PURPOSE OF THE STUDY.
To evaluate the impact of extended boiling on peanut allergenicity and T cell reactivity.
Blood samples were collected from 10 peanut-allergic children ages 8 to 14 years with peanut-specific IgE ranging from 91.8 to >100 kU/L. Skin prick tests using boiled peanut extracts were performed on 20 known peanut-allergic children ages 2 to 16 years. Blood samples were collected for peanut antigen-specific T cell assays from 3 peanut-allergic patients and 3 nonallergic volunteer controls.
Raw peanuts were boiled for 30 minutes, 1 hour, 2 hours, 4 hours, and 12 hours in deionized water. After dehydration, boiled and raw peanuts were ground, defatted in acetone, agitated, centrifuged, and air dried for 24 hours. The resultant pellet was resolubilized in 5 volumes of phosphate-buffered solution, and both the peanut extract and the leachate-containing solubilized peanut proteins were sterilized and retained. SDS-PAGE, Western blot, two-dimensional electrophoresis, IgE-inhibition ELISA, mass spectrometry, and skin prick testing were used to characterize changes to peanut allergens and human IgE reactivity associated with progressive boiling. T cell responses to raw and boiled peanut extracts were determined by proliferation of CD4+/CD25+/CD134+ T cells in peanut-allergic and nonallergic patient blood samples.
Extended boiling caused increasing fragmentation of peanut proteins into lower molecular weight polypeptides, denaturing of conformational epitopes, and transference of proteins to the leachate. Compared with the raw peanut extract, eightfold more 2-hour boiled peanut extract and 19-fold more 12-hour boiled peanut extract were required to achieve 50% inhibition of IgE by inhibition ELISA. Boiling increased the number of unique allergen peptides apparent via mass spectrometry in the boiled peanuts by more than fivefold at 2 hours and by 42-fold at 12 hours. As compared with unboiled raw peanut extract, skin prick testing demonstrated a significant reduction in wheal size to 55% for the 2-hour boiled peanut extracts and to 36% for the 4-hour boiled peanut extracts. Raw peanuts and 2-hour and 12-hour boiled peanut extracts were equivalent in their ability to stimulate T cell activation and proliferation.
Progressive reduction in peanut allergenicity with extended boiling does not affect T cell reactivity. Boiled peanuts may be a candidate for future peanut oral immunotherapy.
Oral immunotherapy using raw peanuts, roasted peanuts, or peanut oil is associated with high rates of adverse events and is therefore not currently recommended for routine clinical practice. A product able to initiate peanut desensitization with fewer adverse events is desirable. Previous investigations of boiled peanut products have studied peanuts boiled for no longer than 1 hour. The current study demonstrates that boiling peanuts for at least 2 hours is required to significantly reduce the allergenicity of Ara h 2, which is stabilized by the presence of 4 disulphide bonds. Extensively boiled peanuts may be an attractive option for future oral immunotherapy secondary to decreased IgE reactivity, with retained peptides capable of stimulating T cell activity.
- Copyright © 2017 by the American Academy of Pediatrics