Conditions: Allergic Rhinitis
Keywords: grass pollen allergy hayfever atopy allergic rhinitis sublingual immunotherapy tablets
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 46
Healthy volunteers: yes

Brief summary:
About 45 million people in Europe have allergic rhinitis (hay fever) - inflammation of the nasal passages causing sneezing, runny nose, nasal congestion, itching and tearing of the eyes. In the United Kingdom (UK), seasonal hay fever due to grass pollen allergy accounts for approximately 7 times more doctors' appointments than asthma. The standard treatment for hay fever consists of treating the symptoms with a nasal spray and an antihistamine. However, in a survey taken in a UK general practice less than 40% of patients with hay fever reported good symptom control with this standard treatment. For those patients with hay fever whose symptoms are not well controlled by treatment with antihistamines and nasal sprays, subcutaneous immunotherapy (SCIT) - (monthly injections of a grass allergen extract for a period of 3-5 years) is an effective alternative, and is approved in the UK on a named patient basis. More recently, allergen immunotherapy tablets (AITs) have been developed, including grass pollen allergen tablets. These have been shown to be highly effective in the treatment of hay fever, with the additional benefit of being convenient for patients, given that they may be taken at home. Grazax® (manufactured by Allergologisk Laboratorium København (ALK)-Abello, Denmark) has UK and European Union (EU) license for use in the treatment of troublesome grass pollen induced hay fever. The aim of this research is to investigate the effects of the AIT treatment on the immune system over time - which changes are taking place and when in the course of treatment. This will provide insight into the complexities of the development of allergen-specific immune tolerance - how harmful allergic responses against innocuous substances such as grass pollen can be overridden.

Detailed description:
The study will be conducted over 44 months. We expect to screen 70 atopic patients in order to enroll up to 50 suitable atopic participants to ensure randomisation of at least 40 atopic participants following their baseline visit. Additionally, we shall recruit 20 healthy, non-atopic volunteers. Individuals with moderate to severe grass pollen hay fever, with or without associated seasonal asthma, will be recruited and screened after the pollen season from September through March 2014. Atopic Participants will undergo baseline assessments in December 2013 to March 2014. All screening assessments will be completed before eligible participants are randomized to active or placebo treatment. Atopic participants will then begin AIT treatment in February-March 2014 and continue treatment for 12 months. All atopic participants will be provided with anti-allergic rescue medications (antihistamine tablets, topical intranasal corticosteroids, and eye-drops) throughout the pollen season. Clinical surrogate endpoint assessments and on specific time Points blood, nasal fluid and nasal brushing sampling, will be performed at baseline (January-March 2014), at 4, 8, 12 and 16 weeks after starting the AIT, and at 6 and at 12 months (January-March 2015) of treatment. Nasal mucosal biopsies will be taken at baseline, during the Peak pollen season, and 12 months of treatment. After 12 months of treatment, unblinding will take place. Those atopic participants receiving active AIT treatment will continue therapy for another 12 months followed by a withdrawal phase of 12 months. Blood samples and nasal biopsies will be taken from these participants again at 24 and at 36 months from the initial start of treatment. Those atopic participants on placebo will be offered 24 months of active treatment after unblinding.

20 healthy, non-atopic participants will also be recruited and studied on a single day before and after nasal challenge at one timepoint at 12 months. They will undergo the same clinical and immunological measurements as for the 46 randomised subjects participating in the clinical trial. The healthy subjects will undergo no therapeutic intervention and serve as controls for the immunological measurements.

The study will receive monitoring by:

* the sponsor, Imperial College London

and audits by:

* Medicines for human use (clinical trials) regulations authority (MHRA)

We will assess the clinical data on paper case report forms and will use Inform as a database in order to verify completeness of data entry.

The Inform database also includes normal ranges of parameters if relevant.

Standard Operating Procedures are in place regarding clinical measures (e.g. nasal allergen challenges, nasal biopsies etc.) as well as data management (e.g. the Trial Master File), reporting of adverse events and data collection. All involved investigators are informed about these procedures and have current Good Clinical Practice (GMC) instructions.

Statistical analysis will be with non-parametric statistics taking into account treatment effect baseline values and visit number. Inclusion of 20 participants per group will give greater than 90% power (p=0.05) to detect a 40% reduction in the early phase response (EPR) after nasal challenge (AUC of the TNSS in the first 60 minutes after challenge, a 40% reduction in the skin late phase response (LPR), and a 50% increase in grass pollen allergen specific immunoglobulin G4 (IgG4) for AIT vs. placebo.

Based on more recent nasal allergen challenge studies (Scadding et al, unpublished data; mean 4.63, standard deviation 1.65 for AUC 0-60 minutes post grass pollen nasal challenge in 14 allergic volunteers), inclusion of 13 patients per group will provide 80% power to detect a 40% reduction in AUC after challenge, whereas inclusion of 22 patients per group will provide 80% power to detect a 30% reduction.

Further based on a recent study (Scadding et al, unpublished data; mean 70.14, standard deviation 14.17 for cross-sectional area in cm2 at 8 hours for skin late phase response to intradermal grass pollen injection), inclusion of 7 participants per group will provide 80% power to detect a 30% reduction, whereas inclusion of 10 participants per group will provide 90% power to detect a 30% reduction.

Intent-to-treat (ITT) sample will be defined as all randomized participants. ITT participants will be analysed with the group to which they were randomized, regardless of the medication actually received. If participants drop out post randomisation, they will be invited to complete study assessments throughout the duration of the trial.

Per-protocol (PP) sample will be defined as ITT sample participants who remain in the study for 12 months and in whom the primary endpoints were assessed. Participants in the PP sample must be compliant with study medication, defined as taking 75% or more of their study medication for the duration of the study. Compliance with study medication will be as assessed by pill count for returned AIT/placebo. Participants in the PP sample will be analysed with the group to which they were randomized. The non-atopics will also be included in the PP sample.

Safety sample (SS) will be defined as all enrolled participants.

Analysis of study data will be conducted to address all objectives of the trial and other interrelationships among all data elements of interest to the investigators and of relevance to the objectives of the study. Primary analysis of treatment effect will be conducted under the intention-to-treat (ITT) principle of eligible patients, whereby outcome data from all eligible patients will be included regardless of treatment compliance. In addition to the analyses described in sections below, summary descriptive statistics will be provided in the following manner: continuous data will be summarized descriptively by mean, standard deviation, median, and range; categorical data will be presented as enumerations and percentages.

Analysis of primary endpoint:

The primary endpoint will be analysed using the ITT and the PP sample. The analysis of the primary endpoint will compare the mean EPR to nasal challenge recorded by the TNSS during the first 60 minutes after the nasal challenge, at 12 months of therapy. Comparison between active and placebo groups will be assessed using ANOVA at the 0.05 level of significance.

Analysis of secondary endpoint:

All secondary analyses will be treated as supportive. P-values will be presented for the secondary endpoints but will not be adjusted for multiplicity and should be interpreted with caution. Findings will be evaluated in the context of the available body of knowledge and with respect to other findings.

Arms:
arm 1: The active treatment arm will receive active grass pollen immunotherapy tablet (AIT), Grazax Oral Lyophilisate 75,000 standardised quality units tablet (SQ-T) once daily. arm 2: This arm will receive Grazax placebo once daily which contains the same composition as in the active Grazax tablet with the only difference being the exclusion of the grass pollen allergen extract.

Interventions: intervention 1: Grazax intervention 2: Grazax Placebo
Intervention descriptions: intervention 1: The active treatment arm will receive active grass pollen immunotherapy tablet (AIT), Grazax Oral Lyophilisate 75,000 SQ-T once daily. intervention 2: This arm will receive Grazax placebo once daily which contains the same composition as in the active Grazax tablet with the only difference being the exclusion of the grass pollen allergen extract.

Protocol document:
ICF_002.pdf: 
13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
1
Royal Brompton & Harefield NHS Foundation Trust & National Heart and Lung 
Institute 
 
PARTICIPANT INFORMATION and CONSENT FORM 
 
Study Title:  ‘Randomised placebo-controlled study of grass pollen allergen 
immunotherapy tablet (AIT) for seasonal rhinitis: time course of nasal, 
cutaneous and immunological outcomes’ 
 
Principal Investigator: Professor Stephen Durham, Head of the Section of Allergy 
and Clinical Immunology 
 
We would like to invite you to take part in a research study. Before you give your 
consent, it is important that you read this information. This sheet describes the study. 
This study will be explained to you and you should also discuss any questions you 
may have with your doctor and other staff members. You will also be given this 
information sheet to keep. This should help you decide whether or not you would like 
to take part in the study. If you sign this consent you agree to take part in this study. 
You will be given a copy of the signed consent for your records. 
 
1- Introduction and Background. About 45 million people in Europe have allergic 
rhinitis (hay fever) – inflammation of the nasal passages causing sneezing, runny 
nose, nasal congestion, itching and tearing of the eyes. In the United Kingdom, 
seasonal hay fever due to grass pollen allergy accounts for approximately 7 times 
more doctors’ appointments than asthma. The standard treatment for hay fever 
consists of treating the symptoms with a nasal spray and an antihistamine.  However, 
in a survey taken in a UK general practice less than 40% of patients with hay fever 
reported good symptom control with this standard treatment. For those patients with 
hay fever whose symptoms are not well controlled by treatment with antihistamines 
and nasal sprays, subcutaneous immunotherapy (SCIT) - (monthly injections of a 
grass allergen extract for a period of 3-5 years) is an effective alternative, and is 
approved in the UK on a named patient basis. More recently, allergen 
immunotherapy tablets (AITs) have been developed, including grass pollen allergen 
tablets. These have been shown to be highly effective in the treatment of hay fever, 
with the additional benefit of being convenient for patients, given that they may be 
taken at home. Grazax® (manufactured by ALK-Abello, Denmark) has UK and EU 
license for use in the treatment of troublesome grass pollen induced hay fever.  
2- What is the purpose of this research study? The aim of this research is to 
investigate the effects of the AIT treatment on the immune system over time – which 
changes are taking place and when in the course of treatment. This will provide 
insight into the complexities of the development of allergen-specific immune 
tolerance – how harmful allergic responses against innocuous substances such as 
grass pollen can be overridden.  
3- Why have I been chosen? You have been asked to take part because you have 
symptoms of hay fever during the grass pollen season - May to July - each summer. 
4- Do I have to take part? You do not have to take part in this study. Your 
participation is completely voluntary. If you decide not to take part your future medical 
care will not be affected in any way. You may also withdraw from the study at any 
time without having to provide a reason for doing so. You cannot participate in this 
study if you are pregnant, breastfeeding or if you are planning to become pregnant 
during the study (see section 15 for more information) 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
2
5- What do I have to do? You will be asked to come to the Allergy Department at 
the Royal Brompton Hospital on several occasions over a period of 1-3 years. This 
will include the following: 
 
 
A screening visit (December 2013 – April 2014) 
 
Two baseline assessments (January – April 2014) 
 
Randomisation and Treatment (January 2014 - April 2015) 
 
Five further assessments during treatment (January 2014 –April 2015) 
 
You may then be required (depending on whether you were randomised to receive 
‘active’ or ‘placebo’ treatment – see below) to continue in the study for another two 
years. This period will involve continuing to take the immunotherapy tablets for 
another year, followed by an assessment at the end of that year. After this, no further 
treatment will be given, but you will be asked to return a further year later for another 
assessment. If you have been assigned to the placebo group your participation in the 
study will end after unblinding at 12 months. You will be offered active treatment for 
24 months free of charge under regular follow-up. 
 
An overview of the first year of the study is shown in the following diagram; details of 
what will happen at each visit are given below.  
 
 
 
 
 
Screening Visit (V-1) 
 
Medical history, allergy history, medication record, and allergy skin prick 
testing 
 
Physical examination, vital signs (blood pressure, heart rate, weight, height 
etc)  
 
Breathing tests (spirometry, peak flow, peak nasal flow) 
 
Questionnaires concerning hay fever symptoms 
 
Nasal allergen challenge (grass pollen extract sprayed into the nose) 
 
Blood sample for full blood count and allergy tests 
(total 20 ml, equivalent to 2 tablespoons) 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
3
 
This visit will take approximately 2 – 3 hours 
 
 
Baseline Visits (V0a and V0b)  
During January to April 2014 you will have your first baseline visit (V0a). At this visit 
you will have the following procedures: 
 
Vital signs 
 
Blood sample of 120 ml (8 tablespoons) 
 
Nasal fluid collection 
 
Nasal brushing 
 
This visit will last approximately 60 minutes 
 
During January to April 2014 you will have your second baseline visit (V0b). This visit 
will be divided into two parts. One will take place in the morning (2 hours), and one in 
the evening (1 hour), after 8 hours. You will be free to leave the hospital in between. 
At this visit you will have the following procedures: 
 
Morning visit: 
 
 
Vital signs 
 
Allergen intradermal skin testing 
 
Nasal fluid collection 
 
Peak nasal inspiratory flow 
 
Peak expiratory flow  
 
Nasal wash 
 
Nasal allergen challenge 
 
Evening visit: 
 
 
Nasal fluid collection 
 
Nasal brushing  
 
Nasal biopsy 
 
Randomisation and start of treatment (V1) 
In January to April 2014, you will be randomly assigned to receive treatment with 
either ‘active’ grass pollen tablets or matched ‘placebo’ (dummy) tablets. This will be 
done by a computer programme. Neither you nor the study team will know which 
treatment group you are assigned to. Following randomisation, you will be asked to 
attend the clinic to take your first tablet under observation. Before starting treatment 
you will have your breathing (peak flow rate) and vital signs (blood pressure, heart 
rate) checked. You will then need to remain in the clinic for 1 hour after taking the 
first tablet. Both the active and placebo treatments are rapidly dissolving tablets, 
placed under the tongue. After this, you will be asked to continue to take your 
assigned tablets once a day, at home, for the following 12 months. (NB: women of 
childbearing age will have a urine pregnancy test before starting treatment/placebo). 
Treatment Visits (V2 – V7) 
Over the next 4 months, further visits will be at around 4 weeks, 8 weeks and 12 
weeks (V2, V3, V4) after starting treatment. The following procedures will be 
undertaken: 
 
Record of current medications 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
4
 
Record of adverse events 
 
Vital signs 
 
Questionnaires 
 
Blood sample of 120 ml (8 tablespoons) 
 
Nasal fluid collection 
 
Nasal wash 
 
Nasal brushing 
 
These visits will take around 60 minutes each. 
 
At the start of the pollen season, around the 1st of May, you will receive a ‘rescue 
medication’ pack (nasal spray, anti-histamine tablets, eye drops) for use as required 
during the season. You will be asked to fill out weekly questionnaires during the 
season, detailing your symptoms and the amount of rescue medication you have 
used.  
 
During the peak of the pollen season (mid-June to mid-July 2014) you will be asked 
to attend the clinic for the following (V5):  
 
 
Record of current medications 
 
Record of adverse events 
 
Vital signs 
 
Questionnaires 
 
Blood sample of 200 ml (16 tablespoons) 
 
Nasal wash 
 
Nasal fluid collection 
 
Nasal brushing 
 
Nasal biopsy 
 
This visit will take around 2 hours. 
 
At around 6 months treatment, shortly after pollen season, you will be asked to fill out 
questionnaires concerning your overall hay fever symptoms during whole of the 
pollen season (V6). Further you will be asked to perform a nasal allergen challenge 
and you will then be asked to record any hay fever symptoms you have at 5, 15, 30, 
and 60 minutes after the spray. This visit will take around 90 minutes.  
 
After 12 months treatment (V7), you will be asked to attend for the same series of 
tests as previously undertaken at V0a and V0b: 
 
Morning visit: 
 
 
Vital signs 
 
Blood take 
 
Allergen intradermal skin testing 
 
Nasal fluid collection 
 
Peak nasal inspiratory flow 
 
Peak expiratory flow  
 
Nasal wash 
 
Nasal allergen challenge 
 
 
Evening visit: 
 
 
Nasal fluid collection 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
5
 
Nasal brushing  
 
Nasal biopsy 
 
This visit will take 2 hours in the morning and 1 hour in the evening. You will be free 
to leave the hospital between these times.  
 
Follow up Visits (V8-V9) 
After 12 months unblinding of treatment will take place, meaning you will be told 
whether you were in the group receiving active or placebo treatment. If you have 
been on active treatment you will continue treatment for another 12 months, after 
which time you will be asked to attend for a blood sample of 200 ml (16 tablespoons), 
nasal brushings, nasal allergen challenge, intradermal skin tests and nasal biopsy 
(V8). Following this, you will not receive any further treatment, but will be asked to 
return in another 12 months, for the same procedures to be repeated (V9).   
If you were receiving placebo treatment, you will be offered 2 years treatment with 
active tablets, but will not be required to re-attend for the above procedures. All 
participants will be contacted periodically by telephone during the 24 months after 
unblinding to allow reporting of any health problems and current medications. 
Description of Procedures 
Nasal Wash (Lavage) (2 minutes) Nasal lavage with salty water (SinuRinse®) will 
be used. After ensuring a suitable temperature, the nozzle of the bottle is placed into 
the volunteer’s nose to form a seal, and, with the volunteer leaning forward over a 
sink, the bottle is then squeezed to allow a jet of solution to pass into one nostril and 
out of the other. The process is then carried out into the other nostril and repeated 
several times until all of the solution has been used.  
Nasal Fluid Collection (2-5 minutes) Small pieces of sponge will be placed into 
each nostril using sterile forceps for 2-5 minutes in order to collect nasal fluid. They 
are then removed and the fluid later extracted and frozen.   
Nasal Allergen Challenge (1 hour) A small amount of purified grass pollen extract 
will be sprayed into your nose. You will then be asked to record any hay fever 
symptoms you have at 5, 15, 30, and 60 minutes after the spray. Additionally, we will 
record how well you can sniff through your nose, using a Peak Nasal Flow meter, 
how well you can breathe in, using a Peak Flow meter, and will collect nasal fluid.  
 
Nasal Biopsy (30 minutes – 1 hour) We shall take 3 nasal biopsies (each ~2mm 
across) from the lining of your nose at baseline, during the season and at 12 months 
of treatment. In those participants on active treatment, biopsies will also be taken at 
24 and 36 months. Before taking the biopsies we will numb the inside of the nose by 
applying a local anesthetic by nasal spray and on cotton wool. Biopsies are taken 
with specialized forceps. The procedure is very well-tolerated and is pain free due to 
the local anaesthetic. About 10% of patients may experience discomfort following the 
procedure when the numbness wears off. This discomfort usually responds to 
paracetamol tablets. Nasal bleeding may also occur in the hours/days after the 
biopsy. This usually responds to simple finger pressure (pinching the bridge of the 
nose). It is very rare for any further intervention to be required; nonetheless, a study 
investigator will be available by telephone 24 hours a day to give further advice 
should this be required.  
 
Nasal brushing (5 minutes) This involves inserting a small brush/probe inside each 
nostril, before brushing/scraping it against the lining of the nose, it is used to collect 
cells. This will be performed on seven occasions: twice during baseline, at 4, 8 and 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
6
16 weeks, and at 7 and 12 months on treatment.  
 
Allergy Skin Prick Testing Skin testing involves putting drops of allergy extracts on 
the forearm and lightly pricking the skin with a sterile lancet through the allergy 
extracts. The results are apparent within 15 minutes.  
Allergy intradermal skin testing This involves injecting a tiny amount of grass 
allergen solution just under the skin of the forearm. After 15 minutes a small skin 
wheal will be apparent in allergic individuals and is recorded with a pen. After 8 
hours, a larger, more diffuse swelling becomes apparent – this is recorded with a 
pencil at 8 hours. 
Lung Function Testing This involves breathing through a tube into a machine to 
measure how well your lungs are working.   
 
Research Tests Blood and biopsy samples are collected during the study. These 
samples are used for research tests. Research tests help us learn more about your 
disease, the immune system (the body’s natural defense system against illness), and 
response to drugs or treatment. The results of the research tests will not be shared 
with you.  These research samples will not identify you. Research tests may include 
genetic tests.  Genetic tests study an individual’s inherited characteristics, found in 
DNA, which is present in each of the cells of your body. DNA contains information 
needed to construct and operate a human body.      
 
6- What is the drug or intervention that is being tested? 
Grazax® Standardized Grass Pollen Tablets contain purified grass pollen extract in a 
fast dissolving pill form taken under the tongue.  
Grazax® contains gelatine (fish source).  
 
7- What are the possible effects of the procedures and interventions being 
tested?  
Study Medication Risks 
Grazax® Standardized Grass Allergy Tablet. Very commonly reported side effects 
(occurring in up to 70% of patients) include itching and mild discomfort within the 
mouth. In a few cases, this may develop into tongue and/or lip swelling, or mild 
blistering in the mouth. Other common side effects of Grazax® include headache, 
fatigue, red, itchy, watery eyes, sneezing, nasal congestion, itchy nose, runny nose, 
irritation and swelling of the roof of the mouth, itchy ears, mild asthma (wheezing, 
cough), itchy skin rash and hives. Abdominal symptoms may include stomach 
irritation, acid reflux, vomiting and diarrhoea.  These side effects are mostly mild to 
moderate in severity, last from minutes to hours after each intake of Grazax®, and 
tend to go away on their own within 1 to 7 days. These side effects are usually well-
tolerated and require no treatment. However, anti-allergic medication is always 
available. 
 
More severe, systemic allergic reactions, including anaphylaxis, to Grazax have been 
reported, but they are exceptionally rare, occurring at a frequency of less than once 
per million tablets taken.  None the less, in view of the potential risks involved, the 
first tablet is taken under medical supervision over 60 minutes and there is a 24-hour 
telephone contact line for advice concerning any side effects at any time.  
 
The usual medication information sheets for Grazax® are available on request. 
 
 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
7
Study Procedure Risks 
 
Intradermal and Skin Prick Tests These tests are routinely performed in clinic. You 
may experience mild to moderate itchiness and discomfort at the sites of the 
intradermal and skin pricks with allergens and the positive control (histamine).  The 
symptoms are usually not bothersome and treatment with topical or oral 
antihistamines is available if needed. In view of the extremely remote risk of a more 
serious reaction a physician is always present and drugs and equipment for 
treatment of anaphylactic reactions are available.  
 
Nasal Allergen Challenge You will experience typical hay fever symptoms including 
nasal itching, nasal blockage, sneezing, nasal discharge, itchy eyes, watery eyes, 
and redness of the lining of the eye. Nose and/or eye symptoms are prominent within 
minutes and fade rapidly, whereas mild nasal congestion may last up to and after 10 
hours. There is a small risk of causing mild asthma symptoms. You will perform a 
breathing test (using a “Peak expiratory flow meter”) before and after the nasal 
challenge and treatment with inhaled bronchodilators and steroids will be 
immediately available. As discussed above, for any intervention using the allergen to 
which you are allergic, there is that small risk of developing an anaphylactic reaction, 
although this has never been observed in studies of several hundred participants 
over the past 20 years in our department. 
 
Nasal Wash (Lavage) Nasal wash with salty water (SinuRinse®) will be used before 
and after the nasal challenge. SinuRinse® is widely available and no adverse effects 
are to be expected. 
 
Blood Samples You will feel the sting of the needle as it enters a vein in your arm.  
There is a small risk of bruising and swelling at the site where the needle is inserted.  
Some people feel faint when they have their blood taken. You will be seated when 
you have your blood sample taken. True faints (‘vaso-vagal’ faints) are rare and 
resolve on lying down. 
 
Nasal Brushing This may provoke discomfort and very rarely mild nasal bleeding. 
Local anaesthetic spray may be applied before the procedure if preferred. 
 
Nasal Biopsy The most frequent side effects are local discomfort, which may persist 
for up to 5-7 days, and nose bleeding. This occurs in about 1 in 10 biopsies taken in 
our centre and responds to conservative measures including application of finger 
pressure below the nasal bridge. In order to minimize discomfort, the inside of the 
nose is pre-treated with a highly effective local anesthetic (numbing medication) 
given by a spray pump directly onto the inner lining of the nose. In order to provide 
complete numbness and to minimize the risk of bleeding, cotton wool soaked in a 
numbing solution is applied to the inner lining of the nose for 10 minutes before the 
procedure. There may be a slightly bitter taste from either the solution or the local 
spray used. 
 
The treatment and procedures in this research study may have risks that are not yet 
known.      
 
8- Who will benefit from the study? Taking part in this trial may or may not be of 
benefit to you. However, your participation in the study may benefit society by helping 
us learn more about better ways to treat allergies in the future. During the pollen 
season you will be provided with medications to alleviate your symptoms in the nose 
and eyes. This “rescue medication” set will include antihistamine tablets, nasal 
corticosteroid sprays and eye drops. Further you will receive 24 months of active 
treatment free of charge either from the beginning of the treatment if you were 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
8
assigned to the active treatment group or after unblinding at 12 months if assigned to 
the placebo arm. We believe this active treatment to have a positive effect on your 
hay fever symptoms during the pollen season.  
 
9- What other choices do I have if I do not take part in this study? Before you 
decide to take part in this study, Prof. Durham or his medical colleagues will talk with 
you about these and other options you may have, which may include just staying on 
the standard treatment for your hay fever. 
 
10.  May I leave this study at any time? You may decide not to take part or to 
withdraw from the study at any time. If you decide to withdraw from the study, there is 
no penalty or loss of benefits in your routine medical care or any other benefit(s) that 
you would otherwise be entitled to receive.   
If you or the study doctor decides that you should stop taking the study medication, 
we would like to have you continue with the study visits and procedures, so as to 
follow your progress after coming off the study medication. This will also be very 
helpful for comparison to those participants who continue on the study medication for 
the duration of the study.  
In addition, you should consult with Prof. Durham who will discuss future treatment 
and procedures for your continued care when you are no longer in this study. 
 
11- Can I be taken off this study without my consent? You may be removed from 
the study without your consent at any time. Reasons why you may be removed from 
the study include, but are not limited to, the following:  
 
Prof. Durham decides that it is best for you not to continue.   
 
You are unable to complete study treatments or tests. 
 
The study is stopped by the Institution, the Sponsor(s), or the Medicines and 
Healthcare products regulatory agency (MHRA), or other Health Authorities. 
 
You meet one of the pre-defined stopping rules as written in the study 
protocol. 
If you are removed from the study Prof. Durham will notify you about treatment or 
procedures for your continued care.  
 
12.  What about new findings? During your participation in this study, Prof. Durham 
will also discuss with you about any new findings so you are up-to-date and to allow 
you to decide whether such findings from this or other research may affect your 
willingness to continue in the study. 
 
13- What if something goes wrong? It is important that you tell your study doctor, 
Prof. Stephen Durham, Dr. Moisés Calderón or Dr. Esther Steveling if you feel that 
you have been injured because of taking part in this study.  You can tell the doctor in 
person or call Prof. Stephen Durham on 020 7351 8024, Dr. Moisés Calderón or one 
of the other study doctors (Dr Esther Steveling) at the Royal Brompton & Harefield 
NHS Foundation Trust. A 24hour telephone contact number 07714 051 248 will be 
available at all times during the study. 
For further support, assistance and advice the Royal Brompton Hospital offers the 
Patient Advice and Liaison Service (PALS). They can help you if you are having 
difficulties, or any complains. You may contact: Patient Advice and Liaison Services 
(PALS), Royal Brompton Hospital, Sydney Street, London, SW3 6NP, Tel: 020 7349 
7715, Email: pals@rbht.nhs.uk. 
 
14- Will my taking part in this study be kept confidential? Your medical and 
research records will be confidential to the extent permitted by law. Every effort is 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
9
made to keep your identity private. You will be identified by a study code, not your 
name. All of your results from the study, including blood and nasal samples, will be 
collected and stored under this number, rather than using your name or other 
personal identifiers. The key to the code is kept in a secured file at the study site.  
All of your results and samples will be stored securely on-site at Imperial College. 
Blood and nasal samples will be analysed at Imperial College, London, at King’s 
College, London, and at ALK Abello, Horsholm, Denmark using only the study code 
number. Only the investigators directly involved in running the study will have access 
to these results. Only the study staff at the Royal Brompton Hospital will have access 
to your personal identifiable information; other investigators, in particular those 
investigators analysing your blood and nasal samples, will only be able to identify you 
by your study code number. Authorised personnel from Imperial College, Royal 
Brompton Hospital NHS R&D offices and regulatory authorities/inspectors may need 
to access to your clinical notes if they need to verify or cross-check data. 
 
15- What about pregnancy, breastfeeding and birth control? You cannot 
participate in this study if:   
 
You are pregnant 
 
You are planning to become pregnant while in the study or 
 
You are breastfeeding 
 
Initiation of treatment with Grazax is not recommended during pregnancy. Additional 
procedures undertaken in this study are also not recommended in pregnancy. If you 
are female, you will be given a pregnancy test before the study. If you join this study, 
you must agree to use birth control during the entire length of the study. If you should 
become pregnant while in this study, or if you think that you have become pregnant, 
you must contact one of the investigators right away.   
16- Will my GP be informed? With your consent we will inform your GP about your 
participation in this study. 
 
17- Ethics Approval This study has been reviewed and approved by the  Research 
Ethics Committee East Midlands – Nottingham 2. 
 
18- What will happen to my samples? Blood and nasal tissue samples will be 
stored and analysed in the laboratories at Imperial College, King’s College,  and ALK 
Abello, (Horsholm, Denmark). The response of certain cells within the samples, 
including numbers of different cell types, mediators released and genes switched on 
or off, will be studied.  
 
19 - Will my blood, and/or tissue samples and information from this study be 
stored for future research use? With your consent any specimens left over at the 
end of the study will be stored securely under your coded number for up to 10 years 
at Imperial College, London, and will be used for future ethically approved research.  
Samples will not be analysed or tested for serious infections, such as HIV or 
Hepatitis B, nor for any inherited genetic conditions. 
The results of tests done on your stored samples will not be given to you or your 
doctor. The results will not be put in your records and will not change your medical 
care. There will be no benefits to you from the storage of these samples and 
information. However, the use of your samples and information may help researchers 
learn more about your disease or help study the genetics related to your disease.   


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
10 
There may be risks in allowing the storage or analysis of samples and information.  
Researchers are required to protect your privacy and to keep your information private 
to the extent permitted by the law.  
Future studies on stored samples and information will be reviewed and approved by 
an Ethics Committee for science and ethics. The samples and information will not be 
sold; however, the results of the tests could lead to the development of commercial 
products. You will not receive any money from research using your stored samples 
and information.   
    
You can change your mind at any time during the study and ask to have your 
samples destroyed. This request should be made in writing to the study doctor. If 
your samples have not been used, they will be destroyed. If your samples have 
already been tested before your request, the information from these tests will be 
used and cannot be destroyed.   
 
20- What will happen to the results of the research study? The results of this 
study will help us to improve our knowledge about hay fever and allergy. We are not 
seeking financial benefits from this study. 
 
21- Who is organising and funding the research? None of the study investigators 
have any personal financial interest in the study. This study is being conducted by 
Imperial College London.  
22 - What if I have a problem or a question?   If you ever have questions about this 
study or have a study-related injury, you should contact Prof. Stephen Durham, Dr. 
Moisés Calderón (or another study doctor) on 020 7351 8024 or 07714 051 248 (24 
hours) at the Royal Brompton & Harefield NHS Foundation Trust. 
For further support, assistance and advice the Royal Brompton Hospital offers the 
Patient Advice and Liaison Service (PALS). They can help you if you are having 
difficulties, or any complains. You may contact: Patient Advice and Liaison Services 
(PALS), Royal Brompton Hospital, Sydney Street, London, SW3 6NP, Tel: 020 7349 
7715, Email: pals@rbht.nhs.uk. 
 
23 - What are the costs of taking part in this study? Taking part in this trial will not 
cost you anything. You will be reimbursed any reasonable travel and parking 
expenses incurred throughout your participation in this clinical study upon providing 
receipts.  
 
24 - Will I be paid for taking part in this study? You will receive no payment for 
your participation in this research study, whereas you will receive £75 compensation 
for each visit. Please be advised, that receiving a payment to take part in the study 
may affect any benefits that you claim.  
 
 


13IC0847 - Information & Consent atopic participants V6.0 (01.10.2015) 
11 
To be printed on departmental headed paper 
 
Study Protocol Number: 13IC0847 V 7.0                    
Study Participant ID:__________ 
 
CONSENT FORM 
 
Full Title of Project: A Prospective Randomised Placebo-Controlled Time Course 
Study Of Grass Pollen Allergen Immunotherapy Tablet (AIT) Treatment For Allergic 
Rhinitis: Study of nasal, cutaneous and immunological outcomes. 
Name of Principal Investigator: Professor Stephen Durham 
If you agree,  
Please initial box 
1. I confirm that I have read and understood the subject information sheet dated  
01.10.2015 version 6.0 for the above study and I have had the opportunity to 
ask questions which have been answered fully. 
 
2. I understand that my participation is voluntary and I am free to withdraw at 
any time, without giving any reason, without my medical care or legal rights 
being affected. 
 
3. I understand that sections of any of my medical notes may be looked at by 
individuals from Imperial College, Royal Brompton & Harefield NHS Foundation 
Trust and other regulatory authorities I give permission for these individuals to 
access my records that are relevant to this research. 
 
4. The compensation arrangements have been discussed with me.  
 
 
5. I agree to take part in the above study.  
 
6. I consent to my blood and tissue samples being used for laboratory research 
tests as described in the protocol.  
 
7. I agree to my samples to be stored and used in future, REC approved 
projects. 
 
 
 
8. I consent to my GP being informed of my participation in the study. 
 
 
 
________________________     
 ________________     ________________ 
Name of Subject                         
Signature                         
 Date 
 
 
_________________________      
________________     ________________ 
Name of Person taking consent   
Signature                          
Date 
 
 
1 copy for subject; 1 copy for Principal Investigator; 1 copy to be kept with hospital notes 

Prot_001.pdf: 
Confidential                                                Page 1 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
 
 
 
Randomised placebo-controlled study of grass pollen allergen immunotherapy tablet 
(AIT) for seasonal rhinitis: time course of nasal, cutaneous and immunological 
outcomes 
 
                    Version 7.0 (01.10.2015) 
 
Sponsor 
 
 
 
Imperial College London 
 
Funder 
 
Wellcome Trust, PhD Programme for  
                                                            Clinicians awarded to Guy Scadding 
 
Study Coordination Centre  
Royal Brompton Hospital 
 
Trial ID 
 
 
 
13IC0847 
 
REC reference: 
 
 
13/EM/0351 
EudraCT reference:  
 
2013-003732-72 
 
 
 
Protocol authorised by: 
 
Stephen Durham, MD 
Chief Investigator            
 
 
 
Signature                                                                                                      Date 
 
 


Confidential                                                Page 2 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
 
 
 
Study Management Group: 
 
Chief Investigator: 
Stephen Durham, MD, MA, FRCP 
Head of Section of Allergy and Clinical Immunology 
Professor of Allergy and Respiratory Medicine 
Royal Brompton & Harefield NHS Foundation Trust 
Imperial College London - NHLI 
Sydney Street - London 
London SW3 6NP 
Tel: 0207-351-8024 Fax: 0207-349-7768 
Email: s.durham@imperial.ac.uk 
 
Co-Investigators:  
Moisés Calderón, MD PhD 
 
 
 
Senior Clinical Fellow 
 
 
 
Department of Allergy and Clinical Immunology 
 
 
 
National Heart and Lung Institute 
 
 
 
Imperial College – NHLI 
 
 
 
Dovehouse Street London 
 
 
 
SW3 6LY 
Tel: 0207-351-8024 Fax: 0273497780 
Email: m.calderon@imperial.ac.uk 
 
Mohamed Shamji, PhD 
Research Associate 
Department of Allergy and Clinical Immunology 
Sir Alexander Fleming Building 
2nd Floor, Room 259 
South Kensington Campus 
SW7 2AZ 
Tel: 0207-594-3476 Mobile 07714051291 
Email: m.shamji@imperial.ac.uk 
 
Esther Steveling, MD 
Clinical Research Fellow 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
 
 
 
Imperial College – NHLI 
 
 
 
Dovehouse Street London 
 
 
 
SW3 6LY 
Tel: 0207-351-8024 Fax: 02073497780 
Email: e.steveling@imperial.ac.uk 
 
Mongkol Lao-araya, MD 
Clinical Research Fellow 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
 
 
 
Imperial College – NHLI 
 
 
 
Dovehouse Street London 
 
 
 
SW3 6LY 
Tel: 0207-351-8024 Fax: 02073497780 
Email: m.lao-araya@imperial.ac.uk 
 
 
 
Guy Scadding, MBBS, MRCP 
Speciality Registrar in Allergy 


Confidential                                                Page 3 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
 
 
 
Imperial College – NHLI 
 
 
 
Dovehouse Street London 
 
 
 
SW3 6LY 
Tel: 0207-351-8024 Fax: 02073497780 
Email: g.scadding@imperial.ac.uk 
 
Aarif Eifan, MD 
Clinical Research Fellow 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
 
 
 
Imperial College – NHLI 
 
 
 
Dovehouse Street London 
 
 
 
SW3 6LY 
Tel: 0207-351-8024 Fax: 02073497780 
Email: a.eifan@imperial.ac.uk 
 
Alina Dumitru, MD 
Clinical Research Fellow 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
 
 
 
Imperial College – NHLI 
 
 
 
Dovehouse Street London 
 
 
 
SW3 6LY 
Tel: 0207-351-8024 Fax: 02073497780 
Email: a.dumitru@imperial.ac.uk 
 
Martin Penagos, MD, MSc 
Research Fellow 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
Imperial College – NHLI 
Dovehouse Street London 
SW3 6LY 
Tel: 0207-351-8024  Fax: 0273497780 
Email: m.penagos@imperial.ac.uk 
 
Christopher Koulias, MD 
Research Fellow 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
Imperial College – NHLI 
Dovehouse Street London 
SW3 6LY 
Tel: 0207-351-8024  Fax: 0273497780 
Email: chris.koulias@gmail.com 
 
 
Miss Rachel Yan, RGN 
Research Nurse 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
Imperial College – NHLI 
Dovehouse Street London 
SW3 6LY 
Tel: 0207-351-8121 
Email: rachel.yan@imperial.ac.uk 
 
 


Confidential                                                Page 4 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
Study Coordination Centre: 
 
For general queries, supply of trial documentation, and collection of data, please 
contact:  
 
Study Coordinator:  
Esther Steveling, MD 
Clinical Research Fellow 
Department of Allergy and Clinical Immunology 
National Heart and Lung Institute 
 
 
 
Imperial College – NHLI 
 
 
 
Dovehouse Street London 
 
 
 
SW3 6LY 
Tel: 0207-351-8024 Fax: 02073497780 
Email: e.steveling@imperial.ac.uk 
 
Clinical Queries: 
 
Clinical queries should be directed to Esther Steveling who will direct the query to the 
appropriate person. 
 
Sponsor:  
 
Imperial College London is the Sponsor for this study. For further information 
regarding the sponsorship conditions, please contact the Head of Regulatory 
Compliance at:  
 
Joint Research Compliance Office  
5L10C, 5th Floor Lab Block  
Charing Cross Hospital  
Fulham Palace Road  
London, W6 8RF  
Tel: 0203 311 0204  
Fax: 0203 311 0203 
 
This protocol describes the prospective grass pollen allergen immunotherapy tablet 
(AIT) study and provides information about procedures for entering participants. The 
protocol should not be used as a guide for the treatment of other participants; every care 
was taken in its drafting, but corrections or amendments may be necessary. These will 
be circulated to investigators in the study. Problems relating to this trial should be 
referred, in the first instance, to the study coordination centre. This trial will adhere to 
the principles outlined in the Medicines for Human Use (Clinical Trials) Regulations 
2004 (SI 2004/1031), amended regulations (SI 2006/1928) and the International 
Conference on Harmonisation Good Clinical Practice (ICH GCP) guidelines. It will be 
conducted in compliance with the protocol, the Data Protection Act and other regulatory 
requirements as appropriate. 
 
 
 
 
 


Confidential                                                Page 5 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
1. Table of Contents 
1. TABLE OF CONTENTS ................................................................................................................ 5 
2. STUDY SUMMARY ....................................................................................................................... 7 
3. ABBREVIATIONS ......................................................................................................................... 9 
BACKGROUND AND RATIONALE .................................................................................................. 12 
5. STUDY OBJECTIVES ...................................................................................................................... 13 
5.1 PRIMARY OBJECTIVE ..................................................................................................................... 13 
5.2 SECONDARY OBJECTIVES ............................................................................................................... 14 
5.3 CLINICAL HYPOTHESES ................................................................................................................. 14 
5.4 MECHANISTIC HYPOTHESES ......................................................................................................... 15 
5.5 EXPLORATORY HYPOTHESES ..................................................................................................... 15 
6. STUDY DESIGN .............................................................................................................................. 16 
6.1 PRIMARY ENDPOINT ..................................................................................................................... 17 
6.2.1 SECONDARY ENDPOINTS ........................................................................................................... 18 
6.2.2 EXPLORATORY ENDPOINTS ....................................................................................................... 18 
6.3 SELECTION AND WITHDRAWAL OF PARTICIPANTS .................................................................... 18 
6.3.1 RECRUITMENT ........................................................................................................................................... 18 
6.3.2 INCLUSION CRITERIA ............................................................................................................................... 19 
6.3.3 EXCLUSION CRITERIA ............................................................................................................................... 20 
6.3.4 WITHDRAWAL CRITERIA......................................................................................................................... 22 
6.3.5 URGENT SAFETY MEASURE ...................................................................................................................... 23 
6.3.6 STOPPING RULES ...................................................................................................................................... 23 
6.3.7 PREMATURE DISCONTINUATION OF STUDY THERAPY ........................................................................ 24 
6.3.8 RANDOMISATION ...................................................................................................................................... 24 
6.3.9 BLINDING ................................................................................................................................................... 25 
6.3.10 UNBLINDING ........................................................................................................................................... 25 
6.4 STUDY MEDICATIONS .................................................................................................................... 25 
6.4.1 ALLERGEN IMMUNOTHERAPY TABLET (AIT) ................................................................................. 25 
6.4.2 FORMULATION AND PACKAGING ........................................................................................................ 26 
6.4.3 ADMINISTRATION ................................................................................................................................. 26 
6.4.4 SPECIAL WARNINGS AND PRECAUTIONS FOR USE .......................................................................... 27 
6.4.5 RECOMMENDED STORAGE CONDITIONS ........................................................................................... 28 
6.4.6 DRUG ACCOUNTABILITY ....................................................................................................................... 28 
6.4.7 ASSESSMENT OF COMPLIANCE WITH STUDY MEDICATION ............................................................. 28 
6.4.8 CONCOMITANT MEDICATION .............................................................................................................. 28 
6.5 STUDY PROCEDURES ..................................................................................................................... 30 
6.5.1 GENERAL ASSESSMENTS .......................................................................................................................... 30 
6.5.2 CLINICAL PROCEDURES ............................................................................................................................ 30 
6.5.3 LOCAL LABORATORY ASSESSMENTS ...................................................................................................... 30 
6.5.4 QUESTIONNAIRES ..................................................................................................................................... 31 
6.5.5 POLLEN COUNT ......................................................................................................................................... 31 
6.5.6 NASAL WASH (LAVAGE) ....................................................................................................................... 31 
6.5.7 NASAL ALLERGEN CHALLENGE ........................................................................................................... 31 
6.5.8 SAMPLING OF NASAL SECRETIONS ...................................................................................................... 32 
6.5.9 INTRADERMAL SKIN TESTS .................................................................................................................. 32 
6.5.10 NASAL EPITHELIAL BRUSHINGS ....................................................................................................... 33 
6.5.11 NASAL BIOPSY ..................................................................................................................................... 35 
6.5.12 VENEPUNCTURE.................................................................................................................................. 37 
6.5.13 CLINICAL RESPONSES ......................................................................................................................... 37 


Confidential                                                Page 6 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
6.5.14 VISITS ................................................................................................................................................... 38 
6.5.15 STUDY FOLLOW UP (12-36 MONTHS POST RANDOMISATION) .................................................. 48 
6.6 BIOMARKERS ................................................................................................................................. 48 
6.6.1 FLOW CYTOMETRY ................................................................................................................................... 49 
6.6.2 DNA METHYLATION STUDIES ................................................................................................................ 49 
6.6.3 T-CELL ASSAYS.......................................................................................................................................... 49 
6.6.4 SERUM ASSAYS .......................................................................................................................................... 49 
6.6.5 SERUM ARCHIVE ....................................................................................................................................... 50 
6.6.6 NASAL FLUID ............................................................................................................................................. 50 
6.6.7 BLOOD SAMPLES ....................................................................................................................................... 51 
6.6.8 NASAL BRUSHINGS .................................................................................................................................... 55 
6.6.9 NASAL BIOPSY ........................................................................................................................................... 56 
6.7 RECORDING, MANAGING AND REPORTING ADVERSE EVENTS ................................................... 58 
6.7.1 DEFINITIONS.............................................................................................................................................. 58 
6.7.2 GRADING OF ADVERSE EVENTS/ADVERSE REACTIONS ....................................................................... 61 
6.7.3 CAUSALITY DEFINITIONS......................................................................................................................... 63 
6.7.4 INVESTIGATOR’S RESPONSIBILITIES ...................................................................................................... 64 
6.7.5 SPONSOR’S RESPONSIBILITIES ............................................................................................................... 65 
6.7.6 COLLECTING AND RECORDING ADVERSE EVENTS .............................................................................. 65 
6.7.6.1 METHODS OF COLLECTION .................................................................................................................. 66 
6.7.6.2 ADVERSE EVENTS AND ADVERSE REACTIONS TO BE COLLECTED ................................................ 66 
6.7.6.3 RECORDING METHOD ........................................................................................................................... 66 
6.7.6.4 REPORTING ............................................................................................................................................ 67 
6.7.6.5 UNBLINDING .......................................................................................................................................... 68 
6.7.6.6 REPORTING PREGNANCY ..................................................................................................................... 68 
6.7.6.7 ANNUAL REPORTS ................................................................................................................................. 69 
6.7.6.8 SAFETY ANALYSIS ................................................................................................................................. 69 
6.8 STATISTICAL METHODS AND POWER CALCULATION................................................................ 69 
6.8.1 STATISTICAL ANALYSIS ........................................................................................................................... 69 
6.8.2 ANALYSIS SAMPLES .................................................................................................................................. 70 
6.8.3. ANALYSIS OF ENDPOINTS ....................................................................................................................... 70 
6.8.3.1. ANALYSIS OF PRIMARY ENDPOINT ................................................................................................... 71 
6.8.3.2 ANALYSIS OF SECONDARY ENDPOINTS ............................................................................................. 71 
6.8.3.3 NASAL BIOPSY ANALYSES .................................................................................................................... 72 
7. INFORMED CONSENT ................................................................................................................. 72 
8. ETHICS APPROVAL ....................................................................................................................... 73 
9. CTA ............................................................................................................................................... 73 
10. CONFIDENTIALITY ...................................................................................................................... 73 
11. INDEMNITY ................................................................................................................................. 73 
12. SPONSOR ..................................................................................................................................... 73 
13. FUNDING ..................................................................................................................................... 73 
14. AUDITS ........................................................................................................................................ 74 
15. PUBLICATION OF RESULTS ........................................................................................................ 74 
16. REFERENCES ............................................................................................................................... 74 
17. APPENDICES ............................................................................................................................... 77 
APPENDIX 1: GLOBAL EVALUATION SCORE 1 - VISUAL ANALOGUE SCALE .............................................. 77 
APPENDIX 2: GLOBAL EVALUATION SCORE 2 ................................................................................................ 78 
APPENDIX 3: MINI RHINOCONJUNCTIVITIS QUALITY OF LIFE QUESTIONNAIRE ...................................... 79 
APPENDIX 4: GLOBAL EVALUATION SCORE 3 – VISUAL ANALOGUE SCALE .............................................. 82 
APPENDIX 5: SYMPTOM AND MEDICATION SCORE ........................................................................................ 82 
APPENDIX 6: VALIDATED SYMPTOM SCORING (TNSS – TOTAL NASAL SYMPTOM SCORE) .................... 83 
APPENDIX 7: VISUAL ANALOGUE SCALE FOR THE EPR AFTER NASAL CHALLENGE ................................ 85 
APPENDIX 8: MEDICATION WASHOUT PERIODS PRIOR TO VISIT-1, 0, 5, AND 7....................................... 86 
APPENDIX 9: DAILY DIARY CARD ..................................................................................................................... 87 


Confidential                                                Page 7 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
 
2. Study summary 
 
Title 
Randomised placebo-controlled study of grass pollen allergen 
immunotherapy tablet (AIT) for seasonal rhinitis: time course of 
nasal, cutaneous and immunological outcomes 
 
Short title 
Grass Pollen Allergen Immunotherapy Tablet (AIT) Time 
Course Study 
 
Conducted by 
Royal Brompton Hospital and Imperial College 
 
Protocol Chair 
Stephen Durham, MD, MA, FRCP 
 
Accrual Objective 
50 participants with moderate-severe grass pollen allergic 
rhinitis; 20 healthy, non-atopic participants 
 
Study Design 
A randomized, double-blind, single-centre, placebo-controlled, 
two-arm time course study comparing grass pollen AIT with 
placebo. 
The study will involve a 4 month recruitment phase, a 12 month 
randomized, blinded treatment phase, and finally a further 12-24 
months open treatment phase. Up to 50 grass pollen allergic 
(atopic) participants will be enrolled to ensure randomisation of 
at least 44. Further we shall recruit 20 healthy, non-atopic 
volunteers.  
Individuals with severe grass pollen hay fever, with or without 
associated seasonal asthma, will be recruited after the 2013 grass 
pollen season, between December 2013 and April 2014. 
Screening will be completed before eligible atopic participants 
are randomized to one of the following two treatment arms in a 
1:1 ratio: 


Confidential                                                Page 8 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
1. Grass pollen AIT 
2. Placebo AIT 
Atopic participants will undergo baseline assessments between 
January 2014 and April 2014.  Atopic participants will then 
begin AIT treatment and continue treatment for 12 months. All 
atopic participants will be provided with anti-allergic rescue 
medications 
(antihistamine 
tablets, 
topical 
intranasal 
corticosteroids, and eye-drops) throughout the pollen season. 
Clinical surrogate endpoint assessments, plus blood, nasal fluid 
and nasal brushing sampling, will be performed at baseline , at 
around 4, 8 and 12 weeks after starting the AIT,  during the peak 
pollen season and at 6 and 12 months of treatment. Nasal 
mucosal biopsies will be taken at baseline, around 5 months, and 
12 months of treatment. After 12 months of treatment, 
unblinding will take place. Those atopic participants receiving 
active AIT treatment will continue therapy for another 12 
months followed by a withdrawal phase of 12 months. All 
measurements and samples taken at 12 months will be repeated  
again at 24 and at 36 months from the initial start of treatment. 
Those atopic participants on placebo will be offered 24 months 
of active treatment after unblinding plus usual clinical follow up.  
The 20 healthy, non-atopic participants will undergo screening 
and one visit only, at 12 months, to coincide with the 12 month 
visit for the atopic participants. This visit will include clinical 
surrogate endpoint assessments, plus blood, nasal fluid, nasal 
brushing sampling, and nasal biopsies. The assessments will 
serve as a control for the results of the atopic participants at 12 
months. 
 
Primary endpoint 
The area under the curve (AUC) of the early phase response 
(total nasal symptom score, TNSS, 0-60 minutes) following 


Confidential                                                Page 9 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
grass pollen nasal allergen challenge in active versus placebo-
treated participants at 12 months. 
 
Study duration 
44 months (4 months recruitment phase, 36 months treatment 
and assessment phase and 4 months conclusion of follow-up) 
 
Treatment description 
Eligible atopic participants will be randomized to one 
of the following two treatment arms in a 1:1 ratio: 
 
AIT (Grazax®, Phleum pretense (Phl p) freeze-dried 
oral lyophilisate/soluble tablet)  
 
AIT placebo  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3. Abbreviations 
 
AIT 
 
Allergy Immunotherapy Tablet 


Confidential                                                Page 10 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
AE 
 
Adverse Event 
AR 
 
Adverse Reaction 
AUC  
area under the curve 
BAU  
Bioequivalent allergy unit 
BU 
 
Bioequivalent Units 
DAIT  
Division of Allergy, Immunology, and Transplantation 
CI 
 
Chief Investigator 
CRF  
electronic case report form 
DAO  
Diamine oxidase 
EPR  
early phase response 
FAB  
facilitated allergen binding 
FDA  
Food and Drug Administration 
FEV1   
forced expiratory volume at 1 second 
GCP  
Good Clinical Practice 
GE 
 
Global Evaluation 
ICH 
 
International Conference on Harmonisation 
ISU 
 
ISAC Standardized Units 
JRCO  
Joint research compliance office 
LPR  
late phase response 
MHRA 
Medicines and Healthcare products regulatory agency 
PEF 
 
peak expiratory flow 
PI 
 
principal investigator 
Phl p  
Phleum pratense (Timothy grass) 
PNIF   
peak nasal inspiratory flow 
REC  
Research Ethics Committee 
SAE   
serious adverse event 
SAP 
 
statistical analysis plan 
SAR  
seasonal allergic rhinitis 
SCIT  
subcutaneous immunotherapy 
SIT 
 
Specific immunotherapy 
SLIT  
sublingual immunotherapy 
SOP 
 
Standard Operating Procedure 
SSAR  
Suspected Serious Adverse Reaction 
SUSAR 
Suspected unexpected serious adverse reaction 


Confidential                                                Page 11 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
SQU  
Standardised quality units 
 
 
TNSS  
total nasal symptom score 
URL  
uniform resource locator 
WAO  
World Allergy Organization 
 
 


Confidential                                                Page 12 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
Background and Rationale  
 
Seasonal Allergic Rhinitis (SAR) is an IgE-mediated disease characterised by itching, 
sneezing, nasal discharge and congestion.  It is characterised by inflammation of the 
nasal mucosa and associated with early and late phase responses (EPR and LPR) upon 
exposure to common aeroallergens. It is believed to affect up to 20-25% of the 
population, with an estimated 80 million sufferers in Europe 1,2. A substantial increase 
in the prevalence of SAR has been reported in industrialised countries, including 
Western Europe 1.  SAR has been shown to impact quality of life and impair learning 
performance in school children 2. SAR and asthma often coexist and entail a higher 
overall disease morbidity3. 
 
The current management of SAR consists of pharmacotherapy such as antihistamines 
and corticosteroids 3. For those patients whose symptoms are not controlled by standard 
medical treatment, allergen specific immunotherapy (SIT) is a therapeutic alternative 4. 
In recent years, the sublingual route has been shown to be effective and induce long-
term remission 5. Although adequately powered head to head studies have not been 
performed, the effects of subcutaneous allergen-specific immunotherapy (SCIT) and 
sublingual allergen specific immunotherapy (SLIT) may be comparable, whereas SLIT 
is more convenient and has a better safety profile such that it may be administered in 
the patient’s home 6.  
  
Successful SCIT has been associated with increases in protective IgG4 and IgA2 
responses and induction/generation of antigen-specific regulatory T-cells, both 
peripherally and within local nasal mucosa 7.  Furthermore, reduced levels of effector 
cells (mast cells, eosinophils, CD4+ T-cells) are recruited to the nasal epithelium after 
grass pollen allergen challenge following immunotherapy 8. Following SLIT, 
peripheral immunologic changes comparable to those seen in SCIT, including the 
generation of regulatory T-cells, IgG4 and IgA induction 9 and decreased eosinophil 
recruitment10, have been reported. However, the local mechanisms involved, in 
particular the interactions between antigen presenting cells, T-cells and local structural 
cells within the oral mucosa are not well understood. The oral mucosa represents a 
privileged pro-tolerogenic site 11. We previously demonstrated that Foxp3-expressing 


Confidential                                                Page 13 
 
13IC0847- AIT Time Course Study                       Version 7.  01.10.2015       
 
cells increase in the sublingual epithelium of SLIT-treated but not placebo-treated 
individuals 9.  
 
Although several small studies have shown changes in immunologic responses 
following treatment with allergy immunotherapy tablets (AITs), a detailed time course 
of early and late cutaneous responses, alongside local and systemic T and B cell 
responses, has yet to be fully determined. We propose to investigate this in a placebo-
controlled, time-course study, using the EPR and LPR to nasal and cutaneous allergen 
provocation, plus changes in serum allergen-specific IgG4 and associated inhibitory 
activity for IgE-facilitated allergen binding (FAB) to B cells, as clinical and 
immunological surrogates of AIT treatment efficacy and allergen-specific tolerance. 
 
The current trial involves a 12-month, randomised, placebo-controlled study, with a 
further 12-24 months open treatment and/or assessment following unblinding at 12 
months. The allergen-induced early nasal response and early and late cutaneous 
responses, plus local and systemic allergic immunologic parameters, will be measured 
at baseline, before commencement of AIT. In order to assess a time-course of action, 
further measurements will take place at around 4, 8 and 12 weeks, at around 5 months 
(peak season), 6 months and 12 months (post season) of treatment. The results will 
build on our understanding of allergen tolerance and have the potential to identify novel 
targets for pharmacotherapy in the treatment of allergic disease. They may also inform 
novel approaches for allergen immunotherapy. A single-centre design will reduce 
variability and allow for detailed assessments including response to nasal allergen 
challenge with sampling of nasal fluid, nasal biopsy and nasal mucosal brushing, and 
measurement of early and late-phase skin responses. We will include 20 healthy, non-
atopic participants to serve as a control group for novel biomarkers in blood samples, 
nasal brushings and nasal biopsies. ALK Abello (Horsholm, Denmark) will supply 
Grazax® tablets and matched placebo tablets. 
5. Study Objectives 
 
5.1 Primary objective 
To understand the time course of clinical and immunological changes after grass pollen 
allergen immunotherapy tablets in the treatment of seasonal allergic rhinitis.  


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5.2 Secondary objectives  
 
1. To identify novel biomarkers for monitoring the clinical efficacy of specific 
allergen immunotherapy and the induction of immunological tolerance.  
2. To investigate the role of local and systemic basophil, T-, B- and dendritic-cell 
responses following AIT. 
3. To document the presence and severity of any local/systemic side effects of AIT 
treatment. 
 
4. To establish a molecular sensitisation profile in untreated allergic individuals 
with SAR and in AIT-treated patients. 
 
5. To follow the changes in molecular sensitisation profile during and following 
specific allergen immunotherapy. 
 
6. To assess the clinical and immunological response to AIT in relation to the 
underlying pre-treatment sensitisation profile of the patient. 
 
7. To study the time-course of induction of Phl p5-specific IgG4 in serum and nasal 
fluids. 
 
8.  To identify a time-course induction of inhibition of B-cell IgE-facilitated 
allergen binding (FAB) in serum and nasal fluid.   
 
5.3 Clinical hypotheses 
Grass pollen AIT treatment compared to placebo results in: 
1. Suppression of the EPR to grass pollen nasal provocation. 
2. Suppression of the EPR and LPR following intradermal grass pollen 
injection.   
3. Reduction in seasonal symptoms and medication use.  


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5.4 Mechanistic hypotheses 
Grass pollen AIT is associated with: 
1. Reduction of grass pollen induced local (nasal mucosa) and systemic (peripheral 
blood) basophil activation as measured by surface expression of CD63, CD203c 
and DAO on CRTH2+ basophils. 
 
2. Increased frequency of naturally occurring CD4CD25FOXP3+T regulatory 
cells (T regs), and/or inducible IL-10+, TGF-β+ and IL-35+ T regs. 
 
3. Induction of local (nasal) and systemic (serum) allergen-specific IgG and/or IgA 
antibodies that inhibit IgE-FAB to B cells, and basophil activation.  
 
 
In addition, that: 
4. The molecular allergen-binding profiles of specific IgE and IgG4 antibodies to 
Phleum pratense (Phl p, Timothy grass pollen) in both serum and nasal fluid are 
altered following AIT and in comparison with placebo treatment, reflecting 
qualitative as well as quantitative effects on humoral immunity.  
 
5. Patients sensitised to Phl p5 show greater clinical benefit effects of AIT 
compared to non Phl p5 sensitised AIT-treated patients.  
 
5.5 Exploratory hypotheses 
 
1. Sequencing of T cell receptor junctional regions will demonstrate the 
deletion/loss of grass pollen-specific T cell clones present at baseline (through 
clonal deletion), and the appearance of distinct grass pollen specific T cell 
clones, during the course of AIT.  
 
2. AIT treatment is associated with changes in local and systemic B cell repertoire 
during and after withdrawal of AIT, as measured by sequencing of expressed B 
cell receptors.  


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3. AIT is associated with inhibition of IL-13 production from innate lymphoid 
cells (ILCs/’neuocytes’). 
 
4. AIT is associated with the induction of regulatory B cells (IL-10+CD19+, 
CD19+CD24hiCD38hi) capable of regulating Th2 cell responses.  
 
5. AIT is associated with induction of IL-27-positive myeloid-derived dendritic 
cells (mDCs) that express PDL-1, induce immune deviation in favour of Th1 
and Tr1 responses that inhibit allergen-driven Th2 responses. 
 
6. Study design 
 
This is a randomized, double-blind, single-centre, placebo-controlled, time course 
study comparing grass pollen AIT with placebo (Figure 1). 
The study will be conducted over 44 months. We expect to screen 70 atopic patients in 
order to enroll up to 50 suitable atopic participants to ensure randomisation of at least 
44 atopic participants following their baseline visit. Additionally, we shall recruit 20 
healthy, non-atopic volunteers. Individuals with severe grass pollen hay fever, with or 
without associated seasonal asthma, will be recruited and screened after the pollen 
season from December 2013 to April 2014. Atopic Participants will undergo baseline 
assessments in January to April 2014. Screening assessments will be completed before 
eligible participants are randomized to active or placebo treatment. Atopic participants 
will then begin AIT treatment in January-April 2014 and continue treatment for 12 
months. All atopic participants will be provided with anti-allergic rescue medications 
(antihistamine tablets, topical intranasal corticosteroids, and eye-drops) throughout the 
pollen season. Clinical surrogate endpoint assessments, plus blood, nasal fluid and nasal 
brushing sampling, will be performed at baseline , at around 4, 8 and 12 weeks after 
starting the AIT, and during the peak pollen season, at 6 monthsand at 12 months  of 
treatment. Nasal mucosal biopsies will be taken at baseline, around 5 months, and 12 
months of treatment. After 12 months of treatment, unblinding will take place. Those 
atopic participants receiving active AIT treatment will continue therapy for another 12 
months followed by a withdrawal phase of 12 months. At 24 and 36 months (visit 8 and 


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9) all procedures and laboratory markers that were performed at 12 months (visit 7) 
(Fig.1) will be repeated for atopic participants on active treatment.  Those atopic 
participants on placebo will be offered 24 months of active treatment after unblinding.  
The healthy, non-atopic participants will undergo screening and one visit only, at 12 
months, to coincide with the 12 month visit for the atopic participants. This visit will 
include clinical surrogate endpoint assessments, plus blood, nasal fluid, nasal brushing 
sampling, and nasal biopsies. The assessments will serve as a control for the results of 
the atopic participants at 12 months. 
Figure 1: Study plan, atopic participants 
 
 
 
6.1 Primary endpoint 
The area under the curve (AUC) of the early phase response (total nasal symptom score, 
TNSS, 0-60 minutes) following grass pollen nasal allergen challenge in active versus 
placebo-treated atopic participants at 12 months. 


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6.2.1 Secondary endpoints 
 
1. Cross-sectional area in cm2 of EPR and LPR skin responses to intradermal 
grass pollen allergen injection at 12 months in active versus placebo treated 
participants. 
 
2. Serum and nasal fluid grass pollen specific IgG4 level and inhibition of B cell 
IgE-facilitated allergen binding in active versus placebo-treated atopic patients 
versus non-atopic controls at 12 months. 
  
3. Allergen-induced peripheral blood basophil activation in active versus 
placebo-treated atopic participants versus non-atopic controls at 12 months.  
 
6.2.2 Exploratory endpoints 
 
1. Mean combined symptom + medication scores over the course of the 2014 
grass pollen season in active versus placebo-treated participants. 
 
2. Proportion of allergen-specific phenotypic Treg cells in peripheral blood as 
measured by flow cytometry in active vs placebo-treated atopic participants 
versus non-atopic controls at 12 months. 
 
Other exploratory endpoints will include, in active versus placebo-treated atopic 
participants versus non-atopic controls at 12 months: the frequency of putative Breg 
cells in peripheral blood; the frequency of T follicular helper cells; the frequency of 
IL-27+ mDCs; the molecular binding profiles of IgE and IgG4 antibodies; T and B 
cell receptor sequencing; frequency of putative innate lymphoid cells (ILCs).  
 
6.3 Selection and Withdrawal of Participants 
Subjects meeting all of the following inclusion criteria and none of the exclusion criteria 
will be considered for admission into the study. 
6.3.1 Recruitment 


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Recruitment of the participants will take place partly out of our existing database of 
eligible atopic and non-atopic patients. If required grass pollen allergic patients and 
non-allergic controls will be recruited through posters, leaflets and flyers placed within 
the Royal Brompton Hospital, on campus of the Imperial College, London, at the 
London tube station, and via local media (for example local newspaper advertisement). 
Further we plan to launch a website for further information.   
6.3.2 Inclusion Criteria 
 Atopic participants:  
 
1. Adults age 18 to 65 years. 
2. A clinical history of grass pollen-induced allergic rhinoconjunctivitis for at 
least 2 years with peak symptoms in mid-May to mid-July. 
3. A clinical history of moderate to severe rhinoconjunctivitis symptoms with 
or without mild seasonal asthma interfering with usual daily activities or 
with sleep. 
4. A clinical history of rhinoconjunctivitis with or without mild seasonal 
asthma that remains troublesome despite treatment with either 
antihistamines or nasal corticosteroids during the grass pollen season. 
5. Positive skin prick test response, defined as wheal diameter ≥ 3 mm, to 
timothy grass pollen. 
6. Positive specific IgE, defined as IgE immunoCAP ≥ 0.7 ISU, against 
timothy grass pollen. 
7. For women of childbearing age, a negative urine pregnancy test at the time 
of screening and willingness to use an effective form of contraception for 
the duration of involvement in the study. 
8. The ability to give informed consent and comply with study procedures. 
9. A positive grass pollen nasal allergen challenge test at screening as defined 
by a total nasal symptom score (TNSS) of at least 7/12 after 5 minutes with 
an allergen dose of 5,000 BU/ml (Appendix 6) (see also section 6.5.7). 
 
Healthy, non-atopic participants: 


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1. Adults age 18 to 65 years. 
2. Negative skin prick test response to timothy grass pollen and panel of 
aeroallergens. 
3. Negative specific IgE, defined as IgE immunoCAP < 0.35 ISU, against 
timothy grass pollen. 
4. For women of childbearing age, a negative urine pregnancy test at the time 
of screening and willingness to use an effective form of contraception for 
the duration of involvement in the study. 
5. The ability to give informed consent and comply with study procedures. 
 
6.3.3 Exclusion Criteria  
 
Atopic participants: 
 
1. Previous grass pollen allergen immunotherapy. 
2. Prebronchodilator FEV1 < 70% of predicted value at screening (out of grass-
pollen season). 
3. A clinical history of symptomatic allergic rhinitis and/or asthma caused by 
an allergen to which the participant is regularly and perennially exposed 
(e.g. cat dander). 
4. Perennial asthma requiring regular inhaled corticosteroids. 
5. Seasonal symptoms outside the grass-pollen season (e.g. hay fever during 
March-April suggestive of birch pollen allergy). 
6. History of emergency visit or hospital admission for asthma in the previous 
12 months. 
7. History of chronic obstructive pulmonary disease. 
8. History of significant recurrent acute sinusitis, defined as 2 episodes per 
year for the last 2 years, all of which required antibiotic treatment. 


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9. History of chronic sinusitis, defined as sinus symptoms lasting greater than 
12 weeks that includes 2 or more major factors or 1 major factor and 2 minor 
factors. Major factors are defined as facial pain or pressure, nasal 
obstruction or blockage, nasal discharge or purulence or discolored 
postnasal discharge, purulence in nasal cavity, or impaired or loss of smell.  
Minor factors are defined as headache, fever, halitosis, fatigue, dental pain, 
cough, ear pain, pressure, or fullness. 
10. At screening visit, current symptoms of, or treatment for, upper respiratory 
tract infection, acute sinusitis, acute otitis media, or other relevant infectious 
process; serous otitis media is not an exclusion criterion.  Participants may 
be re-evaluated for eligibility after symptoms resolve. 
11. Current smokers or a history of ≥ 5 pack years. 
12. History of life-threatening anaphylaxis or angioedema. 
13. Ongoing systemic immunosuppressive treatment.  
14. The use of any investigational drug within 30 days of the screening visit. 
15. The presence of any medical condition that the investigator deems 
incompatible with participation in the study. 
16. History of fish allergy with positive skin test and/or positive specific IgE 
test to vertebrate/finned fish (due to potential fish allergen exposure in AIT). 
17. Contraindications taking Grazax. 
 
 
 
Non-atopic participants: 
1. Previous grass pollen allergen immunotherapy. 
2. Prebronchodilator FEV1 < 70% of predicted value at screening (out of 
grass-pollen season). 


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3. A clinical history of symptomatic allergic rhinitis and/or asthma caused by 
an allergen to which the participant is regularly and perennially exposed 
(e.g. cat dander). 
4. Perennial asthma requiring regular inhaled corticosteroids. 
5. Seasonal symptoms outside or during the grass-pollen season. 
6. History of emergency visit or hospital admission for asthma in the previous 
12 months. 
7. History of chronic obstructive pulmonary disease. 
8. History of significant recurrent acute sinusitis, defined as 2 episodes per 
year for the last 2 years, all of which required antibiotic treatment. 
9. History of chronic sinusitis, defined as sinus symptoms lasting greater than 
12 weeks that includes 2 or more major factors or 1 major factor and 2 minor 
factors. Major factors are defined as facial pain or pressure, nasal 
obstruction or blockage, nasal discharge or purulence or discolored 
postnasal discharge, purulence in nasal cavity, or impaired or loss of smell.  
Minor factors are defined as headache, fever, halitosis, fatigue, dental pain, 
cough, ear pain, pressure, or fullness. 
10. At screening visit, current symptoms of, or treatment for, upper respiratory 
tract infection, acute sinusitis, acute otitis media, or other relevant infectious 
process; serous otitis media is not an exclusion criterion.  Participants may 
be re-evaluated for eligibility after symptoms resolve. 
11. Current smokers or a history of ≥ 5 pack years. 
12. History of life-threatening anaphylaxis or angioedema. 
13. Ongoing systemic immunosuppressive treatment.  
14. The use of any investigational drug within 30 days of the screening visit. 
15. The presence of any medical condition that the investigator deems 
incompatible with participation in the study. 
 
6.3.4 Withdrawal Criteria 


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Participants will be advised in the Informed Consent Form that they have the right to 
withdraw from the study at any time without prejudice. They may also be withdrawn 
from the study at the investigator’s discretion at any time. Reasonable effort should be 
made to contact any participant lost to follow-up during the course of the study in order 
to complete assessments. Participants who prematurely leave the study will be asked to 
undergo an early termination visit. The data and blood, nasal fluids, nasal brushings 
and nasal biopsies collected before the withdrawal will be further processed and 
included in analysis. Participants who prematurely leave the study will not be replaced. 
6.3.5 Urgent safety measure 
The Primary Investigator (PI) will have the authority to deviate from the protocol if 
doing so relates to the immediate safety of a participant, where continuing to follow 
protocol would put that participant at risk. This will be classed as an urgent safety 
measure and must be reported to the Joint research compliance office (JRCO), 
Medicines and Healthcare products regulatory agency (MHRA) and Research Ethics 
Committee (REC) within three calendar days of the occurrence. This may be reported 
verbally in the first instance but must be supported by a written report as soon as 
information is available.  
6.3.6 Stopping Rules 
If any of the criteria listed below are met at any time during the study, study enrolment 
and study therapy will be suspended pending expedited review of all pertinent data by 
the JRCO, MHRA and REC. 
 
 Death in any participant, where death is attributed in any way to study 
therapy or intervention. 
 Grade 4 anaphylaxis attributed in any way to study therapy or intervention. 
Grade 4 anaphylaxis per World Allergy Organization (WAO) criteria35 is 
defined as one or more of the following: 
 Respiratory failure. 
 Life threatening hypotension or cardiovascular collapse. 
 Loss of consciousness resulting from cardiovascular collapse. 
 


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6.3.7 Premature discontinuation of study therapy  
Study therapy is defined as initiation of any of the study medications described in 
Section 6.4 Study therapies will be discontinued for any of the following reasons: 
 
 Two or more occurrences of grade 3 or above systemic allergic reactions after 
administration of study therapy. Grade 3, per World Allergy Organization 
(WAO) criteria, is defined as severe asthma symptoms (over 40% drop in PEF 
or FEV1) not responding easily to an inhaled bronchodilator, and/or laryngeal 
oedema with stridor.35 
 Any adverse event (AE) and adverse reaction (AR) that, in the judgment of the 
principal investigator or the medical monitor, presents an unacceptable 
consequence or risk to the participant. 
 An illness or infection that is not associated with the condition under study and 
that requires treatment not consistent with protocol requirements; or, if a 
participant develops an intercurrent illness that in the judgment of the PI in any 
way justifies discontinuation. 
 An inability or unwillingness to comply with the study protocol, with protocol 
deviations sufficient to jeopardize the participant’s well-being or the integrity 
of the study. 
 Pregnancy occurring during study participation. 
 
Participants who prematurely discontinue study treatment will be invited to complete 
the planned study assessments throughout the duration of the trial. However, if they 
decline they will undergo a follow-up visit 24 hours after discontinuation. One week 
after the follow-up visit, they will be contacted by telephone and discharged from the 
study. 
6.3.8 Randomisation 
After the screening phase eligible participants will be randomised, double-blind, to one 
of the two treatment arms in a 1:1 ratio and assigned a randomisation ID number. 
Additionally, the participant will be assigned a specific investigational medical product 
(IMP) kit, from those available at the site that corresponds to the treatment assignment. 
Each participant will be assigned a kit ID from the site’s inventory whose treatment 


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matches the group to which the participant was randomized. The treatment assignment 
notification will be sent to authorized study personnel and the investigational pharmacy 
via email and/or fax. (The healthy, non-atopic control group will not be randomized to 
a treatment arm but investigators analysing biological samples from these participants 
will be blinded as to their origin.) 
6.3.9 Blinding 
Blinding will be maintained for all study participants throughout the first 12 months of 
the study. Data analysis for primary and secondary endpoints will be performed after 1 
year (efficacy endpoints). 
6.3.10 Unblinding 
Unblinding before the first 12 months of the study is completed will occur only if a 
participant’s well-being is threatened and the investigator believes unblinding is 
necessary to protect the participant. In this event, 24-hour access for emergency 
unblinding will be provided. Unblinding will be provided by on-site Pharmacy 24 hours 
a day/7 days a week after authorisation by the Principal Investigator. A full account of 
the event will be recorded, including the date and time of the emergency, the reason for 
the decision to unblind, and the names of the medical monitor and others who were 
notified of the emergency. During site visits, the site monitor must verify that the 
protocol chair was notified and that a written account was completed. The reasons for 
unblinding of a participant’s treatment will be included in the final study report (see 
also section 6.7.6.5).  
 
6.4 Study medications  
 
6.4.1 Allergen Immunotherapy Tablet (AIT) 
 
Grazax® is a fast-dissolving tablet that is registered throughout Europe for sublingual 
use in patients aged 5–65 years (18–65 years in UK)14, 15, 16.The tablet is administered 
daily for a minimum of 2 months before and during the grass pollen season. In a double-
blind trial of Grazax® that included a withdrawal phase, efficacy was maintained for 
2–3 years with continuous treatment 15, 16 and at 1 year following withdrawal 17, 18. AIT 


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has been shown to be safer than the injection route. It is recommended that the first 
dose of Grazax® is administered in the clinic and that subsequent doses are taken 
independently by the patient. Local side effects of itching and swelling in the mouth 
are common, occurring in 50% to 60% of individuals within minutes and resolving 
within 1 hour with a median half-life of approximately 10 days with daily 
administration14, 15, 16. These responses are, in general, well tolerated and require no 
treatment. In a recent large trial involving over 600 patients, the withdrawal rate was 
5% as compared to 3% in placebo-treated patients14, 15. More severe local and systemic 
reactions have been reported, but they are excessively rare and no fatalities have 
occurred19. 
 
6.4.2 Formulation and Packaging 
GRAZAX® and GRAZAX® PLACEBO 
SQ Standardized Grass Allergy Immunotherapy (Grazax®, ALK- Abello Horsholm, 
Denmark) is formulated as a freeze-dried oral lyophilisate/orally disintegrating tablet 
for oromucosal use. The active pharmaceutical ingredient is a standardized allergen 
extract derived from extraction and purification of grass pollen from timothy grass 
(Phleum pratense). The biological activity of the allergen is expressed in standardized 
quality units (SQ). The recommended dosage is one oral lyophilisate containing 75,000 
SQ Tablet units (SQ-T) or approximately 2800 Bioequivalent allergy units (BAU), a 
measure of Phleum pratense SQ total biological potency defined by the Food and Drug 
Administration (FDA). This dose is equivalent to 15 µg of Phleum pratense major 
allergen. The non-active ingredients consist of fish gelatin, mannitol, sodium hydroxide 
and water. Grazax® placebo is a tablet whose composition is identical to the active 
Grazax® tablet with the only exception being exclusion of the active pharmaceutical 
ingredient, Phleum pratense SQ. Grazax® and Grazax® placebo tablets will be 
supplied in blister packs by ALK-Abello. For the participants the study medication will 
be free of charge.  
6.4.3 Administration 
The first Grazax® or Grazax® placebo will be administered under the supervision of 
the trial physician and the participant observed for one hour thereafter before discharge 
from the clinic. This will take place on Visit 1, which will be conducted 1 day after the 


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second baseline visit (V0b). Thereafter, home sublingual administration of Grazax® or 
Grazax® placebo tablets will occur daily during the 12 months of the trial. In those 
participants who received active treatment a continuation of daily treatment will take 
place for another 12 months. Those participants who received placebo will be offered 
24 months of active treatment after completion of the first 12 months and after 
unblinding. Tablets should be removed from the blister packs with dry fingers and 
should be taken immediately after opening the blister. Each tablet should be placed 
underneath the tongue until fully dissolved (1–2 minutes). Swallowing should be 
avoided for about 1 minute. Food and beverage should not be consumed for the 
following 5 minutes. For additional information on the preparation and administration 
of Grazax®, please refer to the following uniform resource locator (URL): 
 
http://www.grazax.com/SiteCollectionDocuments/Grazax.com/About%20Grazax/Gra 
zax_SmPC_MRP_VAR13_15OCT2010.pdf 
6.4.4 Special Warnings and Precautions for Use  
In case of oral surgery, including dental extraction, treatment with Grazax® should be 
stopped for 7 days to allow healing of the oral cavity. Mild to moderate local allergic 
reactions such as itching or swelling of the mouth may occur during treatment. If 
significant local adverse reactions occur treatment with antihistamines should be 
considered. Rare cases of severe systemic allergic reactions have been reported with 
Grazax® use. Therefore medical supervision at the start of treatment is an important 
precaution. All participants will be provided with an emergency telephone contact 
number for study personnel with 24 hour availability. The onset of systemic symptoms 
may include flushing, itching in the extremities and other areas of the body, general 
discomfort and agitation. In cases of severe systemic reactions including angioedema, 
difficulty swallowing or breathing, throat fullness or hypotension, Grazax® should be 
discontinued and a physician contacted immediately. Severe systemic reactions may be 
treated with adrenaline. Treatment with Grazax® should remain withheld until 
evaluation by a study physician. If participants with concomitant asthma experience 
symptoms and signs indicating asthma deterioration, treatment should be discontinued 
and a study physician consulted immediately. Grazax® contains fish-derived gelatin. 
The available data have not indicated an increased risk of allergic reactions in severe 


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fish allergic patients. However as a precaution, patients will be asked during screening 
whether they are allergic to vertebrate/finned fish. If the history is positive, a skin test 
and/or specific IgE test to vertebrate/finned fish will be requested and if positive the 
subject will not be included in the study. 
 
6.4.5 Recommended Storage Conditions 
Grazax® tablets are sensitive to moisture and blister packs should be stored unopened 
until use. The current formulation has been shown to be stable when stored at room 
temperature in unopened blisters. 
6.4.6 Drug accountability  
The investigator is required to maintain adequate records of the disposition of the 
investigational product, including the date and quantity of drug that was received, the 
participants to whom drug was dispensed (participant by participant accounting), and 
an account of any drug accidentally or deliberately destroyed. Records for receipt, 
storage, use, and disposition of the study drug will be maintained by the study site. A 
drug-dispensing log will be kept current for each participant and will contain the 
identification of each participant and the date and quantity of drug dispensed. All 
records regarding disposition of the investigational product will be available for 
inspection by the clinical trial monitor. 
6.4.7 Assessment of compliance with study medication  
Participants will be asked to return used AIT (Grazax®) and AIT placebo blister packs 
and unused tablets at each study visit. Those participants with less than 75% compliance 
with study medication will be counselled. Use of rescue medications (section 6.4.8.1) 
will be assessed by medication score (Appendix 5). The participant will fill out a daily 
Medication Diary in which he/she will record the Tablet intake (Appendix 9). 
 
 
 
6.4.8 Concomitant medication 


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6.4.8.1 Permitted Medications 
 
The following rescue medications will be provided. These medications are considered 
standard of care and will be provided approximately 2 weeks before the start of the 
pollen season (May 2014) and throughout the pollen season: 
 
 Antihistamine (e.g. desloratidine). 
 Nasal corticosteroid (e.g. fluticasone propionate aqueous nasal spray). 
 Ophthalmic antihistamine (e.g. olopatadine eye drops). 
 
Use of the following rescue medications will be provided by the study only after 
participants are evaluated by the investigator. Evaluations may occur by phone 
contact or clinic visit. 
 
 Oral corticosteroid (e.g. prednisolone). 
 Short-acting beta-agonists (e.g. salbuterol). 
 Inhaled corticosteroids (e.g. fluticasone). 
 Combination long-acting beta-agonists or steroids (e.g. salmeterol/fluticasone). 
 
Refer to Appendix 7 for required medication washout periods prior to screening, nasal 
challenges and biopsies (V-1, V0b, V5, and V7). 
 
 6.4.8.2. Prohibited Medications 
Use of the following medications is prohibited during study participation: 
 β-blockers. 
 Calcium channel blockers. 
 Tricyclic Antidepressants.  
 Monoamine oxidase inhibitors. 
 Anti-IgE monoclonal antibody treatment. 
 
 
 


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6.5 Study Procedures 
 
6.5.1 General assessments 
 
 Informed consent 
 Medical history 
 Allergy history 
 Comprehensive physical examination (includes height and weight) 
 Vital signs: blood pressure, temperature, pulse, and respiratory rate 
 Adverse events 
6.5.2 Clinical procedures 
 
 Nasal challenge 
 Nasal fluid collection 
 Nasal brushing 
 Nasal biopsy 
 Peak nasal inspiratory flow (PNIF) 
 Pulmonary function testing (spirometry) 
 Peak flow testing (PeakFlow) 
 Intradermal skin test: Timothy grass – Phleum pratense 
 Skin prick test: grass pollen (timothy grass), silver birch (Betula verrucosa), 
mugwort (Artemisia vulgaris), house dust mite (Dermatophagoides 
pteronyssinus), cat hair (Felis domesticus), dog hair (Canis familiaris), horse 
hair (Equus caballus), common moulds: Aspergillus fumigatus, Cladosporium 
herbarum, Alternaria alternata, plus negative (diluent)  and positive 
(Histamine dihydrochloride) controls. 
 
6.5.3 Local laboratory assessments   
 Urine pregnancy test 
 Haematology: Complete blood count with differential 
 Total IgE 


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 Timothy grass specific IgE 
 
 
6.5.4 Questionnaires  
 Global evaluation scores (GE) (Appendix 1, 2, 4) 
 miniRhinoconjunctivitis Quality of Life Questionnaire (mRQLQ) (Appendix 
3) 
 Symptom medication score (SMS) (Appendix 5) 
 Total nasal symptom score (TNSS) (Appendix 6) 
 Medication Diary cards for Tablet count (Appendix 9) 
6.5.5 Pollen count 
 
Pollen counts will be quantitated retrospectively for the purposes of analysis. Pollen 
season is defined as follows: 
 
 
Start of season 
First 3 consecutive days at pollen count >10 grains/cm3 
End of season 
First 3 consecutive days at pollen count < 10 grains/cm3 
Start of peak season 
First 3 consecutive days at pollen count >30 grains/cm3 
End of peak season 
First 3 consecutive days at pollen count < 30 grains/cm3 
6.5.6 Nasal wash (lavage) 
Boiled water allowed to cool to lukewarm is added to a purpose made squeezable plastic 
240 ml bottle (Sinus Rinse, NeilMed, USA). A single sachet of pharmaceutical grade 
sodium chloride and sodium bicarbonate is then added to the water and mixed to form 
a solution. After testing again to ensure a suitable temperature, the nozzle of the bottle 
is placed into the volunteer’s nose to form a seal, and, with the volunteer leaning 
forward over a sink, the bottle is then squeezed to allow a jet of solution to pass into 
one nostril and out of the other. The process is then carried out into the other nostril and 
repeated several times until all of the solution has been used. SinuRinse® is widely 
available and no adverse effects are to be expected. 
6.5.7 Nasal allergen challenge 


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Nasal allergen challenge is performed with Aquagen SQ (ALK 225) Timothy grass 
pollen, Phleum pratense, ALK-Abelló freeze dried extract (Cat. no. 1001862, ALK-
Abello, Denmark). Fresh extracts will be reconstituted in albumin-based diluent (ALK-
Abello) on a weekly basis at a concentration of 100,000 SQ-U / 30,000 BU per ml. On 
a daily basis, a fresh dilution of 5,000 BU/ml in normal saline will be prepared from 
the stock solution. This solution will be added to a nasal applicator device (Bidose, 
Aptar Pharma/Pfeiffer, Germany), designed to spray a standard volume (100µL) with 
each metered dose pump application. A single spray will be applied to each nostril, 
giving a total allergen dose of 1,000 BU. Following the nasal challenge the EPR will 
be measured using the TNSS (Appendix 6). A score ≥ 7/12 points after 5 minutes 
during screening will be interpreted as a positive response and patients will be included 
in the study. If during screening the proportion of patients not being included in the 
study because of this inclusion criterion exceeds 20%  the cut-off for inclusion will be 
lowered to ≥ 5 points. 
6.5.8 Sampling of nasal secretions 
Open cell flexible polyurethane foam (Zuschnitt Schaumstoff RG27 grau, Gummi-
Welz GmbH & Co, Germany, ISO 5999, 1982 sponge) pre-cut into rectangular blocks 
20 x 15 x 5 mm will be used to collect secretions. Sponges will be autoclaved for 20 
minutes at 121°C in batches of 20. A fresh batch will be used for each volunteer. The 
patient will be sitting comfortably upright with the head facing forwards. A sponge will 
be placed into each nasal cavity, posterior to the muco-cutaneous junction, under direct 
visualization using blunt-ended forceps and a nasal speculum. Sponges will be left in 
place for up to 5 minutes before removal using the same instruments. Following 
removal, each sponge will then be placed above a microfilter within a centrifuge tube 
and kept on ice. Tubes will then be centrifuged for 10 minutes at 4,500 rcf at 4°C. The 
volume of freed fluid will be calculated by weight using an electronic balance. The fluid 
will then be aliquoted into eppendorf tubes and frozen at -80C. 
6.5.9 Intradermal skin tests 
Intradermal skin tests are performed by injecting 0.02ml of allergen diluted in albumin-
based diluent into the skin of the outer surface of the forearms. 1 BU (0.02ml of 50 


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BU/ml) will be injected into the right and the left forearm. A negative control injection 
of 0.02ml of normal saline will be injected on the outer surface of the wrist on the right.  
 
Skin responses are defined as follows: 
 
Response 
Skin Allergen Challenge  
Early Phase Response (EPR) 
Measured 
15 
Minutes 
after 
Allergen 
Challenge ± 5 min 
Late Phase Response (LPR) 
Measured 8 Hours after Allergen Challenge  
± 1 hour 
 
In pollen sensitized individuals, allergen skin injection provokes a local wheal and flare 
skin response, peaking at about 15 minutes. This is recorded in comparison with the 
negative control (saline) injection. In sensitized, allergic individuals, following 
resolution of the early wheal and flare response, there is a gradual onset mild swelling 
and redness over a larger area around the site of the injection – the LPR. This response 
is measured at 8 hours after injection using a pencil-friction technique to map out the 
extent of skin swelling. Volunteers may experience mild to moderate itchiness or local 
discomfort at the sites of intradermal injections and skin pricks with allergen. The 
symptoms are not bothersome and treatment with oral antihistamines is always 
available and is effective although almost never required. Systemic reactions including 
anaphylaxis after skin tests with standardized aeroallergen extracts are a theoretical risk 
but exceedingly rare. However, a physician is always present and drugs and equipment 
for treatment of anaphylactic reactions available. 
 
6.5.10 Nasal epithelial brushings 
Volunteers will undergo nasal brushings (either with Cyto-Soft Cytology Brushes or 
Rhinoprobes) on each side of the nose. This is a simple procedure and takes less than 5 
minutes.  
 
 
Materials 
 
 Disposable non-latex gloves 
 Tissues 


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 5% lidocaine hydrochloride spray 
 Cytology brush cutters, cleaned thoroughly with DNAZapTM solution before 
the procedure   
 DNAZapTM (Applied Biosystems, Cat# AM9890)  
 Sterile Cytosoft cytology brushes (Cyto-Soft Cytology Brush CYB-1. Cardinal 
Health #S7766-1A)  
 Rhinoprobe curettes (Arlington Scientific, USA, curette 100 SY-96-0905) 
 Nasal Epithelial Cell Collection Tubes with Media (RLT buffer +2-
Mercaptoethanol (β-ME) Qiagen, West Sussex, UK ) 
 
Procedure 
 
1. Explain procedure to patient. 
2. Sit patient comfortably in a chair facing forward, with head tilted slightly back. 
3. Apply local anaesthetic (5% lidocaine hydrochloride spray, 2-3 sprays per 
nostril) to both nostrils. Allow 2 minutes for onset of anaesthesia. (Procedure 
may be performed with or without local anaesthetic, according to participant 
preference, based on pilot studies currently in progress). 
4. Give the participant a tissue for eye tearing or nose running.  
5. If using Cytosoft cytology brushes: insert the cytology brush through the nasal 
vestibule medially along the floor of the nasal cavity and the septum, to the 
depth of the inferior turbinate. 
6. Roll and move the brush laterally, under the inferior turbinate, to the 
anterolateral inferior turbinate.  Rotate while moving the brush in and out, 
applying some pressure to the walls of the turbinate. Brush for 4 seconds. 
7. Roll the brush away from the turbinate and remove from the nose. 
8. Place the brush into a culture tube containing collection medium. 
9. Gently agitate the brush in the culture medium for 4 seconds. 
10. Cut the brush off at its handle so that it fits well in the tube. 
11. Repeat if indicated. 
12. If using Rhinoprobes: gently scrape the inferior turbinate twice, from posterior 
to anterior, remove Rhinoprobe from nose, insert cupped end into culture 
medium and agitate gently for 4 seconds.  
13. Cap the tube and freeze at -80°C. 


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14. Check patient and offer further tissues if required. 
6.5.11 Nasal biopsy 
Biopsy of the mucosa of the inferior turbinates of the nose may be safely performed 
under local anaesthetic and sterile conditions with only minimal discomfort and 
inconvenience to the volunteer. Biopsy allows the capture of mucosal tissue for 
analysis. Nasal biopsy may be associated with slight bleeding and mild discomfort.  
Materials 
 
 Sterile dressing pack. 
 Cotton wool balls. 
 1x Merocel (1cm x 1cm x 0.5 cm) (neurosurgical packing). 
 1x Lidocaine hydrochloride 5% + Phenylepherine hydrochloride 0.5% topical 
solution 2.5mls with nasal spray applicator.  
 1ml 1:1000 adrenaline. 
 2.5ml individual ampule of 10% cocaine hydrochloride.   
 1 x Thuddicum nasal speculum. 
 1 x Crock forceps. 
 2-3 x Fokken’s biopsy forceps.  
 3-4 x Silver Nitrate sticks. 
 2 x 1ml syringe and needle. 
 Direct (forehead-mounted) or indirect (shoulder lamp and forehead mirror) light 
source. 
 Appropriate reagents for immediate storage/fixing of biopsy tissue. 
Procedure 
 
1. Examiner and patient to be sitting upright, leaning slightly forward, immediately 
opposite each other such that the examiner can easily touch the back of the 
patient’s head. Examiner visualises nasal mucosa bilaterally, with attention to 
inferior turbinates, using Thuddicum’s speculum under direct/indirect light. 
2. Lidocaine + Phenylephrine spray applied; 3-4 sprays to each side of the nose. 


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3. Two minutes later, the examiner inspects decongested nasal mucosa and chooses 
an appropriate side for biopsy.  
4. 2-3 small pieces of cotton wool (approximately 2 x 2 cm) are soaked in 2.5ml 
10% cocaine hydrochloride in a sterile tray. Soaked pieces are individually 
placed into the appropriate side of the nose, alongside the inferior turbinate, 
using sterile Crock forceps. 
5. Ten minutes later, the patient is asked to check whether they have an 
abnormal/numb sensation in their upper front teeth. If so, cotton wool can be 
removed; if not, check position of the cotton wool, adjust if required, and then 
leave it in place for a further 5 minutes. 
6. Once cotton wool is removed, check that the turbinate has been anaesthetized by 
prodding it lightly with the tip of Crock forceps – the patient may feel 
pressure/movement but should not feel pain.  
7. Under direct vision, using Fokken’s biopsy forceps a 2 mm bite of the exposed 
edge of the inferior turbinate, approximately 0.5cm back from the anterior edge, 
will be taken. The biopsy forceps will then be passed to an assistant who will 
dislodge the biopsy tissue into an appropriate sterile medium. 
8. The operator immediately takes a second set of Fokken’s forceps and, as before, 
takes another biopsy of the inferior turbinate adjacent to the previous biopsy, on 
the same side of the nose. The sample within the forceps is then passed to the 
assistant as before. 
9. A final third biopsy adjacent to the previous biopsies will then be taken and 
processed. 
10. 0.5 ml of adrenaline is then added, drop-wise, to a loose cotton wool plug (or a 
Merocel neurosurgical sponge) and placed into the nose adjacent to the biopsy 
sites. 
11. After at least 10 minutes the cotton wool/Merocel sponge is removed with Crock 
forceps. 


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12. Under direct vision, the biopsy sites are cauterised using silver nitrate sticks at 
sites of any visible bleeding. 
13. The patient is given 500-1,000mg paracetamol PO and is observed for at least 
15 minutes. 
14. If well, the patient may then be discharged with the following advice: 
a. Avoid nose blowing and picking for the next 5 days. 
b. Avoid straining, heavy lifting and contact sports for the next 5 days. 
c. Avoid alcohol for the next 24 hours. 
d. If bleeding occurs, pinch the cartilaginous area of the nose firmly for 10 
minutes. If bleeding continues, repeat up to 3 times. If bleeding persists 
the patient should attend their local Accident & Emergency department. 
e. Take paracetamol 500-1000mg 6-8 hourly for the next 24 hours; 
continue after this if pain or discomfort persists. 
Patient will be given a 24 hour contact telephone number for advice. 
6.5.12 Venepuncture 
Blood will be drawn from an antecubital vein using a 19 gauge butterfly (or similar) 
sterile cannula. Participants may experience some minor discomfort on skin puncture 
during insertion of the cannula. Blood samples will be taken with the volunteer sitting 
down. If volunteers experience faintness or frank vaso-vagal syncope due to 
venepuncture, they will be assisted to lie flat and blood pressure and vital signs will be 
monitored until symptoms resolve. Oxygen and intravenous fluids will be available in 
the exceptionally unlikely event that they are required. In 6 months we will not take 
more than 500 ml of blood as suggested by the National blood service32.  
6.5.13 Clinical responses 
Participants will be asked to score their previous and current hay fever symptoms at 
different time points before and during treatment (Appendix 1 – 4). During the season 
symptoms and rescue medication scores will be assessed weekly (Appendix 3, 5). 


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Questionnaires will be either all filled out on paper (Appendix 1-5) or all via a 
computerized version of the questionnaires. In case of the latter, SurveyAnalytics 
(Survey Analytics LLC, Seattle, Washington) will be the web-based service used. This 
is a service for conducting surveys and facilitated data collection. Surveys created are 
owned solely by the administrator of that survey. The application allows password 
protection and uses secure encryption. Email addresses uploaded to the system for the 
purpose of sending survey invitations and email communication and the collected data 
are owned solely by the survey administrator and not divulged to a third party. 
6.5.14 Visits 
 
Atopic Participants: 
 
Scheduled Visits (screening, randomisation and blinded treatment phase): 
 
Where possible, all study visits should occur within the time limits specified below: 
 
Screening Visit –Visit- -1, from December 2013 to April 2014. 
Visit – 0a, after screening and before Visit 0b. 
Visit – 0b, from January 2014 to April 2014. 
Visit – 1, after Visit – 0b. 
Visit – 2, from February to  May 2014. 
Visit – 3, from March to May2014. 
Visit – 4, from April  to June2014. 
Visit – 5, from May  to August  2014. 
Visit – 6, from July  to October, 2014 
Visit – 7, from January to April 2015.  
Unblinded treatment phase 
Visit- 8,  January-March 2016 
Visit- 9,  January-March 2017 
 
Unscheduled visits: 


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Unscheduled visits may be performed in the event that participants experience a disease 
exacerbation requiring treatment or because the participant prematurely terminates 
from the study (see section 6.3.3). 
 
Visit -1:  
Screening visit: 
Volunteers will attend the allergy unit at the Royal Brompton Hospital for 
approximately 2 hours. During this visit the following will be undertaken: 
 Written informed consent before any study procedures are performed.  
 Demographic data (sex, date of birth, ethnic origin, medical history).  
 Allergic history - record of allergic symptoms and allergic medication taken by 
the subject during at least the last two years. 
 Physical examination and observations (weight, height, blood pressure, heart 
rate, peak expiratory flow rate, peak nasal inspiratory flow and spirometry). 
 Participants will mark a retrospective Global evaluation (GE) visual analogue 
scale (VAS) (scale 0-100 mm; Appendix 1) and complete a retrospective GE 
chart (Appendix 2) documenting the overall severity of their hay fever 
symptoms during the previous pollen season.  
 
Participants will complete a Mini Juniper Rhinitis Quality of Life Questionnaire 
(mRQLQ) (Appendix 3) and Global evaluation scores (Appendix 4 and 5) 
corresponding to current rhinitis symptoms and medications.  
 Skin prick test - standard allergen test panel as described previously. 
 Blood sampling of up to 20 ml for total IgE, specific IgE to Timothy grass pollen 
and total blood count will be taken.  
 Nasal challenge with 5,000 BU/ml Timothy grass pollen extract with TNSS 
recorded after 5 minutes (Appendix 6).  
 Recording of concomitant medication. 
 Evaluation of inclusion and exclusion criteria. 
 
Visit 0a: 


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1st Baseline Visit: 
 
Volunteers meeting the inclusion and exclusion criteria will be randomised into the 
two arms and will come in for 60 minutes for blood sampling (120 ml), nasal lavage 
(wash), nasal brushings and nasal fluid collections.  
 
Table 1: Visit 0a 
Time 
(hr:min) 
 
 
Procedure 
00:00 
Arrival  
 
00:05 
Blood sampling 
120ml peripheral blood 
00:15 
Pre-lavage  
Secretion collection  
00:20 
Pre-lavage 
Nasal brushings 
00:25 
Nasal-lavage  
 
00:55 
Post-lavage 
Secretion collection  
 
Visit 0b:  
2nd Baseline Visit: 
On the 2nd baseline visit participants will attend the allergy unit for a period of 2 hours 
in the morning and 1 hour in the evening (overall 3 hours). During this visit the 
following will be undertaken (see also Table 2): 
 A period of 15 minutes acclimatization to hospital environment and relative 
inactivity. 
 Participants will receive a nasal wash (lavage). 
 
 Duplicate intradermal injections of 1.0 BU timothy grass allergen into the 
extensor surface of their right and left forearm, and a saline control injection 
also on the right arm will be performed. The EPR will be recorded at 15 minutes 
after injection; the LPR will be measured at 8 hours after injection. 
 The participants will receive a nasal allergen challenge with 5,000 BU/ml 
Timothy grass pollen extract. The EPR after the nasal challenge will be recorded 


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via the TNSS score (Appendix 6) and VAS score (Appendix 7) before 
challenge and after 5, 15, 30 and 60 minutes.  
 Peak nasal inspiratory flow (PNIF) will be assessed shortly before collecting 
the nasal fluid before the nasal challenge and after 5, 15, 30 and 60 minutes after 
the nasal challenge. 
 Peak expiratory flow (Peak Flow) will be assessed before nasal allergen 
challenge and 60 minutes after nasal allergen challenge. 
 Nasal fluid will be collected by inserting a single foam sponge into each nostril 
and will be left in place for 2 minutes before removal. Secretions will be 
collected before challenge, at 5, 15, 30 and 60 minutes after nasal challenge and 
after 8 hours. 
 The volunteers are allowed to leave the hospital (e.g. for work) and will return 
in the evening (after 8 hours) to allow reading of the skin LPR. Additionally, 
volunteers will undergo nasal brushings on both sides and a nasal biopsy. After 
the biopsy, volunteers will be observed for a further 30 minutes to ensure there 
is no nasal bleeding, and then allowed home.  
 Food and non-caffeine-containing beverages (at room temperature) will be 
provided to all participants. 
Table 2: Time line procedures at Visit 0b 
 
Time 
(hr:min) 
*Participants will be 
allowed to leave and 
return at 8hours 
Procedure 
00:00 
Arrival  
 
00:15 
Pre Lavage 
Secretion collection 
00:20 
Nasal Lavage 
Nasal lavage with 240 ml SinuRinse solution 
00:25 
Intradermal test 
1BU intradermal inject to left and right forearm 
+ saline injection 
00:40 
Intradermal test 
EPR recorded at 15 minutes 
00:50 
Pre-Challenge 
TNSS + VAS, PNIF, Secretion collection   


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00:55 
Nasal Challenge 
5,000 BU/ml Timothy grass pollen allergen 
01:00 
Early Nasal response 
TNSS + VAS; PNIF; Secretion collection 
01:10 
Early Nasal response 
TNSS + VAS; PNIF; Secretion collection 
01:25 
Early Nasal response 
TNSS + VAS; PNIF; Secretion collection 
01:55* 
Early Nasal response 
TNSS + VAS; PNIF; PeakFlow; Secretion collection 
08:25* 
Intradermal test 
LPR 
08:30 
 
Secretion collection 
Nasal brushings 
Nasal biopsy 
TNSS; total nasal symptom score 
VAS; visual analogue scale 
EPR; Early phase response 
LPR; Late phase response 
BU; Bioequivalent Unit 
PNIF; Peak nasal inspiratory flow 
 
Visit 1:  
Initiation of treatment will take place after Visit 0 including an inspection of the nasal 
biopsy site. The patients will be required to remain for 60 minutes in the hospital after 
intake of the first AIT/Placebo. 
 
Visit 2, 3 and 4: 
After around 4, 8 and 12 weeks of treatment starting the tablets (AIT/placebo), 
participants will return during the morning for around 60 minutes to have a 120 ml 
blood sample taken. Furthermore the volunteers will fill out questionnaires and undergo 
nasal fluid collection and brushings. (see also Table 3) 
Table 3: Time line procedures at Visit 2, 3 and 4 
Time  
(Hr:min) 
 
Procedure 
00:00 
Arrival  
 
00:05 
Blood  
Blood sample (120ml) 
00:10 
Questionnaires 
mRQLQ, SMS, GE VAS 
00:15 
Pre-lavage 
Secretion collection  
00:20 
Pre-lavage 
Nasal brushings 


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00:25 
Nasal-lavage 
 
00:55 
Post-lavage 
Secretion collection  
GE VAS; Global Evaluation visual analogue scale 
mRQLQ; miniRhinoconjunctivitis Quality of Life Questionnaire 
SMS; Symptom Medication Score 
 
At the start of May 2014, patients will receive the rescue medications (section 6.4.8.1) 
and will be asked to fill out the Global Evaluation (GE) VAS (Appendix 4), Symptom 
Medication Score (SMS) (Appendix 5) and mini-Rhinitis Quality of Life Questionnaire 
(mRQLQ) (Appendix 3) weekly until the end of July. 
Visit 5:  
Around 5 months after begin of AIT/placebo and during the pollen season participants 
will come to the hospital for 1.5 hours. During this visit the following will be 
undertaken (see also Table 4): 
 A period of 15 minutes acclimatization to hospital environment and relative 
inactivity. 
 
Participants will mark questionnaires (Appendix 3-5) corresponding to 
symptoms and rescue medication use during the current pollen season. 
 Furthermore the volunteers will undergo the same procedures as in Visit 0b, 
plus questionnaires and with the exception of the intradermal skin test and nasal 
challenge.  
Table 4: Time line procedures at Visit 5 
Time 
(hr:min) 
 
Procedure 
00:00 
Arrival  
 
00:05 
Blood  
Blood sample (200ml) 
00:15 
Pre-lavage 
GE VAS; mRQLQ; SMS, secretion collection  
00:20 
Pre-lavage 
Nasal brushing 
00:25 
Nasal-lavage 
 
00:55 
Post-lavage 
Secretion collection 


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Nasal biopsy 
GE VAS; Global Evaluation visual analogue scale 
mRQLQ; mini Rhinitis Quality of Life Questionnaires 
SMS; symptom medication score 
 
Visit 6: 
After 6 months of treatment and shortly after the pollen season patients will mark a 
retrospective GE VAS (scale 0-100 mm; Appendix 1), the mRQLQ (Appendix 3) and 
complete a retrospective GE chart (Appendix 2) documenting the overall severity of 
their hay fever symptoms during the previous pollen season. Also a nasal allergen 
challenge with 5.000 BU/ml Timothy grass pollen allergen will be undertaken. We will 
record the Peak Flow as well as the response to challenge for one hour at 5, 15, 30 and 
60 min with TNSS + VAS and PNIF. 
Time 
(hr:min) 
 
Procedure 
-00:15 
Arrival 
TNSS+VAS; GE VAS, mRQLQ, GE,  
PNIF; Peak Flow 
00:00 
Nasal Challenge 
5,000 BU/ml Timothy grass pollen allergen 
00:05 
Early Nasal response 
TNSS + VAS; PNIF 
00:15 
Early Nasal response 
TNSS + VAS; PNIF 
00:30 
Early Nasal response 
TNSS + VAS; PNIF 
01:00 
Early Nasal response 
TNSS + VAS; PNIF; PeakFlow 
 
 
Visit 7: 
After 12 months on treatment volunteers will attend the allergy unit for a period of 2 
hours in the morning and 1 hour during the evening (3 hours). During this visit the same 
procedures as described at Visit 0b plus a blood sample of 200 ml will be undertaken 
(see also Table 2). 
Non-atopic Participants: 
Scheduled Visits (screening (Visit -1), one time point assessment (Visit 1): 
 
Screening Visit – September 1st to November 30th, 2014. 
Visit – 1 coinciding with Visit 7 at 12 months of treatment of the atopic participants, 
from December 1st 2014 to February 28th, 2015.  


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Visit -1:  
Screening visit: 
Volunteers will attend the allergy unit at the Royal Brompton Hospital for 
approximately 2 hours. During this visit the following will be undertaken: 
 Written informed consent before any study procedures are performed.  
 Demographic data (sex, date of birth, ethnic origin, medical history).  
 Allergic history - record of presence/absence of previous allergic symptoms and 
medications taken by the subject. 
 Physical examination and observations (weight, height, blood pressure, heart 
rate, peak expiratory flow rate, peak nasal inspiratory flow and spirometry). 
 
Completion of matching questionnaires to those completed by atopic 
participants at baseline: retrospective Global evaluation (GE) visual analogue 
scale (VAS) (scale 0-100 mm; Appendix 1), retrospective GE chart (Appendix 
2), a Mini Juniper Rhinitis Quality of Life Questionnaire (mRQLQ) (Appendix 
3) and Global evaluation scores (Appendix 4 and 5).  
 Skin prick test - standard allergen test panel as described previously. 
 Blood sampling of up to 20 ml for total IgE, specific IgE to Timothy grass pollen 
and total blood count will be taken.  
 Recording of concomitant medication. 
 Evaluation of inclusion and exclusion criteria. 
 
Visit 1: 
 
Non-atopic participants will be asked to attend for a morning visit (2 hours) and an 
evening visit (1 hour). They will be free to leave the hospital in between. This visit will 
be planned in order to coincide with the 12 month visit for the allergic participants. 
Participants will receive the following tests (see also Table 5): 
 


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 A period of 15 minutes acclimatization to hospital environment and relative 
inactivity. 
 Blood sample of 200 ml.  
 Participants will receive a nasal wash (lavage). 
 
 Duplicate intradermal injections of 1.0 BU timothy grass allergen into the 
extensor surface of their right and left forearm, and a saline control injection 
also on the right arm will be performed. The EPR will be recorded at 15 minutes 
after injection; the LPR will be measured at 8 hours after injection. 
 The participants will receive a nasal allergen challenge with 5,000 BU/ml 
Timothy grass pollen extract. The EPR after the nasal challenge will be recorded 
via the TNSS score (Appendix 6) and VAS score (Appendix 7) before 
challenge and after 5, 15, 30 and 60 minutes.  
 Peak nasal inspiratory flow (PNIF) will be assessed shortly before collecting 
the nasal fluid before the nasal challenge and after 5, 15, 30 and 60 minutes after 
the nasal challenge. 
 Peak expiratory flow (Peak Flow) will be assessed before nasal allergen 
challenge and 60 minutes after nasal allergen challenge. 
 Nasal fluid will be collected by inserting a single foam sponge into each nostril 
and will be left in place for 2 minutes before removal. Secretions will be 
collected before challenge, at 5, 15, 30 and 60 minutes after nasal challenge and 
after 8 hours. 
 The volunteers are allowed to leave the hospital (e.g. for work) and will return 
in the evening (after 8 hours) to allow reading of the skin LPR. Additionally, 
volunteers will undergo nasal brushings on both sides and a nasal biopsy. After 
the biopsy, volunteers will be observed for a further 30 minutes to ensure there 
is no nasal bleeding, and then allowed home.  


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 Food and non-caffeine-containing beverages (at room temperature) will be 
provided to all participants. 
 
 
 
Table 5: Time line procedures at Visit 1 for the non-atopic participants 
 
Time 
(hr:min) 
*Participants will be 
allowed to leave and 
return at 8hours 
Procedure 
00:00 
Arrival  
 
00:10 
Blood  
Blood sample (200 ml) 
00:15 
Pre Lavage 
Nasal secretion collection and brushing 
00:20 
Nasal Lavage 
Nasal lavage with 240 ml SinuRinse solution 
00:25 
Intradermal test 
1BU intradermal inject to left and right forearm 
+ saline injection 
00:40 
Intradermal test 
EPR recorded at 15 minutes 
00:50 
Pre-Challenge 
TNSS + VAS, PNIF, Secretion collection   
00:55 
Nasal Challenge 
5,000 BU/ml Timothy grass pollen allergen 
01:00 
Early Nasal response 
TNSS + VAS; PNIF; Secretion collection 
01:10 
Early Nasal response 
TNSS + VAS; PNIF; Secretion collection 
01:25 
Early Nasal response 
TNSS + VAS; PNIF; Secretion collection 
01:55* 
Early Nasal response 
TNSS + VAS; PNIF; PeakFlow; Secretion collection 
08:25* 
Intradermal test 
LPR 
08:30 
 
Secretion collection 
Nasal brushings 
Nasal biopsy 
TNSS; total nasal symptom score 
VAS; visual analogue scale 
EPR; Early phase response 
LPR; Late phase response 
BU; Bioequivalent Unit 
PNIF; Peak nasal inspiratory flow 
GE VAS; Global Evaluation visual analogue scale 


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mRQLQ; mini Rhinitis Quality of Life Questionnaires 
SMS; symptom medication score 
 
 
 
6.5.15 Study follow up (12-36 months post randomisation) 
After 12 months unblinding of the atopic participants will take place. Participants that 
were included in the active treatment arm (AIT) will continue for a further 24 months 
follow up. The AIT treatment will be continued for a further 12 months after unblinding 
(24 months in total). At 24 months (end of treatment, visit 8) and at 36 months (12 
months after discontinuation, visit 9). At visit 8 and 9 all procedures, tests and markers 
performed at visit 7 (Table 2) will be repeated. Participants who received 12 months 
placebo will be offered 24 months of active AIT following the unblinding; they will not 
undergo further investigational assessments.  
 
6.6 Biomarkers  
 
We will measure known and novel biomarkers locally in the nasal mucosa, and in 
peripheral blood. In atopic participants blood, nasal fluid and nasal brushings will be 
sampled at screening, baseline, after 4, 8 and 16 weeks and at 7 and 12 months of 
treatment. The same sampling will be repeated at 24 and 36 months of the open label 
follow-up for the treatment group. Biopsies will be performed at baseline and at 7 and 
12 months of treatment, and at 24 and 36 months of open label follow-up  At screening, 
20 ml of blood (10 ml blood count, and 10 ml IgE to timothy grass) will be taken.  At 
baseline, after 4, 8 and 16 weeks, 120 ml blood will be taken (100 ml for T-, B- and 
dendritic cell (DC) studies and 20 ml for T- and B-cell repertoire analysis). At 7, 12 
and at 24 and 36 months, 200 ml will be taken (150 ml for T-, B- and DC studies and 
50 ml for T- and B-cell repertoire analysis). The healthy, non-atopic participants will 
present themselves after screening only once coinciding with Visit 7 at 12 months of 
the atopic participants. During this assessment 200 ml of blood will be taken (150 ml 
for T-, B- and DC-studies and 50 ml for T- and B-cell repertoire analysis). Additionally, 


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nasal fluid, nasal brushings and nasal biopsies will be collected. Different analyses will 
be performed as described below. 
6.6.1 Flow Cytometry 
Flow cytometry for analysis of basophils will be performed on fresh blood samples, on 
the same day as venesection. All other  flow cytometry will be performed on stored 
peripheral blood mononuclear cells (PBMCs), isolated  from whole blood by  layering 
over a Ficoll cushion, centrifuging and harvesting the buffy coat layer. PBMCs will be 
stored at -80°C before thawing and analysis in batches to reduce variability. 
6.6.2 DNA Methylation Studies 
Regulatory T cells (Tregs) have been implicated in preventing allergic responses. These 
cells may express the transcription factor FOXP3, the expression of which may be 
controlled by DNA methylation22. Demethylation at a highly conserved region within 
the FOXP3 gene was found to be restricted to natural Tregs when tested against all 
major peripheral blood types and a selection of non-blood cells23, 24. For this study, we 
intend to use a commercial quantitative real-time PCR-based methylation assay to 
enable the specific and sensitive determination of Treg numbers by measuring 
demethylated FOXP3 (e.g. Epionits GMBH, www.epiontis.com). By using the same 
technology to quantify CD3 positive cells, it is possible to calculate Tregs as a 
percentage of total T cells. 
6.6.3 T-cell Assays 
Assays will be performed on thawed PBMCs. 3H-thymidine incorporation and/or 
carboxy fluorescein succinimidyl ester (CFSE) dilution will be used as a measure of 
proliferation in response to allergen. Flow cytometry with surface ± intracellular 
markers will be used to assess T-cell phenotype, to include Th1, Th2 and Treg cells. T-
cell function in vitro will be assessed by specific ELISA (enzyme linked 
immunosorbant assay) for cytokines including interleukin-10 (IL-10) and IL-35, and/or 
by Fluorospot plate assays for IL-4/IL-10 and IL-5/IFN-γ.   
6.6.4 Serum assays 


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ImmunoCAP (Thermo Fisher Scientific, UK) and specific ELISAs will be used to 
identify Timothy grass pollen specific IgE, IgG1, IgG4, IgA1 and IgA2 in serum samples. 
ImmunoCAP ± ISAC microarray (Thermo Fisher Scientific) will be used to assess 
molecular sensitisation profiles.  Additionally, serum will be assessed for its ability to 
inhibit both IgE-mediated facilitated allergen binding (FAB) to B-cells21 and allergen-
induced basophil histamine release.  
6.6.5 Serum Archive 
Serum will be aliquoted and banked from study participants who provide informed 
consent for storage of their samples for a minimum of ten years for future studies as 
novel serum-based assays of immune tolerance become available.  
6.6.6 Nasal fluid 
Nasal fluid will be assayed for inflammatory mediators and local antibodies. 
Measured analyses will include some/all of the following:  
 
 The mast cell degranulation marker tryptase will be measured by ImmunoCAP. 
 
 IFN-γ, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-23, IL-25, 
IL-27, IL-33, MDC, eotaxin, TARC, RANTES, CXCL-8 (IL-8) will be 
measured by MILLIPLEX MAP Human Cytokine/Chemokine assay (Merck 
Millipore, UK). 
 
 TSLP will be measured by MILLIPLEX assay. 
 
 Specific IgE, sIgG1, sIgG4, sIgA1, sIgA2 and their molecular binding profile to 
timothy grass pollen will be assessed by ImmunoCAP and/or ISAC (Thermo 
Fisher Scientific/Phadia, Sweden).  
 
 Inhibitory activity for FcεR1 - dependent basophil activation will be assessed 
by measuring expression of surface CD63, CD203c, and intracellular Diamine 
Oxidase (DAO) by flow cytometry.  
 


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 Additional mediator/antibody/cytokine assays deemed relevant and of scientific 
interest that become apparent during/after the course of the study may be 
performed on the collected samples. 
6.6.7 Blood samples 
Samples will be taken at the time points indicated previously. We plan to perform the 
following analysis: 
 
 In collaboration with Dr Peter Andersen and Dr Peter Wurtzen (ALK-Abello, 
Denmark): investigation of changes in B cell repertoire and receptor editing 
following AIT by advanced gene sequencing techniques.  
 
 With Dr Louisa James (King’s College, London): high-throughput sequencing 
of B cell variable regions, grass pollen-specific antibody cloning and protein 
binding by Surface Plasmon Resonance.   
 
 Ex vivo allergen induced basophil activation and histamine release by flow 
cytometry (BD Biosciences). 
 
 T-cell assays will be performed as described above (section 6.6.3). Additionally, 
48 hour CpG/LPS-stimulated CD4+ cell IL-10 and IL-35 release and ICOS-
Ligand expression will be assessed. 
 A time course analysis of all FoxP3+ T regs, IL-10+, IL-35+ and TGF-+ T regs 
by flow cytometry.  
 
 Cytokine gene methylation assay will be performed on freshly isolated T cells. 
Genomic (g) DNA will be isolated from T cells.  
 
 Investigation of the time course of the effect of grass pollen-AIT on putative 
innate lymphoid cells by 8-colour flow cytometry – lineage negative, CRTH2+ 
CD127+CD25+ST2+IL-13+CD25R+ CD161+cells. 
 


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 Fresh blood will be used for whole blood flow cytometry using monoclonal 
antibodies to identify activated basophils and dendritic cells (see below). 
 
 
 
 
 
 
 
 
 
 
 
 
 
Antibody 
 
Identified cell subset 
Panel 1 
CD1c- APC-Cy7 
CD11c -V450 
CD123- PerCP-Cy5.5 
CD303- FITC 
CD80-PE-Cy7 
CD86-APC 
HLA-DR -V500 
OX40L-PE 
 
Panel 2 
CD1c-APC-Cy7 
CD11c-V450 
CD123-PerCP-Cy5.5 
CD303-FITC 
TSLP-R-APC 
FcRI-PE-Cy7 
ICOS-L-PE 
 
Plasmacytoid dendritic cells and 
activation markers 
CD3-PE-Cy7 
CD294(CRTH2)-PE 
CD203c-PercP-Cy5.5 
CD303-APC 
CD107a-Pacific Blue 
CD63-FITC 
Basophils plus activation and 
degranulation markers 
 


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 Whole blood flow cytometry using monoclonal antibodies will be used for 
phenotypic regulatory T, B and dendritic cells (see below). 
 
 
 
 
 
 
Antibody 
 
Identified cell subset 
CD45RO 
CD3 
CD4 
CD8 
CCR7 
CRTH2 
 
GATA-3 
T-bet 
IL-12Rb2 
IFN-γR 
CD1c 
CD11c 
Th1/Th2 cells 
CD45RO 
CD3 
CD4 
CD69 
 
CRTH2 
CCR7 
CD40L 
GATA-3 
Activated Th2 cells 
CD4 
CD25 
CD45RO 
CD127 
CD8 
CD39 
IL-35 
IL-10 
TGF-b 
IFN-g 
Foxp3 
CD39 
Helios 
pSTAT-5 
Regulatory T cells (panel 1) 
CD62L 
LAG-3 
CD279 (PD1) 
GARP 
Galectin-1 
IL-12p35 
EBI-3 
CTLA-4 
IL-10 
TGF-β 
IFN-γ 
CD161 
SAT1B 
LARP 
Additional regulatory T cell 
markers  


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CD4 
CXCR5 
PD1 
Bcl6 
Blimp 
IL-21 
 
T follicular helper cells 
CD19 
CD5 
CD25 
CD38 
 
CD279 
IL-10 
TGF-β 
B regulatory cell 
CD123 
CD303 
CD80 
CD86 
HLA-DR 
 
DC-SIGN 
OX40L 
ICOS-L 
PD1-L 
IL-27 
Activated dendritic cells 
 
 FluoroSpot assays will be used to determine proportions of IL-10+CD19+CD5+ 
B cells. 
 Phenotypic characterisation of regulatory B cells will be performed on whole 
blood and PBMCs. IL-10-producing B cell subsets will be assessed in the two 
groups. Stimulated B cells will be surface and intracellular stained with anti-
human CD5, CD24, CD38, PD1, CD27 and IL-10 (BD bioscience, USA) and 
analysed by flow cytometry. Further characterisation will as well take place by 
flow cytometry and will be confirmed by RT-PCR. Enumeration of Bregs and 
frequency of allergen-specific B cells will be performed. 
 
 We will perform deep sequencing of the TCRβ repertoire using a previously 
validated Repertoire Sequencing (REP-SEQ) assay26, 27, 28 in PBMCs samples. 
Briefly, multiplex PCR is performed on genomic DNA with primers targeting 
all Vβ and Jβ genes. High-throughput measurement of clonotype, function, and 
abundance of single T cells called TELS (TCRβ-effector linkage sequencing), 
which combines microfluidics and next-generation sequencing to encapsulate 
and genetically analyse hundreds of thousands of single cells per hour. After 
bioinformatics analysis, TELS data provides a simultaneous measure of TCRβ 
CDR3 sequence, effector gene activity, and abundance of T cell subpopulations 
from mixed cell samples with high sensitivity and high throughput. 


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6.6.8 Nasal brushings 
Nasal brushings will be performed according to the timelines mentioned above and used 
for the different analyses described below.  
 
 RNA will be extracted from cells obtained by nasal brushings (RNeasy, 
Qiagen), followed by creation of cDNA and real-time PCR to quantify gene 
expression by automated analysis. Levels of expression of a range of 
allergic/inflammatory genes, regulatory genes and structural genes will be used 
to verify patterns of cytokine recovery from within nasal fluid. Patterns of gene 
expression can be used to identify the presence of different cell types within the 
superficial nasal mucosa. Genes of interest will include those for epithelial-cell 
derived chemokines: MDC, TARC, eotaxin, RANTES; adhesion molecules 
including ICAM-1; cytokines including IL-8 and GM-CSF. Genes of interest 
from migratory effector cells will include IL-4, IL-5, IL-9, IL-13, IFN-γ, IL-10, 
TGF-β, IL-25, IL-22, IL-33, IL-35, IL-27, GATA-3, STAT-6, STAT-5, T-bet, 
and FOXp3. 
 
 Genomic DNA will be isolated from cells using an appropriate isolation kit 
(QIAamp DNA Mini Kit, Qiagen). Global DNA methylation will be recorded 
(Methylamp Global DNA methylation Quantification Kit, Epigentek Group, 
Farmingdale, NY, USA). Individual candidate gene methylation status will be 
measured using methylation specific PCR.  
 
 Cells collected from nasal brushings will be immunostained with anti-human 
CD3, CD303, CD294 (CRTh2), DAO, CD203c, CD63 (BD Biosciences, San 
Jose, CA). Non-activated and activated basophils will be identified as 
CD203cdim CRTh2+ and CD203cbright CRTh2+ cells, respectively. Additionally, 
activated cells will also be identified as CD63+ and CD107a+ CRTh2+ 
basophils. DAO+ CRTH2+ basophil will also be identified. Analyses will be 
performed using BD FACSDiva V6.1.1 software (BD Biosciences). 
 


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 Putative innate lymphoid cells, ILCs, will be Nuocytes will be assessed by 8-
colour flow cytometry – lineage negative, CRTH2+ CD127+CD25+ST2+IL-
13+CD25R+ cells. 
 
6.6.9 Nasal biopsy 
Nasal biopsies will be taken at the time points described. Biopsy specimens will be 
processed as follows: 
 
 In collaboration with Drs Peter Andersen and Peter Wurtzen, we will investigate 
changes in B cell repertoire and receptor editing following AIT. 
  
 Dr Louisa James will carry out high-throughput sequencing of B cell variable 
regions and extract and isolate individual B cells for determining binding 
affinities of peripheral and nasal grass-pollen specific antibodies.  
 
 For immunohistochemistry, one biopsy will be snap-frozen in liquid nitrogen 
and stored at -80°C. 6µm cryostat sections will be cut and stained, and examined 
by immunofluorescence for the following markers: Phl p5, IgA2, CD138 and 
CD20 to detect grass-pollen specific IgA-expressing plasma and memory B 
cells; TGF-1, IL-10, IL-21, IL-35, IL-27, FoxP3, CD3 to detect phenotypic T 
regulatory cells; IL-10, CD19, CD5, ICOS-Ligand as markers of phenotypic B 
regulatory cells.  
 
 For PCR analysis, total mRNA will be extracted from biopsy tissues using a 
suitable tissue mRNA isolation kit (Qiagen tissue mRNA isolation kit, Qiagen). 
Amplified cDNA will be produced from isolated mRNA by RT-PCR. Specific 
DNA primers will be used to assess the expression of the I exon germline gene 
of the IgA2 gene locus (I2) and the C region gene for IgA2 (C2) in the form 
of germline transcripts (GLTs) and switch circle transcripts (SCTs). The origin 
of these transcripts - generated by class switch from IgM, IgG, IgA1 or IgE - 
will also be ascertained according to methods previously described11. Identified 
transcripts will undergo nucleotide sequencing and Southern blot analysis. 


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Activation-induced cytidine deaminase (AID), RAG1 and RAG2 expression 
will also be assessed by PCR as markers of antibody gene class switch 
recombination. 
 
 One biopsy piece will be placed in appropriate medium before undergoing cell 
separation from solid tissue matrix, followed by magnetic bead enrichment and 
single cell FACS sorting to identify grass-pollen specific IgA expressing B 
cells. Cloning of allergen specific IgA, followed by affinity measurement and 
evaluation in functional assays will be carried out as above.  
 
 For High-Throughput sequencing of local and peripheral B cell repertoires RNA 
will be isolated from nasal biopsies and peripheral blood mononuclear cells and 
converted to cDNA. Heavy (IgA, IgG, IgM and IgE) and Light (Ig and Ig) 
chain variable region transcripts will be amplified using semi-nested, isotype-
specific PCR. Sample-specific multiplex identifier (MID) adaptor sequences 
will be incorporated into 2nd round PCR products to allow pooling of samples. 
Pooled PCR products will be purified by gel electrophoresis and sequenced on 
the GS FLX+ system. 
 
 For Isolation of nasal B cells and antibody cloning, single cell suspensions of 
nasal biopsy tissue will be obtained by mechanical dissection and enzymatic 
digestion. Cells will be stained with antibodies against B cell surface markers 
and single B cells will be individually sorted for single-cell RT-PCR. Matched 
heavy and light chain transcripts will be amplified by nested, isotype-specific 
PCR. PCR products will be purified and sequenced by capillary sequencing. 
Selected sequences will be re-amplified by PCR and cloned into a human 
expression vector containing the corresponding heavy and light chain constant 
region genes. Recombinant antibodies will be stably expressed in mammalian 
cells and purified from cell culture supernatant. 
 
 The affinities of recombinant nasal and purified, polyclonal plasma antibodies 
for individual grass pollen allergens will be measured using Surface Plasmon 
Resonance (SPR). Purified antibodies will be captured and immobilised using 
anti-isotype antibodies bound to a SPR sensor chip. Initial experiments will 


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determine their specificity for individual grass pollen allergens at a single 
concentration. Detailed kinetic analysis using a concentration series of the 
relevant individual allergens will then be used to derive association (kon) and 
dissociation (koff) rates. These will be used to calculate antibody binding/affinity 
constants. 
 
6.7 Recording, Managing and Reporting Adverse Events 
 
The PI is responsible for the detection and documentation of events meeting the criteria 
and definition of an adverse event (AE) or adverse reaction (AR) as described under 
sections 6.7.1.1 – 6.7.1.7 in this protocol. All AEs, ARs and severe adverse events 
(SAEs) will be recorded on the appropriate case report forms (CRFs) and the specific 
serious adverse events (SAE) will be reported as soon as possible and within 24 hours 
Data will be entered into MHRA approved Clinical Trial database ‘InForm’. This 
section defines the types of AEs and ARs and outlines the procedures for appropriately 
collecting, grading, recording, and reporting them. Information in this section complies 
with JRCO/SOP/001 SOP concerning Recording, Managing and Reporting Adverse 
Events in the UK31. A 24 hour contact phone number will be given to all participants. 
Day to day management of the study will be coordinated by Dr. Esther Steveling. 
6.7.1 Definitions  
6.7.1.1. Adverse Event 
An AE is any occurrence or worsening of an undesirable or unintended sign, symptom, 
laboratory finding, or disease that occurs during participation in the trial and which does 
not necessarily have a causal relationship with the treatment administered. An AE will 
be followed until it resolves or until 30 days after a participant terminates from the 
study, whichever comes first. All AEs will be reported as specified in section 6.7.6.4. 
During the study it is anticipated that all participants will experience seasonal 
symptoms of itching sneezing, watery discharge, nasal congestion, and eye symptoms, 
with or without wheezing from mid-May through mid-July. These are usual symptoms 
of SAR which represent their entry criteria for the study. In addition, participants will 
have free access to anti-allergic drugs that include antihistamines, intranasal corticoid 


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steroids, and eye-drops for relief of their symptoms. This data will be captured in the 
Questionnaires throughout the pollen season (i.e. Visual Analogue Scale, and Rescue 
Medication Score). These symptoms and requirements for rescue medications will not 
be reported as AEs.  
6.7.1.2. Adverse Reaction (AR)  
 
An adverse reaction (AR) is an untoward and unintended response to the treatment 
administered or the procedure performed. All AEs judged by either the reporting 
investigator or the sponsor as having reasonable causal relationship to 
Grazax®/Grazax® Placebo or the different procedures performed qualify as adverse 
reactions. The expression reasonable causal relationship means to convey in general 
that there is evidence or argument to suggest a causal relationship. 
There are a number of procedures throughout the study which are associated with 
expected symptoms. These include the following:  
 
 AIT involves taking a daily tablet under the tongue. This is frequently associated 
with oral itching and mild swelling. These symptoms may last from minutes to 
hours or longer in some cases. As long as these symptoms are not bothersome 
(i.e. interfere with usual daily activities or sleep) they will not be reported as 
ARs. 
 Non-invasive nasal secretion collection techniques using nasal sponges are not 
expected to cause any significant adverse events. Whilst the sponges are in place 
there is a degree of discomfort and a sensation of nasal blockage felt by the 
participant. Rarely there is mild nasal bleeding. As long as the reactions are not 
bothersome (interfere with sleep or with daily activities) it will not be reported 
as ARs. 
 Intradermal skin tests at the planned concentrations are entirely safe, without 
risk of systemic allergic reactions in grass pollen allergic individuals. As long 
as the reactions are not bothersome (interfere with sleep or with daily activities) 
they will not be reported as ARs.  
 Nasal biopsy is completely painless whereas following the procedure there may 
be slight bleeding and/or discomfort that responds to paracetamol. Bleeding 
responds to gentle local pressure applied bilaterally to the outside of the nose 


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by the volunteer. Discomfort may lastup to 24 hours, very rarely for several 
days, is mild and responds to paracetamol. As long as bleeding responds to 
finger pressure, mild discomfort responds to paracetamol, and consultation with 
a study physician is not required, they will not be reported as ARs. Should 
further measures be required, participants will have 24 hour access to advice 
from members of the study team. 
6.7.1.3. Unexpected Adverse Reaction 
An AR is considered “unexpected” when its nature (specificity) or severity is not 
consistent with applicable product information as described in the safety information 
provided in the package inserts for Grazax® or not consistent with the known AR 
towards the different study procedures described in section 6.5. 
6.7.1.4. Serious Adverse Event or Serious Adverse Reaction 
 
A SAE or SAR is defined as any AE or AR that at any dose suggests a significant 
hazard, contraindication, side effect, or precaution. This includes, but is not limited to, 
any of the following events: 
 
 Death: A death that occurs during the study or that comes to the attention of the 
investigator during the protocol-defined follow-up period must be reported 
whether it is considered treatment related or not. 
 A life-threatening event: Any adverse experience that, in the view of the 
Investigator, places the participant at immediate risk of death. 
 Inpatient hospitalization or prolongation of existing hospitalization. 
 Persistent or significant disability or incapacity. 
 An event that requires intervention to prevent permanent impairment or 
damage. 
 An important medical event that may not result in death, be life threatening, or 
require hospitalization may be considered an SAE when, based on appropriate 
medical judgment, it may jeopardize the participant and may require medical or 
surgical intervention to prevent one of the outcomes listed above. 
 Congenital anomaly or birth defect. 


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Medical judgement should be exercised in deciding whether an AE/AR is serious in 
other situations. Important AE/ARs that are not immediately life-threatening or do not 
result in death or hospitalisation but may jeopardise the subject or may require 
intervention to prevent one of the other outcomes listed in the definition above, should 
also be considered serious.  
6.7.1.5. Suspected Serious Adverse Reaction (SSAR)  
Any AR that is classed as serious and which is consistent with the information given 
about performed study procedures listed in section 6.5 and about Grazax® listed in the 
Summary of Product Characteristics (SmPC) or package inserts. Information on known 
AR of Grazax® can be further found at:  
http://emc.medicines.org.uk/ 
 
6.7.1.6. Suspected Unexpected Serious Adverse Reaction (SUSAR)  
Any SAR related to Grazax®/Grazax Placebo or a procedure performed in the study 
that is both unexpected and serious. Known side effects are listed in section 6.4 and 6.5 
In case of occurrence of a SAR these sections will be checked. If the event is not listed 
as expected, or has occurred in a more serious form than anticipated, this will be 
considered a SUSAR. 
6.7.2 Grading of adverse events/adverse reactions 
The study site will grade the severity of AEs/ARs for the study medication according 
to the following criteria: 
 
 All local reactions not associated with systemic signs or symptoms will be 
graded according to Table 6. 
 All systemic reactions to AIT will be graded according to the WAO SCIT 
Reaction Grading System35. The following modification will apply for AIT: 
Under Grade 1 (Symptoms/signs of 1 organ system present), “throat clearing 
(itchy throat)” is not considered a systemic reaction. 
 
Table 6: Sublingual immunotherapy (local reactions) Grade Description29 


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The study site will grade the severity of AEs/ARs for the following selected study 
procedures according to the following criteria:  
 
 All local reactions to nasal biopsy will be recorded according to Table 7.  
 
 All local reactions to allergen skin testing that are to be reported as AEs and are 
not associated with systemic signs or symptoms will be graded according to 
Table 8. 
 Although very unlikely, systemic reactions related to either AIT tablet 
treatment, nasal challenge or intradermal or skin prick test procedures will be 
graded according to the WAO SCIT Systemic Reaction Grading System35.  
 
Table 7: Nasal biopsy procedure (local reactions) 
Grade Description 
1 Bleeding that does not respond to finger pressure, pain that does not respond to 
paracetamol, or symptoms which require a telephone consultation with a study 
physician. 
2 Bleeding or pain that requires a visit to the doctor for treatment. 
3 Bleeding or pain that requires hospitalization. 
 
Table 8: Intradermal and skin prick test procedure (local reactions) 
Grade Description 
1 Bothersome (interfering with usual daily activities or sleep). 
2 Bothersome (requiring medication). 
3 Bothersome (requiring a visit to the study physician/emergency room for treatment). 
 
 


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The study site will grade the severity of AEs/ARs experienced by study participants not 
covered by Tables 5 through 7 or mentioned in the WAO criteria35 according to the 
criteria set forth in the National Cancer Institute’s Common Terminology Criteria for 
Adverse Events, Version 4.0 (published May 28, 2009 and updated June 14, 2010). This 
document is found at this website: 
 
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-
14_QuickReference_5x7.pdf. 
 
This document (referred to herein as the “NCI-CTCAE manual”) provides a common 
language to describe levels of severity, to analyse and interpret data, and to articulate 
the clinical significance of all AEs. AEs will be graded on a scale from 1 to 5 according 
to the following standards in the NCI-CTCAE manual: 
 Grade 1 = mild adverse event. 
 Grade 2 = moderate adverse event. 
 Grade 3 = severe and undesirable adverse event. 
 Grade 4 = life-threatening or disabling adverse event. 
 Grade 5 = death. 
 
For additional information and a printable version of the NCI-CTCAE manual, go to 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_ 
40 
6.7.3 Causality Definitions 
AEs and ARs will be categorized for their relation to one or more of the following: 
 
 Study medication 
 Study procedures: 
                 Nasal biopsy 
                 Intradermal skin test 
                 All other study procedures 
 


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The investigator will determine the relation, or causality, of an AE or AR to study 
participation and will record the determination on the appropriate CRF and/or SAE 
reporting form. The relation of an AE or AR to study participation will be determined 
using definitions in Table 9. 
 
 
Table 9. Attribution of adverse events Code Descriptor Relationship (to primary 
investigational product and/or other concurrent mandated study therapy)30 
 
Relationship  
Description  
Unrelated  
There is no evidence of any causal 
relationship.  
Unlikely  
There is little evidence to suggest there is 
a causal relationship (e.g. the event did 
not occur within a reasonable time after 
administration of the trial medication). 
There is another reasonable explanation 
for the event (e.g. the participant’s clinical 
condition, other concomitant treatment).  
Possible  
There is some evidence to suggest a 
causal relationship (e.g. because the 
event occurs within a reasonable time 
after administration of the trial 
medication). However, the influence of 
other factors may have contributed to the 
event (e.g. the participant’s clinical 
condition, other concomitant treatments).  
Probable  
There is evidence to suggest a causal 
relationship and the influence of other 
factors is unlikely.  
Definitely  
There is clear evidence to suggest a 
causal relationship and other possible 
Contributing factors can be ruled out.  
 
6.7.4 Investigator’s Responsibilities  
 
The PI will have overall responsibility for the conduct of the study. The PI 
responsibilities in detail are listed below: 
 
1.  
To report all SAEs within agreed timelines  
2.  
To report SUSARs within agreed timelines to Sponsor, MHRA, REC and 
relevant NHS Trust Research and Development Office (R&D)  


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3.  
Provide the Sponsor with details of all AEs identified in the protocol as critical 
to the evaluation of safety within the agreed timeframes.  
4.  
Assess each event for causality and seriousness between the treatment/ 
procedure and the adverse event.  
5.  
Supply the Sponsor, MHRA, REC and relevant NHS Trust R&D with any 
supplementary information they request.  
6.  
On-going safety evaluation of the treatment including trend analyses.  
7.  
Promptly notify all Investigators, REC(s) and MHRA, of any findings that 
may affect the health of subjects.  
8.  
Keep detailed written reports of all AEs and ARs reported and performing an 
evaluation with respect to seriousness, causality and expectedness.  
9. 
Report all relevant safety information to the relevant REC and MHRA.  
10. 
Report all SUSARs to the MHRA, REC and relevant NHS Trust R&D in 
concerned Member States associated with comparator product(s) and 
Marketing Authorisation (MA) holder(s), within given timelines.  
11. 
Break treatment codes before submitting expedited reports to MHRA and REC 
for specific subjects. 
12. 
Submit the annual safety report to Sponsor, MHRA and REC.  
6.7.5 Sponsor’s Responsibilities  
The Sponsor’s Responsibilities are listed as follows: 
 
1. Ensure written SOPs and systems are in place to ensure quality standards are met.  
2. Register users for pharmacovigilance data entry with the European Medicines 
Evaluation Agency (EMEA) if required.  
6.7.6 Collecting and Recording Adverse Events 
All AEs which occur during the course of study participants’ involvement in this 
research project will be appropriately recorded and reported according to the SOP 
Reference: JRCO/SOP/001 31 and according to the Medicines for Human Use (Clinical 
Trials) Regulations 2004 and the Department of Health’s Research Governance 
Framework for Health and Social Care. This will also be recorded on ‘InForm’. Each 
AE will be evaluated for seriousness, causality and expectedness. The recommended 


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flow chart in JRCO/SOP/001 will be used for further assessment of AEs31. They will 
be evaluated as though the patient was on active drug. Cases that are considered serious, 
unexpected and possibly, probably or definitely related (i.e. possible SUSARs) will be 
unblinded. Only those events occurring among patients on the active drug (unless 
thought to be due to the excipient in the placebo) will be considered to be SUSARs 
requiring reporting to the MHRA, RECs and JRCO. Adverse events and reactions not 
excluded in sections 6.7.1.1 and 6.7.1.2 will be described on case report forms (CRFs), 
unless they are classified as serious, in which case, these will be reported on a specific 
SAE form as suggested in SOP JRCO/SOP/00131. The PI will report SAE to the MHRA 
and to the REC. Subject confidentiality and adherence to the Data Protection Act (1998) 
will be maintained on all reports.33 
 
6.7.6.1 Methods of Collection 
All AEs and ARs may be collected as follows: 
 Observing the participant. 
 Questioning the participant in an objective manner. 
 Receiving an unsolicited complaint from the participant. 
6.7.6.2 Adverse Events and Adverse Reactions to be collected 
 
All AEs and ARs whether or not associated to the study medication and study procedure 
and not excluded in section 6.7.1.1 and 6.7.1.2 will be collected from visit -1 
(screening) until the time the participant completes the treatment phase and follow up 
or prematurely withdraws from the study. SAEs will be collected from visit -1 until 30 
days after the participant completes, or prematurely withdraws, from the study. 
6.7.6.3 Recording Method 
 
Throughout the study, the investigator will record AEs on the CRF and on ‘InForm’ 
database regardless of their severity or relation to study participation. The investigator 
will treat participants experiencing AEs and ARs appropriately and observe them at 
suitable intervals until their symptoms resolve or their status stabilizes. SAEs will be 


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recorded on the specific SAE forms suggested in the SOP JRCO/SOP/00131, and the 
appropriate notifications will be performed as outlined in section 6.7.6.4. 
6.7.6.4 Reporting  
All AEs and ARs as defined in section 6.7.1.1 to 6.7.1.6 will be reported. Depending 
on the nature of the event the reporting procedures below will be followed. Any 
questions concerning AEs reporting will be directed to the study coordinator in the first 
instance. Again the recommended flow chart in JRCO/SOP/001 will be used to further 
assess AEs 31. 
 
 
Non serious AR/AEs  
All AR and AEs, whether expected or not, will be recorded on the patient notes and on 
the relevant CRF and will be collected in the study coordination centre. 
 
SARs/SAEs  
If the AR or AEs is considered to be serious (SAEs or SARs) the SAE form will be 
filled out appropriately stating the nature of event, date of onset, severity, corrective 
therapies given, outcome and causality (i.e. unrelated, unlikely, possible, probably, 
definitely). The responsible investigator will sign the causality of the event. Additional 
information will be filled in within 5 days if the reaction has not resolved at the time of 
reporting. The CI will send all SAE reports to the Joint Research Compliance Office, 
Imperial College AHSC as soon as possible after becoming aware of the event. 
Notification will be performed to the MHRA, REC and the Sponsor of all SAEs as soon 
as possible. SAEs will be included in the annual safety report.  
 
SUSARs  
In the case of SUSARs, we will: Complete the SAE case report form signed and dated 
together with relevant treatment forms and anonymised copies of all relevant 
investigations and we will notify expedited the MHRA, REC and the Sponsor of all 
SUSARs according to the following timelines: 
 


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 as soon as possible but not later than 7 calendar days in case of fatal and life 
threatening SUSARs 
 as soon as possible but not later than 15 calendar days in case of non-fatal and 
non-life threatening SUSARs.  
 
Notification will further take place through the e-SUSAR account further described in 
the SPOP JRCO/SOP/00131. All investigators will be informed of all SUSARs 
occurring throughout the study. The CI will include all SSARs and SUSARs in the 
annual safety report.  
 
 
6.7.6.5 Unblinding 
 
SUSAR and SAR reporting will, as far as possible, maintain blinding of individual 
clinicians and of trials staff involved in the day-to-day running of the trial. Assessment 
of seriousness, causality and expectedness will be evaluated as though the patient was 
on active drug. Cases that are considered serious, unexpected and possibly, probably or 
definitely related (i.e. possible SUSARs) would have to be unblinded. Only those events 
occurring among patients on the active drug (unless thought to be due to the excipient 
in the placebo) should be considered to be SUSARs requiring reporting to the MHRA, 
RECs and JRCO. We will ask a colleague of our research team who is not directly 
involved in the management of the trial to perform unblinding (see also section 6.3.9). 
6.7.6.6 Reporting Pregnancy 
 
The investigator should be informed immediately of any pregnancy. The investigator 
should counsel the participant and discuss the risks of continuing with the pregnancy 
and the possible effects on the foetus. Monitoring of the participant should continue 
until the conclusion of the pregnancy. Any premature termination of the pregnancy will 
be reported. While pregnancy itself is not considered to be an AE or SAE, any 
pregnancy complication or elective termination of a pregnancy for medical reasons will 
be recorded as an AE or SAE/SAR as described in section 6.7.1.4. 


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6.7.6.7 Annual reports 
The PI will send annual Development Safety Update Reports (DSUR) for the duration 
of the clinical trial until the regulator has been notified of the end of the trial. This 
process will commence on the anniversary of the first international regulatory approval 
of Grazax® which has been on the 14th of March 2006. Reporting will occur within 60 
days of the 14th of March annually for the duration of the study. The DSUR will be 
submitted to the MHRA, ethics committee and Joint Research Compliance Office 
(JRCO) by the PI. The standard DSUR template will be used as described in SOP 
JRCO/SOP/03534. The PI will conduct regular trend analyses and signal detection to 
determine the continued safety of the drug within the study.  
6.7.6.8 Safety Analysis 
Safety analyses will assess all AEs including laboratory abnormalities, and physical 
examination abnormalities. All participants in the safety sample will be included in all 
safety analyses. Frequency of AEs will be tabulated by system organ class and preferred 
term, as well as by seriousness, severity, and treatment relatedness. Frequency of SAEs 
will be tabulated by system organ class and preferred term. For pertinent laboratory 
measurements, mean and mean change from baseline values will be presented by 
treatment group and visit. Frequency of physical examination abnormalities will be 
tabulated by treatment group, visit, and organ class. Safety data will also be listed by 
treatment group and subject. 
6.8 
Statistical methods and power calculation 
6.8.1 Statistical Analysis 
Analysis will be with non-parametric statistics taking into account treatment effect 
baseline values and visit number. Inclusion of 20 participants per group will give 
greater than 90% power (p=0.05) to detect a 40% reduction in the EPR after nasal 
challenge (AUC of the TNSS in the first 60 minutes after challenge; see Durham et al 
19968), a 40% reduction in the skin LPR (see Durham et al 199936), and a 50% increase 
in grass pollen allergen specific IgG4 for AIT vs. placebo (see Scadding et al 20109).  
 
Based on more recent nasal allergen challenge studies (Scadding et al, unpublished 
data; mean 4.63, standard deviation 1.65 for AUC 0-60 minutes post grass pollen nasal 


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challenge in 14 allergic volunteers), inclusion of 13 patients per group will provide 80% 
power to detect a 40% reduction in AUC after challenge, whereas inclusion of 22 
patients per group will provide 80% power to detect a 30% reduction.  
 
Further based on a recent study (Scadding et al, unpublished data; mean 70.14, standard 
deviation 14.17 for cross-sectional area in cm2 at 8 hours for skin late phase response 
to intradermal grass pollen injection), inclusion of 7 participants per group will provide 
80% power to detect a 30% reduction, whereas inclusion of 10 participants per group 
will provide 90% power to detect a 30% reduction.   
 
 We will include 20 healthy, non-atopic controls to match the two atopic groups.  
6.8.2 Analysis samples   
 Intent-to-treat (ITT) sample will be defined as all randomized participants. 
ITT participants will be analysed with the group to which they were 
randomized, regardless of the medication actually received. If participants drop 
out post randomisation, they will be invited to complete study assessments 
throughout the duration of the trial. 
 Per-protocol (PP) sample will be defined as ITT sample participants who 
remain in the study for 12 months and in whom the primary endpoints were 
assessed. Participants in the PP sample must be compliant with study 
medication, defined as taking 75% or more of their study medication for the 
duration of the study. Compliance with study medication will be as assessed by 
pill count for AIT/placebo. Participants in the PP sample will be analysed with 
the group to which they were randomized. 
 Safety sample (SS) will be defined as all randomized participants who received 
at least one dose of study medication. Participants in the safety sample will be 
analysed with the group according to the medication they actually received, 
regardless of their randomized assignment. 
6.8.3. Analysis of Endpoints  
Analysis of study data will be conducted to address all objectives of the trial and other 
interrelationships among all data elements of interest to the investigators and of 


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relevance to the objectives of the study. Primary analysis of treatment effect will be 
conducted under the intention-to-treat (ITT) principle of eligible patients, whereby 
outcome data from all eligible patients will be included regardless of treatment 
compliance. In addition to the analyses described in sections below, summary 
descriptive statistics will be provided in the following manner: continuous data will be 
summarized descriptively by mean, standard deviation, median, and range; categorical 
data will be presented as enumerations and percentages. 
 
 
6.8.3.1. Analysis of Primary Endpoint 
The primary endpoint is defined in section 6.1 and will be analysed using the ITT 
sample. The analysis of the primary endpoint will compare the mean EPR to nasal 
challenge recorded by the AUC of the TNSS during the first 60 minutes after the nasal 
challenge, at 12 months of therapy. Comparison between active and placebo groups 
will be assessed using ANOVA at the 0.05 level of significance. 
6.8.3.2 Analysis of Secondary Endpoints 
All secondary analyses will be treated as supportive. P-values will be presented for the 
secondary endpoints but will not be adjusted for multiplicity and should be interpreted 
with caution. Findings will be evaluated in the context of the available body of 
knowledge and with respect to other findings.  
 
1. The symptom score and the use of the rescue medications listed in section 
6.4.7.1 will be assessed via Symptom Medication Score (Appendix 5). The 
mean composite score in each treatment group during the peak pollen season 
(approximately mid-June, defined as the max 14 day rolling average pollen 
count during the season) will be computed and compared using ANOVA at the 
0.05 level of significance. 
2. We will compare the mean EPR and LPR to intradermal testing and it will be 
recorded as the mean diameter of the swelling measured at the specified time 


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points - after 15 minutes and 8 hours of allergen challenge respectively - at 
baseline and after 12 months of treatment. 
3. Mini Rhinoconjunctivitis Quality-of-Life Questionnaire (miniRQLQ) scores 
will be collected pre-, peak-, and post-pollen at baseline and after 8 months. 
4. Annual Global Evaluation scores: Global Evaluation Scores 1 and 2 for the nose 
and eye symptoms will be evaluated similarly to the primary outcome, by 
comparing the mean symptom score at screening and after 8 months separately, 
adjusting for baseline symptom score using ANOVA at the 0.05 level of 
significance. Global Evaluation No. 2 will be compared using a nonparametric 
trend test with significance of p ≤ 0.05.  
5. Grass pollen specific immunological markers in serum, nasal fluid and nasal 
brushings at 12 months of treatment will be evaluated. Comparisons will be 
made between atopic active and placebo group and non-atopic participants. 
6.8.3.3 Nasal Biopsy Analyses  
Nasal biopsies will be processed using immunochemistry, multicolour fluorescence 
immunochemistry, and in situ hybridization methods at baseline, at 7 and at 12 months. 
These assays will be conducted in a blinded fashion. Assays will be performed without 
the treatment designation of each group being known. Comparisons of results will be 
made between the 2 treatment groups with strict maintenance of the blinding with 
regard to individual participant treatment assignments. 
7. Informed consent 
 
Consent to enter the study must be sought from each participant only after a full 
explanation has been given, an information leaflet offered and time allowed for 
consideration. Signed participant consent should be obtained. The right of the 
participant to refuse to participate without giving reasons must be respected.  After the 
participant has entered the study the clinician remains free to give alternative treatment 
to that specified in the protocol at any stage if he/she feels it is in the participant’s best 
interest, but the reasons for doing so should be recorded.  In these cases the participants 
remain within the study for the purposes of follow-up and data analysis.  All 
participants are free to withdraw at any time from the protocol treatment without giving 
reasons and without prejudicing further treatment. 


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8. Ethics Approval 
The PI will request approval from a national research ethics committee included as part 
of the National Research Ethics Service (NRES). Site Specific Approval will also be 
required by The Royal Brompton Hospital Research and Development Office prior to 
commencement of the study. The study will be conducted in accordance with the 
recommendations for physicians involved in research on human subjects adopted by 
the 18th World Medical Assembly, Helsinki 1964 and later revisions. 
9. CTA  
This study has Clinical Trials Authorisation (CTA) from the UK Competent Authority; 
MHRA. Reference: 19174/0342/001-0001 
10. 
Confidentiality 
Participants’ identification data will be required for the registration process. The Study 
Coordination Centre will preserve the confidentiality of participants taking part in the 
study and is registered under the Data Protection Act.  
11. 
Indemnity 
Imperial College London holds negligent harm and non-negligent harm insurance 
policies which apply to this study. 
 
12. 
Sponsor 
Imperial College London will act as the main Sponsor for this study. Delegated 
responsibilities will be assigned to the NHS trusts taking part in this study. 
13. 
Funding 
Funding for the study will come from funds awarded to Guy Scadding from the 
Wellcome Trust PhD Programme for Clinicians. Atopic participants will receive £600 
on completion of the study, for their time and inconvenience as well as reasonable 
reimbursement of travel expenses on production of receipts. Non-atopic participants 
will receive 100 £. Participants will not have to pay for procedures or medication related 
to the study. 


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14. 
Audits 
The study may be subject to inspection and audit by Imperial College London under 
their remit as sponsor and other regulatory bodies to ensure adherence to GCP and the 
NHS Research Governance Framework for Health and Social Care (2nd edition). 
15. 
Publication of Results 
The results of the study will be submitted for publication in peer reviewed scientific 
journals. Results may also be presented, in the form of written or oral abstracts, at 
scientific meetings. Participants will be informed of all publications stemming from 
this study, and will be acknowledged in these publications.  
16. 
 References 
 
1. Bauchau V, Durham SR. Prevalence and rate of diagnosis of allergic rhinitis in 
Europe. Eur Respir J. 2004;24:758-64. 
 
2. Walker S, Khan-Wasti S, Fletcher M, et al. Seasonal allergic rhinitis is 
associated with a detrimental effect on examination performance in United 
Kingdom teenagers: case-control study. J Allergy Clin Immunol. 2007;120:381-
7.  
 
3. Brozek JL, Bousquet J, Baena-Cagnani CE, et al Allergic Rhinitis and its Impact 
on Asthma (ARIA) guidelines: 2010 revision. J Allergy Clin Immunol. 
2010;126:466-76. 
 
4. Calderon MA, Alves B, Jacobson M, et al. Allergen injection immunotherapy 
for seasonal allergic rhinitis. Cochrane Database Syst Rev. 2007 Jan 
24;(1):CD001936. 
 
5. Durham SR, Emminger W, Kapp A, et al. SQ-standardized sublingual grass 
immunotherapy: confirmation of disease modification 2 years after 3 years of 
treatment in a randomized trial. J Allergy Clin Immunol. 2012;129:717-725. 
 
6. Canonica GW, Bousquet J, Casale T, et al. Sub-lingual immunotherapy: WAO  
Position Paper. Allergy. 2009;64 Suppl 91:1-59. 
 
7. Shamji MH, Durham SR. Mechanisms of immunotherapy to aeroallergens. Clin 
Exp Allergy. 2011;41:1235-46. 
 
8. Durham SR, Ying S, Varney VA, et al. Grass pollen immunotherapy inhibits 
allergen-induced infiltration of CD4+ T lymphocytes and eosinophils in the 
nasal mucosa and increases the number of cells expressing messenger RNA for 
interferon-gamma. J Allergy Clin Immunol. 1996;97:1356-65. 
 


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9. Scadding GW, Shamji MH, Jacobson MR, et al. Sublingual grass pollen 
immunotherapy is associated with increases in sublingual Foxp3-expressing 
cells and elevated allergen-specific immunoglobulin G4, immunoglobulin A 
and serum inhibitory activity for immunoglobulin E-facilitated allergen binding 
to B cells. Clin Exp Allergy. 2010 ;40:598-606. 
 
10. Passalacqua G, Albano M, Fregonese L. Randomised controlled trial of local 
allergoid 
immunotherapy on allergic inflammation in 
mite-induced 
rhinoconjunctivitis. Lancet. 1998 28;351:629-32. 
 
11. Novak N, Haberstok J, Bieber T, et al. The immune privilege of the oral mucosa.  
Trends Mol Med. 2008;14:191-8. 
 
12. Bousquet J, Lebel B, Khivert H, et al. Nasal challenge with pollen grains, skin-
prick test and specific IgE in patients with grass pollen allergy. Clin Allergy 
1987;17:529-36. 
13. Lent AM, Harbeck R, Strand M, et al. Immunologic response to administration 
of standardized dog allergen extract at differing doses. J Allergy Clin Immunol. 
2006;118(6):1249-56. 
14. Durham SR, Yang WH, Pedersen MR, et al. Sublingual immunotherapy with 
once-daily grass allergen tablets: a randomized controlled trial in seasonal 
allergic rhinoconjunctivitis. J Allergy Clin Immunol. Apr 2006;117(4):802-809. 
15. Dahl R, Kapp A, Colombo G, et al. Efficacy and safety of sublingual 
immunotherapy 
with 
grass 
allergen 
tablets 
for 
seasonal 
allergic 
rhinoconjunctivitis. J Allergy Clin Immunol. Aug 2006;118(2):434-440. 
16. Dahl R, Kapp A, Colombo G, et al. Sublingual grass allergen tablet 
immunotherapy provides sustained clinical benefit with progressive 
immunologic changes over 2 years. J Allergy Clin Immunol. Feb 
2008;121(2):512-518 e512. 
17. Durham SR, Emminger W, Kapp A, et al. Long-term clinical efficacy in grass 
pollen-induced rhino conjunctivitis after treatment with SQ-standardized grass 
allergy immunotherapy tablet. J Allergy Clin Immunol. Jan 2010;125(1):131-
138 e131-137. 
18. Frolund L, Durham SR, Calderon M, et al. Sustained effect of SQ-standardized 
grass allergy immunotherapy tablet on rhinoconjunctivitis quality of life. 
Allergy. Nov 2. 2009. 
19. Cox LS, Larenas Linnemann D, Nolte H, et al. Sublingual immunotherapy: a 
comprehensive review. J Allergy Clin Immunol. May 2006;117(5):1021-1035. 


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20. Pilette C, Nour-Aria KT, Jacobson MR et al. Grass pollen immunotherapy 
induces an allergen-specific IgA2 antibody response associated with TGF-β 
mucosal expression. J Immunol 2007; 178: 4658-4666. 
21. Shamji MH, Wilcock LK, Wachholz PA et al. The IgE-facilitated allergen 
binding (FAB) assay: validation of a novel flow-cytometric based method for 
the detection of inhibitory antibody responses. J Immunol Methods 2006 
20;317(1-2):71-9. 
 
22. Baron U, Floess S, Wieczorek G, et al. DNA demethylation in the human 
FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) 
conventional T cells. Eur J Immunol. Sep 2007; 37(9):2378-2389. 
 
23. Ling EM, Smith T, Nguyen XD, et al. Relation of CD4+CD25+ regulatory T-
cell suppression of allergen-driven T-cell activation to atopic status and 
expression of allergic disease. Lancet. Feb 21 2004;363(9409):608-615. 
 
24. Sakaguchi S, Powrie F. Emerging challenges in regulatory T cell function and 
biology. Science. Aug 3 2007;317(5838):627-629. 
 
25. Powrie F, Carlino J, Leach MW, et al. A critical role for transforming growth 
factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated 
colitis by CD45RB(low) CD4+ T cells. J Exp Med. Jun 1 1996;183(6):2669-
2674. 
 
26. Freeman JD, Warren RL, Webb JR, Nelson BH, Holt RA. Profiling the T-cell 
receptor beta-chain repertoire by massively parallel sequencing. Genome Res. 
2009 Oct;19(10):1817-24.  
 
27. Robins H, Desmarais C, Matthis J, et al. Ultra-sensitive detection of rare T cell 
clones. J Immunol Methods. 2012 Jan 31;375(1-2):14-9. 
 
28. Du G, Chen CY, Shen Y, et al. J Immunol. TCR repertoire, clonal dominance, 
and pulmonary trafficking of mycobacterium-specific CD4+ and CD8+ T 
effector cells in immunity against tuberculosis. 2010 Oct 1;185(7):3940-7.  
 
29. Passalacqua 
G, Baena-Cagnani 
CE, Bousquet 
J, 
et 
al. 
Grading local side effects of sublingual immunotherapy for respiratory allergy: 
Speaking the same language. J Allergy Clin Immunol. 2013 S0091-6749(13)  
 
30. https://workspace.imperial.ac.uk/clinicalresearchgovernanceoffice/Public/JRC
O_SOP_012_Protocol_design%20Final%202012.pdf 
 
31. https://workspace.imperial.ac.uk/clinicalresearchgovernanceoffice/Public/JRC
O_SOP_001%20Safety%20Reporting%20Final2012.pdf 
 
32. http://www.blood.co.uk/can-i-give-blood/who-can-give-blood/ 
 
33.  http://www.opsi.gov.uk/acts/acts1998/ukpga_19980029_en_1 
 


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34. https://workspace.imperial.ac.uk/clinicalresearchgovernanceoffice/Public/JRC
O_SOP_035%20Development_Safety_Update_Report_FINAL%202012.pdf 
 
35. Cox L, Larenas-Linnemann D, Lockey RF, et al. Editors speaking the same 
language: The World Allergy Organization Subcutaneous Immunotherapy 
Systemic Reaction Grading System J Allergy Clin Immunol 2010;125:569-74. 
 
 
36. Durham SR, Walker SM, Varga EM, et al. Long-term clinical efficacy of 
grass-pollen immunotherapy. N Engl J Med. 1999 Aug 12;341(7):468-75. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
17. 
 Appendices 
 
 
Appendix 1: Global Evaluation Score 1 - Visual Analogue Scale 
 
Please complete during screening and after 8 months of treatment.  
 


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Please place a vertical mark along the line which you feel represents the severity of 
your hay fever symptoms. So, if you were to place a mark on the far left of the line, it 
would mean that you are/were completely symptom free. However, if you marked the 
far right of the line, your symptoms are/were as bad as they possibly could be. 
 
 
 
 
 
 
“How has your hay fever been overall during the last pollen season?” 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
Appendix 2: Global Evaluation Score 2  
 
Please complete during screening and after 8 months of treatment.  
 
Please tick only one box. 
 


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“How was your hay fever been this year compared with previous years?” 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Appendix 3: Mini Rhinoconjunctivitis Quality of Life Questionnaire  
Please complete during screening, weekly during the pollen season and after 8 months 
of treatment.  
Please complete all questions by circling the number that best describes how troubled 
you have been during the last week as a result of your nose/eye symptoms. 
 
 


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Appendix 4: Global Evaluation Score 3 – Visual Analogue Scale 
 
Please complete weekly on Wednesday during the pollen season from beginning of 
May until the end of July.  
 
Please place a vertical mark along the line which you feel represents the severity of 
your hay fever symptoms.  So, if you were to place a mark on the far left of the line, it 
would mean that you have been completely symptom free. However, if you marked 
the far right of the line, your symptoms have been as bad as they possibly could be. 
 
 
 
 
 
 
“How has your hay fever been overall during the last week?” 
 
 
 
 
 
 
  
 
 
 
 
 
 
Appendix 5: Symptom and Medication Score 
Please complete weekly on Wednesday during the pollen season from beginning of 
May until the end of July.  


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Symptom Score: 
“How do you assess the severity of your hay fever symptoms when they were at their 
most severe during the last week (tick each single symptom)?” 
 
Rhinoconjunctivitis / 
Hayfever Symptom 
 
Symptoms 
0 (None) 
1 (Mild) 
2 (Moderate) 
3 (Severe) 
Nasal Symptoms 
1 
Runny nose 
 
 
 
 
 
2 
Blocked nose 
 
 
 
 
 
3 
Sneezing 
 
 
 
 
 
4 
Itchy nose 
 
 
 
 
 
Eye Symptoms 
1 
Itchy eyes 
 
 
 
 
 
2 
Watery eyes 
 
 
 
 
 
 
Medication score: 
“On how many days did you require use of rescue medications in the last week (tick 
each single medication)?” 
 
Rescue medication 
Number of days used 
0 days 
1-2 days 
3-4 days 
5 or more days 
Antihistamine tablets 
 
 
 
 
 
Nasal spray 
 
 
 
 
 
Eyedrops 
 
 
 
 
 
 
 
Symptom and Medication Score (SMS) = (Symptom score/6 + Medication 
score/6)/2 
Appendix 6: Validated Symptom scoring (TNSS – total nasal symptom 
score)  
(Bousquet 198712 and Lent 200613)  
 
Symptom 
Score 
Sneezing 
0-3 


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Rhinorrhoea 
0-3 
Nasal congestion/blockage 
0-3 
Pruritus 
0-3 
Total Maximum score 
12 
Positive outcome is an increase of ≥ 5 points from baseline. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix 7: Visual Analogue Scale for the EPR after nasal challenge 
 
Please place a vertical mark along the line where you feel the severity of your 
symptoms lie currently. So, if you were to place a mark on the far left of the line, it 
would mean that you are completely symptom free. However, if you marked the far 
right of the line, your symptoms are as bad as they possibly could be. 
 
 
Overall 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix 8: Medication washout periods prior to visit-1, 0, 5, and 7. 
 
Medication Washout Periods  
 
Medications   
Time 
Inhaled β-agonists 
 
Short acting (e.g., Salbutamol, Ventolin, Salamol Easi-Breathe, 
Terbutaline, Bricanyl) 
6 hours 
Long acting (e.g., Salmeterol, Serevent, Formoterol, Foradil, Oxis) 
2 days 
Oral β-agonists 
 
Conventional release (e.g., Salbutamol, Ventolin) 
12 
hours 
Modified release (e.g., Bricanyl) 
2 days    
Cromolyn drugs (e.g., Intal, Tilade) 
7 days 
Leukotriene modifiers (e.g., Montelukast, Singulair, Zafirlukast, Accolate) 
3 days 
Inhaled corticosteroids (e.g., Beclomethasone, AeroBec, Asmabec 
Clickhaler, Beclanzone Easi-Breathe, Becodisks, Qvar, Budesonide, 
Pulmicort, Symbicort, Flixotide, Seretide, Asmanex)  
14 days 
Oral steroid (e.g., prednisone) 
14 days 
Theophylline product 
14 days 
Short-acting preparation (e.g., Nuelin SA, Slo-Phyllin, Aminophylline) 
24 
hours 
Long-acting preparation (e.g., Uniphyllin Continus, Phyllocintin 
Continus) 
2 days 
Rhinitis Medications 
Time 
Sodium Cromoglicate (e.g., Rynacrom, Vividrin) 
7 days 
Antihistamines (e.g., Cetirizine, Desloratadine, Neoclaritin, Fexofenadine, 
Telfast, Levocetirizine, Xyzal. Loratadine, Chlorphenamine, Piriton, 
Atarax, Zaditen) 
5 days 
Decongestants (e.g. pseudoephedrine, phenylephrine) 
3 days 
Antihistamine-decongestant tablets/liquids (e.g., Zyrtec D, Claritin-D)   
3. days 
Nasal corticosteroids (e.g., Flixonase NS, Flixonase Nasules, Nasonex, 
Nasacort, Flonase, Nasonex, Beclomethasone, Beconase, Betnesol, Vista-
Methasone, Budesonide, Rhinocort Aqua) 
14 days 
 
Médications (Prior to antigen nasal biopsy)  
Time 
Aspirin, NSAIDs,  or other anti-platelet agents 
2 days 
 
 
 
 
 
 
 


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Appendix 9: Daily Diary Card 
• Please answer the following questions once daily 
• Mark an X in case of YES 
• Leave the field empty in case you answer the question with NO 
• If you have not taken the tablet please specify the reason on why  
           you have not taken the tablet in the comment field  


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etc.  
January 2014 
Day Did 
you 
take 
the 
tablet? 
Comment Did you 
feel any 
Itchiness in 
the mouth 
after the 
treatment? 
Did you 
feel any 
swelling 
under the 
tongue 
after the 
treatment? 
Did you 
experience 
any other 
symptoms? 
(please 
specify) 
Where these 
symptoms 
bothersome? 
1. 
 
 
 
 
 
 
2. 
 
 
 
 
 
 
3. 
 
 
 
 
 
 
4. 
 
 
 
 
 
 
5. 
 
 
 
 
 
 
6. 
 
 
 
 
 
 
7. 
 
 
 
 
 
 
8. 
 
 
 
 
 
 
9. 
 
 
 
 
 
 
10. 
 
 
 
 
 
 
11. 
 
 
 
 
 
 
12. 
 
 
 
 
 
 
13. 
 
 
 
 
 
 
14. 
 
 
 
 
 
 
15. 
 
 
 
 
 
 
16. 
 
 
 
 
 
 
17. 
 
 
 
 
 
 
18. 
 
 
 
 
 
 
19. 
 
 
 
 
 
 
20. 
 
 
 
 
 
 
21. 
 
 
 
 
 
 
22. 
 
 
 
 
 
 
23. 
 
 
 
 
 
 
24. 
 
 
 
 
 
 
25. 
 
 
 
 
 
 
26. 
 
 
 
 
 
 
27. 
 
 
 
 
 
 
28. 
 
 
 
 
 
 
29. 
 
 
 
 
 
 
30. 
 
 
 
 
 
 
31. 
 
 
 
 
 
 

SAP_000.pdf: