The effect of extended boiling on peanut allergenicity and its clinical implications

Author: Billy Tao

Tao, Billy, 2018 The effect of extended boiling on peanut allergenicity and its clinical implications, Flinders University, College of Medicine and Public Health

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Peanut allergy is the leading cause of allergy-related emergency attendances at Western world hospitals and its prevalence is still rising. While desensitisation is not a cure, it is nevertheless the only form of proactive and disease-modifying therapeutic approach that we know of in the treatment of peanut allergy. Oral immunotherapy (OIT) is one of several desensitisation methods, and a promising one, but is still not approved for general use other than in a research setting mainly because of its high rate of adverse events (45-93%). An enhanced safety profile is therefore likely to improve its acceptability.

This thesis began with a simple idea of boiling peanuts to make them hypoallergenic. It then provided the scientific evidence to underpin the idea. SDS-PAGE images confirmed progressive fragmentation of peanut proteins and leaching into cooking water as a consequence of increasing boiling time, while Western blot demonstrated corresponding reduction in human IgE allergenicity. Inhibition ELISA, which was capable of detecting conformational (B-cell) epitopes far better than Western blot, demonstrated that 12-hour boiled peanut resulted in a 19-fold reduction of allergenicity while 2-hour boiled resulted in an 8-fold reduction. Mass spectrometry confirmed the fragmentation of peanut proteins as a result of boiling, but also showed that many known T-cell epitopes were actually retained in significant quantities in boiled peanuts, with 12-hour boiled peanut containing 42 times more peptides than raw peanut. Following that, flow cytometry was used to show that T-cell reactivity did not change after extended boiling. This implied that while boiling could reduce allergenicity by altering conformational epitopes, it did not actually affect T-cell epitopes which were responsible for tolerance induction. Such a strategy of differential modification of B- and T-cell epitopes in peanut (and therefore the clinical effect) would be superior to, and more sophisticated than, simple dose-reduction as a means of minimising adverse events during oral immunotherapy. Finally, in vivo skin prick tests were used to demonstrate reduced skin-sensitisation with boiled peanut extracts. The results were published in Clinical and Experimental Allergy in July 2016.

The thesis then made a translation from scientific evidence to a proof-of-concept pilot study, using a novel oral immunotherapy protocol in which hypoallergenic boiled and then roasted peanuts were ingested in a sequential manner. Fourteen subjects were enrolled, two withdrew due to non-compliance and social issues (both had no adverse events during treatment), but 12 were able to ingest 8-10 roasted peanuts at end of desensitisation. Three of 12 subjects showed mild adverse events in the early stages of boiled peanut desensitisation, and two of 12 subjects showed mild adverse event in the first week of roasted peanut desensitisation. This figure was considerably less than those reported in other OIT studies.

The most important message from the pilot study is that up-dosing in oral immunotherapy can be safely performed at home using the sequential method, making hospital-based supervision redundant. This is an important finding and is unique from all other OIT studies. The report was published in Clinical and Experimental Allergy in November 2017.

Keywords: peanut allergy, extended boiling of peanut, reduction of allergenicity, desensitisation, sequential boiled-to-roasted peanut, oral immunotherapy

Subject: Paediatrics thesis

Thesis type: Doctor of Philosophy
Completed: 2018
School: College of Medicine and Public Health
Supervisor: Prof Kevin Forsyth