Allergen exposures

This procedure was performed following the updated European Academy of Allergy and Clinical Immunology (EAACI) guidelines,¹ and comprised the direct intranasal application of 50 μL, with 2 puffs per nostril: 1 in the inferior meatus and 1 on the direction of the middle turbinate. Diluted birch standardized allergen (Allergopharma®) was dispensed using a pump-aerosol spray, by increasing doses until ΔTNSS reached 5. First, a placebo dose was administered, then a maximum of 5 increasing doses were applied at an interval of 10 min until achieving a nasal response. TNSS was assessed every 10 min, after each increasing dose of allergen.

This EEC is 65 m² with 20 seats, and enables the provision of a homogenous distribution of allergens for each participant. This facility was conceived as a clean room, in which the airflow system, environmental conditions, and airborne particles are continuously monitored. Communication between participants inside the EEC and with medical supervisors was enabled using wireless telecommunication.

The same batch of birch allergen extract (Allergopharma®) was used for all exposures. Participants entered the EEC once the plateau of airborne allergen was reached. Before each allergen session, participants received saline nasal lavage. All exposures lasted up to 4 h. Homogeneous allergen distribution was ensured using particle counters, as previously described.^16,25 After each exposure, the Bet v 1 concentration was determined using ELISA (Indoor Biotechnologies®, Charlottesville, VA, USA) by collecting allergen on glass fiber filters located next to the participants’ chairs.

Four rhinitis phenotypes have been defined.^26,27 Early-phase responders (EPRs) were those who experienced at least a 50% drop in TNSS by 7 h post-exposure, and returned to baseline with no second increase in symptoms afterward. Protracted early-phase responders (pEPRs) were defined as those who exhibited no 50% drop in TNSS, as well as no increase in symptoms up to hour 12 post-exposure. Late-phase responders (LPR) exhibited a 50% drop in TNSS by hour 7 post-exposure, followed by stable symptoms without recovery of 2 points in TNSS. Finally, dual responders (DRs) were those who experienced at least a 50% decrease in TNSS by hour 7 post-exposure, with an at least 2-h period of decline in TNSS severity, followed by a plateau of symptoms.

The definition of the birch pollen season—including its peak period, start, and end, and the correlation of patient-reported symptom loads for birch pollen-induced allergic rhinitis—was elaborated by the EAACI. The start of the peak pollen period corresponds to first day of 3 consecutive days, each with ≥100 pollen/m³, and the end is the last day of at least 3 consecutive days, each having ≥100 pollen/m³.^28,29 In Eastern France, the birch pollen season starts at the end of March and peaks between early and mid-April. During the peak period, TNSS was evaluated twice a day, in the morning and evening, and considered positive if the score was ≥5.

This single-center, single-blind, placebo-controlled study was designed to validate use of a EEC for studying allergic rhinitis to birch pollen. We also performed a second analysis of nasal allergen responses during NAC, EEC exposure, and natural exposure in 30 adults allergic to birch.

The primary end-point was defined as the frequency of nasal response assessed by the TNSS during the last 2 h of the 4-h EEC exposure (H2–H4), and was met when at least 60% of participants reached ΔTNSS ≥5 at first allergen exposure (Visit 5), as compared to placebo (Visit 3). The secondary end-points were to assess TNSS over 4 h of exposure (H0–H4), as well as nasal obstruction using PNIF²² and VAS rhinitis.²³ Finally, we measured the TNSS under three different conditions: NAC, EEC, and natural exposure.

The first part of the study consisted of a screening visit to obtain informed written consent to participate, and to conduct a medical history review, skin prick testing, and a blood draw for birch IgE. On the second visit, patients underwent NAC to birch allergen, and only those with positive NAC were selected to participate in the rest of the study. Participants who met the inclusion and exclusion criteria underwent 6 EEC exposures. First, participants attended two 4-h consecutive placebo EEC visits (Visits 3 and 4). After 7 days, participants attended two 4-h consecutive EEC allergen exposures (Visits 5 and 6), which were then repeated two additional times at 14 days intervals (Visits 7 and 8), to assess reproducibility and priming effect (Fig. 1). Exposures were conducted outside of the 2020 tree pollen season in France. The second part of the study involved field evaluations of allergic rhinitis signs and symptoms, and rescue medication use, during the peak birch pollen period. Participants completed the TNSS by the end of March–beginning of April 2021 (with some SARS-CoV-2 pandemic restrictions).

Flow diagram and study design. Abbreviations: PCB, placebo; expo, exposure; NAC, nasal allergen challenge; SPT, skin prick test

This study was approved by an independent ethics committee, and was conducted in accordance with 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 {"type":"clinical-trial","attrs":{"text":"NCT04583202","term_id":"NCT04583202"}}NCT04583202.

Statistical analysis was performed using SAS 9.4 software® (SAS Institute, Cary, NC). Statistical analyses were conducted according to the Statistical Analysis Plan (SAP) version 3.0 July 16, 2021. Since the intention-to-treat (ITT) and per protocol (PP) populations were similar, efficacy analyses were performed on the ITT population. Quantitative variables were described using the usual parameters (total number of data points, missing data, mean, median, minimum, maximum), as well as classical dispersion parameters (standard deviation and interquartile range for each group, period, and time). There were no reliable data regarding the reproducibility of symptoms in participants who developed a nasal reaction in the EEC. The number of participants required was determined pragmatically. We decided to include a total of 30 participants, with an expected nasal reaction proportion of 60%, and a two-sided 95% confidence interval ranging from 41 to 77%. Missing data were not replaced. Continuous variables were described as the number of observed data points, mean and standard deviation of normally distributed values, or median (interquartile range) for non-normally distributed values. Categorical variables were described as the number of participants and percentage in each category. For inferential statistics, P values of < .05 were considered significant. The priming effect was evaluated by the ratio of the scores for the presence of a nasal response at V5 and V6. The same calculation was performed between V7 and V8 for confirmation. Odds ratios and their two-sided 95% confidence intervals were estimated using a logistic model for repeated measures.