Peanut/Tree Nut Desensitization and Induction of Tolerance in Children
Phase: NA
Conditions: Peanut Allergy, Tree Nut Allergy
Sponsor: McGill University Health Centre/Research Institute of the McGill University Health Centre

1  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
1
Peanut/Tree Nut  Desensitization and induction of tolerance in Children 
1 
Proposed Research Questions 
1. What is the rate of desensitization in children allergic to peanuts or tree nuts? 
2. Is the efficacy of a maintenance protocol of 300mg peanut protein similar to the 
efficacy of a maintenance protocol of 30mg peanut protein? 
3. What are the predictors associated with success or failure of desensitization in   
     children? 
4. What are the molecular mechanisms involved in desensitization to peanut/tree nut? 
 
2 
Research Objectives and Significance 
The investigators are proposing to initiate a study assessing a common  
desensitization protocol for peanut/tree nut allergy 
 This study  would enable us to better determine the potential benefit of 
desensitization in individuals with peanut/tree nut allergy. 
More specifically, the investigators will address the following research objectives: 
Objectives 
A. To  develop a protocols for peanut/tree nut desensitization with high (300mg) and 
low (30mg maintenance dose). 
B. To determine the rate of desensitization to peanut/tree nut. 
C. To characterize predictors of successful desensitization.   
D. To characterize molecular mechanisms involved in the process of desensitization. 
 
3. Gaps and Unmet Needs in Existing Knowledge on food desensitization  

2  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
2
3.i Why do the investigators need trials on food desensitization. 
Foods are reported to be primary inciting allergens for anaphylaxis and account for 
33.2% to 56% of all anaphylaxis cases.(1-4) Further, food-induced anaphylaxis hospital 
admissions are reported to have increased, mainly in the first 2 decades of life.(5;6) 
At present, the only treatment for food allergy is to avoid the allergy-causing food, 
while the only treatment for an allergic reaction is prompt administration of intramuscular 
epinephrine. (7;8) 
Primary anaphylaxis prevention is based on allergen desensitization through 
immunotherapy and until recently was not available for food allergies. Although there are 
a few recent research protocols exploring desensitization for foods and although these 
have so far been largely successful, (9;10)  up to this point  there are no  known guidelines 
describing the optimal candidate for desensitization nor are there criteria for the safest 
and most effective dosing schedule. Further, many of these studies have a very small 
sample size and do not use a control group. Hence, the efficacy of these protocols is hard 
to interpret (11-14) and additional larger scale randomized control studies are required.  
3.ii Why peanut/tree nut . 
Peanut  and tree nut allergies are the  most common food allergies in children and 
adults in Canada affecting almost 2%  and 1.5% of the population respectively.(15) and 
recent reports suggest that only 10%-20% will out-grow nut allergy .(16) 
Further, peanut and tree nut allergy are most common trigger for anaphylaxis and the 
most common trigger for recurrent anaphylaxis. (17) 
With the support of AllerGen NCE and CIHR the investigators have initiated in 2014 
the first cross Canada study to assess  desensitization to milk . This study now includes 

3  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
3
almost 50 patients, and our preliminarily results reveal an almost 80% success rate. In 
April 2016 in collaboration with Aimmune  the investigators have initiated the first oral 
desensitization to peanut.(18)The investigators are now proposing to develop a protocol for 
peanut and tree nut desensitization based on the expertise, recruitment skills and data 
collected in our first 2 years of  milk desensitization and more recently peanut 
desensitization.    
Peanut and tree nuts are the most common foods causing acute allergic reactions and 
almost all fatal reactions in North America had been triggered by peanutor tree nuts.  The 
term “tree nut” is collectively used to categorize nuts that grow on trees.  Prevalence of 
individual tree nut allergies varies significantly by region, with hazelnut being the most 
common tree nut allergy in Europe, walnut and cashew the most common in the USA  
and Brazil nut, almond and walnut most commonly reported in Europe.(19) In Canada 
alone, recent research has shown up to 2% of children and 1% of adults have peanut 
allergy. (15;21)  
The main peanut allergens are Arah1 and Arah2 .(22)Mosttree nut allergens identified to 
date are seed storage proteins such as the vicilins, legumins, americ globulins, and 2S 
albumins. Othertree nut allergens include profilins and hevein- related proteins, known 
for their contribution to the allergenicity of a wide variety of pollens, nuts, seeds, fruits 
and vegetables and their propensity for exhibiting a significant degree of IgE- mediated 
cross- reactivity. (23) Cross-reactivity among nuts may occur because of minor constituent 
panallergens such as profilins and lipid transfer proteins, or may involve major nut 
storage protein allergens, including albumins, legumins, and vicilins. (24)Strong cross-
reactivity was present among walnuts, pecans, and hazelnuts, and mild cross- reactivity 
was present among cashews, Brazil nuts, pistachios, and almonds. (202) 
Diagnosis of peanut/tree nut allergy is confirmed by having a convincing clinical history 
as well as the presence of a positive SPT defined as a wheal diameter at least 3 mm larger 
than that elicited by the negative control within 10 to 15 minutes of placement, provided 
by a physician, or an IgE level of at least 0.35 KU/L. (25)If however, exposure to 

4  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
4
peanut/tree nut never occurred, or if clinical history is uncertain, diagnosis is based on 
having a positive SPT and a tree nut- specific IgE above 15 kU/L.  (26) 
Despite the importance of peanut and tree nut  allergy, there are only three  studies 
assessing oral immunotherapy for peanut and there is only one group that published to 
date a research desensitization to hazelnuts.  No protocols have been published so far for 
other tree nuts.  The protocol for hazelnuts reveals encouraging results with significantly 
increased thresholds to food induced allergic reactions after oral immunotherapy in the 
majority of those with hazelnut allergy(18;27;28) In conclusion, the efficacy of the 
immunotherapy, extent of desensitization versus tolerance, the quantity/ frequency of 
allergen consumption required to maintain this effect and molecular mechanisms 
involved in the desensitization process are currently unknown. 
3. iii. Do the investigators achieve only desensitization or do the investigators 
induce also tolerance. 
Desensitization refers to a change in the amount of food antigen needed to cause 
allergic symptoms; this state is dependent on regular antigen exposure. In contrast, 
tolerance refers to long-term immunologic changes associated with the ability to ingest a 
food without symptoms and without ongoing therapy. Desensitization is a worthwhile 
therapeutic goal as it allows individuals freedom from the risk of accidental ingestion in 
everyday settings; achieving long-term clinical tolerance would allow safe food ingestion 
without ongoing therapy by inducing lasting immunologic changes.(28) Trials so far 
suggest that might be   loss of tolerance within 48 h after elimination of the food from the 
diet. (30) However , these trials are flawed by their small sample size (3 patients). (30) In a 
recent study it was reported that prolonged ( at least 4 years) daily immunotherapy is 

5  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
5
crucial for achieving  Sustained Unresponsiveness (SU)=lack of dose limiting symptoms 
during DBPCFC and also 4-6 weeks after stopping OIT .(31)  
 
 
 
3. iv Gaps related to the appropriate maintenance dose of peanut desensitization. 
The threshold  doses of peanut and tree nut that  trigger reactions ranges from 15 mg to 
1000mg (32-34)  and  studies suggest that almost a third of products from 
Western Europe and two thirds of products from eastern Europe without precautionary 
labeling contain detectable levels of peanut or tree nut protein up to 245 mg per liter.(35) 
Hence it is crucial that desensitization protocols will be efficacious in protection against 
reactions to  at least 300mg of peanut or tree nut protein .One previous study on peanut  
had shown similar efficacy in attaining sustained unresponsiveness  (defined as no 
allergic reaction to 5 g of peanut ingested 1 month after stopping oral immunotherapy) 
between a protocol with 3000 mg maintenance dose versus 300mg.(36) .It is of crucial 
importance to assess the efficacy of low dose protocols as trials aiming for high doses of 
peanut maintenance are associated with  up to 40% drop-out due to adverse effects . 
 
3.v Gaps regarding the roles of mast cells in desensitization. 
Given that food desensitization is a relatively rapid process, mast cell are implicated to 
have a key role in this process. Compared to activated cells, desensitized Mast cells were 
shown to have  had impaired degranulation, calcium flux, secretion of arachidonic acid 

6  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
6
products.(37) However, the effect of food desensitization on mast cells was not explored 
so far in clinical trials.   
3vi. Gaps regarding the role of  T regulatory cells  in desensitization. 
Although several studies in humans report that food allergens induce specific effector  
T cells, there are significant controversies between studies regarding the T cell phenotype 
dominating that response.  Some  studies  report  a Th2 dominated response in children  
with food allergies whereas Th1- responses underlie oral tolerance  (38). Other studies 
suggest that food antigens induce a Th2 response  regardless of the presence of food 
allergy(39). Further, it is proposed that Treg cells may be crucial to control food related 
allergic reactions. A potential suppressive role of Treg in food allergies was exemplified 
in both animal models (40-42) and  in human studies. Increased numbers of Treg cells in 
peripheral blood in children who outgrew  allergy was reported (43;44) and decreased Treg 
suppression was associated with allergy in neonates (45). Cord blood from offspring of 
atopic mothers showed fewer innate induced Treg cells and impaired supression(46). In 
line with these, an association with severe food allergy was described in patients with 
profound dysfunction of T reg cells that characterizes IPEX syndrome 
(immunodysregulation polyendocrinopathy enteropathy X-linked syndrome)(47).   
In contrast 1 study looking at the number of Tregs in 10 children undergoing  
desensitization reports no enhancement of CD4+CD25+Foxp3+ T(reg) levels in 
peripheral blood and suggests that there is unlikely a role for long-lasting systemic 
immunologic changes in desensitization. However, this study is limited by a small sample 
size  and relatively short period of follow up ( 6 months). (48)  
3.vii  Gaps regarding the role of demethylation in desensitization. 

7  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
7
The rising incidence of food allergies  is occurring more rapidly than changes to the 
genome sequence would allow(49) and recent studies suggest that epigenetic regulation 
(heritable changes in gene expression that occur in the absence of alterations in DNA 
sequences) may in part mediate the complex gene-by-environment interactions that can 
lead to asthma.(50) Experimental studies provide substantial in vitro data indicating that 
DNA methylation of genes critical to T-helper cell differentiation may induce 
polarization toward or away from an allergic phenotype. Methylation of DNA and 
resulting changes in chromatin structure have been shown to initiate the process by which 
the Th cells lose their plasticity and differentiate productively toward the Th1 versus the 
proallergic Th2 pattern of cytokine gene expression.(51-53) So far no studies have explored 
the role of DNA methylation in short term processes such as desensitization for food 
allergens. 
 
3viii  Effect of Heat Treating Peanuts/Tree Nuts on Allergenicity 
There is ample evidence in the literature that there is a significant increase in the 
allergenicity of peanuts after undergoing thermal processing (i.e. roasting). Glycation at 
high temperatures via the Maillard reaction, the addition of amines on reducing sugars to 
provide Schiff bases and advanced glycation end products (AGEs), is proposed to be the 
primary mechanism of enhancement of allergic responses in roasted peanuts. Our primary 
objective is to develop strategies to decrease glycation and Maillard reaction products in 
the thermal processing of peanuts.  
We propose to use High-Resolution Magic Angle Spinning Nuclear Magnetic 
Resonance Spectroscopy (HR-MAS NMR) to take an array of molecular snapshots of 

8  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
8
intact raw peanuts from room temperature to roasted temperatures (i.e. 20-150oC) and to 
subsequently perform an antigen-specific ELISA, which measures IgE binding, to assess 
the allergenicity at each temperature. We will expand the approach to variable 
temperature studies under conditions where small molecules, such as sugars and amino 
acids, are removed from intact or macerated peanuts prior to heating. 
4. Methodological Approach/Study Design 
Seventy five  males and females between 2 to 40years of age, diagnosed with  peanut 
/tree nut allergy  will be approached by our study group. Participants will be recruited 
from the allergy clinic at the Montreal Children’s Hospital and at collaborating sites in 
Toronto and Vancouver  Participants who meet inclusion criteria will be enrolled in a 
desensitization programme specific to their allergy.  Thus a peanut allergic 
participant will be desensitized to peanut only, while a patient allergic to cashew for 
example, will be desensitized to cashew only.  Patients with more than one peanut or 
nut allergy will be offered the choice of to which allergen they would prefer to be 
desensitized 
Inclusion criteria:  Children and adults between 2 and 40  who satisfy all the 
following criteria will be included: 
 A history suggestive of immediate allergy to peanut/tree nut . A convincing 
clinical history of an IgE mediated reaction to a specific food will be defined 
as a minimum of 2 mild signs/symptoms or 1 moderate or 1 severe 
sign/symptom that was likely IgE mediated and occurred within 120 minutes 
after ingestion or contact (or inhalation in the case of fish and shellfish).  
Reactions will be considered mild if they involve pruritus, urticaria, flushing, 

9  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
9
or rhinoconjunctivitis; moderate if they involve angioedema, throat tightness, 
gastrointestinal complaints, or breathing difficulties (other than wheeze); and 
severe if they involve wheeze, cyanosis, or circulatory collapse.(54-57) 
 The presence of  at least one of the following confirmatory tests: 
o Positive SPT to peanut/tree nut or its proteins (weal diameter 3 mm 
larger than that of the normal saline control). The allergens used will be 
commercial extracts of peanut/tree nut (Omega Labs, Toronto, 
Ontario). 
o Detection of serum  specific IgE (>0.35 kU/L) to peanut/tree nut  or 
any of its proteins, measured by fluorescence enzyme immunoassay 
(Phadia, CAP System, Uppsala, Sweden). 
o Positive oral challenge test to peanut/tree nut. Oral challenges will be 
performed with raw peanut/tree nut according to the recommendations 
of the position paper of the European Academy of Allergology and 
Clinical Immunology (58).  
 Informed consent form signed by the parents or legal guardian (appendix B). 
 
Exclusion criteria. 
 Patients who are unstable from a respiratory point of view .. 
 Patients who  present with  intercurrent disease at the time of starting 
desensitization. 
 Non-IgE-mediated or non-immunological adverse reactions to nuts. 

10  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
10
 Malignant or immunopathological diseases and/or severe primary or 
secondary immune deficiencies. 
 Patients receiving immunosuppressor therapy 
 Patients receiving β-blockers (including topical formulations). 
 Associated diseases contraindicating the use of epinephrine: cardiovascular 
disease or severe hypertension. 
 Patients diagnosed with eosinophilic gastrointestinal disorder . 
 
STUDY PROCEDURES 
The desensitization protocol will be performed at the Center of Innovative medicine 
(CIM ) as is currently done for our  peanut desensitization study, under the direct 
supervision of the medical and nursing staff, and with full cardiopulmonary resuscitation 
measures available for the treatment of possible allergic reactions that could occur during 
the procedure. Oral and intravenous doses of diphenhydramine and intramuscular 
epinephrine were at the bedside at all times.  
 
 
 
Desensitization to peanut (B1 and B2) . 
 
Oral Food Challenge 
Patients will begin with a blinded, placebo-controlled oral food challenge (BPCFC) to 
peanut that will take place over two days, with patients receiving doses of placebo one 

11  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
11
day and peanut/tree nut protein the other.  In all cases, protein will be in the form of 
crushed peanut/tree nut.  All challenges and desensitization procedures will be done with 
commercially available peanut/tree nut proteins.    Patients and evaluating physicians   
will be blinded, but not the study nurses nor the study coordinator .  On both days, 
patients will arrive at the Center for Innovative Medicine (CIM) in the morning.  The 
CIM services include equipment and medications required to monitor and treat potential 
reactions. The CIM nurse that will assist in the desensitization protocol is a nurse that has 
been trained to work in an intensive care unit.  Patients will be reminded beforehand that 
they must be in good health for the challenge.  If they are not in good health, the 
challenge will be rescheduled.  Upon arriving, a physical exam will be performed and 
vital signs taken.  Skin prick test to peanut/tree nut will be performed to establish baseline 
values in both.  An IV catheter will be inserted in the patient’s arm.  The doses will be 
mixed in a food vehicle tolerated by the participant, such as pudding or fruit puree, that 
the participant is known to tolerate.  Doses will be given by the study nurse every 15-20 
minutes, depending on patients’ tolerance of the dose (see Appendix B for challenge 
doses).  If participants tolerate the last, 300 mg dose,  they will be given a dose of food 
that contains the allergen in question – peanut butter in the case of a peanut challenge, 
Nutella in the case of hazelnut allergy.  The study nurse will be with the participant for 
the duration of the challenge and as soon as the participant demonstrates objective signs 
of an allergic reaction, the challenge will be stopped and the reaction treated.  Participants 
will be kept under observation in the CIM for two hours after the resolution of the last 
symptoms. 

12  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
12
Given the similarity of allergens between walnut and pecan and cashew and pistachio 
(202), the investigators will offer challenge with the two cross reacting allergens ( 1 week 
to 4 weeks apart ) for participants who report an allergy to at least one of these allergens . 
 
Randomization 
Through a process of stratified randomization (according to sex), participants will be 
randomly assigned in a 1:1:1 fashion to one of 3 study groups. The first group : group A 
will receive active desensitization with a maintenance dose of 300 mg , the second, group 
B  will receive active desensitization with a maintenance dose of 30 mg , and the third   
group C group will not receive any treatment but will be followed up regularly ( appendix 
B protocols ) .  Patients in groups A and B will be blinded to their group assignment.  
Participants randomized to group C will be offered the opportunity to enter the 
desensitization programme after their year of observation ends. They will be randomized 
in a 1:1 fashion to either the 300 mg protocol or the 30 mg protocol. 
 
 
Rush Desensitization 
The investigators will base our proposed desensitization protocol on previous studies 
by our group on a peanut desensitization protocol  that has been active in the last year at 
the Montreal Children’s Hospital. The modified rush two days will begin with a first 
single dose of 0.5 mg of  peanut/tree nut protein. After the initial dose, subjects will 
receive approximately doubling doses every 30 minutes until a dose of  5 mg. All doses 
will be mixed with an acceptable vehicle food chosen by the subject and his or her parent. 
Those who tolerate 5 mg , will be asked to return the next day for one dose of 3mg.  If 

13  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
13
this is tolerated, participants will be supplied with enough peanut/tree nut protein to be 
able to take a daily dose of 3 mg for the following 2 weeks and will come to the Montreal 
Children’s hospital after at the end of these two weeks for a dose of 6 mg (doses will be 
aliquoted and put in capsules by a professional pharmacist).. Self-administered 
epinephrine will be provided to all patients' caregivers, along with instructions and 
indications for administration and education about the nature of possible reactions to 
OIT.  Patients who are unable to complete the rush desensitization will not be 
eligible to continue the study 
 
Successful desensitization will be defined when the patient will be able to tolerate 
300mg of peanut/tree nut protein. A slow regimen will be used  in those patients who will 
experience repeated moderate reactions or any severe reaction to a given dose; that 
regimen will consist of reducing the dose to the previously tolerated dose for 1-2 weeks 
or splitting the dose into two equal portions according to the discretion of the PI or co-
PI.(11) 
SPT with the allergens listed above will be performed on all patients before starting 
desensitization, and the tests will be  repeated at 3, 6 and 12 months. Blood tests will be 
also  performed to measure sIgE and sIgG, T cell phenotypes (including regulatory 
Tcells), vitamin D levels and DNA methylation before  desensitization, at 3 weeks and at 
3, 6 and 12 months. 
Reactions during the desensitization protocol will be  classified according to the 
categories proposed by Perry et al.(59): mild reactions are defined when symptoms are  
limited to the  oral mucosa or the skin; severe reactions include cardiovascular or 

14  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
14
respiratory symptoms or involvement of any four systems; all other reactions will 
classified as moderate, although the investigators  will consider isolated abdominal 
discomfort as mild when it lasted for 30 min or less. 
The protocol will be  individualized according to each patient's tolerance to 
peanut/tree nut: 
 
. Mild allergic reactions will be treated when necessary and the regimen will be 
continued when the patient becomes asymptomatic. The previously tolerated dose will be 
repeated before resuming the process. 
 
Moderate reactions will be treated and desensitization would be restarted on the 
following day at the previously tolerated dose. 
 
Severe reactions will be treated with the necessary measures and in the 
appropriate department, followed by an assessment of whether to interrupt desensitization 
or reduce the dose on the following day to 1/10 of the dose that caused the reaction. 
 
Build-up phase 
Two weeks after the two-day rush Desensitization, participants will begin the build-up 
phase.  Participants will come to the CIM every two weeks to receive their first dose at 
each level.  Participants will then consume this dose at home for two weeks, then return 
to the CIM for the next dose augmentation (doses are listed in Appendix B).  Home doses 
will be provided in individual preweighed containers, and subjects will be instructed to 
ingest 1 dose daily for 2 weeks. Dose escalations will be made at visits in the research 
unit to ensure that subjects tolerate increases, and subjects will be observed for at least 2 
hours after increases for signs of clinical reactions.  This process will be repeated for 11 

15  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
15
doses (22 weeks) with almost doubling of the dose in each hospital visit till a dose of 
300mg is reached for group A.   For group B participants will attain 30 mg after 6 weeks 
and will continue with the same dose for 16 weeks 
Follow-up / Maintenance phase 
In order to compare the efficacy of a 30 mg peanut protein dose to that of a 300 mg 
dose in inducing peaqnut/tree nut desensitization, participants in Groups A nd B will both 
undergo DBPCFC at similar time points during the maintenance phase.  The first of these 
challenges will take place one year after beginning desensitization.  Participants in either 
group whose progress is delayed because of symptoms during the escalation phase  will 
undergo their DBPCFC three months after attaining their maximal dose (either 30mg or 
300mg).   The subjects will be also instructed to otherwise continue a peanut/ tree nut-
elimination diet for the duration of the study. 
Subjects and families will be asked to complete daily home diaries to document that 
daily doses were taken, as well as to report accidental ingestions, problems with dose 
administration, or related symptoms. Follow-up visits in addition to or in conjunction 
with the biweekly visits for dose escalation will be planned  at 3, 6, 9, 12 months from 
enrollment. Each visit will involve  a medical history and physical examination. SPT, 
serum tree nut-specific IgE and IgG concentrations, T cell penotyping and DNA 
methylation will be  measured at enrollment and at follow-up visits.  At the 9-month 
follow-up visit, subjects from both groups will undergo another DBPCFC, similar to that 
done following escalation. 
The patients' parents will be instructed verbally and in writing about the 
recommendations to be followed after desensitization and how to treat possible allergic 

16  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
16
reactions. They will also given a direct telephone line to members of the study staff for 
consultation. Patients will be  told not to perform physical exercise (60;61) for 2 h after 
eating and not to take non-steroidal anti-inflammatory drugs for 3 h before or after 
ingestion. No special recommendations will be  given for viral infections.(11)  
Food challenges 
As indicated above, subjects who tolerate 300mg (group A) or 30mg (group B) of 
peanut/tree nut will have a two more BPCFC to determine their allergenic reactivity to 
peanut protein. The first of these challenges will take place one year after they begin 
desensitization.  Participants in either group whose progress is delayed because of 
symptoms during the escalation phase  will undergo their DBPCFC three months after 
attaining their maximal dose (either 30mg or 300mg).  .  Subjects will continue OIT 
dosing through the day before the challenge. One challenge will consist of doses of  
peanut/tree nut protein given every 10 to 20 minutes starting at 1 mg in increasing 
amounts up to a total of 2044mg of peanut/tree nut protein (See Appendix B, section A1 
for doses). The other challenge will consist of placebo given also in 6 doses. All negative 
challenge results will be confirmed by means of open challenge, with at least three  
peanuts/tree nuts provided 1 hour after completion of the double-blind portion. The 
second DBPCFC will take place nine months into the Follow-Up/ Maintenance phase.  
Participants who pass these challenges will be instructed to continue to ingest the 
maintenance dose daily to the end of the follow-up period. 
Participants in Group B who have allergic reactions during this oral food challenge 
will re-entered into the Escalation Phase and proceed to the 300 mg dose. 

17  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
17
At the end of the study, oral desensitization will be offered to the control patients who 
had not achieved tolerance. 
Participants who had challenges to both cashew and pistachio will repeat the challenges 
to both nuts at this time point.  This will be done to assess whether desensitization to one 
of these nuts confers protection to the other, antigenically-similar nut 
 
 
Variables measured. The following variables will be measured in each patient: 
A. The primary study variable will be the presence of desensitization defined by the 
ability to toleratepeanut/tree nut 1 year after the start of the trial. This is a qualitative 
variable with three categories: total desensitization ( 300mg of peanut/ nut protein); 
partial desensitization ( 30-300mg of peanut/ nut protein); and failed desensitization (<30 
mg of peanut/ nut protein). Only patients presenting total desensitization will be  regarded 
as being  successfully desensitized. 
B. Secondary variables will be:  
1. 
Number and severity of adverse reactions occurring after oral exposure 
topeanut/tree nut during the desensitization and follow-up phases. 
2. 
Minimum dose ofpeanut/tree nut that triggered symptoms during the 
desensitization protocols or challenge tests. 
3. 
Days until desensitization was achieved, excluding weekends. 
4. 
Indices of desensitization : 
SPT weal size and levels of sIgE and sIgG before desensitization, during follow-up 
and at the end of the study. 
 

18  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
18
5. 
Mast cell activation. 
Mast cell activation will be assessed through a proxy measure :the basophil activation 
test. This test is  based on the percentage of CD63-expressing basophils detected by  
flowcytometry . This test is done by incubating  basophils from highly sensitized atopic 
donors or preferably after priming them with Il-3 ( regardless of basophil source ) (62;63) 
with sera from patients . The investigators will assess the expression of CD63 on 
basophiles after addition of tree nut/ antigens respectively as well as an irrelevant antigen 
( gliadin and tetnus toxoid) The expression of CD63 on these cells is compared to values 
acquired after incubation with healthy controls and the same antigens. Values above the 
mean of CD63+ cells + 2 SD induced will be  considered as positive. (64)   
 
6. 
Indices of T cell tolerance  :  
Levels of Treg and cytokine release at base line and during desensitization. In order to 
characterise molecular pathways associated with desensitization , the investigators will 
draw 5 cc of blood from each participant and check their T cell phenotype including their 
Treg cells and the response to peanut/tree nut protein. As control the response to an 
irrelavent antigen (gliadin) and to tetanus toxoid will be assessed.  The investigators will 
invite participants for annual follow ups in the first 5 years after study entry. The 
investigators will also offer the desensitization to the control  group at the end of the 
study. 
7. Health-Related Quality of Life (HRQL) can be measured in food allergy and was 
validated for both children and adults.(65;66) Given that  HRQL measurements may 
be used to measure the effects of an intervention on the patient's quality of life, 

19  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
19
patients will be asked to fill a validated HRQL for children ( up to 18 years )(65) at 
study entry and after completing 12 months of maintenance 
 
 
 
 
5.   Statistical Considerations  
Given that previous studies in adults have attributed at least 50% improvement in the 
ability to toleratepeanut/tree nut,(67) with an α  of 0.05 and a power (1- β ) of 0.80, a 
sample of 17 cases for group A , 17 for group B   and 17 controls will be recruited. 
Statistical analyses will be performed using paired t-tests. A P value of <0.05 will be 
considered significant. 
Descriptive statistics of the variables including means, standard deviations and 
frequencies will be computed for all study variables. The concentrations of-sIgE and 
sIgG , the weal size , Mast cell, T cell and DNA  methylation status and HQRL scores  
at the different study time-points will be  compared with their baseline values using the 
one-sample paired t-test  and to the control using a 2 sample t-test . 
Multiple regression models will be used to assess factors associated with response to 
desensitization (i.e. baseline demographic characteristics; gender; age; baseline basophile 
activation; T cell status, baseline SPT and specific IgE, DNA methylation status, vitamin 
D status and season). 
 
6.   Ethical Considerations   

20  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
20
In the consent form given at study enrolment, participants will be advised that they 
will be randomly assigned to either desensitization treatment or observation alone for one 
year, (appendix B). Participants will also be advised that their data will be shared only 
among the study members. Participants who will be at the control group will be told that 
they will be offered the desensitization protocol one year after study entry. 
 
7.  Confidentiality and Data Management 
All research documents (case report forms, questionnaires, etc) will be stored in a 
locked cabinet in the Centre for Innovative Medicine (CIM) at the Glen.  This facility has 
limited access, such that non-research personnel cannot access the area. 
Electronic study records will be kept on a password-protected computer in the CIM.  
All participants will be assigned a unique study number and study information will be 
entered under this study number.  The key linking the participant and their study number 
will be kept by the principal investigator. 
Once the study has been completed, all physical study documents will be archived for 
25 years at Iron Mountain.  Electronic information will be stored  by the Principal 
Investigator for 25 years 
 
8.  Research Deliverables and Milestones 
Timeframe 
Deliverables 
Milestones 
08/16 – 03/ 17 
 
Develop sampling frame; ethics approval;  
Ready to recruit participants 
 
05/17 –08/19 
Collect data from participants  
12 weeks of data collection in 

21  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
21
 
 
 
9. Requirement for networking across Disciplines and Sites 
 
Our researchers have a legacy of working across disciplines, across sites, and 
across sectors.  For this study, the investigators have assembled a multi-institutional 
research team with cross-disciplinary expertise Dr Ben-Shoshan  (Montreal Children’s 
Hospital, McGill University) and Dr Mazer and Dr McCusker   (Montreal Children’s 
Hospital  ) bring expertise in clinical immunology to the research team. Dr. Ann Clarke 
(Montreal General Hospital) will bring expertise in the field of clinical allergy and 
together with  Dr. Ben-shoshan, will assisted with the study design and will supervise the 
statistical analyses. Epidemiologic and biostatistician skills are essential to undertaking 
this program of research and are represented in the persons of Moshe Ben-Shoshan 
(McGill) Msc in Epidemiology and Mr . Duncan Lejtenyi. Mr Duncan Lejtenyi will also 
be responsible for the laboratory evaluation of CD63 levels. Finally, as incorporated in 
our protocol, the investigators will share the results of this research with representatives 
of key constituencies involved in the data collection –patients with food allergies  and 
treated participants  completed 
08/19– 12/23 
Knowledge translation activities: 
dissemination workshops & manuscript 
preparation 
 
Preparation of abstracts and 
manuscript 

22  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
22
their families, physicians and allied professionals (e.g. nurses) through scientific 
meetings, our the investigatorsbsite and publications in medical journals.    
 
10. Organization of Research Team 
The nominated principal applicant, Dr. Moshe Ben-Shoshan, and co-principal 
applicants, Dr. Bruce Mazer, will oversee all aspects of the proposed research.   The 
collaborators, Dr Stark , Dr Piccirillo and Dr Bar-Or will aid in the recruitment of 
patients and the assessment T cells and DNA methylation status respectively.  Dr Julia 
Upton from Sick Kid’s Hospital in Toronto and Dr Edmond Chan at BC Children’s 
Hospital are external collaborators and will recruit patients from their respective centers 
Mr Duncan Lejtenyi , who had worked with our team in numerous studies will serve 
as research coordinator. 
 
11. External Research Partnerships and funding 
 
As indicated above, external research partnerships are considered essential to the 
activities of our research team.  The investigators will apply to receive substantial 
financial support from the CIHR and from AllerGen (appendix D budget).  The 
investigators will work closely with client organizations such as the  Anaphylaxis Canada 
, AAIA ( allergy/Asthma Information Association ) , and the AQAA (Association 
québécoise des allergies alimentaires ). These support a cyber-society of people with food 
allergies and will provide support for our research activities from their limited budgets, 
again indicating the relevance of our research efforts to their needs. 
Name of Partner 
Nature of participation 
Contribution (CA$) 
Letter 

23  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
23
yes /no 
 
 
Cash 
In-kind 
 
Anaphyalxis 
Canada  
 
 
1000 
Y 
AAIA 
 
 
1000 
Y 
AQAA 
 
 
1000 
Y 
Total value of partnerships  
 
 
 
References 
 
 
(1)  Decker WW, Campbell RL, Manivannan V, Luke A, St Sauver JL, 
Weaver A et al. The etiology and incidence of anaphylaxis in Rochester, 
Minnesota: a report from the Rochester Epidemiology Project. J Allergy Clin 
Immunol 2008; 122(6):1161-5. 
 
(2)  Bohlke K, Davis RL, DeStefano F, Marcy SM, Braun MM, 
Thompson RS. Epidemiology of anaphylaxis among children and adolescents 
enrolled in a health maintenance organization. J Allergy Clin Immunol 2004; 
113(3):536-42. 
 
(3)  Sheikh A, Alves B. Hospital admissions for acute anaphylaxis: 
time trend study. BMJ 2000; 320(7247):1441. 
 
(4)  Gold MS. EpiPen epidemic or good clinical practice? J Paediatr 
Child Health 2003; 39(5):376-7. 
 
(5)  Liew WK, Williamson E, Tang ML. Anaphylaxis fatalities and 
admissions in Australia. J Allergy Clin Immunol 2009; 123(2):434-42. 
 
(6)  Lin RY, Anderson AS, Shah SN, Nurruzzaman F. Increasing 
anaphylaxis hospitalizations in the first 2 decades of life: New York State, 1990 -
2006. Ann Allergy Asthma Immunol 2008; 101(4):387-93. 
 
(7)  Gold MS, Sainsbury R. First aid anaphylaxis management in 
children who were prescribed an epinephrine autoinjector device (EpiPen). J 
Allergy Clin Immunol 2000; 106(1 Pt 1):171-6. 
 
(8)  Simons FE, Roberts JR, Gu X, Simons KJ. Epinephrine 
absorption in children with a history of anaphylaxis. J Allergy Clin Immunol 1998; 
101(1 Pt 1):33-7. 

24  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
24
 
(9)  Plaut M, Sawyer RT, Fenton MJ. Summary of the 2008 National 
Institute of Allergy and Infectious Diseases-US Food and Drug Administration 
Workshop on Food Allergy Clinical Trial Design. J Allergy Clin Immunol 2009; 
124(4):671-8. 
 
(10)  Ben Shoshan M, Clarke AE. Anaphylaxis: past, present and 
future. Allergy 2010. 
 
(11)  Garcia RR, Urra JM, Feo-Brito F, Galindo PA, Borja J, Gomez E 
et al. Oral rush desensitization to egg: efficacy and safety. Clin Exp Allergy 2011; 
41(9):1289-96. 
 
(12)  Buchanan AD, Green TD, Jones SM, Scurlock AM, Christie L, 
Althage KA et al. Egg oral immunotherapy in nonanaphylactic children with egg 
allergy. J Allergy Clin Immunol 2007; 119(1):199-205. 
 
(13)  Itoh N, Itagaki Y, Kurihara K. Rush specific oral tolerance 
induction in school-age children with severe egg allergy: one year follow up. 
Allergol Int 2010; 59(1):43-51. 
 
(14)  Zapatero L, Alonso E, Fuentes V, Martinez MI. Oral 
desensitization in children with cow's milk allergy. J Investig Allergol Clin 
Immunol 2008; 18(5):389-96. 
 
(15)  Soller L, Ben-Shoshan M, Harrington DW, Knoll M, Fragapane J, 
Joseph L et al. Adjusting for nonresponse bias corrects overestimates of food 
allergy prevalence. J Allergy Clin Immunol Pract 2015; 3(2):291-3. 
 
(16)  Byrne AM, Malka-Rais J, Burks AW, Fleischer DM. How do wee 
know when peanut andallergy have resolved, and how do we keep it resolved? 
Clin Exp Allergy 2010; 40(9):1303-11. 
 
(17)  Hochstadter E, Clarke A, De SS, LaVieille S, Alizadehfar R, 
Joseph L et al. Increasing visits for anaphylaxis and the benefits of early 
epinephrine administration: A 4-year study at a pediatric emergency department 
in Montreal, Canada. J Allergy Clin Immunol 2016. 
 
(18)  Bird JA, Feldman M, Arneson A, Dougherty I, Brown LS, Burk CM 
et al. Modified peanut oral immunotherapy protocol safely and effectively induces 
desensitization. J Allergy Clin Immunol Pract 2015; 3(3):433-5. 
 
(19)  Pumphrey RS. Lessons for management of anaphylaxis from a 
study of fatal reactions. Clin Exp Allergy 2000; 30(8):1144-50. 
 
(20)  McWilliam V, Koplin J, Lodge C, Tang M, Dharmage S, Allen K. 
The Prevalence ofAllergy: A Systematic Review. Curr Allergy Asthma Rep 2015; 
15(9):54. 

25  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
25
 
(21)  Soller L, Ben-Shoshan M, Harrington DW, Knoll M, Fragapane J, 
Joseph L et al. Prevalence and predictors of food allergy in Canada: a focus on 
vulnerable populations. J Allergy Clin Immunol Pract 2015; 3(1):42-9. 
 
(22)  Koppelman SJ, Wensing M, Ertmann M, Knulst AC, Knol EF. 
Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as 
determined by immunoglobulin E Western blotting, basophil-histamine release 
and intracutaneous testing: Ara h2 is the most important peanut allergen. Clin 
Exp Allergy 2004; 34(4):583-90. 
 
(23)  Roux KH, Teuber SS, Sathe SK.allergens. Int Arch Allergy 
Immunol 2003; 131(4):234-44. 
 
(24)  Goetz DW, Whisman BA, Goetz AD. Cross-reactivity among 
edible nuts: double immunodiffusion, crossed immunoelectrophoresis, and 
human specific igE serologic surveys. Ann Allergy Asthma Immunol 2005; 
95(1):45-52. 
 
(25)  Ben Shoshan M, Harrington DW, Soller L, Fragapane J, Joseph 
L, St Pierre Y et al. A population-based study on peanut, tree nut, fish, shellfish, 
and sesame allergy prevalence in Canada. J Allergy Clin Immunol 2010; 
125(6):1327-35. 
 
(26)  Sampson HA, Ho DG. Relationship between food-specific IgE 
concentrations and the risk of positive food challenges in children and 
adolescents. J Allergy Clin Immunol 1997; 100(4):444-51. 
 
(27)  Jones SM, Pons L, Roberts JL, Scurlock AM, Perry TT, Kulis M et 
al. Clinical efficacy and immune regulation with peanut oral immunotherapy. J 
Allergy Clin Immunol 2009; 124(2):292-300, 300. 
 
(28)  Varshney P, Jones SM, Scurlock AM, Perry TT, Kemper A, Steele 
P et al. A randomized controlled study of peanut oral immunotherapy: clinical 
desensitization and modulation of the allergic response. J Allergy Clin Immunol 
2011; 127(3):654-60. 
 
(29)  Enrique E, Malek T, Pineda F, Palacios R, Bartra J, Tella R et al. 
Sublingual immunotherapy for hazelnut food allergy: a follow-up study. Ann 
Allergy Asthma Immunol 2008; 100(3):283-4. 
 
(30)  Rolinck-Werninghaus C, Staden U, Mehl A, Hamelmann E, Beyer 
K, Niggemann B. Specific oral tolerance induction with food in children: transient 
or persistent effect on food allergy? Allergy 2005; 60(10):1320-2. 
 
(31)  Jones SM, Burks AW, Keet C, Vickery BP, Scurlock AM, Wood 
RA et al. Long-term treatment with egg oral immunotherapy enhances sustained 
unresponsiveness that persists after cessation of therapy. J Allergy Clin Immunol 
2016; 137(4):1117-27. 

26  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
26
 
(32)  Blumchen K, Ulbricht H, Staden U, Dobberstein K, Beschorner J, 
de Oliveira LC et al. Oral peanut immunotherapy in children with peanut 
anaphylaxis. J Allergy Clin Immunol 2010; 126(1):83-91. 
 
(33)  Vickery BP, Berglund JP, Burk CM, Fine JP, Kim EH, Kim JI et al. 
Early oral immunotherapy in peanut-allergic preschool children is safe and highly 
effective. J Allergy Clin Immunol 2017; 139(1):173-81. 
 
(34)  Ballmer- Weber BK, Fernandez-Rivas M, Beyer K, Defernez M, 
Sperrin M, Mackie AR et al. How much is too much? Threshold dose distributions 
for 5 food allergens. J Allergy Clin Immunol 2015; 135(4):964-71. 
 
(35)  Vadas P, Perelman B. Presence of undeclared peanut protein in 
chocolate bars imported from Europe. J Food Prot 2003; 66(10):1932-4. 
 
(36)  Vickery BP. Low-Dose Oral Immunotherapy As an Early 
Intervention Strategy for Peanut Allergy. J Allergy Clin Immunol. 131[ 2], AB130. 
1-2-0013.  
Ref Type: Abstract 
 
(37)  Sancho-Serra MC, Simarro M, Castells M. Rapid IgE 
desensitization is antigen specific and impairs early and late mast cell responses 
targeting FcepsilonRI internalization. Eur J Immunol 2011; 41(4):1004-13. 
 
(38)  Turcanu V, Maleki SJ, Lack G. Characterization of lymphocyte 
responses to peanuts in normal children, peanut-allergic children, and allergic 
children who acquired tolerance to peanuts. J Clin Invest 2003; 111(7):1065-72. 
 
(39)  Thottingal TB, Stefura BP, Simons FE, Bannon GA, Burks W, 
HayGlass KT. Human subjects without peanut allergy demonstrate T cell-
dependent, TH2-biased, peanut-specific cytokine and chemokine responses 
independent of TH1 expression. J Allergy Clin Immunol 2006; 118(4):905-14. 
 
(40)  Takayama N, Igarashi O, Keon MN, Kiyono H. Regulatory role of 
Peyer's patches for the inhibition of OVA-induced allergic diarrhea. Clin Immunol 
2007; 123(2):199-208. 
 
(41)  van Wijk F, Wehrens EJ, Nierkens S, Boon L, Kasran A, Pieters R 
et al. CD4+CD25+ T cells regulate the intensity of hypersensitivity responses to 
peanut, but are not decisive in the induction of oral sensitization. Clin Exp Allergy 
2007; 37(4):572-81. 
 
(42)  Cardoso CR, Teixeira G, Provinciatto PR, Godoi DF, Ferreira BR, 
Milanezi CM et al. Modulation of mucosal immunity in a murine model of food-
induced intestinal inflammation. Clin Exp Allergy 2008; 38(2):338-49. 

27  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
27
 
(43)  Karlsson MR, Rugtveit J, Brandtzaeg P. Allergen-responsive 
CD4+CD25+ regulatory T cells in children who have outgrown cow's milk allergy. 
J Exp Med 2004; 199(12):1679-88. 
 
(44)  Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzen A, 
Sampson HA. Association of allergen-specific regulatory T cells with the onset of 
clinical tolerance to milk protein. J Allergy Clin Immunol 2009; 123(1):43-52. 
 
(45)  Smith M, Tourigny MR, Noakes P, Thornton CA, Tulic MK, 
Prescott SL. Children with egg allergy have evidence of reduced neonatal 
CD4(+)CD25(+)CD127(lo/-) regulatory T cell function. J Allergy Clin Immunol 
2008; 121(6):1460-6, 1466. 
 
(46)  Schaub B, Liu J, Hoppler S, Haug S, Sattler C, Lluis A et al. 
Impairment of T-regulatory cells in cord blood of atopic mothers. J Allergy Clin 
Immunol 2008; 121(6):1491-9, 1499. 
 
(47)  Torgerson TR, Linane A, Moes N, Anover S, Mateo V, Rieux-
Laucat F et al. Severe food allergy as a variant of IPEX syndrome caused by a 
deletion in a noncoding region of the FOXP3 gene. Gastroenterology 2007; 
132(5):1705-17. 
 
(48)  Mori F, Bianchi L, Pucci N, Azzari C, De MM, Novembre E. 
CD4+CD25+Foxp3+ T regulatory cells are not involved in oral desensitization. Int 
J Immunopathol Pharmacol 2010; 23(1):359-61. 
 
(49)  Tan TH, Ellis JA, Saffery R, Allen KJ. The role of genetics and 
environment in the rise of childhood food allergy. Clin Exp Allergy 2011. 
 
(50)  Miller RL, Ho SM. Environmental epigenetics and asthma: current 
concepts and call for studies. Am J Respir Crit Care Med 2008; 177(6):567-73. 
 
(51)  Lee DU, Agarwal S, Rao A. Th2 lineage commitment and efficient 
IL-4 production involves extended demethylation of the IL-4 gene. Immunity 
2002; 16(5):649-60. 
 
(52)  Jones B, Chen J. Inhibition of IFN-gamma transcription by site-
specific methylation during T helper cell development. EMBO J 2006; 
25(11):2443-52. 
 
(53)  Tykocinski LO, Hajkova P, Chang HD, Stamm T, Sozeri O, 
Lohning M et al. A critical control element for interleukin-4 memory expression in 
T helper lymphocytes. J Biol Chem 2005; 280(31):28177-85. 
 
(54)  Ben-Shoshan M, Kagan RS, Alizadehfar R, Joseph L, Turnbull E, 
St.Pierre Yvan et al. Is the prevalence of peanut allergy increasing? A five-year 
follow-up study on the prevalence of peanut allergy in primary school children in 
Montreal. Journal of Allergy and Clinical Immunology 123[4], 783-8. 2009.  

28  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
28
Ref Type: Journal (Full) 
 
(55)  Hourihane JO, Kilburn SA, Dean P, Warner JO. Clinical 
characteristics of peanut allergy. Clin Exp Allergy 1997; 27(6):634-9. 
 
(56)  Brown SG. Clinical features and severity grading of anaphylaxis. J 
Allergy Clin Immunol 2004; 114(2):371-6. 
 
(57)  Sicherer SH, Burks AW, Sampson HA. Clinical features of acute 
allergic reactions to peanut and tree nuts in children. Pediatr 1998; 102(1):e6. 
 
(58)  Bindslev-Jensen C, Ballmer-Weber BK, Bengtsson U, Blanco C, 
Ebner C, Hourihane J et al. Standardization of food challenges in patients with 
immediate reactions to foods--position paper from the European Academy of 
Allergology and Clinical Immunology. Allergy 2004; 59(7):690-7. 
 
(59)  Perry TT, Matsui EC, Conover-Walker MK, Wood RA. Risk of oral 
food challenges. J Allergy Clin Immunol 2004; 114(5):1164-8. 
 
(60)  Calvani M, Sopo SM. Exercise-induced anaphylaxis caused by 
wheat during specific oral tolerance induction. Ann Allergy Asthma Immunol 
2007; 98(1):98-9. 
 
(61)  Caminiti L, Passalacqua G, Vita D, Ruggeri P, Barberio G, Pajno 
GB. Food-exercise-induced anaphylaxis in a boy successfully desensitized to 
cow milk. Allergy 2007; 62(3):335-6. 
 
(62)  Wedi B, Novacovic V, Koerner M, Kapp A. Chronic urticaria 
serum induces histamine release, leukotriene production, and basophil CD63 
surface expression--inhibitory effects ofanti-inflammatory drugs. J Allergy Clin 
Immunol 2000; 105(3):552-60. 
 
(63)  Gentinetta T, Pecaric-Petkovic T, Wan D, Falcone FH, Dahinden 
CA, Pichler WJ et al. Individual IL-3 priming is crucial for consistent in vitro 
activation of donor basophils in patients with chronic urticaria. J Allergy Clin 
Immunol 2011. 
 
(64)  Gyimesi E, Sipka S, Danko K, Kiss E, Hidvegi B, Gal M et al. 
Basophil CD63 expression assay on highly sensitized atopic donor leucocytes-a 
useful method in chronic autoimmune urticaria. Br J Dermatol 2004; 151(2):388-
96. 
 
(65)  Flokstra-de Blok BM, Dubois AE. Quality of life in food allergy: 
valid scales for children and adults. Curr Opin Allergy Clin Immunol 2009; 
9(3):214-21. 

29  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
29
 
(66)  Saleh-Langenberg J, Goossens NJ, Flokstra-de Blok BM, Kollen 
BJ, van der Meulen GN, Le TM et al. Predictors of health-related quality of life of 
European food-allergic patients. Allergy 2015; 70(6):616-24. 
 
(67)  Shertzer CL, Lookingbill DP. Effects of relaxation therapy and 
hypnotizability in chronic urticaria. Arch Dermatol 1987; 123(7):913-6. 
 
(68)  Skripak JM, Nash SD, Rowley H, Brereton NH, Oh S, Hamilton 
RG et al. A randomized, double-blind, placebo-controlled study of milk oral 
immunotherapy for cow's milk allergy. J Allergy Clin Immunol 2008; 122(6):1154-
60. 
 
(69)  Caminiti L, Passalacqua G, Barberi S, Vita D, Barberio G, De 
Luca R et al. A new protocol for specific oral tolerance induction in children with 
IgE-mediated cow's milk allergy. Allergy Asthma Proc 2009; 30(4):443-8. 
 
(70)  Martorell A, De la Hoz B, Ibanez MD, Bone J, Terrados MS, 
Michavila A et al. Oral desensitization as a useful treatment in 2-year-old children 
with cow's milk allergy. Clin Exp Allergy 2011; 41(9):1297-304. 
 
(71)  Fernandez-Rivas M, Garrido FS, Nadal JA, Diaz dD, Garcia BE, 
Gonzalez-Mancebo E et al. Randomized double-blind, placebo-controlled trial of 
sublingual immunotherapy with a Pru p 3 quantified peach extract. Allergy 2009; 
64(6):876-83. 
 
(72)  Vassallo MF, Banerji A, Rudders SA, Clark S, Mullins RJ, 
Camargo CA, Jr. Season of birth and food allergy in children. Ann Allergy 
Asthma Immunol 2010; 104(4):307-13. 
 
(73)  Pali-Scholl I, Jensen-Jarolim E. Anti-acid medication as a risk 
factor for food allergy. Allergy 2010. 
 
(74)  Gourbeyre P, Denery S, Bodinier M. Probiotics, prebiotics, and 
synbiotics: impact on the gut immune system and allergic reactions. J Leukoc 
Biol 2011; 89(5):685-95. 
 
(75)  Poole JA, Barriga K, Leung DY, Hoffman M, Eisenbarth GS, Rers 
M et al. Timing of initial exposure to cereal grains and the risk of wheat allergy. 
Pediatrics 2006; 117(6):2175-82. 
 
(76)  Du Toit G, Katz Y, Sasieni P, Mesher D, Maleki SJ, Fisher HR et 
al. Early consumption of peanuts in infancy is associated with a low prevalence 
of peanut allergy. J Allergy Clin Immunol 2008; 122(5):984-91. 
 
(77)  Katz Y, Rajuan N, Goldberg MR, Eisenberg E, Heyman E, Cohen 
A et al. Early exposure to cow's milk protein is protective against IgE-mediated 
cow's milk protein allergy. J Allergy Clin Immunol 2010; 126(1):77-82. 

30  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
30
 
(78)  Joseph CL, Ownby DR, Havstad SL, Woodcroft KJ, Wegienka G, 
Mackechnie H et al. Early complementary feeding and risk of food sensitization in 
a birth cohort. J Allergy Clin Immunol 2011. 
 
(79)  Koplin JJ, Osborne NJ, Wake M, Martin PE, Gurrin LC, Robinson 
MN et al. Can early introduction of egg prevent egg allergy in infants? A 
population-based study. J Allergy Clin Immunol 2010; 126(4):807-13. 
 
(80)  DesRoches A, Infante-Rivard C, Paradis L, Paradis J, Haddad E. 
Peanut allergy: is maternal transmission of antigens during pregnancy and 
breastfeeding a risk factor? J Investig Allergol Clin Immunol 2010; 20(4):289-94. 
 
(81)  Sicherer SH, Wood RA, Stablein D, Lindblad R, Burks AW, Liu 
AH et al. Maternal consumption of peanut during pregnancy is associated with 
peanut sensitization in atopic infants. J Allergy Clin Immunol 2010; 126(6):1191-
7. 
 
(82)  Chung SY, Butts CL, Maleki SJ, Champagne ET. Linking peanut 
allergenicity to the processes of maturation, curing, and roasting. J Agric Food 
Chem 2003; 51(15):4273-7. 
 
(83)  Liu GM, Cheng H, Nesbit JB, Su WJ, Cao MJ, Maleki SJ. Effects 
of boiling on the IgE-binding properties of tropomyosin of shrimp (Litopenaeus 
vannamei). J Food Sci 2010; 75(1):T1-T5. 
 
(84)  Samson KT, Chen FH, Miura K, Odajima Y, Iikura Y, Naval RM et 
al. IgE binding to raw and boiled shrimp proteins in atopic and nonatopic patients 
with adverse reactions to shrimp. Int Arch Allergy Immunol 2004; 133(3):225-32. 
 
(85)  Milner JD, Stein DM, McCarter R, Moon RY. Early infant 
multivitamin supplementation is associated with increased risk for food allergy 
and asthma. Pediatrics 2004; 114(1):27-32. 
 
(86)  Camargo CA, Jr., Clark S, Kaplan MS, Lieberman P, Wood RA. 
Regional differences in EpiPen prescriptions in the United States: the potential 
role of vitamin D. J Allergy Clin Immunol 2007; 120(1):131-6. 
 
(87)  Mullins RJ, Clark S, Camargo Jr CA. Regional variation in infant 
hypoallergenic formula prescriptions in Australia. Pediatr Allergy Immunol 2009. 
 
(88)  Mullins RJ, Clark S, Camargo CA, Jr. Regional variation in 
epinephrine autoinjector prescriptions in Australia: more evidence for the vitamin 
D-anaphylaxis hypothesis. Ann Allergy Asthma Immunol 2009; 103(6):488-95. 
 
 
 

31  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
31
Appendix A : Disease burden and gaps. 
Table 2. Primary and secondary prevention measures for food allergiesa 
 
Trigg
er 
Primary 
prevention 
Route of 
desensitization 
Reference number 
Secondary pr
Foods 
 
PO, SL 
(14;68-70) 
Avoidance of 
education of aller
their care-givers 
avoidance, impro
prepackaged food
wearing of Medic
specific food alle
Tree nut 
PO 
(11-13) 
Peanut 
PO 
(27) 
Tree nut 
SL 
(29) 
Peach 
SL 
(71) 
a only the most common foods , drugs and insect desensitization approaches are 
mentioned.  
PO, Per os; SL, Sublingual; SC,  
 
 
 
 
Table 2.Environmental factors associated with food allergy. 
Factor 
study 
Type of study 
Effect 

32  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
32
Season 
Vassall
o MF  
Case control 
Children younger than 5 years born in fall or winter had
higher odds of food allergy compared with controls. 
Drugs 
Palli-
Scholl  
Case control 
The relative risk to develop food-specific IgE after anti-
therapy was 10.5 (95% CI,1.44,76.48).  
Microbial 
exposure 
Gourbe
yre  
Review of case 
control and cohort 
studies 
No clear conclusion regarding probiotic beneficial effec
the prevention or treatment of allergy .  
Food 
consumption 
(quantity 
and timing) 
Poole 
JA  
Cohort 
After adjusting for breastfeeding duration, introduction o
cereal, family history of allergy, and history of food allergy
6 months of age, age at initial exposure to cereal grains con
to be strongly associated with wheat allergy ( 7 months: ad
OR: 3.8; 95% CI, 1.18,12.28) 
Du Toit  
Case control 
After adjustment for atopy, other food allergies, age, and
the RR for peanut allergy in the UK vs Israel is  5.8 (95% C
2.8,11.8), and  largest and most significant difference in an
between the UK and Israel was observed in the age of intro
of peanut (P < .0001). By 9 months of age, 69% of Israelis 
eating peanut compared with only 10% of UK infants. 
Katz   
Cohort 
The OR was 19.3 (95% CI, 6.0,62.1) for development o
mediated CMA among infants with exposure to cow  prote
age of 15 days or more (P < .001) vs those introduced to co
protein before 15 days.  

33  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
33
Joseph   
Cohort 
Early feeding reduced the risk of peanut sensitization am
children with a parental history [adjusted OR, 0.2 (95% CI,
0.1,0.7); P = .007]. The relationship also became significan
forwhen a cutoff for IgE of ≥0.70 IU/mL was used [adjuste
0.5 (95% CI, 0.3,0.9)]. 
Koplin  
Case control 
Introduction of cooked at age 4 to 6 months, vs later exp
reduced the risk of allergy [OR, 0.2 (95% CI, 0.06-0.71)]. 
Des 
Roches  
Case control 
The reported consumption of peanuts during pregnancy 
breastfeeding was higher in the case group (those who deve
peanut allergy and associated with an increased risk of pean
allergy in offspring [OR, 4.22 (95%CI, 1.57,11.30) and OR
(95% CI, 1.31,3.97) for pregnancy and breastfeeding, 
respectively]. 
Sichere
r  
Case control 
Multivariate analysis including clinical, laboratory, and 
demographic variables showed frequent peanut consumptio
during pregnancy {OR, 2.9( 95% CI, 1.7,4.9)] to be associa
with peanut IgE ≥5 kUA/L. 
Food 
processing 
Chung  
Laboratory 
analysis 
After curing and roasting, mature peanuts exhibited 
approximately 20% higher levels of advanced glycation en
adducts and higher IgE binding   vs immature peanuts. 
Yadzir  
Laboratory 
analysis 
Extracts from raw shrimp bound higher IgE than extract
boiled shrimp, but the purified boiled tropomysoin (the ma
shrimp allergen) demonstrates higher IgE binding vs raw sh

34  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
34
Samson  
Laboratory  
analysis 
Thermal processing can lead to the formation of new an
structures. 
Vitamin 
D 
Milner  
Cohort  
Early vitamin D use (within the first 6 months of life) w
associated with a higher risk for food allergies in the exclus
formula-fed population [OR,1.63( 95% CI,1.21,2.20)]. Vita
use at 3 years of age was associated with increased risk for 
allergies but not asthma in both breastfed [OR,1.62(95% 
CI,1.19,2.21) ]and exclusively formula-fed infants [OR, 1.3
CI,1.03,1.88)]. 
 
Cramag
o  
Ecologic  study 
Strong north-south gradient for the prescription of EpiPe
the United States, with the highest rates found in New Engl
[adjusted β for New England vs the rest of the US, 4.07 (95
2.77,5.36)] 
Mulins 
et al  
Ecologic  study 
Using multivariate analysis , EpiPen prescription rates th
higher in southern latitudes (less sunlight) compared with n
regions [β, −19.22( 95% CI, −26.71 , −11.73)]. 
Mulins 
et al 
Ecologic  study 
Southern latitudes were associated with higher hypoaller
formulae prescription rates [beta, -147.98( 95% CI,-281.83
14.14)]. 
OR, odds ratio;RR, Relative Risk; CI,confidence interval ;CMA, Cow’s  Allergy 
 
 

35  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
35
 
 
 
 
 
 
 
 
Appendix B. protocols. 
 
A1. BPCFC 
Double-blind, placebo-controlled food challenge (BPCFC) up to 100 mg (144 mg 
cumulative)done at screening and 3 months post OIT 
Dose 
Cumulative 
1 mg 
1 mg 
 3 mg 
4 mg 
 10 mg 
 14 mg 
 30 mg 
 44 mg 
100 mg 
144 mg 
300 
444 
 
 
 
A 2. Scale for Grading Reaction Severity 
Score  
Symptom 
Action 

36  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
36
Mild 
Pruritus, 
Urticaria, 
Flushing, 
Rhinoconjunctivitis 
Observe 
May 
give 
Antihistamine 
(e.g. 
Benadryl or Reactin as prescribed 
Call Research Team  
Research team will evaluate if dose 
adjustment is needed and if next dose 
will be given at home or in hospital.  
Moderate  
Angioedema, 
 Throat tightness, 
 Gastrointestinal complaints (cramping, 
. pain,vomiting,diarrhea) 
 Respiratory symptoms (Cough, Mucous 
production)  
Give epinephrine IM as per protocol  
Give Antihistamine (e.g. Benadryl or 
Reactin as prescribed 
Seek urgent care  (hospital emergency 
room) 
Call Research team 
To give next adjusted dose in hospital 
research unit (CIM) 
Severe 
Wheeze, Respiratory Distress 
Hypoxia, 
Cyanosis,  
Hypotension 
Circulatory collapse (Shock) 
Give epinephrine IM as per protocol  
Give Antihistamine (e.g. Benadryl or 
Reactin as prescribed 
Call 911 
Seek urgent care (transfer to hospital 
emergency room) 
Call Research team; if the symptoms 
are not improving within 10 minutes 
of the first dose, instructions will be 
given from the team regarding use of 
a second dose of epinephrine.  
 
 
 
 
 
 
B1. Initial Escalation: Day 1 and 2 
• 
Verify no wheezing, flare of atopic disease, or other intercurrent illness 
• 
Update history and con meds 
• 
Vital signs and PE 
• 
PEFR 
• 
Dose escalation at 20 minute intervals 
• 
Post-dose vital signs 

37  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
37
• 
Vital signs q30min x3 post last dose 
• 
Monitoring for AEs (see  
Day 1 
Day 1 
Day 2  
Day 2  
Dose ( raw 
peanut/ tree nut) 
Dose ( crushed 
peanut/ tree nut) 
Dose( raw  peanut/ 
tree nut protein) 
Dose( crushed raw  
peanut/ tree nut) 
0.5mg 
2.0 mg 
3mg 
 
1mg 
4.0 mg 
 
 
1.5mg 
6.0 mg 
 
 
2.5 mg 
10.0 mg 
 
 
3mg 
12 mg 
 
 
3.5mg  
14 mg 
 
 
4mg  
16 mg 
 
 
4.5mg 
18 mg 
 
 
5mg 
20 mg 
 
 
 
 
B2a. Build up phase for 300mg (group A) 
 
Dose Number 
Dose 
(peanut/tree 
nut protein) 
(mg) 
 
Dose  
(crushed 
peanut/tree nut 
      (mg) 
Interval  
(weeks) 
Percent 
Increase from 
Previous Dose 

38  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
38
1 
3 
 
2 
  
2 
6 
24 
2 
100% 
3 
12 
48 
2 
100% 
4 
20 
80 
2 
67% 
5 
40 
160 
2 
100% 
6 
80 
320 
2 
100% 
7 
120 
480 
2 
50% 
8 
160 
640 
2 
33% 
9 
200 
800 
2 
25% 
10 
240 
960 
2 
20% 
11 
300 
1200 
Enter 
Maintenance Period 
25% 
  
 
 
B2a. Build up phase for 30mg (group B) 
 
Dose Number 
Dose 
peanut/tree 
nut protein 
(mg) 
Dose  
crushed 
peanut/tree nut  
(mg) 
Interval  
(weeks) 
Percent 
Increase from 
Previous Dose 
1 
3 
12 
2 
  

39  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
39
2 
6 
24 
2 
100% 
3 
12 
48 
2 
100% 
4 
20 
80 
2 
67% 
5 
30 
120 
2 
50% 
6 
30 
120 
2 
00% 
7 
30 
120 
2 
0% 
8 
30 
120 
2 
0% 
9 
30 
120 
2 
0% 
10 
30 
120 
2 
0% 
11 
30 
120 
Enter 
Maintenance Period 
0% 
 
If the subject does not tolerate a given dose and symptoms are mild, then that dose or 
the previously tolerated one is repeated, and the protocol proceeds as outlined. If the 
subject experiences significant symptoms, then the protocol is stopped, and the highest 
tolerated dose is used as the starting daily dose. 
 All doses will be pre-weighed and encapsulated to blind both patients and evaluating 
physicians    
 
 
After achieving a dose of 300 mg(group A) or 30 mg (group B) , the subject will 
return for a first maintenance visit 2 weeks later.  
• 
Visits are then every 4 weeks until Exit Visit 

40  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
40
 
 
 
 
 B3.Challenges during maintenance period ( for group A or B): 
Dose  
(peanut protein) 
Dose  
(crushed peanut) 
Cumulative  
(peanut protein)  
Cumulative  
(crushed peanut) 
3 mg 
12 mg 
3 mg 
12 mg 
10 mg 
40 mg 
13 mg 
52 mg 
30 mg 
120 mg 
43 mg 
172 mg 
100 mg 
400 mg 
143 mg 
572 mg 
300 mg 
1200 mg 
443 mg 
1772 mg 
600 mg 
2400 mg 
1043 mg 
4172 mg 
1000 mg 
4000 mg 
2043 mg 
8172 mg 
 
 
 
 
 
 
 
Appendix C. Budget 
Total budget estimate: 78,000CAD per year including: 

41  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
41
 
 
 
Estimate (CAD) 
comment 
Yearly salary for an 
undergraduate student for salary  
that will support  training in Lab 
evaluation mechanisms for 
tolerance detection  
35,000 
 
Study coordinator 
40,000 
Yearly salary with 
benefits 
Paediatric test center (70CAD 
for each blood sample) 
3000 
 
Staining with monoclonal Abs 
3000 
 
Use of FACS 
3000 
 
CBA 
3500 
 
Culture materials and stimulating 
material 
3500 
 
Travel expenses for presentation 
of results in scientific conferences  
5000 
 
 
78,000 
yearly 

42  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
42
Appendix D: Flow chart of study procedures 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Particpants  
Group A 
rush 
Group B 
rush 
Group C 
challenge 
Potential 
Participants 
Randomization 
300mg 
52 wks 
30mg 
30mg 
52 wks 
300mg 
Challenge 
Challenge 
Challenge 
52 wks 
Challenge 
Challenge 
36wks 
36wks 

43  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
43
 

44  Ben -Shoshan PN 
 
Version 4 
Aug 7, 2019 
44