Investigating Modified Protocols of Oral Immunotherapy to Validate Efficacy and Safety
Phase: NA
Conditions: Peanut Allergy, Milk Allergy, Cow's, Egg Allergy
Sponsor: McGill University Health Centre/Research Institute of the McGill University Health Centre

 
1 
IMPROVES Protocol Version 1 
November 22, 2021 
 
 
IMPROVES : Investigating  Modified Protocols of Oral immunotherapy to validate Efficacy and 
Safety  
 
Principal Investigator: Moshe Ben-Shoshan 
 
Sub-Investigators: Bruce Mazer, Christine McCusker 
 
Funding Source: Teal Wheel Foundation 
 
 
1. Background, knowledge gaps and Impact of the research: (our publications marked *) 
1.1 Food allergy is a common problem with substantial societal, nutritional and economic 
impact. 
Almost 3,500,000 Canadians are reported to have food allergy, a life threatening condition associated 
with substantial social and economic burden.1-5* Studies suggest a worrisome increase in food allergy 
prevalence in the last decade mainly in North America.*6,7 Up to 20% of households in Canada have at 
least one member with a food allergy while another 30% consider food allergies when purchasing or 
preparing foods (e.g., their school age child attends a school where certain allergenic foods are 
prohibited). 4,6,8* Further, food-induced anaphylaxis, the most severe manifestation of food allergy, has 
almost doubled in the last decade among Canadian children presenting to the emergency department 
(ED).2,9* It is estimated that the annual cost (direct & indirect) for Canadians with food allergies is 2.9 
billion dollars.10-12  
The most common food allergy affecting Canadian children and the major cause of anaphylaxis is 
peanut,2,4,13* while milk allergy is associated with the highest annual risk for accidental allergic 
reactions (up to 40%).14,15 Hence, managing these food allergies appropriately is of crucial importance. 
Until recently the only management for food allergy was avoidance.16* However, elimination of peanut 
and milk in children’s diets has substantial nutritional and health implications. Peanuts are one of the 
most widely consumed legumes globally due to their nutritional value, taste, and affordability.17 
Studies have shown that consumption of dairy products are beneficial for building muscle and bone, 
lowering blood pressure and low-density lipoprotein cholesterol, and preventing tooth decay, diabetes, 
cancer, and obesity.18 Indeed an expanding body of research suggests that food allergy, mainly milk 
allergy, and elimination diets can negatively affect childhood growth and nutrition.19,20 For egg, the  
perfect balance and diversity in  nutrients along with its high digestibility and its affordable price has 
highlighted  egg as a basic food for humans. Further, given the need for strict avoidance, high risk of 
accidental reactions,21-24* paying meticulous attention to food labelling25,26* and perpetual vigilance for 
potential undeclared sources of contamination, it is unsurprising that individuals with food allergies and 
their families have higher levels of anxiety and a lower quality of life than those with other chronic 
conditions such as diabetes mellitus and inflammatory bowel disease.27 We must develop management 
strategies that replace avoidance of these important sources of nutrients, and that are highly accessible, 
in order to improve the health of Canadians with food allergies.  
1.2 The shift of paradigm in food allergy management and the use of oral immunotherapy 
While only 10 years ago management relied solely on avoidance of the offending allergen and rescue 
treatment for accidental reactions with epinephrine injection,16* it is now agreed that almost 80% of 
children28-30* can be desensitized and protected from accidental reactions through gradual and 
medically supervised introduction of the allergic food, via oral immunotherapy (OIT).30,31* OIT has a 

 
2 
IMPROVES Protocol Version 1 
November 22, 2021 
marked effect on immunological and clinical markers and allows for many fold increases in the 
threshold of food which elicits an allergic reaction.21,30* This increase in tolerance protects children 
from reactions after accidental exposure to their food allergen and in some cases allows normal 
introduction into their diet.30 Accordingly, protocols for OIT for the main food allergens have been 
recently incorporated in clinical practice and their clinical benefits have been acknowledged in 
European and Canadian official guidelines.30,32 
Adaption of OIT as a management for food allergy has been slow for two main reasons. Firstly, the 
most widely used OIT protocols create more allergic reactions than they avoid.29,33* Secondly, in the 
USA, the FDA treats peanut OIT as a drug because it is treating a disease (peanut allergy). The clinical 
efficacy of OIT for food allergy is further attested by the FDA approval 
( https://www.wsj.com/articles/fda-approves-first-drug-for-peanut-allergy-11580510666) of the very 
first pharmaceutical peanut allergy treatment, Palforzia(https://www.fda.gov/media/134838/download), 
encapsulated peanut protein manufactured from defatted peanut flour. However, the prescription of 
Palfozia requires both the patient and physician to agree to a risk mitigation strategy, deterring potential 
users. Further, while thousands of children have undergone OIT treatment with regular, store bought 
foods and this approach is endorsed by the Canadian30 and European32 Allergy associations, it requires 
intense medical and nursing management which limits its accessibility. 
Hence, while the clinical benefits of OIT inducing desensitization are promising, safer protocols 
using regular, store-bought foods are required. 
 
 
2. Overarching Objective: Using a randomized trial design, the investigators aim to develop, 
through our IMPROVES study, new OIT protocols that will promote a safer, effective strategy for 
OIT. This proposal will focus on peanut, milk and egg allergy given their frequency and effect on the 
health of Canadian children. 
 
 
3. Hypothesis: We hypothesize the proportion of anaphylactic reactions among all therapeutic doses 
will be at least five times lower among participants managed through our two modified protocols when 
compared to the standard protocol in use today. We also anticipate that immune biomarkers will change 
similarly over time in the three OIT protocols assessed indicating similar efficacies between the three 
protocols. Finally, we expect that accidental reactions due to inadvertent exposure after the 
maintenance dose is attained will occur in less than 5 % of our patients over a three-year period. 
 
 
4. Experimental aims: We will test our hypothesis using the following three Aims: 
Aim 1. Develop and evaluate modified protocols that will improve the safety of OIT.  
Aim 2: Determine the long-term adherence and protective effect of OIT for all protocols used. 
Aim 3: Explore changes in immune biomarkers over the course of desensitization.  
 
 
5. Research plan/Methodological approach/Study design  
 
AIM1: DEVELOP AND EVALUATE MODIFIED PROTOCOLS THAT WILL IMPROVE 
THE SAFETY OF  
Background and Rationale: Gaps related to current oral immunotherapy protocols 
Would lower target doses be as efficacious while safer?  

 
3 
IMPROVES Protocol Version 1 
November 22, 2021 
The threshold doses provoking reactions to peanut, the most common food allergy in Canadian 
children, range from 15mg to 1000mg,34,35 and it is reported that eliciting doses for milk proteins are as 
low as 0.1 mg for sensitized individuals.36 Alarmingly, almost a third of products from 
Western Europe and two thirds of products from eastern Europe without precautionary labeling contain 
detectable levels of peanut up to 245 mg per liter.37 It was reported that products marked as milk free 
can contain up to 15 mg of milk protein31.  Hence it is crucial that desensitization protocols cover 
threshold doses of at least 300mg of food protein, a dose that is equivalent approximately to 1.5 
peanuts and was reported as a target safe dose by our group and by others30,31. Interestingly, studies 
reveal that OIT using doses that are 10 times lower still offer protection against this target dose.31* A 
recent small sample study reveals that children undergoing OIT using peanut powder up to 133 mg/day 
tolerated a challenge of 300 mg with a good safety profile.38 Preliminary data from our group on the 
use of low dose protocols clearly indicate the feasibility and safety of these protocols in  a small 
group of  14 children with peanut allergy who were randomized  to either high dose of 
maintenance (300mg ) or low dose (30mg ) . Our preliminary findings reveal that children 
desensitized to 30 mg versus 300mg tolerated similar amount of peanut at the exit challenge 
(5522.5mg versus 5795.2mg on average respectively) and that the rate of anaphylaxis was three 
times higher in the higher dose group (45 versus 15 cases of anaphylaxis). Further, there were six 
severe anaphylactic reactions in the high dose group (defined as the development of hypoxia or 
hypotension) and no severe reactions in the low dose group. 
 Similarly for milk, it was reported that low doses (e.g. 20 ml of milk)39,40 were as effective as high 
doses (100ml)40-42 and, that the risk of severe symptoms in the maintenance phase was five times lower 
in the low-dose group.39,43 Using lower doses (e.g. 10 times lower than currently used protocols) if still 
effective, is clearly of advantage given the better safety profile.29,30  
Would OIT using processed forms be as effective while safer? 
Studies suggest that a large subset of children who react to uncooked foods, mainly milk, can tolerate 
cooked forms of these foods.44,45 Further, immunologic changes induced by a diet containing well-
cooked milk are reported to be similar to changes that have been observed during OIT.45 Similarly, 
there is ample evidence that boiled peanut versus raw and roasted have decreased the risk of  allergic 
reactions to peanut.46 It was also reported in two small sample studies that desensitization to fresh milk 
can be achieved with gradual exposure to well-cooked milk in baked goods in almost half of the 
cases.47,48 However, the safety and efficacy of this approach compared to current protocols using 
uncooked milk  was not yet been established. Such a comparison is crucial in order to determine the 
best strategy to desensitize children. In addition, the peanut snack used for the LEAP study was a 
processed form consisting of corn puff/stick containing approximately 50% peanut protein (Bamba; 
Osem Food Industries, Shoham, Israel), and was reported to be an effective OIT strategy.49 Given the 
practicality and safety of OIT using processed/cooked forms of food allergens, it is of particular interest 
to compare protocols incorporating initially processed/cooked forms  and then transitioning to 
unprocessed/uncooked forms of peanut/milk to currently used protocols.  
 
Primary objective: Compare the rate of anaphylaxis in different OIT protocols.  
Secondary objective: Compare the efficacy of protocols A to C 
 
Study Procedures: (Figure 1): Three OIT protocols (A, B, C) will be assessed each for milk, egg or 
peanut. Each protocol will use a group of 20 males and 20 females, between 2 to 40 years, with 
physician diagnosed food allergy. Participants will be recruited from the allergy clinic at the Montreal 
Children’s Hospital and at the collaborating site The Hospital for Sick Kids in Toronto. We will base 
our proposed desensitization protocol on previous studies by our group on milk, egg and peanut 

 
4 
IMPROVES Protocol Version 1 
November 22, 2021 
desensitization protocols that have been active in the last year at the McGill University Health 
Centre29,31* and at the Hospital for Sick Children29,31*  and on current existing guidelines.30
  
Figure 1. Flow chart for study procedures: Protocols A to C for milk and peanut   
Inclusion criteria:  
 Patients between 2 and 40 who satisfy all the following criteria will be included: 
 
A history suggestive of immediate allergy to the food. 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. 29,50*  
 
The presence of at least one of the following confirmatory tests: 
o 
Positive SPT to the culprit food allergen (weal diameter 3 mm larger than that of the normal 
saline control). The allergens used will be commercial extracts of the foods (Omega Labs, Toronto, 
Ontario). 
o 
Detection of serum specific IgE (>0.35 kU/L) to the culprit food or any of its proteins, 
measured by fluorescence enzyme immunoassay (Phadia, CAP System, Uppsala, Sweden). 
o 
Informed consent form signed by the parents or legal guardian. 
 
Exclusion criteria: 
 
Patients who have uncontrolled respiratory disease (asthma, cystic fibrosis, etc.)  
 
Patients who present with intercurrent disease active at the time of starting desensitization. 
 
Non IgE mediated or non-immunological adverse reactions to milk or peanuts. 
 
Malignant or immunopathological diseases and/or severe primary or secondary immune   
deficiencies. 
 
Patients receiving oral immunosuppressor therapy. 
 
Patients receiving β-blockers (including topical formulations), or who receive daily doses of 
NSAIDs, aspirin or ACE inhibitors for cardiac issues. 

 
5 
IMPROVES Protocol Version 1 
November 22, 2021 
 
Associated diseases contraindicating the use of epinephrine: cardiovascular disease, severe 
hypertension or hypotension. 
 
Patients diagnosed with eosinophilic gastrointestinal disorders, including patients with a history 
of antacid use for reflux related to food impaction or with a history of esophageal spasm. 
 
Patients already tolerating processed/cooked forms of the food (e.g.,baked goods with milk). 
 
 
Asthmatic patients will be eligible to participate in the study.  These patients will have pulmonary 
function testing at the beginning of the study.  Their asthma must be very well controlled: they must be 
symptom-free, and must have been on a stable dose of asthma medication for at least the previous three 
months.  Potential subjects who do not meet these criteria at screening will will have their asthma 
control assessed by one of the study physicians. Their screening visit will be rescheduled for three 
months later.  At this visit, they will have a second pulmonary function test.  If the study physician 
decides that the subject’s asthma is now well controlled, they will be able to proceed with the screening 
visit. 
 
 Once these criteria are assessed qualifying participants take the following steps: A qualifying oral 
food challenge, a two-day rush period to ensure they are able to tolerate the minimum allergen intake 
requirements (for peanut and milk desensitization only), an up-dosing period (also referred to as build-
up phase) of 21 to 44 weeks depending upon the protocol, a maintenance period of 52 weeks followed 
by a final exit oral food challenge. 
 
Qualifying Oral Food Challenge (OFC)  
Studies exemplify the need for oral food challenges prior to OIT to establish the food allergy given 
high false positive rates for skin tests and IgE levels alone.30,51,52 Further, conducting food challenges at 
study entry also allow us to determine the tolerated cumulative dose of the culprit food and allow us to 
evaluate the effect of OIT on this threshold. Patients will begin with a double blinded, placebo-
controlled oral food challenge (DBPCFC) to the culprit food that will take place over two days, with 
patients receiving doses of placebo one day and the food protein the other. For peanut, the protein will 
be in the form of crushed peanut, powdered egg white for egg protein for milk the protein will be in the 
form of fresh cow’s milk.  Patients, study nurses and evaluating physicians will be blinded, but not the 
study coordinator.  On challenge 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 study nurses that assist in the desensitization protocol have been trained to 
work in an intensive care unit.  Patients will be reminded beforehand that they must be in good health 
for the challenge and must not have taken anti-histamines in the last seven days.  If they are not in good 
health, the challenge will be rescheduled.  Upon arriving, a physical exam will be performed and vital 
signs taken.  A complete allergic history will be 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.  In 
the case of egg and peanut/tree nut, the doses will be mixed in a food vehicle tolerated by the 
participant, such as pudding, fruit puree or crumbled crackers.  On the placebo days, a substance that 
the participant is known to tolerate, such as wheat flour, will be used.  Doses will be given by the study 
nurse every 15-30 minutes, depending on patients’ tolerance of the dose (see Appendix B for challenge 
doses).  If participants tolerate the last dose, they will be given a dose of food that contains the allergen 
in question – peanut butter in the case of a peanut challenge, or a softly coked omelet for egg allergy.  
For the milk challenges, to maintain the blind as well as making the doses more palatable, the doses 
will be mixed with chocolate syrup. The placebo doses of the milk challenges will be a milk substitute 

 
6 
IMPROVES Protocol Version 1 
November 22, 2021 
that the participant is known to tolerate, such as soy, oat, rice or almond milk.  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 a minimum of two hours after a dose of 
epinephrine and/or one hour after the resolution of the last allergic symptoms. 
 
Participants will then be randomly assigned in a 1:1:1 fashion to one of 3 study groups (A, B, C) 
using a process of stratified randomization (according to sex) using opaque envelops.  Participants 
tolerating a minimum cumulative dose of 154.4 ml of milk, or the 300 mg dose of peanut/egg will be 
considered screen fails and NOT be included in the study. 
 
Two-Day Rush Desensitization Period 
Milk: The two-day rush desensitization will begin with a first single dose of milk diluted 1/1000 and 
will advance in 10 increments over 2 days until the participant can tolerate 2.5 ml 1:10 diluted milk on 
day 2. Patients will be instructed on how to make the diluted milk doses by a member of the study 
team.  They will then continue this dosage daily at home for 2 weeks (all Doses for Rush 
Desensitization are in Appendix B). 
Peanut: The two-day rush desensitization period will begin with 0.5 mg of peanut protein. After the 
initial dose, subjects will receive approximately doubling doses every 30 minutes until a dose of 5 mg. 
Those who tolerate 5 mg, will be asked to return the next day for one dose of 3mg. They will continue 
this dosage daily at home for 2 weeks. 
For these allergens, if a participant develops allergic symptoms during the Rush desensitization 
phase, the procedure will be stopped.  The participant will begin their home dosing with the last 
tolerated dose during the Rush Desensitization that caused no allergic symptoms. 
Egg: No Rush Desensitization will take place for egg desensitization.  Rather, participants will begin 
their desensitization dosing with the last dose of egg powder that was tolerated during the challenge 
(for example, if the participant tolerated the third dose of egg powder during the challenge, but reacted 
to the fourth dose, desensitization will begin with the amount of egg powder present in the third dose).  
Participants will come to the CIM and take this dose under nursing supervision.  If the dose is well 
tolerated, they will begin taking this dose at home. 
 
Up Dosing period 
Two weeks after the two-day rush desensitization, participants will begin the build-up phase. 
Participants will come to the clinic every two weeks to receive their first new dose at each up-dose 
level. Subsequent home doses of milk are measured by the participant or their parent/caregiver using a 
syringe. The study staff will train them in the use of the syringe, and supply all syringes required.  
Home doses of peanut and egg will be provided in individual pre-weighed containers. 
  
Group A ( the standard protocol currently used in our centers and by other groups21,29,30*): 
Milk: Beginning with a dose of 2.5 ml (1:10 dilution) subsequent doses are incrementally increased 
every two weeks until the maintenance dose of 200 ml is tolerated. This phase will take approximately 
38 weeks. 
Peanut: Beginning with a dose of 3 mg subsequent doses are incrementally increased every two 
weeks until the maintenance dose of 300 mg is tolerated. This phase will take approximately 22 weeks. 
Egg : Beginning with the dose established during the Screening Challenge,  subsequent doses are 
incrementally increased every two weeks until the maintenance dose of  1200mg egg protein (~1/5 of 
an egg) is tolerated. This phase will take approximately 24 weeks. 

 
7 
IMPROVES Protocol Version 1 
November 22, 2021 
 
 
Group B: 
Milk: Beginning with a dose of dose of 2.5 ml (1:10 dilution) subsequent doses are incrementally 
increased every two weeks until the maintenance dose of 50 ml is tolerated. This phase will take 
approximately 28 weeks.  
Peanut: Beginning with a dose of 3 mg subsequent doses are incrementally increased every two 
weeks until the maintenance dose of 30 mg is tolerated. This phase will take approximately 18 weeks. 
 
  
 
Egg: Beginning with the dose established during the Screening Challenge, subsequent doses are 
incrementally increased every two weeks until the maintenance dose of  300mg egg protein ( ~1/20 of 
an egg ) is tolerated. This phase will take approximately 22 weeks. 
 
 
Group C:  
Participants in group C will start the up-dose phase with a processed form of the allergen.  
Milk: Participants randomized to Group C will be given a recipe (Appendix D) for baked goods and 
will use a portion of a cupcake containing 50 mg protein respectively (e.g., 1/16 of a muffin).  Because 
allergenic protein levels are based on weights of ingredients, participants will be instructed that the 
recipe is to be strictly followed.  This start dose has been chosen as it is  reported to be well tolerated in 
studies using well-cooked forms of milk.48 Participants will take these portions daily and will double 
the portion every two weeks until 800mg protein equivalent in baked goods is reached ~ 1 muffin). 
Once participants have reached this dose, they will transition to 40mg of milk protein (equivalent to a 
dose of 1 ml of pure milk).  They will double this dose every 2 weeks until 4 ml are reached. At this 
point the subject will continue from week 7 according to protocol A (Figure 1). This phase will take 
approximately 44 weeks. 
Peanut: will start with 10 mg of peanut puff (one Bamba, a peanut-butter-flavored snack, one 
peanut puff = 80 mg protein). The dose will be doubled every 2 weeks and once 4 puffs are reached 
(320 mg processed protein) and then we will transition to 80 mg crushed standard peanut protein. The 
protocol will continue from week 7 according to protocol A. Total duration of the up-dosing phase is 
24 weeks. 
Egg Participants randomized to Group C will be given a recipe (Appendix D)  and will use a portion 
of a muffin 75 mg protein (e.g. 1/8 of a muffin). Because allergenic protein levels are based on weights 
of ingredients, participants will be instructed that the recipe is to be strictly followed. Participants will 
take this portion daily and will double the portion every two weeks until 600mg protein equivalent in 
baked goods is reached (~ 1 muffin).  Once participants have reached this dose, they will transition to 
50 mg of egg protein and will continue according to protocol A.  This phase will take approximately 26 
weeks. 
 
In addition, participants will continue to consume the tolerated baked/processed forms at least 3 times 
a week after the transition. 
 
For all groups, 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 

 
8 
IMPROVES Protocol Version 1 
November 22, 2021 
previously tolerated dose for 1-2 weeks followed by an increase of the dose under supervision, when at 
least 3-4 days without symptoms.29*  
 
Maintenance dose period 
Once subjects have reached their group’s maximum dose, they will take that dose daily for 52 
weeks.  During this period, 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 recent medical history and physical examination. A complete nursing assessment of the 
symptoms and reactions at home, as well as the management of the co-factors and adherence to the 
study will be assessed at enrollment and at follow-up visits.   
The patients' parents will be instructed verbally and in writing about the recommendations to be 
followed during desensitization and how to treat possible allergic reactions. They will also be given a 
direct telephone line to members of the study staff for consultation. Patients will be instructed not to 
perform physical exercise for 2 h before or 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. 
  
Group A (Standard protocol): will receive a daily maintenance dose of 200 ml milk, 300 mg peanut 
or 1200 mg egg for 52 weeks. 
 
Group B (low dose protocol): will receive a daily maintenance dose of 50 ml milk, 30 mg peanut or 
300 mg egg or 52 weeks.  
 
Group C (baked /processed protocol): will receive a daily maintenance dose of 200 ml milk, or 300 
mg peanut, or 1200mg egg for 52 weeks. 
 
Study Exit oral challenge 
A single oral challenge will be conducted immediately at the end of the 52-week maintenance period.  
The challenge will be conducted in a similar fashion to the entry challenge (IV inserted, physical exam, 
etc).  Doses are specified in Appendix B. 
 
Documentation of intake doses adverse events: Subjects and families will be asked to complete 
daily home diaries to document that doses were taken, as well as to report accidental ingestions, 
problems with dose administration, or adverse symptoms. The patients' parents will be instructed 
verbally and in writing about the recommendations to be followed during desensitization and how to 
treat possible allergic reactions. They will also be given a direct telephone line to members of the study 
staff for consultation. In addition patients will be able to contact our team through a dedicated email 
and text messages. 
Classification of adverse reactions: Reactions during the desensitization protocol will be classified 
according to the categories proposed by Perry et al.53 Mild reactions are defined when symptoms are 
limited to the oral mucosa or the skin; severe reactions include cardiovascular or 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.29* 
Anaphylaxis will be defined when reactions involve at least 2 organ systems or hypotension 
develops.54 A modified grading system described by Muraro et al. will be used to classify anaphylaxis 
severity to mild, moderate and severe.55  

 
9 
IMPROVES Protocol Version 1 
November 22, 2021 
Definition of successful desensitization: Successful OIT will be defined when the patient will be 
able to tolerate 200ml ( 8000mg protein of milk ) , one egg ( 6000mg egg protein) 300 mg of peanut 
protein( ~ one peanut). Partial desensitization will be defined when a dose greater than 100ml milk/ 
1000mg egg / 100 mg of the peanut protein is tolerated. Patients who do not tolerate 100ml milk / 
1000mg egg protein / 100 mg of peanut protein  at the exit challenge will be considered as study 
failure. Participants in Group B-C who have allergic reactions during the exit challenge will be offered 
protocol A if they wish to continue. 
  
 
Successful OIT will be defined when the patient will be able to tolerate 200ml (8000mg) of milk 
protein or 300mg of peanut protein. Partial desensitization will be defined when a dose greater than 
100ml/100mg respectively of the culprit protein is tolerated. Patients who do not tolerate 100ml of the 
milk or 100mg peanut protein at the exit challenge will be considered as study failure. Participants in 
Group B-C who have allergic reactions during the exit challenge will be offered protocol A if they wish 
to continue. 
 
Discontinuation of study treatment: Discontinuation of study treatment for a patient occurs when 
the study treatment is stopped earlier than the protocol planned duration.  This can be initiated by the 
participant or by the investigator at any time. Participants can withdraw their consent to participate at 
any time for any reason. Participants who do withdraw will be asked to attend a last visit at the study 
site in order to review their allergic status.  Discontinuation can also be initiated by the investigator if 
they believe that continuation would negatively impact the risk/benefit of study participation, such as 
pregnancy or pregnancy planning. 
Attrition and failure: Studies by our group and by others report that almost 10% of participants in 
protocol A will withdraw from the study mainly due to anaphylactic reactions occurring during the 
buildup phase, while 20% will complete the program but fail the oral food challenge at the end of the 
therapy.29*30. 
 
Statistical Considerations: We will base our sample size calculations on our primary objective, i.e., 
the number of anaphylactic reactions among participants in the test protocols - B, C versus the non- 
modified protocol A. Given that preliminary data by our group (unpublished) and previous published 
studies by others suggest that modified protocols reduce by the risk of severe side effects compared to 
the high dose non modified protocol (protocol A)43 by at least 5-fold, and given previous research by 
our group reveals that 80% of participants will report anaphylactic reactions, a sample size of 40 
participants per group (a total of 120) will provide > 90% statistical power at a significance level of 
0.05 using chi-squared tests, with Bonferroni correction accounting for multiple comparison to show 
increased safety. This sample size will also provide adequate statistical power for exploratory analyses 
in our secondary objectives.  
Descriptive statistics, including means and standard deviations for continuous data, and proportions 
for categorical data, will be computed for all study variables. multiple logistic regression models will 
be used to assess factors associated with increased risk of anaphylaxis (i.e. baseline demographic 
characteristics; gender; sex, age, presence of co-morbidities such as asthma or eczema). 
For the secondary objective(Compare the efficacy of protocols A to C) , we will be able to determine 
an efficacy of 70% with a 95% confidence interval of 53.3% to 82.9%. 
 
AIM 2: DETERMINE THE LONG-TERM ADHERENCE AND PROTECTIVE EFFECTS OF 
OIT 

 
10 
IMPROVES Protocol Version 1 
November 22, 2021 
Background and rational:Do patients adhere to OIT protocols after maintenance achieved? 
Only two studies have so far attempted to determine long term adherence to OIT.56,57 The first study 
followed for 6 months 145 children reaching peanut OIT maintenance. The researchers reported that 
adherence to treatment was significantly higher in patients consuming 1200 mg (96.1%) versus those 
consuming 3000 mg (72.2%), (P =.001).56 The second study followed 101 children going through egg 
OIT for one year and revealed that in children  with daily consumption the risk of allergic events 
decreased five folds from build-up phase to maintenance ( from 5.3 on average per patient to 1.3 on 
average ) and that this risk was higher in those consuming egg every two days  ( [0.76 ± 1.85 (0‐7) with 
daily consumption vs 2.1 ± 3.49 (0‐7) when consumed every two days (P < 0.05) respectively].57 No 
study so far assessed long term adherence to milk OIT. It is crucial to assess longer follow up periods 
using different protocols in order to determine which protocol is more likely to be followed in the long 
term and to determine the annual risk of allergic reactions. Such a comparison between protocols will 
contribute to determine which protocol will have a higher long term social and health impact.  
What is the risk of accidental reactions after achieving OIT maintenance? 
 It is estimated that up to 50% of children with food allergy will experience an accidental reaction 
annually14 despite continuous vigilance22,58* and that these reactions may be fatal.59,60 Although several 
studies reported on high rate of allergic reactions during the build-up phase of OIT,29,30* no studies so 
far assessed long term (years) effect of OIT on the incidence of allergic reactions after maintenance is 
attained.  
Objectives: 1.To determine long term (4 years) adherence to OIT 2. To assess the annual risk of 
allergic reactions over a 4 year period. 
Study Procedures: Long term compliance with OIT and the risk of future reactions will be assessed 
through a follow up questionnaire that will be administered annually to patients after the exit challenge 
for 4 years (please see Appendix D for questionnaires).  Patients will be queried on the continuous 
consumption (how often do they consume the maintenance dose per week), the occurrence of allergic 
reactions (the suspected culprit, the clinical presentation defined as mild, moderate, severe or 
anaphylaxis as previously defined in this proposal and based on previous publications by our group 29*) 
and their management.  
Statistical Considerations: The proportion of patients adhering to the OIT protocol and factors 
associated with lack of compliance will be assessed with a cox model and Kaplan-Meier curve. For 
each time interval, cumulative compliance probability will be calculated as the number of subjects 
compliant with the protocol divided by the number of patients at risk. Similarly, the incidence of 
patients experiencing allergic reactions over the 4 years follow up will be calculated.  
 
AIM 3: EXPLORE CHANGES IN IMMUNE BIOMARKERS OVER THE COURSE OF 
DESENSITIZATION   
Background and rationale: Are there significant long-term immunologic changes that would 
predict sustained desensitization?   
The symptoms of food allergy are initiated by the presence of IgE antibodies against foods. In 
individuals without food allergies, the level of IgE antibodies is low, and in fact although individuals 
make IgG against foods, this is of unknown significance.61 The exception to this is IgG4, which we and 
others have shown slowly increases as OIT progresses, and correlates with improvement in clinical 
performance.62 At the same time, production of IgE against the food used in OIT actually increases and 
does not decrease significantly until well after maintenance is initiated, 6-9 months after starting 
therapy. The balance between increasing IgG4 despite maintaining IgE has led to the concept that IgG4 
plays the role of a “blocking antibody” to counteract the adverse functions of IgE. It is not known if the 
lower dose OIT protocols will induce the same incremental changes in IgG4 within the same time 

 
11 
IMPROVES Protocol Version 1 
November 22, 2021 
frames, or if there will be delays in the diminution of IgE.63 Based on our preliminary work with milk 
we expect at least a 50% increase in the values of specific IgG4, and a 50% decrease in milk 
component specific IgE over one year (Figure 2 A and B respectively).  
 
 
 
 
 
 
 
 
Figure 2: A. IgG4 increases in 10 patients undergoing Milk OIT using the standard protocol (A:200 ml 
maintenance dose) and in B. temporal changes in specific IgE in 8 patients undergoing Milk OIT using 
the standard protocol over 1 year (CAS: Casein, BLG: betalactoglobulin, ALA-alphalactalbumin) 
What influences the production of IgG4? B-lymphocytes are cells specialized for producing 
antibodies, and play a crucial role in the pathogenesis of food allergy by producing IgE. In addition to 
this already important role, B-cells likely play other crucial roles in the ability tolerate food proteins. 
While B-cells can produce cytokines such as IL-4 and IL-13, which contribute to allergic inflammation 
in lungs and sinuses 64 and increase IgE production. However, B-cells can play an active role as 
regulatory cells in various infectious and inflammatory processes. B-regulatory(Breg) cells produce 
regulatory cytokines, such as IL-10 and TGF-. The production of IgG4 is highly influenced by the 
presence of IL-10.65 B-cells make both IgE which causes allergies and IgG4 (which regulates 
allergies). We have shown in preliminary data that OIT can we understanding the contribution of Breg 
to food desensitization is an important facet of clarifying the immunological network that is in play in 
this disease. Thus, we will examine the total numbers of Breg (designated by the surface markers CD19 
and CD38 as well as the intracellular cytokines IL-10) and determine changes in these cells in children 
undergoing different OIT protocols.  
What are the changes regulatory T (Treg) and regulatory B cells in different OIT protocols?  
Another key cell that is crucial for inducing tolerance in allergy is the regulatory T-cell.Regulatory T 
cells (Treg) expressing the FOXP3 transcription factor are a subset of CD4+ T cells that play a key role 
in the induction and maintenance of tolerance to a plethora of self and non-self-antigens in murine 
models and humans. Importantly, FOXP3+ Treg cells are implicated in the control of allergen-specific 
effector T (Teff) cells, and as such, serve as a key cellular mechanism for the induction and 
maintenance of oral tolerance to food.A potential suppressive role of Treg in food allergies was 
exemplified both in animal models 66,67 and in human studies. At least two studies showed increased 
Treg cells in peripheral blood in children who spontaneously outgrew milk allergy68,69. We have shown 
that antigen-specific Treg attenuate airway inflammation in a mouse model of asthma.70,71*, in part due 
to production of regulatory cytokines like IL-10 and TGF-. It is not known if increases in Treg during 
the natural outgrowth of peanut/milk allergy are mirrored by similar increased in Treg following in 
active oral immunotherapy. In a pilot study in millk-allergic children, we reported that the systemic 
frequency of milk allergen (casein)-specific Treg cells correlated with successful milk OIT, while a 
similar study showed no correlation in similar settings when evaluating polyclonal Treg cells. Similarly 
for peanut72 it was reported that oral immunotherapy results in increased antigen-induced Treg cell 
function.72,73 Thus, we will investigate the effect of immunotherapy to peanut and milk on the 
circulating frequency and function of allergen-specific Teff and Treg cells.  
We will track Breg from peripheral blood mononuclear cells taken from children at the indicated time 
points; we predict that as IgG4 increases, so will the percentage of CD19+IL-10+ positive cells in their 

 
12 
IMPROVES Protocol Version 1 
November 22, 2021 
blood, as detected by flow cytometry. We also predict that the Breg population will be stable over the 3 
years of follow up, leading to maintenance of IgG4 and tolerance to milk or peanut.  
We will carefully monitor and quantify antigen-specific, CD4+ T cell subsets in blood cells by multi-
parametric flow cytometry. To this end, we will identify antigen-activated Treg and Teff directly ex 
vivo through the differential expression of CD137 (Treg) and CD154 (Teff) in conjunction with 
reference Treg signature markers CD25, FOXP3 and Helios, as we have previously published. We will 
establish a correlation between cycling (intranuclear Ki67 expression) and function (cytokine 
expression by intracellular staining: allergic [IL-4] and non-allergic [IFNg]), activated Teff cells in 
various groups and correlate these responses with the frequency of antigen-specific Treg cells. We will 
confirm the function of these allergen-specific CD4+ T cell subsets following in vitro re-stimulation 
with designated allergens and quantitation of suppressive or inflammatory functions, as we have 
shown.74,75* We will also determine if children who have undergone OIT through protocols A-C have 
decreased T-eff responses to food in different OIT protocols, and determine the cytokines that are 
produced by the Treg. It is yet known if changes in Treg cells will occur to the same extent in protocols 
based on low doses or baked /processed forms. 
 
Objective: To assess the change in immune biomarkers over the course of OIT. 
 
Study Procedures: We will aim to assess biomarkers including Skin Prick Tests (SPT) at study 
entry, after the up-dose period is complete, and after the 52 week maintenance cycle is complete and 
then annually through the entire follow up period in 12 consecutive participants for each group A to C. 
Milk and Peanut component specific IgE and IgG4 will be measured according to our published ELISA 
protocols for milk29* and recent protocols for peanut (Levy, Cohen et al submitted). At these visits, 30 
mls of blood will be collected from participants. We will also assess biomarkers including tryptase 
levels and specific IgE levels at study entry one week and one month post challenge to assess the 
temporal accuracy of this biomarkers. As negative controls we will assess specific IgE and skin tests 
for a food allergen tolerated by the patient (typically wheat. Soy will be used as the negative control 
allergen in the case of a participant with wheat allergy). 
  
Statistical Considerations: Descriptive statistics, including means and standard deviations for 
continuous data, and proportions for categorical data, will be computed for all study variables. The 
concentrations of specific IgE and IgG ,Breg and  Treg at the different study time-points will be 
compared with their baseline values and between treatment groups using a linear mixed-effects 
regression model. 
 
6. Knowledge Translation (KT) plan 
Our integrated KT plans target patients and their families, caregivers and policy makers. We will 
involve all members of our transdisciplinary team. This collaborative and participatory approach 
focuses on impactful and meaningful solutions. To this end, we will continue to use social media (e.g. 
Facebook, Twitter) to distribute our findings effectively to all Canadians76,77* Going forward, we will 
also hold physicians and patient-focused webinars, and when the situation permits, in-person Café 
Scientifique-style presentations, where families can raise questions and expect meaningful answers. We 
will also engage with national and local patient advocacy organizations (e.g, Food Allergy Canada, 
Allergies Quebec), with whom we already enjoy strong collaborations, and, who participate in all 
stages of our study design. We highlight that, consistent with CIHR’s mission of knowledge 
mobilization, knowledge users including clinicians, research members of the CSACI and patient 
representatives (Food Allergy Canada) have been active partners at all stages of preparing this 

 
13 
IMPROVES Protocol Version 1 
November 22, 2021 
proposal. Hence, the findings from our studies will be distributed to care givers and patients and 
contribute to reincorporate allergenic foods in infancy and childhood, ultimately avoiding nutritional 
deficiencies,78 morbidity and fatality.78,79  
 
 7. Potential challenges and mitigation strategies 
The OIT protocols require substantial commitment from patients and family which may lead to high 
rate of attrition. However, in our experience patients and families with food allergy are highly 
motivated and follow diligently the protocol. In fact, in our last 10 years of experience with OIT, less 
than 15% withdrew from OIT mainly due to adverse effects. Further, we have currently more 500 
families on the waiting list for OIT. Hence, we are confident we will be able to address our study aims. 
 
 
8. Equity diversity and inclusion  
At study entry data are collected on age, sex, and allergen. Studies by our group and by others do not 
suggest that these factors affect the efficacy of OIT or increase the risk of allergic reactions. However, 
as discussed above (Statistical Considerations section, aim 1) we will evaluate these factors through 
comparing uni- and multivariate logistic regressions. 
 
9. Significance and Implications 
Safer approaches of OIT are required to be able to allow patients to access the therapeutic effects of 
OIT. Some approaches of interest include using biological therapies to protect against anaphylaxis80 
and certainly adjuvant approaches may be needed in some very highly allergic individuals. However, 
these strategies are often associated with high costs and increased risk of adverse events after weaning 
from adjuvant therapies.81 We maintain that there remain significant opportunities to improve safety of 
OIT with natural food through protocol modification as elaborated in our proposal. 
 The proposed research program will be the first to strategically examine the effectiveness of different 
OIT protocols and will contribute to future safe and effective protocols that will be incorporated in 
clinical practice. The study will also be one of the first to examine the safety and efficacy of oral 
immunotherapy in adults.  Improved practices and incorporation of safe protocols will save 
enormous amounts of money in both direct and indirect costs to the system and reductions in 
emergency room visits, will contribute to better health and will save lives. Further, improved 
protocols will also have important nutritional impact that includes a ready source of protein, 
minerals and vitamins, which are crucial components for growth and health. Most importantly, 
given the safety concerns related to current protocols, developing safe and effective protocol 
using regular, store bought foods will allow use of OIT outside of the hospital setting and hence 
increase OIT accessibility to all Canadians.  
 
10. Expertise, Experience & Resources  
The McGill University Health center is a multi-disciplinary research institute that is an integral part 
of the Montreal Children’s Hospital facilitating the collaboration between clinicians, epidemiologists 
and basic science researchers. The nominated principal applicant, Dr. Moshe Ben-Shoshan will oversee 
all aspects of the proposed research and will dedicate 15 hours per week (h/w) for the IMPROVES 
study. Dr. Ben-Shoshan is an epidemiologist and allergist at the Montreal Children’s Hospital and has 
extensive experience managing cohorts/registries, and OIT trials29,31* whose findings have been 
published in more than 100 publications in major allergy and paediatrics journals. Dr. McCusker 
(Montreal Children’s Hospital), Dr Mazer (Montreal Children’s Hospital) (8h /W each) bring expertise 
in clinical immunology to the research team and will assist in patient recruitment and in supervising the 

 
14 
IMPROVES Protocol Version 1 
November 22, 2021 
OIT procedures and laboratory analysis of IgE, and IgG4. Dr Xun Zhang, a biostatistician in McGill 
university is a collaborator in this initiative (1h /W). He has assisted in the development of this protocol 
and will supervise, together with Dr Ben-Shoshan, the statistical analysis. The collaborator, Dr 
Piccirillo who is a leader in the field of Regulatory T-cells, and has collaborated with our group 
previously, will co-supervise the analysis of these antibodies (5h /W).74,75,82-84 Dr Julia Upton (Sick 
Kid’s Hospital, Toronto, 10h/w) is an external applicant and will recruit patients from Sick kids 
Hospital. Mr Duncan Lejtenyi who has been an integral part of our team in numerous studies, will 
serve as research coordinator and data entry. We will continue and work with our medical nurse Mrs 
Liane Beaudette who has been part of the OIT team over the last 5 years and gained tremendous skills 
and experience in managing individuals with OIT. In addition, as in previous studies our team will 
include an MSc student in experimental medicine who will base their thesis on the results on this 
analysis. The students will dedicate at least 8 hours a week to help supervise data collection, data entry 
and analysis.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
11. References 
 
1. 
Soller L, Ben-Shoshan M, Harrington DW, et al. Prevalence and predictors of food allergy in Canada: a 
focus on vulnerable populations. JAllergy ClinImmunolPract. 2015;3(1):42-49. 

 
15 
IMPROVES Protocol Version 1 
November 22, 2021 
2. 
Hochstadter E, Clarke A, De SS, 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. 
JAllergy ClinImmunol. 2016. 
3. 
Gabrielli S, Clarke A, Morris J, et al. Evaluation of Prehospital Management in a Canadian Emergency 
Department Anaphylaxis Cohort. J Allergy Clin Immunol Pract. 2019;7(7):2232-2238 e2233. 
4. 
Clarke AE, Elliott SJ, St Pierre Y, Soller L, La Vieille S, Ben-Shoshan M. Comparing food allergy prevalence 
in vulnerable and nonvulnerable Canadians. J Allergy Clin Immunol Pract. 2020. 
5. 
Ben Shoshan M, Clarke AE. Anaphylaxis: past, present and future. Allergy. 2010. 
6. 
Ben-Shoshan M, Turnbull E, Clarke A. Food allergy: temporal trends and determinants. CurrAllergy 
Asthma Rep. 2012;12(4):346-372. 
7. 
Sicherer SH, Munoz-Furlong A, Godbold JH, Sampson HA. US prevalence of self-reported peanut, tree 
nut, and sesame allergy: 11-year follow-up. JAllergy ClinImmunol. 2010;125(6):1322-1326. 
8. 
Soller L, Ben-Shoshan M, Harrington DW, et al. Adjusting for nonresponse bias corrects overestimates 
of food allergy prevalence. JAllergy ClinImmunolPract. 2015;3(2):291-293. 
9. 
Miles BT, Gabrielli S, Clarke A, Eisman H, Shand G, Ben-Shoshan M. Rates of anaphylaxis for the most 
common food allergies. J Allergy Clin Immunol Pract. 2020. 
10. 
Warren CM, Jiang J, Gupta RS. Epidemiology and Burden of Food Allergy. Curr Allergy Asthma Rep. 
2020;20(2):6. 
11. 
Protudjer JL, Jansson SA, Heibert Arnlind M, et al. Household costs associated with objectively 
diagnosed allergy to staple foods in children and adolescents. J Allergy Clin Immunol Pract. 
2015;3(1):68-75. 
12. 
Bilaver LA, Chadha AS, Doshi P, O'Dwyer L, Gupta RS. Economic burden of food allergy: A systematic 
review. Ann Allergy Asthma Immunol. 2019;122(4):373-380 e371. 
13. 
Lee A, Enarson P, Clarke A, et al. Anaphylaxis across two Canadian pediatric centers: Evaluating 
management disparities. journal of asthma and allergy. 2016. 
14. 
Blom WM, Michelsen-Huisman AD, van Os-Medendorp H, et al. Accidental food allergy reactions: 
Products and undeclared ingredients. J Allergy Clin Immunol. 2018;142(3):865-875. 
15. 
Boyano-Martinez T, Garcia-Ara C, Pedrosa M, Diaz-Pena JM, Quirce S. Accidental allergic reactions in 
children allergic to cow's milk proteins. JAllergy ClinImmunol. 2009;123(4):883-888. 
16. 
O'Keefe AW, De SS, Mill J, Mill C, Dery A, Ben-Shoshan M. Diagnosis and management of food allergies: 
new and emerging options: a systematic review. JAsthma Allergy. 2014;7:141-164. 
17. 
Toomer OT. Nutritional chemistry of the peanut (Arachis hypogaea). Crit Rev Food Sci Nutr. 
2018;58(17):3042-3053. 
18. 
Tunick MH, Van Hekken DL. Dairy Products and Health: Recent Insights. J Agric Food Chem. 
2015;63(43):9381-9388. 
19. 
Robbins KA, Wood RA, Keet CA. Persistent cow's milk allergy is associated with decreased childhood 
growth: A longitudinal study. J Allergy Clin Immunol. 2020;145(2):713-716 e714. 
20. 
Flammarion S, Santos C, Guimber D, et al. Diet and nutritional status of children with food allergies. 
Pediatr Allergy Immunol. 2011;22(2):161-165. 
21. 
Yeung JP, Kloda LA, McDevitt J, Ben-Shoshan M, Alizadehfar R. Oral immunotherapy for milk allergy. 
CochraneDatabaseSystRev. 2012;11:CD009542. 
22. 
Segal L, Ben-Shoshan M, Alizadehfar R, et al. Initial and accidental reactions are managed inadequately 
in children with sesame allergy. J Allergy Clin Immunol Pract. 2017;5(2):482-485. 
23. 
O'Keefe AW, St PY, Mill J, et al. Recurrence rates of anaphylaxis in children. Journal of Allergy and 
Clinical Immunology. 2014. 
24. 
Nguyen-Luu NU, Ben-Shoshan M, Alizadehfar R, et al. Inadvertent exposures in children with peanut 
allergy. PediatrAllergy Immunol. 2011. 
25. 
De Schryver S, Clarke A, La Vieille S, et al. Food-induced anaphylaxis to a known food allergen in 
children often occurs despite adult supervision. Pediatr Allergy Immunol. 2017;28(7):715-717. 

 
16 
IMPROVES Protocol Version 1 
November 22, 2021 
26. 
Ben-Shoshan M, Sheth S, Harrington D, et al. Effect of precautionary statements on the purchasing 
practices of Canadians directly and indirectly affected by food allergies. J Allergy ClinImmunol. 
2012;129(5):1401-1404. 
27. 
Primeau MN, Kagan R, Joseph L, et al. The psychological burden of peanut allergy as perceived by 
adults with peanut allergy and the parents of peanut-allergic children. ClinExpAllergy. 2000;30(8):1135-
1143. 
28. 
Wasserman RL, Factor J, Windom HH, et al. An Approach to the Office-Based Practice of Food Oral 
Immunotherapy. J Allergy Clin Immunol Pract. 2021. 
29. 
De Schryver S, Mazer B, Clarke AE, et al. Adverse Events in Oral Immunotherapy for the Desensitization 
of Cow's Milk Allergy in Children: A Randomized Controlled Trial. J Allergy Clin Immunol Pract. 
2019;7(6):1912-1919. 
30. 
Begin P, Chan ES, Kim H, et al. CSACI guidelines for the ethical, evidence-based and patient-oriented 
clinical practice of oral immunotherapy in IgE-mediated food allergy. Allergy Asthma Clin Immunol. 
2020;16:20. 
31. 
Investigators PGoC, Vickery BP, Vereda A, et al. AR101 Oral Immunotherapy for Peanut Allergy. N Engl J 
Med. 2018;379(21):1991-2001. 
32. 
Pajno GB, Fernandez-Rivas M, Arasi S, et al. EAACI Guidelines on allergen immunotherapy: IgE-
mediated food allergy. Allergy. 2018;73(4):799-815. 
33. 
Chu DK, Wood RA, French S, et al. Oral immunotherapy for peanut allergy (PACE): a systematic review 
and meta-analysis of efficacy and safety. Lancet. 2019;393(10187):2222-2232. 
34. 
Vickery BP, Berglund JP, Burk CM, et al. Early oral immunotherapy in peanut-allergic preschool children 
is safe and highly effective. J Allergy Clin Immunol. 2017;139(1):173-181 e178. 
35. 
Ballmer-Weber BK, Fernandez-Rivas M, Beyer K, et al. How much is too much? Threshold dose 
distributions for 5 food allergens. J Allergy Clin Immunol. 2015;135(4):964-971. 
36. 
Bianchi DM, Adriano D, Astegiano S, Gallina S, Caramelli M, Decastelli L. Egg and Milk Proteins as 
Hidden Allergens in Food: 5-Year (2010 to 2014) Results of Food Allergen Monitoring in Piedmont, Italy. 
J Food Prot. 2016;79(9):1583-1587. 
37. 
Vadas P, Perelman B. Presence of undeclared peanut protein in chocolate bars imported from Europe. 
JFood Prot. 2003;66(10):1932-1934. 
38. 
Blumchen K, Trendelenburg V, Ahrens F, et al. Efficacy, Safety, and Quality of Life in a Multicenter, 
Randomized, Placebo-Controlled Trial of Low-Dose Peanut Oral Immunotherapy in Children with 
Peanut Allergy. J Allergy Clin Immunol Pract. 2019;7(2):479-491 e410. 
39. 
Yanagida N, Sato S, Asaumi T, Okada Y, Ogura K, Ebisawa M. A Single-Center, Case-Control Study of 
Low-Dose-Induction Oral Immunotherapy with Cow's Milk. Int Arch Allergy Immunol. 2015;168(2):131-
137. 
40. 
Yanagida N, Sato S, Asaumi T, Nagakura K, Ogura K, Ebisawa M. Safety and Efficacy of Low-Dose Oral 
Immunotherapy for Hen's Egg Allergy in Children. Int Arch Allergy Immunol. 2016;171(3-4):265-268. 
41. 
Itoh-Nagato N, Inoue Y, Nagao M, et al. Desensitization to a whole egg by rush oral immunotherapy 
improves the quality of life of guardians: A multicenter, randomized, parallel-group, delayed-start 
design study. Allergol Int. 2018;67(2):209-216. 
42. 
Buchanan AD, Green TD, Jones SM, et al. Egg oral immunotherapy in nonanaphylactic children with egg 
allergy. JAllergy ClinImmunol. 2007;119(1):199-205. 
43. 
Takaoka Y, Yajima Y, Ito YM, et al. Single-Center Noninferiority Randomized Trial on the Efficacy and 
Safety of Low- and High-Dose Rush Oral Milk Immunotherapy for Severe Milk Allergy. Int Arch Allergy 
Immunol. 2020;181(9):699-705. 
44. 
Leonard SA, Sampson HA, Sicherer SH, et al. Dietary baked egg accelerates resolution of egg allergy in 
children. JAllergy ClinImmunol. 2012;130(2):473-480. 
45. 
Huang F, Nowak-Wegrzyn A. Extensively heated milk and egg as oral immunotherapy. Curr Opin Allergy 
Clin Immunol. 2012;12(3):283-292. 

 
17 
IMPROVES Protocol Version 1 
November 22, 2021 
46. 
Zhang T, Shi Y, Zhao Y, Tang G, Niu B, Chen Q. Boiling and roasting treatment affecting the peanut 
allergenicity. Ann Transl Med. 2018;6(18):357. 
47. 
Gruzelle V, Juchet A, Martin-Blondel A, Michelet M, Chabbert-Broue A, Didier A. Benefits of baked milk 
oral immunotherapy in French children with cow's milk allergy. Pediatr Allergy Immunol. 
2020;31(4):364-370. 
48. 
Ball HB, Luyt D. Home-based cow's milk reintroduction using a milk ladder in children less than 3 years 
old with IgE-mediated cow's milk allergy. Clin Exp Allergy. 2019;49(6):911-920. 
49. 
Hindley JP, Filep S, Block DS, King EM, Chapman MD. Dose of allergens in a peanut snack (Bamba) 
associated with prevention of peanut allergy. J Allergy Clin Immunol. 2018;141(2):780-782. 
50. 
Hourihane JO, Kilburn SA, Dean P, Warner JO. Clinical characteristics of peanut allergy. ClinExpAllergy. 
1997;27(6):634-639. 
51. 
Peters RL, Allen KJ, Dharmage SC, et al. Skin prick test responses and allergen-specific IgE levels as 
predictors of peanut, egg, and sesame allergy in infants. JAllergy ClinImmunol. 2013;132(4):874-880. 
52. 
Yanagida N, Okada Y, Sato S, Ebisawa M. New approach for food allergy management using low-dose 
oral food challenges and low-dose oral immunotherapies. Allergol Int. 2016;65(2):135-140. 
53. 
Perry TT, Matsui EC, Conover-Walker MK, Wood RA. Risk of oral food challenges. JAllergy ClinImmunol. 
2004;114(5):1164-1168. 
54. 
Sampson HA, Munoz-Furlong A, Campbell RL, et al. Second symposium on the definition and 
management of anaphylaxis: summary report--second National Institute of Allergy and Infectious 
Disease/Food Allergy and Anaphylaxis Network symposium. AnnEmergMed. 2006;47(4):373-380. 
55. 
Muraro A, Roberts G, Clark A, et al. The management of anaphylaxis in childhood: position paper of the 
European academy of allergology and clinical immunology. Allergy. 2007;62(8):857-871. 
56. 
Nachshon L, Goldberg MR, Katz Y, Levy MB, Elizur A. Long-term outcome of peanut oral 
immunotherapy-Real-life experience. Pediatr Allergy Immunol. 2018;29(5):519-526. 
57. 
Martin-Munoz MF, Alonso Lebrero E, Zapatero L, et al. Egg OIT in clinical practice (SEICAP II): 
Maintenance patterns and desensitization state after normalizing the diet. Pediatr Allergy Immunol. 
2019;30(2):214-224. 
58. 
Nguyen-Luu NU, Ben-Shoshan M, Alizadehfar R, et al. Inadvertent exposures in children with peanut 
allergy. Pediatr Allergy Immunol. 2012;23(2):133-139. 
59. 
Wuthrich B. Lethal or life-threatening allergic reactions to food. JInvestigAllergolClinImmunol. 
2000;10(2):59-65. 
60. 
Vogel NM, Katz HT, Lopez R, Lang DM. Food allergy is associated with potentially fatal childhood 
asthma. JAsthma. 2008;45(10):862-866. 
61. 
Lavine E. Blood testing for sensitivity, allergy or intolerance to food. CMAJ. 2012;184(6):666-668. 
62. 
Wang J, Lin J, Bardina L, et al. Correlation of IgE/IgG4 milk epitopes and affinity of milk-specific IgE 
antibodies with different phenotypes of clinical milk allergy. J Allergy Clin Immunol. 2010;125(3):695-
702, 702 e691-702 e696. 
63. 
Kulis M, Yue X, Guo R, et al. High- and low-dose oral immunotherapy similarly suppress pro-allergic 
cytokines and basophil activation in young children. Clin Exp Allergy. 2019;49(2):180-189. 
64. 
Hajoui O, Janani R, Tulic M, et al. Synthesis of IL-13 by human B lymphocytes: regulation and role in IgE 
production. J Allergy Clin Immunol. 2004;114(3):657-663. 
65. 
Rieger A, Bar-Or A. B-cell-derived interleukin-10 in autoimmune disease: regulating the regulators. Nat 
Rev Immunol. 2008;8(6):486-487. 
66. 
Adel-Patient K, Wavrin S, Bernard H, Meziti N, Ah-Leung S, Wal JM. Oral tolerance and Treg cells are 
induced in BALB/c mice after gavage with bovine beta-lactoglobulin. Allergy. 2011;66(10):1312-1321. 
67. 
Schouten B, van Esch BC, Hofman GA, et al. Oligosaccharide-induced whey-specific CD25(+) regulatory 
T-cells are involved in the suppression of cow milk allergy in mice. J Nutr. 2010;140(4):835-841. 
68. 
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):1679-1688. 

 
18 
IMPROVES Protocol Version 1 
November 22, 2021 
69. 
Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn 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 e47. 
70. 
Kaufman GN, Massoud AH, Audusseau S, et al. Intravenous Immunoglobulin attenuates airway 
hyperresponsiveness in a murine model of allergic asthma. Clinical and experimental allergy : journal of 
the British Society for Allergy and Clinical Immunology. 2011;41:718-728. 
71. 
Massoud AH, Guay J, Shalaby KH, et al. Intravenous Immunoglobulin attenuates airway inflammation 
disease via induction of Foxp3+ regulatory T-cells. The Journal of allergy and clinical immunology. 
2012;in press. 
72. 
Syed A, Garcia MA, Lyu SC, et al. Peanut oral immunotherapy results in increased antigen-induced 
regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3). J Allergy Clin 
Immunol. 2014;133(2):500-510. 
73. 
Fuentes-Aparicio V, Alonso-Lebrero E, Zapatero L, et al. Induction of Treg cells after oral 
immunotherapy in hen's egg-allergic children. Pediatr Allergy Immunol. 2014;25(1):103-106. 
74. 
Dhuban KB, d'Hennezel E, Ben-Shoshan M, et al. Altered T helper 17 responses in children with food 
allergy. Int Arch Allergy Immunol. 2013;162(4):318-322. 
75. 
d'Hennezel E, Ben Shoshan M, Ochs HD, et al. FOXP3 forkhead domain mutation and regulatory T cells 
in the IPEX syndrome. NEnglJMed. 2009;361(17):1710-1713. 
76. 
Facebook live with Dr. Ben-Shoshan. 2016; https://www.facebook.com/events/facebook-
live/facebook-live-with-dr-ben-shoshan/104054010052237/. 
77. 
Ben-Shoshan M. Creating a Better Future Webinar Series: Understanding When to Use Epinephrine. 
2020. 
78. 
Skypala IJ, McKenzie R. Nutritional Issues in Food Allergy. Clin Rev Allergy Immunol. 2019;57(2):166-
178. 
79. 
Chinthrajah RS, Cao S, Dunham T, et al. Oral immunotherapy for peanut allergy: The pro argument. 
World Allergy Organ J. 2020;13(8):100455. 
80. 
Loh W, Tang M. Adjuvant Therapies in Food Immunotherapy. Immunol Allergy Clin North Am. 
2018;38(1):89-101. 
81. 
Azzano P, Paquin M, Langlois A, et al. Determinants of omalizumab dose-related efficacy in oral 
immunotherapy: Evidence from a cohort of 181 patients. J Allergy Clin Immunol. 2020. 
82. 
Massoud AH, Yona M, Xue D, et al. Dendritic cell immunoreceptor: a novel receptor for intravenous 
immunoglobulin mediates induction of regulatory T cells. J Allergy Clin Immunol. 2014;133(3):853-863 
e855. 
83. 
Massoud AH, Kaufman GN, Xue D, et al. Peripherally Generated Foxp3(+) Regulatory T Cells Mediate 
the Immunomodulatory Effects of IVIg in Allergic Airways Disease. J Immunol. 2017;198(7):2760-2771. 
84. 
Bin Dhuban K, Piccirillo CA. The immunological and genetic basis of immune dysregulation, 
polyendocrinopathy, enteropathy, X-linked syndrome. Curr Opin Allergy Clin Immunol. 2015;15(6):525-
532. 
 
 
APPENDICES 
Appendix A : Disease burden and gaps. 
Table 1. Primary and secondary prevention measures for food allergiesa 
 

 
19 
IMPROVES Protocol Version 1 
November 22, 2021 
Trigg
er 
Primary 
prevention 
Route of 
desensitization 
Reference number 
Secondary preventio
Foods 
 
PO, SL 
(14;68-70) 
Avoidance of allerge
education of allergic ind
their care-givers on imp
avoidance, improved lab
prepackaged foods for a
wearing of Medic-Alert 
specific food allergy 
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 
Season 
Vassall
o MF  
Case control 
Children younger than 5 years born in fall or winter had a 53%
higher odds of food allergy compared with controls. 
Drugs 
Palli-
Scholl  
Case control 
The relative risk to develop food-specific IgE after anti-acid 
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 effects on 
the prevention or treatment of allergy .  

 
20 
IMPROVES Protocol Version 1 
November 22, 2021 
Food 
consumption 
(quantity 
and timing) 
Poole 
JA  
Cohort 
After adjusting for breastfeeding duration, introduction of rice 
cereal, family history of allergy, and history of food allergy before
6 months of age, age at initial exposure to cereal grains continued 
to be strongly associated with wheat allergy ( 7 months: adjusted 
OR: 3.8; 95% CI, 1.18,12.28) 
Du Toit  
Case control 
After adjustment for atopy, other food allergies, age, and sex, 
the RR for peanut allergy in the UK vs Israel is  5.8 (95% CI, 
2.8,11.8), and  largest and most significant difference in aning 
between the UK and Israel was observed in the age of introduction
of peanut (P < .0001). By 9 months of age, 69% of Israelis were 
eating peanut compared with only 10% of UK infants. 
Katz   
Cohort 
The OR was 19.3 (95% CI, 6.0,62.1) for development of IgE 
mediated CMA among infants with exposure to cow  protein at th
age of 15 days or more (P < .001) vs those introduced to cow  
protein before 15 days.  
Joseph   
Cohort 
Early feeding reduced the risk of peanut sensitization among 
children with a parental history [adjusted OR, 0.2 (95% CI, 
0.1,0.7); P = .007]. The relationship also became significant 
forwhen a cutoff for IgE of ≥0.70 IU/mL was used [adjustedOR, 
0.5 (95% CI, 0.3,0.9)]. 
Koplin  
Case control 
Introduction of cooked at age 4 to 6 months, vs later exposure  
reduced the risk of allergy [OR, 0.2 (95% CI, 0.06-0.71)]. 
Des 
Case control 
The reported consumption of peanuts during pregnancy and 

 
21 
IMPROVES Protocol Version 1 
November 22, 2021 
Roches  
breastfeeding was higher in the case group (those who developed 
peanut allergy and associated with an increased risk of peanut 
allergy in offspring [OR, 4.22 (95%CI, 1.57,11.30) and OR, 2.28 
(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 consumption 
during pregnancy {OR, 2.9( 95% CI, 1.7,4.9)] to be associated 
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 end 
adducts and higher IgE binding   vs immature peanuts. 
Yadzir  
Laboratory 
analysis 
Extracts from raw shrimp bound higher IgE than extracts from 
boiled shrimp, but the purified boiled tropomysoin (the main 
shrimp allergen) demonstrates higher IgE binding vs raw shrimp. 
Samson  
Laboratory  
analysis 
Thermal processing can lead to the formation of new antigenic 
structures. 
Vitamin 
D 
Milner  
Cohort  
Early vitamin D use (within the first 6 months of life) was 
associated with a higher risk for food allergies in the exclusively 
formula-fed population [OR,1.63( 95% CI,1.21,2.20)]. Vitamin 
use at 3 years of age was associated with increased risk for food 
allergies but not asthma in both breastfed [OR,1.62(95% 
CI,1.19,2.21) ]and exclusively formula-fed infants [OR, 1.39( 95%

 
22 
IMPROVES Protocol Version 1 
November 22, 2021 
CI,1.03,1.88)]. 
 
Cramag
o  
Ecologic  study 
Strong north-south gradient for the prescription of EpiPens in 
the United States, with the highest rates found in New England. 
[adjusted β for New England vs the rest of the US, 4.07 (95%CI, 
2.77,5.36)] 
Mulins 
et al  
Ecologic  study 
Using multivariate analysis , EpiPen prescription rates the were
higher in southern latitudes (less sunlight) compared with northern
regions [β, −19.22( 95% CI, −26.71 , −11.73)]. 
Mulins 
et al 
Ecologic  study 
Southern latitudes were associated with higher hypoallergenic 
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 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Appendix B. Procedures 
 
I. 
Food Challenges 
 
1. 
Screening Double-Blind, Placebo Controlled Oral Food Challenges (DBPCFC) 
a) Peanut  

 
23 
IMPROVES Protocol Version 1 
November 22, 2021 
Double-blind, placebo-controlled food challenge (BPCFC) up to 100 mg (444 mg cumulative) done 
at screening  
 
Dose (n:0) 
Dose 
(mg peanut protein) 
 
Cumulative 
(mg peanut protein) 
1 
1  
1  
2 
 3  
4  
3 
 10  
 14  
4 
 30  
 44  
5 
100  
144  
6 
300  
444  
For placebo day, a substance that the participant is known to tolerate 
 
b) Milk 
Double-blind, placebo-controlled food challenge (BPCFC) up to 154.4 ml done at screening  
 
Dose (n:0) 
Dose (l milk 
Cumulative dose in ml 
1 
0.1 
0.1  
2 
0.3 
0.4 
3 
1 
1.4 
4 
3 
4.4 
5 
5 
9.4 
6 
10 
19.4 
7 
30 
49.4 
8 
45 
94.4 
9 
60 
154.4 
 
c) Egg  
Double-blind, placebo-controlled food challenge (BPCFC) up to 845.1 ml done at screening 
 
Dose (n:0) 
Egg protein/placebo (mg) 
Cumulative egg dose (mg) 

 
24 
IMPROVES Protocol Version 1 
November 22, 2021 
1 
0.2 mg placebo 
0 
2 
0.2 mg egg 
0.2 
3 
0.4 placebo 
0.2 
4  
0.4 mg egg 
0.6 
5 
1.5 placebo 
0.6 
6  
1.5 egg 
2.1 
7 
6 egg 
8.1 
8 
12 egg 
20.1 
9 
25 egg 
45.1 
10 
50 egg 
95.1 
11 
100 egg 
195.1 
12 
150 egg 
345.1 
13 
200 egg 
545.1 
14 
300 egg 
845.1 
 
 
 
 
2. Exit Challenges 
 
a.  Peanut exit challenge 
Dose  
 
Dose  
(mg peanut protein) 
Cumulative  
(peanut protein)  
1 
3  
3  
2 
10  
13  
3 
30  
43  
4 
100  
143  
5 
300  
443  
6 
600  
1043  
7 
1000  
2043 
 b.Milk exit challenge 
 
Dose 
Dose ml milk 
Cumulative dose ml milk 
1 
200 ml 
200 ml 
2 
100 ml 
300 ml 

 
25 
IMPROVES Protocol Version 1 
November 22, 2021 
 
 
c. Egg exit challenge 
Dose  
Egg protein mg dose  
Cumulative dose  
1 
100 
100  
2 
300 
400  
3 
600 
1000  
4 
1000 
2000  
5 
2000 
4000  
6 
2000 
6000  
 
.  
Scale for Grading Reaction Severity( non anaphylactic reactions ) 
 
Score  
Symptom 
Action 
Mild 
Pruritus, 
Urticaria, 
Flushing, 
Rhinoconjunctivitis 
Observe 
May give Antihistamine (e.g. 
Rupatidine 
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. Reactin 
or Rupatidine  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. Reactin 
or Rupatidine  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, 

 
26 
IMPROVES Protocol Version 1 
November 22, 2021 
instructions will be given from the 
team regarding use of a second 
dose of epinephrine.  
 
 
 
 
Anaphylaxis is defined as involvement of 2 organ systems and/or hypotension in response to a 
potential allergen and anaphylaxis.81 
 
 
 
Anaphylaxis  severity is classified according to a modified grading system published by Muraro. 82 
a. 
Mild anaphylaxis: characterized by the presence of skin and subcutaneous tissues symptoms 
(urticaria, erythema, and angioedema) as well as oral pruritus, nausea (i.e. gastrointestinal involvement 
and cutaneous) or nasal congestion, sneezing, rhinorrhea or throat tightness (i.e. respiratory 
involvement).  
 
b. 
Moderate anaphylaxis: characterized by the presence of any of the previous symptoms as well 
as crampy abdominal pain, diarrhea or recurrent vomiting, hoarseness, “barky” cough, difficulty 
swallowing, dyspnea, moderate wheezing, and or “light headedness.”  
 
c. 
Severe anaphylaxis: characterized by cyanosis, hypoxia (saturation<92%), respiratory arrest, 
hypotension, dysrhythmia, confusion or loss of consciousness. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
II. 
Rush Desensitization 
 
Day 1 and 2 common for group A, B, C  
• 
Verify no wheezing, flare of atopic disease, or other intercurrent illness 
• 
Update history and con meds 

 
27 
IMPROVES Protocol Version 1 
November 22, 2021 
• 
Vital signs and PE 
• 
PEFR 
• 
Dose escalation at 20 minute intervals 
• 
Post-dose vital signs 
• 
Vital signs q30min x3 post last dose 
• 
Monitoring for AEs (see  
 
 
1 Peanut /tree nut: groups A/B/C 
  
Day 1 
Day 1 
Day 2  
Day 2  
Dose (mg peanut 
protein) 
Dose (mg crushed 
peanut) 
Dose(mg peanut 
protein) 
Dose (mg crushed 
peanut) 
0.5 
2.0  
3 
12  
1.0 
4.0 
 
 
1.5 
6.0  
 
 
2.5  
10.0  
 
 
3.0 
12  
 
 
3.5  
14  
 
 
4.0  
16 
 
 
4.5 
18  
 
 
5.0 
20  
 
 
 
2 Milk: Groups A/B/C 
 
Time Point 
Milk Dilution/dose  
Volume 
(ml) 
Day 1 
1/1000 
1 
 
1/1000 
2 
 
1/1000 
4 
 
1/1000 
8 
 
1/100 
1.6 
Day 2 
1/100 
1.6 
 
1/100 
3.2 
 
1/100 
6.4 
 
1/100 
12 
 
1/10 to continiue 
2.5 
 
 
 
 
 
3 Egg: No Rush Desensitization will be done for egg desensitization – for participants in Groups 
A and B, the highest tolerated dose established during the Screening Challenge will be used to 
determine the first dose for the buildup phase.  
 

 
28 
IMPROVES Protocol Version 1 
November 22, 2021 
  
III. 
Escalation Phase 
 
1. Peanut Group A 
Time Point 
Dose peanut protein (mg) 
Dose crushed peanut (mg) 
Week 2 
3 
12 
Week 4 
6 
24 
Week 6 
12 
48 
Week 8 
20 
80 
Week 10 
40 
160 
Week 12  
80 
320 
Week 14 
120 
480 
Week 16 
160 
640 
Week 18 
200 
800 
Week 20 
240 
960 
Week 22 
300 
1200 
 
Peanut/ tree nut: Build up phase for-group B 
 
Time Point 
Dose peanut protein (mg) 
Dose crushed peanut (mg) 
Week 2 
3 
12 
Week 4 
6 
24 
Week 6 
12 
48 
Week 8 
20 
80 
Week 10 
30 
120 
Week 12  
30 
120 
Week 14 
30 
120 
Week 16 
30 
120 
Week 18 
30 
120 
Peanut/ tree nut: Build up phase for processed -group C 
 
Time Point 
Dose peanut protein (mg) 
Dose crushed peanut (mg) 
Week 2 
10mg * 
1/8 puff 

 
29 
IMPROVES Protocol Version 1 
November 22, 2021 
Week 4 
20mg * 
1/4 
Week 6 
40mg* 
1/2 
Week 8 
80mg* 
1 
Week 10 
160mg* 
2puffs 
Week 12  
320mg* 
4 puffs 
Week 14 
80** 
320 
Week 16 
120** 
480 
Week 18 
160** 
640 
Week 20 
200** 
800 
Week 22 
240** 
960 
Week 24 
300** 
1200 
*Processed form “ Bamba” puffs for peanut 
** crushed form of the peanut/ tree nut 
 
 
Milk :Build up phase -group A 
1ml= 35mg milk protein 
Desensitization protocol 
Time Point 
Dose  
Volume (ml) Milk protein (mg) 
Week 2 
1/10 
5 
3.5 
Week 4 
Undiluted 
1 
35 
Week 6 
Undiluted 
2 
70 
Week 8 
Undiluted 
4 
140 
Week 10 
Undiluted 
6 
210 
Week 12  
Undiluted 
8 
280 
Week 14 
Undiluted 
10 
350 
Week 16 
Undiluted 
12 
420 
Week 18 
Undiluted 
15 
525 
Week 20 
Undiluted 
20 
700 
Week 22 
Undiluted 
25 
875 
Week 24 
Undiluted 
30 
1050 
Week 26 
Undiluted 
40 
1400 

 
30 
IMPROVES Protocol Version 1 
November 22, 2021 
Week 28 
Undiluted 
50 
1750 
Week 30 
Undiluted 
50 
1750 
Week 32 
Undiluted 
50 
1750 
Week 34 
Undiluted 
50 
1750 
Week 36 
Undiluted 
150 
5250  
Week 38 
Undiluted 
200 
7000 
 
 
 
 
Milk: Build up phase -group B 
Desensitization protocol 
Time Point 
Dose  
Volume (ml) Milk protein (mg) 
Week 2 
1/10 
5 
3.5 
Week 4 
Undiluted 
1 
35 
Week 6 
Undiluted 
2 
70 
Week 8 
Undiluted 
4 
140 
Week 10 
Undiluted 
6 
210 
Week 12  
Undiluted 
8 
280 
Week 14 
Undiluted 
10 
350 
Week 16 
Undiluted 
12 
420 
Week 18 
Undiluted 
15 
525 
Week 20 
Undiluted 
20 
700 
Week 22 
Undiluted 
25 
875 
Week 24 
Undiluted 
30 
1050 
Week 26 
Undiluted 
40 
1400 
Week 28 
Undiluted 
50 
1750 
 
 
 
 
Milk : Build up phase for processed -group C 
 
Time Point 
Dose  
Volume /form 
Milk protein (mg) 

 
31 
IMPROVES Protocol Version 1 
November 22, 2021 
Week 2 
1/16 muffin 
muffin 
50mg 
Week 4 
1/8 muffin 
muffin 
100mg 
Week 6 
1/4 muffin 
muffin 
200mg 
Week 8 
1/2 muffin  
muffin 
400mg 
Week 10 
1 muffin  
muffin 
800mg 
Week 12 
Undiluted 
1 
35mg 
Week 14 
Undiluted 
2 
70mg 
Week 16 
Undiluted 
4 
140mg 
Week 18  
Undiluted 
6 
210mg 
Week 20 
Undiluted 
8 
280mg 
Week 22 
Undiluted 
10 
350mg 
Week 24 
Undiluted 
12 
420mg 
Week 26 
Undiluted 
15 
525mg 
Week 28 
Undiluted 
20 
700mg 
Week 30 
Undiluted 
25 
875mg 
Week 32 
Undiluted 
30 
1050mg 
Week 34 
Undiluted 
40 
1400mg 
Week 36 
Undiluted 
50 
1750mg 
Week 38 
Undiluted 
50 
1750mg 
Week 40 
Undiluted 
50 
1750mg 
Week 42 
Undiluted 
50 
1750mg 
Week 44 
Undiluted 
150 
1750mg  
Week 46 
Undiluted 
200 
1750mg 
 
1ml= 35mg milk protein 
 
 
 
 
Egg :Build up phase -group A 
 
Note: the highest tolerated dose established during the Screening Challenge will be used to 
determine the first dose for the buildup phase  

 
32 
IMPROVES Protocol Version 1 
November 22, 2021 
 
 
 
 
Time Point 
Dose egg powder (mg) 
Dose egg protein (mg) 
Week 2 
0.2 
0.2 
Week 4 
0.4 
0.4 
Week 6 
1.5 
1.5 
Week 8 
6 
6 
Week 10 
12 
12 
Week 12 
25 
25 
Week 14 
50 
50 
Week 16 
100 
100 
Week 18 
150 
150 
Week 20 
200 
200 
Week 22 
300 
300 
Week 24 
600 
600 
Week 26 
1200 
1200 
 
Egg :Build up phase -group B 
 
Note: the highest tolerated dose established during the Screening Challenge will be used to 
determine the first dose for the buildup phase  
 
 
Time Point 
Dose egg powder (mg) 
Dose egg protein (mg) 
Week 2 
0.2 
0.2 
Week 4 
0.4 
0.4 
Week 6 
1.5 
1.5 
Week 8 
6 
6 
Week 10 
12 
12 
Week 12 
25 
25 
Week 14 
50 
50 

 
33 
IMPROVES Protocol Version 1 
November 22, 2021 
Week 16 
100 
100 
Week 18 
150 
150 
Week 20 
200 
200 
Week 22 
300 
300 
 
Egg :Build up phase -group C 
 
All participants randomized to this group will begin with 1/8 muffin, irrespective of amount of 
egg powder tolerated at Screening Challenge. 
 
Time Point 
Dose muffin/egg powder (mg) 
Dose egg protein (mg) 
Week 2 
1/8 muffin  
75 
Week 4 
¼ muffin 
150 
Week 6 
½ muffin  
300 
Week 8 
1 muffin  
600  
Week 10 
50 (mg powder) 
50 
Week 12 
75 (mg powder) 
75 
Week 14 
100 (mg powder) 
100 
Week 16 
150 (mg powder) 
150 
Week 18 
200 (mg powder) 
200 
Week 20 
250 (mg powder) 
250 
Week 22 
300 (mg powder) 
300 
Week 24 
600 (mg powder) 
600 
Week 26 
1200 (mg powder) 
1200 
 
 
 
 
 
 
 
 
 
 
 
 

 
34 
IMPROVES Protocol Version 1 
November 22, 2021 
Appendix C. Recipes 
1. Milk baked  
Muffin Recipe for Baked Milk  
For 10 muffins (each muffin is 800 mg milk protein 1/16 muffin = 50mg )  
• Follow this recipe carefully and use all the batter. 
• Following baking time and temperature carefully. 
• Please bring at least four (4) regular size cupcakes with you. 
• Do not use jumbo or mini muffin/cupcake pans. 
Ingredients: 
200 of 2% Natrel Lactose-Free milk (~8000mg milk protein) 
1 tablespoon of vegetable oil 
1 large egg* 
*If egg allergic: 1⁄4 cup of applesauce 
1 teaspoon vanilla extract 
1 1⁄4 cups white flour 
1⁄2 cup sugar 
2 teaspoon baking powder 
1⁄4 teaspoon salt 
 
Directions: 
1. Preheat oven to 350 degrees F 
2. Line muffin/cupcake pan(s) with standard size paper liners 
3. Mix liquid ingredients: milk, vegetable oil, vanilla extract, egg or applesauce 
4. In separate bowl, mix the dry ingredients: flour, sugar, salt, and baking powder 
5. Add the liquid ingredients to the dry ingredients and mix thoroughly. 
6. Pour mixture into pan dividing equally, scraping bowl so to use ALL the batter. 
Bake for 30-35 minutes (not less). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2. Egg baked  
Muffin Recipe for Baked egg - 10 muffins ( each with 600mg protein of egg – will start with 1/8 
=75mg) 

 
35 
IMPROVES Protocol Version 1 
November 22, 2021 
 
• Follow this recipe carefully and use all the batter. 
• Following baking time and temperature carefully. 
• Please bring at least four (4) regular size cupcakes with you. 
• Do not use jumbo or mini muffin/cupcake pans. 
 
Ingredients: 
1 cup of 2% Natrel Lactose Free milk* 
‘if allergic to milk can replace milk rice milk/ soy milk/ coconut milk 
1 tablespoon of vegetable oil 
1 large egg ( 6000mg protein)  
1 teaspoon vanilla extract 
1 1⁄4 cups white flour 
1⁄2 cup sugar 
2 teaspoon baking powder 
1⁄4 teaspoon salt 
 
Directions: 
1. Preheat oven to 350 degrees F 
2. Line muffin/cupcake pan(s) with paper liners 
3. Mix liquid ingredients: milk, vegetable oil, vanilla extract, egg or applesauce 
4. In separate bowl, mix the dry ingredients: flour, sugar, salt, and baking powder 
5. Add the liquid ingredients to the dry ingredients and mix thoroughly. 
6. Pour mixture into pan dividing equally, scraping bowl so to use ALL the batter. 
Bake for 30-35 minutes (not less). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
36 
IMPROVES Protocol Version 1 
November 22, 2021