Lung Immune Challenge Study Controlled Exposure to Inhaled Resiquimod (R848) to Study Mechanisms of Inflammation
Phase: NA
Conditions: Innate Immunity, Mucosal Immunity, Asthma, Viral Infection
Sponsor: Akhilesh Jha

 
     
    
       
 
         
 
Lung Immune Challenge Study: Controlled Exposure to Inhaled Resiquimod (R848) to Study Mechanisms of Inflammation 
Study Protocol 
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Version 1.0 
IRAS ID 340091 
Page 1 of 35 
 
 
 
Study Protocol 
 
 
Lung Immune Challenge Study 
Controlled Exposure to Inhaled Resiquimod (R848) 
to Study Mechanisms of Inflammation 
 
 
 

 
     
    
       
 
         
 
Lung Immune Challenge Study: Controlled Exposure to Inhaled Resiquimod (R848) to Study Mechanisms of Inflammation 
Study Protocol 
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Version 1.0 
IRAS ID 340091 
Page 2 of 35 
 
1. 
STUDY DETAILS 
Sponsor Reference Number:  
   R&D A096886 
IRAS Reference Number:  
 
   340091 
Date and Version No:  
 
   14.03.2024 Version 1.0 
Principal Investigator: 
 
Dr Akhilesh Jha 
MRC Clinician Scientist and Consultant in Respiratory Medicine 
University of Cambridge 
Co-Investigator: 
 
Dr Marie Fisk 
Consultant in Clinical Pharmacology and Acute Medicine 
Cambridge University Hospital NHS Foundation Trust 
 
Protocol Contributors: 
 
Mrs Jacqui Galloway  
Lead Clinical Nurse Specialist 
Cambridge University Hospital NHS Foundation Trust 
 
Mr Jamie Forrester 
Lead Physiotherapist in Respiratory Medicine 
Cambridge University Hospital NHS Foundation Trust 
 
Dr Karl Sylvester  
Head of Respiratory Physiology Services 
Cambridge University Hospital NHS Foundation Trust 
Study Pharmacist 
 
Dr Robyn Staples 
Cambridge University Hospitals NHS Foundation Trust 
Sponsors: 
 
Cambridge University Hospitals NHS Foundation Trust 
University of Cambridge 
Funders: 
 
Medical Research Council 
Reference MR/Y000935/1 
Signatures: 
 
 
Dr Akhilesh Jha 
14th March 2024 
 
Confidentiality Statement 
All information contained within this protocol is regarded as and must be kept confidential.  No part 
of it may be disclosed by any Receiving Party to any Third Party, at any time, or in any form without 
the express written permission from the Principal Investigator and / or Sponsor. 

 
     
    
       
 
         
 
Lung Immune Challenge Study: Controlled Exposure to Inhaled Resiquimod (R848) to Study Mechanisms of Inflammation 
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Table of Contents 
1. 
STUDY DETAILS ................................................................................................................ 2 
2. 
AMENDMENT HISTORY ................................................................................................... 6 
3. 
SYNOPSIS ........................................................................................................................ 7 
4. 
ABBREVIATIONS .............................................................................................................. 8 
5. 
BACKGROUND AND RATIONALE ...................................................................................... 9 
5.1 
Background ............................................................................................................................. 9 
5.2 
Rationale ................................................................................................................................ 9 
6. 
OBJECTIVES ................................................................................................................... 11 
6.1 
Primary Objectives ................................................................................................................ 11 
6.2 
Secondary Objectives ............................................................................................................ 11 
7. 
STUDY DESIGN .............................................................................................................. 12 
7.1 
Summary of Trial Design ........................................................................................................ 12 
7.2 
Primary Outcome Measures .................................................................................................. 12 
7.3 
Secondary Outcome Measures .............................................................................................. 12 
8. 
TRIAL PARTICIPANTS ..................................................................................................... 13 
8.1 
Overall Description of Trial Participants ................................................................................. 13 
8.2 
Inclusion Criteria ................................................................................................................... 13 
8.3 
Exclusion Criteria ................................................................................................................... 13 
8.4 
Treatment Assignment and Stratification ............................................................................... 14 
9. 
STUDY PROCEDURES AND ASSESSMENTS ....................................................................... 15 
9.1 
Study Procedures .................................................................................................................. 15 
9.1.1 
Nasosorption ............................................................................................................................................ 15 
9.1.2 
Nasal Brushing ......................................................................................................................................... 15 
9.1.3 
Blood ........................................................................................................................................................ 15 
9.1.4 
Induced sputum ....................................................................................................................................... 15 
9.1.5 
Spirometry ............................................................................................................................................... 15 
9.1.6 
Bronchodilator Reversibility or Methacholine Challenge or Exercise Testing ........................................ 15 

 
     
    
       
 
         
 
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9.2 
Informed Consent.................................................................................................................. 15 
9.3 
Screening and Eligibility Assessment ...................................................................................... 16 
9.4 
Subsequent Assessments ....................................................................................................... 17 
9.4.1 
Visit 1 (Day 1) ........................................................................................................................................... 17 
9.4.2 
Visit 2 (Day 2) ........................................................................................................................................... 17 
9.4.3 
Visit 3 (Day 8) ........................................................................................................................................... 17 
9.5 
Definition of End of Trial ........................................................................................................ 17 
9.6 
Discontinuation/Withdrawal of Participants from Study Treatment ........................................ 17 
9.7 
Source Data........................................................................................................................... 18 
10. 
TREATMENT OF TRIAL PARTICIPANTS ............................................................................ 19 
10.1 
Description of Study Treatment ............................................................................................. 19 
10.2 
Dose Escalation ..................................................................................................................... 19 
10.3 
Storage of Study Equipment or Related apparatus ................................................................. 20 
10.4 
Compliance with Study Treatment ......................................................................................... 20 
11. 
SAFETY REPORTING DEFINITIONS .................................................................................. 21 
11.1 
Adverse Event (AE) ................................................................................................................ 21 
11.2 
Serious Adverse Event (SAE) .................................................................................................. 21 
11.3 
Expected Adverse Events and Serious Adverse Events Exempt from Recording........................ 21 
11.4 
Suspected Unexpected Serious Adverse Reactions (SUSAR) .................................................... 22 
11.5 
Reporting Procedures for All Adverse Events .......................................................................... 22 
11.6 
Reporting Procedures for Serious Adverse Events................................................................... 22 
12. 
STATISTICS .................................................................................................................... 24 
12.1 
Description of Statistical Methods ......................................................................................... 24 
12.2 
The Number of Participants ................................................................................................... 24 
12.3 
The Level of Statistical Significance ........................................................................................ 24 
12.4 
Procedure for Accounting for Missing, Unused, and Spurious Data. ........................................ 24 
13. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ............................................................ 25 

 
     
    
       
 
         
 
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14. 
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES ......................................... 26 
15. 
CODES OF PRACTICE AND REGULATIONS ........................................................................ 27 
15.1 
Participant Confidentiality ..................................................................................................... 27 
15.2 
Other Ethical Considerations.................................................................................................. 27 
15.3 
Informed Consent.................................................................................................................. 27 
15.4 
Declaration of Helsinki .......................................................................................................... 27 
15.5 
ICH Guidelines for Good Clinical Practice ................................................................................ 28 
16. 
DATA HANDLING AND RECORD KEEPING ....................................................................... 29 
16.1 
Data Handling ....................................................................................................................... 29 
16.2 
Data Collection Forms ........................................................................................................... 29 
16.3 
Data Quality and Security ...................................................................................................... 29 
16.4 
Sample handling .................................................................................................................... 29 
17. 
FINANCING AND INSURANCE ......................................................................................... 30 
17.1 
Insurance and Liability ........................................................................................................... 30 
17.2 
Research Costs ...................................................................................................................... 30 
17.3 
Service Support Costs ............................................................................................................ 30 
17.4 
Study Sponsorship ................................................................................................................. 30 
18. 
PUBLICATION POLICY .................................................................................................... 31 
19. 
REFERENCES .................................................................................................................. 32 
20. 
APPENDIX A: STUDY FLOWCHART .................................................................................. 34 
21. 
APPENDIX B: SCHEDULE OF PROCEDURES ...............................Error! Bookmark not defined. 
 

 
     
    
       
 
         
 
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2. 
AMENDMENT HISTORY 
Amendment 
No. 
Protocol 
Version No. 
Date 
issued 
Author(s) of 
changes 
Details of Changes made 
 
 
 

 
     
    
       
 
         
 
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3. 
SYNOPSIS 
 
Title 
Lung Immune Challenge Study:  
Controlled Exposure to Inhaled Resiquimod (R848) to Study 
Mechanisms of Inflammation  
 
Internal Reference No. 
A096886 
 
Design 
Randomised single ascending dose inhaled exposure to Toll-like 
receptor (TLR)7/8 agonist R848 
 
Participants 
Participants without asthma 
Participants with asthma 
 
Sample Size 
36 
 
Duration 
3 years 
 
Aims 
1. To identify a suitable dose of inhaled R848 that induces innate 
immune responses in the lungs and blood 
 
2. To identify a suitable dose of inhaled R848 that is clinically 
tolerable 
 
Outcome Measures 
Primary Outcome 
i. 
Change in CXCL10 in serum or induced sputum 24 
hours after single ascending dose inhaled R848 
 
Secondary Outcomes 
i. 
Change in FEV1 after 1, 4, 8, 24 and 48 hours of single 
ascending dose inhaled R848 
 
ii. 
Change in physiological observations (temperature, 
pulse rate and blood pressure) after 1, 4, 8, 24 and 48 
hours of single ascending dose inhaled R848 
 
iii. 
Change in peripheral eosinophil and lymphocyte counts 
after 4, 24 and 48 hours of single ascending dose 
inhaled R848 
 
iv. 
Change in serum C-Reactive Protein (CRP) after 4, 24 
and 48 hours of single ascending dose inhaled R848 
 
Eligibility 
Adult males and females between 18 and 60 years of age meeting 
inclusion and exclusion criteria. 
 
 
 

 
     
    
       
 
         
 
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4. 
ABBREVIATIONS 
AE 
Adverse event 
AR 
Allergic Rhinitis 
ARwA 
Allergic Rhinitis with Asthma 
CUH 
Cambridge University Hospitals 
CCRC 
Cambridge Clinical Research Centre 
CRF 
 
Case Report Form 
CRP 
C-Reactive Protein 
CT 
 
Clinical Trials 
FEV1 
 
Forced Expiratory Volume in First Second 
FVC 
 
Forced Vital Capacity 
GCP 
Good Clinical Practice 
HLRI 
Heart and Lung Research Institute 
ICF 
Informed Consent Form 
LPS 
Lipopolysaccharide 
NHS 
National Health Service 
PBMC 
Peripheral Blood Mononuclear Cell 
PI 
Principal Investigator 
PIS 
Participant Information Sheet 
R&D 
NHS Trust R&D Department 
R848 
Resiquimod 
REC 
Research Ethics Committee 
REDCap 
Research Electronic Data Capture 
RPH 
Royal Papworth Hospital 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reactions 
TMF 
Trial Master File 
 
 
 
 

 
     
    
       
 
         
 
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5. 
BACKGROUND AND RATIONALE 
5.1 Background 
Acute respiratory viral infections cause significant morbidity, especially in vulnerable individuals 
and has been a topic of immense significance during the recent COVID-19 global pandemic. 
Respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) 
involve inflammation of the airways and viruses are a major cause of exacerbations (1). Whilst 
new biological drugs targeting allergic and eosinophilic immune pathways have been 
developed for chronic allergic asthma, therapeutic options for treating acute asthma attacks 
remain limited and rely on decades-old blunt tools such as oral corticosteroids. 
 
Development of new therapies and vaccines to combat viral respiratory tract infections is slow, 
partly because of the limited understanding of immune responses at the respiratory mucosal 
site of disease (2). Detailed characterization of such responses can facilitate biomarker 
definition for respiratory diseases, providing mechanistic insights and a platform for the testing 
of novel therapeutics. 
 
The innate immune system is crucial for effectively detecting and mounting responses to 
respiratory viruses. Toll-like receptors (TLRs) inside cells play a critical role in the initial 
detection of viral nucleic acid. TLR7/8 specifically detects RNA viruses such as influenza, 
rhinovirus, and coronavirus. Airway epithelial and immune cells express TLR7/8 and are 
therefore vital in mounting appropriate innate immune responses to viral infections (3-5).  
 
Resiquimod (R848) is a synthetic compound that binds to TLR7/8. As R848 activates anti-viral 
responses, it has been given systemically to treat viral hepatitis C infection (6) and employed 
topically as a skin cream for actinic keratosis (7). Related TLR7 agonists have been developed 
for therapeutic use by repeated dose intranasal administration to target allergic inflammation in 
healthy volunteers and those with allergic rhinitis (8, 9). A TLR7 agonist has also been trialled 
by the nebulised route and found to activate anti-viral innate immune responses (as evidenced 
by a rise in the biomarker CXCL10) in the lungs and blood (10). R848 can therefore be used as 
a suitable translational approach for studying innate immune responses in humans. 
 
5.2 Rationale 
An intranasal R848 human challenge model has been established by the Principal Investigator 
(PI). In total, it has been administered intranasally to 54 volunteers at different doses and is 
well tolerated with no reported serious adverse events (SAE) (11, 12). Nasal R848 induces 
anti-viral innate immune responses (interferons and chemokines) in a similar manner to 
respiratory viruses. It therefore represents a very useful research tool for studying innate 
immune responses to viral infections in the respiratory tract without needing to use live viral 
challenge, which is significantly more technically and resource intensive. 
 
Intranasal R848 has been given to healthy volunteers and those with allergic rhinitis (AR) or 
allergic rhinitis with asthma (ARwA). Individuals with AR and ARwA have increased nasal 
mucosal interferon and chemokine responses compared to healthy volunteers highlighting that 
dysregulated respiratory mucosal innate immune responses are likely to be important in 
determining the clinical outcome of viral triggers in individuals with allergy and asthma (12).  
 
The nose however has a different microenvironment, structure and composition of cells 
compared to the lungs (13). Furthermore, in those with asthma, distinct changes occur in the 
lungs such as airway remodelling and airway smooth muscle hypertrophy (14). It is therefore 
crucial to study host innate immune responses directly at the site of disease. 
 
Defining the cellular basis and molecular drivers of innate immune responses to respiratory 

 
     
    
       
 
         
 
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viral infections in the lungs will help provide mechanistic insights into how these responses 
might be dysregulated in diseases and help identify potential therapeutic targets.  
 
The goal of this study is to develop a human lung immune challenge model using controlled 
exposure to inhaled R848. The aim is to build upon the already established nasal challenge 
model to identify a clinically tolerable dose for use in the lungs that induces anti-viral innate 
immune responses. It will first be trialled in healthy individuals and subsequently in volunteers 
with asthma.  
 
Once established, inhaled R848 challenge is a clinical research approach that will enable: 
 
• Study of the effect of different host states on respiratory innate immune responses  
 
• Understanding of the molecular and immune drivers of asthma exacerbations 
 
• A platform for testing the efficacy of anti-inflammatory compounds 
 
• Significant practical advantages over live viral challenge models 
 

 
     
    
       
 
         
 
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6. 
OBJECTIVES 
6.1 Primary Objectives 
i. 
To identify an appropriate dose of inhaled R848 which induces innate immune 
responses in serum and lungs 
 
 
6.2 Secondary Objectives 
i. 
To assess clinical tolerability of inhaled R848 with regards to FEV1 
 
ii. 
To assess clinical tolerability of inhaled R848 with regards to physiological 
observations (temperature, pulse, blood pressure) 
 
iii. 
To assess change in peripheral eosinophil and lymphocyte counts after inhaled R848 
 
iv. 
To assess change in serum CRP after inhaled R848 
 
 

 
     
    
       
 
         
 
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7. 
STUDY DESIGN 
7.1 Summary of Trial Design 
Nasal administration of R848 has been shown to cause dose-dependent (10 to 100 μg/mL) 
induction of mucosal innate immune responses (11, 12). The current study will aim to nebulise 
R848 into the lower airways using a similar dosing regimen and using a nebulisation technique 
well established using inhaled lipopolysaccharide (LPS) challenge (15). The design of the study 
is broadly based on a previously published clinical trial of an inhaled TLR7 agonist (10).  
 
Single ascending dose (0.1, 1, 10, 100 μg/mL) nebulisation of R848 will be performed in 
healthy volunteers (up to n = 24) and subsequently in volunteers with asthma (n = 6).  
 
All volunteers will receive only a single dose and the starting dose cohort for nebulised 
challenge will be 100-fold lower than the dose administered in the nose to ensure clinical 
tolerability.   
 
 For each dose-cohort (n = 6), participants will be randomised to either R848 (n = 4) or saline (n 
= 2).  
 
A summary of the study design is outlined in Appendix A.  
 
The study will start with a small “sample only’ cohort of six participants without asthma who will 
be recruited for the collection of nasal brush, sputum, and blood samples to optimise sample 
processing protocols prior to the main study.  
 
7.2 Primary Outcome Measures 
i. 
Change in CXCL10 in serum or induced sputum 24 hours after single ascending 
dose inhaled R848 
 
 
7.3 Secondary Outcome Measures 
i. 
Change in FEV1 after 1, 4, 8, 24 and 48 hours of single ascending dose inhaled R848 
 
ii. 
Change in physiological observations (temperature, pulse rate and blood pressure) 
after 1, 4, 8, 24 and 48 hours of single ascending dose inhaled R848 
 
iii. 
Change in peripheral eosinophil and lymphocyte counts after 4, 24 and 48 hours of 
single ascending dose inhaled R848 
 
iv. 
Change in serum CRP after 4, 24 and 48 hours of single ascending dose inhaled 
R848 
 

 
     
    
       
 
         
 
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8. 
TRIAL PARTICIPANTS 
8.1 Overall Description of Trial Participants 
The study will start with a small “sample only’ cohort of six participants without asthma who will 
be recruited for the collection of nasal brush, sputum, and blood samples to optimise sample 
processing protocols prior to the main study.  
 
The interventional part of the study will be performed in participants without asthma (n = 24), 
and subsequently in participants with asthma (n = 6). 
 
8.2 Inclusion Criteria 
• 
Male or female aged between 18 and 60 years. 
• 
Willing and able to give informed consent for participation in the study. 
• 
Female participants of child-bearing potential and male participants whose partner is 
of child-bearing potential must be willing to ensure that they or their partner use 
effective contraception during the study. 
• 
Clinically acceptable laboratory measurements and ECG at enrolment. 
• 
Ability to expectorate sputum. 
• 
Optional additional swab for SARS-CoV-2 testing will be collected from participants 
if required by local or/and national health and safety policies at the time of sampling. 
 
For volunteers without asthma: 
• 
No clinical history of asthma 
• 
Normal baseline spirometry i.e. FEV1/Forced Vital Capacity (FVC) ratio z-score 
greater than the lower limit of normal. 
 
For volunteers with asthma: 
• 
Physician-diagnosed mild to moderate asthma which is not poorly controlled as 
evidenced by an Asthma Control Questionnaire (ACQ-5) score of ≤1.5. 
• 
They are permitted to be on inhaled corticosteroids (ICS), long-acting beta agonist 
(LABA) and long-acting muscarinic antagonists (LAMA). 
• 
Pre-bronchodilator FEV1 ≥70% predicted. 
• 
Evidence of bronchial hyperreactivity as evidenced by either (i) Bronchodilator 
reversibility (increase FEV1 ≥12% and 200 mL); (ii) Positive methacholine challenge 
(PC20 < 8mg/ml), or (iii) Positive challenge test as per current CUH policy. 
 
8.3 Exclusion Criteria 
• 
Upper respiratory tract infection in preceding 14 days. 
• 
Lower respiratory tract infection in preceding 28 days. 
• 
Female participants who are pregnant, lactating or planning pregnancy. 
• 
Respiratory diseases (other than asthma where specified). 
• 
Significant extrapulmonary medical conditions. 
• 
Extreme obesity (BMI >40). 
• 
Any other significant disease or disorder which, in the opinion of the Investigator, 
may either put the participants at risk because of participation in the study, or may 
influence the result of the study, or the participant’s ability to participate in the study. 
• 
Participants who have participated in another research study involving an 
investigational product in the past 12 weeks. 
• 
No newly prescribed courses of medication including corticosteroids in the four 
weeks before first study dose other than mild analgesia, vitamins, and supplements. 
• 
Smoking tobacco or vaping products in previous 6 months. 

 
     
    
       
 
         
 
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• 
Smoking history of >5 pack years. 
 
8.4 Treatment Assignment and Stratification  
A web-based randomisation and treatment allocation system using REDCap (Research 
Electronic Data Capture) will be used. Participants will be stratified by sex to ensure equal 
allocation to active and saline groups. 
 
For each dose cohort of 6 participants, individuals will be randomised (2:1) to either: 
- 
Nebulised R848  
- 
Nebulised Saline  
 
The PI (or delegate) will prepare the solution for nebulisation in a location away from the 
participant. When it is administered to the participant, they will be unaware of the 
intervention to which they have been allocated. 

 
     
    
       
 
         
 
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9. 
STUDY PROCEDURES AND ASSESSMENTS 
A schedule of study procedures in relation to study visits is outlined in Appendix B. 
 
9.1 Study Procedures 
9.1.1 
Nasosorption 
Nasal mucosal lining fluid will be collected non-invasively using nasosorption at baseline (0 
hours) and 24 hours after intervention to assess whether immune changes in the lungs can 
also be detected in the nose. 
 
9.1.2 
Nasal Brushing 
• 
A small cytology brush will be used to gently and non-invasively collect epithelial cells 
from the nasal inferior turbinate to enable epithelial cell culture in the laboratory.  
 
9.1.3 
Blood 
Blood will be collected on the following occasions: 
• 
Screening (31.5 mL): For routine safety and eligibility (9 mL), DNA testing (2.5 mL) and 
blood for isolation of peripheral blood mononuclear cells (PBMCs) (20 mL). 
 
• 
Baseline (14 mL): At 0 hours for full blood count (FBC) (4 mL), CRP (5 mL) and serum 
collection (5 mL). 
 
• 
Post intervention (52 mL): At 15 minutes, 1 hour, 4 hours, 24 hours and 48 hours for 
serum collection (25 mL); At 4 hours, 24 hours, and 48 hours for full blood count (FBC) 
(12 mL) and CRP (15 mL) measurement. 
 
Therefore, a total volume of approximately 100 mL blood will be collected from each volunteer 
throughout the whole trial including the screening and main study period. 
 
9.1.4 
Induced sputum 
This will be performed using hypertonic saline at the screening visit and 24 hours after 
intervention using standardised techniques (16) over a 15-20 minute period to obtain sputum 
that can be analysed for CXCL10 levels. Salbutamol will be co-administered as a 
bronchodilator to reduce the chances of bronchoconstriction. 
 
9.1.5 
Spirometry 
Volunteers will perform spirometry at the screening visit and throughout the study period.   
 
9.1.6 
Bronchodilator Reversibility or Methacholine Challenge or Exercise Testing 
For volunteers with asthma, as part of their screening they will be asked to perform either 
bronchodilator reversibility, methacholine challenge or challenge testing as per latest CUH 
policy (e.g. exercise testing), to confirm bronchial hyperreactivity. If their baseline FEV1 is 
80%, they will be asked to perform spirometry before and after inhalation of salbutamol to 
check for reversibility by 12% and 200 mL. If their baseline FEV1 is 80% they will instead 
perform a methacholine challenge or challenge test as per CUH policy (e.g. exercise test). 
 
9.2 Informed Consent 
A written or electronic version of the Participant Information Sheet (PIS) will be provided to 
participants detailing the nature of the study and any known side effects and risks involved in 
taking part. The participant will be free to withdraw from the study at any time for any reason 
without prejudice to future care, and with no obligation to give the reason for withdrawal. 
 

 
     
    
       
 
         
 
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The participant will be allowed time to consider the information, and the opportunity to question 
the Investigator, to decide on participation in the study. Electronic Informed Consent 
(eConsent) will then be obtained by means of participant dated and investigator dated 
electronic signatures. The person obtaining consent will be suitably qualified and experienced 
and have been authorised to do so by the PI as detailed on the Delegation log for the study. A 
copy of the signed form will be retained in the Trial Master File (TMF). A written or electronic 
copy of the signed consent form will also be provided to participants. 
 
9.3 Screening and Eligibility Assessment 
Participants in this study will be recruited from Cambridge University Hospitals (CUH), Royal 
Papworth Hospital (RPH), Heart and Lung Research Institute (HLRI), University of Cambridge, 
volunteer databases, local advertising in the Cambridge area and wider East of England region, 
as well through online platforms. Participants from CUH may be recruited from outpatients (in 
particular, departments of allergy and asthma) or comprise of staff working there. Employees or 
students at the University of Cambridge who express an interest in taking part are also eligible. 
Advertisements for the study will be through organisational posters, newsletters, online 
bulletins, local newspapers, online platforms, and social media channels.  
 
Pre-screening of interested participants will primarily involve interaction with a dedicated study 
website (www.lungchallenge.org.uk) or by email and telephone screening. They will be able to 
download the Participant Information Sheet (PIS) from the study website.  
 
An online pre-screening questionnaire hosted using REDCap software will be utilised to 
ascertain basic eligibility for the study in an anonymised form. If participants are not eligible no 
data will be stored. If participants may be eligible, they will be asked for further demographic 
and medical details to help ascertain eligibility and their data will be stored using the University 
of Cambridge’s Secure Data Hosting Service which has a Safe Haven for Personal Identifiable 
Data https://cscs.medschl.cam.ac.uk/server-services/secure-data-hosting-service/. 
 
Subsequently, for potentially eligible participants, a screening visit will be undertaken at the 
Cambridge Clinical Research Centre (CCRC) to evaluate the following:  
 
Demographics: date of birth, gender, race, smoking status. 
 
Medical History: Details of any history of disease or surgical interventions and systems review 
to determine inclusion/exclusion criteria. 
 
Concomitant Medication: All over the counter or prescription medication, vitamins, and/or 
herbal supplements will be recorded. 
 
Physical Examination as well as physiological observations, height, weight, and temperature 
will be recorded. 
 
ECG Test: A 12-lead ECG will be undertaken for each participant. 
 
Laboratory Tests: Biochemistry, full blood count, liver function test, total IgE, PBMC isolation, 
skin prick allergy testing, and a pregnancy test (for women) will be performed.  
 
Nasal Brushing: At the discretion of the Investigator, this will be done to collect nasal epithelial 
cells. 
 
Induced Sputum: Volunteers will undergo induced sputum sampling. For volunteers with 
asthma this will take place at a second screening visit approximately 7 days (+/- 3 days) after 

 
     
    
       
 
         
 
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the reversibility testing is done at the screening visit. This is to limit any confounding effects of 
reversibility testing on cellular counts in the induced sputum.  
 
All screening results will be reviewed by the Investigator to determine participant eligibility. 
 
For participants in the “sample only” part of the study, their screening will briefly involve 
collection of data on demographics, medical history, medication usage, spirometry, and 
physiological observations to ensure they meet the inclusion and exclusion criteria. They will 
not require other screening tests. Sample collection will involve a nasal brushing, induced 
sputum, and blood sampling (20 mL – for isolation of peripheral blood mononuclear cells). This 
will be the end of the study for them. 
 
9.4 Subsequent Assessments 
For volunteers who are eligible for the study, the following study visits and procedures will take 
place and are outlined in Appendix B. 
 
9.4.1 
Visit 1 (Day 1) 
This will take place over 24 hours including an overnight stay in the CCRC. Baseline 
assessments will include:  
• 
Spirometry 
 
• 
Physiological observations (temperature, pulse, and blood pressure) 
 
• 
Nasosorption - to determine baseline nasal immune profile 
 
• 
Blood tests (FBC, CRP, Serum) 
 
Volunteers will have been randomised to receive either R848 or saline via a breath-actuated 
nebuliser (Aeroeclipse II). After administration, they will be monitored at regular intervals by the 
clinical research team to check for clinical tolerability and repeat blood tests. The following 
morning after 24 hours, volunteers will subsequently have repeat blood tests as well as a 
repeat induced sputum performed. 
 
9.4.2 
Visit 2 (Day 2) 
This will take place at 48 hours to monitor physiological observations, spirometry and perform 
blood tests. 
 
9.4.3 
Visit 3 (Day 8) 
This will take place at 7 days after challenge to monitor physiological observations, spirometry 
and perform blood tests. This will then be the end of the study for participating volunteers.  
 
9.5 Definition of End of Trial 
The end of trial is the date of the last visit of the last participant.  
9.6 Discontinuation/Withdrawal of Participants from Study Treatment 
Each participant has the right to withdraw from the study at any time.  In addition, the 
investigator may discontinue a participant from the study at any time if the investigator 
considers it necessary for any reason including:  
 
• 
Ineligibility (either arising during the study or retrospective having been overlooked at 
screening) 

 
     
    
       
 
         
 
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• 
Significant protocol deviation 
• 
Significant non-compliance with treatment regimen or study requirements 
• 
An adverse event which requires discontinuation of the study or results in inability to 
continue to comply with study procedures 
• 
Consent withdrawn 
 
The reason for withdrawal will be recorded in the CRF. If the participant is withdrawn due to an 
adverse event, the investigator will arrange for follow-up visits or telephone calls until the 
adverse event has resolved or stabilised.   
 
9.7 Source Data 
Source documents are original documents, data, and records from which participants’ CRF 
data are obtained.  These include, but are not limited to, hospital records (from which medical 
and medicine history may be summarised), clinical and office charts, laboratory and pharmacy 
records, diaries, microfiches, radiographs, and correspondence. 
 
CRF entries will be considered source data if the CRF is the site of the original recording (e.g., 
there is no other written or electronic record of data). All documents will be stored safely in 
confidential conditions. On all study-specific documents, other than the signed consent, the 
participant will be referred to by the study participant number and not by name.  

 
     
    
       
 
         
 
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10. 
TREATMENT OF TRIAL PARTICIPANTS 
10.1 Description of Study Treatment  
 
   
    
     
R848 has been administered in-vivo in pre-clinical models and humans in a wide variety of 
formulations and routes at significantly higher doses than the proposed study, with extensive 
available data on tolerability: 
• 
R848 was well tolerated in pre-clinical models in mice and rats (17, 18). 
 
• 
Oral R848 has been given to patients with chronic hepatitis C infection and was well-
tolerated at doses of 0.01mg/kg, equating to 700 g for a 70 kg human (6). 
 
• 
Topical dermal R848 is safe and effective as a skin cream for the treatment of 
anogenital warts, actinic keratosis and skin cancer. Human studies have been carried 
out giving topical R848 to inflamed skin with actinic keratoses at doses up to 0.25%, 
which is equivalent to 2500 g/mL (19). A related compound, the TLR7 agonist 
Imiquimod (marketed as Aldara), is approved for clinical use as a topical application 
for genital warts. 
 
• 
Nasal R848 at doses between 10-100 g/mL has been given by the PI to 54 
participants ranging from those who are healthy to those with allergic rhinitis and 
asthma. This was effective at inducing mucosal innate immune responses whilst 
being clinical tolerable (11, 12).  
 
For the present study, R848 will be reconstituted as per the manufacturer’s instructions and 
diluted to the appropriate doses at CUH. A sample of the prepared dose vials will be sent for 
external quality control sterility testing to British Pharmacopeia standards before use in the 
clinical study.   
 
Saline will be obtained from commercial medicine stock from within the hospital. It will be 
manufactured to GMP and considered sterile therefore no additional sterility testing will be 
required.  
 
10.2 Dose Escalation  
The following doses of nebulised R848 will be tested using a single dose escalation design: 
0.1, 1, 10, 100 μg/mL. Six volunteers will be involved in each dose cohort with only a single 
dose given to each participant.  
 
Each participant will be administered a dose of R848 or saline in a volume of approximately 
1 mL (optimal volume to be confirmed after testing) using a breath-actuated nebuliser.   
 
 
At the end of each dose cohort, CXCL10 levels in sputum and blood will be assessed and 
compared between saline and R848. If there are no statistically significant differences 
Challenge Agent 
Manufacturer 
Catalogue Dose 
Production Purity 
Resiquimod (R848) 
VacciGrade 
TLR7/8 agonist 
 
Synthetic vaccine 
grade 
Invivogen, California 
3950 Sorrento Valley 
Boulevard, Suite 
100, San Diego, CA 
92121, USA 
www.invivogen.com 
5 mg lyophilized 
R848 
Reconstitute in 
endotoxin-free 
physiological water 
CAS 144875-48-9 
Non-biological 
origin: does not 
contain any animal 
nor human 
components. 
 
Endotoxin <1.25 
EU/mg 

 
     
    
       
 
         
 
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between groups, and the dose is tolerable for volunteers, then the study will proceed to the 
next dose cohort. 
 
Spirometry will be monitored regularly after inhaled challenge. Bronchodilator drugs will be 
available on standby for administration if volunteers have symptoms of breathlessness or 
tight chest associated with a significant drop in FEV1 (20%) from baseline. 
 
10.3 Storage of Study Equipment or Related apparatus 
R848 will be stored in labelled storage vials in a -20C freezer within CUH in PI’s laboratory 
and defrosted on the day of usage. Saline will be stored at room temperature. All other clinical 
equipment will be stored either in the PI’s laboratory or the CRF. 
 
10.4 Compliance with Study Treatment 
Participants will be administered R848 or saline via a nebuliser directly by the research team 
and therefore compliance will be directly assessed. 

 
     
    
       
 
         
 
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11. 
SAFETY REPORTING DEFINITIONS 
11.1 
Adverse Event (AE) 
An AE or adverse experience is any untoward medical occurrence in a participant, 
which does not necessarily have a causal relationship with the intervention. An AE can 
therefore be any unfavourable and unintended sign (including an abnormal laboratory 
finding), symptom or disease temporally associated with the study, whether or not 
considered related to the study. 
 
11.2 
Serious Adverse Event (SAE) 
A serious adverse event or reaction is any untoward medical occurrence that at any 
dose: 
 
• 
Results in death, 
• 
Is life-threatening, 
 
NOTE: The term "life-threatening" in the definition of "serious" refers to an event in 
which the participant was at risk of death at the time of the event; it does not refer to 
an event which hypothetically might have caused death if it were more severe, 
including an event which: 
 
• 
Requires inpatient hospitalisation or prolongation of existing hospitalisation, 
• 
Results in persistent or significant disability/incapacity, or 
• 
Is a congenital anomaly/birth defect. 
• 
Other important medical events* 
 
*Other events that may not result in death, are not life threatening, or do not require 
hospitalisation, may be considered a serious adverse event when, based upon 
appropriate medical judgement, the event may jeopardise the patient and may require 
medical or surgical intervention to prevent one of the outcomes listed above. 
 
Planned admission to hospital for a pre-existing condition will not be considered an 
SAE. 
 
11.3 
Expected Adverse Events and Serious Adverse Events Exempt from Recording 
Nasal R848 challenge administered at doses between 10 - 100 μg/mL in healthy 
participants and those with asthma has previously been well tolerated with no reported 
SAEs (11, 12).  
 
R848 is a synthetic substance that when given to in the respiratory tract is designed to 
mimic the effects of a viral infection such as influenza. Therefore, expected AEs that 
participants may experience include transient flu-like symptoms such as: 
- 
Cough  
- 
Runny nose  
- 
Mild headache  
- 
Low grade fever 
- 
Lethargy  
- 
Myalgia  
 
If a high-grade fever occurs that is more sustained or causes symptoms, then 
volunteers will be offered an antipyretic such as paracetamol.  
 

 
     
    
       
 
         
 
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Participants with asthma will be carefully monitored after R848 dosing. If their FEV1 
drops by >20% then they will be administered nebulised salbutamol as a 
bronchodilator.  
 
      
Nasal sampling using nasosorption and nasal cytology brush may cause: 
- 
Nasal irritation and runny nose  
- 
Minor epistaxis  
- 
Watering of the eyes  
- 
Sneezing 
 
There are minimal if any side effects anticipated from the induced sputum procedure 
and salbutamol will be given as a bronchodilator at the same time of administration in 
healthy and asthma participants, but recognised side effects of the procedure include:  
- 
Cough  
- 
Tight chest  
 
   
Expected AEs from blood sampling procedures may include: 
- 
Mild discomfort during venepuncture 
- 
Bruising at venepuncture site  
 
11.4 
Suspected Unexpected Serious Adverse Reactions (SUSAR) 
A serious adverse reaction, the nature or severity of which is not consistent with the 
applicable product information  
 
 
 
11.5 
Reporting Procedures for All Adverse Events 
All AEs occurring during the study observed by the investigator or reported by the 
participant, whether or not attributed to study, will be recorded on the CRF. The 
following information will be recorded: description, date of onset and end date, severity, 
assessment of relatedness to study and action taken. Follow-up information should be 
provided as necessary. 
 
AEs considered related to the study as judged by a medically qualified investigator or 
the sponsor will be followed until resolution, or the event is considered stable. All 
related AEs that result in a participant’s withdrawal from the study or are present at the 
end of the study, should be followed up until a satisfactory resolution occurs. 
 
It will be left to the investigator’s clinical judgment whether or not an AE is of sufficient 
severity to require the participant’s removal from treatment. A participant may also 
voluntarily withdraw from treatment due to what he or she perceives as an intolerable 
AE. If either of these occurs, the participant must undergo an end of study assessment 
and be given appropriate care under medical supervision until symptoms cease or the 
condition becomes stable. The relationship of AEs to the study will be assessed by a 
medically qualified investigator.  
 
11.6 
Reporting Procedures for Serious Adverse Events 
All SAEs, except those expected ones defined in section 10.3 that do not require 
immediate reporting, must be reported to the Sponsor within one working day of 
discovery or notification of the event.  The Sponsor will perform an initial check of the 
information and ensure that it is reviewed at the next R&D Management meeting. All 
SAE information must be recorded on an SAE form and sent to the Sponsor using the 
appropriate reporting form and the contact details on there. Additional information 

 
     
    
       
 
         
 
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received for a case (follow-up or corrections to the original case) needs to be detailed 
on a new SAE form which must be sent to the Sponsor using the appropriate reporting 
form and the contact details on there.  
 
The Sponsor will report all SUSARs to the Research Ethics Committee concerned. 
Fatal or life-threatening SUSARs must be reported within 7 days and all other SUSARs 
within 15 days. In addition to the expedited reporting above, the PI shall submit once a 
year throughout the study or on request an Annual Report to the Ethics Committee 
which lists all SAEs / SUSARs that have occurred during the preceding 12 months. 

 
     
    
       
 
         
 
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12. 
STATISTICS 
12.1 
Description of Statistical Methods 
CXCL10 will be measured in sputum and serum 24 hours after each ascending dose of inhaled 
R848 (n = 4) and compared to inhaled saline (n = 2). CXCL10 levels will be compared between 
groups using unpaired t-tests. 
 
Spirometry (FEV1), physiological assessments (temperature, pulse, blood pressure) and 
leukocyte differential counts from FBC samples measured after challenge will be compared to 
baseline values using one-way analysis of variance (ANOVA). 
 
 
12.2 
The Number of Participants 
Nasal R848 is a suitable method of inducing nasal innate immune responses (evidenced by a 
rise in interferon levels) in healthy individuals and those with asthma (11, 12). Therefore, it is 
anticipated that inhaled R848 will have a similar effect in inducing lung and peripheral innate 
immune responses even in a small number of participants. 
 
The number of participants in each dose cohort of the dose finding phase of the study (n = 4 
R848, n = 2 saline) is based on AstraZeneca’s clinical trial of their inhaled TLR7 agonist (a 
compound with similar effects) (10), and was a suitable number of participants for assessing 
innate immune responses as evidenced by a rise in CXCL10 in sputum and serum. 
 
12.3 
The Level of Statistical Significance 
A P-value of < 0.05 will be taken to indicate statistical significance. 
 
12.4 
Procedure for Accounting for Missing, Unused, and Spurious Data. 
Missing clinical data points will be imputed as the mean of values from that volunteer. 
 

 
     
    
       
 
         
 
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13. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
Direct access will be granted to authorised representatives from the sponsor, host institution 
and the regulatory authorities to permit trial-related monitoring, audits, and inspections. 
 

 
     
    
       
 
         
 
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14. 
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
The study will be conducted in accordance with the current approved protocol, ICH GCP, 
relevant regulations, and standard operating procedures. The University of Cambridge 
operate a risk-based audit program to which this study will be subject.  
 
 
 

 
     
    
       
 
         
 
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15. 
CODES OF PRACTICE AND REGULATIONS 
15.1 
Participant Confidentiality 
The study staff will ensure that the participants’ anonymity is maintained.  The participants 
will be identified only by initials and a participant’s ID number on the CRF and electronic 
databases.  All documents will be stored securely and only accessible by study staff and 
authorised personnel. The study will comply with the Data Protection Act 2018 which 
requires data to be anonymised as soon as it is practical to do so. 
 
15.2 
Other Ethical Considerations 
The study will not be initiated before the protocol, and all study relevant material such as 
the PIS and ICF have received a favourable opinion from the Research Ethics Committee 
(REC) and the HRA, and Cambridge University Hospitals NHS Foundation Trust has 
issued trust confirmation of capacity and capability.  Any changes to protocol or relevant 
study documents will be approved by the Sponsor. Should an amendment be made that 
requires REC approval, as defined by REC as a substantial amendment, the changes will 
not be instituted until the amendment has been reviewed and received favourable opinion 
from the REC and the HRA, and Cambridge University Hospitals NHS Foundation Trust 
have issued confirmation of continuing capacity and capability. A protocol amendment 
intended to eliminate an apparent immediate hazard to participants may be implemented 
immediately providing that the REC is notified as soon as possible, and an approval is 
requested. Minor amendments as defined by REC as a non-substantial amendment may 
be implemented upon receipt of HRA approval. 
 
15.3 
Informed Consent 
It is the responsibility of the PI, or a person designated by the PI (if acceptable by local 
regulations), to obtain signed informed consent from each patient participating in this 
study, after adequate explanation of the aims, methods, anticipated benefits, and potential 
hazards of the study using the PIS. 
 
The process for obtaining participant informed consent will be under the REC guidance, 
and GCP and any other regulatory requirements that might be introduced. The PI or 
delegate and the participant shall both sign and date the ICF before the person can 
participate in the study. 
 
The participant will receive a written or electronic copy of the PIS and a signed and dated 
ICF. The ICF will also be printed and retained. 
 
The decision regarding participation in the study is entirely voluntary. The investigator or 
their nominee shall emphasize to the participant that consent regarding study participation 
may be withdrawn at any time without penalty or affecting the quality or quantity of their 
future medical care, or loss of benefits to which the participant is otherwise entitled. 
 
15.4 
Declaration of Helsinki 
The Investigator will ensure that this study is conducted in full conformity with the current    
revision of the Declaration of Helsinki (last amended in October 2013). 
 

 
     
    
       
 
         
 
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15.5 
ICH Guidelines for Good Clinical Practice 
The Investigator will ensure that this study is conducted in full conformity with relevant 
regulations and with the ICH Guidelines for Good Clinical Practice: ICH E6(R3). 
 

 
     
    
       
 
         
 
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16. 
DATA HANDLING AND RECORD KEEPING 
16.1  
Data Handling 
All data will be handled within the conditions of the Data Protection Act 2018 
 
16.2 
Data Collection Forms  
A record of a participant’s involvement in the study will be made in their electronic healthcare 
record. Data collection will comprise of an electronic case report form using REDCap and will 
include participant characteristics, disease severity, medication lists, clinical examination, 
pulmonary and blood test results. 
 
16.3  
Data Quality and Security 
The data will be securely stored in line with GCP standards and the data protection principles. 
Standard Operating Procedures (SOPs) will be followed to ensure quality control. Only staff 
authorised to work on this study will have access to participants' data. The PI will facilitate 
access to study records for monitoring, audits, and regulatory inspections. Participant's consent 
to this will be sought at the time of enrolment into the study.  
 
The Cambridge Integrated Data Environment (CAM:IDE) System will support digital 
management of data within the study. It will utilise the REDCap Safe Haven, which is suitable 
for personally identifiable data and anonymised research data and hosted on the Cambridge 
University Information Services (UIS) ISO 27001 accredited environment which is also NHS 
Toolkit Compliant. 
 
16.4 
Sample handling 
Induced sputum and serum will be processed and analysed in laboratory facilities on the 
Cambridge Biomedical Campus.  
Nasal epithelial cells will be collected, stored, and cultured in a laboratory on the Cambridge 
Biomedical Campus or within the University of Cambridge.  
 
 

 
     
    
       
 
         
 
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17. 
FINANCING AND INSURANCE 
17.1 Insurance and Liability  
CUH, as a member of the NHS Clinical Negligence Scheme for Trusts, will accept full financial 
liability for harm caused to participants in the clinical trial caused through the negligence of its 
employees and honorary contract holders. There are no specific arrangements for 
compensation should a participant be harmed through participation in the trial, but no-one has 
acted negligently.  
 
 
The University of Cambridge will arrange insurance for negligent harm caused because of 
protocol design and for non-negligent harm arising through participation in the clinical study.   
 
17.2 Research Costs 
The research is funded by the Medical Research Council (MRC; Reference Number 
MR/Y000935/1).  
 
17.3 Service Support Costs 
Not applicable 
 
17.4 Study Sponsorship 
This study is being jointly sponsored by the University of Cambridge and Cambridge University 
Hospitals NHS Foundation Trust.  
 

 
     
    
       
 
         
 
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18. 
PUBLICATION POLICY 
Results of this study will be submitted for publication in a peer-reviewed journal. The 
manuscript will be prepared by the PI in conjunction with the study team; authorship will be 
determined by agreement. 
 
Anonymised data containing genetic information will be deposited on publicly accessible 
databases in order to share and archive the data for the benefit of the wider scientific 
community. For example, data may be deposited with The European Genome-phenome 
Archive 
(EGA) 
(https://ega-archive.org/) 
or/and 
the 
Human 
Cell 
Atlas 
(https://www.humancellatlas.org/). 
 
 
 

 
     
    
       
 
         
 
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19. 
REFERENCES 
1. 
Papi A, Brightling C, Pedersen SE, Reddel HK. Asthma. The Lancet. 
2018;391(10122):783-800. 
2. 
Iwasaki A, Foxman EF, Molony RD. Early local immune defences in the respiratory 
tract. Nature Reviews Immunology. 2017;17(1):7-20. 
3. 
Ioannidis I, Ye F, McNally B, Willette M, Flaño E. Toll-Like Receptor Expression and 
Induction of Type I and Type III Interferons in Primary Airway Epithelial Cells. Journal of 
Virology. 2013;87(6):3261-70. 
4. 
Tengroth L, Millrud CR, Kvarnhammar AM, Kumlien Georén S, Latif L, Cardell L-O. 
Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal 
Epithelial Cells. PLOS ONE. 2014;9(6):e98239. 
5. 
Junt T, Barchet W. Translating nucleic acid-sensing pathways into therapies. Nature 
Reviews Immunology. 2015;15(9):529-44. 
6. 
Pockros PJ, Guyader D, Patton H, Tong MJ, Wright T, McHutchison JG, Meng T-C. 
Oral resiquimod in chronic HCV infection: Safety and efficacy in 2 placebo-controlled, double-
blind phase IIa studies. Journal of Hepatology. 2007;47(2):174-82. 
7. 
Mark Karen E, Corey L, Meng TC, Magaret Amalia S, Huang ML, Selke S, et al. 
Topical Resiquimod 0.01% Gel Decreases Herpes Simplex Virus Type 2 Genital Shedding: 
A Randomized, Controlled Trial. The Journal of Infectious Diseases. 2007;195(9):1324-31. 
8. 
Greiff L, Ahlström-Emanuelsson C, Alenäs M, Almqvist G, Andersson M, Cervin A, et 
al. Biological effects and clinical efficacy of a topical Toll-like receptor 7 agonist in seasonal 
allergic rhinitis: a parallel group controlled phase IIa study. Inflammation Research. 
2015;64(11):903-15. 
9. 
Tsitoura D, Ambery C, Price M, Powley W, Garthside S, Biggadike K, Quint D. Early 
clinical evaluation of the intranasal TLR7 agonist GSK2245035: Use of translational 
biomarkers to guide dosing and confirm target engagement. Clinical Pharmacology & 
Therapeutics. 2015;98(4):369-80. 
10. 
Delaney S, Biffen M, Maltby J, Bell J, Asimus S, Aggarwal A, et al. Tolerability in man 
following inhalation dosing of the selective TLR7 agonist, AZD8848. BMJ Open Respiratory 
Research. 2016;3(1):e000113. 
11. 
Progatzky F, Jha A, Wane M, Thwaites RS, Makris S, Shattock RJ, et al. Induction of 
innate cytokine responses by respiratory mucosal challenge with R848 in zebrafish, mice, 
and humans. J Allergy Clin Immunol. 2019;144(1):342-5.e7. 

 
     
    
       
 
         
 
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12. 
Jha A, Thwaites RS, Tunstall T, Kon OM, Shattock RJ, Hansel TT, Openshaw PJM. 
Increased nasal mucosal interferon and CCL13 response to a TLR7/8 agonist in asthma and 
allergic rhinitis. Journal of Allergy and Clinical Immunology. 2021;147(2):694-703.e12. 
13. 
Deprez M, Zaragosi L-E, Truchi M, Becavin C, Ruiz García S, Arguel M-J, et al. A 
Single-Cell Atlas of the Human Healthy Airways. American Journal of Respiratory and Critical 
Care Medicine. 2020;202(12):1636-45. 
14. 
Heijink IH, Nawijn MC, Hackett TL. Airway epithelial barrier function regulates the 
pathogenesis of allergic asthma. Clinical & Experimental Allergy. 2014;44(5):620-30. 
15. 
Jardine L, Wiscombe S, Reynolds G, McDonald D, Fuller A, Green K, et al. 
Lipopolysaccharide inhalation recruits monocytes and dendritic cell subsets to the alveolar 
airspace. Nature Communications. 2019;10(1):1999. 
16. 
Weiszhar Z, Horvath I. Induced sputum analysis: step by step. Breathe. 
2013;9(4):300-6. 
17. 
Camateros P, Tamaoka M, Hassan M, Marino R, Moisan J, Marion D, et al. Chronic 
Asthma–induced Airway Remodeling Is Prevented by Toll-like Receptor-7/8 Ligand S28463. 
American Journal of Respiratory and Critical Care Medicine. 2007;175(12):1241-9. 
18. 
Reuter S, Dehzad N, Martin H, Bohm L, Becker M, Buhl R, et al. TLR3 but Not TLR7/8 
Ligand Induces Allergic Sensitization to Inhaled Allergen. The Journal of Immunology. 
2012;188(10):5123-31. 
19. 
Meyer T, Surber C, French LE, Stockfleth E. Resiquimod, a topical drug for viral skin 
lesions and skin cancer. Expert Opinion on Investigational Drugs. 2013;22(1):149-59. 
 

 
     
    
       
 
         
 
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20. 
APPENDIX A: STUDY FLOWCHART 

 
     
    
       
 
         
 
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21. 
APPENDIX B: SCHEDULE OF PROCEDURES 
 
 
 
 
Procedure 
Screening 
Healthy 
(n = 24) 
Screening 
Asthma 
(n = 6) 
Day 1 
Day 2 
Day 8 
Visit 1 
Visit 2 
Visit 3 
0h 
Challenge 
15 min  
1 h 
4 h 
8 h 
24 h 
48 h 
Day 7 
Informed consent 
X 
X 
 
 
 
 
 
 
 
 
 
Demographics, Medical History, 
Medications 
X 
X 
 
 
 
 
 
 
 
 
 
General physical examination 
X 
X 
 
 
 
 
 
 
 
 
 
ECG and baseline laboratory tests 
X 
X 
 
 
 
 
 
 
 
 
 
DNA blood test + PBMC isolation 
X 
X 
 
 
 
 
 
 
 
 
 
Nasal brush 
X 
X 
 
 
 
 
 
 
 
 
 
Skin prick testing for aeroallergens 
X 
X 
 
 
 
 
 
 
 
 
 
Bronchodilator reversibility, methacholine 
challenge or test as per CUH policy 
 
X 
 
 
 
 
 
 
 
 
 
Induced sputum  
X 
7 Days Post 
Reversibility 
 
 
 
 
 
 
X 
 
 
Nasosorption 
 
 
X 
 
 
 
 
 
X 
 
 
Spirometry (FEV1) and Observations 
(temperature, pulse, blood pressure) 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
Serum 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Full blood count + C-Reactive Protein 
 
 
X 
 
 
 
X 
 
X 
X 
X 
Nebulisation with R848 or Saline 
 
 
 
X