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\begin{document}

\title{Birch allergen challenges in allergic conjunctivitis using unitary tests
and an exposure chamber}



\author[1]{Alina Gherasim}%
\author[2]{Jean Luc Fauquert}%
\author[1]{Nathalie Domis}%
\author[3]{Xavier SIOMBOING}%
\author[1]{Nicole Beck}%
\author[4]{Tristan Bourcier}%
\author[5]{Frédéric De Blay}%
\affil[1]{Alyatec}%
\affil[2]{University Hospital}%
\affil[3]{SOLADIS }%
\affil[4]{University Hospitals Strasbourg}%
\affil[5]{University Hospital Strasbourg}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Background: Environmental exposure chambers (EECs) have been used
extensively to study allergic rhinoconjunctivitis. Few studies have been
published using EECs in conjunctivitis only, and none have used
conjunctival allergen challenge as a selection criterion. The present
study validated ALYATEC EEC in allergic conjunctivitis to birch
allergens. Methods: Sixteen patients with a positive conjunctival
allergen challenge (CAC) were ex-posed to 60 ng/m3 of Bet v 1 in an EEC
on 2 consecutive days for a maximum of 4 hours. Re-producibility was
tested among seven of the patients. A positive conjunctival response
during the CAC and the EEC exposure was defined as a Total Ocular
Symptom Score (TOSS) [?] 5. Results: Fifty percent of patients had a
positive conjunctival response during the first expo-sure and 75\%
during the second. The mean time to a positive conjunctival response was
81.2\selectlanguage{ngerman}±33.9 minutes and 101.6±57 (P\textgreater{}0.05) during the first
and second exposure, respectively. No difference in the TOSS occurred
between the two exposures. The time necessary to ob-tain a positive
response during the CAC was significantly shorter than with the EEC. The
es-timated quantity of Bet v 1 inducing a positive response was
0.07±0.03 ng (exposure 1), 0.07±0.07 ng (exposure 2), 980±784 ng (CAC).
The frequency of conjunctival responses and quantity of Bet v 1 was
reproducible in all six EEC exposures. Conclusions: Birch allergen
exposures inducing early conjunctival responses were different than
those identified with direct installation during CAC. EEC appears to be
closer to natural exposure than CAC.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Birch allergen challenges in allergic conjunctivitis using
individual tests and an exposure chamber}

\textbf{Short title: Allergen challenges in conjunctivitis to birch}

Alina Gherasim \textsuperscript{1}, Jean-Luc
Fauquert\textsuperscript{2}, Nathalie Domis \textsuperscript{1}, Xavier
Siomboing \textsuperscript{3}, Nicole Beck \textsuperscript{1,} Tristan
Bourcier\textsuperscript{4}, Frederic de Blay F \textsuperscript{1,5,6}

\textsuperscript{1} ALYATEC Environmental Exposure Chamber, 1 place de
l'H\selectlanguage{ngerman}ôpital, Strasbourg, France

\textsuperscript{2} Consultation Ophtalmo-Allergo Unité d'Allergologie
de l'enfant, CHU Montpied, Clermont-Ferrand Cedex 1, France

\textsuperscript{3} Soladis Clinical Studies, Roubaix, France

\textsuperscript{4} Department of Ophthalmology, Strasbourg University
Hospital, FMTS, Strasbourg University, Strasbourg, France

\textsuperscript{5} Chest Diseases Department, Strasbourg University
Hospital, Strasbourg, France

\textsuperscript{6} Federation of translational medicine EA 3070,
University of Strasbourg, Strasbourg, France

\textbf{Correspondence}

Alina Gherasim, ALYATEC® Environmental Exposure Chamber,

1 place de l'Hôpital, 67000 Strasbourg, France.

Email :\emph{alina.gherasim@alyatec.com}

\textbf{Abstract}

\textbf{Background:} Environmental exposure chambers (EECs) have been
used extensively to study allergic rhinoconjunctivitis. Few studies have
been published using EECs in conjunctivitis only, and none have used
conjunctival allergen challenge as a selection criterion. The present
study validated ALYATEC EEC in allergic conjunctivitis to birch
allergens.

\textbf{Methods} : Sixteen patients with a positive conjunctival
allergen challenge (CAC) were exposed to 60 ng/m\textsuperscript{3} of
Bet v 1 in an EEC on 2 consecutive days for a maximum of 4 hours.
Reproducibility was tested among seven of the patients. A positive
conjunctival response during the CAC and the EEC exposure was defined as
a Total Ocular Symptom Score (TOSS) \selectlanguage{english}[?] 5.

\textbf{Results} : Fifty percent of patients had a positive conjunctival
response during the first exposure and 75\% during the second. The mean
time to a positive conjunctival response was 81.2+-33.9 minutes and
101.6+-57 (P\textgreater{}0.05) during the first and second exposure,
respectively. No difference in the TOSS occurred between the two
exposures. The time necessary to obtain a positive response during the
CAC was significantly shorter than with the EEC. The estimated quantity
of Bet v 1 inducing a positive response was 0.07+-0.03 ng (exposure 1),
0.07+-0.07 ng (exposure 2), 980+-784 ng (CAC). The frequency of
conjunctival responses and quantity of Bet v 1 was reproducible in all
six EEC exposures.

\textbf{Conclusions} : Birch allergen exposures inducing early
conjunctival responses were different than those identified with direct
instillation during CAC. EEC appears to be closer to natural exposure
than CAC.

\textbf{(245 words)}

\textbf{Key words} : birch allergy, conjunctivitis, conjunctival
allergen challenge, environmental exposure chamber

ABBREVIATIONS

Bet v 1 Betula verrucose major allergen

CAC Conjunctival allergen challenge

EAACI European Academy of Allergy and Clinical Immunology

EEC Environmental exposure chamber

Expo Exposure

FEV1 Forced expiratory volume in one second

GCP Good Clinical Practice

GINA Global Initiative for Asthma

ICH International Conference on Harmonization

IgE Immunoglobulin E

TOSS Total Ocular Symptom Score

TNSS Total Nasal Symptom Score

\textbf{Introduction}

Allergic conjunctivitis occurs in atopic individuals exposed to specific
antigens and manifests as an early reaction, within minutes or hours of
exposure to allergens, and may or not be associated with allergic
rhinitis.\textsuperscript{1} IgE-mediated hypersensitivity inducing
conjunctivitis is frequent, from 15-20\% in general
practice\textsuperscript{2, 3} to 40\% in the US population when
examined in an ophthalmological survey.\textsuperscript{4}

In France, the prevalence of seasonal allergic rhinoconjunctivitis (SAR)
was found to be 13\% among 10-year-old children and approximately 20\%
among adults.\textsuperscript{5} Birch is one of the most frequent
sources of allergens that induce
rhinoconjunctivitis.\textsuperscript{6}In the general French adult
population, the prevalence of birch sensitization was
4.7\%.\textsuperscript{7} A relationship between pollen counts and the
occurrence of symptoms was shown recently.\textsuperscript{8} The
frequency of the ocular response to natural exposure to birch pollen in
sensitized patients was linear until birch daily average concentrations
reached a plateau of 110 grains/m\textsuperscript{3}, with a cutoff of
ocular symptoms at 70 grains/m\textsuperscript{3} at the beginning of
the season.\textsuperscript{9}

In 1990, Abelson \emph{et al} demonstrated a correlation between skin
sensitization (grass pollen, ragweed pollen, and cat allergen) and
positivity to conjunctival allergen challenge (CAC). This tool confirms
allergen involvement in the diagnosis of allergic conjunctivitis,
allowing precise selection of the patients in clinical studies. The CAC
model is the only clinically validated method recognized by the Food and
Drug Administration (FDA) for testing the efficacy of eye anti-allergic
molecules.\textsuperscript{10} The practical aspects were described in a
position paper of the European Academy of Allergy and Clinical
Immunology (EAACI) Task Force.\textsuperscript{11}

Environmental exposure chambers (EECs) have been in development since
1985 to study new therapeutics for allergic pathologies, including
conjunctivitis.\textsuperscript{12, 13} EECs have the advantage of
achieving reproducible and safe exposure with controlled levels of
allergen for several hours in several subjects simultaneously by
avoiding confounding factors during exposure.\textsuperscript{14, 15}The
ALYATEC EEC has been validated in mite and cat-induced
asthma.\textsuperscript{16, 17} These studies demonstrated that the
allergen exposures are standardized with an inter-test coefficient of
variation of less than 30\%.

To validate the ALYATEC EEC with birch pollen, we exposed patients
affected by seasonal allergic conjunctivitis caused by birch pollen to
airborne birch allergen. We investigated the amount of Bet v 1 and the
time necessary to induce a conjunctival response in at least 50\% of
patients. We also evaluated the reproducibility of the exposures.

\textbf{Material and methods}

\textbf{Patients}

Patients aged 18 to 65 years were selected for eligibility based on
having a history of \textgreater{}2 years of moderate allergic
conjunctivitis during birch pollen season.\textsuperscript{1,
11}Allergic sensitizations were documented by a positive skin prick test
to birch allergen with a wheal diameter \selectlanguage{english}[?] 6 mm compared to negative
control and positive birch-specific IgE (\textgreater{} 0.10 kU/l). The
main inclusion criterion was a positive conjunctival response during an
individual conjunctival allergen challenge.\textsuperscript{10, 11} The
study was performed outside the pollen season in France. A 7\selectlanguage{english}-day washout
period was required for topical or systemic anti\selectlanguage{english}-histamines or other
ophthalmic treatment. Exclusion criteria were evaluated prior to
inclusion and were as follows: patients experiencing a single ocular
symptom in the previous week; patients who received long\selectlanguage{english}-acting
corticosteroids within the past 4\selectlanguage{english} weeks; ocular laser treatment within
the past 3 months; ocular surgery within the last 6 months; abnormality
or clinically significant ocular disorder, including symptoms of
allergic conjunctivitis; ongoing immunotherapy to any allergen or,
within the last 5 years, to birch allergen.

\textbf{Interventions}

This was an open, single-center study designed to determine the
concentration of airborne Bet v 1 inducing an allergic conjunctivitis
response in patients allergic to birch pollen during allergen exposures
to birch pollen extracts in the EEC. During the first screening visit,
the patient gave informed written consent and underwent the following
procedures and assessments: medical history review, skin prick testing
to birch pollen allergen (ALK\emph{-Abello(r)} ), and a blood draw for
specific IgE to birch (\emph{Betula verrucose,} Phadia ImmunoCap,
Thermofisher(r)). The second screening visit was for an individual
conjunctival allergen challenge (CAC) to birch allergen. All responders
in the CAC were included in the present study.

The CAC was performed according to the updated EAACI
guidelines.\textsuperscript{1, 11} This procedure consisted of the
instillation of 20 \selectlanguage{greek}µ\selectlanguage{english}l of diluted birch allergen extract (100 IR
lyophilized extract, Stallergenes Greer\selectlanguage{ngerman}®) in the inferior-external
quadrant of the bulbar conjunctiva in incremented dilutions at 10-min
intervals: 3, 6, 12, 25, 50, and 100 IR/ml.\textsuperscript{11} The
clinical response was assessed by the Total Ocular Symptom Score (TOSS)
with the same cumulated positivity criteria for the CAC and EEC
exposure. If the TOSS was \textless{}5 at 10 minutes after each
instillation, the test was considered negative. The next concentrated
dose was then instilled until a positive response was reached.

Step 1 of the study consisted of two consecutive EEC exposures to the
same birch pollen extract (Expo 1 and Expo 2). The primary endpoint of
the study was met when 50\% of the patients were positive. The main
judgment criteria were the amount of Bet v 1 inducing a positive
conjunctival response after EEC exposure. In addition, we compared the
intensity of the clinical response induced in patients exposed to birch
pollens using the TOSS and the mean time to reach a positive ocular
response (i.e., TOSS \selectlanguage{english}[?] 5). In step 2, we studied the reproducibility of
the allergen exposure. Patients who responded to Expo 1 and Expo 2 were
enrolled in step 2 and exposed two additional times on 2 consecutive
days (from Expo 3 to Expo 6). Each double EEC exposure test was
separated by 7 days.

\textbf{Clinical assessments}

The TOSS was used during both the CAC and EEC exposures. This score was
first described\textsuperscript{10} as the sum of four conjunctival
symptom scores: itching, redness, chemosis, and tearing (range: 0-13).
It was evaluated after instillation of the allergen on one side, with
the other eye serving as a negative control after instillation of the
physiological serum. A slit lamp examination was used to score redness
and chemosis only. Itching was assessed by the patient using a 5-point
severity scale from 0 (none) to 4 (very severe: incapacitating itch with
irresistible urge to rub), with 1=mild (intermittent tickling
sensation), 2=moderate (continual awareness but without the desire to
rub), and 3=severe (continual awareness with the desire to rub). For
ocular redness, ratings were collected for the nasal and temporal area
of each eye and averaged by the study physician using a 4-point severity
score (0=absent, 1=mild, 2=moderate, 3=severe). Tearing was also rated
by the physician using a 4-point severity score (0=absent; 1=mild, eyes
feel slightly watery; 2=moderate, blows nose occasionally; and 3=severe,
tears rolling down cheeks). Chemosis was rated by the study physician as
follows: 0=absent, 1=mild (detectable with slit lamp, conjunctiva
separated from sclera), 2=moderate (visually evident, raised
conjunctiva, especially at the limbal area), and 3=severe (ballooning of
conjunctiva). The patient left the EEC when the mean TOSS of both eyes
was \selectlanguage{english}[?] 5.

Safety monitoring of pulmonary function was performed by clinical
survey. The Total Nasal Symptom Score (TNSS) and portable spirometry
were performed every 20 minutes during exposure. Early asthma response
was defined as a drop in the forced expiratory volume in 1 second
(FEV\textsubscript{1}) of 20\%. Patients with this asthma response
during allergen exposures were discharged. At the end of an exposure,
all patients were treated as needed with topical antihistamines, eye
drops, and oral second generation H1-antihistamines according to the
persistence and severity of the conjunctival or rhinitis symptoms. When
an early asthmatic response occurred, patients remained under
supervision for 6 hours. Thereafter, they were discharged with a rescue
therapy kit containing oral antihistamines, topical mast cell
stabilizers, and short-acting beta 2 agonist inhaler.

This study was approved by an independent ethics committee and was
conducted according to Good Clinical Practice (GCP) standards using the
guidance documents and practices offered by the International Conference
on Harmonization (ICH) and European directive 2001/20/CE. The study was
registered at ClinicalTrials.gov under number NCT04641130.

\textbf{Environmental exposure chamber}

The ALYATEC EEC is a new generation EEC located in Strasbourg, France.
Its capacity and allergen exposure conditions are described
elsewhere.\textsuperscript{16, 17} In this study, patients were exposed
to the same batch of lyophilized birch allergen GMP extract (100
IR;\emph{Stallergenes Greer} (r)), diluted in saline serum, as the one
used for the CAC. Before the patients entered the EEC, the exposure was
initiated in order to reach a plateau of airborne birch allergen and
then maintained for a maximum of 4 hours. A homogeneous allergen
concentration was ensured by using particle counters and online
measurements of the temperature, relative humidity, and air exchange as
described previously.\textsuperscript{16, 17} The birch allergen was
collected on five glass fiber filters located next to the patients'
chairs during exposure to determine the Bet v 1 concentration using
ELISA (Indoor Biotechnologies(r), Charlottesville, VA, USA), after each
allergen exposure. The concentration of the Bet v 1 airborne exposure
was estimated to be 60 ng/m\textsuperscript{3}. During EEC allergen
exposure, the conjunctival response was assessed every 10 minutes during
the first hour and then every 20 minutes.

\textbf{Quantity of Bet v 1 (Q) inducing a conjunctival response}

As the allergen affected the ocular surface, we used the tear film
renewal rate (TRR) to estimate the amount of extract applied during EEC
exposure. The TRR was calculated to be
154x10\textsuperscript{6}mm\textsuperscript{3}/min by Beaudouin et
al.\textsuperscript{18} Thus, the quantity (Q) of extract applied was
calculated in nanograms as follows: Q = C x Time x TRR, where C is the
concentration of airborne Bet v 1 in ng/m\textsuperscript{3} and Time is
the time required to induce the conjunctival response in minutes.

\textbf{Statistical analysis}

All statistical analyses were performed using
SAS\textsuperscript{(r)}guide Enterprise software (SAS Institute, Cary,
NC, USA). Missing data were not replaced. Continuous variables were
described as the number of observed data, mean and standard deviation of
normally distributed values, or median (interquartile range)\textbf{.}
Categorical variables were described as the patients' size and
percentage in each category. For inferential statistics, P-values
\textless{} 0.05 were considered significant. Tests were two-tailed.
Reproducibility was tested using a Pearson correlation between exposure
days.

\textbf{Results}

Patients

Among 23 screened participants, 16 met the inclusion criteria and
performed two consecutive EEC exposures. Eight patients who responded at
Expo 2 were included in step 2 (Figure 1). Patient characteristics are
given in Table 1. Roughly, sensitization to birch pollen was evidenced
by the skin prick test wheal diameter of 7.4 +- 1.6 mm and specific IgE
value of 61.3 +- 109.3 kUI/L for the total study population.
Co-sensitizations were frequent, as only one patient was mono-sensitized
to birch pollen. Approximately 90\% of patients had concomitant
rhinitis, and 43\% presented with asthma according to GINA 1
classification.

Conjunctival outcomes during CAC and EEC exposures (step 1)

Among the 16 patients included in step 1, 12 presented a conjunctival
response during allergen exposure in the EEC during Expo 1 or Expo 2. In
the EEC, the mean TOSS was 5.7 +- 0.8 in Expo 1 and 5.5 +- 0.6 in Expo 2,
whereas the mean TOSS during the CAC was 6.2 +- 1.1. No correlation was
observed between the TOSS in the EEC versus CAC (r= 0.05). The maximal
TOSS was 9 for both types of exposure (CAC and EEC). The mean time
needed to obtain a positive conjunctival challenge was not significantly
different between Expo 1 (81.2 +- 33.9 min) and Expo 2 (101.6 +- 57 min).
During the CAC, positivity was obtained in 36 +- 15 min. The estimated
quantity of Bet v 1 inducing a conjunctival response was 980 ng in the
CAC and 0.07 +- 0.03 ng during Expo 1 and 0.07 +- 0.07 ng during Expo 2.
This level was significantly lower with the EEC than the CAC (Table 2).

Reproducibility of EEC exposures (step 2)

The eight patients included in step 2 had identical characteristics to
the whole cohort from step 1 (Table 1). One patient dropped out of the
study before the last exposure and was not analyzed here. This patient
left the EEC before reaching a positive TOSS due to an early asthma
response. Among the seven remaining patients, all except one exhibited a
positive conjunctival response to the entire course of six designed
exposures (Expo 1 to Expo 6; Table 3). The clinical response was
identical throughout the six exposures in terms of TOSS. Moreover, the
time necessary to reach TOSS \selectlanguage{english}[?] 5 was \textless{}2 hours for all
exposures. The amount of Bet v 1 inducing a positive conjunctival
response was similar in all six exposures. Reproducibility was studied
regarding the time and quantity of allergen necessary for a positive
challenge. Time exposition was highly reproducible (Table 3) with a
Pearson correlation coefficient of 0.78 (p\textless{}0.05) between Expo
1 and Expo 4. As for the quantity of allergen inducing a positive
response, reproducibility was also assessed between Expo 1 and Expo 4
with a Pearson correlation coefficient 0.81 (p= 0.028).

\textbf{Kinetics of ocular symptoms after exposure in the EEC}

Time of onset and intensity of each symptom in the seven patients
following step 2 are reported in Figure 2. During all six exposures,
redness was the first symptom to appear, with a mean time of 16 +- 6.8
min, reaching maximum intensity in 55 +- 20.2 minutes. Tearing and
itching appeared second, with a mean of 25 +- 3.4 min and 35 +- 16.9 min,
respectively. Thus, reproducibility of redness, tearing, and itching
occurred in 100\% of individuals completing all six exposures. Chemosis
was observed in Expo 4, 5, and 6 in six patients with a mean time of 28
+- 16.3 min after the patient entered the EEC. The maximum TOSS was 9 and
occurred in three patients. No severe conjunctivitis was induced during
exposure. All observed ocular reactions were considered mild and were
controlled with a topical rescue treatment.

Nine patients (60\% of the patients included) had a positive nasal
response on the first two exposures (Expo 1 and Expo 2), confirmed by a
positive TNSS \selectlanguage{english}[?] 6 with a mean TNSS of 8.17 +- 1.47. This mean score was
reached in 61 min. Five out of 16 patients developed an early asthma
response during allergen exposure, with a mean decrease in
FEV\textsubscript{1} of 21.9\% in 65 min on average (min 30 minutes; max
100 min). All early asthma reactions were treated by inhaled
short-acting beta-2 agonist. Among patients presenting an early
asthmatic response in the EEC, only one had a late asthmatic response,
which was treated by oral corticosteroids and inhaled beta 2 agonist. No
prolonged observation period was needed. No severe asthma reactions were
observed during the study. No patients used the emergency kit provided
during the test.

Discussion

Different clinical studies have assessed the effect of allergen exposure
in EECs on rhinoconjunctivitis, but very few have focused on allergic
conjunctivitis. The time course of allergic signs and symptoms differed
between the CAC and EEC sessions. In a previous study evaluating 13
patients with a history of ragweed allergy who underwent CAC and EEC
exposure, the response time was different but the intensity of the
maximal response was similar.\textsuperscript{19} In the present study,
when comparing ocular symptoms of patients exposed to birch pollen in
the EEC compared to the reference CAC, we achieved the primary endpoint
of 50\% positivity in 16 patients during the first exposure. The
following day, 75\% of patients had a positive response. Furthermore,
the airborne concentration of birch pollen inducing the response was
very low, reaching a mean 60 ng/m\textsuperscript{3} of airborne Bet v 1
in the ALYATEC EEC.

The main inclusion criteria were a positive CAC, which is considered the
gold standard for objectively evaluating conjunctival reactivity to a
specific allergen at the mucosal surface. We chose a TOSS \selectlanguage{english}[?] 5 during the
CAC as the threshold for a positive conjunctival response according to
European guidelines.\textsuperscript{11} This threshold has been
demonstrated to allow a specificity and sensitivity of 100\% and 90\%,
respectively, in mite allergic conjunctivitis.\textsuperscript{20} We
used the same clinical positivity criteria for EEC exposure. After EEC
exposure, the mean TOSS was not significantly different after CAC and
EEC exposure. The maximal TOSS was 9 in both the CAC and EEC. In
contrast, the time necessary to obtain a positive response was
significantly longer in the EEC than the CAC. To the best of our
knowledge, studies have not reported time between natural exposure and
the occurrence of ocular symptoms. Patients do not describe significant
conjunctivitis symptoms in day-to-day life within 30 minutes after being
exposed to birch pollen. Consequently, the duration to obtain a
significant clinical response to birch pollen in the EEC appeared to be
closer to natural exposure than after a CAC. Moreover, the quantity of
birch allergen inducing a positive conjunctival response was
dramatically different between these two exposures. During CAC, positive
responses were obtained with a mean cumulative dose of 980 ng of Bet v
1, whereas it was calculated to be 0.07 ng with the EEC. According to
the HIALINE study,\textsuperscript{21} the amount of Bet v 1 per pollen
grain can vary from 3.2 to 32 pg. Therefore, 980 ng of Bet v 1
corresponds to approximately 30,000 to 300,000 pollen grains. In the
EEC, the amount of Bet v 1 inducing a positive response corresponds to 2
to 21 pollen grains. The literature assumed that patients allergic to
pollen had symptoms as soon as the pollen grain threshold reached 22 to
30 grains/m\textsuperscript{3} for grass pollens\textsuperscript{22} and
70 grains/m\textsuperscript{3} for birch pollen.\textsuperscript{9} Even
though the manner of exposure is different, the results of natural and
EEC exposure are similar, whereas challenge of the ocular surface
through CAC exposes the individual to a much greater amount of allergen.
Moreover, exposure in the CAC is performed through diluted allergen in
physiological serum instilled onto the ocular surface, whereas in EECs
the allergens are nebulized in the air, which is a modality closer to
naturel exposure. This triangular comparison enhances the clinical
significance of the EEC challenge.

The positive conjunctival response in three-fourths of patients during
the second exposure in the EEC suggests that a priming effect occurs.
However, we did not observe a difference in the severity of the TOSS
between Expo 1 and Expo 2. Jacobs et al.\textsuperscript{22} suggested
that no priming effect exists when exposures are performed on 2
consecutive days and that the conjunctivitis reaction that occurred on
the second day may be a simple manifestation of a late phase reaction
captured within a 24-hour period after
exposure.\textsuperscript{23}Prior studies suggested that two, or even
three, priming visits may be required to obtain high levels of
symptoms.\textsuperscript{24-25}However, the priming effect leads to
rapid onset of symptoms and signs rather than a greater allergic
response.\textsuperscript{26}

We observed reproducibility of ocular response frequency during all
exposures. Reproducibility was assumed when challenging the ocular
surface in the EEC for the time and the quantity of allergen inducing a
clinical reaction.

Symptoms and signs induced in the EEC were comparable to those induced
by CAC. Ocular redness was the first sign to appear in the EEC and
lasted until the end of exposure. Its reproducibility was consistent
across six allergen exposures. Our findings were in line with Jacobs et
al.,\textsuperscript{22} who investigated phenotypes of allergic
conjunctivitis. Other clinical symptoms of conjunctivitis, such as
tearing and itching, occur rapidly. The kinetics of the appearance of
the three main signs and symptoms is the same as in real life. Chemosis
has also been associated with allergic conjunctivitis. When mild or
moderate, chemosis requires slit-lamp examination, which was used in
both CAC and EEC exposure. Chemosis was not observed in our patients who
submitted to CAC but was mild during EEC sessions. We observed good
reproducibility of the kinetics of conjunctival symptoms: ocular
redness, tearing, and ocular itching followed by chemosis. The latter
can be considered a sign of severity, occurring when conjunctivitis was
clearly present. The mild intensity of the chemosis when it was observed
enhances the safety aspect of EEC exposure. The absence of loco-regional
symptoms, such as rhinitis, is another argument that reinforces the
safety of the technique. Nevertheless, in five patients, we observed
mild asthma symptoms during EEC exposure. This makes it possible to
enroll mild asthma patients with allergic conjunctivitis when
investigating ocular allergy. A control of spirometry parameters before,
during, and after EEC challenge remains necessary.

The limitation of this study is the small number of patients. However,
we could demonstrate the clinical validity and good reproducibility of
the method. The estimation of the amount of allergen deposition on the
ocular surface could be discussed, but the calculation took into account
the different physiological factors involved in the pathophysiology of
conjunctivitis.

\textbf{Conclusion}

We demonstrated that exposure to 60 ng/m\textsuperscript{3} of Bet v 1
in the ALYATEC EEC induces conjunctival responses in more than 50\% of
patients with birch allergic conjunctivitis. Birch allergen exposures
inducing early conjunctival responses were different than those
identified with direct instillation during CAC, demonstrating that EECs
more closely mimic a natural exposure during high pollinating days.

\textbf{(2482 words)}

AUTHORS CONTRIBUTIONS

All authors reviewed the manuscript. GA provided supervision,
interpreted the data, and drafted the manuscript. dBF conceived and
designed the study, and interpreted the data. DN conceived and designed
the study, and interpreted the data. NB provided clinical study
supervision and interpreted the data. JLF provided supervision and
reviewed the manuscript. SX provided the statistical analysis.

CONFLICTS OF INTEREST

GA and NB are ALYATEC employees. DN is cofounder and ALYATEC employee.
dBF is medical expert, cofounder, and shareholder of ALYATEC. dBF
reports grants from STALLERGENES GREER, grants from CHIESI, and personal
fees from ALK ABELLO, MUNDIPHARMA,

NOVARTIS, and REGENERON and is a member of the Board at STALLERGENES
GREER, NOVARTIS, ALK ABELLO, MUNDIPHARMA, MEDAPHARMA, BOEHRINGER,
ASTRAZENECA, and CALOR. JLF: none\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. Flow diagram of patient inclusion\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2a. Mean time to TOSS occurrence after beginning EEC challenge
(n=7).

Time in minutes; Error bars are presented as standard deviation (SD)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 2b. Time to reach maximal intensity of TOSS during exposures
(Expos) in the EEC (n=7). In Expo 2, only one patient experienced mild
chemosis.

Time in minutes; Error bars are presented as standard deviation (SD)\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
STEP 1\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
STEP 2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Number of patients\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Age, years\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
26.4 \selectlanguage{ngerman}± 6.8\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
26.6 \selectlanguage{ngerman}± 7.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Gender (male)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
10 (62.5\%)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5 (62.5\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Skin prick test to birch, mm\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.4 \selectlanguage{ngerman}± 1.6\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.7 \selectlanguage{ngerman}± 1.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Birch specific IgE, kU/I\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
61.3 \selectlanguage{ngerman}± 109.3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
54.6 \selectlanguage{ngerman}± 114.1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Allergic comorbidities Positive skin prick test\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
1/16 (6.2\%) +\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
0/16 (0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
15/16 (93.7\%) ++\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
16/16 (100\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Asthmatic patients\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
9/16 (57\%)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
8/16 (50\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Mean predictive FEV1, \%\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
103.7 \selectlanguage{ngerman}± 9.6\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
101.63 (7.1)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
CAC Provocative dose of Bet v 1, ng\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
507.5 \selectlanguage{ngerman}± 392.2\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
385 \selectlanguage{ngerman}± 349\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
CAC cumulative dose of Bet v 1, ng\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
980.0 \selectlanguage{ngerman}± 784.5\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
735.0 \selectlanguage{ngerman}± 698.0~§\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 1. Study population

Data are given as mean ± standard deviation or n (\%).
\selectlanguage{english}+Mono-sensitization to birch; \selectlanguage{english}++ Poly-sensitization; SS CAC cumulative
dose for 8 patients\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Positivity threshold} \textbf{TOSS [?] 5}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
CAC N=16\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
EEC Day 1 N=8 +\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
EEC Day 2 N=12 ++\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
TOSS units\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
6.2 \selectlanguage{ngerman}± 1.1\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
5.7 \selectlanguage{ngerman}± 0.8 n.s.\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
5.5 \selectlanguage{ngerman}± 0.6 n.s.\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
TOSS {[}Min; Max{]}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
{[}0; 9{]}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
{[}0; 7{]}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
{[}0; 7{]}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Time until positivity, min\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
35 \selectlanguage{ngerman}± 15.06\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
81.2 \selectlanguage{ngerman}± 33.9 p\textless{}0.05\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
101.6 \selectlanguage{ngerman}± 57.3 p\textless{}0.05\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Cumulative dose of birch allergen exposure, ng\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
980 \selectlanguage{ngerman}± 784.5\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.07 \selectlanguage{ngerman}± 0.03 p\textless{}0.001\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.07 \selectlanguage{ngerman}± 0.07 p\textless{}0.001\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 2. Positive conjunctival responses after CAC and EEC.

Data are given as mean ± standard deviation unless otherwise noted. Step
1 (EEC versus CAC) n=16. \selectlanguage{english}+Exposure 1: 8 out of 16 patients; \selectlanguage{english}++ exposure
2: 12 out of 16 patients had a positive response in the EEC. EEC =
environmental exposure chamber; CAC = conjunctival antigen challenge;
TOSS = Total Ocular Symptom Score; n.s. = not significant.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Positivity threshold} \textbf{TOSS [?] 5}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Expo 1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Expo 2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Expo 3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Expo 4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Expo 5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Expo 6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TOSS Scoring units\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.7 \selectlanguage{ngerman}± 0.8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.5 \selectlanguage{ngerman}± 0.6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.8 \selectlanguage{ngerman}± 1.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.7 \selectlanguage{ngerman}± 1.1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6.8 \selectlanguage{ngerman}± 1.5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6.3 \selectlanguage{ngerman}± 1.9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TOSS Scoring Units {[}Min; Max{]}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}0; 7{]}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}0; 7{]}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}0; 8{]}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}0; 8{]}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}0; 9{]}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}0; 9{]}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Time until positivity, min\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
68.3 \selectlanguage{ngerman}± 25.6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
91.4 \selectlanguage{ngerman}± 62.03*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
77.1 \selectlanguage{ngerman}± 34.9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
82.8 \selectlanguage{ngerman}± 31.4 *\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
72.8 \selectlanguage{ngerman}± 28.1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
65.7 \selectlanguage{ngerman}± 21.4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Cumulative dose of birch allergen exposure, ng\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.06 \selectlanguage{ngerman}± 0.02\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.08 \selectlanguage{ngerman}± 0.06**\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.07 \selectlanguage{ngerman}± 0.03\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.08 \selectlanguage{ngerman}± 0.03**\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.07 \selectlanguage{ngerman}± 0.03\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.06 \selectlanguage{ngerman}± 0.02\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 3. Reproducibility of results with EEC exposure in seven patients
(step 2)

Data are given as mean ± standard deviation unless otherwise noted. EEC
= environmental exposure chamber; Expo = exposure; TOSS = Total Ocular
Symptom Score. * P = 0.045, **P =

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