Comparison of Innate Immune Responses Induced by Allergy Immunotherapy (AIT) With Different Adjuvants
Conditions: Allergic Rhinitis
Sponsor: University of Zurich

 
 
HFV Research Project - Project Plan 
 
 
 
COMPARISON OF INNATE IMMUNE RESPONSES 
INDUCED BY ALLERGY IMMUNOTHERAPY (AIT) WITH 
DIFFERENT ADJUVANTS  
USZ-AZW_MCT001 
 
 
Sponsor and Project Leader 
University Zurich 
Department of Dermatology 
Prof. Pål Johansen 
University Hospital Zurich 
Gloriastrasse 31  
8091 ZURICH  
Switzerland 
HFV Category 
HFV A: Non–clinical research project with sampling and fur-
ther use of samples and health related data 
Version Number and Date 
Version 2, 8.8.2019 
Confidential 
The information contained in this document is confidential and the property of the Department of Der-
matology, University Hospital Zurich. This document serves to inform the project leaders, further pro-
ject personnel and the Independent Ethics Committee(s). The information may not - in full or in part - 
be transmitted, reproduced, published, or disclosed to others without prior written authorization from 
the Department of Dermatology, University Hospital Zurich]. 
 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 2 of 18   
Project Synopsis 
Sponsor /  
Project Leader: 
Pål Johansen 
Department of Dermatology,  
University Hospital Zurich & University of Zurich 
HFV category: 
A – research project – sampling and subsequent use of samples 
Title: 
Comparison of innate immune responses induced by allergy immunother-
apy with different adjuvants 
Short Title: 
USZ-AZW_MCT001 
Project Popula-
tion: 
Adults, males and females 
Coding: 
Coded samples and data 
Objectives: 
Analysis of inflammatory responses in blood of patients receiving allergen 
immunotherapy (AIT) with different adjuvants 
Outcomes: 
Inflammatory cytokines, acute-phase proteins, and antibodies are meas-
ured in serum from patients receiving AIT 
Number of Sub-
jects: 
24 
Inclusion Criteria: 
• 
Indication: Scheduled AIT  
• 
Male and Female subjects 18 years to 50 years of age 
• 
Written informed consent to the research project by the participant af-
ter information about the project  
Exclusion Criteria: 
• 
Objection of subsequent use of biological samples and personal 
health data 
• 
Systemic or local immunosuppressive treatment last 30 or next 7 
days 
• 
Previous AIT 
• 
Chronic inflammatory diseases 
• 
Infections 
• 
Blood donations last 30 or next 7 days 
• 
Drug or alcohol abuse 
• 
Pregnancy or breast feeding 
Project Duration: 
Planned start: 09/2019 
Planned project close: 09/2020 
Statistical Method-
ology: 
The study is a pilot study and it aims to obtain descriptive information on 
the type of inflammatory immune responses measured in serum after AIT 
and will be analysed using descriptive statistics. 
Statement: 
This project will be conducted in compliance with the protocol, the current 
version of the Declaration of Helsinki as well as all Swiss and German legal 
and regulatory requirements, in particular data protection regulations. 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 3 of 18   
Table of Content  
 
 
 
 
                  Page 
Project Synopsis ........................................................................................................................... 2 
Abbreviations ................................................................................................................................ 4 
Project Personnel and Administration ......................................................................................... 4 
1. 
Introduction .......................................................................................................................... 5 
1.1 
Background and purpose .............................................................................................. 5 
1.2 
Project site Centre for Rhinology and Allergology, Wiesbaden (AZW) ........................... 6 
1.3 
Project site University Hospital Zurich (USZ) ................................................................. 7 
2 
Project Objectives ................................................................................................................ 7 
2.1 
Project Objectives ......................................................................................................... 7 
2.2 
Project Outcomes .......................................................................................................... 7 
3 
Project Design ...................................................................................................................... 8 
3.1 
Project Schedule ........................................................................................................... 9 
4 
Population of Biological Samples ....................................................................................... 9 
4.1 
Inclusion Criteria ........................................................................................................... 9 
4.2 
Exclusion Criteria ........................................................................................................ 10 
4.3 
Premature withdrawal criteria ...................................................................................... 10 
5 
Subject Information and Informed Consent ..................................................................... 10 
6 
Project Data and Samples ................................................................................................. 10 
6.1 
Sample generation and processing ............................................................................. 11 
6.2 
Analysis of samples and data ...................................................................................... 11 
6.3 
Code Administration .................................................................................................... 13 
7 
Statistical Plan .................................................................................................................... 13 
7.1 
Sample Size Determination ......................................................................................... 13 
7.2 
Statistical Methods ...................................................................................................... 13 
8 
Independent Ethic Committee ........................................................................................... 14 
9 
Confidentiality and Safe Handling of Samples and Data ................................................. 15 
9.1 
Proof of secure handling of samples and data and the storage thereof ....................... 15 
9.2 
Coding of samples and data ........................................................................................ 15 
9.3 
Storage period after finalization of the research project ............................................... 16 
10 
Funding and Support ......................................................................................................... 16 
11 
Publication .......................................................................................................................... 16 
12 
Signatures .......................................................................................................................... 17 
References ................................................................................................................................... 18 
 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 4 of 18   
Abbreviations 
AIT 
Allergen Immunotherapy 
ALAT 
Alanine aminotransferase (or Ala transaminase) 
Alum 
Aluminium hydroxide or phosphate (adjuvant) 
ASAT 
Aspartate-aminotransferase (or Asp transaminase) 
AZW 
Centre for rhinology and allergology (Allergiezentrum Wiesbaden) 
CRF 
Case report form 
CRP 
C-reactive protein 
EDTA 
Ethylene diamine tetra acetic acid (blood anticoagulant)  
Gamma-GT 
Gamma-glutamate-transferase 
HFV 
Human research ordinance (German: Humanforschungs-Verordnung) 
HRO 
Human Research Ordinance (HFV – Humanforschungs-Verordnung) 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IgG, IgE, IgM 
Immunoglobulin G, Immunoglobulin E, Immunoglobulin M 
IRB 
Independent review board 
MCT 
Microcrystalline tyrosine (adjuvant) 
MPLA 
Monophosphoryl lipid A (adjuvant) 
USZ 
University Hospital Zurich (German: UniversitätsSpital Zürich)  
 
Project Personnel and Administration 
Sponsor and Project Leader:  
Prof. Dr. Pål Johansen 
University Hospital Zurich 
Department of Dermatology 
Gloriastrasse 31, 8091 Zurich 
Switzerland  
EMail: pal.johansen@usz.ch 
Phone: +41 044 255 86 16  
 
Department of Dermatology 
University Hospital Zurich 
Gloriastrasse 31, 8091 Zurich 
Switzerland 
Dr. med. Deborah Leuthard 
Dermatology, USZ 
Email: deborah.leuthard@usz.ch 
Phone: 043 253 05 68 
 
 
Dr. med. Sara Micaletto 
Allergy Unit, Dermatology, USZ 
Email: sara.micaletto@usz.ch 
Phone: 043 253 76 30 
 
 
Dr. med. Eugen Bersuch 
Allergy Unit, Dermatology, USZ 
Email: eugen.bersuch@usz.ch 
Phone: 043 253 88 50 
 
 
Elvira Schmid Schwotzer 
Allergy Unit, Dermatology, USZ 
Email: elvira.schmid@usz.ch 
Phone: 044 255 24 41 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 5 of 18   
 
Master med. Alina Müller 
Dermatology, USZ 
Email: alina.mueller@usz.ch 
Phone: 076 391 18 35 
 
Allergiezentrum Wiesbaden 
An den Quellen 10,  
65183 Wiesbaden 
Germany 
Prof. Dr. med. Ludger Klimek 
Allergiezentrum Wiesbaden 
Email: ludger.klimek@allergiezentrum.org 
Phone: +49 611 308 608 0 
 
 
Dr. med. Annette Sperl 
Allergiezentrum Wiesbaden 
Email: annette.sperl@allergiezentrum.org 
Phone: +49 611 308 608-0 
 
 
Master med. Alina Müller 
Allergiezentrum Wiesbaden 
Email: alinamueller@gmx.net 
Phone: +41 76 391 18 35 
 
 
Dr. Ingrid Casper 
Allergiezentrum Wiesbaden 
Email: Ingrid.Casper@allergiezentrum.org 
Phone: +49 611 308 608-0 
 
Biostatistics 
Dr. Nicole Graf 
Amelenweg 5, 8400 Winterthur 
Email: graf@biostatistics.ch 
Phone: +41 79 270 6772 
 
Data codes 
Séverine Buffoni 
Dermatology, USZ 
severine.buffoni@usz.ch 
 
1. Introduction 
This research plan describes a scientific research project (Swiss HFV category A) and includes the 
re-usage of human blood serum samples collected from patients receiving allergen immunotherapy 
(AIT) at University Hospital Zurich and at the Centre for Allergy and Rhinology in Wiesbaden (Aller-
giezentrum, AZW). The USZ research project is led by Prof. Pål Johansen, USZ, while Prof. Ludger 
Klimek holds the lead at AZW; Prof. Klimek is also professor at the University Mannheim/Heidelberg. 
All serum samples from USZ and AZW will be all assessed at the USZ.  
Ethical approval for the blood sampling at AZW will be obtained by Prof. Ludger Klimek from the 
Independent Ethics Committee (IEC) in Mannheim (Ethik-Kommission der Medizinischen Fakultät 
Mannheim/Medizinische Ethikkommission der Universität Heidelberg). 
1.1 Background and purpose 
IgE-mediated allergy represents a serious health problem in industrialized countries with about 30% 
of the population suffering from allergic symptoms such as rhinitis, conjunctivitis, bronchial asthma, 
or atopic dermatitis 1. Allergy also represent a frequent cause of morbidity and disability when it 
manifests with bronchial asthma and anaphylaxis. These atopic diseases are characterized by pro-
duction of IgE from B cells and a T-cell population that preferentially produces T-helper like 2 (Th2) 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 6 of 18   
cytokines such as interleukin (IL)-4, IL-5, and IL-13. In contrast, protective biomarkers in allergy is 
thought to be Th1-like cytokines, regulatory T cells (Tregs), and allergen-neutralising IgG antibodies 
2. 
Since more than 100 years, the only causative treatment of IgE-mediated allergy was allergen im-
munotherapy (AIT) with allergen extracts, or more lately in clinical trials, purified, synthetic, or re-
combinant allergen proteins 3,4. In Europe, but not in the USA, the AIT products usually contain 
adjuvants 5. The adjuvant should provide a depot of the therapeutic allergen, facilitate protective 
immune responses, and in part to improve the AIT safety by adsorption of allergen. For a long time, 
hydroxide or phosphate salts of aluminium (Alum) were the only registered adjuvants for use in hu-
man 6. However, new adjuvants that more specifically stimulate the inflammatory responses required 
for protection in allergy is ascribed an important role in the development of better and safer AIT 
regimens.  
The adjuvant effect of Alum has been ascribed to its particle nature and its depot formation, but more 
lately, the mechanism of action has been shown to include the activation of inflammasomes. Inflam-
masome activation results in the immediate release of inflammatory cytokines such as IL-1β and IL-
18 6. It is also well recognised that Alum is a Th2-polarising adjuvant and facilitates IgE production 
7. Hence, it is an immunological paradox that the Th2-biasing Alum is used to cure a Th2-mediated 
allergy, because improved AIT regimens are thought to be associated with Th1-polarising immune 
responses. To what extent Th1 or Th2 immune responses are triggered, very much depends on the 
initial and innate inflammatory reaction which can immune responses. Hence, the development of 
more beneficial adjuvants is a focused research area in allergy. 
More lately, microcrystalline tyrosine (MCT) and combinations of MCT and Monophosphoryl Lipid A 
(MPLA) have been introduced as alternative AIT adjuvants 5. MPLA is known to mediate the release 
of inflammatory immune responses through stimulation of Toll-like receptor 4 (TLR4) on antigen 
presenting cells (APCs) 8. Mechanistic knowledge on MCT is by comparison incomplete, although 
we recently demonstrated that mice immunised with antigen and Alum or MCT produced comparable 
IgG antibody responses, but with less IgE antibodies and more Th1 cytokines in MCT-treated mice 
9. In humans, a side-by-side comparison of the inflammatory responses induced by AIT with the 
different adjuvants is totally missing.  
The primary purpose of the current project is therefore to collect blood from patients that receive 
grass pollen AIT with Alum (Allergovit®), MCT (PolvacTM), or MCT-MPLA (Pollinex QuattroTM) as part 
of their standard AIT. How and to what extent the adjuvant system contributes to the stimulation of 
innate immune responses will subsequently be measured by means of antibody arrays and other 
immunological methods on patient serum samples obtained prior to AIT as well as 1, 7, and 49 (±3) 
days after AIT. This study will be the first in its kind, and the results of this analysis may provide clues 
to the mechanism of action and safety of the various adjuvants used in AIT, which again should allow 
us to optimize such adjuvants. The study should also allow us to correlate inflammatory responses 
with historical data on efficacy, which again may help the rational development of new and better 
AIT and AIT adjuvants. Bearing in mind that 90% of the allergy patients are currently reluctant to 
receive treatment by the only causative and disease-modifying method, safer and more efficient AIT 
is probably the most important goal as to make AIT more attractive to the vast number untreated 
allergy patients. 
1.2 Project site Centre for Rhinology and Allergology, Wiesbaden (AZW) 
Blood samples and data from patients that receive AIT with Pollinex QuattroTM (8 patients) is obtained 
from the AZW. The AZW is one of the leading German institutions in the area of research and treat-
ment of diseases of the nose and the upper respiratory tracts and the only institute specializing 
exclusively in this area. Scientifically, AZW is part of the University Mannheim/Heidelberg. It has a 
special focus on the diagnosis and treatment of chronic inflammatory diseases of the nose and the 
paranasal sinuses – in particular also the identification of allergic causes. 
http://www.allergiezentrum.org/de/ 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 7 of 18   
The Pollinex QuattroTM patients are not available in Switzerland, since Pollinex QuattroTM is not gen-
erally approved for use in Switzerland.  
Although the current study is non-interventional, the study participants will receive AIT between the 
first (V1) and the second (V2) visits. The AIT is performed by well-trained and experienced allergol-
ogists, and AZW has all required infrastructure for treating patients that present with serious adverse 
event (SAEs) within the timeframe of the duration of the study. In case of SEAs because of the 
venous blood collection, the AZW has all infrastructure and medical personnel required to care for 
these study subjects. 
Laboratories 
• Labor Fleischauer: since 2006, accredited by DAKKS (Deutsche Akkreditierungsstelle) ac-
cording to DIN EN ISO 15189  
1.3 Project site University Hospital Zurich (USZ) 
Blood samples and data from patients that receive AIT with Allergovit® or with POLVACTM is obtained 
from patients treated in USZ (16 patients) All the blood samples are analysed at the USZ. The USZ 
has accredited and otherwise certified laboratories that can perform the serological analysis of acute-
phase proteins and antibodies with high quality. Moreover, the Department of Dermatology is one of 
the most active dermatological research centres in Europe with a strong focus on skin cancer, in-
flammatory skin diseases, allergology, and immunotherapy. Here, the Allergy Unit is part of a net-
work of 19 EU allergy centres joined in the so-called Global Allergy Asthma European Network 
(GA2LEN), which strive and commit to high quality standards in patient care, training, and quality 
management, and are regularly reviewed through external audits. 
Laboratories 
• USZ Haematology: accreditation no. STS 0445 
• USZ Dermatology: accreditation no. STS 0610 
• USZ Immunology: accreditation no. STS 0227  
• USZ Clinical chemistry: accreditation no. STS 0206 
 
2 Project Objectives 
2.1 Project Objectives  
The primary objective is to compare inflammatory responses in blood sera from patients receiving 
first AIT with Alum, MCT, or MCT-MPLA as adjuvants. The AIT products are containing allergen 
extracts of non-specified origin (pollen, dander, dust mite, venom) and provided by Allergopharma 
(with Alum) or Bencard Allergie (with MCT or MCT-MPLA).  
Since no MPLA-containing allergy vaccines or AIT preparations are approved for use in Switzerland, 
the study subjects enrolled into this study will be recruited among AIT patients at the AZW. 
2.2 Project Outcomes 
The following secondary objectives will be investigated on all study subjects, but not on all time 
points: 
• the composition of blood leukocytes is measured on days 0, 1, and 7 
• allergen-specific antibodies are measured on days 0, 7, and 49 
• acute-phase proteins including liver enzymes are measured on day 0, 1, and 7. 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 8 of 18   
3 Project Design 
This study is an observational study with subsequent use of coded biological material. The blood 
sera are collected and prepared at the Allergy Units at USZ (Zürich) or AZW (Wiesbaden). The AZW 
serum samples will be transferred to Zurich for serological analysis at USZ. The flow chart in Figure 
1 illustrates the overall design, content, and schedule of the study.  
The AIT patients will be allocated to three arms based on their scheduled AIT and as indicated in 
Figure 1. Two arms will be allocated to patients at the USZ Allergy Unit, while the last arm is allo-
cated to patients at AZW Wiesbaden. 
Sixteen study subjects will be recruited from allergy patients that visit the Allergy Unit at USZ to 
receive AIT as part of standard of care treatment for their allergy. If the patients receive AIT, they 
can be included in the study. The decision on performing AIT is done by the USZ allergologist, and 
the therapy itself is not part of the current research project. The 16 USZ patients are split in two study 
arms. One arm of study subject is scheduled for AIT with Allergovit® and the second arm is sched-
uled to receive AIT with POLVACTM.  
Eight study subjects will be recruited from allergy patients that visit the AZW to receive AIT as part 
of standard of care treatment for their allergy. If the patients receive AIT, they can be included in the 
study. The decision on performing AIT is done by the AZW allergologist, and the therapy itself is not 
part of the current research project. The 8 patients comprise the third arm of the overall study, and 
they receive AIT with Pollinex QuattroTM. The Pollinex QuattroTM patients are not available in Swit-
zerland.  
Figure 1. Flow chart showing the study design, the allocation of the groups of patients, and the study pro-
gression included into the trial at the ASW study site in Wiesbaden. Patient sera are transferred to Zurich 
for assessment of inflammatory proteins and antibodies at USZ. 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 9 of 18   
The trial requires four visits (Fig. 1). Screening visit (V1) takes place on the day of the scheduled 
AIT. Prior to AIT (not part of the study), patient blood is drawn. Three subsequent visits are planned 
one (V2), seven (V3), and 49 (V4) days after the first visit. Patient blood is drawn on all visits. 
3.1 Project Schedule 
The individual duration of study participation for patients completing the study is 49 days. The total 
number of patients required for the study is expected to be recruited an enrolled within two months. 
The estimated time to complete the subsequent lab analysis of blood from patients included in the 
trial is ca. 6 months. 
• 
Duration of the recruitment phase: Sep 2019 to Jan 2020 
• 
Planned project start (first sampling of blood): Sep 2019 
• 
Planned project end (last sampling of blood): Jan 2020 
• 
Definition of the project end (finalised lab analysis): Sep 2020 
• 
Duration of the project in total: Aug 2019 to Sep 2020 
 
4 Population of Biological Samples  
Blood samples will be obtained from a total of 24 patients with IgE-mediated allergy as diagnosed or 
confirmed by an allergologists at the Allergy Units of USZ (16 samples) and AZW (8 samples).  
For blood obtained from patients at the USZ, all samples will be processed and analysed at USZ. 
For blood obtained from patients at AZW, whole blood will be directly analysed at Labor Fleischauer 
in Wiesbaden for leukocyte composition, while part of the blood will be centrifuged for preparation of 
serum that is frozen (-20°C) and transferred to USZ for further analysis.  
Patients that will be considered for inclusion into the study should have been scheduled for treatment 
with AIT by means of subcutaneous injections of Allergovit® (USZ), PolvacTM (USZ), or Pollinex 
QuattroTM, (AZW), the goal being to include eight patients from each of the three treatment regimens. 
Important, the study subjects are only recruited among patients that are to receive their very first 
AIT. The AIT is not part of the study, but takes place due to prior decisions by the allergologists Dr. 
med. Sara Micaletto and Dr. med. Eugen Bersuch at the Allergy Unit USZ or by Dr. med. Annette 
Sperl at AZW.  
Patients that will be considered for inclusion into the study are scheduled for treatment with AIT. The 
study subjects are primarily recruited by medical doctors at USZ (16 subjects) and AZW (8 subjects) 
during allergological consultation. The method of recruitment both at USZ and AZW is by interviews 
with the allergologist during pre-AIT consultancy. Alternatively, telephone interviews of patients 
scheduled for an AIT appointment may be applied. An USZ study doctor (Alina Müller) will join the 
team at AZW and help recruiting and supervising the study subjects. 
4.1 Inclusion Criteria 
Subjects, who will fulfil all the following inclusion criteria, may be included into this project: 
• 
Males and females of age 18 to 50 years 
• 
History of allergy due to IgE sensitisation to any allergen that is treatable by means of AIT, 
e.g. grass pollen allergens, three pollen allergens, animal dander allergens, dust mite aller-
gens, or insect venom allergens.  
• 
Scheduled to receive first AIT with Allergovit® oder PolvacTM or to receive Pollinex QuattroTM 
at AZW. 
• 
Signed written informed consent for subsequent use of coded blood samples including blood 
leukocytes data and serological data. 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 10 of 18   
4.2 Exclusion Criteria 
If a subject fulfils any of the following exclusion criteria, will not be included in the project: 
• 
Previous AIT 
• 
Chronic inflammatory diseases (rheumatic diseases, pyelonephritis, osteomyelitis or others) 
• 
Acute infections 
• 
Drug or alcohol abuse within the last 5 years 
• 
Relevant anaemia (as judged by investigator) 
• 
Blood donation within the last 30 days or during the next 7 days  
• 
Pregnancy or breast feeding 
• 
Systemic glucocorticoid or antihistamine therapy within the last 30 days or during the next 7 
days. 
• 
Systemic or local immune drug therapy within the last 30 days during the next 7 days. 
• 
For linguistic and/or cognitive reasons unable to understand the study procedures  
4.3 Premature withdrawal criteria 
Samples will be withdrawn from the trial if the patients, from whom the blood samples were drawn, 
meet any of the following criteria: 
• 
marked deterioration of their allergic status as judged by the investigator 
• 
patient or investigator decision 
• 
protocol violation 
The samples will then be replaced by samples from another patients who fulfil the inclusion criteria 
but none of the exclusion criteria.  
 
5 Subject Information and Informed Consent  
The patients are informed in writing and verbally on: 
• 
The purpose and duration of, and procedure for, the research project. 
• 
Their right to withhold or to revoke their consent at any time without giving reasons. 
• 
The consequences of revocation of consent for the biological material and personal data 
used up to this point. 
• 
Their right to receive information at any time in response to further questions relating to the 
research project. 
• 
Their right to be informed of results concerning their health, as well as their right to disclaim 
that information; or to designate a person who is to take this decision for them. 
• 
Measures to protect the biological material and the personal data. 
• 
The main sources of financing for the research project. 
 
6 Project Data and Samples 
Upon enrolment in the trial, a patient will undergo a short physical examination and demographic 
details will be obtained and recorded in the CRF. Blood is then sampled for baseline determination 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 11 of 18   
of test parameters (blood and serum analysis). On the next experimental study days (1 and 7 days 
later), a short examination with questionnaire is performed and blood is again drawn for analysis. 
6.1 Sample generation and processing 
Visit 1 (V1; screening and baseline laboratory) 
A signed “Informed Consent” will be obtained from each patient prior to initiating any trial procedure. 
The patient eligibility will be determined in accordance with the inclusion/exclusion criteria. Further, 
a medical history and medication use will be obtained and recorded, and a short physical examina-
tion will be performed. Venous blood will be taken for the determination of lab parameters: two red-
cap vials (serum) of each approx. 6 ml for serum analysis, one green-cap vial (heparin) of approx. 3 
ml for acute-phase protein analysis and one violet-cap vial (EDTA) of ca. 2 ml for whole blood anal-
ysis. The study subjects will then be dismissed to receive AIT with their allergologists and asked to 
return next day at the same time point. The study subjects will also be asked to consume foods and 
drinks similar to that of the visit 1 day.  
Visit 2 (V2; one day post V1) 
Visit 2 will be performed 24 hours after visit 1. It is important that the patient present at the same 
time of the day as for visit 1. Venous blood will be sampled for the determination of lab parameters 
as for the previous day: two red-cap vials (serum) of each approx. 6 ml for serum analysis, one 
green-cap vial (heparin) of approx. 3 ml for acute-phase protein analysis and one violet-cap vial 
(EDTA) of ca. 2 ml for whole blood analysis. The patients will be asked to return six days later at the 
same time point and to consume foods and drinks on the morning of the visit similar to that of the 
days of visits 1 and 2. 
Visit 3 (V3, seven days post V1) 
Visit 3 will be performed seven days after visit 1 and six days after visit 2. It is important that the 
study subjects present at the same time of the day as for visits 1 and 2. Venous blood will be sampled 
for the determination of lab parameters: two red-cap vials (serum) of each approx. 6 ml for serum 
analysis, one green-cap vial (heparin) of approx. 3 ml for acute-phase protein analysis and one 
violet-cap vial (EDTA) of ca. 2 ml for whole blood analysis. 
Visit 4 (V4, 49 day post V1, final visit) 
Visit 4 will be performed ca. 49 (±3 days) after visit 1. It is important that the study subjects present 
at the same time of the day as for the previous visits. Venous blood will be sampled for the determi-
nation of lab parameters: one red-cap vials (serum) of 10 ml for serum analysis. 
Physical examination and questionnaire 
Mild adverse events may follow AIT, and although not part of the study and not mandatory for ob-
servational studies, the study doctor will assess by examination of the site of AIT injection and by 
questionnaire, to what extent the study subjects reacted to the AIT and whether the reactions varied 
across the three AIT regimens applied. The parameters assessed are local reactions such as pain, 
itch, erythema, and swelling, as well as systemic reactions such as fever, irritability, drowsiness, loss 
of appetite, vomiting. In addition, a photograph of the site of AIT injection is made. Questionnaires 
and the photographs are stored in the CRF. 
6.2 Analysis of samples and data 
Whole blood and serum will be sampled and analysed. The volume of blood sampled from patients 
completing the whole study is approximately 85 ml within seven days (25 ml on each of the visits V1, 
V2, and V3 and 10 ml on V4). EDTA analysis is submitted for whole blood analysis to Haematology 
at USZ or Labor Fleischauer in Wiesbaden on the day of sampling. Serum blood is processed for 
production of serum at USZ or AZW on the day of sampling and stored at -20 °C for later protein and 
antibody analysis. 
Blood leukocyte composition 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 12 of 18   
The composition of blood leukocytes from whole blood is measured by standardized and certified 
methods. The blood collection is made in EDTA vacutainers and the analysis is made directly after 
sampling. Total leukocyte and total white leukocytes counts are determined as well as the frequency 
of leukocyte sub-populations such as T and B lymphocytes, neutrophils, monocytes, eosinophils, 
and basophils. 
Protein array 
Briefly, inflammatory cytokines and other proteins will be measured in serum by means of a quanti-
tative protein array according to a protocol provided by the producer (RayBiotech®). The array is a 
multiplexed sandwich ELISA-based quantitative array that combines the advantages of the high de-
tection sensitivity & specificity of ELISA and the high throughput of arrays (Fig. 2). We will use glass 
slides that are pre-coated with antibodies against 120-200 inflammatory cytokines, chemokines, and 
growth factors. 
Enzyme-linked ImmunoSorbent Assay (ELISA) 
Proteins, which secretion into blood is notably reduced or increased as suggested from the results 
of the protein array, will be re-tested for confirmation by using specific quantitative ELISAs accord-
ing to protocols provided by the producer (eBioscience®). These ELISA methods a well standard-
ized and established in the laboratory at USZ. 
Acute-phase protein analysis 
Inflammation is accompanied by many changes, far from the site of inflammation and involving many 
organ systems 10. We do not expect the secretion of acute-phase proteins to be highly elevated upon 
AIT. However, AIT and adjuvants are by nature inflammatory, hence, changes may be detectable, 
but such data is typically not collected for AIT in clinical testing or use. Therefore, we plan to analyse 
the secretion of acute-phase proteins such as, but not exclusively, aspartate-aminotransferase 
(ASAT), alanine aminotransferase (ALAT), gamma-glutamyl transferase (GGT), C-reactive protein 
(CRP), and IL-6 by accredited methods at the USZ Department of Clinical Chemistry. 
 
 
 
Figure 2. Scheme showing the principle how the protein array works (left panel) and the list of anti-
proteins antibodies contained in the array (right panel). 
 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 13 of 18   
Antibody ELISA 
The sera will also be analysed to measure changes in allergen-specific antibodies. Total IgM and 
allergen-specific IgE are measured by accredited methods at the USZ Department of Clinical Chem-
istry. Allergen-specific IgM, IgG, IgG1, and IgG4 are measured by standardized and in part accred-
ited methods at the USZ Department of Dermatology. Typically, strong induction of antigen-specific 
antibody responses may not expected within one week of AIT and by consequent, allergen-specific 
antibodies are normally monitored at later time points after AIT 11. Nonetheless, since the AIT pa-
tients are already primed through natural sensitisation, the reactivity of the immune system is higher 
and antibody responses may be detectable earlier after AIT. However, while early detection of IgG 
or IgE is a result of boosting pre-existing immune responses, the induction of IgM is a primary im-
mune response, precedes IgG and IgE iso-class switching, and typically peaks within 5-7 days of 
immunization 12. Since the targeted clinical effect of adjuvants is the early activation of the innate 
immune system, the different adjuvants tested in the current study may be discriminated in their 
activation of IgM and other allergen-specific antibody responses. 
6.3 Code Administration 
When un-coded blood or serum samples are submitted to clinical chemistry (IKC), clinical immunol-
ogy (AKI), haematology (HAD) or dermatology (DER) for analysis, this carried out internally within 
the USZ using the clinical information system (KISIM). The results of the analysis is subsequently 
available in KISIM. The access to KISIM is restricted and secured by password.  
All data from analysis will be coded before entered into an Excel database specifically created for 
this project. Hence, the data analysis is only performed with coded data and patient identities are 
any more available for persons performing data analysis. The Excel database will be created ac-
cording to the format and content of the CRF, the study protocol, and the blood and serum analysis 
performed. 
When samples and data are transferred from AZW to USZ, only coded samples and data are trans-
ferred. The code for patients at AZW remains with the study-coordinator at AZW (Ingrid Casper). 
The code for patients at USZ is secured by the QM officer for clinical research at the USZ Department 
of Dermatology (Séverine Buffoni). 
The coded data will be accessible to the project leader at USZ (Pål Johansen), two medical research-
ers at USZ (Deborah Leuthard and Alina Müller), and the statistician (Nicole Graf). 
7 Statistical Plan  
7.1 Sample Size Determination 
The primary objective of this experimental study is to compare inflammatory responses in blood 
serum from patients receiving AIT with Alum, MCT, or MCT-MPLA as adjuvants. The study is purely 
descriptive with regards to an array of 120-200 inflammatory proteins that will be determined in blood 
serum. Moreover, no hypothesis is or can be made as to how the differential production of these 
inflammatory proteins can be expected. Therefore, this study does not satisfy any statistical require-
ment. Twenty-four allergy patients (3 groups of 8) is considered sufficient to acquire the necessary 
scientific screening. 
7.2 Statistical Methods 
The investigators and the appointed external biostatistics service group will be responsible for the 
statistical analysis of the collected data. The statistical software used will be Graph Pad Prism®, 
SPSS®, or open-source software “The R Project”.  
The results from the array analysis will used to generate heat maps, and for the hierarchical cluster-
ing of AIT-adjuvant and protein-secretion cluster phenotypes, we will apply The R Project with Eu-
clidean metrics. 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 14 of 18   
Data from all clinical assessments whether explicitly referred to in the statistics section or not, will 
be presented in summary tables. Data will be summarised primarily with respect to the type of AIT 
received. In addition, the data may be summarized with regards to demographic characteristics or 
with regards to clinical observations, e.g. reported side effects of AIT. 
• 
Standard descriptive summary statistics will be calculated for continuous variables, e.g. se-
rum concentration of proteins and antibodies (arithmetic mean, standard deviation, minimum 
value, lower quartile, median, upper quartile, maximum value, number of non-missing values). 
• 
Heat map and cluster analysis will be applied for the original protein array data after normal-
isation of the data for appropriate heat-map analysis.  
• 
Categorical data will be presented in frequency tables using counts and percentages.  
• 
Individual patient data listings will be presented by parameter and will be sorted by treatment 
group, patient number, and visit number. Summary tables will be displayed by treatment 
group and for the total of the sample.  
• 
All p-values and confidence levels of inferential statistical methods will be interpreted in the 
exploratory sense.  
Analysis sets: A criterion to enter the study analysis is that the study subject completed the study 
with four blood samples drawn according to protocol (no protocol violation), and that all samples 
could be analysed according to plan. Hence, only one data set will be analysed. This full analysis 
set will consist of all patients for whom values for inflammatory protein assessment and specific-
antibody assessment have been obtained. The data set will be divided in three subgroups, namely 
patients that originally reived AIT with (i) Allergovit®, (ii) PolvacTM, and (iii) Pollinex QuattroTM.  
Baseline and demographic characteristics: Assessments made at the screening (V1) and the 
experimental visits (V2+V3+V4) will be summarised. These assessments will include demographic 
characteristics and other relevant parameters, e.g. medical history and physical examination.  
Summary tables will be provided for the full analysis set by means of descriptive statistics and fre-
quency tables where appropriate. 
 
8 Independent Ethic Committee 
Before this project will be conducted, the protocol, the proposed participant information, consent 
form, and other project-specific documents will be submitted to a properly constituted Independent 
Ethics Committee (IEC), for formal approval, the IEC being the Kantonale Ethikkommission Zürich. 
The start of the project will not be commenced until after the decisive approval of the IEC is achieved 
in writing to the Project Leader. For blood samples obtained from patients visiting AZW, ethical ap-
proval is obtained from the IEC in Mannheim (Ethik-Kommission der Medizinischen Fakultät Mann-
heim/ Medizinische Ethikkommission der Universität Heidelberg). 
Significant changes to an authorized research project must be approved by the IEC before being 
implemented. Exempt from this requirement are measures which have to be taken immediately in 
order to protect the participants. 
The following are considered to be significant changes: 
• changes affecting the participants’ safety and health, or their rights and obligations; 
• a change of research site or conducting the research project at an additional site; or 
• a change of the project leader or sponsor. 
The IEC will be informed within 90 days regarding the termination or completion of the project. 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 15 of 18   
9 Confidentiality and Safe Handling of Samples and Data 
Data generation, transmission, archiving, and analysis of health related personal data within this 
project strictly follow the current Swiss legal requirements for data protection and according to the 
Ordinance HFV Art. 5. Prerequisite is the voluntary approval of the participant given by signing the 
informed consent prior start of participation of the research project.  
Health related personal data captured during this project from participants are strictly confidential, 
and the disclosure of such data to third parties is prohibited. Confidentiality will be ensured by using 
coded data. The code will be protected against unauthorized access and will be stored appropriately 
with the QM clinical research officer at USZ Dept. Dermatology (Séverine Buffoni), or with the study-
coordinator at AZW (Ingrid Casper). 
The anonymised serum samples will be stored at USZ for 5 years after termination of the project 
and then disposed. 
9.1 Proof of secure handling of samples and data and the storage thereof 
Health-related personal data captured during this project from participants are strictly confidential 
and disclosure to third parties is prohibited. Coding will safeguard participants' confidentiality.  
The electronic case report forms (CRFs) will be entered into an excel sheet, which is secured using 
a password. The excel document is kept on stored on a USZ Server with restricted access. \\fs-
group\der_forschungen\Der_FL_Immunotherapie 
The project site will retain all essential documents according to national legal and regulatory require-
ments, including source data and CRF. Source data are all information in original records and certi-
fied copies of original records of clinical findings, observations, or other activities necessary for the 
reconstruction and evaluation of this project. All essential documents are stored until 10 years after 
the termination of the project, during which time the project leader guarantees access and availability 
of the data at any time. 
Confidentiality will be ensured by entering only coded data into the analysis set. This coded data is 
entered into an Excel-based database. The coded data will be password-protected and accessible 
to the project leader at USZ (Pål Johansen) and two investigators two researchers at USZ (Deborah 
Leuthard and Alina Müller). The statistician will obtain copies of the Excel database after the data 
entry has been completed. Coded research data are not considered part of the essential documents 
and will be stored on a central data server at the USZ with restricted access: \\fs-group\der_for-
schungen\Der_FL_Immunotherapie 
This project also concerns the re-usage of sera data generated at AZW in Wiesbaden. Patient names 
or details that identify the AZW patients will not follow the sera transfer to USZ in Zurich, i.e., only 
coded samples and data are transferred. The code remains in at AZW. 
The code for data generated at USZ is not available for any researchers in this project, but is kept 
with the QM clinical research officer at the Department of Dermatology, USZ (Séverine Buffoni). 
Processed or analysed coded data will be stored in the analysis software (Prism®, SPSS®, and The 
R project). This coded data will be accessible to the project leader at USZ (Pål Johansen), two 
medical researchers at USZ (Deborah Leuthard and Alina Müller), and the statistician, but the data 
will not be password-coded. 
9.2 Coding of samples and data  
At UZH, the code of the samples and the data is hold by the Clinical research Quality Manager at 
Department of Dermatology, USZ (Séverine Buffoni). At AZW, the code of the samples and the 
data is hold by the study-coordinator at AZW (Ingrid Casper).  

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 16 of 18   
The code holders will annotate numerical codes in a non-randomized way (at AZW: USZ-AZW 
001-008; at USZ: USZ-AZW 009-0024) as patients enters the study blood and first blood is col-
lected, i.e., first patient donating blood gets the code number “USZ-AZW 001” and last patient do-
nating blood getting the number “USZ-AZW 024”. 
9.3 Storage period after finalization of the research project 
The anonymised serum samples will be stored at USZ for 10 years and then disposed.  
The anonymised analysed data will be stored at USZ for 10 years and then erased.  
The CRFs will be stored at USZ for 10 years and then disposed.  
 
10 Funding and Support 
All personnel and material are available with support by University Hospital Zurich (USZ) and the 
University of Zurich (UZH) and by Allergiezentrum Wiesbaden (AZW). 
 
11 Publication 
After the statistical analysis of this trial the project leader will make every endeavour to publish the 
data in a medical journal. 
 
 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 17 of 18   
12 Signatures 
The following people agree with the content of this project by signing this protocol. Changes con-
cerning the responsibilities of every person signing this protocol need to be announced immedi-
ately. 
 
 
Sponsor and Project Leader 
 
 
I hereby confirm that this project plan was subjected to a critical review according the moral, ethical 
and scientific principles governing clinical research and approved/released by myself. I confirm that 
samples and data will be treated and used according to national legal requirements of data secu-
rity. 
 
Prof. Dr. Pål Johansen 
University Hospital Zurich 
Department of Dermatology 
 
 
Zurich, 5.7.2019  
Place, Date 
 
Signature 
 
 

 
USZ-AZW_MCT001, Version 2, 8.8.2019 
 
Page 18 of 18   
13 References 
1. Gunawardana & Durham. New approaches to allergen immunotherapy. Ann Allergy Asthma 
Immunol 121, 293-305 (2018). 
2. Shamji, et al. Biomarkers for monitoring clinical efficacy of allergen immunotherapy for allergic 
rhinoconjunctivitis and allergic asthma: an EAACI Position Paper. Allergy 72, 1156-1173 
(2017). 
3. Calderon, et al. One hundred years of allergen immunotherapy European Academy of Allergy 
and Clinical Immunology celebration: review of unanswered questions. Allergy 67, 462-476 
(2012). 
4. Larsen, et al. Allergy immunotherapy: the future of allergy treatment. Drug Discov Today 21, 
26-37 (2016). 
5. Klimek, et al. Clinical use of adjuvants in allergen-immunotherapy. Expert Rev Clin Immunol 13, 
599-610 (2017). 
6. Ghimire. The mechanisms of action of vaccines containing aluminum adjuvants: an in vitro vs in 
vivo paradigm. Springerplus 4, 181 (2015). 
7. Jensen-Jarolim. Aluminium in Allergies and Allergen immunotherapy. World Allergy Organ J 8, 
7 (2015). 
8. Del Giudice, et al. Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. 
Semin Immunol 39, 14-21 (2018). 
9. Leuthard, et al. Microcrystalline tyrosine and aluminum as adjuvants in allergen-specific immu-
notherapy protect from IgE-mediated reactivity in mouse models and act independently of in-
flammasome and TLR signaling. J Immunol 200, 3151-3159 (2018). 
10. Gabay & Kushner. Acute-phase proteins and other systemic responses to inflammation. N Engl 
J Med 340, 448-454 (1999). 
11. Senti, et al. Epicutaneous allergen-specific immunotherapy ameliorates grass pollen-induced 
rhinoconjunctivitis: A double-blind, placebo-controlled dose escalation study. J Allergy Clin Im-
munol 129, 128-135 (2012). 
12. Colino & Outschoorn. Dynamics of the murine humoral immune response to Neisseria meningi-
tis group B capsular polysaccharide. Infect Immun 66, 505-513 (1998).