Egg allergy is among the most common food allergies in children (1) with overall estimate of
prevalence ranging from 0.5 – 2% and even up to 8.9% of infants (11-15 months) were found
to be allergic to raw egg in Australia (HealthNuts study) (2). The prevalence of egg allergy
confirmed by food challenge has been estimated at 1.6% at 2.5 years (3). Management of
food allergies is currently based on avoidance of the offending food and prompt recognition
and treatment of allergic reactions, resulting from accidental exposure. However, total
avoidance is practically difficult since egg is used as an ingredient in many food products. It
could lead to nutritional deficiencies, and impact quality of life of the child and the parents
(4). Additionally, accidental exposure to ‘hidden’ egg allergens can impose a risk for the
child.
In recent years, there is increasing clinical and scientific interest in oral
immunotherapy (OIT) to food with the aim to re-establish permanent tolerance, to desensitize
or to increase the threshold level of reaction to the food, in order to reduce the risk of severe
allergic reactions after accidental exposure. Two reviews summarizing the recent advances
made in OIT for egg allergy, suggested efficacy of this approach (5, 6), which was
substantiated by a recent multicentre, randomized clinical trial in the US (7). However, a
major drawback of OIT using standard food products is the risk of severe side effects.
Additionally, OIT classically involves a hospital visit not only at the initial escalation (with
typically 6-8 doses given during day 1), but also every 1 to 2 weeks during the build-up
dosing phase, over 6-12 months (8).
We designed a hydrolysed egg (HydE) product, which was earlier shown to have low
allergenicity (9), but could still induce oral tolerance in mice (10).
Due to the low allergenicity of HydE, after having passed the food challenge to assess
tolerance at the start of the trial, the product was directly given at the maintenance dose from
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the first day, thereby minimizing the number of necessary hospital visits as well as
expectantly decreasing the risk of adverse allergic reactions during therapy. Here, we present
the results of a double blind placebo controlled pilot study in which we assessed the safety of
use of HydE for OIT as well as its efficacy for desensitisation in children allergic to egg.
Methods
Study population
Twenty-nine children recruited from 3 study sites in Europe (Athens, Greece, Davos,
Switzerland and Padua, Italy) were admitted to the study. All fulfilled the inclusion criteria of
being 1 – 5.5 years old, diagnosed with an IgE-mediated egg allergy based on a positive skin
prick test (SPT) to egg white within the last 3 months as well as a positive oral challenge or a
convincing history, defined as an immediate (<1h) reaction following isolated ingestion of
egg and positive sIgE (>0.35 kU/L) for at least one of the following: egg, egg white,
ovalbumin or ovomucoid, within the last 12 months. All ethics committees of the hospitals
approved the study, and an informed consent was obtained from the parents of each child.
The trial was registered at ClinialTrials.gov (registration identifier NCT01526863) and the
study design is shown in Fig. 1.
The randomization at 1:1 HydE:placebo ratio was applied by using the software
TrialSys (developed at Nestlé, Lausanne). Stratification was performed by gender (male or
female), centre (Greece, Italy or Switzerland) and age (12-35 months or 36-66 months). All
randomized subjects were included and analysed in the intention-to-treat group. The PP
dataset was constructed using the following criteria of exclusion: less than 70% days of
product consumption or >10 consecutive days off the product.
Study products were packaged, labelled and stored at Eurofins, France. A study box
containing 110 sachets of the randomized product was sent to the study site (and provided to
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the parents) directly after randomization of the child, and after 2.5 months. Each centre
received at the start of the study “SPT kits” containing each a sachet of HydE and a sachet of
placebo.
HydE (hydrolysed egg) and placebo preparation
The process of HydE preparation has been described in detail before (9) and was adopted
with minor adjustments as described by Hacini-Rachinel et al. (10). The obtained HydE
powder was mixed 1:1 with the placebo product, consisting of a mix of maltodextrin (89.2%),
caramel (2.2%), palm oil (5.2%) and water (3.4%), to obtain the final test product (HydE).
Whole egg (WholE) used in the basophil activation test was obtained by spray drying
of the pasteurized liquid WholE (ABCD S.A., Ploërmel, France) used to produce the HydE
powder.
Assessment of product tolerance and OIT protocol
On day 1, tolerance of the randomized product was assessed by an oral food challenge. 9±1g
of product was diluted in liquid (1/5 orange juice and 4/5 carrot juice) to a final volume of 50
ml. Every 20-30 minutes the final product was administered with increasing doses as follows:
Step 1: 1.5 ml; Step 2: 3.5 ml; Step 3: 10 ml; Step 4: 15 ml and step 5: 20 ml. Both objective
and subjective symptoms were recorded. The patient passed the tolerance assessment test if
no symptoms occurred after having consumed all doses.
In case no adverse reaction occurred, the subject was administered 1 day later with a
full dose (9g±1g) at once incorporated in a solid meal and again adverse reactions were
recorded. It was planned that any child with an allergic reaction to the study product during
the tolerance assessment would be withdrawn from the study.
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In case of no adverse reactions, a sachet containing 9±1g HydE or placebo was
administered daily for 6 months (see study design, Fig. 1). Parents were instructed to mix the
content of the sachet into a prepared meal and not to heat or cook the meal after the addition
of the study product. Parents were also instructed that the children should not consume any
other food preparation or product that contain raw or heat-treated egg.
Adverse events were recorded throughout the study and compared between the 2
groups. Compliance of product intake was evaluated by analysing the records filled in daily
by the parents.
Skin prick test (SPT) protocol
Skin prick tests were performed at enrolment using the HydE product, placebo product, egg
white (ALK-Abello, Hørsholm, Denmark), egg yolk (ALK-Abello), saline and histamine
control (ALK-Abello). The wheal size was calculated as (D+d)/2, in which ´D´ is the longest
diameter of the wheal, and ´d´ the longest diameter orthogonal to D. A cut-off of ≥3 mm was
used for a positive SPT.
Oral food challenge (OFC)
A food challenge to boiled egg (7 minutes boiling of a 50-60g egg, using a standardized
protocol) was done at the final visit. Every 20-30 minutes the product was administered with
increasing doses as follows: Step 1: 0.3 g; Step 2: 0.9 g; Step 3: 3 g; Step 4: 9 g and step 5: 30
g (total amount of 43.2g). Any occurrence of objective or persistent (45 min) subjective
symptoms was considered as a positive result to the challenge test. Besides the primary
outcome of having a positive or negative result of the challenge test, also the cumulative dose
ingested without reaction (maximum cumulative dose tolerated) was calculated.
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Additional post hoc analysis was performed to look further into the severity of the
allergic reactions at the final OFC. An overall score was calculated by multiplying two
scores; maximum dose tolerated (0g: grade 3, <1g: grade 2, >1g: grade 1, equal to maximum
dose tolerated: grade 0) and severity of symptoms (no reaction: grade 0, mild: grade 1,
moderate: grade 2, severe: grade 3).
Quantification of total and allergen specific IgE and IgG4
Total IgE and egg-specific IgE and IgG4 antibodies (anti-egg white, anti-egg yolk, anti-
ovomucoid (OVO), anti-ovalbumin (OVA)) were determined using ImmunoCAP (Thermo
Fisher, Uppsala, Sweden) according to the instructions of the manufacturer.
Basophil activation test (BAT)
Basophil activation was assessed at the start (V0) and end (V5) of the study, following the
instructions of the allergenicity kit (Beckman Coulter, Brea, CA, USA), with minor
modifications. Peripheral blood was stimulated with 0.01 and 0.1 µg/ml of WholE, OVO,
OVA, HydE, placebo, PBS or 10 µg/ml anti-IgE, in the presence of an antibody mix (CD3,
CRTH2, CD203c from the allergenicity kit, plus CD63), for 15 minutes at 37°C. Cells were
lysed, fixed at the study sites and then sent in PBS at 4°C to the Nestlé Research Center
(Lausanne, Switzerland). After washing, both CD63 and CD203c upregulation was measured
by flow cytometry using a BD Fortessa machine (BD, San Jose, CA, USA). Raw data were
analysed by FlowJo software (Treestar, Ashland, OR, USA). Comparisons were made of
values after subtracting the medium control.
Statistics
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The software SAS 9.2 and R 3.0.1 were used for the analysis and all outcomes were assessed
by intention-to-treat analysis. Frequencies were calculated for categorical variables, and
Fisher´s exact test was calculated for between-group comparisons for the positive or negative
outcome of the OFC.
The power of the study has been calculated 80% with 16 subjects per arm in order to
identify an effect of 40% as being significant at a 5% level for a one-sided test.
For all immunological parameters, the change score from baseline (V5-V0) was
analysed with the ANCOVA model when distribution was normal, and with the non-
parametric Wilcoxon rank sum test if the distribution was not normal. For the SPT, a paired t-
test was performed to compare the wheals (measured in mm) between the groups. When P <
0.05, the difference was interpreted as significant.
Results
Baseline clinical characteristics
From January 2012 until December 2013, 29 subjects were enrolled in the study and thus
included in the intention-to-treat group (Fig. 1). Ten, 10 and 9 patients were included and
randomized from Greece, Italy and Switzerland, respectively; 14 received placebo and 15
HydE. Median age of the subjects was 29 months (range, 15 - 65 months) and 76% (22/29)
were male (Table 1). There were no significant differences between the study groups (Table
1).
Twenty-five patients completed the study; there were 4 dropouts in the HydE group, 1
due to aversion to the product, 1 due to anxiety of the parent to the OFC and 2 due to non-
compliance.
Safety aspects of HydE
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Clinical
For the tolerance assessment at the start, all egg allergic patients randomized to HydE (n=15)
tolerated the full dose in liquid at V1 and 14 out of 15 tolerated the full dose in the solid meal
at V2 (Table 1). The patient who did not tolerate the full dose presented one wheal and
sneezed once 45 minutes after ingestion without any other symptoms. No medication was
taken and it was decided to include the patient in the study.
Of all the 29 patients tested in the SPT, 1 had a positive SPT to HydE as compared to
28 and 22 to egg white and egg yolk, respectively (Table 1). The SPT size of the positive
patients were 5.5 mm for HydE, 7.35±2.69 mm for egg white and 5.97±1.38 mm for egg
yolk.
All patients, except 1 in the HydE group, experienced at least 1 adverse event during
the course of the treatment. There was no significant difference between the HydE and
placebo group with respect to the type of adverse events and there were no serious adverse
events or need for epinephrine use related to the intake of the study product (data not shown).
Excluding the adverse events that were judged as being ´unrelated to the product´, 7
adverse events occurred during the 6 months of intervention in the active group and 2 in the
placebo group. In the active group, adverse events occurred once in 5 patients and only 1
patient experienced an adverse event twice during the course of the study. Four adverse
events were ´probably related to the product´ (flush around the mouth; wheal/sneezing/atopic
dermatitis flare; constipation; dermatitis) and 3 were ´unlikely related to the product´
(vomiting: aversion to product; cough/runny nose/eczema; diarrhea). In the placebo group,
the 2 adverse events occurred in 2 different subjects; they were both ´unlikely related to the
product´) (respiratory infection and bad skin).
Immunological (BAT)
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At the start (V0) and end (V5) of the study, for 27 patients, a BAT could be performed
comparing the response of cells to stimulation with HydE or WholE. Four % of the subjects
were considered as non-responders (non-responding to the positive control or to any stimuli
tested). The median (range) percentage of CD63+ activated basophils after stimulation with
0.01 and 0.1 µg/ml was 0.9% (-6.3 – 17.7) and 1.0% (-4.8 – 60.0) for HydE, respectively and
8.2% (-3.6 – 66.7) and 27.5% (5.4 – 86.5) for WholE, respectively. For CD203c+ activated
basophils this was 1.3% (-4.2 – 29.6) and 2.2% (-3.7 – 71.7) for HydE, respectively and
12.0% (-1.0 – 66.6) and 37.5% (9.4 – 85.2) for WholE, respectively. For both concentrations
of stimuli, basophil activation was significantly less (P < 0.001) for HydE compared to
WholE (Table 1).
Oral food challenge
Oral food challenge was performed at the end of the study with boiled egg as standard
introductory form of egg and because its preparation can be well standardized. Of the 25
patients who underwent an OFC, no significant difference (P = 0.66) was observed on this
primary outcome, comparing the 2 groups. Thirty-six % (4/11) and 21% (3/14) had a negative
OFC to egg in the treatment and placebo group, respectively (Table 2). Results were also not
significant when ITT population (negative OFC in 4/15 for treatment group vs 3/14 for
placebo group (27% vs 21% respectively; P = 1) and PP population (negative OFC in 3/9 for
treatment group vs 3/11 for placebo group (33% vs 27% respectively; P = 1) was analysed
(Table 2). No significant difference (P = 0.35) was found for the maximum dose tolerated by
the subjects between the active (median = 1.2 g) and the placebo (median = 0.3g) group (data
not shown).
Even though there was no significant difference (P = 0.33) for the overall grading
between the active and the placebo group; no reactions with a grade higher than 4 were
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observed in the treatment group, while 4 of the 14 patients (28.6%) in the placebo group had
a grade ≥6 (Table 3).
Specific IgE and IgG4 and basophil activation
While no significant difference was observed regarding egg specific IgE levels (Fig. 2B) ,
IgG4 to egg white, egg yolk and ovalbumin increased significantly more over time (V0-V5)
in the active compared to the placebo group (P = 0.07, P = 0.01 and P = 0.04, respectively;
Fig. 2A). A higher increase over time in the active group compared to the placebo group was
also observed for specific IgG4/specific IgE (data not shown).
In the basophil activation test, a significant decrease in percentage CD203c+ cells (P
= 0.04, Table 4) and a trend for a lower percentage CD63+ cells (P = 0.07, data not shown)
was observed after stimulation with 0.01 µg/ml OVO in the treatment group, as compared to
an increase over time in the placebo group (Fig. 3). For other stimuli tested (OVA, WholE,
HydE and placebo), there was no difference comparing the 2 groups (Table 4).
Discussion
This is the first double-blind placebo controlled randomized study using well-characterized,
low allergenic hydrolysed egg for oral immunotherapy in egg allergic children. For ethical
reasons, oral challenge was not systematically performed at the beginning of the study; the
identification of egge allergic children was based on clinical history, SPT and sIgE. Results
showed a high safety of the product to be used in infants allergic to egg. Furthermore,
tolerogenic IgG4 antibodies were induced and basophil activation was reduced over the
course of the treatment with HydE.
The rationale of using a low allergenic product was twofold: better safety by fewer
side effects, as well as a lower burden protocol by providing the maintenance dose from the
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start, thereby minimizing the number of hospital visits. We showed that using HydE was a
safe way of performing OIT. All patients tolerated the product, no adverse events were
observed during the tolerance assessment and therefore all patients succeeded in up-dosing to
the maintenance dose in only 1 day. Safety was further underlined by an overall negative SPT
to HydE, as well as very low basophil activation after in vitro basophil stimulation with
HydE.
OIT is traditionally performed by giving gradually increasing doses of the food, in
which the dose escalations occur in a controlled setting (build-up phase), followed by a
maintenance phase (11). This often entails standard visits to the clinic for the dose escalations
depending on the protocol followed, which can be a high burden for the patient as well as for
the medical team. By using HydE, after the initial visits for the tolerance assessments, no
further hospital visits were needed until the end of the study for the final evaluation, as the
maintenance dose could be administered from the start. Additionally, only few allergic
reactions possibly related to the product took place during the 6 months of intervention (7 in
the active group versus 2 in the placebo group), which corresponds to less than 0.4% of the
total doses of OIT with egg or placebo administered. This is remarkably low compared to
other studies, i.e. compared to the study of Burks et al. (7) in which adverse events were
associated with 25% and 3.9% of the doses of egg and placebo, respectively. Of note, allergic
reactions to OIT doses have been shown to impact negatively on children’s health-related
quality of life (HRQL) as reported by their parents (12), and is the main reason to quit during
OIT, reinforcing the importance of improving safety during OIT.
Efficacy was assessed by an oral food challenge to boiled egg at the final visit, by the
maximum dose tolerated during the challenge test to boiled egg, as well as by the change
from baseline to V5 on the immune parameters. Even though a higher percentage of patients
passed the OFC at the end in the active group (36%) compared to the placebo group (21%),
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this was not significantly different. Even though not significant, grading the reactions
suggested that the reactions to the OFC in the active group were overall less severe compared
to the reactions in the placebo group. In our study, 64% (7/11) could tolerate >1g in the active
group compared to 43% (6/14) in the placebo group. Increase in the egg protein amounts that
can be tolerated by an egg allergic child could minimize allergic reactions during accidental
exposure. Nevertheless, the major weakness of this pilot study is the small number of
patients.
Despite the lack of significant alterations in symptoms, significant changes were
observed in both specific levels of IgG4 as well as in the BAT between the placebo and the
active groups. Several other egg OIT studies showed an increase from baseline in egg-
specific IgG4 antibody levels during therapy following successful OIT (7, 13, 14). The
relatively low increase in IgG4 observed in our study could be explained by the lower
immunogenicity of HydE compared to whole egg usually used in other studies. The timing of
assessment of IgG4 levels (only 6 months after treatment) could also be part of the
explanation. Even though a decreased reactivity of basophils has been associated with
desensitization, only 1 other study on egg OIT performed the BAT and showed reduced egg
white-induced basophil activation overtime in the group who followed OIT compared to the
placebo group (7). Our study included testing of several egg components (whole egg, OVO
and OVA). Basophil activation was decreased in the treatment group during the course of the
treatment for all 3 components tested, while it was increased for the placebo group. However,
this difference between the two groups was only significant for OVO, which is well known to
be a dominant allergen and allergic children sensitized to it tend to have a more severe and
prolonged course of egg allergy (15). The lack of significant difference between the groups,
in basophil activation after stimulation by whole egg could be possibly due to the fact that
OVO represents only 11% of egg proteins. Mechanistically, it was recently shown that IgG4
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antibodies are important in blocking basophil and mast cell activation in peanut-sensitized but
tolerant children (16).
The study of Burks et al. (7) and our study are currently the only studies that use a placebo
group which enables to exclude the bias of natural outgrowth of the allergy during the course
of the trial and to control for a possible placebo effect. Although the study focuses on
desensitisation to egg rather than permanent tolerance, and the results shown are not
statistically significant, all numerical parameters tend towards favourable direction. Failing to
have significant differences might be attributed to smaller number of children than planned
and/or the short course of intervention (6 months). This short intervention period was chosen
to ensure minimal compliance in such a heavy study and has previously been used in
previous studies with positive outcomes (17-20).
Our study opens also new avenues for the OIT field like baked egg where the product
should initially be tested under medical supervision and then can be given at home. Recent
studies indicate that regular exposure of heat-modified egg protein in egg allergic patients is
not only well tolerated in up to 70% of allergic patients but might be clinically beneficial (17,
21, 22). However, there is some debate on how baked egg should be introduced, at home or
in a supervised setting, and the specific foods that should be allowed to be consumed
afterwards, depending on the amount of the allergen and the extend of heat-treatment.
According to the British Society of Allergy and Clinical Immunology guidelines for the
management of egg allergy, children who have only mild symptoms on significant exposure
of egg are advised to introduce heat-treated egg products at home from the age of 2-3 years
(23). An advantage of our HydE is that it is well characterized and standardized with respect
to the extent of hydrolysis.
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In conclusion, HydE would be a safe and standardized product to use for regular
consumption to presumably speed up tolerance development. This first proof of concept is
encouraging (safe product and immune modulation) but clinical efficacy still needs to be
confirmed. A larger study considering a longer treatment period and/or a higher dose could
improve the clinical outcome.
Acknowledgements
We thank R. Fritsché, R. Schaller, and C Blanchard for their scientific inputs. We thank E.
Bersuch and N. Celegato for their help in performing the clinical part of the study and V.
Zisaki, C. Chliva, T. Stefanaki, P. Korovesi, N. Douladiris and E. Manousakis for the
recruitment of patients. We thank T. Bourdeau, S. Moille, I. Montagner, M. Passioti, S.
Megremis, M.E. Barcos and S. Lefebvre for their technical help and J. Sauser, N. Emami, L.
Rincon Kohli, W. Sauret and M. Beaumont for their help in the operational part of the
clinical trial.
Author contribution
SG, YMV, Amu, RL, AMe, AW, SN, NGP: Designed the study / study product
RF: Performed the laboratory studies
SG, Amu, RL, APK, FL RB, SS, NGP: Performed the clinical part of the study
SG, YVM, AMu, RL, SN, NGP: Interpreted the data and wrote the paper
Conflict of interest
Several authors (YMV, AM, SN, AW) are or were employees of Nestec Ltd.
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egg can affect the natural course of egg allergy: Hypothesis-generating observations. Journal of Allergy and Clinical Immunology 2008;122(2):414-415.18. Dello Iacono I, Tripodi S, Calvani M, Panetta V, Verga MC, Miceli Sopo S. Specific oral tolerance induction with raw hen's egg in children with very severe egg allergy: A randomized controlled trial. Pediatric Allergy and Immunology 2013;24(1):66-74.19. Patriarca G, Nucera E, Pollastrini E, Roncallo C, Pasquale TD, Lombardo C, et al. Oral specific desensitization in food-allergic children. Digestive Diseases and Sciences 2007;52(7):1662-1672.20. Morisset M, Moneret-Vautrin DA, Guenard L, Cuny JM, Frentz P, Hatahet R, et al. Oral desensitization in children with milk and egg allergies obtains recovery in a significant proportion of cases. A randomized study in 60 children with cow's milk allergy and 90 children with egg allergy. European Annals of Allergy and Clinical Immunology 2007;39(1):12-19.21. Leonard SA, Caubet JC, Kim JS, Groetch M, Nowak-Wegrzyn A. Baked milk- and egg-containing diet in the management of milk and egg allergy. Journal of Allergy and Clinical Immunology: In Practice 2015;3(1):13-23.22. Sampson HA, Aceves S, Bock SA, James J, Jones S, Lang D, et al. Food allergy: A practice parameter update - 2014. Journal of Allergy and Clinical Immunology 2014;134(5):1016-1025.e1043.23. Clark AT, Skypala I, Leech SC, Ewan PW, Dugué P, Brathwaite N, et al. British Society for Allergy and Clinical Immunology guidelines for the management of egg allergy. Clinical and Experimental Allergy 2010;40(8):1116-1129.
Tolerance at V1 Number that passed (%) 13e (92.9%) 15 (100%)
Tolerance at V2 Number that passed (%) 14 (100%) 14 (93.3%)
D: largest diameter of wheal (length); d: longest diameter orthogonal to D (width); BAT: basophil activation test; NA: not applicable; a Number of SPT where (D+d)/2 ≥ 3 mm; b P < 0.001 (HydE vs Egg white & HydE vs Egg yolk); c P < 0.001 (HydE vs WholE); d n=12; e n=13; f n=14; g
n=24; h n=26; i n=28; j Wilcoxon rank sum test; k Chi-square test; l t-test
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Table 2 Result of the challenge test to boiled egg after the intervention for ITT and PP population
ITT PlaceboN=14
Test productN=15 P-valuea
Missing 0 (0.0%) 4 (26.7%)
n 14 11
Negative 3 (21.4%) 4 (36.4%) P = 0.66
Positive 11 (78.6%) 7 (63.6%)
Odds ratio 2.1 (0.36 – 12.32)b P = 0.41a Fisher's exact; b 95% confidence interval
PP PlaceboN=11
Test productN=9 P-valuea
n 11 9
Negative 3 (27.3%) 3 (33.3%) P = 1.00
Positive 8 (72.7%) 6 (66.7%)
Odds ratio 1.3 (0.2 – 9.08)b P = 0.77a Fisher's exact; b 95% confidence interval
a Change (V5 – V0) of Test product – Change (V5 – V0) of Placebo; b Because distribution was not normal, the non-parametric Wilcoxon rank sum test was applied, based on mean results
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Figure legends
Figures
Figure 1: Consort flow diagram for the clinical trial. Twenty-nine subjects met the inclusion
criteria, signed informed consent forms and were included at V0. Subjects were randomly
assigned to either placebo or HydE OIT at the baseline visit (V0). Tolerance assessment was
performed the next days (V1) with stepwise increasing doses of the randomized products. In
case no adverse reaction occurred, the subjects were administered 1 day later (V2) with a full
dose at once (9±1g) incorporated in a solid meal. All subjects passed the tolerance assessment
test, continued the study and HydE or placebo was administered daily for 6 months. Twenty-
five patients completed the study; there were 4 dropouts in the HydE group, 1 due to aversion
to the product, 1 due to anxiety to the OFC and 2 due to non-compliance. All patients in the
placebo group and 11 in the HydE group underwent the OFC after 6 months.
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Figure 2 A. Change over time of IgG4 antibody levels in placebo (upper panels) and HydE
(lower panels) groups. (Dotted lines are patients who passed the OFC, while patients with the
full line did not pass the OFC). B. Change over time of IgE antibody levels.
