effects* / Q000737:chemistry; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D005260:Female; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D008297:Male; D021183:Peanut Hypersensitivity / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500890:Ara h 2 allergen, Arachis hypogaea; C492737:Ara h 6 allergen, Arachis hypogaea; D006023:Glycoproteins; D007073:Immunoglobulin E; D001619:beta-N-Acetylhexosaminidases