Virtual Issue 1

Received: 29 January 2019 | Revised: 8 July 2019 | Accepted: 14 July 2019 DOI: 10.1111/all.14020
ORIGINAL ARTICLE Asthma and Lower Airway Disease
The moderating role of allergy immunotherapy in asthma progression: Results of a population‐based cohort study
Jochen Schmitt1,2 | Eike Wüstenberg2,3,4 | Niels Serup‐Hansen5 | Falko Tesch1
1Center for Evidence-Based Healthcare, TU Dresden, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany
2University Allergy Center, TU Dresden University Hospital Carl Gustav Carus, Dresden, Germany
3ALK-Abelló, Hamburg, Germany
4Department for Otorhinolaryngology, TU Dresden, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany
5ALK-Abelló, Hørsholm, Denmark
Correspondence
Jochen Schmitt, Center for Evidence-Based Healthcare, TU Dresden, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany.
Email: Jochen.Schmitt@uniklinikum- dresden.de
Funding information
ALK-Abelló, Grant/Award Number: /
Denise Küster1 |
Victoria Mücke3 |
Abstract
Background: Allergic asthma causes substantial morbidity and constitutes a public health burden, which increases with asthma severity. There is evidence that allergy immunotherapy (AIT) prevents the progression of allergic rhinitis (AR) to asthma. However, evidence is missing on the potential of AIT to prevent progression from milder to more severe asthma.
Methods: This population‐based cohort study utilized healthcare data (2005 to 2014) from a statutory health insurance in Germany. The severity of asthma was classified according to the treatment steps recommended by the global initiative for asthma (GINA). The effect of AIT on the transition between the GINA steps was analyzed using multivariable Cox regression models adjusted for age and sex.
Results: From the total cohort of 1,739,440 patients, 39,167 individuals aged 14 years or older were classified as having incident asthma during the observation period and were included in the study. From these, 4111 patients (10.5%) received AIT. AIT expo- sure was associated with a significantly decreased likelihood of asthma progression from GINA step 1 to GINA step 3 (HR 0.87; 95% CI 0.80‐0.95) and GINA step 3 to GINA step 4 (HR 0.66; 95% CI 0.60‐0.74). GINA medication for step 2 and step 5 was rarely prescribed.
Conclusions: This observational study in a real-world setting indicates that patients with allergic asthma who receive AIT are less likely to experience progression of asthma severity than asthma patients not receiving AIT.
KEYWORDS
allergy immunotherapy, asthma progression, cox regression, secondary data analysis
1 | INTRODUCTION
Allergic rhinitis (AR) has been recognized as the most important risk factor for the development of asthma.1 AR and asthma cause sub- stantial public health burden due to high prevalence, adverse impact on quality of life, and high direct and indirect costs for the healthcare system. Costs attributable to asthma substantially increase with disease severity.2-5 Therefore, medications that prevent the pro- gression from AR to asthma, and/or disease progression of existing
asthma are of particular relevance to tackle the current public health burden of allergic diseases.
Allergen-specific immunotherapy (AIT) is currently the only causal treatment modality that not only reduces the symptoms of prevalent AR and asthma. AIT modifies the course of these allergic diseases in restoring allergen tolerance and reducing the tendency to produce immunoglobulin E (IgE).6 A large cohort study demon- strated that AIT with native unmodified allergens led to a significant risk reduction in asthma development.7 Similar data were obtained
596 | © 2019 EAACI and John Wiley and Sons A/S. wileyonlinelibrary.com/journal/all Allergy. 2020;75:596–602. Published by John Wiley and Sons Ltd.


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GRAPHICAL ABSTRACT
In a large cohort study of 39,167 asthma patients, those with AIT had a decreased likelihood for disease progression, measured in symptomatic medication according to GINA. Younger patients benefited most from AIT, which is reflected in prevention or delay of asthma
progression. Inhaled corticosteroids (ICS) monotherapy (GINA stage
for grass tablet immunotherapy.8 A large, double-blind, placebo- controlled 5-year trial with a SQ standardized grass immunotherapy tablet showed a significant preventive effect on the development of asthma symptoms and/or use of asthma medications.9 Despite the beneficial potential of AIT, only 7% of patients with AR and 5% of patients with asthma receive AIT in Germany, which has been inter- preted as an underutilization of this treatment method.10
While there is evidence that AIT prevents the development/ man- ifestation of asthma in patients with prevalent AR,7 studies on the ef- fect of AIT on the course of disease in patients with prevalent asthma are missing. The value of AIT would be even higher if it not only prevented the progression from AR to asthma (AA), but also modi- fied the course of asthma, that is, prevented asthma progression. To generate new evidence for this highly relevant research question, we undertook a large cohort study. We hypothesized that patients with asthma, who are exposed to AIT, are less likely to experience progres- sion of asthma severity than patients not receiving AIT.
2 | METHODS
2.1 | Study design and participants
We undertook a longitudinal cohort study based on comprehensive routine healthcare data from Germany. In Germany, approximately 90% of the population is covered by statutory health insurances. We used routine healthcare claims data from AOK PLUS, a large statutory health insurance in Saxony (area ~ 18 000 km2, population ~ 4 Mio.), which covers approximately half of the local population. The data- base was used previously for several analyses in the field of allergy and other disease areas.11-13 The database includes information on
2) is rarely prescribed in Germany for asthma patients over 14 years.
outpatient care using diagnosis based on the International Statistical Classification of Diseases, 10th edition (ICD-10 Code), prescriptions according to the Anatomical Therapeutic Chemical classification (ATC code) as well as information on age and sex of the insured population.
The study population consisted of patients aged 12 years or older who were continuously insured from January 1, 2005, until December 31, 2014, or until death, and who were classified as hav- ing incident asthma during the observation period. Case validation methods were applied as suggested by the Guidelines “Good Practice Secondary Data Analysis” of the German Society of Epidemiology.14 Accordingly, an individual was classified as having asthma if asthma had been diagnosed at least twice (ICD‐10 Code J45) in outpatient care by a physician (both primary and/or specialized physician) and if the subject had received at least two prescriptions of short-acting beta agonists (SABA), inhaled corticosteroids (ICS) or combinations of inhaled corticosteroids and long-acting beta agonists (LABA) within four consecutive quarters (For further details please refer to Table S1). Individuals who did not meet this definition in the years 2005-06, but did in the following years, were classified as incident cases of asthma. These patients with incident asthma constituted the study population. The study population was further stratified into three sub-cohorts according to their age in 2005: adolescents 12-17 years, younger adults 18‐50 years, and older adults and elderly ≥50 years. Children younger than 12 years were not included as asthma severity could not be adequately defined based on the available data.
2.2 | Primary outcome
Progression of disease severity in asthma over time was the pri- mary outcome. The prescribed asthma medication for each patient


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Whole cohort (n = 1 739 440)
Prevalent Asthma (n = 34 362) No Asthma (n = 1 656 319)
Patients with incident Asthma
(n = 48 759)
Younger than 12 y in 2005 (n = 9592)
Study cohort (n = 39 167)
Patients included exposed to AIT (n = 4111)
Patients included not exposed to AIT (n = 35 056)
FIGURE 1 Patient flow chart
according the Global Initiative for Asthma (GINA) classification was used as a marker for asthma severity.15 The GINA guidelines are ac- cepted worldwide and provide recommendations on how to diagnose and treat asthma with a strong focus on disease control. GINA defines 5 treatment steps ranging from step 1 to step 5. If a patient is not well controlled, treatment escalation is recommended. If a patient is well controlled, a step down in asthma medication is recommended.15 All asthma medications prescribed for individuals of the study popula- tion were classified according to these 5 GINA steps. Allocation to low and moderate/high ICS medication was done by a professional pharmacist. An increase of asthma severity was defined as a step up in asthma medication according to the GINA recommendations.
2.3 | Exposure to AIT and confounding variables
AIT exposure was defined as receiving at least one AIT prescription. A step up in symptomatic asthma treatment according to the GINA steps was only attributed to the AIT group, if the corresponding change in asthma treatment occurred after AIT initiation. Likewise, transitions between GINA steps were allocated to the non-AIT group if AIT was initiated after or at the same time as the corresponding change in symptomatic asthma treatment. There was no adjustment for the duration of AIT therapy prior to a change in asthma severity.
Age (continuously) and sex were considered as confounders. One potential limitation to internal validity in nonrandomized trials is confounding by indication. Patients with nonallergic asthma should not receive or benefit from AIT. As part of our sensitivity analyses, the population was limited to individuals who had at least two out- patient diagnoses for allergic rhinitis (ICD-10 J30) within 12 months in 2005-06.
2.4 | Statistical analysis
In addition to standard descriptive statistics, we applied time-to- event analyses with nonparametric cumulative event curves and semiparametric Cox proportional hazard models.16 Time in days since January 1st, 2007, was used as the process variable. A step up in asthma medication was handled as the failure variable. The end of the observation period on December 31, 2014, or death was modeled as censored events. Follow-up could therefore be up to 8 years. Results were displayed as Hazard Ratios (HR) with corre- sponding 95% confidence intervals and adjusted for age and sex. A corresponding number needed to treat (NNT) after 5 years was cal- culated. Analysis was conducted using the program R, version 3.3.2 with the packages survival and survminer.17,18
3 | RESULTS
The study database included a total of 1 739 440 patients, with 54% being female and an average age of 49 years. A total of 39 167 pa- tients (63% female; mean age: 49 years) with incident asthma met all eligibility criteria and were included in the study (Figure 1). A total of 4111 patients (10.5%) were exposed to AIT during the observation period. Patients receiving AIT were younger and more frequently suffering from comorbid allergic rhinitis than patients not exposed to AIT (Table 1).
Medications of GINA step 2 (3.5%) and GINA step 5 (0.03%) were rarely prescribed so that the transition between GINA steps 1 and 2, step 2 and 3, and step 4 and 5 could not be analyzed. A total of 8726 patients had at least one transition between GINA steps 1, 3, or 4, and 1085 had two. Not all 39 167 patients were under risk of progression into all GINA steps. Some patients did not receive a GINA step 3 med- ication within the observation period and were therefore not at risk for step 4 medication. Others initially received treatments allocated to GINA step 3 or step 4 so that progression to these steps was impossi- ble. Table 2 describes the populations at risk for the transition between the different GINA steps and proportions of patients transitioning
TABLE 1 Baseline characteristics of cohort of patients with incident asthma
Variable
All subjects (percent)
AIT group (percent)
non‐AIT group (percent)
Total
Female Young adults
Allergic rhinitis
39 167 (100) 14 544 (63) 16 950 (43)
1733 (44)
4111 (100) 2563 (62) 2916 (71)
3871 (94)
35 056 (100) 22 060 (63) 14 034 (40)
13 461 (38)
Male
24 623 (37)
1548 (38)
12 996 (37)
Adolescents
2586 (7)
653 (16)
1933 (6)
Older adults/ elderly
19 631 (50)
542 (13)
19 089 (54)
No allergic rhinitis
21 835 (56)
240 (6)
21 595 (62)


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TABLE 2 Transition between GINA steps stratified by AIT exposure and age group
AIT exposure
Transition 1‐3
Transition 3‐4
At risk for transition (n)
With transi‐ tion (n/ %)
Without tran‐ sition (n/ %)
At risk for transition (n)
With transi‐ tion (n/ %)
Without tran‐ sition (n/ %)
All Yes
No 20891 4865 23
1896 75 2022 16 026 77 14 646
1348 75 1413 6462 74 6335
395 20 3921 27
266 19 1719 27
1627 80 10 725 73
1147 81 4616 73
2526 630 25
Adolescents
Yes
427
110
26
317
74
337
68
20
269
80
No
1344
450
33
894
67
965
243
25
722
75
Young adults Yes
No 8752
1792 444 25 2290 26
Older adults/elderly
Yes
307
76
25
231
75
272
61
22
211
78
No
10 795
2125
20
8670
80
7346
1959
27
5387
73
FIGURE 2 Cumulative event curves from step 1 to step 3 GINA medication for incident Asthma with 95% CI. 2007‐14 Data source AOK PLUS
between GINA steps stratified by AIT exposure. Generally, the pro- portions of patients experiencing a step up in asthma therapy as an in- dicator of asthma progression were lower in the subgroup of patients exposed to AIT than in patients not exposed to AIT (Table 2, Figure 2).
Multivariable Cox regression analyses indicated that AIT expo- sure was associated with a significantly decreased risk of asthma progression from GINA step 1 to GINA step 3 with a HR of 0.87 (95% CI 0.80‐0.95). Male sex and higher age were also related to a lower likelihood of transition to step 3 GINA medications. When stratifying the cumulative event curves (S3-S5) and models by age group the effect of AIT was strongest in adolescents (HR 0.72: 0.58‐0.88), followed by young adults (HR 0.89: 0.80‐0.98). For the patients 50 years and older, no risk reduction was observed (HR 1.09: 0.87‐1.38). In this age group, a high proportion (37.8%) of pa- tients had comorbid chronic obstructive pulmonary disease (COPD).
For the transition from GINA step 3 to step 4, the preventive ef- fect of AIT was more pronounced and resulted in a HR of 0.66 (95% CI 0.60‐0.74) for the total cohort. Significant preventive effects of AIT on asthma progression from GINA step 3 to GINA step 4 were observed in all age groups. Effect estimates were similar in the three
age groups (Table 3 Figure 3). The number needed to treat (NNT) to prevent 1 patient from moving from GINA step 3 to GINA step 4 after 5 years was 10.9 (95% CI 8.2‐16.2).
As part of the predefined sensitivity analysis, the cohort was restricted to those with incident asthma and allergic rhinitis (n = 17 332). The results were similar to the ones from the overall cohort indicating robustness of our findings. The HR for the transi- tion from GINA step 1 to GINA step 3 was 0.81 (CI 0.74‐0.89) with an HR of 0.68, 0.81, and 1.01 for adolescents, younger adults, and elderly, respectively. For the transition from GINA step 3 to GINA step 4 medication, the HR was 0.70 (CI 0.62‐0.78) in the total cohort of patients with comorbid allergic rhinitis and 0.76, 0.65, and 0.82 for the three age groups (Table S2).
4 | DISCUSSION
This study is the first to investigate the effect of AIT on asthma progression in a large population-based cohort. Although this study was observational and conclusions have to be drawn with caution,


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TABLE 3 Results of the Cox regression model on the association between AIT exposure and asthma progression
Hazard ratio (95% CI)
ALL
Adolescents
Young adults
Older adults/elderly
Disease progression (GINA step 1 to step 3) stratified for different age groups Male (Ref. Female) 0.86 (0.82‐0.91) 0.84 (0.71‐1.00) Disease progression (GINA step 3 to step 4) stratified for different age groups Male (Ref. Female) 1.01 (0.95‐1.08) 1.07 (0.85‐1.35)
0.86 (0.79‐0.93)
0.96 (0.88‐1.06)
0.88 (0.81‐0.96)
1.05 (0.96-1.16)
AIT
0.87 (0.80‐0.95)
0.72 (0.58‐0.88)
0.89 (0.80‐0.98)
1.09 (0.87‐1.38)
Age (y)
0.99 (0.99-0.99)
0.96 (0.91-1.01)
0.99 (0.99-1.00)
0.98 (0.98‐0.99)
AIT
0.66 (0.60‐0.74)
0.76 (0.58‐0.99)
0.63 (0.56-0.72)
0.72 (0.56‐0.94)
Age (y)
1.00 (1.00-1.00)
0.99 (0.93-1.06)
1.01 (1.00-1.01)
1.00 (0.99-1.00)
FIGURE 3 Cumulative event curves step 3 to step 4 GINA medication for incident Asthma with 95% CI. 2007‐14 Data source AOK PLUS
our findings consistently indicate that AIT exposure is associated with a decreased risk of asthma progression, particularly in younger patients. Our study therefore suggests that AIT prevents the pro- gression of incident asthma under routine care conditions.
In the younger age group, the effect for the prevention of pro- gression from GINA step 1 to GINA step 3 was more pronounced. This result corresponds well with the results of a large double- blind placebo‐controlled asthma prevention trial on 812 children. Patients participating in this trial had a history of grass pollen aller- gic rhinoconjunctivitis (no asthma diagnosis at start of the study). Furthermore, this study demonstrated that children treated with a SQ standardized grass pollen tablet had a significantly reduced risk of asthma symptoms and/or the need for asthma medication at the end of the five years trial.8 The NNT to prevent asthma symptoms and asthma medication in the two treatment-free follow-up years was decreased with the age of the children at randomization. The results of this study and the results presented in this paper therefore consistently confirm the current recommendations from AIT guide- lines to start AIT as early as possible after disease onset.19
The limited effect in elderly on progression from GINA step 1 to GINA step 3 may be explained by a significant proportion of comorbid COPD. The diagnosis of asthma in primary care is prone to overdiag- nosis,20 and asthma medication is also used to treat chronic obstruc- tive pulmonary disease (COPD). About 46% of patients with asthma medication in a Swedish study neither had a diagnosis of asthma nor of COPD. Major indications for off-label prescriptions of asthma med- ication were cough, airway obstruction, dyspnea, and acute bronchi- tis.21 In addition to that, disease prevalence might be overestimated and diagnostic and pharmacological treatments for asthma and COPD might be underestimated when relying only on diagnosis codes.22 In our cohort, the proportion of comorbid asthma and COPD was 37% among patients age 50 years or older, and thus larger than the pro- portion of 20% reported elsewhere.23 AIT intervention is not effec- tive if the progression of symptoms is driven by COPD. Particularly, in elderly patients, it is possible that asthma occurrence was not inci- dent in some patients despite our efforts for a valid definition of inci- dent cases. We do not have any information on morbidity and clinical care before 2005, and it may be possible that some patients without


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asthma treatment and diagnosis in 2006 and 2007 had asthma man- ifestation before the observation period of this study. If this was the case, it can be assumed that the remodeling of the pulmonary tissue is more advanced due to more chronic inflammation.24
Only few patients were prescribed GINA step 2 medications, so that it was not possible to investigate the effect of AIT on progres- sion from GINA step 1 to GINA step 2. The low rate of GINA step 2 prescriptions indicates that, when a patient first develops asthma symptoms, SABA is prescribed (GINA 1). If asthma is not fully con- trolled an immediate step up to a GINA step 3 medication seems to be common, which seems to be only rarely reduced to GINA 2 even if patients have full asthma control. The German guideline “Diagnostics and therapy of patients with asthma” recommends low dose ICS alone and low dose ICS in combination with LABA as inter- changeable alternatives for adult asthma patients.25
AIT effectively prevented a step up from GINA step 3 to GINA step 4 therapy with a number needed to treat of 10.9. This indicates that, despite restrictive guidelines for the use of AIT in severe and uncontrolled asthma,19 a preventive effect of AIT even in these pa- tients seems to be possible. There is only one large, double-blind, placebo-controlled trial investigating the treatment effect of a SQ standardized sublingual house dust mite (HDM) SLIT tablet on mod- erate-to-severe asthma exacerbations in patients having uncon- trolled asthma despite the use of asthma medication corresponding to GINA step 2‐4. This trial demonstrated a significant reduction in the risk of having a moderate-to-severe asthma exacerbations com- bined with a good safety profile even in patients with uncontrolled asthma.26 Based on this study, GINA recommended the use of HDM SLIT for asthmatic patients.27
Meta-analyses investigating the clinical efficacy of AIT products revealed a substantial heterogeneity between different studies. Therefore, the World Allergy Organization recommends a product- based evaluation.28 This was not possible in the current study as the sample size for individual products would have become too small. Therefore, the results of this study should be considered supportive of product-specific efficacy documented in randomized clinical trials.
4.1 | Study strengths and limitations
An important strength of our study is that it is based on a large and relatively unselected proportion of individuals from the general pop- ulation in Saxony, Germany. Patients switching their health insurance within the observation period were excluded which may have re- sulted in a disproportional underrepresentation of young people who moved into or out of the federal state of Saxony within the obser- vation period. Another major strength of our study is its robustness against selection bias due to missing data. Neither a nonresponse nor recall bias is possible due to the study design and the use of rou- tine healthcare data. Confounding by age or sex was prevented by multivariable adjustments in the Cox models. Co-medication, non- compliance or comorbidities, which could enhance or reduce the ef- fectiveness of AIT, were taken into account, ensuring that the results are not attributable to an artificial setting but reflect routine care.
Established methods of internal case validation of administrative healthcare data as recommended by the guidelines for good practice secondary data analysis have been utilized.14 However, not all cases are incident cases according to epidemiologic standards. Access to care by an allergy/respiratory care specialist is most likely associated with a higher probability that existing asthma is diagnosed. With rou- tine healthcare data, it is not possible to rule out misclassification of individual patients (patients suffering from the investigated disease, who do not consult a physician or who are falsely diagnosed with a disease). No data on the results of neither spirometry, peak expira- tory flow (PEF), nor bronchial challenge testing were available in the healthcare database. The accuracy of the severity classification of a patient depends on the prescription behavior of his treating physi- cian. If present, the resulting misclassification is most likely nondiffer- ential and may therefore not explain the overall findings of our study. Furthermore, the type of allergy and the history of asthma disease treatment before the year 2005 are unknown.
4.2 | Implications for clinicians and health policymakers
In accordance with findings from clinical trials which demonstrated effectiveness of AIT to treat AR and asthma symptoms, this study indicates that AIT may modify the course of asthma. Our study supports the assumption that treatment with AIT may prevent the progression from mild to more severe asthma. In addition to that, the results are also suitable to support product-specific efficacy documented in randomized clinical trials. In concordance with exist- ing literature, our data also show stronger benefits in the younger age groups which supports current recommendations to start causal treatment with AIT in the early stage of the disease.
Unfortunately, the present study design lacks an alternative measure of asthma severity as lung function or the number of pa- tient-reported exacerbations. We highly recommend the collection of relevant data on the potential preventive effect on AIT on asthma progression during future clinical trials. Due to the positive effects of AIT on the reduction in disease symptoms and its potential to pre- vent disease progression, AIT should be used to a greater extent. Furthermore, interventions for effective treatment compliance need to be evaluated and eventually implemented.
FUNDING
This study was independently planned and undertaken by the center of Evidence-based Healthcare, TU Dresden, Germany. The study was financially supported by ALK-Abelló. The views expressed are those of the authors, and not necessarily those of ALK-Abelló.
ACKNOWLEDGMENTS
The authors thank the Compulsory Health Insurance AOK PLUS for their cooperation in data use and technical support. We also thank Luise Mocke for conducting data queries.


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CONFLICT OF INTEREST
E. G. Wüstenberg, V. Mücke and N.-S.-Hansen were all employees of ALK-Abelló while conducting this work. E. G. Wüstenberg has stock/ stock options in ALK-Abelló. J. Schmitt has received institutional funding for investigator initiated research from ALK-Abelló, during the conduct of the study, and MSD, Novartis, Pfizer, Sanofi outside the submitted work. D. B. Küster and F. Tesch report grants from ALK-Abelló, during the conduct of the study.
15. From the Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA);2006.
16. Cox DR. Regression Models and Life-Tables. J Roy Stat Soc: Ser B (Methodol). 1972;34(2):187‐220.
17. A Package for Survival Analysis in S [computer program]. Version 2.382015.
18. Survminer: Drawing Survival Curves using 'ggplot2' [computer pro- gram]. 2017.
19. Pfaar O, Bachert C, Bufe A, et al. Guideline on allergen-specific immunotherapy in IgE-mediated allergic diseases: S2k Guideline of the German Society for Allergology and Clinical Immunology (DGAKI), the Society for Pediatric Allergy and Environmental Medicine (GPA), the Medical Association of German Allergologists (AeDA), the Austrian Society for Allergy and Immunology (OGAI), the Swiss Society for Allergy and Immunology (SGAI), the German Society of Dermatology (DDG), the German Society of Oto- Rhino-Laryngology, Head and Neck Surgery (DGHNO-KHC), the German Society of Pediatrics and Adolescent Medicine (DGKJ), the Society for Pediatric Pneumology (GPP), the German Respiratory Society (DGP), the German Association of ENT Surgeons (BV- HNO), the Professional Federation of Paediatricians and Youth Doctors (BVKJ), the Federal Association of Pulmonologists (BDP) and the German Dermatologists Association (BVDD). Allergo J Int. 2014;23(8):282‐319.
20. Aaron SD, Vandemheen KL, FitzGerald JM, et al. Reevaluation of diagnosis in adults with physician-diagnosed asthma. JAMA. 2017;317(3):269-279.
21. Weidinger P, Nilsson JL, Lindblad U. Medication prescribing for asthma and COPD: a register-based cross-sectional study in Swedish primary care. BMC Fam Pract. 2014;15:54.
22. Schneider A, Gantner L, Maag I, Borst MM, Wensing M, Szecsenyi J. Are ICD-10 codes appropriate for performance assessment in asthma and COPD in general practice? Results of a cross sectional observational study. BMC Health Serv Res. 2005;5(1):11.
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SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Schmitt J, Wüstenberg E, Küster D, Mücke V, Serup-Hansen N, Tesch F. The moderating role of allergy immunotherapy in asthma progression: Results of a population-based cohort study. Allergy. 2020;75:596–602. https://doi.org/10.1111/all.14020
ORCID
Jochen Schmitt Falko Tesch
https://orcid.org/0000‐0003‐0264‐0960 https://orcid.org/0000‐0001‐8933‐643X
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(GPS): Leitlinien und Empfehlungen. Gesundheitswesen. 2015;77(02):120-126.


Received: 16 September 2019 | Revised: 28 October 2019 | Accepted: 2 November 2019 DOI: 10.1111/all.14113
REVIEW ARTICLE
30 years of sublingual immunotherapy
Giovanni Passalacqua1 |
1Allergy and Respiratory Diseases, IRCCS Policlinico San Martino -University of Genoa, Genoa, Italy
2Personalized Medicine Clinic Asthma and Allergy, Humanitas Clinical and Research Center IRCCS, Department of Biomedical Sciences, Humanitas University, Rozzano- Milan, Italy
Correspondence
Giovanni Passalacqua, Allergy & Respiratory Diseases, DIMI, Padiglione Maragliano, L.go R. Benzi 10, 16132 Genoa, Italy.
Email: [email protected]
Diego Bagnasco1 |
Giorgio Walter Canonica1,2
Abstract
Allergen Immunotherapy (AIT) was introduced in clinical practice on an empirical basis more than 100 years ago. Since the first attempts, AIT was administered sub- cutaneously. Indeed, other routes of administration were proposed and studied, in particular to improve the safety, but only the sublingual route (SLIT) achieved a cred- ibility based on evidence and was then accepted as a viable “alternative” option to the subcutaneous route. SLIT was largely used in clinical trials and clinical practice in this last 30 years. Thus, a large amount of data is available, coming from either controlled trials and postmarketing surveillance studies. It is clear that SLIT is overall effective, but it is also clear that the efficacy is not “class-related,” as derived from meta-anal- yses, but restricted to each specific product. The 30-year lasting use of SLIT allowed to clarify many clinical aspects, such as efficacy, safety, use in asthma, regimens of administration, and optimal doses. In parallel, the mechanisms of action of AIT were elucidated, and new indications were proposed (eg food allergy, atopic dermatitis). In addition, the introduction of molecular-based diagnosis, allowed to better refine the prescription of SLIT, based on specific sensitization profiles. The present article will describe the origin and evolution of SLIT for respiratory allergy, taking into account the clinical context that suggested this form of treatment, the recently developed aspects, the future perspectives and unmet needs, This is not, therefore, a systematic review, rather a narrative historical description of the past history, and a look forward to the future opportunities.
KEYWORDS
efficacy, history, perspectives, respiratory allergy, sublingual immunotherapy
1 | FAR HISTORY AND CLINICAL CONTEXT
Allergen-specific immunotherapy (AIT) is the practice of adminis- tering to allergic subjects increasing amounts of allergen(s) (the al- lergenic extract) to achieve a hyposensitization thus reducing the symptoms during the natural exposure to the allergen(s). Since its discovery, immunotherapy was only given subcutaneously (SCIT). Nevertheless, other modalities of administration were proposed and
Abbreviations: AIT, Allergen-specific immunothreapy; RDBPC, Randomized double-blind placebo-controlled; SCIT, Subcutaneous immunotherapy; SLIT, Sublingual immunotherapy.
investigated during the 2nd half of 1900, involving the gastrointes- tinal, nasal and bronchial route. Those routes were variously named: alternative, nonparenteral, noninjections or local. Presently, only the term “local” survived, although progressively less utilized.
As mentioned above, the first empirical attempts of AIT was per- formed by Dr Noon in 1911,1 with the aim of “vaccinating” against some hypothesized “aerogenic toxins.” Despite the rationale was wrong, the subcutaneous administration of pollen extracts re- sulted effective in reducing hay fever symptoms. Thus, the use of SCIT gradually increased and was progressively extended to other allergens. SCIT was the only mode of administration for more than 70 years, but its use remained totally empirical until 1965, when IgE
Allergy. 2019;00:1–14. wileyonlinelibrary.com/journal/all © 2019 EAACI and John Wiley and Sons A/S. | 1 Published by John Wiley and Sons Ltd.


2 |
was discovered.2 Of note, the first randomized double-blind pla- cebo-controlled (RDBPC) study on SCIT was published in 1954 by Frankland et al,3 and few years later, Johnstone suggested that SCIT could modify the natural history of respiratory allergy.4 In 1978, the first RDBPC trial with SCIT for hymenoptera venom allergy appeared.5
AIT represented a first and concrete attempt to “personalize” a therapy, since it's intent was to target in some way the identified “sentisizing allergen” in the candidate patients.6
Indeed, the idea of administering allergenic extracts orally is not so recent as commonly believed: the oral route was first suggested in 19007 and the first clinical attempts were made a few years later.8 Subsequently, other routes of administration for respiratory allergy were proposed, with various evolutions (Figure 1): the local bron- chial (LBIT) during the 1950s,9 the local nasal (LNIT)10,11 during the 1970s, and the oral (OIT) at the beginning of the 1980s12 OIT and LBIT were abandoned, due to the scarce efficacy and side effects. Only LNIT survived for a short period,13 then it was abandoned as well, because SCIT was well established.
In 1986, the British Committee for the Safety of Medicines (CSM)14 described 26 deaths caused by SCIT, that had to be added to the deaths reported in the USA surveys.15,16 This raised seri- ous concerns about the safety and the risk/benefit ratio of SCIT, also because cheap, safe and effective drugs had become avail- able for asthma and rhinitis. Indeed, it was subsequently acknowl- edged that, in most cases, life-threatening or fatalities were due to avoidable human errors (wrong dose, incorrect administration)17,18 Nonetheless, after the CSM report, the interest in “alternative” routes of administration rapidly increased and studies with the gas- troenteric-based routes appeared. In addition, it was felt that an ex- tensive and evidence-based evaluation of the literature was urgently needed.
Within the above mentioned context, in 1986 the first positive RDBPC trial with a sublingual preparation of dust mite was pub- lished by Scadding et al,19 with encouraging results. It involved a small sample of patients, but it prompted the research interest, and new studies rapidly appeared. The first SLIT clinical trial (20 patients) with HDM allergoid tablets dates 1998,20 confirming the clinical ef- ficacy and the downregulation of allergic inflammation. SLIT was first mentioned as possible alternative to SCIT in the World Health Organization position paper in 1998 with 4 RDBPC trials,21 and its role in clinical practice was confirmed in the subsequent official doc- uments22-30 (summarized in Table 1). After 25 years, the Food and Drug Administration, approved 3 SLIT products to be marketed in the USA.31
Finally, the treatment was included as possible add-on therapeu- tic option for allergic asthma in the GINA document.32 A summary of the history of SLIT can be found in a recent review.33
So far, the experimental evidence shows that when the extract is kept under the tongue for 1-2 minutes and then swallowed (sub- lingual-swallow), is effective, according to biodistribution studies. Those studies, which used a radiolabelled allergen/allergoid (never replicated for ethical reasons), showed that there it was no direct
PASSALACQUA et AL.
Major milestone discoveries
The first controlled trial with sublingual immunotherapy (SLIT), by G. Scadding, dates 1986. This route was explored to improve the safety of allergen immunotherapy. Few years later, tablets were introduced. In 1998, the World Health Organization accepted SLIT as a viable alterna- tive to the subcutaneous route and SLIT was therefore included in official documents. There are now two World Allergy Organization position papers (2009 and 2014) spe- cifically dedicated to SLIT, and one EAACI position paper (2018), also largely dealing with SLIT available. SLIT was admitted as add-on therapy also for asthma in the 2017 GINA document.
Future research perspectives
SLIT is now accepted as a viable alternative option in res- piratory allergy. Its historical evolution, supported by nu- merous clinical trials, confirmed its potential role. Looking back to the far history, and prospectively, there is still room for improvement, including optimization of the administra- tion regimen, standardization of trials, real-life studies, new indications and better definition of the eligible pa- tients (responder vs nonresponder).
sublingual absorption into the blood-flow, but that a fraction of the allergen remained confined into the mouth cavity for hours34- 36 (Figure 2). In addition, after swallowing the allergen is degraded in the gastrointestinal tract, and only traces of the native protein (if chemically modified) pass into the bloodstream. Also, the sub- lingual-spit method was suggested but, presently, on the basis of biodistribution considerations, only the sublingual-swallow route is used. Throughout the text, SLIT will indicate the sublingual-swallow modality. To conclude this general summary of the historical aspects, and for completeness, it has to be mentioned that in the last decade two new noninjection routes were proposed: the intralymphatic immunotherapy (ILIT) and the epicutaneous immunotherapy (EPIT), which are currently under development.37,38 A summary of the his- torical development of SLIT is depicted in Figure 3, and the main milestones are summarized in Table 2.
2 | THE RECENT HISTORY OF SLIT
The official acceptance of SLIT came in 2009 with the publication of a first position paper by the World Allergy Organization,26 includ- ing 60 RDBPC, followed by an updated version in 2014 with 77 tri- als.27 Currently, there are more than 100 RDBPC trials available in the literature, and the official documents and guidelines presently accept SLIT (Table 1). Certainly, most of the earliest trials included


PASSALACQUA et AL.
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F I G U R E 1 The historical evolution of allergen immunotherapy in its various forms (ILIT = intralymphatic immunotherapy;
EPIT = epicutaneous immunotherapy; LBIT = local bronchial immunotherapy; LNIT = local nasal immunotherapy; OIT = oral immunotherapy; SCIT = subcutaneous immunotherapy; SLIT = sublingual immunotherapy)
small samples and were affected by a large variability (type of ex- tract, doses, outcome, assessment, regimens, and inclusion cri- teria), so that a comparison among them was virtually impossible. Nonetheless, an impressive number of meta-analyses on the clinical efficacy of SLIT were published, starting with the one by Wilson et al in 2003,39 until the more recent one by Dhami et al40 (see 33,41). Almost all of the meta-analyses agree on the favourable effect of SLIT vs placebo, and some of them were performed only for a single selected allergen source,42,43 but the main weakness of meta-analy- ses is the large heterogeneity of the included studies. It is true that the results are overall positive, and this lead to define the efficacy of SLIT as a “class effect,” to be extended virtually to all SLIT products. It was recently acknowledged that a claim for efficacy must not be generic, but must refer to each single defined product, with robust experimental evidence.44,45
Other important achievements in the more recent history of SLIT were the RDBPC trials involving hundreds of patients (so-called “big trials”). These were conducted with a quite uniform methodology, with well-defined and standardized extracts (allergen content clearly declared).27 Those studies allowed to identify for each product the optimal maintenance dose (major allergen's content). This was done only anecdotally before, so that the optimal doses remained for a long time largely empirical and variable. A summary of the dose-find- ing aspects of the big trials is reported in Table 3.26,27,31
In addition, the big trials, conducted with quite homogeneous methods, demonstrated the dose-dependency of the clinical effect
of SLIT,46-49 at least for the more relevant allergens, and this is a very robust proof in favour of each studied product, as stated in the GRADE recommendations.50 This aspect had been previously stud- ied also for SCIT (9 trials), but the studies were heterogeneous in outcome, patients and allergen preparation,51 with only one large dose-ranging study.52
In the recent history of SLIT, one of the most debated aspects was the comparison of the clinical efficacy vs SCIT. This should be assessed only by head-to-head comparisons,53-55 and there are, so far, very few studies conducted accordingly. The two studies in double-dummy fashion,56,57 failed to show a significant differ- ence between the two routes, and this was confirmed indirectly by a recent comprehensive meta-analysis.58 None of those trials assessed the long-lasting effect. The choice between SLIT and SCIT does not rely only on the proof of efficacy (equivalent for both routes), but also on the personal culture. We have now avail- able two different routes of administration for AIT, that should be chosen on the basis of personal experience and skills, and accord- ing to the patient's preferences.59 In this latter case, the patient's age maybe relevant, and in many countries, SLIT is the preferred choice in paediatric ages. It is true, nonetheless, that differences between the two routes exist.53 The onset of action seems to be more rapid with SCIT than with SLIT: in a recent double-dummy study, it was shown that SCIT but not SLIT was effective since the first pollen season, although the two treatments became equal at the second season.60 Of note, in this study, 2 years of AIT failed


4 | 1998
2001
2016
2014
2017
21 Allergy 1998;53(suppl)
23 J Allergy Clin Immunol 2001; 108 (5 Suppl):S147-S334
25
27 World Allergy Organ J. 2014 Mar 28;7(1):6
29 Ann Allergy Asthma Immunol. 2017; 118:276-282
PASSALACQUA et AL.
TABLE 1 The main Documents and Position papers and guidelines on SLIT
Year
Organization
Type of AIT
Reference
World Health Organization (WHO)
Allergic Rhinitis and its Impact on Asthma (ARIA)
Allergic Rhinitis and its Impact on Asthma (ARIA)
World Allergy Organization (WAO)
American Academy Of Allergy Asthma and Immunology (ACAAI)
SCIT/SLIT
SCIT/SLIT
SCIT/SLIT
SLIT
SLIT
1998
European Academy of Allergy and Clinical Immunology (EAACI)
Non injection routes
22 Allergy. 1998; 53:933-44
2008
Allergic Rhinitis and its Impact on Asthma (ARIA)
SCIT/SLIT
24 Allergy. 2008; 63 Suppl 86:8-160
2009
World Allergy Organization (WAO)
SLIT
26 Allergy. 2009;64 Suppl 91:1-59
2011
American Academy of Allergy Asthma and Immunology/ American College of Allergy Asthma and Immunology (AAAAI/ACAAI)
SCIT
28 J Allergy Clin Immunol. 2011; 127(1 Suppl):S1-55
2018
European Academy of Allergy and Clinical Immunology (EAACI)
SCIT/SLIT
30 Allergy. 2018 Apr;73(4):739-743
to achieve a persistent clinical effect. Also, the immunological response seems to be different, being the increases in IgG4 usu- ally more pronounced with SCIT.61 A similar debate in the recent history of SLIT in seasonal allergies, concerned the comparison between the continuous (all-year lasting) and the precoseasonal regimens of administration. Also in this case, only few direct head- to-head comparison studies are available, showing that at least for the licensed products there is no difference in efficacy and safety between the two regimens.62 Finally, the combination of SCIT up- dosing with SLIT maintenance, to increase the speed of action has been proposed with encouraging results.63
The more recent history of SLIT had to face its use in asthma, since asthma has been ever regarded as a possible contraindica- tion to AIT. In addition, most of the RDBPC trials were designed using rhinitis as primary outcome, leaving asthma as ancillary out- come. Thus, two relevant questions emerged; (a) is asthma a risk factor for adverse events due to AIT?; (b) does AIT elicit or worsen asthma?.64,65 The most important meta-analyses (with their intrin- sic limitations) suggested that AIT, and SLIT, are clinically effective in reducing asthma symptoms and medication score,66-68 without an increase in adverse events. It has to be considered that asthma is more frequent in mite-sensitized patients, where it is more dif- ficult to assess the exposure and to evaluate outcomes69 (Table 4). During the last decade, some SLIT studies were designed specif- ically for asthma, taking into account the exacerbation rate and/
or the use of inhaled corticosteroids.70-72 The study conducted on more than 800 asthmatic patients with dust mite allergy,70 clearly proved the reduction in asthma exacerbations even when inhaled corticosteroids were tapered down or even stopped. Of note, in this study, about 20% of patients were classified as GINA step 4. On the other hand another study72 did not reach the overall out- come, but showed that SLIT is effective in moderate asthma, not in mild, where symptoms are by definitions already controlled. These studies lead to include SLIT as an add-on option in asthma in the GINA document.32 A recent review of the literature, performed by the European Academy of Allergy and Clinical Immunology73 evidenced that only uncontrolled severe asthma74 is an absolute contraindication to SLIT. There is no evidence that SLIT can ex- acerbate asthma or that asthma, if controlled, is a risk factor for adverse events.
The overall safety of SLIT was one of the most important aspects to be carefully investigated, since safety was the primary reason to use this route. Looking at the available literature it appears that the majority of side effects with SLIT are “local”, that is limited to the route of administration (lip/tongue swelling, oral pruritus, mouth irritation, stomach-ache, nausea). These local side effects tend to disappear after the first administrations (for review see 75). After the publication of a proposed classification and grading of systemic side effects due to AIT in general,76 also a specific system of classifica- tion/grading for local side effects due to SLIT was also published,


PASSALACQUA et AL.
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FIGURE 2 The persistence of a radiolabelled allergen (I-123 Par j 1) in the sublingual region 1 and 2 h after the administration (scintiscan)
with the aim of standardizing the description of local side effects.77 Concerning the safety aspects, the historical view included recently also eosinophilic oesophagitis. In this context there are only case-re- ports, and no evidence from large groups, that SLIT can elicit the disease or worsen it.78-80
A part of the history of SLIT that should not be neglected is the possible disease-modifying effect: (a) the reduction in the risk of developing asthma in subjects (usually children) with allergic rhini- tis only and (b) the persistence of the effect after stopping the ad- ministration. The preventative effect was hypothesized more than 50 years ago with SCIT4 in an open prospective study. Then, the re- duction in the risk of asthma onset was described in two additional randomized (non–placebo-controlled) SLIT trials with grasses and various allergens.81,82 In those trials, showing consistent results, SLIT significantly prevented asthma onset in the active groups.83 The most recent trial concerning this aspect of SLIT84 was conducted in >800 children in a RDBPC trial (3 years of grass-SLIT +2 years fol- low-up). Although the (ambitious) primary outcome was not reached, all the secondary outcomes (use of bronchodilators, symptoms of asthma, exacerbations), clearly demonstrated the effectiveness of SLIT in the long-term period, on the evolution of the allergic disease. Thus, the role in prevention of the allergic march remains a current attractive objective.85,86
The long-lasting, or persisting, effect after AIT stopping was demonstrated with SCIT in a RDBPC trial in 1999.87 This was
then evidenced again with SLIT in an open controlled trial lasting 15 years, with an house-dust mite extracts.88 In this long-term trial, it was seen that, dependently on the duration of SLIT, there was a 3-5 years lasting effect followed by a worsening that could be rapidly controlled again with a new course of SLIT. The long-term effect was subsequently confirmed in the follow-up observations of RDBPC big trials.89-92 Of note, a recent trial with the nasal provocation chal- lenge, showed 2 years of treatment were not sufficient to achieve a long-standing effect.60
Further challenges in the recent history of SLIT are the use in polysensitized patients and the use in the elderly. It has been shown that SLIT maintains its clinical efficacy also in the presence of polysensitization, provided that the truly responsible allergen(s) are clearly identified.93,94 It was shown that the use of 2-3 aller- gens (without mixing them) did not increase the occurrence rate of adverse events as compared to a single allergen.95 The same was seen, with demonstration of efficacy administering at the same time Japanese cedar and mite tablets96 and mite and grass solutions.97 Interestingly, in a randomized not double-blind trial, the administration of grass and birch SLIT was more effective than each individual allergen in patients with the dual sensitization, and a significant efficacy was seen for birch-allergic subjects also in the grass season and vice-versa.98 Finally, SLIT was demonstrated to be effective also in elderly patients,99-101 although this was not expected.


6 | PASSALACQUA et AL.
FIGURE 3 Themilestonesofsublingualimmunotherapy


PASSALACQUA et AL.
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TABLE 2 Milestones in SLIT Efficacy and safety
Biodistribution of SLIT
“Big trials”
Long-lasting effect Preventive effect Asthma
Molecular-based diagnosis Real-life studies
Predictive biomarkers Standardization and regulatory
Several side effects with SCIT. Search for more convenient routes of administration. 1986 first rand- omized controlled trial with SLIT
According to the few studies available, no direct absorption through sublingual mucosa is demonstrated
Dose-dependency of the effect
Identification of the optimal maintenance dose for selected products
Ascertained at least 2 y after discontinuation for some products. A 3-y course seems to be necessary to achieve long-lasting effects
Reduction in the risk of developing asthma in children with allergic rhinitis
Clinical trials specifically designed for asthma. Reduction in inhaled steroids and/or exacerbations. SLIT is safe in controlled asthma. SLIT accepted in GINA document
Refined prescription of SLIT
Such studies would better define the safety and efficacy profile of SLIT, that cannot be established in clinical trials
There is no current biomarker available to predict at individual level if SLIT will work
Only few allergen products for AIT are adequately standardized and approved. The prescription rules and practice largely varies among countries
Far history (1986-2000)
Comments
Efficacy and safety
SLIT accepted as a viable alternative option to SCIT. Confirmed in guidelines and documents
Use of tablets
Tablets introduced in a randomized trial (1998)
Recent history (2000-2015)
Comments
Use of multiple allergens
Few controlled studies available with multiple allergens, but with positive results with 2-3 allergens. The efficacy and safety of allergen mixtures VS single allergens is still debated
Populations
SLIT also effective in children and elderly
Meta-analyses
All consistently confirmed the efficacy of SLIT. These reports remain seriously affected by a large heterogeneity
SLIT vs SCIT
Head-to-head comparisons and meta-analyses showed no clinical difference. SCIT has faster onset of action
Present and future 2015 -
Comments
New indications
Food allergy, extrinsic atopic dermatitis (investigational)
Challenge/exposure chambers
Some trials performed with this methodology. Useful to have a standardized exposure and monitoring
Main unmet needs
Comments
When to start SLIT in children?
The safety is well ascertained also in preschool children, but there is no consensus on the age to prescribe
3 | PRESENT, FUTURE AND UNMET NEEDS OF SLIT
The prescription of SLIT (and AIT in general) requires a clear dem- onstration of the role of genuine components (allergenic molecules) from the allergenic sources in the allergen-IgE-mast cell reaction (Table 2). The introduction in clinical practice of the molecular-based diagnosis approaches, allowed to refine the prescription of AIT in general, and of SLIT in particular.102-106 The ability of defining the genuine sensitization profile (thus to rule out the confounding role of pan-allergens as cause of false positivity at standard tests) also al- lowed to obtain a more and more detailed prescription profile. It was clearly documented that the molecular-based diagnosis could lead to a significant change, estimated in about 40%-50% in the SLIT pre- scription as compared to the standard diagnostic approaches.107-109 Notably, it was shown that as larger is the sensitization profile, as greater is the AIT failure.110 It is also true that an immunotherapy
“built” for each individual according to his/her specific sensitization profile would be too complex and expensive, due to the large vari- ability of profiles themselves.111
In the recent years, there has been a considerable interest into the use of SLIT (and the old OIT) in food allergy (oral desen- sitization, specific oral tolerance induction). This aspect is not pertinent to the present article, but numerous reviews are avail- able.112-115 The studies consistently show that a desensitization can be achieved in a proportion of children, but it is not clear if a permanent tolerance can be induced, and the high rate of side ef- fects has also to be considered. Under a more general perspective, favourable reports with epicutaneous immunotherapy in food al- lergy are now available, but the results are still not conclusive116 The results are more controversial with SLIT for atopic dermati- tis,117,118 although this field remains an intriguing opportunity. A summary of the possible developments of SLIT and AIT in general, is summarized in Figure 4.


8 |
TABLE 3 Optimal maintenance doses in the main dose-finding studies (see 27,64)
PASSALACQUA et AL.
ALK-Abellò MSD
ALK-Abellò ALK-Abellò
Author, year
Dose
Allergen source
Manufacturer
Durham, 2006 Creticos, 2013
Mosbech, 2014 Virchow, 2016
15 mcg Phl p 5/d 12 mcg Amb a 1/d
6 SQ*/d (15 mcg/d) 6 or 12 HDM-SQ/d*
Grass Ragweed
Dust Mite Dust mite
Didier, 2007
25 mcg Group 5/d
Grass
Stallergenes
Bergmann, 2014
(500IR) 300IR – approved: 16/68 Der p 1/Der f 1/d
Dust mite
Stallergenes
Nolte, 2015
12 DU/d (=6 SQ/d)
Ragweed
MSD
Demoly, 2017
6 or 12 HDM-SQ/d*
Dust mite
ALK-Abellò
Note: Der p 1/Der f 1 plus Der p 2/Der f 2 in 1:1 ratio. European dose = 12SQ; Japanese dose = 6 SQ.
TABLE 4 SLIT and Asthma
Special problems with slit and asthma
Asthma is usually more prevalent in house dust mite (HDM) allergic patients. A careful selection of patients is needed (HDM as certain aetiological agent of asthma)
The objective measurement of HDM exposure is impractical in real- life settings. Exposure chambers could represent a reasonable sur- rogate, provided that the “average natural exposure” is established and reproduced
Which primary outcome would be optimal?
• Respiratory function
• Exhaled nitric oxide
• Daily symptom score
• As needed bronchodilators’ use
• Inhaled corticosteroid (ICS) dose
Fix the dose of ICS to achieve control, then give SLIT and try to escalate ICS considering if the control is maintained (eg symptoms, exacerbations and bronchodilators)
Evaluation of symptoms, respiratory function or exhaled nitric oxide after exposure in challenge chamber
Despite the amount of clinical and mechanistic data on AIT, and its consolidated use, the regulatory aspects (pharmacologi- cal classification of products, marketing authorization, national and supranational approval, deputy regulatory authorities) re- main sometime vague and largely differ among Countries. If in the USA and in the European Community, there are well-defined regulatory authorities (namely Food and Drug Administration, European Medical Agency and Paul Ehrlich Institute) in other countries, such as Latin America, there is no uniform regulation.119
In Europe, numerous official regulatory documents have been re- leased,120,121 mainly concerning the Good Manufacturing Practice. Those impose to all members of the EC specific standards for the production of allergen extracts. Within the EC, apart few excep- tions, allergen extracts are considered as named patient products (NPP). This would mean that AIT is prepared for each single patient according to the prescription of the physician. Indeed, this is not true since almost all extracts are manufactured by industrial pro- cedures, and the extract preparation for each allergen is the same for all patients. There is a general effort to abolish the possibility to have NPP, with exceptions for rare allergens or special sensiti- zation profiles, whereas the single preparation should contain in the near future only allergens from “homologous groups”. For each new product, a registrative dossier (from phase I to III) is required for the marketing authorization.
Some of the recent pharmaco-economic studies suggested that SLIT can be cost-effective in the long-term period,122-124 while others provided opposite results.125 This is still a matter of debate, due to the large variability in mathematical models, assumptions, time horizon etc126 It is in general accepted that, in the long period, both SCIT and SLIT have an economic advantage over pharmacotherapy alone.127
Of note, the use of the challenge (exposure) chambers is consid- ered currently, as a promising methodological option for the near fu- ture. Challenge chambers have been used in several well-conducted clinical trials.128-131 Certainly, challenge chambers allow to have a totally controlled environment, with a stable concentration of aller- gen. This is important to simplify the trials with pollens, where the outdoor concentration is measurable and easy to reproduce. With house-dust mite, that have a largely variable concentration over long time periods, it may be difficult to assess which is the concentration to be used to reproduce the natural exposure. In addition, exposure chambers are expensive in functioning and maintenance, and are not available everywhere.132
There is still a large variability in administration schedules, dosages and duration of SLIT (Figure 5). Only few products rep- resent an exception: OralairTM (Stallergenes), Grazax-GrastekTM (Alk-Abellò), Acarizax (Alk-Abellò) and RagwitekTM (MSD). Another critical point is the standardization. Almost all AIT vaccines
The exposure to HDM is largely variable over time and external physical conditions, thus appropriate “windows” or long periods of observation are required
• Exacerbations
Possible approach es to evaluate the efficacy
Fix the dose of ICS, add SLIT and follow-up over time for control of asthma in comparison with placebo


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FIGURE 4 The near-future and far- future possible experimental evolutions of allergen immunotherapy (those possibly involving SLIT are boxed)
FIGURE 5 The available options of administration for SLIT
commercialized are standardized either biologically or immuno- logically, based on in-house references. Extracts are labelled in units that differ from one manufacturer to another, the allergen content (in mcg) largely vary among extracts,133,134 thus the com- parison among trials and products remains difficult, and for most products a dose-response relationship is not demonstrated. In ad- dition, there is still a gap between USA and Europe standards in the administration/choice of AIT: the methods, concentration of extracts, mixing of allergens still differ profoundly.135,136 This re- mains a fundamental part of the “history” that persists in the pres- ent. The use of SLIT in children is well accepted by paediatricians, at least in those countries which are familial with this route.137-139 The main unsolved problem is when to start SLIT, and on which
basis. The only clear fact is that the previously suggested limita- tion of >5 years is no more valid in an absolute way and probably, the age of 4 years or greater would be the best choice especially to achieve the optimal adherence.139
Also, the adherence or compliance with SLIT still remains a pivotal point, and probably an unmet need as for most chronic treatments, since SLIT is self-managed and self-administered. The adherence is high in clinical trials, but in real life the adherence is largely variable, from <50% to up to 90%.140-143 When the sales data from manufacturer are considered, at the 3rd year of SLIT, only about 15% of patients are still persisting.144 As a promising aspect, especially from an immunological viewpoint is the associ- ation of SLIT with the new biological treatments. This association


10 |
PASSALACQUA et AL.
FIGURE 6 SLIT as a model of “precision medicine”
has been shown very advantageous in clinical terms, but the asso- ciation of the two therapeutic approaches has the major limitation of the costs (for review see 145).
As a final consideration, the main unmet need in the long-last- ing history of SLIT (ad this keeps true also for SCIT) is the absence of predictive biomarkers of efficacy. We have numerous clinical biological markers of current efficacy (eg symptoms, IgG4, cyto- kines),146,147 but we still cannot predict at individual level who will favourably respond, even if SLIT is correctly prescribed. This will be the main challenge of the future history.
4 | CONCLUSION
AIT is a cornerstone in the management of respiratory allergic dis- eases since it is allergen-specific, immunomodulating, and may af- fect disease progression. SLIT, represented a significant advance, offering to patients an excellent safety and convenience profile. From an historical viewpoint, in the last three decades, there was an impressive development of SLIT, against the overall century history of AIT in general. Many aspects have been clarified through the his- torical development: efficacy, safety, role of standardized allergen preparations.. In parallel, many questions emerged, that were only partially clarified (polyallergic patients, use of mixtures, preventative effects of SLIT etc).30
When a historical perspective is considered, the starting point is “why?”, and this is explained by the safety concerns raised more than 30 years ago by SCIT. The intermediate historical points are the investigations performed on different clinical aspects, and the
clinical questions that emerged along the way. The future aspects are the new emerging questions and the space for improvement. So far SLIT, after 30 years of rapid development and evolution re- mains a good paradigm of precision medicine.6,30,148 After about 30 years of clinical trials and practical use, SLIT remains an ex- ample of how clinical intuitions can lead to effective treatments (Figure 6).
CONFLICT OF INTEREST
None to declare for all authors.
ORCID
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140. Senna G, Ridolo E, Calderon M, Lombardi C, Canonica GW, Passalacqua G. Evidence of adherence to allergen-specific immu- notherapy. Curr Opin Allergy Clin Immunol. 2009;9:544-548.
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141. Turkalj M, Banic I, Anzic SA. A review of clinical efficacy, safety, new developments and adherence to allergen-specific immu- notherapy in patients with allergic rhinitis caused by allergy to ragweed pollen (Ambrosia artemisiifolia). Patient Prefer Adherence. 2017;11:247-257.
142. Lemberg ML, Berk T, Shah-Hosseini K, Kasche EM, Mösges R. Sublingual versus subcutaneous immunotherapy: patient adher- ence at a large German allergy center. Patient Prefer Adherence. 2017;11:63-70.
143. Incorvaia C, Mauro M, Leo G, Ridolo E. Adherence to Sublingual Immunotherapy. Curr Allergy Asthma Rep. 2016;16(2):12.
144. Senna G, Lombardi C, Canonica GW, Passalacqua G. How adherent to sublingual immunotherapy prescriptions are patients? The man- ufacturers' viewpoint. J Allergy Clin Immunol. 2010;126:668-669.
145. Lombardi C, Canonica GW, Passalacqua G. Allergen immunother- apy as add-on to biologic agents. Curr Opin Allergy Clin Immunol. 2018;18:502-550.
146. Senna G, Calderon M, Makatsori M, Ridolo E, Passalacqua G. An evidence-based appraisal of the surrogate markers of effi- cacy of allergen immunotherapy. Curr Opin Allergy Clin Immunol. 2011;11:375-380.
147. Shamji MH, Kappen JH, Akdis M, et al. Biomarkers for monitoring clinical efficacy of allergen immunotherapy for allergic rhinocon- junctivitis and allergic asthma: an EAACI Position Paper. Allergy. 2017;72:1156-1173.
148. Passalacqua G, Bagnasco D, Ferrando M, Heffler E, Puggioni F, Canonica GW. Current insights in allergen immunotherapy. Ann Allergy Asthma Immunol. 2018;120:152-154.
How to cite this article: Passalacqua G, Bagnasco D, Canonica GW. 30 years of sublingual immunotherapy. Allergy. 2019;00:1–14. https://doi.org/10.1111/all.14113


Received: 21 May 2019 | Revised: 26 July 2019 | Accepted: 16 August 2019 DOI: 10.1111/all.14036
ORIGINAL ARTICLE Allergen‐Specific Immunotherapy and Biologics
Alum impairs tolerogenic properties induced by allergoid‐ mannan conjugates inhibiting mTOR and metabolic reprogramming in human DCs
Cristina Benito‐Villalvilla1 | Enrique Fernández‐Caldas2,3 |
1Department of Biochemistry and Molecular Biology, School of Chemistry, Complutense University, Madrid, Spain
2Inmunotek, Alcalá de Henares, Madrid, Spain
3University of South Florida, College of Medicine, Tampa, FL, USA
Correspondence
Oscar Palomares, Department of Biochemistry and Molecular Biology, School of Chemistry, Complutense University of Madrid, Avenida Complutense s/n, 28040 Madrid, Spain.
Email: [email protected]
Funding information
Ministerio de Economía y Competitividad, Grant/Award Number: SAF‐2017‐84978‐R; Centre for Industrial Technological Development, Grant/Award Number: IDI‐20110410 and IDI‐20141131
Irene Soria2 | Mario Pérez‐Diego1 |
José Luis Subiza2
| Oscar Palomares1
Abstract
Background: Polymerized allergoids conjugated to mannan (PM) are suitable vaccines for allergen‐specific immunotherapy (AIT). Alum remains the most widely used ad‐ juvant in AIT, but its way of action is not completely elucidated. The better under‐ standing of the mechanisms underlying alum adjuvanticity could help to improve AIT vaccine formulations.
Objective: We sought to investigate the potential influence of alum in the tolerogenic properties imprinted by PM at the molecular level.
Methods: Flow cytometry, ELISAs, cocultures, intracellular staining and suppression assays were performed to assess alum and PM effects in human dendritic cells (DCs). BALB/c mice were immunized with PM alone or adsorbed to alum. Allergen‐specific an‐ tibodies, splenocyte cytokine production and splenic forkhead box P3 (FOXP3)+ regula‐ tory T (Treg) cells were quantified. Metabolic and immune pathways were also studied in human DCs.
Results: Alum decreases PD‐L1 expression and IL‐10 production induced by PM in human DCs and increases pro‐inflammatory cytokine production. Alum impairs PM‐induced functional FOXP3+ Treg cells and promotes Th1/Th2/Th17 responses. Subcutaneous immunization of mice with PM plus alum inhibits in vivo induction of Treg cells promoted by PM without altering the capacity to induce functional allergen‐specific blocking an‐ tibodies. Alum inhibits mTOR activation and alters metabolic reprogramming by shifting glycolytic pathways and inhibiting reactive oxygen species (ROS) production in PM‐acti‐ vated DCs, impairing their capacity to generate functional Treg cells.
Conclusion: We uncover novel mechanisms by which alum impairs the tolerogenic properties induced by PM, which might well contribute to improve the formulation of novel vaccines for AIT.
Abbreviations: 2‐DG, 2‐deoxy‐D‐glucose; AIT, allergen‐specific immunotherapy; Alum, aluminium hydroxide; Breg, regulatory B; CLRs, C‐type lectin receptors; DCs, dendritic cells; FOXP3, forkhead box P3; HmoDC, human monocyte‐derived dendritic cell; mDC, myeloid dendritic cell; mTOR, mammalian target of rapamycin; N, native grass pollen extract; NAC, N‐acetyl‐L‐Cysteine; OX40L, OX40 ligand; pDC, plasmacytoid dendritic cell; PD‐L1, programmed death‐ligand 1; PM, polymerized allergoids conjugated to mannan; PTEN, phosphatase and tensin homologue; ROS, reactive oxygen species; Treg, regulatory T; Δψ, membrane potential.
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2019 The Authors. Allergy published by John Wiley & Sons Ltd.
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KEYWORDS
allergen‐specific immunotherapy, alum, dendritic cells, polymerized allergoids conjugated to mannan, regulatory T cells
GRAPHICAL ABSTRACT
This study investigated the potential influence of alum in the tolerogenic properties imprinted by PM. Alum decreased expression of PD‐L1 and production of IL‐10, IL‐6 and lactate, increased IL‐23 release and inhibited mTOR activation in PM‐activated DCs. Alum suppressed PM‐ induced functional FOXP3+ Treg cells and promoted Th1/Th2/Th17 responses.
Abbreviations: Akt, protein kinase B; Alum, aluminium hydroxide; FOXP3, forkhead box P3; mTOR, mammalian target of rapamycin; P70S6K, 70 kDa ribosomal protein S6 kinase; PD‐L1, programmed death ligand 1; PM, polymerized‐allergoids conjugated to mannan; ROS, reactive oxygen species
1 | INTRODUCTION
Allergen‐specific immunotherapy (AIT) is the only treatment with po‐ tential long‐lasting disease‐modifying effects for allergic diseases.1‐3 Successful AIT is associated with a very rapid desensitization of mast cells and basophils and with the inhibition of type 2 immune‐mediated responses. Sustained tolerance after AIT discontinuation requires the generation and preservation of functional allergen‐specific regula‐ tory T (Treg) and B (Breg) cells as well as allergen‐specific blocking antibodies.4‐6 Clinical trials and real‐life practice demonstrated AIT as an effective treatment; however, it still faces several drawbacks regarding the long treatment duration, patient compliance, side ef‐ fects or low efficacy for some patients.3,7,8 Polymerized allergoids conjugated to mannan (PM) have been recently reported as next‐gen‐ eration vaccines targeting dendritic cells (DCs) that might well con‐ tribute to overcome such inconveniences.9‐12 PM are captured very efficiently by human DCs via mannose receptor and other C‐type lectin receptors (CLRs). PM activates CLRs and promotes IL‐10‐pro‐ ducing tolerogenic DCs that generate functional forkhead box P3 (FOXP3)+ Treg cells through programmed death‐ligand 1 (PD‐L1).9,10 In mice, subcutaneous or sublingual immunization with PM increases
the frequency of splenic FOXP3+ Treg cells and induces healthy im‐ mune responses to allergens.9,11,13 All these beneficial effects depend on the structural integrity of the conjugated mannan.9,12‐14
Aluminium hydroxide (alum) remains the most widely used ad‐ juvant in AIT.15‐17 For long time, the main effects attributed to alum have been related to its depot effect leading to the slow release of the adsorbed allergens.15,18 Alum also activates innate immune responses in DCs and macrophages. Alum induces the release of uric acid and double‐stranded DNA, which activate NLRP3 inflam‐ masome and IRF3 leading to the production of pro‐inflammatory cytokines.19,20 In addition, alum activates Syk‐PI3kδ pathways and induces PGE2 production, which is involved in Th2 polarization.19,20 However, the detailed molecular mechanisms driving alum adjuvan‐ ticity still remain largely unknown.15,18
Metabolic reprogramming in immune cells refers to the changes occurring after activation in the intracellular metabolic pathways that lead to functional regulation.21 DCs’ function is finely regulated by metabolic reprogramming in cooperation with immune pathways.22 The change in metabolism towards glycolysis with high lactate production despite the availability of oxygen is known as the Warburg effect, which represents a key event in the


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regulation of DCs’ function.21,22 Mammalian target of rapamycin (mTOR) is an important regulator of metabolic reprogramming, and its activation state is highly responsive to intracellular and extra‐ cellular signals.21,22 How DCs simultaneously activated with alum and CLR ligands drive immune responses and the potential con‐ sequences of this cross‐talk in metabolic reprogramming remains fully elusive.
The better understanding of the mechanisms involved in alum adjuvanticity might well contribute to improve vaccine formulations for AIT. Herein, we show for the first time that alum impairs the tolerogenic features imprinted by allergoids conjugated to mannan in human DCs. Alum inhibits PM‐induced functional FOXP3+ Treg cells and promotes Th1/Th2/Th17 responses. In vivo, subcutaneous immunization of mice with PM adsorbed to alum inhibits the healthy immune responses to allergens induced by PM alone. Alum shifts glycolytic pathways, inhibits mTOR activation and impairs reactive oxygen species (ROS) production in PM‐activated DCs, thus inhib‐ iting their capacity to generate functional Treg cells. Collectively, we uncover novel mechanisms by which alum impairs the tolerogenic properties induced by PM, which might well pave the way not only for the future rational design of novel AIT vaccines but also for other vaccine formulations.
2 | METHODS
2.1 | Material, media and reagents
For cell cultures, we used RPMI 1640 (Lonza) supplemented with 10% heat‐inactivated foetal bovine serum, 100 μg/mL normocin (InvivoGen), 50 μg/mL penicillin‐streptomycin, 1% nonessential amino acids, 1% MEM vitamins and 1 mmol/L sodium pyruvate (Life Technologies). Glutaraldehyde‐polymerized grass pollen (Phleum pratense) allergoids conjugated to nonoxidized mannan (PM) and na‐ tive grass pollen P pratense allergens (N) were provided by Inmunotek SL. Aluminium hydroxide gel (Alhydrogel) was from InvivoGen. Inhibitors for mTOR (rapamycin) (InvivoGen), ROS (N‐acetyl‐cysteine (NAC)) or glycolysis (2‐Deoxy‐D‐glucose (2‐DG)) (Sigma‐Aldrich) were used for the inhibition experiments.
2.2 | Cell cultures
Immature hmoDCs or human total blood DCs from healthy donors or allergic patients (106 cells per mL) were stimulated with medium (Ctrl ‐), alum (0.1 mg/mL), PM (50 μg/mL) or PM with alum for 18 hours. PM were adsorbed to alum with continuous stirring for 2 hours. Cell pellets were used to analyse their phenotype by flow cytometry and cell‐free supernatants to quantify IL‐6, IL‐23, IL‐12, IL‐4 and IL‐10 by ELISA. For inhibition experiments, hmoDCs were preincubated for 1 hour with 2‐DG (10 mmo/L) or NAC (25 mmo/L), or for 30 min‐ utes with rapamycin (100 nmo/L) (or corresponding vehicle controls) prior to activation. Then, the cells were stimulated with the stimu‐ lus for 18 hours in the presence of the corresponding inhibitors to quantify IL‐10 by ELISA or PD‐L1 by flow cytometry. Cell viability
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was analysed in all the cases by trypan blue exclusion with a light microscope.
2.3 | Statistical analysis
In all experiments, data represent the mean ± SEM of the indicated parameters. Statistical differences were determined with the paired or unpaired Student t test using Prism software 6.0 (GraphPad Software). Significance is indicated in each figure.
All procedures used in this study are fully described in the Methods section in this article's Data S1.
3 | RESULTS
3.1 | Alum impairs tolerogenic features imprinted by
PM in human DCs
To analyse the impact of alum on the expression pattern of differ‐ ent surface molecules and cytokine production induced by PM in human DCs, we treated human monocyte‐derived dendritic cells (hmoDCs) or an enriched fraction of total DCs with PM alone or with PM plus alum. The expression of the inhibitory molecule PD‐L1 was significantly reduced in PM‐stimulated hmoDCs in the presence of alum (Figure 1A). In contrast, alum significantly increased the ex‐ pression of CD‐83 and OX40 ligand (OX40‐L), which are molecules associated with mature DCs and amplification of Th2 cell responses, respectively.23,24 There were no significant differences in HLA‐DR expression (Figure 1A). Representative histograms are displayed in Figure 1B. HmoDCs activated by PM in the presence of alum pro‐ duced significantly higher levels of the pro‐inflammatory cytokine IL‐23 than PM‐stimulated hmoDCs (Figure 1C). We did not detect IL‐12 or significant differences in IL‐4 production (Figure 1C). Alum significantly reduced the production of IL‐6 and the anti‐inflamma‐ tory cytokine IL‐10 in PM‐activated hmoDCs (Figure 1C). Remarkably, alum alone only induced significant production of IL‐23 but not any of the other assayed cytokines. Next, we isolated an enriched frac‐ tion of human blood total DCs containing both plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) (Figure 1D). Supporting our data in hmoDCs, alum significantly increases the production of IL‐23 by PM‐treated total blood DCs. IL‐10 and IL‐6 production also tends to decrease in the presence of alum in PM‐activated total blood DCs (Figure 1E).
3.2 | Alum promotes Th1, Th2 and Th17 responses and decreases IL‐10‐producing T cells induction by PM‐activated human DCs
To determine the capacity of human DCs stimulated with PM in the presence of alum to polarize CD4+ T‐cell responses, we performed coculture experiments. HmoDCs activated with PM plus alum gen‐ erated T cells producing higher levels of IFN‐γ, IL‐5 and IL‐17A than hmoDCs stimulated with PM alone (Figure 2A). In contrast, PM‐ac‐ tivated hmoDCs in the presence of alum generated T cells producing


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F I G U R E 1 Alum alters the phenotype and function induced by PM in human DCs from healthy donors. A, Percentage of positive cells after stimulation of hmoDCs with medium (Ctrl ‐), alum, PM or PM with alum for 18 h (n = 5‐7). B, Flow cytometry representative histograms. C, Cytokine production after stimulation of hmoDCs with the indicated stimulus for 18 h (n = 7). D, Representative dot plots for pDCs and mDCs in PBMCs and the enriched total DC fraction. E, Cytokine production after stimulation of total blood DCs with the indicated stimulus for 18 h (n = 6). Paired Student t test: *P < .05, **P < .01 and ***P < .001
less IL‐10 than PM‐activated hmoDCs (Figure 2A). The IFN‐γ/IL‐10, IL‐5/IL‐10 and IL‐17A/IL‐10 ratios were significantly higher when T cells were primed by hmoDCs activated with PM plus alum than with PM alone (Figure 2B). To verify these data at the single‐cell level, we performed intracellular staining experiments. Primed CD4+ T cells were restimulated with PMA/ionomycin in the presence of brefel‐ din A. Supporting our results, the percentages of IFN‐γ‐, IL‐5‐ and IL‐17A‐producing CD4+ T cells generated by hmoDCs activated with PM in the presence of alum were significantly higher than with PM alone. The percentage of IL‐10‐producing CD4+ T cells
was significantly decreased in the presence of alum (Figure 2C). Representative dot plots are displayed in Figure 2D. Coculture ex‐ periments using an enriched fraction of total blood DCs demon‐ strated that T cells primed by blood DCs activated with PM in the presence of alum produced higher amounts of IFN‐γ, IL‐5 and IL‐17A and significantly lower levels of IL‐10 than T cells generated by total DCs activated with PM alone (Figure 2E). The IFN‐γ/IL‐10, IL‐5/IL‐10 and IL‐17A/IL‐10 ratios were significantly increased in the presence of alum (Figure 2F). Similar results were obtained in longer cocul‐ tures performed during 10 days (data not shown).


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F I G U R E 2 Alum promotes the generation of Th1, Th2 and Th17 cells and impairs the induction of IL‐10‐producing T cells by PM. A and B, Cytokines (A) or cytokine ratios (B) produced by allogeneic naïve CD4+ T cells primed by PM‐ or PM and alum‐activated hmoDCs from healthy donors after 5 d (n = 6). C, After 5 d of coculture, primed T cells were washed and stimulated for 6 h with PMA/ionomycin in the presence of brefeldin A. Percentage of CD3+CD4+ T cells producing the indicated cytokines after intracellular staining and flow cytometry analysis (n = 4). D, Representative dot plots for the intracellular staining after flow cytometry analysis. E and F, Cytokines (E) or cytokine ratios (F) produced by allogeneic naïve CD4+ T cells primed by PM‐ or PM and alum‐activated total blood DCs from healthy donors after 5 d (n = 5). Paired Student t test: *P < .05 and ** P < .01
3.3 | Alum impairs the generation of functional FOXP3+ Treg cells by PM‐activated DCs
PM‐treated hmoDCs induced significantly higher numbers of CD4+CD25highCD127‒FOXP3+ Treg cells than medium‐ or alum‐ treated hmoDCs. In the presence of alum, the number of FOXP3+ Treg cells induced by PM‐activated hmoDCs was significantly de‐ creased (Figure 3A). For functional experiments, we sorted the gen‐ erated Treg cells (CD4+CD25highCD127‒) (Figure S1A). Purified Treg cells generated by PM‐activated hmoDCs suppressed autologous
PBMCs in a dose‐dependent manner (Figure S1B) and displayed a more potent suppression capacity than those generated in the pres‐ ence of alum (Figure 3B). Purified non‐Treg cells (CD4+CD25‒CD127‒) from both conditions showed no suppression capacity (Figure 3B). Interestingly, PM‐activated total DCs also generated a large number of CD4+CD25highCD127‒FOXP3+ Treg cells, which was significantly reduced in the presence of alum, thus confirming the results ob‐ tained with hmoDCs (Figure 3C). We obtained the same results when hmoDCs from allergic patients were employed (Figure S2A). The cy‐ tokine signature of PBMCs from allergic patients and healthy donors


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F I G U R E 3 Alum reduces PM‐induced functional FOXP3+ Treg cells. A, Percentage of induced CD4+CD25highCD127‒FOXP3+ Treg cells by allogeneic medium (Ctrl ‐)‐, alum‐, PM‐ or PM and alum‐treated hmoDCs from healthy donors after 5 d (gating in lymphocytes, n = 9). B, Proliferation of CFSE‐labelled PBMCs gated on CD4+ T cells after 5 d of coculture with autologous purified FOXP3+ Treg cells generated by allogeneic PM‐ or PM and alum‐treated hmoDCs (ratio 1:1). The percentage of proliferating responder PBMCs stimulated with anti‐CD3 and anti‐CD28 for each condition at a 1:1 ratio is shown (n = 2). C, CD4+CD25highCD127‒FOXP3+ Treg cells generated by allogeneic medium (Ctrl ‐)‐, alum‐, PM‐ or PM and alum‐treated total blood DCs after 5 d (gating in lymphocytes, n = 4). Paired Student t test: *P < .05, **P < .01 and ***P < .001
stimulated with PM alone or with PM in the presence of alum was also comparable and characterized by reduced IL‐10 production and increased INF‐γ/IL‐10 ratio in the presence of alum (Figure S2B,C). These results confirm our previous data showing that PM is able to induce similar responses in allergic patients and healthy donors.9
3.4 | Immunization of BALB/c mice with PM
in the presence of alum impairs the healthy immune responses to allergens imprinted by PM
To assess the in vivo relevance of our findings, we immunized sub‐ cutaneously BALB/c mice with diluent (Ctrl ‐), alum, PM or PM plus alum, following the protocol showed in Figure 4A and analysed the indicated parameters. Splenocytes from mice immunized with PM plus alum produced significantly higher levels of IFN‐γ, IL‐5 and IL‐4 but less IL‐10 after in vitro stimulation with native grass pollen ex‐ tract (N) than those from mice immunized with PM alone (Figure 4B).
The ratios IL‐10/IFN‐γ and IL‐10/IL‐5 were significantly lower in the presence of alum (Figure 4C). Interestingly, the percentage of splenic FOXP3+ Treg cells was significantly higher in mice immunized with PM than diluent (Ctrl ‐), which was significantly impaired in the presence of alum (Figure 4D). The levels of serum IgG1 and IgE anti‐ bodies specific for native grass pollen allergens were higher in mice immunized with PM plus alum than in mice immunized only with PM, whereas the levels of IgG2a were not significantly different between these two groups (Figure 4E). The IgG1/IgG2a ratio was significantly higher in mice immunized with PM plus alum than with PM alone (Figure 4F). Next, we assessed the capacity of the antibodies gener‐ ated in mice after each immunization to block patients’ IgE binding to native grass pollen extract. Although the antibodies induced with PM in the presence of alum display a slight higher blocking activ‐ ity than those generated by PM alone, significant differences were not observed, indicating that alum does not significantly enhance the capacity of PM to generate allergen‐specific blocking antibodies.


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F I G U R E 4 Alum impairs in vivo tolerogenic properties induced by PM in BALB/c mice. A, Scheme of the subcutaneous immunization protocol and analysis of induced systemic responses. B and C, Cytokine production (B) or cytokine ratios (C) by splenocytes from mice immunized subcutaneously with diluent (Ctrl ‐), alum, PM or PM and alum, and stimulated in vitro with native grass pollen extract. D, Percentage of CD4+CD25highFOXP3+ Treg cells in spleens of mice immunized with diluent (Ctrl ‐), alum, PM or PM and alum. Representative dot plots are shown. E and F, Serum grass pollen‐specific antibodies (E) or ratio of serum grass pollen‐specific IgG1/IgG2a (F) from mice immunized with diluent (Ctrl ‐), alum, PM or PM and alum. G, Inhibition of serum IgE binding to native grass pollen allergens by blocking antibodies from mice immunized with diluent (Ctrl ‐), alum, PM or PM and alum (mean ± SEM of n = 5 grass pollen allergic patients). For panels B‐F, values are mean ± SEM of 18 individual mice per condition of 3 independent experiments. Unpaired Student t test: *P < .05,
**P < .01 and ***P < .001
3.5 | Alum alters metabolic reprogramming induced by PM and inhibits mTOR activation in human DCs
We studied changes in the metabolic state of DCs activated with PM and how this could be influenced by the presence of alum. HmoDCs activated with PM increased the production of lactate (Warburg effect) compared with unstimulated hmoDCs, which was significantly reduced when hmoDCs were stimulated with PM plus alum (Figure 5A,B). The stimulation of hmoDCs with PM significantly increased the consumption of glucose from the culture medium (Figure 5C), thus significantly enhancing the
metabolic activity (Figure 5D). We did not detect significant dif‐ ferences in glucose consumption or metabolic rates when DCs were stimulated with PM in the presence of alum (Figure 5C,D), suggesting that alum did not alter glucose uptake but its final metabolic fate. To test whether PM alone or in combination with alum affect mitochondrial activity in hmoDCs, we moni‐ tored changes in mitochondrial membrane potential (Δψ) with MitoTracker Red CMXRos. The mitochondrial Δψ was signifi‐ cantly increased by PM plus alum (Figure 5E), suggesting that alum has influence on the glucose metabolic fate by favouring mitochondrial oxidative phosphorylation. Further analysis of the


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F I G U R E 5 Alum alters the metabolic reprogramming induced by PM in human DCs from healthy donors. A, Lactate content in cell‐free supernatants from PM‐ or PM and alum‐stimulated hmoDCs after 18 h relative to the unstimulated (Ctrl ‐) condition (n = 6). B, Quantification of the induced Warburg effect relative to the unstimulated (Ctrl ‐) condition (n = 6). C, Glucose consumption by stimulated DCs and (D) calculated metabolic rate (n = 6). E, Fluorescence intensity of stimulated hmoDCs stained with MitoTracker Red (n = 5). F, Cellular redox status analysed based on the determination of NAD+, NADH and total NAD levels (NADt) (n = 5). G, NAD+/NADH ratios. H, Western blot analysis of protein extracts from hmoDCs stimulated for 30 min in the indicated conditions, and one representative example out of 4 is shown. I, Quantification of the reactive phosphorylated bands by scanning densitometry (mean ± SEM of n = 4 independent experiments). J, Intracellular ROS quantification in hmoDCs after 18 h of stimulation with PM or PM and alum (n = 6). Paired Student t test: *P < .05, **P < .01 and ***P < .001
redox status showed that the NADH accumulation induced by PM in hmoDCs was significantly reduced in the presence of alum (Figure 5F). Consequently, the NAD+/NADH ratio was signifi‐ cantly higher in hmoDCs stimulated with PM plus alum than with PM alone (Figure 5G). Next, we studied the potential implication
of mTOR pathway in the observed effects as a key regulator of metabolic responses. PM induced the rapid activation of mTOR pathway in hmoDCs as determined by the phosphorylation of its downstream substrate p70S6 kinase (Thr389) and its upstream activator Akt (Ser473) (Figure 5H,I). The phosphorylation of both


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F I G U R E 6 Influence of mTOR pathway, glycolysis and ROS production in the induction of human tolerogenic DCs by PM. A‐C, IL‐10 production (n = 6; A), percentage of hmoDCs expressing PD‐L1 (n = 6; B) and flow cytometry representative histogram of PD‐L1 expression (C) of hmoDCs after stimulation with PM for 18 h in the presence of 2‐DG, NAC or rapamycin. D, CD4+CD25highCD127‒FOXP3+ Treg
cells generated after 5 d by allogeneic hmoDCs stimulated in the indicated conditions (gating in lymphocytes, n = 5). E, Flow cytometry representative dot plots of the generated CD4+CD25highCD127‒FOXP3+ Treg cells under the indicated conditions. Paired Student t test:
*P < .05, **P < .01 and ***P < .001
p70S6 kinase and Akt was inhibited when hmoDCs were acti‐ vated by PM plus alum (Figure 5H,I), indicating that alum inhib‐ its the PM‐induced activation of mTOR. Supporting these data, alum also inhibited the phosphorylation of phosphate and tensin homologue (PTEN) (Ser380/Thr382/383), an inhibitor of mTOR signalling pathway that remains inactive while it is phosphoryl‐ ated (Figure 5H,I). In addition to its role in glycolytic metabo‐ lism, previous studies showed a positive interplay between PI3K/ Akt/mTOR signalling pathway and the regulation of ROS produc‐ tion.25 PM treatment significantly increased ROS production in hmoDCs, which was significantly inhibited in the presence of alum (Figure 5J). Collectively, our results demonstrate that alum impairs the PM‐induced activation of mTOR and metabolic repro‐ gramming in hmoDCs.
3.6 | Glycolytic metabolism, mTOR activation and ROS production contribute to the tolerogenic properties imprinted by PM in hmoDCs that are impaired by alum
The inhibition of glycolysis with 2‐DG, mTOR with rapamycin and ROS activity with NAC in PM‐activated hmoDCs significantly suppressed the production of the anti‐inflammatory cytokine IL‐10 (Figure 6A). Similarly, the inhibition of glycolysis, mTOR and ROS activity significantly decreased the expression of PD‐L1 in
PM‐activated hmoDCs (Figure 6B). Representative histograms of PD‐L1 expression in PM‐activated hmoDCs under the different conditions are displayed (Figure 6C). To assess the relevance of these findings in the generation of FOXP3+ Treg cells by PM, we performed coculture experiments with PM‐activated hmoDCs in the presence of the different inhibitors. As expected by the pre‐ vious experiments, alum significantly reduced the frequency of PM‐induced FOXP3+ Treg cells (Figure 6D). Similarly, inhibition of glycolysis with 2‐DG, mTOR with rapamycin and ROS activ‐ ity with NAC in PM‐activated hmoDCs significantly reduced the percentage of induced FOXP3+ Treg cells (Figure 6D,E). These data suggest that (i) these pathways contribute to the induction of FOXP3+ Treg cells by PM‐activated DCs and (ii) alum impairs such induction through its inhibitory effect on these signalling pathways.
4 | DISCUSSION
In this study, we show that alum impairs the tolerogenic properties imprinted by allergoids conjugated to nonoxidized mannan. Alum decreases the expression of PD‐L1 and IL‐10 production in PM‐ac‐ tivated human DCs, reducing their capacity to generate functional FOXP3+ Treg cells. Mechanistically, we demonstrated that alum inhibits mTOR activation, shifts glycolysis metabolism and reduces


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ROS production in human PM‐activated DCs, thus impairing the tolerogenic properties promoted by PM. Subcutaneous immuniza‐ tions of mice with PM adsorbed to alum induces a shift to allergic responses and impairs the induction of splenic FOXP3+ Treg cells supporting the in vitro results with human cells. Remarkably, alum did not alter the capacity of PM to produce IgG‐blocking antibodies with similar functional activity than those generated by PM in the absence of alum. Overall, our data point against the use of alum in order to preserve the tolerogenic properties imprinted by allergoids conjugated to mannan in DCs. This should be also taken into account when considering novel vaccination approaches aimed to induce tolerogenic DCs,26,27 especially when the mTOR pathway is involved in such effects.28 This might be also relevance for those vaccines based on triggering trained immunity, in which the Akt/mTOR acti‐ vation is essential.29,30
This is a paradigmatic study revealing the importance of the bet‐ ter understanding of the molecular mechanisms driving alum adju‐ vanticity in the context of allergy, especially in humans, to continue improving allergen vaccine formulations for AIT. Allergoid‐mannan conjugates represent a major development in the search of novel vaccine approaches to improve AIT,6,9‐11 and Alum was introduced as a vaccine adjuvant in 1926 by Glenny et al,31 and since 1937, it is used in AIT. Although there are different adjuvant preparations that can be used in AIT as immunostimulants (ie, mineral salts or TLR‐tar‐ geting adjuvants) or delivery systems (ie, different types of micro‐ and nanoparticles),11 nowadays most of the subcutaneous vaccines for AIT include alum as adjuvant.15 Although it is considered safe in terms of acute local or systemic effects,18 the molecular mecha‐ nisms driving alum adjuvanticity are not completely understood.20 Alum polarizes responses into Th2 cells in mice, whereas in humans there is still controversy.32‐34 Recent findings showed that alum can also activate innate immune responses in DCs and macrophages.19,20 Here, we performed a comprehensive immunologic study of the po‐ tential influence of alum in the tolerogenic effects imprinted by PM targeting CLRs in DCs.9 Alum significantly decreases the production of IL‐10 and PD‐L1 expression in human PM‐activated DCs, which are molecules involved in the generation of functional Treg cells.35,36 In contrast, the OX40‐L expression, a molecule favouring Th2 polar‐ ization,37,38 is significantly increased in the presence of alum. At the T‐cell level, we showed that in humans, alum (in the presence of PM) not only promotes Th2 responses but also a broad pro‐inflammatory profile, with Th1 and Th17 cells being also enhanced, whereas inhib‐ iting the generation of functional FOXP3+ Treg cells. Up to now, no data showing cross‐talk between CLR‐ and alum‐mediated signalling pathways have been reported. Alum is sensed by membrane lipids on DCs inducing activation in a receptor‐independent manner that is mediated by the Syk‐PI3K pathway.20,39 PM trigger CLRs such as Syk‐coupled Dectin‐2 or DC‐SIGN, both involved in PD‐L1 expres‐ sion and cytokine signature in human PM‐activated DCs.9 Our data clearly suggest that alum interferes with the CLR‐mediated signalling pathways activated by PM in DCs.
Metabolic reprogramming plays a very important role in the control of DCs’ function by regulating tolerogenicity vs. immunogenicity.21,22
mTOR is a central regulator of cell metabolism, growth, proliferation and survival.40,41 To gain insight into potential novel molecular mecha‐ nisms involved in the observed effects, we studied metabolic changes induced by PM in DCs and how alum could interfere on them. PM rap‐ idly activates mTOR signalling pathway and PM‐activated DCs display a high rate of glycolysis and lactic acid fermentation (Warburg effect),40,42 features that were impaired by alum. Increased mTOR pathway activ‐ ity is correlated with enhanced glycolysis, which appears to be associ‐ ated with the generation of peripherally induced Treg cells.21 Lactate has been also shown to enhance IL‐10 production in macrophages and DCs.43 It is well‐recognized that TLR activation in DCs and macro‐ phages induces a glycolytic burst,44,45 but data for CLRs are mostly lim‐ ited to Dectin‐1 activated by β‐glucan.29 We demonstrate that mTOR activation is implicated in the induction of IL‐10 and PD‐L1 in human PM‐activated DCs. Schülke et al also described that in mDCs activated by the fusion protein rFlaA:Betv1, mTOR regulates IL‐10 production.28 Similarly, the expression of PD‐L1, a key molecule for the induction of functional FOXP3+ Treg cells,36,46,47 has been shown to be regulated by mTOR in DCs.48 Herein, we show that the inhibition of PM‐induced mTOR signalling and the reduction in PD‐L1 expression in DCs by alum correlate with a reduction on the generation of FOXP3+ Treg cells. Supporting this view, SH2 domain‐containing inositol 5’‐phosphatase (SHIP)‐deficient mice, in which the Akt/mTOR pathway is upregulated on myeloid cells,49 show a remarkable increase in FOXP3+ Treg cells.50
Tolerogenic DCs display enhanced glycolytic capacity and ROS production with respect to mature pro‐inflammatory DCs.51 PM signifi‐ cantly increases intracellular ROS in human DCs, which were inhibited by alum. Recently, D‐mannose treatment increased ROS production in T cells compared with TCR stimulation alone.52 In line with our results, blockade of ROS activity by NAC significantly reduced numbers of D‐ mannose‐induced Treg cells.52 Although ROS have been considered toxic products of cellular metabolism, increasing evidence supports the idea that low amounts of ROS are positive contributors to normal sig‐ nalling pathways.42,53 Previous studies demonstrated that ROS further activates Akt/mTOR pathway, enabling optimal T‐cell proliferation and glycolysis.53‐55 Therefore, our results confirm that the inhibition of ROS production and the blockade of mTOR signalling by alum in PM‐acti‐ vated DCs represent novel mechanisms by which this adjuvant impairs the induction of Treg cells.
Interestingly, mice immunized with PM in the presence of alum display significantly lower numbers of splenic FOXP3+ Treg cells and higher IFN‐γ, IL‐5 and IL‐4 levels than mice immunized only with PM. The ratio of serum IgG1/IgG2a is significantly higher in mice immu‐ nized with PM plus alum, indicating that alum also impairs in vivo tolerogenic responses to PM. Remarkably, alum did not enhance the capacity of PM to induce allergen‐specific antibodies with blocking activity, indicating that the absence of alum in PM vaccine formula‐ tions would not significantly modify this feature.
In conclusion, this study provides novel insights into molecular pathways that might be affected by alum as adjuvant in AIT. We uncover novel molecular mechanisms involving mTOR, glycolysis and ROS production by which alum interferes with CLR‐mediated signalling pathways activated by polymerized allergoids conjugated


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to nonoxidized mannan in human DCs, thus impairing the imprinted tolerogenic properties. This study demonstrates the importance of understanding the influence of adjuvants such as alum in novel vac‐ cine formulations for AIT. Future studies on how other approved adjuvants for AIT might influence the tolerogenic features imprinted by allergoids conjugated to mannan could also contribute to pro‐ vide novel mechanistic insights into their way of action. Finally, our findings might well go beyond AIT formulations and could be also relevant for other types of vaccines such as those promoting innate trained immunity, in which mTOR activation and metabolic repro‐ gramming represent key mechanistic events in their mode of action.
ACKNOWLEDGEMENTS
This work was supported by grants SAF‐2017‐84978‐R to OP from MINECO, Spain, and by grants IDI‐20110410 and IDI‐20141131 to Inmunotek SL from CDTI and MINECO. CB‐V. and MP are re‐ cipients of FPU and FPI fellowships, respectively, from MINECO. IS is recipient of Torres Quevedo grant (PTQ‐12‐05785), from MINECO, Spain.
CONFLICTS OF INTEREST
OP has received payment for lectures and participation in Advisory Boards from Allergy Therapeutics, Amgen, AstraZeneca, Inmunotek SL, Novartis, Sanofi Genzyme and Stallergenes. OP has received re‐ search grants from Inmunotek SL and Novartis SL. JLS is the founder and CEO of Inmunotek SL. EF‐C. is a shareholder and employee of Inmunotek SL. IS is an employee of Inmunotek. The rest of the au‐ thors declare no competing financial interests.
AUTHOR CONTRIBUTIONS
OP and JLS conceived and designed the study. C. B.‐V. (human ex‐ periments), IS (mice experiments) and M. P.‐D. (technical support for human experiments) performed the experiments. E. F.‐C, JLS and O.P provided reagents. C. B.‐V., IS, M. P.‐D., EF‐C., JLS and O.P ana‐ lysed and discussed the data. OP and C. B.‐V wrote the paper. All the authors read and approved the final version of the manuscript.
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11. Benito‐Villalvilla C, Soria I, Subiza JL, Palomares O. Novel vaccines targeting dendritic cells by coupling allergoids to mannan. Allergo J Int. 2018;27:256‐262.
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ORCID
Cristina Benito‐Villalvilla Enrique Fernández‐Caldas
https://orcid.org/0000‐0002‐5544‐0199 https://orcid.org/0000‐0001‐5873‐6932
José Luis Subiza Oscar Palomares
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SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Benito‐Villalvilla C, Soria I, Pérez‐ Diego M, Fernández‐Caldas E, Subiza JL, Palomares O. Alum impairs tolerogenic properties induced by allergoid‐mannan conjugates inhibiting mTOR and metabolic reprogramming in human DCs. Allergy. 2020;75:648–659. https://doi. org/10.1111/all.14036


Received: 18 March 2019 | Revised: 9 May 2019 | Accepted: 26 May 2019 DOI: 10.1111/all.13980
ORIGINAL ARTICLE
Drug Allergy, Insect Sting Allergy, and Anaphylaxis
Venom immunotherapy in patients with clonal mast cell disorders: IgG4 correlates with protection
Jesper Jarkvist1,2 | Clara Salehi1 |
1Department of Respiratory Medicine and Allergy, Karolinska University Hospital Huddinge, Stockholm, Sweden
2Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet, and Karolinska University Hospital, Stockholm, Sweden
3Division of Allergy and Clinical Immunology, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI, USA
4Mastocytosis Center Karolinska, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
Correspondence
Theo Gulen, Department of Respiratory Medicine and Allergy, K85, Karolinska University Hospital Huddinge, SE‐141 86 Stockholm, Sweden.
Email: [email protected]
Funding information
Konsul TH C Bergh Foundation, Sweden; The regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet.
1 | INTRODUCTION
Clonal mast cell disorders (cMCD) comprise systemic mastocyto‐ sis (SM) and monoclonal mast cell activation syndrome (MMAS).1,2 Common to these two conditions is the presence of mast cell (MC) clonality, as reflected in a mutation in codon 816 of KIT and/or occurrence of immunophenotypically aberrant MCs expressing CD25.3 In patients with MMAS, the WHO criteria for SM are not fully met.3
Cem Akin3 |
Theo Gülen1,2,4
Abstract
Background: Patients with clonal mast cell disorders (cMCD), systemic mastocytosis (SM) and monoclonal mast cell activation syndrome (MMAS), represent an increased risk for Hymenoptera venom anaphylaxis (HVA). Lifelong venom immunotherapy (VIT) is recommended; however, its efficacy and safety are controversial. Hence, we sought to evaluate the efficacy and safety of VIT in HVA patients with cMCD. Methods: A retrospective study was conducted among 46 patients with Vespula venom allergy who had experienced severe HVA, 32 cMCD (22 with SM and 10 with MMAS) and 14 controls. There were no differences between cMCD patients and controls in age (58 vs 66) and duration of VIT (47 vs 48 months), respectively. Results: During VIT, 11 (34%) cMCD patients experienced adverse reactions (ARs) (7% in controls), including 1 anaphylaxis. There were 23 re‐stings in 17 (53%) patients during VIT. Of episodes, four (17%) presented with anaphylaxis, 14 (60%) presented with local reaction, and five (23%) were asymptomatic. In 11 episodes (48%), the patient did not take epinephrine, of these 8 (73%) presented with local reaction, and 3 (27%) were asymptomatic. Patient‐based protection from anaphylaxis was 76% (4/17) in cMCD vs. 100% in controls during VIT. The venom‐specific IgG4 concentra‐ tions increased during VIT (P < .001) although tryptase and IgE were unaltered. Conclusion: Both safety and efficacy of VIT in cMCD patients were slightly reduced than controls. Severe ARs were rare. The elevated IgG4 levels may be a biomarker for efficacy of VIT in cMCD patients, as it correlates with protection from re‐stings.
KEYWORDS
D816V mutation, hymenoptera venom anaphylaxis, IgG4, mastocytosis, venom immunotherapy
Allergy. 2020;75:169–177. wileyonlinelibrary.com/journal/all © 2019 EAACI and John Wiley and Sons A/S. | 169 Published by John Wiley and Sons Ltd.
Anaphylaxis is a well‐known feature of cMCD; particularly, venom allergy represents an increased risk of severe, even fatal, sting anaphylaxis in these patients.4,5 Although the overall prev‐ alence of Hymenoptera venom‐induced anaphylaxis (HVA) is ap‐ proximately 25% in patients with SM,6 the underlying reason(s) for this association remains elusive. The aggravated risk of severe HVA might be due to increased MC burden, perivascular aggre‐ gation of MCs and an amplified IgE reaction due to the presence of D816V KIT mutation.7 These findings stress the importance of


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GRAPHICAL ABSTRACT
Patients with clonal mast cell disorders (cMCD) convey increased risk for venom‐induced anaphylaxis and lifelong venom‐immunotherapy (VIT) is recommended, when appropriate. VIT appears to be rather safe and effective in cMCD patients, as the risk of severe adverse
reactions are rare. Elevated levels of venom‐specific IgG4 correlates
accurate diagnostics; therefore, underlying cMCD should be con‐ sidered in patients with HVA that have elevated serum baseline tryptase levels (sBT) (≥11.4 ng/mL). Additionally, sensitization against Hymenoptera venom components should be confirmed by skin prick test and/or serum‐specific IgE assays. However, it must be noted that patients with cMCD and HVA may lack sensitization to venoms.4,8
Venom immunotherapy (VIT) has been used for treatment of patients with diagnosed cMCD and HVA since 1990s; however, increased adverse reactions (ARs) and reduced efficacy have been main concerns in earlier studies.9 As many as 6 in 7 patients with cMCD had reactions to field re‐stings, despite ongoing VIT treat‐ ment.9 This raised concerns and controversies about current rec‐ ommendations regarding necessity and duration of VIT in patients with cMCD. Interestingly, more recent studies found VIT to be safe and effective in patients with cMCD but acknowledged a reduced efficacy and more frequent ARs during the administration of VIT compared with the general population.10‐13 At present, there is no evidence that VIT induces sustainable tolerance in patients with cMCD. Hence, the current recommendation is to proceed lifelong VIT in these patients.14 Additionally, the known markers of success‐ ful VIT (IgG4, IL‐10, regulatory T cells) have not been specifically studied in cMCD patients.
Thus, there is a continuing unmet need for further studies regard‐ ing VIT in patients with cMCD, as the available observations are based on limited number of reports. Here, we sought to determine the safety and efficacy of VIT by evaluating ARs during the administration of VIT and assessing the severity of field re‐sting reactions. Furthermore, we
with protection from re‐sting anaphylaxis in cMCD patients during VIT.
also analysed the efficacy by monitoring certain biomarkers before and during ongoing treatment in patients with cMCD.
2 | METHODS
2.1 | Patients and clinical procedures
Between January 2006 and December 2018, 396 consecutive adult patients (≥18 years old) have been referred to the Mastocytosis Centre Karolinska due to clinically suspected cMCD including pa‐ tients with mastocytosis in the skin, patients with severe anaphylaxis or patients with elevated baseline tryptase levels of unknown origin. The final diagnoses, for example, SM or MMAS, were obtained after a comprehensive medical evaluation and bone marrow investigation following WHO criteria.3 Moreover, sBT levels (Thermo Fisher) were measured.
Anaphylactic reactions were diagnosed in accordance with NIH clinical criteria, when either reduced blood pressure or as‐ sociated symptoms such as syncope/presyncope and/or respira‐ tory compromise were present accompanied by the involvement of the skin‐mucosal tissue and/or gastrointestinal symptoms.15 In cases where assessments were difficult because of insufficient documentation, only patients who had syncope episodes after ex‐ posure to a likely trigger (eg insect sting) were assessed to have anaphylaxis. When available, serum tryptase levels during acute episodes were applied to confirm anaphylaxis. The diagnosis of HVA was based on clinical history, skin prick test and/or allergen‐ specific lgE.16


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F I G U R E 1 The flow chart illustrates the selection process of the study subjects. *35 patients were excluded due to various reasons (15 received VIT at other clinics, 7 had elevated baseline tryptase levels without underlying cMCD, 6 declined to undergo VIT, 4
had comorbidities with cancer, two patients were investigated during study start, and 1 patient was sensitized for honeybee only). Abbreviations: w/o, without; VIT, venom immunotherapy; MCD, mast cell disorders
2.2 | Allergy work‐up
As previously described,4 all patients went through a complete al‐ lergic work‐up at Karolinska University Hospital Huddinge, allergy outpatient clinic including medical history, skin prick testing (SPT) with commercial extracts (ALK‐Abelló A/S, Horsholm, Denmark) of standard aeroallergens, food allergens and allergen of Hymenoptera venom (honeybee and common wasp). The venom‐specific lgE antibody test for honeybee and Vespula venom (ImmunoCAP Phadiatop®, Thermo Fisher, Uppsala, Sweden) was also performed and considered positive for values >0.10 kU/L. Moreover, serum concentrations of component‐specific venom lgE (Ves r5, Ves r1, Api m1 and Api m10); venom‐specific lgG4; and serum total lgE were determined by ImmunoCAP® (Thermo Fisher).
2.3 | Study design and subjects
A retrospective study was conducted. Data were collected through re‐ view of electronic patient records. Of 396 investigated patients, 178 had experienced at least one anaphylactic reaction (Figure 1). Among these, 97 patients with HVA were identified. After excluding 51 patients, 46 pa‐ tients with Vespula venom allergy who fulfilled the criteria for VIT14 were enrolled in this study (Figure 1). Of study subjects, 32 had diagnoses of cMCD and HVA. Additionally, 14 patients with HVA and normal sBT lev‐ els (<11.4 ng/mL) were included as controls to compare the safety and efficacy of VIT. The study was approved by Stockholm's Ethics Review Board (Dnr: 2011/1750/31/3 and Dnr: 2018/2621‐31), and all enrolled patients provided their written informed consent to participate.
2.4 | Venom immunotherapy and follow‐up
Venom immunotherapy was started with Vespula extract (ALK‐ Abelló) according to a 7‐week traditional build‐up schedule at the al‐ lergy outpatient clinic. Patients received incremental, weekly doses of depot venom extract subcutaneously until a maintenance dose (1 mL of 100 000 SQ‐U/mL, corresponding to 100 μg) was reached. The achieved maintenance dose was then given every four (between May and October; high‐risk season) or 6 weeks (between November and April; low‐risk season for stings), and follow‐up ended on 31 December 2018. All patients received premedication with HI block‐ ers, 1‐2 hours prior to VIT, and were observed 45 minutes after each injection. Extra drugs were given in case of acute reactions. Additionally, simultaneous treatment with omalizumab (Xolair®) during VIT was documented, when applied.
Blood samples were collected as part of routine patient care, and biological markers were in general analysed before VIT started and at different time points during VIT. Information about possible ARs was documented during the routine VIT visits, or as in few cases, patients reported late ARs by phone. Data about field re‐stings during VIT and their outcomes were documented at the time of next follow‐up visit and confirmed by the emergency room (ER) reports, when available.
2.5 | Statistical analysis
All analyses were performed using IBM SPSS Statistics 24.0 (IBM). Values of P < 0.05 were considered statistically significant. Frequencies were reported for categorical variables, and group


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differences were analysed by using Fisher's exact test. Continuous variables were presented as median values and ranges. Because the distribution of the data was not normal according to Shapiro‐Wilk test, the nonparametric Mann‐Whitney U test or Kruskal‐Wallis test was used to compare the group distributions, when appropriate. Additionally, when crude data analysis was significant, a post hoc analysis was performed using the Wilcoxon's matched pair rank sum test to detect alterations at different time points within groups. We used Spearman's rank correlation coefficient to demonstrate clinical relevance of venom‐specific IgG4 during re‐stings. Since re‐stings were unpredictable, and it was impossible to assess the IgG4 on the day of re‐sting, we used IgG4 concentrations closest to the day of re‐sting occasions.
3 | RESULTS
3.1 | Patient characteristics and clinical data prior to
venom immunotherapy
Of 32 patients with cMCD, 22 were diagnosed with indolent SM; 64% were males and had a median age of 61 (range 40‐80) at diagnosis. Additionally, 45% of patients with SM (10/22) had cutaneous involve‐ ment, 36% had MC clusters in bone marrow biopsy, and 86% carried a KIT D816V mutation. They started VIT at a median age of 62 (40‐81) years and received the treatment for a median period of 45 months (7‐154). The remaining 10 cMCD patients were diagnosed with MMAS; 56% were males and had a median age of 55 (range 38‐71). All MMAS patients had MCs expressing CD25+ aberrant immunophe‐ notype, whereas 22% concomitantly carried a KIT D816V mutation. They started VIT at a median age of 52 (38‐66) years and received VIT for a median period of 77 months (9‐104). Bone marrow examinations were offered to all but performed only in 4 of 14 controls, since most refused to undertake the procedure. Additionally, peripheral blood KIT D816V mutation was negative in all analysed controls (n = 11).
Subjects with cMCD were more often males (63%) than in the con‐ trol group (43%), although this finding was not statistically significant (Table 1). Serum baseline tryptase levels were significantly higher in patients with cMCD, whereas total IgE were significantly higher in con‐ trols (Table 1). Moreover, controls presented with significantly higher levels of venom‐specific IgE and component rVes v5 (P = .001; Table 1). To note, 57% of patients in control group suffered from cardiovascular comorbidities including hypertension and/or angina pectoris prior to the culprit sting reaction (P < .001; Table 1). Regarding the severity of culprit reactions, both cMCD patients and controls presented with severe HVA, where syncope frequently occurred (82% of the patients with SM, 56% with MMAS patients and 57% in controls). Additionally, controls frequently presented with skin symptoms (P < .001); other‐ wise, there were no significant differences between the two groups in terms of reaction severity (Figure 2).
VIT started in subjects with cMCD at an earlier age with a me‐ dian of 58 years compared to 66 years in controls (Table 2). The du‐ ration of VIT was similar between the two groups and varied among individual patients due to the retrospective nature of the study. A
TABLE 1 Comparison of demographic, clinical and laboratory characteristics of cMCD patients with HVA compared to controls with HVA prior to VIT
Total, n = 46 Age ≥ 18 y
Clonal MCD (n = 32)
Controls (n = 14)
P‐value
Male gender, n (%)
sBT levels (ng/mL), median (range)
Presence of atopy, n (%)
Positive ImmunoCAP for wasp, n (%)
Component rVes v 5 (kU/L), median (range)
Wasp‐specific IgG4 (mg/L), median (range)
Comorbidity with CVD prior to first sting,
n (%)
20/32 (63)
18 (3.2‐68) (7 NA)
10/32 (31)
26/28 (93) (4 NA)
0.30 (0.1‐25) (13 NA)
0.52 (0.04‐8.9) (22 NA)
2/32 (6)
6/14 (43) 5.3 (2.4‐11)
3/14 (21)
12/12 (100) (2 NA)
6.2 (0.11‐60) (1 NA)
2.20 (13 NA)
8/14 (57)
.333a <.001b
.724a 1.000a
.001b
ND
<.001a
Age at diagnosis, me‐ dian (range)
59 (38‐80)
66 (46‐78)
.129b
Total IgE (kU/L), median (range)
25 (2.6‐1000) (11 NA)
77 (28‐790) (2 NA)
.033b
Positive SPT for wasp, n (%)
22/31 (71) (1 NA)
10/13 (77) (1 NA)
1.000a
Wasp‐specific IgE (kU/L), median (range)
0.54 (0.09‐48) (10 NA)
5.3 (0.12‐50) (3 NA)
.007b
Component rVes v 1 (kU/L), median (range)
0.10 (0.1‐75) (15 NA)
0.10 (0.1 −0.16) (11 NA)
ND
Syncope from wasp sting prior to VIT
24/32 (75)
8/14 (57)
.301a
Abbreviations: cMCD, clonal mast cell disorders; CVD, cardiovascular disease; HVA, Hymenoptera venom anaphylaxis; NA, not analysed; ND, not done; sBT, serum baseline tryptase; SPT, skin prick test; VIT, venom immunotherapy.
aP‐values were calculated using Fisher's exact test;
bP‐values were calculated using a 2‐tailed Mann‐Whitney U test; bold indicates statistical significance (P < .05).
total of seven patients discontinued VIT mainly due to the appear‐ ance of comorbidities (including cancer) or were referred to home clinics for practical reasons.
3.2 | Adverse reactions during VIT
Among patients with cMCD, 11 (34%) experienced ARs, eight (73%) during the induction phase and three (27%) during maintenance. The total number of episodes was 16, and epinephrine was administered twice (Table 2). Nine of these episodes were only local reactions, and six involved milder systemic reactions (without respiratory/cardio‐ vascular symptoms). Conversely, ARs in controls were limited to one patient (7%) who reacted with milder systemic reaction (Table 2). Anaphylaxis was observed only in one patient with SM who re‐ ceived simultaneous immunotherapy against wasp and honeybee and occurred nine months after the maintenance phase of VIT had started. The patient presented with flush and general weakness a


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FIGURE 2 Clinical symptoms in patients with cMCD and controls during the culprit anaphylactic reactions to wasp sting prior to VIT. Statistical analysis performed by Fisher's exact
test. *Hypotension, objectively verified. Abbreviations: F/U, faecal and/or urinary incontinence; GI, gastrointestinal cramps, nausea, vomiting and diarrhoea; RESP, respiratory symptoms; SKIN, local swelling and redness, itching
TABLE 2 Group comparison of adverse reactions during VIT
Clonal MCD (n = 32)
Controls (n = 14)
P‐value
Age at start of VIT, median (range)
Patients with adverse reactions from VIT, n (%) Mild systemic adverse reactions, n (%)
Total number of injections, n
Number of injections needed per anaphylaxis, n
58 (38‐81) 11/32 (34) 7/16 (44) 1 781
1 781
66 (45‐79) 1/14 (7) 1/6 (17) 493
ND
.127a .073b ND ND ND
Total VIT duration (months), median (range)
47 (7‐154)
48 (13‐65)
.277a
Total number of episodes with adverse reactions (n)
16
1
ND
Patients with anaphylactic reaction from VIT, n (%)
1/32 (3)
0/6 (0)
ND
Number of injections needed per adverse reaction, n
111
1
ND
Use of adrenaline in adverse reaction, n (%)
2/16 (18)
0/1 (0)
ND
few minutes after receiving both VIT injections. The blood pressure was initially normal (122/70 mm Hg). Despite receiving immediate treatment with epinephrine, 3‐4 minutes later the patient had docu‐ mented hypotension (80/44 mm Hg). He received another dose of epinephrine and intravenous fluid before he was transferred to the ER. Afterwards, VIT could be resumed, however, solely with wasp extract and in conjunction with omalizumab (Xolair@) treatment (300 mg q2weeks). The omalizumab treatment has been continued, and no further incidences occurred since then. Omalizumab pro‐ tection was applied in two other patients (diagnosed with SM and MMAS, respectively), and the treatment was discontinued after 54 and 20 months, respectively. Thereby, VIT was tolerated, as these patients still receive VIT. We needed to apply omalizumab in these patients to achieve maintenance doses since it has been reported in the literature with successful results to allow administration of VIT.
3.3 | Protection from re‐sting reactions while ongoing VIT
A total of 17 (53%) patients with cMCD were re‐stung in 23 separate ep‐ isodes. Epinephrine was used in 12 episodes. One sting occurred during
the induction phase and the remaining during the maintenance phase. Six episodes were asymptomatic, 10 resulted in local reaction, and three resulted in milder systemic reactions. Four episodes (in four separate patients) were assessed to be anaphylactic reactions, and all four pa‐ tients used intramuscular epinephrine and sought emergency care (Table 3). Table S1 shows the main characteristics of 5 patients with ana‐ phylaxis during VIT (four anaphylaxis during re‐stings and one anaphy‐ laxis as AR). Interestingly, these five patients had positive SPT for wasp at baseline (Table S1), whereas only 22 patients in overall cohort (71%) (Table 1). Nevertheless, this was not clinically significant. Meanwhile, five (35%) controls were re‐stung in a total of eight episodes, which oc‐ curred during the maintenance phase of VIT (Table 3). Epinephrine was used in two episodes, although no anaphylaxis was observed.
During VIT, clinical symptoms from re‐stings were found to be less severe compared to culprit reactions. Most re‐sting reactions in pa‐ tients with cMCD were limited only to skin; moreover, 23% were as‐ ymptomatic. In contrast to the culprit reactions, where 75% of patients with cMCD had syncope, only one patient (5%) had syncope during re‐stings (P < .001). Consequently, we observed 76% (13/17) patient‐ based and 83% (19/23) episode‐based protection from anaphylaxis in patients with cMCD during field re‐sting reactions (P < .001).
Abbreviations: cMCD, clonal mast cell disorders; ND, not done; VIT, venom immunotherapy. aP‐values were calculated using a two‐tailed Mann‐Whitney U test.
bP‐value was calculated using Fisher's exact test.


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Clonal MCD (n = 32)
Controls (n = 14)
P‐value
Patients who were re‐stung during VIT, n (%) Protection from anaphylaxis per subject, n (%) Episodes with anaphylactic reaction to re‐sting, n (%) Use of adrenaline in episode of re‐sting, n (%)
17/32 (53) 13/17 (76) 4/23 (17) 12/23 (52)
5/14 (35) .346 0/5 (100) ND 0/8 (0) ND 1/8 (13) .095
TABLE 3 Comparison of patients with cMCD and control subjects regarding re‐ sting reactions during VIT
Number of patients with anaphylactic reaction, n (%)
4/17 (24)
0 (0)
ND
Number of episodes of re‐stings (n)
23
8
ND
Protection from anaphylaxis per episode, n (%)
19/23 (83)
8/8 (100)
ND
Abbreviations: cMCD, clonal mast cell disorder; ND, not done; VIT, venom immunotherapy. P‐values were calculated using Fisher's exact test.
3.4 | Dynamics of biomarkers in patients with cMCD during VIT
Serum concentration of wasp‐specific lgG4 increased significantly in patients with cMCD over the period of VIT (P < .001; Table S2). In con‐ trast, repeated measures of other biomarkers including plasma levels of sBT, total IgE, wasp venom‐specific IgE or venom component rVes v5 did not show significant dynamics during VIT compared to baseline levels. Calculation of specific ratios incorporating IgG4 did not pro‐ vide any additional significance beyond IgG4 levels alone (Figure S2).
On group level, the median of wasp‐specific lgG4 before VIT was 0.52 mg/L (range 0.04‐8.9) in cMCD (Table S2). Two outliers were identified (2.4 and 8.9 mg/L, respectively) at baseline, and both previously completed a 5‐year VIT course (Figure 3A). Afterwards, VIT was restarted when both patients were diagnosed with cMCD. We also evaluated wasp‐specific IgG4 levels in six cMCD patients in relation to nine re‐sting reactions and found an inverse correla‐ tion between IgG4 concentrations and reaction severity (P < .01; Figure 3B). Since IgG4 could not be obtained on the day of the re‐stings, we analysed the closest IgG4 values in relation to day of reactions (median 4 months, range 0‐10). Two patients who were as‐ ymptomatic in three episodes when re‐stung had wasp‐specific IgG4 levels between 21 and 25 mg/L. Conversely, the only patient who reacted in two episodes during the same summer with mild systemic reactions presented with lower wasp‐specific IgG4 levels, 10 and 11 mg/L prior to and after the stings. The remaining five patients who only experienced local reactions (<10 cm in diameter) had IgG4 concentrations between 8 and 21 mg/L.
4 | DISCUSSION
Our results support that VIT appears to be rather safe and effective in patients with cMCD and HVA as the risk of severe systemic ARs was rare. Additionally, significantly increased serum concentrations of wasp‐specific IgG4 were detected; this is, to our knowledge, the first report demonstrating this phenomenon in cMCD patients.
While allergic reactions to foods and drugs have been reported in cMCD, the primary trigger for IgE‐mediated severe, even fatal,
anaphylactic reactions remains Hymenoptera stings.17,18 In general, VIT induces protection from severe sting reactions in HVA patients during and after discontinuation of therapy.19‐22 Nevertheless, there have been controversies regarding its safety and efficacy in patients with cMCD. ARs to VIT have been reported in 29% of patients with cMCD compared to 14% in general HVA population.12,23 VIT protocols applied during induction phase are also essential since increased frequencies of ARs were reported in rush‐ or ultrarush protocols.24 Interestingly, no ARs were observed in a recent study of eight mastocytosis patients receiving VIT by ultrarush protocol.25 Our study supports previous findings as we found a fivefold increased risk for ARs in cMCD patients compared to controls (34% vs 7%, respectively). Additionally, the ARs occurred mainly during the build‐up phase, of which 44% were milder systemic reactions. Only one patient suffered from an anaphylactic re‐ action (3%). Of note, this patient was treated with both honeybee and wasp venom simultaneously, a procedure reported to increase the risk of ARs.12 No patients in the current study had to be discontinued; how‐ ever, VIT in patients with cMCD is not risk‐free.
Efficacy of VIT is typically evaluated by sting challenges and reports from field re‐stings; however, sting challenges are not per‐ formed in all clinics.14 Several studies have reported on efficacy as the rate of protection from systemic reactions; nevertheless, no uni‐ versally accepted grading system exists to classify the severity of systemic reactions.26 This complicates comparison of the efficacy of VIT across different studies.27 In patients with mastocytosis, the protection rate varied from 14% to 85% in a review of 10 studies with 201 patients.12 Our results support VIT being an efficient treatment to prevent anaphylaxis, since only four episodes (17%) from field re‐ stings were classified as anaphylaxis. That implies a protection rate of 83%, in line with a previous report (86%).10 Only one patient with cMCD reacted with syncope when re‐stung compared to 75% during culprit reactions (P < .001). There were no fatalities in our series. However, since this protection can only be sustained during VIT, the current guidelines recommend lifelong VIT in patients with cMCD.
Notably, the severity of culprit reactions in patients with cMCD appears to be independent from concomitant cardiovascu‐ lar diseases (CVDs) and use of beta blockers and/or ACE inhibitors since the control subjects more frequently presented with CVDs (P < .001) without experiencing more severe reactions. Thus, the


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F I G U R E 3 A, Dynamic of wasp‐specific IgG4 levels in cMCD patients during VIT. Two outliers were identified (2.4 and 8.9 mg/L, respectively) at baseline, and both patients previously received
VIT. P‐values were analysed with Wilcoxon's matched pair rank sum test. A significant increase was noted between baseline and years 1‐2 (P = .028) and between years 3‐4 and years 5‐6 (P = .018). n; number of matched patients at different time points. Different colours represent different time periods. B, Correlation between wasp‐specific IgG4 levels and severity of re‐sting reaction. The reactions were classified as asymptomatic, local reactions (<10 cm in diameter) and mild systemic reactions (without respiratory
or circulatory compromise). Correlation was quantified by using Spearman's rank correlation coefficient
mechanisms leading to severe HVA in cMCD patients may be dif‐ ferent from those of controls; for instance, they might be due to the inherent mast cell hyperreactivity. Additionally, the serum concen‐ tration of the venom‐specific IgE and component‐specific venom IgE rVes v5 (which is the dominating allergic epitope in Sweden) levels at baseline was lower in patients with cMCD compared to controls (P = .013). It is known that patients with SM typically exhibit lower levels of total and specific IgE, presumably due to the adsorption of specific IgE by the expanded MC burden28; however, whether this is true also for rVes v5 has not been previously investigated.
The immunological mechanisms underlying VIT efficacy have not been fully elucidated, although induction of peripheral tolerance and the generation of allergen‐specific regulatory T (Treg) and B (Breg) cells
appear to be cardinal features. Treg cells are characterized by IL‐10 se‐ cretion that directly or indirectly suppresses effector cells including mast cells, and also have influence on B cells, suppressing IgE produc‐ tion and inducing the production of blocking‐type IgG4 antibodies against venom allergens.29‐33 The earlier studies concerned patients from the general HVA population and acknowledged that specific IgG levels increased significantly during VIT but also decreased signifi‐ cantly when VIT was discontinued.21,34‐36 Since a protective effect was still evident even after VIT was discontinued, they concluded other im‐ munological mechanisms rather than specific IgG were likely responsi‐ ble. Nevertheless, these studies did not involve the IgG subclass, IgG4. Interestingly, another investigation reported that specific honeybee IgG4 concentrations remained increased two years after discontinuing VIT against honeybee, suggesting a long‐lasting protection of specific IgG4.37 This issue has been re‐evaluated in a recent study in patients with HVA and demonstrated increased levels of wasp‐specific IgG4 during VIT course, but concentrations declined substantially at 3‐ and 8‐year follow‐up after discontinuation of VIT.38 Additionally, Golden et al21 found that 88% of patients who reacted systemically to a sting during VIT had venom‐specific IgG antibody levels ≤3 mg/L, thereby recommended that monitoring sIgG levels during VIT might be pre‐ dicting residual risk of systemic reactions after a sting. Similarly, a later study reported that monitoring VIT efficacy was only possible in ves‐ pid‐venom allergy, and the authors proposed that sIgG4 threshold for rVes v5 had the highest sensitivity to confirm tolerance.39
Currently, no studies have demonstrated sustained tolerance development in patients with cMCD. Because severe or fatal ana‐ phylactic reactions with re‐stings occur only in patients with cMCD after discontinuation of VIT, the immunological mechanism behind clinical efficacy may differ from nonclonal population.12,14 However, the dynamic of IgG4 during VIT has never been analysed in patients with cMCD. We demonstrated that venom‐specific IgG4 levels con‐ stantly increased during VIT and reached a 20‐fold increase during the first 2 years of treatment (Figure 3A). Additionally, when we ob‐ served wasp‐specific IgG4 in relation to re‐sting reactions, we found an inverse correlation between IgG4 levels and reaction severity sug‐ gesting that IgG4 concentrations might reflect the clinical efficacy (Figure 3B). Furthermore, we also identified two patients who had anaphylaxis when re‐stung, 4 and 11 years after having discontinued VIT. Both patients were later diagnosed with cMCD. The serum con‐ centrations of wasp‐specific IgG4 were clearly reduced in both (8.9 and 0.72 mg/L, respectively) before VIT was restarted compared to patients with cMCD who had been continued VIT (median IgG4 con‐ centration 20.5 mg/L at years 5‐6). Hence, treatment failure may be related to inadequate levels of IgG4. VIT could be administered more frequently in these patients to attempt to raise the serum concentra‐ tion of venom‐specific IgG4, thereby increasing efficacy.
Conversely, no significant alterations during different time points of VIT were observed regarding the levels of tryptase, total IgE, wasp venom‐specific IgE and component‐specific venom IgE. Notably, previous studies demonstrated both decreased and un‐ changed levels of venom‐specific IgE during VIT.23,40,41 In the study by Gonzales et al23 with SM patients and HVA, venom‐specific IgE


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levels decreased for the entire group during VIT. However, only 6 of 21 patients had Vespula venom allergy.23 Remarkably, their patients had relatively high median levels of specific IgE before VIT compared to our patients (4.15 KU/L vs 0.54kU/L). These might be contributing to the contradictory results. Meanwhile, tryptase levels remained unchanged during VIT in both studies.23
The main strength of this single‐centre study was the homo‐ geneity of the subjects enrolled, since all patients received VIT against wasp, with extract from the same manufacturer and fol‐ lowed by similar VIT protocols. We could therefore compare pa‐ tients with cMCD to control patients in the same clinical settings. By contrast, paucity of study subjects and retrospective nature of the study lacking relevant data from all patients at all time points analysed were our limitations making difficult to generalize re‐ sults. When reactions to re‐stings were assessed, patient‐reported data were unavoidably used and could include a recall bias. Use of epinephrine is a confounding factor that could not be avoided. Finally, several symptoms from systemic allergic reactions and panic attacks overlap, for example anxiety and tachycardia, and all these factors complicate clinicians' assessment of reactions.
In conclusion, our results suggest that both efficacy and safety of VIT are somewhat less reliable in cMCD patients, but the over‐ whelming benefit justifies the relatively small increase in risk, as severe ARs are rare. The patient‐based and episode‐based protec‐ tive rate from field re‐sting anaphylaxis were 76% and 83%, respec‐ tively; nevertheless, the efficacy of treatment was only measured during VIT course. Therefore, we support the notion that patients with cMCD should continue VIT indefinitely. Over the course of VIT, venom‐specific IgG4 antibodies were increased significantly in pa‐ tients with cMCD and an inverse correlation between IgG4 levels and reaction severity to field re‐stings appears to exist. Using wasp‐ specific IgG4 to monitor clinical efficacy may allow us to schedule more individualized administration of VIT; however, this issue needs to be further explored.
ACKNOWLEDGMENTS
This study was supported by grants from the Konsul TH C Bergh Foundation, Sweden, and through the regional agreement on medi‐ cal training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet.
CONFLICT OF INTEREST
T. Gülen has received lecture fees from Thermo Fisher. C. Akin has received consultancy fees from Blueprint Medicines and Novartis and has a patent for LAD2 cells. J. Jarkvist and C. Salehi declare no relevant conflicts of interest.
AUTHOR CONTRIBUTIONS
JJ took active part in the acquisition, analysis and interpretation of the data, and drafting and revising of the manuscript. CS took active
part in the acquisition and analysis of the data. CA analysed and interpreted the data and revised the manuscript critically. TG con‐ ceptualized and designed the study; collected, analysed and inter‐ preted the data; and wrote and revised the manuscript. All authors approved the final submitted manuscript.
ORCID
Theo Gülen
https://orcid.org/0000‐0002‐1683‐8882
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SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.
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28. Vos B, van Anrooij B, van Doormaal JJ, Dubois A, Oude Elberink J. Fatal anaphylaxis to yellow jacket stings in mastocytosis: options for identification and treatment of at‐risk patients. J Allergy Clin Immunol Pract. 2017;5:1264‐1271.
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Lieberman P, Platts‐ alpha‐gal sensitivity anaphylaxis. Allergy.
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Received: 25 May 2019 | Revised: 22 August 2019 | Accepted: 7 September 2019 DOI: 10.1111/all.14080
ORIGINAL ARTICLE
Atopic Dermatitis, Urticaria and Skin Disease
Dupilumab is very effective in a large cohort of difficult- to-treat adult atopic dermatitis patients: First clinical and biomarker results from the BioDay registry
Lieneke F. M. Ariëns1 | Jorien van der Schaft1 | Daphne S. Bakker1 | Deepak Balak1 | Margreet L. E. Romeijn2 | Tessa Kouwenhoven3 | Marijke Kamsteeg3 |
Barbara Giovannone1 | Julia Drylewicz4 | Cynthia Catalina Aurora van Amerongen2 | Evelien M. Delemarre4 | Edward F. Knol1,4 | Femke van Wijk4 | Stefan Nierkens4 |
Judith L. Thijs1 | Marie L. A. Schuttelaar3 |
1Department of Dermatology and Allergology, National Expertise Center for Atopic Dermatitis, University Medical Center Utrecht, Utrecht, The Netherlands
2Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
3Department of Dermatology, Radboud University Medical Center Nijmegen, Nijmegen, The Netherlands
4Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands
Correspondence
Lieneke F. M. Ariëns, Department of Dermatology and Allergology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Email: [email protected]
Funding information
The BioDay registry is sponsored by Sanofi and Regeneron Pharmaceuticals.
Marjolein S. de Bruin-Weller1
Abstract
Introduction: Dupilumab has recently been approved for the treatment of moderate to severe atopic dermatitis (AD) in adults. Daily practice data on dupilumab treatment are scarce.
Objective: To study the effect of 16-week treatment with dupilumab on clinical re- sponse and serum biomarkers in adult patients with moderate-severe AD in daily practice.
Methods: Data were extracted from the BioDay registry, a prospective multicenter registry. Sixteen-week clinical effectiveness of dupilumab was expressed as num- ber of patients achieving EASI-50 (Eczema Area and Severity Index) or EASI-75, as well as patient-reported outcomes measures (Patient-Oriented Eczema Measure, Dermatology Life Quality Index, Numeric Rating Scale pruritus). Twenty-one bio- markers were measured in patients treated with dupilumab without concomitant use of oral immunosuppressive drugs at five different time points (baseline, 4, 8, 12, and 16 weeks).
Results: In total, 138 patients treated with dupilumab in daily practice were included. This cohort consisted of patients with very difficult-to-treat AD, including 84 (61%) patients who failed treatment on ≥2 immunosuppressive drugs. At week 16, the mean percent change in EASI score was 73%. The EASI-50 and EASI-75 were achieved by 114 (86%) and 82 (62%) patients after 16 weeks of treatment. The most reported side effect was conjunctivitis, occurring in 47 (34%) patients. During dupilumab
Abbreviations: AD, atopic dermatitis; DLQI, Dermatology Life Quality Index; EASI, Eczema Area and Severity Index; Generic five-dimension five-level EQ-5D-5L, EuroQoL scale; HrQoL, health-related quality of life; IL, interleukin; IQR, interquartile range; MCID, minimal clinically important difference; METC, Medical Research Ethics Committee; NRS, Numeric Rating Scale; PARC, pulmonary and activation-regulated chemokine; PMD, pellucid marginal degeneration; POEM, Patient-Oriented Eczema Measure; TARC, thymus- and activation-regulated chemokine; TCS, topical corticosteroids; TNF, tumor necrosis factor; TSLP, thymic stromal lymphopoietin.
Lieneke F. M. Ariëns, Jorien van der Schaft, Marie L. A. Schuttelaar and Marjolein S de Bruin-Weller contributed equally to this manuscript.
116 | © 2019 EAACI and John Wiley and Sons A/S. wileyonlinelibrary.com/journal/all Allergy. 2019;75:116–126. Published by John Wiley and Sons Ltd.


ARIËNS et Al. | 117
treatment, disease severity-related serum biomarkers (TARC, PARC, periostin, and IL-22), eotaxin-1, and eotaxin-3 significantly decreased.
Conclusion: Treatment with dupilumab significantly improved disease severity and decreased severity-related serum biomarkers in patients with very difficult-to-treat AD in a daily practice setting.
KEYWORDS
atopic dermatitis, biomarkers, daily practice, disease severity, dupilumab
GRAPHICAL ABSTRACT
This study evaluated the clinical effectiveness and safety of 16-weeks of dupilumab treatment in adults with AD. Dupilumab treatment significantly suppressed disease severity-related serum biomarkers and eosinophil chemokines. By the end of the treatment, the EASI-50 and EASI-75 was achieved by 86% and 62% of patients, respectively.
Abbreviations: AD, Atopic dermatitis; DLQI, Dermatology life quality index; EASI, Eczema area and severity index; IGA, Investigators global assessment; NRS, Numeric rating scale; PARC, Pulmonary and activation-regulated chemokine; POEM, Patient-oriented eczema measure; TARC, Thymus- and activation-regulated chemokine
1 | INTRODUCTION
Dupilumab is a fully human monoclonal antibody directed against the interleukin (IL)-4 receptor alpha that blocks the binding of IL-4 and IL-13, which are the key drivers of Th2 immune diseases includ- ing AD. IL-4 and IL-13 have a direct effect on the epidermis by ef- fecting the keratinocyte differentiation, production of filaggrin, and cell adhesion molecules. Furthermore, IL-4 and IL-13 induce Th2 cell activation and survival, promote IgE class switching, and stimulate eosinophil recruitment. Dupilumab is the first biologic agent that has been approved in the EU, USA, Japan, and other countries for the treatment of patients with inadequately controlled moderate to severe AD. The clinical efficacy and safety of dupilumab ± topical corticosteroids (TCS) has been demonstrated in phase 3 clinical tri- als at 16 weeks and 52 weeks in adult patients with moderate to severe AD.1-3 Overall, dupilumab has shown a favorable safety pro- file in clinical trials. However, higher rates of conjunctivitis (9%-28%)
have been reported in patients treated with dupilumab compared to placebo.1-3
Limited data on dupilumab treatment in a daily practice setting are available. Patients participating in randomized controlled tri- als are often carefully screened based on predefined inclusion and exclusion criteria. In contrast, patients treated in a real-life setting are unselected and therefore probably less compliant and may have more comorbidities.4 Therefore, data derived from clinical trials might not be generalizable to a population treated with dupilumab in a real-life setting. In a daily practice setting, the balance between effectiveness and side effects determines whether treatment will be continued or not.
In this study, we evaluated the clinical effectiveness and safety of 16 weeks of dupilumab in adult patients with difficult-to-treat AD in a real-life setting. Our secondary aim was to study which bio- markers are affected by dupilumab treatment and if they correlate to pathways involved in the pathogenesis of AD.


118 | 2 | METHODS 2.1 | Study design
A prospective, observational cohort study was performed includ- ing patients who started dupilumab treatment from October 2017 to February 2018 at the National Expertise Center for Atopic Dermatitis from the University Medical Center Utrecht (UMCU), the department of Dermatology, University Medical Center Groningen (UMCG) and the department of Dermatology, Radboud University Medical Center Nijmegen (Radboud UMC). All patients were aged ≥18 years and fulfilled the criteria for dupilumab treatment es- tablished by the Dutch Society of Dermatology and Venereology (NVDV). Data were extracted from an online Good Clinical Practice database called BioDay registry. The BioDay registry includes a prospective cohort of adult patients with moderate to severe AD treated with dupilumab in daily practice. Patients included in the BioDay registry gave written informed consent. Physicians in the participating hospitals were trained by members of the registry team in clinical scoring. This study did not fall under the scope of the Medical Research Involving Human Subjects Act which was confirmed by the local Medical Research Ethics Committee (METC 18/239). The study has been performed according to the declara- tion of Helsinki.
2.2 | Patients and outcome measures
All 138 patients were assed prior to initiation and for 16 weeks dur- ing dupilumab treatment. At baseline, all patients received a load- ing dose of dupilumab 600 mg subcutaneously administered by a clinician, followed by subcutaneous dupilumab 300 mg every other week (mostly self-administered). Systemic immunosuppressive treatment was discontinued before starting dupilumab treatment in most patients, or a shared decision on continuation of systemic im- munosuppressive treatment during dupilumab treatment was made. Concomitant treatment with TCS was allowed. At baseline, and after 4, 8, 12, and 16 weeks of treatment, disease severity was assessed by the Eczema Area and Severity Index (EASI). Additionally, patient- reported outcomes including the Patient-Oriented Eczema Measure (POEM), weekly average Numeric Rating Scale (NRS) pruritus, Dermatology Life Quality Index (DLQI), and generic five-dimension five-level EuroQoL scale (EQ-5D-5L) were collected. Clinical end- points (all at weeks 4, 8, 12, and 16, unless otherwise indicated) in- cluded the mean percent change from baseline in EASI, NRS pruritus, DLQI (at week 16), and POEM, proportions of patients with ≥50%, ≥75%, or ≥90% improvement from baseline in EASI score (EASI-50, EASI-75 or EASI-90), achieving ≥4-point reduction in weekly aver- age NRS pruritus, reporting “no problem” on the EQ-5D-5L pain/ discomfort and anxiety/depression subscales (week 16), achieving ≥4-point improvement in DLQI score (minimal clinically important difference (MCID) at week 16), and achieving ≥4-point improvement in POEM score (MCID) and change over time for number of days
ARIËNS et Al.
with itch because of eczema (POEM item 1) and number of nights that sleep was disturbed in the past week (POEM item 2). In addi- tion, the proportion of patients using systemic immunosuppressive drugs during dupilumab treatment was monitored.
2.3 | Clinically relevant response
A clinically relevant response was defined based on thresholds in one or more outcomes of the three major AD domains (signs, symp- toms, and quality of life).5 Clinically relevant response was meas- ured via analysis of proportion of patients who achieved EASI-75 or improvement (reduction) in weekly average NRS pruritus ≥4 points from baseline or improvement (reduction) in DLQI score ≥4 points from baseline.
2.4 | Safety
Side effects during the use of dupilumab were evaluated every visit. Patients were asked whether they had experienced subjective side effects and safety laboratory parameters (blood count, serum creati- nine, liver enzymes) were monitored.
2.5 | Serum biomarkers
Patients using oral immunosuppressive drugs at one of the five time points and patients using oral immunosuppressive drugs within 2 (fast-acting drugs) or 4 (slow-acting drugs) weeks before screening were excluded. Twenty-one biomarkers associated with different disease pathways were measured: severity-associated markers (IL- 22, thymus- and activation-regulated chemokine [TARC], pulmonary and activation-regulated chemokine [PARC], and periostin), Th2- associated markers (IL-4, IL-13, and thymic stromal lymphopoietin [TSLP]), Th17-associated markers (IL-6, IL-17, IL-21, IL-22, IL-23, and IL-26), Th22-associated markers (IL-20, IL-22, IL-26), a Th1-related marker (IL-12), an inflammation-related marker (tumor necrosis fac- tor [TNF] alpha), a pruritus-related marker (IL-31), eosinophil markers (IL-5, eotaxin-1, eotaxin-3), and neutrophil markers (elastase, IL-8) (Table S1). Biomarkers were measured before initiation of dupilumab treatment (screening) and after 4, 8, 12, and 16 weeks of treatment using multiplex immunoassays as previously described.6
2.6 | Super-responders and development of conjunctivitis at week 16
Patients were stratified by the achievement of a clinically relevant improvement in all of the three key domains at week 16 (super-re- sponders) and the development of conjunctivitis. Clinical character- istics were compared in the total group between patients who did or did not achieve a clinically relevant improvement at week 16 and


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TABLE 1 Baseline characteristics
Age (y), mean (SD)
Atopic/allergic diseases at baseline, n (%)
Asthma
Allergic conjunctivitis
History of ≥2 oral immunosuppressive treatments, n (%)
Previous use of methotrexate, n (%)
Previous use of mycophenolate mofetil/ enteric-coated mycophenolate sodium, n (%) Missing, n (%)
POEM score, median (IQR) DLQI score, median (IQR) EQ-5D-5L dimension, n (%)
Mobility
Self-care
Usual activity
Pain/discomfort
Anxiety/depression
aIndependent sample t test. bchi-square test. cMann-Whitney U test.
patients with and without conjunctivitis at week 16. Baseline and changes over time in serum biomarkers were compared between pa- tients included in the biomarker subgroup who did or did not achieve a clinically relevant improvement at week 16 and patients with and without conjunctivitis at week 16.
43.4 (15.4)
90 (65.2)
89 (64.5)
84 (60.9)
55 (39.9)
40 (39.0)
3 (2.2)
20 (16.0-23.0)
12.5 (8.0-19.0)
39.8 (13.1) .12a
24 (68.6) .63b 27 (77.1) .18b 22 (62.9) .78b 11 (31.4) .24b 11 (31.4) .71b
2 (5.7) – 20 (16.0-25.0) .86c 10 (7.5-19.0) .52c
Total group (n = 138)
Biomarker subgroup (n = 35)
P-value
Men, n (%)
86 (62.3)
25 (71.4)
.20b
Allergic rhinitis
100 (72.5)
27 (77.1)
.47b
Food allergy
70 (50.7)
21 (60.0)
.20b
Previous use of systemic immunosuppressants for atopic dermatitis, n (%)
136 (98.6)
35 (100.0)
.68b
Previous use of cyclosporin A, n (%)
131 (94.9)
34 (97.1)
.49b
Previous use of azathioprine, n (%)
46 (33.3)
13 (37.1)
.58b
EASI score, median (IQR)
19.9 (13.6-28.3)
24.4 (16.8-31.9)
.19c
Weekly average pruritus NRS, median (IQR)
7 (6.0-8.0)
7 (5.0-8.0)
.57c
Missing, n (%)
3 (2.2)
1 (2.9)
–
Missing, n (%)
2 (1.4)
2 (5.7)
–
Missing, n (%)
2 (2.2)
1 (2.9)
–
No problems
106 (77.9)
27 (79.4)
.81b
Problems
30 (22.1)
7 (20.6)
No problems
114 (84.4)
28 (82.4)
.70b
Problems
21 (15.6)
6 (17.6)
No problems
48 (35.3)
15 (44.1)
.21b
Problems
88 (64.7)
19 (55.9)
No problems
20 (14.7)
5 (14.7)
1.00b
Problems
116 (85.3)
29 (85.3)
No problems
58 (42.6)
15 (44.1)
.84b
Problems
78 (57.4)
19 (55.9)
2.7 | Statistical analysis
Data were analyzed at baseline and 4, 8, 12, and 16 weeks after initiation of dupilumab treatment, except for patients with discon- tinuation of dupilumab treatment, which are described separately.


120 |
TABLE 2 Effectiveness outcomes during dupilumab treatment in 138 patients
ARIËNS et Al.
9 (6.6)
4.0 (2.0-7.6)* −16.3 (10.9)
114 (85.7) 32 (24.1)
3.0 (1.0-5.0)* −53.5 (35.0)
3.0 (2.0-6.0)* −9.2 (6.3)
7.0 (3.0-12.0)* −12.0 (6.6)
−1.9 (1.5)
63 (48.1
92 (70.2)
Baseline (n = 138)
Week 4 (n = 138)
Week 8 (n = 138)
Week 12 (n = 138)
Week 16 (n = 136)
Concomitant use of systemic prednisone, n (%)
EASI score, median (IQR)
ΔEASI from baseline, mean (SD)
EASI-50, n (%)
EASI-90, n (%)
Weekly average pruritus NRS, median (IQR)
∆Weekly average pruritus NRS % from baseline, mean (SD)
DLQI score, median (IQR) ∆DLQI from baseline, mean (SD)
POEM score, median (IQR) ∆POEM from baseline, mean (SD)
∆POEM item 1 (itch) from baseline, mean (SD)
EQ-5D item 4 (pain/discomfort): proportion of patients reporting “no problem”, n (%)
EQ-5D item 5 (anxiety/depression): proportion of patients reporting “no problem”, n (%)
37 (26.8)
19.9 (13.6-28.3) –
–
–
7.0 (6.0-8.0) –
12.5 (8.0-19.0) –
20.0 (16.0-23.0) –
–
20 (14.7)
58 (42.6)
23 (16.8)
7.8 (5.6-13.5)* −12.1 (9.9)
84 (62.7)
4 (3.0)
4.0 (2.0-6.0)* −38.3 (41.3)
– –
10.0 (5.0-15.5)* −8.9 (6.0)
−1.4 (1.4)
–
–
14 (10.2)
6.2 (3.1-9.1)* −15.0 (10.6) 107 (81.7) 17 (13.0)
3.0 (2.0-5.0)* −45.3 (46.2)
– –
8.0 (4.0-13.0)* −11.0 (7.0)
−1.7 (1.5)
–
–
11 (8.0)
4.5 (2.2-8.5)* −15.7 (10.7) 110 (82.7) 31 (23.3)
3.0 (1.0-5.0)* −48.8 (48.0)
– –
7.0 (3.0-12.5)* −11.6 (7.0)
−1.8 (1.5)
–
–
Accumulated dose of systemic prednisone (mg), median (IQR)
12.5 (7.5-25.0)
10.0 (5.0-11.3)*
5.0 (3.5-10.0)*
5.0 (3.5-10.0)*
2.5 (2.0-10.0)*
Missing, n (%)
3 (2.2)
1 (0.7)
4 (2.9)
2 (1.4)
0 (0)
ΔEASI % from baseline, mean (SD)
–
−51.1 (31.4)
−64.8 (32.2)
−68.4 (35.5)
−73.1 (26.5)
EASI-75, n (%)
–
27 (20.1)
58 (44.3)
74 (55.6)
82 (61.7)
EASI ≤ 7, n (%)
8 (5.9)
57 (41.6)
78 (58.2)
94 (69.1)
96 (70.6)
Missing, n (%)
1 (0.7)
1 (0.7)
2 (1.4)
1 (0.7)
0 (0)
Weekly average pruritus NRS, proportion of patients who achieved improvement (reduction) ≥4 points from baseline, n (%)
–
52 (37.7)
67 (48.6)
75 (54.3)
79 (57.2)
Missing, n (%)
2 (1.4)
–
–
–
1 (0.7)
Proportion of patients with ≥ 4-point improvement in DLQI score, n (%)
–
–
–
–
102 (77.9)
Missing, n (%)
3 (2.2)
9 (6.5)
4 (2.9)
5 (3.6)
0 (0)
Proportion of patients with ≥4-point improvement in POEM score, n (%)
–
102 (83.6)
117 (92.9)
116 (93.5)
119 (93.0)
∆POEM item 2 (sleep) from baseline, mean (SD)
–
−1.5 (1.5)
−1.7 (1.5)
−1.8 (1.5)
−1.8 (1.6)
Missing, n (%)
2 (1.4)
–
–
–
5 (3.7)
Missing, n (%)
2 (1.4)
–
–
–
5 (3.7)
Note: Data were analyzed by using a Wilcoxon matched-pairs signed-rank test. *Statistically significant (P < .05) compared to baseline.
Clinical outcome measures at each follow-up time point were com- pared using the Wilcoxon signed-rank test. Serum biomarker levels were normalized by a log-transformation. Differences in biomarker
levels between T0 (baseline) and T1 (4 weeks), and between T1 (4 weeks) and T4 (16 weeks) were compared using the Wilcoxon signed-rank test. P-values <.05 were considered statistically


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| 121
FIGURE 1 Characterization of the patients with a clinical relevant response: proportion of patients achieving EASI-75 or NRS ≥4-point improvement or DLQI ≥4-point improvement after 16 weeks of dupilumab treatment (outcomes available in 129 patients) [Color figure can be viewed at wileyonlinelibrary.com]
13 (10)
18(14)
Week 16 9(7)
52 (40) 17 (13)
14 (11)
6 (5)
0 (0)
EASI-75 (n(%))
NRS≥4 improvement (n(%))
DLQI≥4 improvement (n(%))
No clinical relevant response (n(%))
Outcomes were available in 129 patients.
EASI, Eczema Area and Severity Index; EASI-75, ≥75% improvement in EASI score; NRS, Numeric Rating Scale; DLQI, Dermatology Life Quality Index.
significant. Differences in baseline characteristics and serum bio- markers between subgroups stratified by treatment response and development of conjunctivitis at week 16 were analyzed by using a t test for normally distributed data, Mann-Whitney U test for variables with a non-normal distribution, and chi-square test for categorical variables. All statistical analyses were conducted using SPSS (for Windows, version 25.0, SPSS Inc) and Prism (version 7.4; GraphPad).
3 | RESULTS
3.1 | Patients and baseline characteristics
Between November 2017 and September 2018, 138 consecu- tive patients treated with dupilumab were included with a me- dian EASI score of 19.9 (interquartile range [IQR 13.6-28.3]) at baseline (Table 1). Weekly average pruritus NRS was 7 (IQR 6.0-8.0). Patients reported high scores on the POEM (median 20.0 [IQR 16.0-23.0]) and DLQI (median 12.5 [IQR 8.0-19.0]). Most patients reported problems on usual activity (88 patients [64.7%]), pain/discomfort (116 patients [85.3%]), and anxiety/ depression (78 patients [57.4%]) dimensions of the EQ-5D-5L questionnaire. Before initiation of dupilumab treatment, 136 patients (99%) were treated with oral immunosuppressants for AD (Table 1). Most patients (84 [61%]) had a history of ≥2 oral immunosuppressive treatments before starting dupilumab treatment indicating difficult-to-treat AD. None of the patients were previously treated with dupilumab in clinical trials or daily practice.
3.2 | Effectiveness
After 16 weeks of treatment, the mean percent change in EASI score was −73%. At week 16, the EASI-50, EASI-75, and EASI-90 were achieved by 114 (86%), 82 (62%), and 32 (24%) patients, respectively (Figure S1). The proportion of patients achieving EASI ≤7 (clear-mild AD) at week 16 was 71% (Table 2). The weekly average NRS pruritus significantly decreased from baseline (NRS pruritus mean = 6.9, SD = 2.1) to week 16 (NRS pruritus mean = 3.1, SD = 2.2; P < .001) (Figure S1). At week 16, 79 patients (57%) achieved ≥4 points improvement (reduction) in weekly average pruritus NRS. Treatment with dupilumab also improved other patient-reported outcomes including the health-related quality of life, symptoms of AD, pain/discomfort, sleep and symptoms of anxi- ety and depression (Table 2). The DLQI score significantly decreased from baseline (mean = 13.4, SD = 7.2) to week 16 (mean = 4.3, SD = 4.2; P < .001) with 78% of the patients achieving a ≥4-point improvement in DLQI score after 16 weeks of treatment. The POEM score significantly decreased from baseline (mean = 19.7, SD = 5.5) to week 16 (mean = 7.7, SD = 5.9; P < .001). The proportion of patients reporting “no problems” on the EQ-5D-5L pain/discomfort and anxiety/depression subscale in- creased from baseline (15% and 43%) to week 16 (48% and 70%).
In 129 patients, data including the NRS pruritus, EASI, and DLQI score after 16 weeks of treatment with dupilumab were available to define whether a clinical relevant response was achieved. The proportion of patients achieving a clinically relevant improvement in at least one of the three key domains (EASI-75 or NRS ≥4 point improvement or DLQI ≥4 point improvement) after 16 weeks of dup- ilumab treatment was 89% (115 out of 129 patients). In 11% (14 out of 129 patients), no clinically relevant improvement in at least one of the key domains was achieved (Figure 1).


122 |
TABLE 3 Side effects during dupilumab treatment in 138
ARIËNS et Al.
Other relatively frequently reported side effects included head- ache in 14 patients (10%), injection site reaction in 7 patients (5%), and gastro-intestinal complaints in 7 patients (5%).
The proportion of patients with a blood eosinophilia (≥0.45 × 10 × 9/L) increased from screening (45 patients [33%]) to week 16 (78 pa- tients [57%]). Increased blood eosinophil levels were not associated with symptoms and did not result in dose adjustment or treatment discontinuation of dupilumab. No other laboratory abnormalities were observed during treatment with dupilumab in this study.
3.4 | Biomarkers
For the biomarker analysis, twenty-one biomarkers (Table S1) were measured in a subgroup of 35 patients without concomitant use of oral immunosuppressive drugs at 5 different time points (screening, after 4, 8, 12, and 16 weeks of treatment with dupilumab). Baseline characteristics were not significantly different between the patients included for the biomarker analysis and the total group of patients (Table 1).
Dupilumab treatment significantly reduced severity-related serum biomarkers TARC, PARC, periostin, and IL-22 from screening through week 4. TARC and periostin further decreased from week 4 through week 16 (Figure 2). IL-4 showed a significant increase from screening (median 0.27 pg/mL, IQR 0.27-0.27) through week 4 (me- dian 1.44 pg/mL, IQR 0.90-1.88) (P < .0001). IL-13 was stable from screening to week 4, but then increased significantly from week 4 (median 7.16 pg/mL, IQR 3.00-15.61) through week 16 (median 9.13 pg/mL, IQR 3.02-19.18) (P = .037). Dupilumab treatment sig- nificantly decreased serum levels of eotaxin-1 and eotaxin-3 from screening through week 4 (from median 103.33 pg/mL [IQR 78.33- 130.01] to 83.52 pg/mL [IQR 63.57-133.55] at week 4 P = .038, and from median 5.51 pg/mL [IQR 3.54-8.89] to 1.91 pg/mL [IQR 1.70- 2.52], P < .0001, respectively).
No significant changes were found in the levels IL-5, IL-6, IL-8, IL-12, IL-17, IL-20, IL-21, IL-23, IL-26, IL-31, TNF-a, TSLP, and elastase during dupilumab treatment (Figure S2).
3.5 | Super-responders and development of conjunctivitis at week 16
The baseline EASI score was significantly higher among patients who achieved a clinically relevant improvement in all of the 3 key domains of the clinically relevant response compared to patients who did not achieve a clinically relevant improvement in all of the three domains (median EASI [IQR] 23.5 [16.5-31.8] vs 18.3 [12.6- 26.5], P = .024). Other baseline characteristics (total group and biomarker subgroup and baseline serum biomarkers (biomarker subgroup)) did not significantly differ between patients who did or did not achieve a clinically relevant improvement in all of the three key domains of the clinically relevant response and patients with or without conjunctivitis (Tables S2-S6). Changes over time in EASI
patients
Conjunctivitis (total)
Moderate-severe conjunctivitis (ophthalmologist- confirmed, anti-inflammatory eye drops/ointment)
Blood eosinophilia (≥0.45 × 10 × 9/L)
Screening 8 wk
16 wk
Headache
Gastro-intestinal complaints Hair loss
Blepharitis
3.3 | Safety
47 (34.1) 27 (19.6)
45 (32.6) 78 (56.5) 78 (56.5) 14 (10.1) 7 (5.1)
5 (3.6) 4 (2.9)
Number of patients with, n (%)
Mild conjunctivitis
20 (14.5)
4 wk
67 (48.6)
12 wk
76 (55.1)
Eye irritation
34 (24.6)
Injection site reaction
7 (5.1)
Fatigue
6 (4.3)
Herpes Simplex
4 (2.9)
Flu like symptoms
3 (2.2)
Two patients discontinued dupilumab treatment during the 16-week follow-up period. One patient with a history of pellucid marginal degeneration (PMD) of both eyes and amblyopia of the left eye de- veloped conjunctivitis of both eyes during dpilumab treatment. Due to the development of a limbal stem cell deficiency in this predis- posed patient, treatment with dupilumab was discontinued after 12 weeks. Another patient with a history of atopic keratoconjuncti- vitis developed hyperemia of the conjunctiva with visual complaints. Ophthalmological examination showed a progressive PMD of the left eye. Since involvement of dupilumab in the development of this rapid progressive eye disorder could not be excluded, dupilumab treatment was discontinued after 12 weeks.
The most reported side effects during dupilumab treatment were eye irritation in 34 patients (25%) (including symptoms of dry eyes, itch, and tearing) and conjunctivitis in 47 patients (34%) (symptoms and signs including hyperemia of the conjunctiva) (Table 3). Patients were diagnosed with mild conjunctivitis when signs and symptoms could be controlled with artificial tears, antihistamine eye drops, or topical treatment with anti-inflammatory ointment on the eye- lids. Patients who needed treatment with ocular anti-inflammatory eyedrops or ointment were diagnosed with a moderate to severe conjunctivitis by an ophthalmologist. Out of the 47 patients devel- oping conjunctivitis during treatment with dupilumab, 20 patients (15%) had mild conjunctivitis, and 27 patients (20%) had moderate to severe conjunctivitis. Treatment characteristics are described in Table 4.


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TABLE 4 Treatment characteristics of patients developing (allergic) conjunctivitis during dupilumab treatment
into a real-world setting. In clinical trials, patients are often carefully screened based on strict inclusion and exclusion criteria. Patient characteristics including comorbidities, susceptibility to side effects, and earlier treatment failure may influence the treatment success in a real-world setting. In this study, 61% of the patients had a history of ≥2 oral immunosuppressive treatments which implies that these patients have a very difficult-to-treat AD. Despite earlier treatment failure, dupilumab was still very effective in this patient group with comparable results to clinical trials.
Recently, Faiz et al described the effectiveness of dupilumab treatment in a cohort of 241 AD patients treated with dupilumab in daily practice.8 Characteristics of the patients included in the study of Faiz et al were similar to our patients in terms of age, sex, atopic comorbidities, disease severity, and previous systemic treatments. The EASI-75 was achieved by 48.8% of the patients which is lower compared to 62% of the patients included in our study. Our study confirms the effectiveness of dupilumab treatment in a cohort of dif- ficult-to-treat AD patients in daily practice. A limitation of the study by Faiz et al is the retrospective study design leading to missing data concerning outcome measures including the EASI score which was only measured in 34% of the patients. In the BioDay registry, we collect high-quality prospective data including a large set of vali- dated outcome measures with limited missing data. Moreover, we measured serum biomarkers reflecting several biomarker pathways, which have not been studied in a daily practice cohort before.
The main outcomes in dupilumab AD clinical trials were fixed endpoints such as the proportion of patients achieving EASI-75. However, since dupilumab treatment affects both clinician-reported outcomes (EASI) and patient-reported outcomes (pruritus, HrQoL), these endpoints do not capture the full range of clinical benefits in daily practice. For instance, patients might be considered as non- responders based on EASI scores, while they experience clinical relevant improvement in patient-reported outcomes including pru- ritus and HrQoL. In our view, a combination of clinical scores and patient-reported outcomes should be used to decide on treatment continuation. We defined clinically relevant responses based on thresholds of commonly used tools to assess the major AD domains: signs, symptoms, and HrQoL. A large majority of the dupilum- ab-treated patients (89%) reported clinically relevant improvement in at least one of the three domains (EASI-75 or NRS ≥4-point im- provement or DLQI ≥4-point improvement). The use of a clinically relevant response may help to identify super-responders (improve- ment in all domains) and nonresponders (improvement in none of the domains) to treatment. In future, the clinically relevant response may also help to differentiate between very good responders and nonre- sponders based on biomarker profile. Due to the small sample size of nonresponders, a responder nonresponder comparative analysis was not possible in the present study.
The proportion of patients developing new onset or worsening of conjunctivitis (34%) was higher compared to previous phase 3 clinical trials (9%-28%).1,2,7 This might be explained by an increased awareness of conjunctivitis. In the study by Faiz et al, conjunctivitis was also the most reported side effect in 38.2% of the patients which
n (%)
Conjunctivitis, n (%)
Presence of preexisting conjunctivitis, n (%) Mild conjunctivitis, n (%)
a
Presence of preexisting conjunctivitis, n (%) Dexamethasone 1 mg/mL eye drops
Tobramycin 3 mg/mL and dexamethasone 1 mg/ mL eye drops
Tacrolimus 0.3 mg/g eye ointment aMultiple treatments per patient.
47 (34.1)
35 (76.1) 20 (42.6)
3 (15.0) 8 (40.0)
22 (84.6) 16 (59.3)
1 (3.7) 7 (25.9)
Time to registration of conjunctivitis as adverse event (days), median (IQR)
56 (31-84)
Missing, n (%)
1 (2.1)
Presence of preexisting conjunctivitis, n (%)
13 (65.0)
Conjunctivitis treatment, n (%) Antibiotic eye drops
No treatment/artificial tears
Ketotifen 0.025 mg/mL eye drops
7 (35.0)
Tacrolimus 1 mg/g ointment eyelids
3 (30.0)
Moderate-severe conjunctivitis (treated with anti- inflammatory eyedrops/ointment)
27 (57.4)
Conjunctivitis treatment, n (%)a
Oxytetracycline 5 mg/g and hydrocortisone 10 mg/g eye ointment
3 (11.1)
Fluorometholone Liquifilm 1 mg/mL eye drops
11 (40.7)
Cyclosporin A 1 mg/mL eye drops
3 (11.1)
and serum biomarkers did also not significantly differ between pa- tients with or without conjunctivitis at week 16 (Table S6).
4 | DISCUSSION
This study demonstrates that treatment with dupilumab significantly improves signs and symptoms of AD as well as patient-reported out- comes including pain/discomfort, itch, anxiety and depression and HrQoL (health-related quality of life) in a vast majority of difficult- to-treat AD patients in a daily practice setting. Treatment with dupilumab also significantly suppressed disease severity-related serum biomarkers TARC, PARC, periostin, and IL-22, and eosinophil- related markers eotaxin-1 and eotaxin-3.
The clinical effectiveness of dupilumab treatment in this daily practice cohort was consistent with the results observed in clinical phase 3 AD trials.1,2,7 The primary outcome EASI-75 used in phase 3 clinical trials was achieved by 62% of the patients after 16 weeks of treatment in this daily practice cohort. In the phase 3 clinical trials, the EASI-75 was achieved by 44%-69% after 16 weeks of dupilumab. Results derived from prospective daily practice registries such as the BioDay registry are important to translate clinical trial results