Justicia et al. World Allergy Organization Journal (2021) 14:100545 http://doi.org/10.1016/j.waojou.2021.100545 Open Access Immunological parameters as biomarkers of response to MicroCrystalline Tyrosine- adjuvanted mite immunotherapy José L. Justiciaa*, Clara Padrób, Albert Rogerb, Francisco Morenoc, Manuel J. Riald, Antonio Parrad, Antonio Valeroe, Alfons Maletf, Aina Tenienteg,h, Anna Boronata and Carla Torán-Baronaa ABSTRACT Background: Despite the effectiveness of allergen immunotherapy (AIT), some patients are unresponsive for reasons still unknown; yet validated response biomarkers remain unavailable. Objective: To analyze immunological parameters as biomarkers to monitor and predict clinical response to a MicroCrystalline Tyrosine-adjuvanted house dust mite (HDM) AIT in patients with allergic rhinitis (AR). Methods: Observational, prospective, multicenter study including adult patients (aged 18–65 years) with AR, with and without asthma, sensitized to the HDM Dermatophagoides pteronyssinus (DP) and prescribed Acarovac Plusâ DP 100% in the routine practice. Serum concentrations of total IgE, specific IgE, specific IgG4, IL-4, IL-5, IL-10, IL-13, and IFN-g were compared between baseline and 12 months after AIT. The relationship between patients’ baseline immunological profiles and classification as low, high, and non-responders and between their sensitization profile to DP allergens and effectiveness were analyzed. Results: Of 141 patients recruited, 118 (mean [SD] age of 33.6 [9.5] years) were evaluable. One year after treatment, Der p 1-specific IgE, DP-specific IgG4, and IL-10 increased by a mean (SD) of 3.4 (13.6) kU/L (p ¼ 0.016), 0.43 (0.55) mg/L (p < 0.0001), and 1.35 (7.56) pg/mL (p ¼ 0.033), respectively. Non-responders showed increased baseline levels of IL-13 compared to high re- sponders (p ¼ 0.037). Changes in effectiveness variables between baseline and after AIT were similar regardless of the sensitization profile. Conclusion: Non-responsive patients to AIT showed increased baseline IL-13 concentrations, suggesting its value as prognostic biomarker. DP-specific AIT increased Der p 1-specific IgE, DP- specific IgG4, and IL-10 concentrations in patients with AR. All patients benefited from treatment regardless of their sensitization profile to major DP allergens. Keywords: House dust mite, Allergic rhinitis, Subcutaneous allergen immunotherapy, Micro- crystalline Tyrosine, Immunological biomarkers, Sensitization profile, Allergoid, Adjuvant a Allergy Therapeutics Ibérica, Sant Joan Despí, Barcelona, Spain Online publication date xxx *Corresponding author. E-mails: joseluis.justicia@gmail.com; 1939-4551/© 2021 The Author(s). Published by Elsevier Inc. on behalf of scervantesroldan@gmail.com World Allergy Organization. This is an open access article under the CC BY Full list of author information is available at the end of the article https://doi. license (http://creativecommons.org/licenses/by/4.0/). org/10.1016/j.waojou.2021.100545. Received 14 January 2021; Received in revised from 7 April 2021; Accepted 20 April 2021 2 Justicia et al. World Allergy Organization Journal (2021) 14:100545 http://doi.org/10.1016/j.waojou.2021.100545 INTRODUCTION patients with different predominant profiles of sensitization to major DP allergens. Allergic rhinitis (AR) is one of the most prevalent allergic diseases, with an estimated prevalence of 10%–40%.1 Its high symptom burden, together MATERIAL AND METHODS with that of its associated allergic conditions, Study design and population such as asthma, impact patients’ quality of life and daily activities, and generating high direct This observational, prospective, multicenter and indirect costs.1,2 Of the multiple allergens study was a sub-analysis of the immunological involved in AR, mites are one of the most variables obtained from a study in adult patients frequent, and, in Spain, they are the second (aged 18–65 years) diagnosed with allergic rhinitis cause of AR after pollen.3,4 The house dust mite for  1 year, with and without allergic asthma, (HDM) Dermatophagoides pteronyssinus (DP) is a caused by sensitization to HDM DP.18 A total of common indoor allergen comprising at least 23 141 patients, who attended visits at 10 Spanish different variants, of which Der p 1 and Der p 2 allergy centers between June 2015 and June are the most sensitizing.5–7 2016 and who were prescribed AIT with Acarovac PlusÒ DP 100% according to the routine practice, were consecutively included in Effective management of AR entails adequate the study. symptom control and, to this end, current guide- lines recommend allergen avoidance and phar- Acarovac PlusÒ is a purified allergen extract macological treatment.2,8 However, in many from the HDM DP modified with glutaraldehyde patients conventional pharmacological treatment and associated with MicroCrystalline Tyrosine may be insufficient; it induces side effects and (MCT) as an adjuvant. Treatment with Acarovac fails to provide long-term benefits.9 In contrast, PlusÒ was carried out in 2 phases: an up-dosing allergen immunotherapy (AIT) targets the phase consisting of 4 injections of 0.05, 0.1, 0.3, underlying cause of the disease by inducing and 0.5 mL at one-to two-week intervals and a immune tolerance to specific allergens, providing maintenance phase of 8 injections of 0.5 mL at six- long-term effects and potentially modifying the week intervals. Treatment administration started at course of the disease.10–12 Despite the growing Visit 1 (V1), and assessments were performed evidence about the clinical benefits of AIT, the during the Selection Visit (V0), V1 (4 weeks after degree of clinical response varies, being V0), Visit 2 (V2) (6  1 months after V1), and Final suboptimal in some patients for reasons still Visit (FV) (12  1 months after V1). The total dura- unknown.13,14 In this context, understanding the tion of the study was 13 months. All participating immunological mechanisms of immune tolerance patients provided written informed consent during induced by AIT may enable to identify the Selection Visit (V0). The study was conducted in biomarkers associated with clinical response. accordance with the Helsinki Declaration and the local Personal Data Protection Law (LOPD 15/ Despite the growing knowledge about the 1999); the study protocol was approved by the immunological mechanisms of AIT, investigators ethics committee of Hospital Germans Trias i Pujol claim the availability of validated biomarkers to (Barcelona, Spain) (EPA-14-023). monitor and predict the efficacy of AIT treatments Variables and clinical assessments at an individual level with the goal of improving patients’ clinical management.15–17 In this sub- Demographic and clinical variables, including analysis of the immunological results obtained in history of allergic disease, previous and concomi- a previous prospective study,18 we assessed the tant diseases, and concomitant medications were changes observed in immunological parameters recorded from patients’ medical records during in response to subcutaneous AIT (SCIT) with V0. Patients were also provided with a diary to Acarovac PlusÒ and evaluated the baseline record their symptoms (presence and intensity) immunological profile to identify predictive and use of medication to treat allergy symptoms biomarkers of clinical response. In addition, we for 4 weeks prior to V1, V2, and FV, and in- analyzed the effectiveness of Acarovac PlusÒ in vestigators collected the information at the Volume 14, No. 6, Month 2021 3 corresponding visit. Additionally, patients with available data regarding baseline serum values of asthma underwent a functional respiratory test immunoglobulins and cytokines and changes in (spirometry) during V0. The primary endpoint of CSMS one year after AIT were categorized ac- this study was to evaluate the effectiveness of AIT cording to their response (ie, CSMS reduction). by assessing changes between V1 (ie, symptoms Patients with a CSMS reduction <20%, 20%–50%, and use of medication during the 4 weeks be- and 50% were categorized as non-responders tween V0 and V1) and FV (ie, symptoms and use of (NRs), low responders (LRs), and high responders medication during the 4 weeks prior to FV) in the (HRs), respectively. Combined Symptom and Medication Score (CSMS), a 0-6-point scale described by the Euro- Patients who met the selection criteria and pean Academy of Allergy and Clinical Immu- received at least 1 treatment dose were evaluated. nology.19 Additional effectiveness variables Categorical variables were described as fre- assessed, included daily symptom and quencies and percentages, and quantitative vari- medication scores, “well” and “bad” days, clinical ables as the mean and standard deviation (SD) evaluation of symptoms, and Nasal Provocation and/or the median and interquartile range (IQR). Test results.18 Categorical variables were compared using the McNemar test and Fisher’s test and quantitative Immunological assessments variables were compared using the non- Serum concentrations of total IgE, specific IgE, parametric Mann-Whitney U and Wilcoxon tests, specific IgG4, and cytokines, including IL-4, IL-5, IL- for paired and unpaired data, respectively. The 10, IL-13 and IFN-g, were determined from blood baseline immunological parameters among the samples obtained at V0 and FV. DP-specific IgE different response groups were compared using (DP-sIgE), Der p 1-specific IgE (Der p 1-sIgE), Der p the one-way ANOVA test and a multivariate anal- 2-specific IgE (Der p 2-sIgE), and DP-specific IgG4 ysis (logistic regression), including the variables (DP-sIgG4) were determined according to the that yielded significant p values in the bivariate routine clinical practice using ImmunoCAPÒ analysis. ROC curves were used to assess the (Thermo Fisher Scientific, Massachusetts, USA), ability of baseline IL-13 levels to predict response whereas cytokines were determined by cytometric to AIT treatment by measuring their area under the bead array (BD Biosciences). receiver-operating curve (AUC). The significance threshold for all bivariate and multivariate analyses Statistical analysis was set at a two-sided a ¼ 0.05. All analyses were In order to analyze treatment effectiveness ac- performed using the statistical package support cording to patients’ predominant sensitization (Der PASS 2011 version and Prism GraphPad v6.0. p 1 vs. Der p 2), patients were categorized based on 7 classes of serum concentrations of specific RESULTS IgE to Der p 1 or Der p 2 (ie, Class 0: <0.35; Class 1: 0.35–0.70; Class 2: 0.71–3.50; Class 3: 3.51– Demographic, clinical and treatment 17.50; Class 4: 17.51–50.0; Class 5: 50.1–100; Class characteristics of study patients 6: >100 kU/L), allowing the definition of 3 groups: Of 141 patients initially recruited, 12 failed to (1) Predominant Sensitization to Der p 1 (PS to Der meet the inclusion/exclusion criteria and 11 p1), defined by Der p 1-sIgE class > Der p 2-sIgE remained untreated, resulting in an evaluable class; (2) Predominant Sensitization to Der p 2 study population of 118 patients with a mean (SD) (PS to Der p 2), defined by Der p 2-sIgE class > Der age of 33.6 (9.5) years.18 The demographic, p 1-sIgE class; and (3) Same Class/No Predominant clinical, and treatment characteristics of the study Sensitization (SC/NP), defined by equal sensitiza- population are summarized in Table 1. Mean (SD) tion to Der p 1 and Der p 2. time of rhinitis evolution was 11.72 (10.15) and In order to screen the immunological profile to 14.11 (18.77) years for the complete study identify immunological parameters as predictive population and the 55 patients with rhinitis and biomarkers of treatment response, patients with asthma, respectively. 4 Justicia et al. World Allergy Organization Journal (2021) 14:100545 http://doi.org/10.1016/j.waojou.2021.100545 2-sIgE and total IgE (tIgE) remained unchanged 1 Demographic Characteristics year after AIT (n ¼ 67 for both, p ¼ 0.297 and p ¼ 0.859, respectively) (Fig. 1A, B, D). Regarding Sex cytokines, IL-10 significantly increased by a mean Male 51 (43.2) (SD) of 1.35 (7.56) pg/mL (n ¼ 65, p ¼ 0.033) be- Female 67 (56.8) tween baseline and 1 year after treatment and IL-5 Clinical Characteristics showed a trend to decrease, albeit not significantly (p ¼ 0.065); whereas concentrations of IL-4, IL-13, Asthma diagnosis 55 (46.6) and IFN-g remained unchanged (n ¼ 65 for all, Other allergies 3 (2.5) p ¼ 0.216, p ¼ 0.982, and p ¼ 0.841, respectively) Food allergy 1 (0.8) (Fig. 1F–J). Conjunctivitis 1 (0.8) Treatment Characteristics Immunological profile as biomarker of treatment response Previous immunotherapy 2 (1.7) Immunotherapy for mites 1 (0.8) Of the 118 patients included in the study, 79 had available data for changes in CSMS between Medication for allergic rhinitis 103 (87.3) baseline and 1 year after AIT as well as baseline Oral antihistamines 92 (89.3) serum levels of immunoglobulins (tIgE, DP-sIgE, Nasal corticosteroids 71 (68.9) DP-sIgG4, Der p 1-sIgE, and Der p 2-sIgE) and Other concomitant medication 36 (30.5) cytokines (IL-4, IL-5, IL-13, IL-10 and IFN-g), and were categorized according to their response as Table 1. Demographic, clinical, and treatment characteristics of study patients, n (%) N ¼ 118 NRs, LRs, and HRs. A comparison of the baseline immunological parameters of NRs and HRs showed no significant differences between them. Immunological changes in response to AIT Likewise, the subset of patients with more severe Of the immunoglobulins analyzed, Der p 1-sIgE disease (n ¼ 40), identified as those with a baseline and DP-sIgG4 significantly increased by a mean CSMS 1.33—median baseline CSMS in the study (SD) of 3.4 (13.57) kU/L (n ¼ 67, p ¼ 0.0160) and population—were categorized as NRs (n ¼ 8), LRs 0.43 (0.55) mg/L (n ¼ 68, p < 0.0001), respectively, (n ¼ 9), and HRs (n ¼ 23) according to their clinical between baseline and 1 year after treatment response (Fig. 2). Of those, 32 patients had (Fig. 1C, E). The concentration of DP-sIgE showed a available baseline data for all cytokines (NRs, trend towards an increase from a mean (SD) of n ¼ 6; LRs, n ¼ 4; HRs, n ¼ 22) and were 26.5 (27.5) to 32.8 (31.6) kU/L, albeit not statisti- included in univariate and bivariate analyses. cally significant (n ¼ 67, p ¼ 0.092), whereas Der p Baseline serum levels of IL-13, IL-10, and TH2 Fig. 1 Serum concentrations of total IgE (A), DP-sIgE (B), Der p 1-sIgE (C), Der p 2-sIgE (D), DP-sIgG4 (E), IL-4 (F), IL-5 (G), IL-13 (H), IL-10 (I), and IFNg (J) at the indicated timepoints. Columns and error bars represent the median and interquartile range, respectively. Wilcoxon test *p < 0.05, **p < 0.01 Volume 14, No. 6, Month 2021 5 Fig. 2 Categorization of patients according to their response. Each data point corresponds to a patient with severe disease (CSMS 1.33). Lines separate patients’ categories according to treatment response, measured as the relative decrease of CSMS after AIT. AIT, Allergen immunotherapy; CSMS, Combined Symptom and Medication Scores profile (combined IL-4, IL-5, and IL-13) were significantly higher in NRs compared to HRs (p ¼ 0.019, p ¼ 0.043, and p ¼ 0.044, respectively, One-way ANOVA) (Fig. 3, Table 2). The remaining absolute and relative baseline immunological parameters evaluated, including tIgE, DP-sIgE, Der p 1-sIgE, Der p 2-sIgE, Der p 1þp 2-sIgE, DP- sIgG4, DP-sIgE/tIgE, Der p1-sIgE/tIgE, Der p2-sIgE/ tIgE, Der p 1þp 2-sIgE/tIgE, Der p 1-sIgE/DP-sIgE, Der p 2-sIgE/DP-sIgE, Der p 1 þ p 2-sIgE/DP-sIgE, DP-sIgG4/DP-sIgE, IL-4, IL-5, IFN-ɣ, TH1 (IFN-ɣ þ IL- 10), and TH1/TH2, showed no significant differ- ences between response categories (Table 2). The 3 immunological parameters that yielded significant differences were included in a multivariate analysis, being baseline IL-13 levels the only parameter that was statistically significant Fig. 3 Baseline serum concentrations of IL-13 (A), IL-10 (B), and TH2 (p ¼ 0.037). Consequently, patients with higher (IL-4 þ IL-5 þ IL-13) according to clinical response in patients with baseline IL-13 levels were less likely to respond to severe disease. Columns and error bars represent the mean and SD, respectively. Non-responders, n ¼ 6 and high responders, AIT (OR ¼ 0.475, 95% CI 0.236–0.957). The ROC n ¼ 22. SD, standard deviation *One-way ANOVA p < 0.05 for analysis of baseline IL-13 levels showed and AUC comparisons between groups of 0.788 (95% CI:0.562–1.01) (p ¼ 0.033). Regarding the sensitivity and specificity of IL-13 as prognosis factor of response, the most sensitive patients were sensitized to Der p 2 but not to Der p and specific cut-off was 1.335, showing a speci- 1, and 70 (70.7%) patients were sensitized to Der p ficity of 0.83 and a sensitivity of 0.73. 1 and Der p 2 (Table 3). Regarding the sensitization groups, 11 (11.1%) and 37 (37.4%) patients were predominantly sensitized to Der p Treatment effectiveness according to 1 and Der p 2, respectively, whereas 51 (51.5%) predominant sensitization patients showed no predominant sensitization Of the 99 patients with available data at baseline (i.e., equal sensitization to Der p 1 and Der p 2) for specific IgE to Der p 1 and Der p 2, 12 (12.1%) and were included in the Same Class/No were not sensitized to Der p 1 or Der p 2 (specific Predominant Sensitization (SC/NP) group IgE <0.35 kU/L, class 0), 5 (5.1%) patients were (Table 3). At baseline, CSMS, daily symptom sensitized to Der p 1 but not to Der p 2, 12 (12.1%) score, % of well and bad days, NPT symptom 6 Justicia et al. World Allergy Organization Journal (2021) 14:100545 http://doi.org/10.1016/j.waojou.2021.100545 Non-Responders High Responders P Valuea n¼6 n ¼ 22 tIgE (kU/L) 326.2 (414.2) 164.9 (209.8) 0.193 DP-sIgE (kU/L) 25.08 (18.26) 22.22 (23.11) 0.782 DP-sIgG4 (mg/L) 0.46 (0.30) 0.31 (0.23) 0.179 Der p 1-sIgE (kU/L) 9.90 (8.85) 8.63 (12.07) 0.812 Der p 2-sIgE (kU/L) 9.61 (6.94) 11.73 (12.71) 0.700 Der p 1þ p 2-sIgE (kU/L) 19.52 (14.24) 20.36 (23.50) 0.935 DP-sIgE/tIgE 17.66 (16.98) 18.38 (11.77) 0.906 Der p 1-sIgE/tIgE 6.00 (7.56) 6.04 (5.13) 0.985 Der p 1-sIgE/DP-sIgE 31.64 (22.87) 31.19 (18.82) 0.961 Der p 2-sIgE/tIgE 8.08 (10.91) 10.24 (9.05) 0.622 Der p 2-sIgE/DP-sIgE 33.17 (21.69) 52.91 (38.31) 0.241 Der p 1þ p 2-sIgE/tIgE 14.07 (15.33) 16.29 (11.14) 0.693 Der p 1 þ p 2-sIgE/DP-sIgE 64.81 (34.70) 84.11 (41.94) 0.312 DP-sIgG4/DP-sIgE 30.23 (23.87) 36.25 (41.81) 0.740 IL-4 (pg/ml) 2.92 (2.48) 1.43 (1.56) 0.080 IL-5 (pg/ml) 1.80 (1.42) 1.06 (1.09) 0.178 IL-13 (pg/ml) 2.37 (1.54) 0.95 (1.15) 0.019 IL-10 (pg/ml) 4.49 (3.62) 1.70 (2.62) 0.043 IFN-ɣ (pg/ml) 3.68 (4.55) 1.50 (3.23) 0.191 TH1 (IFN-ɣ þ IL-10) (pg/ml) 8.17 (8.07) 3.20 (5.71) 0.095 TH2 (IL-4 þ IL-5 þ IL-13) (pg/ml) 7.10 (4.73) 3.45 (4.73) 0.044 TH1/TH2 Ratio 0.95 (0.40) 0.76 (0.75) 0.565 Table 2. Baseline immunological parameters according to treatment response, mean (SD). Abbreviations: DP, Dermatophagoides pteronyssinus; sIgE, specific IgE; sIgG4, specific IgG4; tIgE, total IgE. a. One-way ANOVA. Statistically significant differences are shown in bold type. score, and ESPRINT-15 questionnaire scores were Changes in the effectiveness variables 1 year similar between groups, whereas daily medication after AIT were assessed in those patients with score, NPT (% drop of NIPF), and visual analog available data, were similar between the predom- scale (VAS) scores showed statistically significant inant sensitization groups and showed no signifi- differences between groups (Table S1). The cant differences, suggesting that treatment with distribution of patients according to baseline the DP allergoid was effective regardless of the rhinitis classification, including frequency predominant sensitization (Table 4). Remarkably, (persistent or intermittent), severity (mild and the 12 patients who were not sensitized to Der p moderate/severe), and control (not controlled, 1 and Der p 2 showed a mean (SD) reduction in partially controlled, and controlled) was similar CSMS of 0.60 (0.89), indicating a clinical benefit between predominant sensitization groups in patients sensitized to DP allergens other than (Fig. S1). Der p 1 and Der p 2. Similar to the quantitative Volume 14, No. 6, Month 2021 7 Der p 1 Der p 2 Class 0 Class 1 Class 2 Class 3 Class 4 Class 5 Class 0 12 (12.12) 0 (0) 1 (1.01) 3 (3.03) 1 (1.01) 0 (0) Class 1 2 (2.02) 0 (0) 1 (1.01) 0 (0) 0 (0) 0 (0) Class 2 3 (3.03) 1 (1.01) 7 (7.07) 1 (1.01) 0 (0) 0 (0) Class 3 5 (5.05) 0 (0) 10 (10.1) 19 (19.19) 3 (3.03) 0 (0) Class 4 0 (0) 0 (0) 0 (0) 12 (12.12) 10 (10.1) 1 (1.01) Class 5 2 (2.02) 0 (0) 0 (0) 0 (0) 2 (2.02) 3 (3.03) Table 3. Distribution of patients according to baseline levels of specific IgE for Der p 1 and Der p 2, n (%) n ¼ 99 Change Baseline - One Year After AIT P Valuea Der p 1 Der p 2 SC/NP Der p 1 vs. Der p 1 vs. Der p 2 vs. n ¼ 11b n ¼ 37 n ¼ 51 Der p 2 SC/NP SC/NP CSMS n ¼ 66 1.09 0.58 0.79 0.332 0.400 0.815 (1.22) (1.37) (1.05) n¼8 n¼28 n¼30 Daily symptom 0.31 0.23 0.53 0.897 0.485 0.187 score n ¼ 67 (0.79) (0.81) (0.69) n¼8 n¼29 n¼30 Daily medication 0.69 0.37 0.24 0.354 0.102 0.259 score n ¼ 72 (0.71) (0.86) (0.60) n¼9 n¼29 n¼34 % Well days n ¼ 73 26.6 14.6 15.1 0.368 0.570 0.995 (54.5) (44.5) (46.6) n¼9 n¼30 n¼34 % Bad days n ¼ 69 4.76 0.74 6.68 0.509 0.879 0.408 (34.4) (26.7) (18.3) n¼9 n¼29 n¼31 ESPRINT-15 global 1.63 1.04 1.58 0.181 0.683 0.155 score n ¼ 69 (0.92) (1.90) (1.64) n¼8 n¼28 n¼33 Visual Analog Scale 3.33 4.41 3.49 0.367 0.709 0.258 score n ¼ 71 (1.65) (3.29) (2.74) n¼9 n¼28 n¼34 NPT (% drop of 23.2 12.5 8.0 0.764 0.285 0.516 nasal inspiratory (23.7) (42.2) (28.0) peak flow) n ¼ 52 n¼5 n¼23 n¼24 NPT (symptom 1.33 2.09 2.08 0.765 0.773 0.949 score) n ¼ 53 (2.42) (2.78) (2.47) n¼6 n¼23 median), serum levels of DP-sIgE and significantly increased those with poor or no response to treatment had Der p 1-sIgE shown by the results of this study significantly higher baseline levels of IL-13 most likely reflected allergen exposure during AIT compared to highly responsive patients. Addition- administration. Despite the heterogeneous results ally, the analysis of effectiveness variables according of previous studies, overall, it is accepted that DP- to patients’ sensitization profiles showed similar sIgE levels transiently increase during AIT treat- outcomes, indicating that treatment with Acarovac ment without clinical or functional relevance.17,21– 27 PlusÒ was effective regardless of the predominant While previous studies have mostly focused on sensitization. DP-sIgE, our study further distinguished between the 2 major HDM allergens, Der p 1 and Der p 2, Immunological parameters can serve as surro- showing increased Der p 1-sIgE but not Der p 2- gate measures of clinical response to AIT, playing sIgE. In this regard, previous studies assessing a role as biomarkers to guide diagnosis and conventional aluminum-formulated AIT reported management, including treatment start, discontin- transient increases in Der p 1-, Der p 2-, and Der p uation, and reinstatement, and predict response to 23-sIgE after SCIT, suggesting higher allergenicity of this formulation compared to the MCT- associated allergoid used in this study.7,28 In addition, similar to previous studies assessing AIT with HDM allergens, our results showing a significant 2.2-fold increase in DP-sIgG4 one year after AIT confirm the accepted notion that AIT re- sults in increased DP-sIgG4 and reflect the immu- nogenicity of the allergoid used in this study.22,26– 29 In this regard, the use of an allergoid instead of a native extract may have reduced the allergenicity Fig. 4 Combined symptom and medication score (CSMS) while maintaining the immunogenicity of the according to predominant sensitization at baseline. Columns and allergen, resulting in modest increases of some, error bars represent the median and interquartile range, respectively. SC/NP, same class/no predominant sensitization. albeit not all, IgE and a concomitant increase in Wilcoxon test *p < 0.05 and **p < 0.01, for differences between DP-sIgG4. Likewise, the use of MCT as an adju- timepoints vant, which, unlike other adjuvants (ie, aluminum), Volume 14, No. 6, Month 2021 9 has been shown to curb the allergen-mediated restrained the action of AIT-induced regulatory induction of IgE, may explain the modest in- cytokines, limiting AIT clinical benefit. crease in IgE.30 Despite our comprehensive evaluation of Regarding cytokines as biomarkers of treatment immunological parameters as biomarkers of clin- effectiveness, the results from this study, which ical response, serum immunoglobulins lacked a show a significant increase in IL-10 and a decrease, predictive value. Previous studies have shown that albeit not significant, in IL-5, are in line with the DP-sIgE, DP-sIgE/tIgE, and Der p 1-sIgE/Der p 1- expected increase in TH1 responses at the expense sIgG4 may be useful biomarkers to predict clin- of TH2 responses. Similarly, previous studies ical responses to AIT for HDM allergy, even though assessing AIT with HDM allergens have shown that these results are yet to be confirmed.22,25,27,36,37 allergen-induced IL-10 production paralleled a In this regard, our study and previous ones global suppression of TH2 proliferative responses assessed immunoglobulin and cytokine levels and cytokine production.31 Importantly, IL-10 has using different samples (ie, serum and isolated been shown to suppress allergen-specific IgE peripheral blood mononuclear cells) and secretion and increase sIgG4 production.32–34 methods with different sensitivity thresholds. Even though the relationship between changes in These methodological differences preclude these parameters and clinical outcomes remains direct comparisons among studies and may to be analyzed, the immunological changes partially explain the heterogeneous results from observed in this study agree with the role of IL- the different studies, hampering the identification 10 and sIgG4 as mediators of immune tolerance, of validated biomarkers. supporting the notion that immunotherapy re- stores a tolerant T-cell response.31 Marketed AIT products differ in contents, con- centrations, and ratios of individual allergens, Due to the current indication of AIT in patients particularly those obtained from natural extracts, with moderate/severe symptoms and/or unre- likely influencing the degree of response.38,39 In AIT sponsive to pharmacological treatment, we used for HDM, which contains different allergen the corresponding population of patients (ie, those molecules, the differences in the allergen with more severe symptoms) to evaluate immu- composition of marketed AITs may further nological parameters as predictors of treatment influence their ability to induce immune tolerance response.13 Considering the exploratory nature of in patients with different sensitization profiles. these analyses and to increase the ability of IL-13 Results obtained from this study, which show no to predict patients’ response, we excluded low differences between changes in effectiveness responders (LRs) from the analysis and focused on variables according to the predominant the most extreme responders, non-responders sensitization, indicate that Acarovac PlusÒ is (NRs) and high responders (HRs). We found that effective regardless of the sensitization profiles increased baseline levels of IL-13, IL-10, and TH2 represented in this study. Similar to this, previous cytokines (combined IL-4, IL-5, and IL-13) were studies assessing other AITs also reported similar associated with no response, albeit only IL-13 effectiveness in patients with different sensitization remained significantly different in the multivariate profiles.7,40 analysis, showing the potential value of IL-13 as predictor of treatment response. The production of Despite its prospective nature, the results from IL-13 by allergen-specific TH2 CD4þ cells plays a this study should be interpreted in the context of role in allergen sensitization by switching antibody some limitations related to its real-life setting. The production to allergen-specific IgE. Conversely, inclusion of all patients starting AIT regardless of during AIT-induced immune tolerance, the action their immunological profile resulted in an uneven of IL-13—together with that of other TH2 cytokines— distribution of patient groups for analysis, with a is blunted by regulatory cytokines, including IL-10 reduced number of patients in some categories and TGFb.16,35 Considering the regulatory role of (ie, predominant sensitization groups). Addition- increased IL-10 in antagonizing TH2-mediated IgE ally, the detection of serum levels of cytokines, responses during AIT-induced immune tolerance, which are typically low, was constrained by the increased baseline levels of IL-13 in NRs likely methods used in real life, precluding the use of 10 Justicia et al. World Allergy Organization Journal (2021) 14:100545 http://doi.org/10.1016/j.waojou.2021.100545 more sensitive methods frequently used in clinical Abbreviations trials and potentially limiting the sensitivity of our AIT, allergen immunotherapy; ANOVA, analysis of variance; analyses. Likewise, Der p 23-IgE, Der p 1-sIgG4 AR, allergic rhinitis; CI, confidence interval; CSMS, combined symptom and medication score (CSMS); Der p and Der p 2-sIgG4 are not routinely assessed in the 1-sIgE, Der p 1-specific IgE; Der p 2-sIgE, Der p 2-specific Spanish clinical practice and few centers have their IgE; DP, Dermatophagoides pteronyssinus; DP-sIgE, Der- laboratories equipped to determine serum con- matophagoides pteronyssinus-specific IgE; DP-sIgG4, centrations of these immunoglobulins. For this Dermatophagoides pteronyssinus-specific IgG4; HDM, reason, IgG4 specific to the DP allergens and house dust mite; HRs, high responders; IQR, interquartile sensitization to Der p 23, despite being a major range (Q1, Q3); LRs, low responders; MCT, MicroCrystal- line Tyrosine; NIPF, nasal inspiratory peak flow; NPT, nasal allergen, were not assessed in this study. Further- provocation test; NRs, non-responders; OR, odds ratio; SC/ more, while SCIT with HDM allergen extracts is NP, same class/no predominant sensitization; SD, standard typically administered for 3 years, patients were deviation; VAS, visual analog scale. assessed after one year of AIT, precluding the analysis of immunological parameters after pro- Agreement to publish the work longed periods of treatment. In spite of these All authors have reviewed and approved the final version of limitations, the size of the study was large and was the manuscript. able to capture immunological response in a population mirroring the sensitization profiles of Author contributions patients from different centers, encompassing JLJ, AB and CT-B made substantial contributions to the different geographical areas. Unlike clinical trials, conception and design of the work; and the analysis, and this study lacked strict selection criteria, allowing interpretation of data for the work. CP, AR, FM, MJR, AP, to capture the effects of Acarovac PlusÒ in the AV, AM and AT made substantial contributions to the acquisition of data for the work. JLJ and CT-B drafted the heterogeneous population found in routine clinical article and CP, AR, FM, MJR, AP, AV, AM, AT and AB practice. This study confirms that Acarovac PlusÒ contributed to revising the work critically for important in- partly summarizes previously described immuno- tellectual content. All authors gave their final approval of logical responses to other AIT treatments and the version to be published, and agree in their account- provides useful information for clinicians to stratify ability for all aspects of the work in ensuring that questions patients and optimize AIT prescription and clinical related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. management of patients with allergy to HDM. Furthermore, our finding that IL-13 may be a potentially valuable biomarker to predict treat- Ethics approval and consent to participate ment response in non-responsive and hyper- All included patients and legal representatives of patients <18 years signed a written informed consent before any responsive patients warrants future larger studies. data was recorded. The study was conducted in accordance with the Helsinki Declaration and the local Personal Data Protection Law (LOPD 15/1999); the study protocol was CONCLUSION approved by the Ethics Committee of Hospital Germans Trias i Pujol (Barcelona, Spain) (EPA-14-023). Our results obtained from a cohort of patients with allergic rhinitis with and without asthma treated in the real-world setting provide evidence Editorial policy confirmation and agreement of increased levels of DP-sIgG4, Der p 1 sIgE, and I hereby state that the manuscript, including related data, figures and tables, has not been previously published and IL-10 as mechanisms of immune tolerance induced has not been submitted to any other journal (in any other by AIT specific for HDMs. In addition, increased language or any other type of publication), either by me or baseline levels of IL-13 in non-responders suggest any of my co-authors. that this immunological parameter might serve as biomarker to predict clinical response. Finally, our Availability of data and materials study suggests that patients sensitized to different The anonymized datasets collected during the current DP allergens may benefit from treatment with an study are available from the corresponding author on MCT-associated house dust mite allergoid. reasonable request. Volume 14, No. 6, Month 2021 11 Declaration of competing interest 7. Rodríguez-Domínguez A, Berings M, Rohrbach A, et al. This work was supported by Allergy Therapeutics Ibérica Molecular profiling of allergen-specific antibody responses (Sant Joan Despí, Barcelona, Spain). JLJ, AB and CT-B are may enhance success of specific immunotherapy. J Allergy Clin Allergy Therapeutics employees; CP has collaborated with Immunol. 2020;146(5):1097–1108. https://doi.org/10.1016/j. jaci.2020.03.029. Allergy Therapeutics Ibérica to attend courses/congresses/ medical training; AR reports personal fees from Allergy 8. Bro_zek JL, Bousquet J, Agache I, et al. Allergic rhinitis and its Therapeutics, during the conduct of the study; personal impact on asthma (ARIA) guidelines—2016 revision. J Allergy fees from Leti, Stallergenes, Roxall, Diater, and Hal, outside Clin Immunol. 2017;140(4):950–958. https://doi.org/10.1016/j. the submitted work; MJR reports personal fees from Astra- jaci.2017.03.050. Zeneca, GSK, Allergy Therapeutics, and Chiesi, outside the 9. Wheatley LM, Togias A. Allergic rhinitis. Solomon CG, ed submitted work; FM, AP, AV, AM and AT have nothing to N Engl J Med. 2015;372(5):456–463. https://doi.org/10.1056/ disclose. NEJMcp1412282. 10. Bousquet J, Lockey R, Malling H. Allergen immunotherapy: Acknowledgements therapeutic vaccines for allergic diseases A WHO position The authors would like to thank i2e3 Biomedical Research paper. J Allergy Clin Immunol. 1998;102(4):558–562. https:// Institute for providing medical writing support during the doi.org/10.1016/S0091-6749(98)70271-4. preparation of the manuscript. 11. 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