Birch allergen challenges in allergic conjunctivitis using standard conjunctival allergen challenge and environmental exposure chamber

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 pollen. Methods Sixteen patients with a positive conjunctival allergen challenge (CAC) were exposed to 60 ng/m3 of Bet v 1 in an EEC on two consecutive days for a maximum of 4 h to validate EEC exposure to birch. Reproducibility was tested among seven of the patients. A conjunctival positive scoring during the CAC and the EEC exposure was defined as a Total Ocular Symptom Score (TOSS) ≥ 5. Results Fifty percent of patients had a conjunctival positive scoring during first exposure and 75% during second exposure. The mean time to a conjunctival response was 81.2 ± 33.9 min and 101.6 ± 57 (P > 0.05) during first and second exposure, respectively. No difference in 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). Conjunctival positive scoring and quantity of Bet v 1 was reproducible in all six EEC exposures. Conclusions Early conjunctival responses induced by birch allergen exposures in EEC were different than from those identified with direct instillation during CAC. EEC appears to be closer to natural exposure than CAC.


| BACKGROUND
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 may not be associated with allergic rhinitis. 1 IgE-mediated hypersensitivity inducing conjunctivitis is frequent, from 15% to 20% in general practice 2,3 to 40% in the US population when examined in an ophthalmological survey. 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. 5,6 Birch is one of the most frequent sources of allergens that induce rhinoconjunctivitis. 7 In the general French adult population, the prevalence of birch sensitization was 4.7%. 8 In a recent analysis, a statistically significant correlation has been found between the birch pollen concentration levels during the pollen season as well as the peak pollen period and the pollen-induced symptoms such as Total Nasal Symptom and Medication Score (TNSMS). 9 In another study, the frequency of the ocular response to natural exposure to birch pollen in sensitized participants followed a liner progression un- (FDA) for testing the efficacy of eye anti-allergic molecules. 10 The practical aspects were described in a position paper of the European Academy of Allergy and Clinical Immunology (EAACI) Task Force. 11 Environmental exposure chambers (EECs) have been in development since 1985 to study new therapeutics for allergic pathologies, including conjunctivitis. 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. 14,15 The ALYATEC EEC has been validated in mite and cat-induced allergic asthma. 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 (Betula verrucose major allergen) and the time necessary to induce a conjunctival response in at least 50% of participants evaluated on TOSS scoring of at least 5 (primary endpoint). We also evaluated the reproducibility of the EEC method.

| Demographic characteristics
Participants aged from 18 to 65 years were selected for eligibility based on having a history of more than two years of moderate allergic conjunctivitis during birch pollen season. 1,11 Allergic sensitizations were documented by a positive skin prick test to birch allergen with a wheal diameter ≥6 mm compared to negative control and positive birch-specific IgE (>0.10 kU/l). The main inclusion criterion was a conjunctival positive scoring (defined as TOSS scoring reaching up to five points 15 min after instillation of the culprit allergen) during an individual CAC. 10,11 The study was performed outside the pollen season (September-December) in France. A 7-days washout period was required for topical or systemic antihistamines or other ophthalmic treatment. Exclusion criteria were evaluated prior to inclusion and were as follows: participants experiencing moderate ocular symptoms in the previous week; participants who received systemic long-acting corticosteroids within the past 4 weeks; ocular laser treatment within the past 3 months; ocular surgery within the last 6 months; abnormality or clinically significant current ocular disorder, including symptoms of allergic conjunctivitis; ongoing immunotherapy to any allergen or, within the last 5 years, to birch allergen.

| Interventions
This was an open, single-center study designed to determine the concentration of airborne Bet v 1 inducing an allergic conjunctivitis response in participants 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-Abello®), and a blood draw for investigating specific IgE to birch (Betula verrucose, Phadia ImmunoCap, Thermofisher®). The second screening visit was for an individual CAC to birch allergen. All responders in the CAC were included in the present study ( Figure 1).
The CAC was performed according to the updated EAACI guidelines. 1,11 This procedure consisted of the instillation of 20 µl of diluted birch allergen extract (100 IR lyophilized extract, Stallergenes Greer®) 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. 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 <5 at 10 min after each instillation, the test was considered negative. The next concentrated dose was then instilled until a positive response was reached. One week after CAC, patients were enrolled for EEC visits.
Step 1 of the study consisted of two EEC exposures in consecutive days to the same birch pollen extract (Expo 1 and Expo 2). This was sustained to mimic daily life, and to reproduce an eventual priming effect. The primary endpoint of the study was met when at least 50% of the participants had a conjunctival positive scoring. The main judgment criteria were the amount of Bet v 1 inducing a conjunctival response after EEC exposure. In addition, we compared the intensity of the clinical response induced in participants exposed to birch pollens using the TOSS and the mean time to reach a positive ocular response (i.e., TOSS ≥ 5). In step 2, we studied the reproducibility of the allergen exposure. Participants who responded to Expo 1 and Expo 2 were enrolled in step 2 and exposed two additional times on two consecutive days (from Expo 3 to Expo 6). Each double EEC exposure test was separated by 14 days.

| Clinical assessments
The TOSS was used during both the CAC and EEC exposures. This score was first described 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 NaCl solution. 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 four-point severity score (0 = absent; 1 = mild: perhaps localized within some quadrant, 2 = moderate: more marked and diffuse reddening in the quadrants; 3 = severe: very marked and diffuse reddening in the quadrants). 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 ≥5 (Table 1).

ALYATEC EEC is a new generation EEC located in Strasbourg,
France. It was conceived as a clean room with low-emitting, nonadherent and easily-cleaned materials. The absence of endotoxins and VOCs levels is periodically evaluated. The volume of the EEC is 147 m 3, (area = 65 m 2 ) with a capacity up to 20 seats. During allergen exposures, airborne particles number and size are continuously monitored in order to ensure homogenous distribution of allergen 16,17 In this study, participants were exposed to the same batch of lyophilized birch allergen extract (100 IR; Stallergenes Greer®), diluted in a NaCl solution, as the one used for the CAC. Before each EEC exposure, lyophilized birch allergen was prepared in a NaCl solution and nebulized in the EEC. The concentration of Bet v 1 in the allergen extract was measured before and after nebulization, using ELISA. Nebulization of birch allergen was initiated 30 min before patients entered the EEC, allowing to reach a stable plateau of airborne particles. The homogeneous distribution of allergen was ensured by using 10 particle counters in the EEC with real-time monitoring. Therefore, the size and number of particles were continuously monitored during 4 h of exposure.
Online measurements of the temperature, relative humidity, and air exchange were provided as described in the previous studies. 16,17 After each allergen exposure, allergen was collected on five glass fiber filters located next to the participants' chairs during exposure to determine the Bet v 1 concentration using ELISA (Indoor Bio-technologies®). The concentration of the Bet v 1 airborne allergen was estimated to be 60 ng/m 3 . During each EEC allergen exposure, the conjunctival scoring was assessed every 10 min during the first hour and then every 20 min.

| Quantity of Bet v 1 (Q) inducing a conjunctival positive scoring
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 154 � 10 6 mm 3 /min by Beaudouin et al. 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 3 and Time is the time required to induce the conjunctival positive scoring in minutes.

| Statistical methods
All statistical analyses were performed using SAS ® guide Enterprise software (SAS Institute). 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). Categorical variables were described as the participants' size and percentage in each category. For inferential statistics, P-values < 0.05 were considered significant. Tests were two-tailed.
Reproducibility was tested using a Pearson correlation between exposure days.

| Participants
Among 23 screened participants, 16 met the inclusion criteria and performed two consecutive EEC exposures. Eight participants who responded at Expo 2 were included in step 2 ( Figure 1). Patient characteristics are given in Table 2

| Reproducibility of EEC exposures (step 2)
The eight participants included in step 2 had identical characteristics to the whole cohort from step 1 ( Table 2). One patient dropped out of

| DISCUSSION
In the present study, when comparing ocular symptoms of partici- 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 ≥ 5 during the CAC as the threshold for a conjunctival response according to European guidelines. 11 This threshold has been demonstrated to allow a specificity and sensitivity of 100% and 90%, respectively, in mite allergic conjunctivitis. 20 We used the same clinical positivity criteria for EEC exposure to birch. In our study, the mean TOSS was not significantly different after CAC and EEC exposure. The maximal TOSS was 9 in both the CAC and EEC. In  Cumulative dose of birch allergen exposure, ng 0.06 ± 0.02 0.08 ± 0.06** 0.07 ± 0.03 0.08 ± 0.03** 0.07 ± 0.03 0.06 ± 0.02 Note: Data are given as mean ± standard deviation unless otherwise noted.
Abbreviations: EEC, environmental exposure chamber; Expo, exposure; TOSS, Total Ocular Symptom Score; *P = 0.045, **P = 0.028 (Pearson correlations between Expo 2 and 4). and the occurrence of ocular symptoms. In daily practice, patients do not describe significant conjunctivitis symptoms in day-to-day life within 30 min after being exposed to birch pollen. When comparing patients with and without conjunctival positive scoring after EEC exposure, we observed no difference in terms of level of sensitization to birch pollens and also in terms of comorbidities. 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 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-h period after exposure. 23 Prior studies suggested that two, or even three, priming visits may be required to obtain high levels of symptoms. 24,25,26 However, the priming effect leads to rapid onset of symptoms and signs rather than a greater allergic response. 27 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., 23 who investigated phenotypes of allergic conjunctivitis.
Other clinical symptoms of conjunctivitis, such as tearing and itching, In our study, atopic status was evidenced by positive SPT to birch pollen and a high level of serum specific IgE. At inclusion, they reported birch pollen-induced allergic conjunctivitis with or without rhinitis for at least 2 years with peak symptoms in March-April. All have a positive CAC at the inclusion. Nevertheless, 25% among them did not obtained a TOSS ≥ 5. These results could be explained by the difference of allergen amount during the two procedures. Allergen exposures in the EEC appear to be closer to natural exposure.
The limitation of this study is the small number of participants.
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. Nevertheless, one can suggest that EEC exposures is closer to natural exposure than the CAC exposure.

| CONCLUSION
We demonstrated that exposure to 60 ng/m 3 of Bet v 1 in the ALYATEC EEC induces conjunctival positive scoring in more than 50% of participants with birch allergic conjunctivitis. Birch allergen exposures inducing early conjunctival responses were different than those identified with direct instillation during CAC, suggesting that EEC is closer to natural exposure during birch pollen season.

ACKNOWLEDGMENTS
Clinical data collection and data analysis provided by Nicole Beck from ALYATEC. Expertise on slit lamp examination provided by Pr.
The author(s) received no financial support for the research, authorship, and/or publication of this article.