Nasal Immune Challenge Study
Phase: NA
Conditions: Innate Inflammatory Response, Asthma, Nasal Allergy
Sponsor: Cambridge University Hospitals NHS Foundation Trust

 
 
    
 
 
 
         
 
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Nasal Immune Challenge Study 
 
Protocol 
 
Sponsor Reference Number:  
    R&D A095696 
 
Ethics Reference Number:  
 
    21/NE/0136 
Date and Version No:  
 
    04.07.22  Version 1.2 
Principal Investigator: 
Dr Akhilesh Jha 
Co-investigators: 
(in alphabetical order) 
Professor Clare Bryant 
Professor Menna Clatworthy 
Mr Matthew Coates 
Professor Andres Floto 
Dr Mattia Frontini 
Dr Martin Knolle 
 
Sponsors: 
             
Cambridge University Hospitals NHS Foundation Trust 
University of Cambridge 
 
 
Funders: 
Wellcome Trust 
Cambridge Academy of Therapeutic Sciences 
The Evelyn Trust 
Signatures: 
Dr Akhilesh Jha  
04th July 2022  
 
 
 
 
 
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 Chief Author/Investigator and / or Sponsor. 

 
 
    
 
 
 
         
 
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TABLE OF CONTENTS 
1. 
AMENDMENT HISTORY ......................................................................................................... 5 
2. 
SYNOPSIS .............................................................................................................................. 6 
3. 
ABBREVIATIONS .................................................................................................................... 7 
4. 
BACKGROUND AND RATIONALE .......................................................................................... 8 
5. 
OBJECTIVES ......................................................................................................................... 10 
5.1 
Primary Objectives .......................................................................................................... 10 
5.2 
Secondary Objectives ..................................................................................................... 10 
5.3 
Exploratory Objectives .................................................................................................... 10 
6. 
STUDY DESIGN .................................................................................................................... 11 
6.1 
Summary of Trial Design ................................................................................................ 11 
6.2 
Primary Outcome Measure ............................................................................................. 11 
7. 
TRIAL PARTICIPANTS .......................................................................................................... 13 
7.1 
Overall Description of Trial Participants .......................................................................... 13 
7.2 
Inclusion Criteria ............................................................................................................. 13 
7.3 
Exclusion Criteria ............................................................................................................ 14 
8. 
STUDY PROCEDURES ......................................................................................................... 15 
8.1 
Informed Consent ........................................................................................................... 16 
8.2 
Screening and Eligibility Assessment .............................................................................. 16 
8.3 
Subsequent Assessments .............................................................................................. 17 
8.4  
Definition of End of Trial .................................................................................................. 17 
8.5 
Discontinuation/Withdrawal of Participants from Study Treatment .................................. 17 
8.6 
Source Data .................................................................................................................... 18 
9. 
TREATMENT OF TRIAL PARTICIPANTS ............................................................................. 19 
9.1 
Description of Study Treatment ....................................................................................... 19 
9.2 
Storage of Study Equipment or Related apparatus ......................................................... 19 
9.3 
Compliance with Study Treatment .................................................................................. 19 
10. SAFETY REPORTING ........................................................................................................... 20 

 
 
    
 
 
 
         
 
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10.1 Definitions ....................................................................................................................... 20 
10.2 Reporting Procedures for All Adverse Events ................................................................. 21 
10.3 Reporting Procedures for Serious Adverse Events ......................................................... 21 
11. STATISTICS .......................................................................................................................... 23 
11.1 Description of Statistical Methods ................................................................................... 23 
11.2 The Number of Participants ............................................................................................ 23 
11.3 The Level of Statistical Significance ................................................................................ 23 
11.4 Procedure for Accounting for Missing, Unused, and Spurious Data. ............................... 23 
12. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS .......................................................... 24 
13. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES .................................... 25 
14. CODES OF PRACTICE AND REGULATIONS ....................................................................... 26 
14.1 Participant Confidentiality ............................................................................................... 26 
14.2 Other Ethical Considerations .......................................................................................... 26 
14.3 Informed Consent ........................................................................................................... 26 
14.4 Declaration of Helsinki .................................................................................................... 27 
14.5 ICH Guidelines for Good Clinical Practice ....................................................................... 27 
15. DATA HANDLING AND RECORD KEEPING ......................................................................... 28 
15.1  Data Handling ................................................................................................................. 28 
15.2 Data Collection Forms .................................................................................................... 28 
15.3  Data Quality and Security ............................................................................................... 28 
15.4 Sample handling ............................................................................................................. 28 
Nasal epithelial cells may also be collected, stored and cultured in a laboratory on the Cambridge 
Biomedical Campus or at other sites within the University of Cambridge. ................................. 28 
16. FINANCING AND INSURANCE ............................................................................................. 29 
16.1 Insurance and Liability ...................................................................................................... 29 
16.2 Research Costs .............................................................................................................. 29 
16.3 Service Support Costs .................................................................................................... 29 
16.4 Study Sponsorship .......................................................................................................... 29 

 
 
    
 
 
 
         
 
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17. PUBLICATION POLICY ......................................................................................................... 30 
18. REFERENCES ...................................................................................................................... 31 
19. APPENDIX A: STUDY FLOWCHART .................................................................................... 33 
20. APPENDIX B: SCHEDULE OF PROCEDURES .................................................................... 35 
21. Appendix C: TABLE of Symptoms from previous challenge study .......................................... 37 
 
 
 

 
 
    
 
 
 
         
 
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1. 
AMENDMENT HISTORY 
Amendment 
No. 
Protocol 
Version No. 
Date 
issued 
Author(s) of 
changes 
Details of Changes made 
1 
(non-
substantial) 
1.1 
18.05.22 
Akhilesh Jha 
Altering wording of post-nasal 
space biopsy to “a minimum of 6 
weeks prior to nasal challenge 
(pages 15, 17 and appendix B) 
and added in section 15.4 to 
clarify epithelial cells will be 
cultured 
and 
stored 
within 
Cambridge. 
2 
(non-
substantial) 
1.2 
04.07.22 
Akhilesh Jha 
Confirmed that only SARS-CoV-2 
lateral flow (rather than PCR) is 
required prior to nasal sampling 
(as per University Department of 
Medicine 
Biological 
Safety 
Committee Review). Changed 
wording on pages 13, 16 and 35  
 

 
 
    
 
 
 
         
 
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2. 
SYNOPSIS 
 
Title 
Nasal Immune Challenge Study 
Internal reference no 
A095696 
Design 
Open label design using nasal challenge with a topical spray using 
saline or a Toll-like receptor (TLR)7/8 agonist and viral analogue (R848) 
Participants 
Healthy non-allergic volunteers 
Volunteers with allergic rhinitis with/without asthma 
Sample Size 
6 volunteers for sample collection to enable optimisation of protocols 
24 for challenge (12 healthy and 12 allergic rhinitis with/without asthma) 
Duration 
2 years 
Aims 
1. To compare changes in gene expression at single-cell resolution in 
nasal mucosal samples and blood before and 24 hours after nasal 
TLR7/8 agonist (R848) challenge 
 
2. To compare differences in nasal mucosal and blood single-cell gene 
expression between healthy and allergic participants 24 hours after 
R848 nasal challenge. 
 
3. To assess systemic physiological measures and clinical tolerability of 
repeat R848 nasal challenge two weeks apart in healthy participants 
and those with allergic disease. 
 
4. To measure change in levels of cytokines and chemokines in nasal 
mucosal lining fluid after repeat R848 nasal challenge. 
 
5. To measure changes in peripheral immune (blood) responses to ex-
vivo stimulation with lipopolysaccharide (LPS) before and after nasal 
challenge with R848. 
 
Outcome Measures 
1. Increase in CXCL10 nasal mucosal gene expression at 24 hours 
after nasal challenge with R848 compared to baseline.  
 
2. Difference in CXCL10 nasal mucosal gene expression at 24 hours 
after R848 challenge between healthy subjects and those with 
allergic rhinitis. 
 
3. Change in CXCL10 nasal mucosal protein in nasal mucosal lining 
fluid over 24 hours expressed as area under curve (AUC) between 
first and second nasal challenge with R848. 
 
4. Change in participant’s temperature between first and second nasal 
challenge with R848 (as a marker of clinical tolerability). 
 
Eligibility 
Adult males and females aged 18 years and above will need to meet 
inclusion and exclusion criteria. Participants in this study will be 
recruited from Cambridge University Hospital, University of Cambridge, 
volunteer databases and advertising in the Cambridge area. 

 
 
    
 
 
 
         
 
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3. 
ABBREVIATIONS 
AE 
Adverse event 
AR 
Allergic Rhinitis 
ARwA 
Allergic Rhinitis with Asthma 
CUHFT 
Cambridge University Hospital Foundation Trust 
CI 
Chief Investigator 
CRA 
Clinical Research Associate (Monitor) 
CCRC 
Cambridge Clinical Research Centre 
CRF 
 
Case Report Form 
CRO  
Contract Research Organisation 
CT 
 
Clinical Trials 
EC 
 
Ethics Committee (see REC) 
GCP 
Good Clinical Practice 
GP 
General Practitioner 
ICF 
Informed Consent Form 
LPS 
Lipopolysaccharide 
NHS 
National Health Service 
NRES 
National Research Ethics Service  
PI 
Principal Investigator 
PIL/S 
Participant/ Patient Information Leaflet/Sheet 
PNIF 
Peak Nasal Inspiratory Flow 
R&D 
NHS Trust R&D Department 
REC 
Research Ethics Committee 
R848 
Resiquimod 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reactions 
TMF 
Trial Master File 
TNSS 
Total Nasal Symptom Score 
 
 

 
 
    
 
 
 
         
 
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4. 
BACKGROUND AND RATIONALE 
 
Background 
Acute respiratory viral infections cause significant morbidity, especially in vulnerable 
individuals and is a topic of immense significance during the current 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 allergic asthma, therapeutic options for treating asthma exacerbations 
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 innate 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.   
 
Our understanding of the role of innate immunity to infection has recently undergone a 
paradigm shift as it is now recognized that innate cells (and not just adaptive immune cells) 
have the capacity to undergo “training” after infection via metabolic changes and epigenetic 
reprogramming (3). Pathogens can therefore modulate chromatin structure and the 
transcriptional programme of innate immune cells, which can result in altered but non-specific 
responses upon reinfection either with the same or heterologous stimuli. These epigenetic 
mechanisms may be particularly relevant in understanding how viral triggers (e.g. influenza) 
can lead to increased susceptibility to bacterial infections and cause pneumonia (e.g. due to 
streptococcus spp.) (4). 
 
Most studies have focused on peripheral immune cells such as macrophages, but the concept 
of innate immune training has been extended to long-lived non-immune skin epithelial stem 
cells, which have the capacity for “inflammatory memory” (5). Allergic inflammatory memory 
has also recently been established in respiratory progenitor cells (6). Murine studies have 
highlighted the potential for structural cells (such as epithelial cells) to have the potential to 
mount immune responses (7). However, mechanisms of innate immune “memory” in human 
respiratory mucosal epithelial and immune cells in response to microbes and pathogen 
associated molecular patterns (PAMPs) are not well understood despite the respiratory tract 
being under a constant barrage of infectious stimuli.  
 
Toll-like receptors (TLRs) play a critical role in the initial detection of microbes and PAMPs. 
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 the 
innate immune response to viral infections, as well as helping shape adaptive immunity (8–
10). Resiquimod (R848) is a single-stranded RNA viral analogue that binds to TLR7/8. When 
given systemically to treat human hepatitis C infection, it causes cytokine release and a flu-
like syndrome (11). However, R848 has been successfully employed topically as a skin cream 
for actinic keratosis (12). 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 (13, 14). 
 
Rationale for current study 
Intranasal R848 administration has now been established in a human challenge model in 
healthy volunteers and in those with allergic rhinitis (AR) and allergic rhinitis with asthma 
(ARwA) by the chief investigator (15). In total, 44 volunteers have received intranasal R848 at 
a range of doses; 9 healthy volunteers received 10µg per nostril (n=9) with one person 
receiving 100µg per nostril (n=1); subsequently 35 volunteers received between 1-2µg per 
nostril based on weight (0.02µg/kg, healthy n=12, AR n=12, ARwA n=11). This dose was well 

 
 
    
 
 
 
         
 
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tolerated by all volunteers with minimal nasal symptoms and no significant change in airflow 
obstruction (FEV1) in volunteers with asthma. Intranasal R848 was well tolerated systemically 
with no change in physiological measures or serum inflammatory mediators compared to 
saline challenge. The study demonstrated that individuals with AR and ARwA have increased 
nasal mucosal interferon and chemokine responses compared to healthy volunteers. This is 
coupled with the observation that there is a small and transient reduction in peripheral 
eosinophil counts at 4 hours and in lymphocyte counts at 24 hours, a finding limited to 
volunteers with AR and ARwA. This suggests that leukocyte trafficking may be occurring from 
the peripheral blood to the nasal mucosa. These findings highlight that dysregulated mucosal 
innate immune responses are likely to be important in determining the clinical outcome of viral 
triggers.  
 
R848 nasal challenge (up to 2µg per nostril) is therefore a practical, tolerable, and non-
invasive method to stimulate and investigate TLR-driven respiratory mucosal innate immune 
responses in both health and disease.  
 
Defining the cellular basis for TLR-driven innate immunity will help provide mechanistic 
insights into how these responses differ between individuals, its dysregulation in disease (such 
as during an asthma exacerbation) and susceptibility to secondary infections. Understanding 
the epigenetic mechanisms which regulate interferon and chemokine production may help 
identify the molecular drivers of excessive or dampened inflammation, helping to refine 
potential therapeutic targets. Given the current COVID-19 pandemic, the clinical translational 
model utilised in this study allows for a practical and straightforward method to assess the 
host factors which determine respiratory mucosal innate immunity.  
 
The goal of this project is to utilize an established tolerable human nasal TLR agonist 
challenge model combined with single-cell transcriptomic and epigenetic techniques to 
precisely characterize innate immune responses and mechanisms of innate immune training 
in airway epithelial and immune cells. Samples will be collected from the nasal mucosa and 
blood before and after saline and R848 challenge. Cell cultures from airway epithelium and 
blood will also be established to assess the functional impact of R848 on cellular response to 
repeat stimulation with viral and bacterial ligands. Study populations will include healthy 
participants and those with allergic rhinitis with/without asthma in order to investigate how in-
vivo innate immunity is altered in these populations. Some participants will also undergo 
repeat nasal challenge to assess mechanisms of innate immune memory in the respiratory 
tract. 

 
 
    
 
 
 
         
 
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5. 
OBJECTIVES  
 
5.1 
Primary Objectives 
 
To compare changes in gene expression at single-cell resolution in nasal mucosal samples 
and blood before and 24 hours after nasal TLR7/8 agonist (R848) challenge 
 
 
5.2 
Secondary Objectives 
 
To compare differences in nasal mucosal and blood single-cell gene expression between 
healthy and allergic participants 24 hours after R848 nasal challenge. 
 
To assess systemic physiological measures and clinical tolerability of repeat R848 nasal 
challenge two weeks apart in healthy participants and those with allergic disease. 
 
To measure change in levels of cytokines and chemokines in nasal mucosal lining fluid after 
repeat R848 nasal challenge. 
 
To measure changes in peripheral immune (blood) responses to ex-vivo stimulation with 
lipopolysaccharide (LPS) before and after nasal challenge with R848. 
 
 
5.3 
Exploratory Objectives 
 
To establish cell culture models from primary nasal epithelium and immune cells to understand 
the functional impact of R848 on cellular responses to repeat stimulation with viral and 
bacterial ligands. 
 
 

 
 
    
 
 
 
         
 
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6. 
STUDY DESIGN 
6.1 
Summary of Trial Design 
 
The first stage of the study will include up to 6 participants (healthy or allergic disease) to 
undergo nasal mucosal tissue and blood collection without any nasal challenge. These 
samples will be used to optimise the protocols required for downstream processing, e.g. for 
single cell and epigenetic analysis. 
 
The main component of the study is an open label design employing nasal challenge with a 
topical spray using saline or a TLR7/8 agonist R848. The aim is to perform deep 
immunophenotyping of nasal mucosal tissue and peripheral blood before and after nasal 
challenge with R848 and compare responses between non-allergic and allergic individuals. 
Some participants will undergo repeat challenge two weeks later with assessment of cytokine, 
chemokine, and gene expression profiles to determine which cells contribute to innate immune 
memory of previous exposures and how this may be altered in disease.  
 
After an initial screening visit to determine eligibility, participants constituting equal numbers 
of non-allergic and allergic individuals will undergo nasal challenge with R848 (n=24). They 
will have sampling performed for up to 24 hours and can choose to go home or stay overnight 
in the Cambridge Clinical Research Centre (CCRC). This will be the end of the study for half 
of the participants.  
 
The remaining half will be invited to subsequently undergo repeat saline (n=2) or R848 (n=10) 
challenge approximately two weeks later to determine clinical tolerability, assess for 
exaggerated or dampened nasal mucosal immune responses and investigate mechanisms of 
innate immune memory, and how they may differ between the groups.  
 
The maximum duration for participants in the study are as follows (Appendix A): 
Single challenge study (n=12): one day  
Double challenge study (n=12): two days spread two weeks apart 
 
6.2 
Primary Outcome Measure 
 
1. Increase in CXCL10 nasal mucosal gene expression at 24 hours after nasal 
challenge with R848 compared to baseline  
 
 
Secondary Outcome Measures 
 
1. Difference in CXCL10 nasal mucosal gene expression at 24 hours after R848 
challenge between healthy subjects and those with allergic rhinitis  
 
2. Change in participant’s temperature between first and second nasal challenge 
with R848 (as a marker of clinical tolerability) 
 
3. Difference in CXCL10 nasal mucosal protein in nasal mucosal lining fluid over 
24 hours expressed as area under curve (AUC) between first and second nasal 
challenge with R848. 
 
 
 
 
Exploratory Outcome Measures 
 

 
 
    
 
 
 
         
 
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1. Difference in CXCL10 after ex-vivo stimulation of blood samples with LPS before 
and 24 hours after nasal challenge with R848 
 
2. Single-cell gene expression and epigenetic profiles in mucosal tissue and blood 
before and after nasal challenge with R848  
 
3. Comparison of single-cell and epigenetic profiles between healthy and allergic 
disease groups  
 

 
 
    
 
 
 
         
 
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7. 
TRIAL PARTICIPANTS 
7.1 
Overall Description of Trial Participants 
 
Adults will be considered for inclusion in this study. They will either be healthy individuals 
with no history of allergy, or they will have a history of allergic rhinitis (hay fever) 
with/without physician diagnosis of asthma.  
 
For the sample optimisation phase of the study (n=6), volunteers will only be required to 
provide nasal mucosal tissue (using curettage and or brush) and a blood sample and will 
therefore not undergo a detailed medical screening visit. 
 
For participants undergoing nasal challenge, they will be recruited within the parameters of 
the following criteria: 
 
7.2 
Inclusion Criteria 
 
• 
Participant is willing and able to give informed consent for participation in the study. 
• 
Male or female aged 18 years and above 
• 
Able (in the Investigators opinion) and willing to comply with all study requirements. 
• 
Willing to allow his or her General Practitioner and consultant, if appropriate, to be 
notified of 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 
• 
Participant has clinically acceptable laboratory and ECG at enrolment.  
• 
Negative lateral flow or polymerase chain reaction (PCR) test for SARS-CoV-2 
• 
For healthy volunteers: 
- 
No clinical history of allergic rhinitis, asthma or eczema 
- 
Negative skin prick tests or specific IgE response to a panel of common 
aeroallergens: cat, dog, grass pollen, tree pollen, house dust mite, fungal 
spores 
- 
Normal blood eosinophil count (< 300 cells/μL) 
- 
Normal baseline forced expiratory volume (FEV1) i.e. ≥80% 
• 
For volunteers with allergic rhinitis with or without asthma: 
- 
A clinical history of allergic rhinitis symptoms (sneezing, runny or itchy nose) 
in response to aeroallergens 
- 
At least one positive skin-prick test or specific IgE response to a panel of 
common aeroallergens: cat, dog, grass pollen, tree pollen, house dust mite, 
fungal spores 
- 
Pre-bronchodilator FEV1 ≥50% predicted 
- 
Participants are permitted to have physician-diagnosed mild to moderate 
asthma which is not poorly controlled as evidenced by an Asthma Control 
Questionnaire (ACQ-5) score of ≤1.5. 
- 
If they have asthma, they are permitted to be on inhaled corticosteroid (ICS) 
and a long-acting beta agonist (LABA), but no other controller medication.  
• 
Have had no other courses of medication including nasal and systemic 
corticosteroids, whether prescribed or over-the-counter, in the four weeks before first 
study dose other than mild analgesia, vitamins and mineral supplements or, for 
females, oral contraceptives. 
 

 
 
    
 
 
 
         
 
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7.3 
Exclusion Criteria 
• 
Recent infections in past 14 days before screening:  especially upper respiratory 
tract illnesses (including colds and influenza), sore throats, sinusitis, infective 
conjunctivitis.  
• 
Lower respiratory tract infection in past 28 days 
• 
Nasal anatomical defects, precluding use of nasal sampling techniques  
• 
The participant may not enter the study if any of the following apply: 
- 
Female participants who are pregnant, lactating or planning pregnancy during the 
study. 
- 
Respiratory diseases (other than hay fever or asthma where specified) 
- 
Significant medical history of hepatic, cardiovascular, gastrointestinal, renal, 
endocrine, infective, haematological, autoimmune, metabolic, rheumatological, 
neurological, dermatological or neoplastic conditions 
- 
Extreme obesity (BMI >40) 
- 
Depression and psychiatric disorders 
• 
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 
• 
Smoking in previous 6 months 
 
 

 
 
    
 
 
 
         
 
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8. 
STUDY PROCEDURES 
Pictures of the study procedures are outlined in appendix A and an outline of study procedures 
in relation to study visits is outlined in appendix B. 
 
Nasal lavage: 10ml of normal saline will be administered and collected gently through a 
disposable plastic olive-shaped device into both nostrils prior to each challenge. This acts to 
“clean” the nasal mucosa of nasal debris prior to challenge and the nasal washing will be 
discarded. 
 
Nasosorption: Nasal mucosal lining fluid will be collected non-invasively using nasosorption 
initially regularly (0min, 15min, 30min, 1h, 2h) then at two-hourly intervals (4h, 6h, 8h, 24h) 
after each challenge to assess the kinetics of interferon, cytokine and chemokine production.  
 
Nasal mucosal epithelium: Nasal mucosal epithelial cells will be collected at the 
screening visit, baseline (0h) and 24 hours after each challenge to assess 
transcriptional and epigenetic signatures and to culture epithelial cells in the lab. 
Therefore, a maximum of up to three sets of samples per volunteer will be taken from 
half of the group undergoing single challenge (n=12), whilst the remainder of the 
group undergoing double challenge will have a total of five sets of samples taken 
(n=12). Up to five sets of nasal curettage samples were taken per volunteer in a prior 
study using R848 with good tolerability and no withdrawal of participants from the 
study15. Topical anaesthetic spray may be offered prior to procedures. 
- 
Nasal brushings are taken using a small cytology brush from the nasal 
inferior turbinate – up to two samples will be taken (one from each nostril).  
- 
Nasal curettage involves the use of a small plastic curette to gently “scrape” 
a small amount of tissue from the nasal inferior turbinate – up to four samples 
will be taken (two from each nostril). 
Post-nasal space biopsy will be an optional procedure in order to obtain a small 
sample of tissue (0.5cm) taken from the postnasal space using endoscope and 
forceps under topical local anaesthetic. It will be performed by a trained Ear, Nose 
and Throat (ENT) surgeon (Mr Coates) in a specific area of Addenbrookes hospital 
with dedicated facilities for the procedure. The purpose of this is to obtain a larger 
and deeper volume of tissue than the nasal curettage/brushings to facilitate detailed 
molecular phenotyping particularly of immune cell subsets, which are found 
underneath the epithelial layer. The procedure is generally well tolerated, and 
complications are rare. This procedure is more invasive than performing nasal 
curettage/brushings and will therefore only be performed twice in up to 12 people. 
These participants will only undergo a single nasal challenge and not the second 
repeat challenge. Volunteers who agree to have the procedure will have the first 
sample collected at the screening visit. There will then be a minimum of 6-weeks (to 
ensure wound healing) before performing nasal challenge with saline (up to n=2) or 
R848 (up to n=10), and a subsequent post-nasal space biopsy performed 24 hours 
after challenge. 
 
Blood: Blood will be collected on the following occassions: 
- Screening visit for routine safety and eligibility (9ml) and DNA analysis (8.5ml) 
- Before (0h) and after (24h) each nasal challenge for single cell analysis and Truculture (13ml 
each time-point)  
- Before (0h) and twice after (4h, 24h) nasal challenge for serum and full blood count (9ml 
each time point).  
 
This means that half of the volunteers undergoing single challenge (n=12) will provide a total 
of approximately 70ml blood over three visits and the remaining half undergoing 2 challenges 
(n=12) will provide a total of approximately 125ml blood over five visits. 

 
 
    
 
 
 
         
 
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8.1 
Informed Consent 
 
The participant must personally sign and date the latest approved version of the informed 
consent form before any study specific procedures are performed. 
 
Written and verbal versions of the participant information and Informed consent will be 
presented to the participants detailing no less than: the exact nature of the study; the 
implications and constraints of the protocol; the known side effects and any risks involved in 
taking part.  It will be clearly stated that the participant is 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. 
 
The participant will be allowed as much time as wished to consider the information, and the 
opportunity to question the Investigator, their GP or other independent partiefs to decide 
whether they will participate in the study.  Written Informed Consent will then be obtained by 
means of participant dated signature and dated signature of the person who presented and 
obtained the informed consent. The person who obtained the consent must be suitably 
qualified and experienced and have been authorised to do so by the Chief/Principal 
Investigator as detailed on the Delegation of Authority and Signature log for the study. The 
original signed form will be retained at the study site within the Trial Master File (TMF) or 
Investigator Site File (ISF). A copy of the signed Informed Consent will be given to participants 
and a copy retained in the participant medical notes.   
 
8.2 
Screening and Eligibility Assessment 
 
Participants in this study will be recruited from Cambridge University Hospital (CUHFT), 
University of Cambridge, volunteer databases and advertising in the Cambridge area. 
Participants from CUHFT 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 poster. Pre-screening of interested 
potential participants will involve telephone screening and if potentially eligible they will receive 
the participant information sheet and consent form by email at least 24 hours prior to the 
screening visit. 
 
A comprehensive  screening visit will be undertaken at the CCRC to evaluate the following:  
Demographics: date of birth, gender, race, smoking and drinking habits  
Medical History: Details of any history of disease or surgical interventions and a systems 
review to determine inclusion/exclusion criteria 
Concomitant Medication: All over-the-counter or prescription medication, vitamins, and/or 
herbal supplements will be recorded on CRFs.  
Physical Examination as well as physiological observations, height, weight and oral 
temperature will be recorded. 
ECG Test: A 12-lead ECG will be taken for each participant.  At least the following ECG 
parameters will be recorded: heart rate (HR), PR, QT and QRS intervals and QTC.  The report 
will be signed by the Investigator who will record in the CRF whether it is normal, abnormal 
but not clinically significant, or abnormal AND clinically significant.  In the latter case the 
eligibility of the participants will be reviewed. 
Laboratory Tests: Biochemistry, full blood count, liver function test, total IgE, skin prick 
allergy testing, pregnancy test (for women) will be performed.  
COVID Testing: All participants will be required to be free of any potential symptoms of 
COVID (fever, cough, anosmia) for at least 14 days prior to enrolment and not have any history 
of long-term symptoms due to COVID. A negative SARS-CoV-2 lateral flow or PCR test will 
be required prior to nasal sampling procedures. Staff and students from the University of 

 
 
    
 
 
 
         
 
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Cambridge as well as staff from CUHFT who are undergoing routine surveillance screening 
for COVID-19 and who test negative for the disease will also be invited to participate in the 
study. 
 
All laboratory results will be reviewed by the Investigator who will record in the CRF whether 
they are normal, abnormal but not clinically significant, or abnormal AND clinically significant.  
In the latter case the eligibility of the participants will be reviewed. 
 
 
Nasal curettage and brushings: This will be collected from all volunteers 
 
Post-nasal space biopsy: For up to half the group (n=12) who volunteer for post-nasal space 
biopsies to be collected, a negative COVID test is required prior to sampling. These 
participants will undergo nasal challenge experiments a minimum of 6-weeks after the biopsy 
to allow for adequate time for wound healing.  
 
 
8.3 Subsequent Assessments 
 
All study visits and procedures are outlined in appendix B – the following details pertain to 
participants undergoing nasal challenge: 
 
Visit 1 This visit will take place as close to a negative COVID test result. They will undergo a 
brief medical assessment including baseline physiological observations. They will then 
undergo nasal lavage followed by nasal curettage/brushing and blood sampling.  
 
Subsequently, nasal challenge with saline (n=4) or R848 (n=20) will then be performed and 
non-invasive nasosorption samples will be taken every 2 hours until 8 hours after challenge. 
Physiological observations, total nasal symptom scores (TNSS) and peak nasal inspiratory 
flow (PNIF) will also be assessed 2-hourly. Blood will be collected for white blood cell 
differential and serum 4 hours after nasal challenge. 
 
Participants can then choose to go home or have the option of remaining at the CCRC 
overnight as it has the facilities to provide this support (e.g. if the patient has travelled from 
further away).  
 
Visit 2 This will take place at 24h with collection of nasosorption, curettage/brushing and a 
blood sample along with assessment of physiological observations, TNSS and PNIF. 
 
For up to half the group who volunteered for an initial post-nasal space biopsy (6 weeks 
earlier), a second post-nasal biopsy will be undertaken. For these participants this will be the 
end of the study. 
 
Visit 3 For the remaining half of the participants (n=12), they will undergo a second nasal 
challenge with saline (n=2) or R848 (n=10) to investigate mechanisms of innate immune 
memory in epithelial cells. All assessments and procedures will be identical to visit 1. 
 
Visit 4 All study assessments and procedures will the same as visit 2. 
 
 
8.4  Definition of End of Trial 
 
The end of trial is the date of the last visit  of the last participant.  
 
 
8.5 Discontinuation/Withdrawal of Participants from Study Treatment 
 
 

 
 
    
 
 
 
         
 
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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) 
• 
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.   
 
 
8.6 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 
history and previous and concurrent medication may be summarised into the CRF), 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/code, not by name. 
 
 

 
 
    
 
 
 
         
 
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9. 
TREATMENT OF TRIAL PARTICIPANTS 
9.1 
Description of Study Treatment  
 
 
 
 
9.2 
Storage of Study Equipment or Related apparatus 
 
R848 will be stored in labelled storage vials in the laboratory freezer 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. 
 
9.3 
Compliance with Study Treatment 
 
Participants will be administered saline or R848 via a nasal spray directly by the investigators 
and therefore there will be no issues in assessing compliance. 
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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10. 
SAFETY REPORTING 
10.1 
Definitions 
 
10.1.1 Adverse Event (AE) 
An AE or adverse experience is any untoward medical occurrence in a participant, 
which does not necessarily have to 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. 
 
10.1.2 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. 
 
10.1.3 Expected Adverse Events and Serious Adverse Events Exempt from Recording 
 
The nasal sampling procedures outlined in this document have been performed on 
numerous occasions by the PI (>300 times by Akhilesh Jha, AJ) and Co-I (ENT 
surgeon Matthew Coates, MC) and are very well tolerated. Nasal curettage has also 
been performed in >1200 young people including babies as young as 5 days old and 
was well tolerated (16).  
 
Expected Adverse Events from nasal procedures include the following: 
• 
Nasal irritation and runny nose 
• 
Minor epistaxis  
• 
Watering of the eyes  
• 
Sneezing  
• 
Transient bitter taste if topical anaesthesia is used 
 
Obtaining a post-nasal space biopsy is more invasive than performing nasal 
curettage/brushings but the use of topical local anaesthetic means that it is generally 

 
 
    
 
 
 
         
 
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well tolerated. Prolonged epistaxis, requiring nasal packing and/or admission to 
hospital, is rare following this procedure. 
 
Nasal R848 challenge (higher dose 10µg/nostril n=9, and low dose 1-2µg/nostril 
n=35) is well tolerated with no reported SAEs (a mixture of healthy, AR and ARwA 
participants). Given that R848 is a substance that binds to the same pattern 
recognition receptors as influenza, some of these participants experienced transient 
flu-like symptoms such as: 
 
• 
Runny nose 
• 
Mild headache 
• 
Lethargy  
• 
Myalgia  
 
Symptoms diminished over a 24 to 36-hour period and there were no changes in 
systemic physiological observations (temperature, pulse and blood pressure) at 
multiple time-points after R848 challenge15. A table of symptoms from the previous 
nasal challenge study is provided in appendix C. 
 
The current study will assess the impact of two nasal challenges with R848 
administered two weeks apart. The second nasal challenge is expected to induce 
similar flu-like symptoms to the first challenge. However, one of the main outcomes 
of the study is to assess the tolerability of the second challenge. Therefore, a small 
increase in the severity of flu-like symptoms (e.g. runny nose, headache, lethargy, 
myalgia, fever) may be expected.  
 
10.1.4 Suspected Unexpected Serious Adverse Reactions (SUSAR) 
A serious adverse reaction, the nature or severity of which is not consistent with the 
applicable product information  
 
10.2 
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, other suspect device 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 (see section 7.7).  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.  
 
10.3 
Reporting Procedures for Serious Adverse Events 
All SAEs, except those expected ones defined in section 10.1.3 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 

 
 
    
 
 
 
         
 
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the appropriate reporting form and the contact details on there. Additional information 
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. The PI will inform all investigators concerned of relevant 
information about SUSARs that could adversely affect the safety of participants. 
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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11. 
STATISTICS 
11.1 
Description of Statistical Methods 
 
 
This is an open label interventional trial to investigate basic mechanisms of innate immunity. 
The following data outputs will be generated: 
 
- Single-cell transcriptomic signatures 
 
- Epigenetic signatures  
 
- Nasal and blood cytokine levels  
 
This data is typically of a non-parametric distribution and the statistical plan will follow a similar 
template to the previous publication using R848 challenge (15). For the single-cell and 
epigenetic datasets, a series of pre-processing quality control checks will take place followed 
by cell and gene‐level downstream analysis with the support of specialised bioinformaticians.  
 
For protein analysis, data will be plotted for individual cytokines using raw data at an individual 
level. For the repeat R848 challenge, area under curve (AUC) values will be calculated for 
each cytokine and paired Wilcoxon signed-rank test will be used to compare differences 
between each challenge with the hypothesis that repeat R848 challenge causes increased 
responses on secondary challenge.  
 
AUC values of cytokine production after R848 challenge will also be compared between 
healthy and allergic participants using unpaired Mann-Whitney test. 
 
11.2 
The Number of Participants 
 
 
In the prior publication using R848 nasal challenge (15), 12 participants in each group (healthy, 
allergic rhinitis and asthma) were sufficient to see differences in bulk gene expression and 
immune mediators in the nasal mucosa. The present study is based on utilising a powerful 
single-cell transcriptomic approach which is able to distinguish differences in immune 
responses at an individual cellular level. It may therefore be possible to see differences 
between groups in fewer numbers of participants but will allow recruitment of up to 12 
participants in each group.   
 
11.3 
The Level of Statistical Significance 
 
 
 
A P-value of < 0.05 will be taken to indicate statistical significance. 
 
11.4 
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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12. 
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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13. 
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 programme to which this study will be subject.  
 
 
 

 
 
    
 
 
 
         
 
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14. 
CODES OF PRACTICE AND REGULATIONS 
14.1 
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 an electronic database.  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. 
 
14.2 
Other Ethical Considerations 
 
The study will not be initiated before the protocol, and all study relevant material such 
as the informed consent forms and participant information sheets 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. 
 
14.3 
Informed Consent 
 
 
 
It is the responsibility of the Chief Investigator and Principal Investigator, or a person 
designated by the CI or PI (if acceptable by local regulations), to obtain written 
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 consent process will be documented in the patient’s notes.  
 
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 CI/PI/delegate and the participant shall both sign and date the Informed Consent 
Form before the person can participate in the study. 
 
The participant will keep a copy of the PIS and a signed and dated Consent Form. 
The original will be retained in the Trial Master File. A second copy, along with the 
PIS, will be filed in the participant's medical notes and a signed and dated note made 
in the notes of when the PIS was provided, and that informed consent was obtained 
for the study. 
 
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. 
 

 
 
    
 
 
 
         
 
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14.4 
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 October 2000, with additional footnotes 
added 2002 and 2004). 
 
14.5 
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 (CPMP/ICH/135/95) July 
1996. 
 

 
 
    
 
 
 
         
 
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15. 
DATA HANDLING AND RECORD KEEPING 
15.1  Data Handling 
 
 
 
All data will be handled within the conditions of the Data Protection Act 2018 
 
15.2 
Data Collection Forms  
 
Enrolment into the study will be documented in each participants’ medical notes.  
Data collection will comprise of a paper case report form, including participant 
characteristics, disease severity, medication lists, clinical examination, pulmonary 
and blood test results. 
 
15.3  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 Principal 
Investigator 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.  
 
15.4 
Sample handling 
 
Nasal epithelial cells may also be collected, stored and cultured in a laboratory on the 
Cambridge Biomedical Campus or at other sites within the University of Cambridge. 
 
 
 
 
 
 

 
 
    
 
 
 
         
 
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16. 
FINANCING AND INSURANCE 
16.1 Insurance and Liability  
 
CUHFT, 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.   
 
16.2 
Research Costs 
 
The research is funded by Wellcome Trust and Cambridge Academy of Therapeutic 
Sciences 
via 
a 
Developing 
Concept 
Fund 
grant 
(Reference 
number 
RG93172/BRYANT/34636), Wellcome Trust Investigator Award to Professor 
Clatworthy (220268/Z/20/Z) and through the Evelyn Trust (21/05). 
 
16.3 
Service Support Costs 
 
Not applicable 
 
16.4 
Study Sponsorship 
 
   
This study is being jointly sponsored by the University of Cambridge and CUHFT.  
 
 

 
 
    
 
 
 
         
 
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17. 
PUBLICATION POLICY 
Results of this study will be submitted for publication in a peer-reviewed journal. The 
manuscript will be prepared by the Principal Investigator 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 will 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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18. 
REFERENCES 
 
1. Papi A, Brightling C, Pedersen SE, Reddel HK. Asthma. The Lancet 2018;391:783–800. 
2. Iwasaki A, Foxman EF, Molony RD. Early local immune defences in the respiratory tract. 
Nature Reviews Immunology 2017;17:7–20. 
3. Netea MG, Schlitzer A, Placek K, Joosten LAB, Schultze JL. Innate and Adaptive Immune 
Memory: an Evolutionary Continuum in the Host’s Response to Pathogens. Cell Host & 
Microbe 2019;25:13–26. 
4. Aguilera ER, Lenz LL. Inflammation as a Modulator of Host Susceptibility to Pulmonary 
Influenza, Pneumococcal, and Co-Infections. Front Immunol 2020;11:. 
5. Naik S, Larsen SB, Gomez NC, Alaverdyan K, Sendoel A, Yuan S, Polak L, Kulukian A, 
Chai S, Fuchs E. Inflammatory memory sensitizes skin epithelial stem cells to tissue 
damage. Nature 2017;550:475–480. 
6. Ordovas-Montanes J, Dwyer DF, Nyquist SK, Buchheit KM, Vukovic M, Deb C, Wadsworth 
MH, Hughes TK, Kazer SW, Yoshimoto E, Cahill KN, Bhattacharyya N, Katz HR, Berger B, 
Laidlaw TM, Boyce JA, Barrett NA, Shalek AK. Allergic inflammatory memory in human 
respiratory epithelial progenitor cells. Nature 2018;560:649. 
7. Krausgruber T, Fortelny N, Fife-Gernedl V, Senekowitsch M, Schuster LC, Lercher A, 
Nemc A, Schmidl C, Rendeiro AF, Bergthaler A, Bock C. Structural cells are key regulators 
of organ-specific immune responses. Nature 2020;583:296–302. 
8. Ioannidis I, Ye F, McNally B, Willette M, Flano E. Toll-Like Receptor Expression and 
Induction of Type I and Type III Interferons in Primary Airway Epithelial Cells. Journal of 
Virology 2013;87:3261–3270. 
9. 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. In: Alexopoulou L, editor. PLoS ONE 2014;9:e98239. 
10. Junt T, Barchet W. Translating nucleic acid-sensing pathways into therapies. Nature 
Reviews Immunology 2015;15:529–544. 
11. 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;47:174–182. 
12. Mark KE, Corey L, Meng T, Magaret AS, Huang M, Selke S, Slade HB, Tyring SK, 
Warren T, Sacks SL, Leone P, Bergland VA, Wald A. Topical Resiquimod 0.01% Gel 

 
 
    
 
 
 
         
 
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Decreases Herpes Simplex Virus Type 2 Genital Shedding: A Randomized, Controlled 
Trial. The Journal of Infectious Diseases 2007;195:1324–1331. 
13. Greiff L, Ahlström-Emanuelsson C, Alenäs M, Almqvist G, Andersson M, Cervin A, Dolata 
J, Lindgren S, Mårtensson A, Young B, Widegren H. 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;64:903–915. 
14. 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;98:369–380. 
15. Jha A, Thwaites RS, Tunstall T, Kon OM, Shattock RJ, Hansel TT, Openshaw PJ. 
Increased nasal mucosal interferon and CCL13 response to a TLR7/8 agonist in asthma 
and allergic rhinitis, Journal of Allergy and Clinical Immunology; 2020; In Press, 
doi.org/10.1016/j.jaci.2020.07.012 
16. Jalowayski AA, Walpita P, Puryear BA, Connor JD. Rapid detection of respiratory 
syncytial virus in nasopharyngeal specimens obtained with the rhinoprobe scraper. Journal 
of Clinical Microbiology 1990;28:738–741. 

 
 
    
 
 
 
         
 
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19. 
APPENDIX A: STUDY FLOWCHART 
 
 
 
               
 
 

 
 
    
 
 
 
         
 
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20. 
APPENDIX B: SCHEDULE OF PROCEDURES 
TNSS Total Nasal Symptom Score; PNIF Peak Nasal Inspiratory Flow 
Procedures 
Visits 
Screening 
Visit 1 (duration 10h)  
1st nasal challenge 
Visit 2 (at 24h)  
1st nasal challenge 
Visit 3 (duration 10h) 
2nd nasal challenge 
Visit 4 (at 24h) 
2nd nasal challenge 
Informed consent 
X 
 
 
 
 
Demographics, Medical History and Medications 
X 
 
 
 
 
General physical examination 
X 
 
 
 
 
ECG and Laboratory tests 
X 
 
 
 
 
DNA blood test (up to 8.5mL) 
X 
 
 
 
 
SARS-CoV-2 Lateral Flow or PCR nasal swab 
 
X (negative test prior to 
nasal sampling) 
 
X (negative test as close 
as possible to sampling) 
 
1st Nasal challenge with saline (n=4) or R848 (n=20) 
 
X 
 
 
 
2nd Nasal challenge with saline (n=2) or R848 (n=10) 
 
 
 
X 
 
Vital signs, clinical assessment and symptom scores 
 
X (2 hourly) 
X 
X (2 hourly) 
X 
TNSS and PNIF 
 
X (2 hourly) 
X 
X (2 hourly) 
X 
Nasal lavage 
 
X (0h) 
 
X (0h) 
 
Nasosorption (filter strips to absorb mucosal fluid) 
 
X (15min, 30min, 1hr, 
2hr, then 2 hourly) 
X 
X (15min, 30min, 1hr, 
2hr, then 2 hourly) 
X 
Nasal curettage 
X 
X 
X 
X 
X 
Nasal brushing 
X 
X 
X 
X 
X 
Post-nasal space biopsy (a minimum of 6 weeks prior to 
challenge) and at visit 2 
X 
 
X 
 
 
Blood (9ml FBC, serum 0h, 4h, 24h), 
(13ml single cell analysis and Truculture at 0h and 24h) 
 
X (0h and 4h) 
X 
X (0h and 4h) 
X 

 
 
    
 
 
 
         
 
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Nasal Immune Challenge Study: Study protocol IRAS ID 291653 
 
21. 
APPENDIX C: TABLE OF SYMPTOMS FROM PREVIOUS CHALLENGE STUDY 
The following symptoms were recorded from the previously established R848 (1-2µg/nostril) 
nasal challenge study performed in 35 healthy, AR and ARwA volunteers (reference 15). All 
were mild and none were serious adverse events. 
 
Symptom 
Number of cases  
(out of total 35 volunteers) 
Nasal blockage/rhinitis 
11 
Lethargy 
6 
Headache 
3 
Myalgia 
1 
Sore throat  
1