Development of Human Nasal Challenge Models With Microbial Constituents and Grass Pollen: Monophosphoryl lipidA, Poly-inosine-cytosine, Poly-inosine-cytosine Stabilised With Poly-L-lysine and Carboxymethylcellulose, Resiquimod, Tuberculin and Timothy Grass Pollen
Phase: NA
Conditions: Allergic Rhinitis, Asthma, Latent Tuberculosis
Sponsor: Imperial College London

 
 
 
 
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Study Protocol  
Nasal Challenge with  
Microbial Constituents and Allergen  
 
Development of Human Nasal Challenge Models with 
Microbial Constituents and Grass Pollen: 
• monophosphoryl lipid A (MPLA) 
• poly-inosine-cytosine (poly-IC) 
• poly-inosine-cytosine stabilised with poly-L-lysine and 
carboxymethylcellulose  (poly-ICLC) 
• resiquimod 
• tuberculin 
• Timothy grass pollen and mechanistic study with Vitamin D 
 
Study Number: 13SM1837 
 
CHIEF INVESTIGATOR: Dr Trevor Hansel 
 
CO-INVESTIGATORS 
• Prof. Peter Openshaw and Prof Robin Shattock: TLR-agonists (MPLA, poly-
IC, poly-ICLC, resiquimod) 
• Prof Ajit Lalvani, Dr Onn Min Kon, Dr Robert Davidson: tuberculin 
• Prof Sebastian Johnston: allergen 
• Dr Zoltan Takats: mass spectrometry and metabolomics 
• Prof Kasia Hawrylowicz on vitamin D mechanistic study with grass pollen 
 
MAIN SPONSOR: Imperial College London 
FUNDER: National Institute of Health Research (NIHR) Imperial College and 
Imperial NHS Trust Biomedical Research Centre (BRC) 
 
STUDY CENTRES:  
Imperial Clinical Respiratory Research Unit (ICRRU) 
St. Mary’s Hospital 
Mint Wing, First Floor 
Paddington, Imperial College Healthcare NHS Trust,   
London W2 1NY 
London North West Healthcare NHS Trust   
Northwick Park Hospital  
Watford Road  
Harrow, HA1 3UJ 
 
NRES reference: 13/LO/1899 

 
 
 
 
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Protocol authorised by: 
Name & Role 
Date 
Signature 
 
 
 
Study Management Group  
Principal Investigator: Dr Trevor Hansel 
 
Co-Investigators: 
• 
Prof. Robin Shattock & Peter Openshaw: TLR-agonists 
• 
Prof Ajit Lalvani, Dr Onn Min Kon & Dr Robert Davidson: tuberculin 
• 
Prof Sebastian Johnston: allergen 
• 
Prof Kasia Hawrylowicz on vitamin D mechanistic study with grass pollen 
 
 
Statistician: Mrs Jackie Turner 
 
Study Management: Mrs Tanushree Tunstall/Dr Akhilesh Jha 
 
Study Coordination Centre:  
For general queries, supply of study documentation, and collection of data, please contact: 
Study Coordinator: Mrs Tanushree Tunstall 
Address: ICCRU, Mint Wing, St Mary’s Hospital, Paddington London W2 1NY 
 
E-mail: Tanushree.tunstall@imperial.nhs.trust 
 
Clinical Queries 
Clinical queries should be directed to Dr Akhilesh Jha, Clinical Research Fellow and Dr 
Trevor T. Hansel, Medical Director of the ICRRU. 
Clinical queries for the ViDAR part of the study should be directed to Dr Natasha 
Gunawardana and Dr Trevor T. Hansel, Medical Director of the ICRRU. 
 
 

 
 
 
 
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Sponsor 
Imperial College London is the main research Sponsor for this study.  For further information 
regarding the sponsorship conditions, please contact the Head of Regulatory Compliance at: 
 
 
 
Joint Research Compliance Office 
Imperial College London and Imperial College Healthcare NHS 
Trust 
Room 5L10B 
5th Floor, Lab Block 
Charing Cross Hospital 
Fulham Palace Road 
London 
W6 8RF 
 
Tel: 0203 311 0212 
Fax: 0203 311 0203 
 
Funder 
National Institute of Health Research (NIHR) Imperial College and Imperial NHS Trust 
Biomedical Research Centre (BRC) 
 
This protocol describes the “Nasal challenge with microbial constituents and allergen” study 
and provides information about procedures for entering participants.  Every care was taken 
in writing this protocol and this has been reviewed by the co-investigators. Problems relating 
to this study should be referred, in the first instance, to the Chief Investigator.  
This study will adhere to the principles outlined in the NHS Research Governance 
Framework for Health and Social Care (2nd edition). It will be conducted in compliance with 
the protocol, the Data Protection Act and other regulatory requirements as appropriate.  
 
 

 
 
 
 
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Table of Contents 
Table of Contents 
INTRODUCTION 
1.1 
BACKGROUND 
2.  STUDY OBJECTIVES 
2.1 
PRIMARY AND SECONDARY OBJECTIVES 
3.  STUDY DESIGN   (SEE FIGURES ABOVE) 
3.1 
STUDY OUTCOME MEASURES 
4. PARTICIPANT ENTRY 
4.1 
PRE-REGISTRATION EVALUATIONS: SCREENING HISTORY AND TESTS 
4.2 
MIXED INCLUSION & EXCLUSION CRITERIA (FOR TLR AGONIST & ALLERGEN CHALLENGE 
ARMS ONLY) 
4.3 
MIXED INCLUSION & EXCLUSION CRITERIA FOR TUBERCULIN CHALLENGE   ARM ONLY 
4.4 
WITHDRAWAL CRITERIA 
5. ADVERSE EVENTS 
5.1 
DEFINITIONS 
5.2 
REPORTING PROCEDURES 
5.2.1 Non serious AEs 
5.2.2 Serious AEs 
6. ASSESSMENT AND FOLLOW-UP: 
DOSAGE JUSTIFICATION 
SAFETY MEASURES 
SAMPLING SCHEDULE 
7. STATISTICS AND DATA ANALYSIS 
8. REGULATORY ISSUES 
8.1 
ETHICS APPROVAL 
8.2 
CONSENT 
8.3 
CONFIDENTIALITY 

 
 
 
 
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8.4 
INDEMNITY 
8.5 
SPONSOR 
8.6 
FUNDING 
8.7 
AUDITS 
9. STUDY MANAGEMENT 
10. 
PUBLICATION POLICY 
11. 
REFERENCES 
APPENDICES 
Appendix 1: Schedule of Events: Summary of Screening Procedures 
Appendix 2a: Summary of Procedures for Nasal Toll-Like Receptor (TLR) Agonist Challenge 
Appendix 2b: Summary of Procedures for Nasal Toll-Like Receptor (TLR) Agonist Challenge 
Appendix 3a: Part A Summary of Procedures for Tuberculin Nasal Challenge 
Appendix 3b: Part B Summary of Procedures for Tuberculin Nasal Challenge 
Appendix 4A:  Vitamin D3 in Nasal Allergic Rhinitis (ViDAR) 
Appendix 4B: Summary of Procedures for Nasal Allergen Challenge (NAC) 
Appendix 5: Pharmaceutical Quality of Agents delivered by Nasal Spray as Nasal Challenges 
or taken orally 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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GLOSSARY OF ABBREVIATIONS 
ACQ 
asthma control questionnaire 
AE 
adverse events 
BRC 
Biomedical Research Centre 
CC 
chemokine of cysteine-cysteine pattern 
CXC 
chemokine of cysteine-X-cysteine pattern 
CRF 
case report form 
FEV1 
forced expiratory volume in 1st second 
IGRA 
interferon-γ release assay 
IL 
interleukin 
LPS 
lipopolysaccharide 
LTBI 
latent tuberculosis infection 
MLF 
mucosal lining fluid 
MPLA 
monophosphoryl lipid A 
NIHR 
National Institute of Healthcare Research 
NAC 
nasal allergen challenge 
NC 
nasal challenge 
PBMC 
peripheral blood mononuclear cell 
PNIF 
peak nasal inspiratory flow 
Poly-IC 
poly inosine-cytosine 
Poly-ICLC 
poly-IC stabilised with poly-L-lysine and 
carboxymethycellulose 
QFT-it 
Quantiferon TB Gold-in-Tube  
SAM 
synthetic absorptive matrix 
SAE 
serious adverse event 
SSP 
special sampling probe 
TB 
tuberculosis 
TNSS 
total nasal symptom scores 
TU 
tuberculin unit 
ViDAR 
vitamin D nasal allergen challenge study 
 
KEYWORDS 
 
human challenge study,  
allergy, asthma, hay fever, tuberculosis, respiratory tract infection, nasal, respiratory,  
lipopolysaccharide, virus, grass pollen, inflammation, innate immune response  
biomarkers, mucosal lining fluid, nasosorption 
cytokines, chemokines, gene expression, eosinophil, T cells 
 
 

 
 
 
 
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STUDY SUMMARY 
 
TITLES Development of Human Nasal Challenge Models with Microbial 
Constituents and Grass Pollen:  
• monophosphoryl lipid A (MPLA) 
• poly-inosine-cytosine (poly-IC) 
• poly-inosine-cytosine stabilised with poly-L-lysine and 
carboxymethylcelluylose (poly-ICLC) 
• resiquimod 
• tuberculin 
• Timothy grass pollen 
 
ViDAR: A mechanistic study of Vitamin D supplementation in allergic 
rhinitis  
 
DESIGN Nasal challenges with a topical spray to the nostrils, with serial mucosal 
samples taken before and after the challenge. Open design, incremental 
ascending dose with strict safety precautions. 
 
AIMS To characterise the molecular and cellular basis of the nasal mucosal 
immune response to a variety of challenges 
 
OUTCOME 
MEASURES 
Tolerability and clinical symptoms 
Levels of nasal mucosal lining fluid (MLF) cytokines and chemokines 
Metabolites assessed on mass spectrometry 
Transcriptomics and flow cytometry on nasal curettage specimens 
 
POPULATIONS Healthy volunteers (non-atopic) 
Atopic subjects with allergic rhinitis 
Atopic subjects with mild allergic asthma 
Subjects with latent tuberculosis (TB) :  
Control healthy volunteers with negative interferon-gamma release assay 
(IGRA)  
Vitamin D insufficient atopic subjects 
 
ELIGIBILITY Adults (aged 18 to 60 years) will need to meet strict inclusion and 
exclusion criteria. Participants in this study will be obtained from clinics at 
Imperial College Healthcare Trust and other Northwest London Hospitals 
and by newspaper advertising 
 
DURATION 3 years 

 
 
 
 
 
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REFERENCE DIAGRAMS 
APPENDIX 2A 
Nasal Challenge with TLR Agonists: 
Poly-IC or poly-ICLC (TLR3) or Resiquimod (TLR7/8) or  MPLA (TLR4) 
Part A: Incremental Ascending Dose Study based on Tolerability and SAM
4 Cohorts of 8 Healthy Volunteers (4 atopic; 4 non-atopic) One cohort per agonist
Each subject given saline control and 3 ascending doses
Safety & Dosing Tolerability
Nasal mucosa examination, nasal & systemic symptoms checked at all timepoints
•
1st subject challenged (observed for 8h)
•
If dose well tolerated, 2nd & 3rd subjects challenged (observed until 8h) 
•
If dose well tolerated, then subjects 4-8 challenged (observed until 8h)
Nasosorption analysis will be undertaken after completing Part A of study to ensure viability of dosing regime
Nasal Challenge
Saline
Nasal Challenge
10μg/ 100μl
Nasal Challenge
100μg/ 100μl
Screening
•
skin AG pricks
•
ECG
•
Bloods
Nasal Challenge
500μg/ 100μl
d-7
d0
d7
d14
d21
Nasal SAM, neat:
•
Mass Spectrometry
•
Lipidomics
•
Prostanoids 
Nasal SAM, eluate:
•
Cytokines & chemokines 
Nasal Challenge Procedures
•
Initial bilateral lavage at -20m
•
PNIF & nasal symptoms: -30m, 15m, 30m, 45m, 1-8h
•
Nasal SAM: -30m,-10m, 15m, 30m, 45m, 1-8h
Appendix 2A
UPDATE FEB 2016: RESIQUIMOD WILL NOT BE GIVEN AT A HIGHER DOSE THAN 10μg/100μl PER NOSTRIL 

 
 
 
 
 
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APPENDIX 2B 
Nasal Challenge with TLR Agonists: 
Poly-IC or poly-ICLC (TLR3) or Resiquimod (TLR7/8) or MPLA (TLR4) 
Part B. Single Top Dose with Additional Special Sampling Probe (SSP) 
and Curettage
Single Cohort of up to 16 Healthy Volunteers (8 atopic; 8 non-atopic)
Each subject given saline control and single top dose (determined by Part A results)
Nasal special sampling probe (SSP)  for real time mass spectrometry
Safety Observations 
Nasal mucosa examination, nasal & systemic symptoms checked at all timepoint
Nasal SAM, neat:
•
Mass Spectrometry
•
Lipidomics
•
Prostanoids 
Nasal SAM, eluate:
•
Cytokines & chemokines
Nasal curettage: 
•
Flow cytometry & 
transcriptomics
Blood
•
Flow cytometry
Nasal Challenge Procedures
•
Initial bilateral lavage at -20m
•
PNIF and nasal symptoms: -30m, 15m, 30m, 45m, 1-24h
•
Nasal SAM: -30m, -10m, 15m, 30m, 45m, 1-24h
•
Nasal curettage: d-7, 8h and 24h (up to 4/timepoint)
•
Blood 20ml x4: -20m & 24h
Nasal Challenge
Saline
Nasal Challenge
Top dose (up to 1000μg)
d-7
d0
d7
Screening
•
skin AG pricks                                
•
ECG
•
Nasal curettage
•
bloods
Appendix 2B
UPDATE FEB 2016: RESIQUIMOD WILL NOT BE GIVEN AT A HIGHER DOSE THAN 10μg/100μl PER NOSTRIL 

 
 
 
 
 
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APPENDIX 2C 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nasal Challenge with Resiquimod (TLR7/8) 
Part B. 0.02μg/kg to 0.05μg/kg with Blood Sampling and Nasal Curettage
Single Cohort of up to 46 Volunteers 
(12 non-atopic; 12 atopic, 16 allergic asthma, 6 non-allergic asthma)
Each subject given saline control and between resiquimod 0.02μg/kg to 0.05μg/kg per nostril
4 subjects with asthma initially given half the dose of healthy volunteers
Safety Observations 
Nasal mucosal exam, nasal & systemic symptoms and spirometry checked hourly until 10h and at 24h
Nasal SAM, neat:
•
Mass Spectrometry
•
Lipidomics
•
Prostanoids 
Nasal SAM, eluate:
•
Cytokines & chemokines
Nasal curettage: 
•
Flow cytometry & 
transcriptomics
Blood
•
Flow cytometry
Nasal Challenge Procedures
•
Initial bilateral lavage at -20m
•
PNIF and nasal symptoms: -30m, 15m, 30m, 45m, 1-24h
•
Nasal SAM: -30m, -10m, 15m, 30m, 45m, 1-24h
•
Nasal curettage: d-7, 8h and 24h (up to 4/timepoint)
•
Blood up to 20ml x4: -20m, 4h, 8h & 24h
Appendix 2C
Nasal Challenge
Saline
Nasal Challenge
Resiquimod
0.02μg/kg to 
0.05μg/kg
d-7
d0
d7
Screening
•
Skin AG pricks                                
•
ECG
•
Blood DNA
•
Nasal curettage
•
Bloods
•
Nasal/pharyngeal swab
Repeat Nasal Challenge
(up to 6 from each group)
0.02μg/kg to 
0.05μg/kg
>6 weeks

 
 
 
 
 
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APPENDIX 3A 
Nasal Challenge with Tuberculin
Part A: Tuberculin nasal challenge ascending dose study based on clinical tolerability 
and levels of soluble inflammatory mediators 
Nasal challenge RT23 PPD
Dosing regime
•
0.1 TU in 100µl, n=4
•
1.0 TU in 100µl, n=4
•
2.0 TU in 100µl, n=4
•
5.0 TU in 100µl, n=4
Nasosorption mediator analysis after clinical phase of study up to 5.0TU. Curettage for transcriptomics only.
4 Cohorts of 4 latent TB subjects   One cohort per dose, single dose per subject
Safety & Dosing Tolerability
Nasal mucosa examination, nasal & systemic symptoms checked every 30m for 2h after challenge
•
1st subject challenged & observed for 4d
•
If dose well tolerated, 2nd subject challenged & observed for 2d
•
If dose well tolerated, then 3rd & 4th subjects challenged, observed for 4d before next cohort
Nasal 
Challenge
100μl
d-7
d7
Screening
•
Blood QFT-it +ve
•
TST ≥6mm to ≤ 25mm
•
CXR
•
Nasal curettage
•
ECG
•
Bloods
d0
d1
d2
d3
d4
Clinic 
Visit
Clinic 
Visit
Clinic 
Visit
Clinic 
Visit
Clinic 
Visit
Nasal SAM, eluate:
•
Cytokines & chemokines 
Nasal SAM, neat:
•
Mass Spectrometry
•
Lipidomics
•
Prostanoids 
Nasal Challenge Procedures
•
Initial bilateral lavage
•
PNIF and nasal symptoms: d-7, -30m, 1-8h, d1-d4, d7
•
Nasal SAM, neat & eluate: d-7, -30m, -10m, 1-8h, d1-d4, d7
•
Nasal curettage: d-7, d2, d4, d7 (up to 4/timepoint)
Appendix 3A

 
 
 
 
 
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APPENDIX 3B 
Nasal Challenge with Tuberculin
Part B: Tuberculin nasal challenge at a selected dose with placebo control 
in latent TB and healthy subjects
1 Cohort of 8 healthy volunteers; 1 Cohort of 8 latent TB subjects
Single dose per subject
Safety Observations
Nasal mucosa examination, nasal & systemic symptoms checked every 30m for 3h after challenge & at clinic visits
d-7
d7
Screening
•
Blood QFT-it
•
TST 
•
CXR
•
Nasal curettage
•
ECG
•
Bloods
d0
d1
d2
d3
d4
Clinic 
Visit
Clinic 
Visit
Clinic 
Visit
Clinic 
Visit
Clinic 
Visit
Nasal 
Challenge
Top dose/ 
placebo
Nasal Challenge Procedures
•
Initial bilateral lavage 
•
PNIF and nasal symptoms: d-7, -30m, 1-8h, d1-d4, d7
•
Nasal SAM, neat & eluate: d-7, -30m, -10m, 1-8h, d1-d4, d7
•
Nasal Special Sampling Probe (SSP) : left in place for up to 3h
•
Nasal curettage: d-7, d2, d4, d7 (up to 4/timepoint)
•
Blood 20ml x4: -60m, d2, d4, d7
Nasal Challenge Dosing Regime
Healthy
Top dose n=6
Placebo n=2
Latent TB
Top dose n=6
Placebo n=2
Nasal SAM, neat:
•
Mass Spectrometry
•
Lipidomics
•
Prostanoids 
Nasal SAM, eluate:
•
Cytokines & chemokines
Nasal curettage: 
•
Flow cytometry & 
transcriptomics
Blood
•
Flow cytometry
Nasal special sampling probe (SSP)  for real time mass spectrometry
Appendix 3B
 

 
 
 
 
 
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APPENDIX 4A 
Nasal Challenge with Allergen: Mechanistic study of Vitamin D (VIDNA)
Single Cohort of 24 subjects with a history of Hayfever
Vitamin D3 group: 18 subjects  Placebo group: 6 subjects 
Nasal Challenge with Timothy Grass Pollen before and after supplementation
Nasal Challenge Procedures
•
Initial bilateral lavage 
•
PNIF and nasal symptoms: -30m,-15m, 5m, 15m, 30m, 45m, 1-8h
•
Nasal SAM, neat & eluate: -30m,-15m, 5m, 15m, 30m, 45m, 1-8h
•
Nasal curettage: taken at 5pm
•
Blood 75ml: 50ml PBMC isolation, 10ml serum, 10ml transcriptomics, 
5ml Full Blood Counts
Nasosorption samples
•
Cytokines & chemokines
•
Prostanoids & resolvins
Nasal curettage
•
Transcriptomics & epigenetics
Blood & Serum
•
PBMC in-vitro assays
•
Transcriptomics
•
Genotyping of atopy assoc. 
genes
Safety Observations 
Nasal mucosa examination, nasal & systemic symptoms checked at all timepoints
Nasal Challenge
5000SQ-U/100µl
Nasal Challenge
5000SQ-U/100µl
d-7
d0
d28
Screening
•
skin AG pricks
•
ECG
•
Serum IgE levels
•
Nasal curettage & SAM
•
Bloods
•
25-OH Vit. D <75nMol/L
Vitamin D3 4000IU or 
Placebo
Daily dietary 
supplementation
4 weeks
This trial is registered at  Clinicaltrials.gov

 
 
 
 
 
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APPENDIX 4B 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nasal Challenge with Allergen: Timothy Grass Pollen
3 Cohorts: 12 Hay Fever Subjects; 12 Allergic Asthmatics;  6 Healthy Volunteers 
Safety Observations 
Nasal mucosa examination, nasal & systemic symptoms checked at all timepoint
Appendix 4
Nasal Challenge Procedures
•
Initial bilateral lavage at -20m
•
PNIF and nasal symptoms: -30m, 5m, 15m, 30m, 45m, 1-6h
•
Nasal SAM, neat: -30m, -10m, 5m, 10m, 15m, 30m, 45m, 1-6h
•
Nasal SAM, eluate: -30m, -10m, 5m, 10m, 15m, 30m, 45m, 1-6h
•
Nasal Special Sampling Probe (SSP) : left in place pre-challenge  up to 6h
•
Nasal curettage: 6h (up to 4 samples)
•
Blood 20ml x2: -60m, 6h 
Nasal special sampling probe (SSP)  for real time mass spectrometry in up to 16 ADDITIONAL SUBJECTS 
Nasal SAM, neat:
•
Mass Spectrometry
•
Lipidomics
•
Prostanoids 
Nasal SAM, eluate:
•
Cytokines & chemokines
Nasal curettage: 
•
Epithelial culture
•
Flow cytometry & 
transcriptomics
Blood
•
Flow cytometry
Nasal Challenge
Saline
Nasal Challenge
5000 SQ-U/ 100μl
d-7
d0
d7
Screening
• skin AG pricks
• ECG
• Nasal curettage
• bloods
Cohorts
Hay fever (n=12) 
TGP skin test positive
Outside May-August
Allergic Asthma (n=12) 
TGP skin test positive
•
6 on ICS/LABA
•
6 no ICS
Healthy (n=6)
Non-atopic
TGP skin test negative

 
 
 
 
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INTRODUCTION  
 
1.1 
BACKGROUND 
 
Summary 
 
• 
Respiratory diseases such as asthma and COPD involve inflammation of the airways, 
and microbes are increasingly recognised as being related to the pathogenesis of these 
diseases, with viruses the major cause of exacerbations (1). We propose the development of 
a range of topical nasal spray challenge models to study the upper respiratory tract mucosal 
immune response. We will develop nasal challenge with bacterial and viral pathogen 
associated molecular patterns (PAMPS) that stimulate Toll-Like Receptors (TLRs), tuberculin 
purified protein derivative (PPD), and allergen (AG: Timothy grass pollen). In this way we 
challenge the nasal upper respiratory tract mucosa with stimuli of the innate immune system 
(TLR agonists), as well as with a specific T helper type 1 (Th1) stimulus (tuberculin PPD) and 
a specific Th2 stimulus (AG).  
 
• 
Following challenge we shall employ non-invasive methods of taking samples from the 
nasal mucosal surface. Synthetic absorptive matrix (SAM) strips are like blotting paper, and 
will be inserted onto the airway mucosal surfaces within a nostril to absorb concentrated 
nasal secretions, inflammatory mediators in the mucosal lining fluid (MLF) reflect underlying 
inflammation. 
 
• 
Nasosorption is painless, and MLF can be obtained even from non-inflamed noses at 
frequent intervals, without the need for local anaesthetic. We have performed successful pilot 
studies with nasal strips in adults after nasal allergen challenge (NAC), and in babies and in 
young children. There is minimal protein binding to the SAM strip, and fluid can be eluted by 
spin filtration. High levels of mediators of inflammation can then be measured in the MLF: 
higher than detectable by nasal lavage. 
 
• 
In addition, a novel probe will be placed on the surface of the nasal mucosa and we shall 
carry out real time mass spectrometry.  We shall also perform nasal epithelial curettage with 
a Rhinoprobe device. This takes a pinhead of nasal tissue, again without requiring a local 
anaesthetic, and the sample can then be used for analysis of cells by flow cytometry and to 
determine gene expression (transcriptomics). 
 
• 
Disease-specific signatures (including metabolomics and cytokines / chemokines) from 
the upper airways (nose) and lower airways (bronchi) of patients with asthma have been 
studied. Using nasosorption to obtain samples and identify molecular signatures of 
respiratory disease, we can achieve fundamental insights into airway diseases. This will 
refine the diagnosis, stratification and monitoring of a variety of airway diseases, and may 
provide targets and biomarkers for new therapy. 
 
• 
Additionally we wish to study of the effects of vitamin D on nasal mucosal mediators of 
inflammation produced after nasal allergen challenge (NAC) with Timothy grass pollen. 
Vitamin D deficiency is a common condition and may be an important environmental factor in 
the development of allergy and asthma (2). In particular, vitamin D has been shown to have 
a range of anti-inflammatory effects on the immune system (3), and is thought to have some 
shared actions with corticosteroids (4). We shall study the actions of vitamin D on hay fever, 
since this is an allergic inflammatory disease of the upper airways that is caused by airborne 

 
 
 
 
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grass pollen during the British summer. We propose giving oral vitamin D supplementation 
for 4 weeks to selected subjects with vitamin D insufficiency in the British winter, and 
assessing whether there are effects on levels of mediators of inflammation produced after 
nasal allergen challenge (NAC) with a nasal spray consisting of grass pollen extract. 
 
Challenge Models and Sampling Nasal Mucosal Lining Fluid (MLF) with Synthetic 
Absorptive Matrix (SAM) by Nasososorption 
 
 The inhaled allergen challenge model has been extensively employed as the classical 
means to study effects of anti-inflammatory agents on asthma (5) (6), while the allergen 
chamber is especially useful for immunotherapy (7). In the last 15 years a series of clinical 
studies have been carried out with specific monoclonal antibodies (mABs) in patients with 
asthma, generally beginning with inhaled allergen challenge studies, that have given insights 
into the heterogeneity of asthma phenotypes (8) (9) (10). These studies with mABs against 
soluble IgE and cytokines produced by Th2 cells (IL-4, IL-5, IL-13) have established the 
principle of personalised medicine: that a highly specific mAB tends to be more effective in 
selected stratified asthmatics that express higher levels of a relevant biomarker (11) (12). 
Nasal challenge models have certain advantages over inhaled models, especially because 
the nose is much more accessible than the airways to obtain respiratory samples (13). 
Therefore repeat non-invasive sampling can be performed to study in detail the cellular and 
molecular basis of the mucosal upper respiratory tract (URT) immune response. Nasal and 
bronchial epithelial mucosal lining fluid (MLF) is a compartment that can be utilised to study 
inflammatory mediators in a variety of respiratory diseases.  
 
We have developed a novel system that utilises strips of synthetic absorptive matrices (SAM) 
or synthetic sponge to sample MLF, before eluting the fluid and measuring levels of 
mediators that cause and are a feature of inflammation. Filter paper consisting of natural 
cellulose from the cotton plant has been widely used to absorb nasal secretions (14-18), and 
ophthalmic Weck-Cel sponges composed of natural cellulose have been used to sample 
saliva, cervical and vaginal secretions (19). Our group originally performed nasosorption with 
filter paper after nasal allergen challenge (NAC), but found in an initial study that detectable 
levels of cytokines such as IL-5 and IL-13 are generally low (20). Following this we found that 
different batches of filter paper vary in their degree of protein binding, and thus decided to 
identify a suitable SAM. 
 
As a response to problems using filter paper of natural origin from the cotton plant, different 
SAMs have been developed:  Accuwik Ultra (21) (22) (23) (24), Leukosorb (Pall Life 
Sciences), polyvinyl acetate (PVA) sponge (25), and polyurethane foam (26)[(27),(28)26) 
Different absorptive materials have been compared for sampling saliva prior to measuring 
antibodies (29). Recent studies have demonstrated that polyurethane foam performs well as 
a SAM for collecting nasal MLF  (26;27).  
 
Our experience with SAM for nasosorption has been published with regard to children with 
allergic rhinitis (21), infants with a family history of atopy (23), and in atopic adults after nasal 
allergen challenge (NAC) (24). We have also successfully used leukosorb after experimental 
human rhinovirus (HRV experimental challenge in asthma, when both nasosorption and 
bronchosorption were performed (Jackson D et al., manuscript in preparation).  
 
Surface nasal sampling and real time mass spectrometry  
Dr Zoltan Takats of Imperial College has developed special ways of sampling droplets or 
electrosurgical smoke of tissue for mass spectrometry (30-32). Using rapid evaporative 

 
 
 
 
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ionization mass spectrometry (REIMS) in conjunction with electrosurgery (the intelligent knife 
or iknife) enables real time feedback during surgery as to whether tissue contains cancerous 
cells (31). Building on this experience with molecular characterisation of many thousands of 
lower molecular weight metabolites, we hope to test a new surface probe on the nasal 
mucosa following the different challenges. This probe will bound minute volumes of water 
molecules against the mucosal surface lining fluid. 
 
Role of host microbiome in innate immune response to resiquimod 
Viral respiratory tract illness is often complicated by secondary bacterial infections, 
especially in those with compromised host defences. One mechanism may be due to the 
presence of virus in the nose altering epithelial cell receptor expression allowing any resident 
bacteria present in the associated mucosal biofilm to become pathogenic. Low bacterial 
diversity rather than bacterial load appear to be related to disease severity in those with 
chronic rhinosinusitis.  
 
The nose has a greater biomass of commensal bacteria than other parts of the respiratory 
tract and likely affects the composition of the lower airways. Asthmatic bronchi have a 
disordered microbiome with a greater prevalence of Haemophilus species and in this 
population, viral and bacterial co-infection leads to greater chances of being hospitalized. In 
murine models, respiratory (and possibly gut) commensal bacteria play a role in regulating T 
cell function and other immune defences against acute respiratory viral infections (such as 
influenza). It has been demonstrated that lower respiratory tract immune responses to 
bacterial TLR ligands are attenuated for at least 6 weeks after infection. Our recent work with 
lipopolysaccharide (LPS, a component of the outer wall of Gram negative bacteria) given 
nasally suggests that baseline mucosal immune tone may be affected by environmental 
factors such as host microbiota.  
 
To investigate how the host respiratory microbiome correlates with innate immune 
responses to viruses in asthmatic, allergic and healthy participants, we propose collecting 
anterior nasal and oropharyngeal swab samples from volunteers at their screening visit 
using sterile dry cotton-headed swabs and synthetic absorptive matrix strips (SAM) and 
analyse the bacterial profile using traditional laboratory culture techniques as well as 
microbiome analysis. The findings will be correlated with subsequent innate immune 
functional assays.  
 
1.2 
RATIONALE FOR CURRENT STUDY 
 
There is now the need to extend nasal challenge from allergen to a range of other agents, 
utilising the novel SAM methodology to take serial nasosorption samples and then measure 
inflammatory mediators. Hence our aim in this proposal is to develop nasal challenge with a 
range of immunostimulatory agents. In parallel, we shall also develop challenges against 
specific/adaptive immune responses: tuberculin (Th1 and cell-mediated immune stimulus) 
nasal challenge will be developed in subjects with latent TB, and Timothy grass pollen will be 
employed as an allergen in subjects with hay fever outside the grass pollen season. 
1. We shall begin by performing ascending dose studies with innate Toll-like receptor 
(TLR)-agonists. At St Mary’s Hospital there is considerable experience under 
Professor Sebastian Johnston with the Human Rhinovirus (HRV, common cold virus) 
experimental challenge model in adults (33), while Professor Peter Openshaw is 
developing an adult human experimental challenge model with respiratory syncytial 

 
 
 
 
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virus (RSV) (34), (35). Instead of challenging with live viruses we shall give nasal 
topical challenge with key immunogenic viral components:  
A. Monophosphoryl lipid A (MPLA) is an analogue of bacterial lipopolysaccharide 
(LPS) that is now licensed as an adjuvant and stimulates TLR4 and TLR2, but 
with much lower toxicity than LPS (36) (37) (38). In addition, a range of TLR4 
antagonists are in development (39;40). Nasal challenge has been performed 
with LPS but without using nasosorption (41-44). In particular, it has recently been 
found that nasal allergen challenge in allergic subjects, when followed 24h later 
by LPS, causes an amplified cytokine response 6h later (44). Following nasal LPS 
challenge we have taken serial nasosorption samples and documented the innate 
immune response (Dhariwal J et al., manuscript in preparation).  
Nasal MPLA challenge followed by serial nasosorption sampling has not been 
performed to our knowledge. However MPLA has been extensively used as an 
adjuvant in human clinical trials (45), and as an adjuvant for subcutaneous and 
sublingual allergen immunotherapy (46;47). 
B. Poly-inosine-cytosine (poly-IC) resembles double-stranded viral RNA and is an 
immunogenic agonist for TLR3 (48). Many of the major respiratory viral infections 
have an RNA genome: so this model is relevant to infection with HRV, RSV and 
influenza.  We shall use a highly purified high molecular weight poly-IC from 
Invivogen. Ampligen is a poly-IC derivative with regular repeats of uridylic acid, 
and clinical trials have been carried out in man by Hemispherx (49-52). Recently it 
has also been given nasally in seven volunteers with allergy to birch pollen and 
tolerated well. The study group sampled nasal tissue using an interdental brush to 
measure mRNA expression. However, we plan to study both atopic and non-
atopic volunteers and sample the mucosal lining fluid directly to measure the 
protein mediators that are secreted in addition to measuring RNA expression (53). 
C. Poly-ICLC is poly-IC stabilised with poly-L-lysine and carboxymethylcellulose. It is 
more RNAse resistant than poly-IC, and better able to stimulate intracellular 
endosomal TLR3. Poly-ICLC has been developed by a Pharmaceutical Company 
(Oncovir of Washington DC, USA) and has been administered to man by injection 
in a range of clinical studies, including as a vaccine adjuvant (48;54).  
D. Resiquimod stimulates TLR7/8 that is also stimulated by respiratory viruses (40).  
Resiquimod is an immune response modifier that has been successfully 
employed topically as a skin cream for actinic keratosis (55). Resiquimod is 
closely related to imiquimod, but is more potent (56), and can block airway 
remodelling in a rat asthma model (57). Resiquimod when given systemically to 
treat human hepatitis C infection causes cytokine release and a flu-like syndrome. 
We are unaware of studies with topical resiquimod given to the respiratory tract. 
2. Tuberculin challenge employing purified protein derivative (PPD) has been studied in 
skin test PPD-positive subjects by Richard F. Silver and colleagues in Cleveland 
following bronchosegmental challenge (58) (59) (60) (61).  Silver and colleagues 
performed BAL at a first bronchoscopy, before doing local broncho-segmental 
challenge with 0.5 tuberculin units of PPD in normal saline (10ml) instilled into a 
subsegment of the lingual. Repeat bronchoscopy and BALs were performed 48h 
later.  
• 
PPD challenge caused a BAL lymphocytosis with increased numbers of CD4+ 
T cells and IFN-γ producing cells (58).  
• 
The IFN-γ producing cells were then shown to be CD4+ Th1 cells of effector 
memory phenotype (CD45RO+, CCR7-) (59). 

 
 
 
 
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• 
The α4β1 integrin is involved in localization of these M. tuberculosis-specific 
Th1 cells (60). 
We are also aware of a study that safely developed nasal challenge with leprosin A in 
leprosy (62), but are unaware of a nasal challenge study in man with TB components. 
In PPD-positive skin test individuals with latent TB, we hope to perform an ascending 
dose tolerability study with up to 2.0 tuberculin units in 100µg of saline nasal spray to 
each nostril. If local nasal inflammation is caused by the PPD spray challenge, the 
study will be discontinued, and the individual can be treated with local and systemic 
corticosteroids. 
 
3. Allergen (Timothy grass pollen):  
A. Mechanistic study with Vitamin D: Our group has studied IL-5 and IL-13 nasal 
responses in nasal MLF after NAC (24).  We have also taken nasal epithelial 
curette samples and looked at transcriptomics (Trevor Hansel with Merck 
Pharmaceuticals, manuscript in preparation) and developed a method of flow 
cytometry for assessment of mechanically-dispersed leukocytes from the 
nasal epithelial curette sample (Ross Walton, Jaideep Dhariwal and 
colleagues). We now wish to investigate the mechanistic processes that 
underlie vitamin D supplementation in allergic disorders and in particular after 
nasal challenge with grass pollen. A number of studies have shown a 
correlation between serum vitamin D levels and the severity of asthma (63) 
(64) (3). However, vitamin D supplementation failed to show clinical benefit in 
a recently published randomized trial of vitamin D supplementation in adults 
with symptomatic asthma: the VIDA (vitamin D add-on therapy enhances 
corticosteroid responsiveness in asthma) study  (65). Nevertheless, in studies 
from the Hawrylowicz group it has been showed that a short course of vitamin 
D can increase responsiveness to corticosteroids (66) and that IL-17A 
production in severe asthma is decreased by vitamin D (67). It is now 
recognised that suboptimal levels of vitamin D are present in many people in 
the UK during the winter, and dietary supplementation is recommended for 
pregnant women, children and high risk groups  (68). Taking serum levels of 
25-hydroxy-vitamin D it has been found that 87% of UK adults have 
suboptimal vitamin D levels at <75nmoles/l in winter (69) 
In nasal micro-curettage samples it is possible to measure gene expression, 
which assesses levels of thousands of mRNA molecules. The Hansel group 
has recently found that there are profound changes in circadian-associated 
genes in the nasal mucosa after NAC, and that these genes are inhibited by 
corticosteroids. Circadian genes regulate innate aspects of the immune 
system by actions involving the BMAL1-CLOCK complex of nuclear 
transcription factors, receptors and enzymes (70) (71;72).  The circadian 
clock was first identified with regard to lipopolysaccharide(LPS) responses 
varying with the time of the day, and these circadian responses have been 
shown to be present in murine macrophage responses to endotoxin (73;74) 
and more recently in murine inflammatory monocytes (75). In addition, a 
bronchiolar 
epithelial 
clock 
controls 
pulmonary 
inflammation 
and 
glucocorticoid action in mice, as shown by genetic ablation of the mouse 
clock gene Bmal1 in bronchiolar epithelial cells (76).  
Our demonstration in the human nose of allergen-induced changes in 
circadian-associated genes in the presence of innate inflammatory pathways, 
suggests that the circadian-associate genes could be controlling gene 
expression and protein synthesis involved in these innate inflammatory 
pathways. Furthermore, these allergen–induced circadian changes are 
reversed by a single preceding oral dose of prednisone, consistent with 

 
 
 
 
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known actions of endogenous cortisol (77) (76) implying that the 
corticosteroid may be acting on circadian-associated genes in order to 
resolve inflammation. 
Since the NAC model with grass pollen is exquisitely sensitive to studying the 
effects of therapy, we have designed a clinical study to assess whether 
vitamin D supplementation can alter NAC responses through effects on 
circadian–associated genes and pathways of inflammation in asthma (78). 
After a review of the literature we propose a dose of 4000 IU per day for 4 
weeks, since this level of dietary supplementation has been safely used in 
pregnant women and in a large clinical study in asthma (79;80) (65). We shall 
assess whether there are effects of vitamin D on inflammation produced after 
NAC: looking at levels of cytokines, chemokines, cells and gene expression, 
with a focus on circadian–associated genes (78).  
 
B. Allergen (Timothy Grass Pollen): An additional aim is to identify the cells that 
are responsible for production of IL-5 and IL-13 in MLF, suspecting that these 
may be Th2 cells or natural helper cells (nuocytes). We wish to perform NAC 
with appropriate saline control challenge in allergic and non-allergic subjects. 
Serial samples will be taken to assess chemokines and cytokines in nasal 
MLF, with measurement of transcriptomics and leukocyte flow cytometry on 
nasal epithelial curette samples. 
 
 
Research Question and Hypotheses 
 
Following nasal spray challenge with various agents can we develop a safe system that 
mimics the response of the airways to microbes (pathogen-associated molecular patterns) 
and allergens? 
 
Will different challenge agents cause characteristic and differing nasal mucosal responses? 
Following nasal challenge we shall take serial non-invasive samples and be able to 
document the inflammatory response to challenges in terms of cytokines and chemokines in 
mucosal lining fluid, and in terms of cells and gene expression in nasal epithelial curettage 
samples. 
 
Will supplementary Vitamin D alter levels of inflammatory mediators, gene expression and 
modulate circadian-associated gene expression following nasal allergen challenge (NAC) 
with Timothy grass pollen extract? 
 
By understanding the nasal response to challenge agents, we will have a system for 
identifying biomarkers of disease, targets for new drugs, and be able to provide a clinical 
challenge test for testing the efficacy of new drugs. 
 
 
2.  STUDY OBJECTIVES 
 
2.1 
PRIMARY AND SECONDARY OBJECTIVES 
 
• 
To develop a set of well-tolerated, robust and reproducible (validated) nasal 
challenge systems with microbial constituents and allergen. To identify biomarker 
signatures following different challenges. 

 
 
 
 
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• 
To document a dose response and time course to nasal challenge in terms of 
cytokine and chemokine release, and nasal epithelial curettage (Rhinoprobe) mRNA 
expression, cell influx by flow cytometry. 
 
• 
To provide a set of nasal challenge methods to enable a “proof of target 
pharmacology” for a novel therapy or vaccine. This may also permit dosage 
optimisation in relation to clinical efficacy studies. 
 
• 
To develop real time sampling utilising a special probe that touches the surface of the 
nasal lining and is linked to a mass spectrometer. 
 
To assess nasal cytokines/chemokines in the nasal mucosa and assess levels of 
circadian–associated gene expression (BMAL-CLOCK genes) in curettage samples 
after NAC before and after Vitamin D supplementation. 
 
3.  STUDY DESIGN   (See Figures above) 
 
Group 1: Nasal challenge with TLR Agonists: This part of the study has a total of 96 
subjects divided into two parts; Part A and B.  
Part A: We shall have 3 cohorts of 8 healthy volunteers (4 of the 8 having atopy). There will 
be a separate cohort of 8 subjects for each challenge agent (MPLA, poly-ICLC, and 
resiquimod). 
A careful incremental ascending dose tolerability study will be performed: starting with saline 
and then giving single doses of 10, 100,500μg and up to 1000µg of a given challenge agent 
in volumes ranging from 100µl to 500µl per nostril. 
 
Part A, N= 24 
a. MPLA 
 
n = 8 
b. Poly-ICLC  
n = 8 
c. Resiquimod 
n = 8 
For each challenge agent we shall initially take serial SAM samples over 8 hours, and 
assess the cytokine and chemokine response. 
 
Part B: When we have defined the top safe dose, we shall study up to a further 16 subjects 
for each challenge agent (up to 46 for resiquimod): and include extra sampling for nasal 
curettage and blood flow cytometry. For those volunteers in the resiquimod arm of the study, 
we will invite some of the participants (up to 6 in each group) for a repeat challenge at least 
6 weeks after the prior challenge. In January 2016 we updated the protocol to move to a 
weight based dosing regimen for administering resiquimod. We intend to challenge 6 
volunteers (mix of healthy volunteers an those with allergic rhinitis) at a dose of 0.02μg/kg 
per nostril, which equates to 1μg/100μl per nostril in a 50kg volunteer. For the asthma group, 
we intend to challenge 4 people at half the dose given to the healthy group. The maximum 
tolerated dose will then be used for the remaining subjects in the study, We will not use 
resiquimod at a dose higher than 10μg/100μl per nostril for the remainder of the study. 
 
Part B, N = 90 
a. MPLA 
 
n = 16 
b. Poly-IC 
 
n = 16 
c. Poly-ICLC  
n = 16 
d. Resiquimod 
n =  46 (12 healthy, 12 atopic, 16 allergic asthmatic, 6 non-
allergic asthma) 

 
 
 
 
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Group 2: Nasal challenge with tuberculin: This part of the study has a total of 32 subjects 
divided into two parts; Part A and B.  
Part A: This involves 4 cohorts of subjects with defined latent TB.  
A careful incremental ascending dose tolerability study will be performed: starting 
with single doses of: 
 
Part A, N=16 
A. Subjects with Latent TB 
a. 0.1 TU/100µl/nostril,  
n = 4 
b. 1.0 TU /100µl/nostril,  
n = 4  
c. 2.0 TU /100µl/nostril,  
n = 4 
d. 5.0 TU /100µl/nostril,  
n = 4 
For each agent we shall look at tolerability and initially take serial SAM samples over the 
next 7 days to assess the cytokine and chemokine response. 
 
Part B: When we have defined the top safe dose, we shall study up to a further 16 subjects 
with the top tuberculin dose: and include extra sampling for nasal real time mass 
spectrometry, nasal curettage and blood flow cytometry. 
 
Part B, N=16 
A. Subjects with Latent TB,   
a. 10.0 TU /100µl/nostril, 
n = 8 
B. Healthy volunteers,             
b. 10.0 TU /100µl/nostril, 
n = 8 
 
Group 3: Nasal challenge with allergen (Timothy grass pollen): This part of the study 
has a total of 70 subjects divided into two parts; Part A and B. 
 
Part A Nasal challenge with allergen and vitamin D3 supplementation or placebo:  
This involves a total of 24 subjects who are allergic to grass pollen and are vitamin D 
insufficient. Participants are divided into 2 groups (vitamin D supplementation and placebo 
arms).This is a randomized, double-blind, placebo-controlled study. They will have two NAC; 
one before and one after supplementation. At each NAC visit, subjects will be given a single 
dose of Timothy grass pollen (Phleum P5) allergen:  
The Phleum P5 is given as 5000 SQ-U = 1μg/100μl per nostril. 
 
After being randomized into one of the two groups they will receive their first NAC and 
subsequently take vitamin D3, 4000IU (n=18) or placebo (n=6) orally once daily at 
approximately the same time each morning until their second NAC. This second NAC will 
take place between 28-35 days. During both days of NAC, serial SAM samples will be taken 
and nasal curettage performed. Blood samples will also be taken for routine safety tests, 
peripheral blood mononuclear cells (PBMC) in vitro assays, vitamin D, calcium, cortisol, 
melatonin and whole blood transcriptomics (circadian-associated genes). 
 
  
Part A, N=24 
A. Subjects with grass pollen allergy and insufficient Vitamin D3 
a. Subjects taking Vitamin D3 supplementation 
n = 18 
b. Subjects on placebo   
 
 
 
n = 6 

 
 
 
 
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Part B: Nasal challenge with allergen involves a total of 46 subjects: 
This is divided into 4 groups of subjects given a single dose of Timothy grass pollen (Phleum 
P5) allergen:  
The Phleum P5 is given as 5000 SQ-U = 1μg/100μl/nostril 
 
Subjects with hay fever out of season (n=12) 
Subjects with allergic asthma out of season (n=12): half taking no anti-inflammatory drugs, 
half receiving combined inhaled corticosteroids and long-acting beta-agonists (ICS/LABA) 
Non-atopic healthy controls (n=6) 
Subjects with hay fever out of season who will trial a novel nasal special sampling probe 
(SSP) for real time mass spectrometry (n=16) 
 
N= 46 
a. People with hay fever, 
n = 12 
b. People with allergic asthma, n = 12 
c. Healthy, non-atopic people, n =  6 
d. People with hay fever,  
n =16 
 
3.1 
STUDY OUTCOME MEASURES 
 
• 
Clinical symptoms and tolerability 
• 
Inspection of nasal mucosa by a clinician: erythema, discharge, ulceration 
• 
Mucosal lining fluid cytokines and chemokines 
• 
Blood and nasal flow cytometry and transcriptomics 
• 
Nasal lining fluid metabolites 
 
4. 
PARTICIPANT ENTRY  
 
4.1 
PRE-REGISTRATION EVALUATIONS: SCREENING HISTORY AND 
TESTS 
 
Before patients are considered for inclusion in the study we shall consider whether they have 
asthma or hay fever, or a history of latent tuberculosis (TB), or whether they are healthy 
volunteers (HVs) with none of these conditions. 
 
Screening tests may include intra-epidermal skin allergen prick tests and an intradermal 
tuberculin skin test.  
 
 
4.2 
MIXED INCLUSION & EXCLUSION CRITERIA (FOR ALLERGEN 
CHALLENGE ARMS ONLY) 
 
General: for all subjects  
 
• 
Males and females aged 18 to 60 years  
• 
Current non-smokers for last year, maximum of 10 cigs per month, with a smoking 
history of <5 pack years 

 
 
 
 
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• 
Body mass index in the range18-39 
• 
No systemic illnesses that might affect nasal immune responses 
• 
No recent viral infections in past month: including colds and flu. 
• 
No nasal anatomical abnormalities, sinus infections, polyps, nasal mucosal 
abnormalities 
• 
No prescribed anti-inflammatory drug therapy  
A. 
Healthy non-atopic volunteers 
• 
No clinical history of allergic rhinitis, allergic asthma or eczema. 
• 
Negative skin prick tests to a range of 6 common aeroallergens: cat, dog, grass 
pollen, tree pollen, house dust mite, fungal spores 
• 
Normal blood eosinophil count 
 
B.  Atopic subjects with Timothy Grass Pollen sensitivity & Vitamin D 
Insufficiency  
 
• 
A clinical history of seasonal grass pollen allergic rhinitis: sneezing, running and itching 
nose, nasal drip in the UK grass pollen summer season (May-July).  
• 
Specific allergy confirmed by positive intra-epidermal skin prick test to Timothy grass 
pollen extract (Soluprick, Phleum pratense; ALK, Horsholm, Denmark), a positive 
reaction being a raised wheal of diameter >3mm larger than a negative saline control. 
• 
Vitamin D insufficiency: serum levels of 25-hydroxy vitaminD3 (25-OHD3) <30ng/ml or 
<30µg/L or <75nmoles/L 
• 
No use of sun lamps, vitamin D supplements (including fish oils), no holidays abroad 
in sunny locations (including beach holidays and skiing) for the duration of 
participation 
• 
No clinical history of tree pollen allergy from February to April 
 
C. Asthmatic subjects with Timothy Grass Pollen sensitivity 
• 
Seasonal grass pollen allergic rhinitis: sneezing, running and itching nose, nasal drip in 
the UK grass pollen summer season (May-July). 
• 
Specific allergy confirmed by positive intra-epidermal skin prick test to Timothy grass 
pollen extract (Soluprick, Phleum pratense; ALK, Horsholm, Denmark), a positive 
reaction being a raised wheal of diameter >3mm larger than a negative saline control. 
• 
Half the asthmatics have clinical history and diagnosis of asthma, requiring therapy with 
occasional inhaled beta-agonists, but no inhaled corticosteroids for the past 28 days. 
Half the asthmatics receive regular combined inhaled corticosteroids and long-acting 
beta-agonists (ICS/LABA) 
 
4.3  
MIXED INCLUSION & EXCLUSION CRITERIA (FOR TLR AGONIST ARM  
ONLY) 
General: for all subjects  
 
• 
Males and females aged 18 to 60 years  
• 
Current non-smokers for last year, maximum of 10 cigs per month, with a smoking 
history of <5 pack years 
• 
Body mass index in the range18-39 
• 
No systemic illnesses that might affect nasal immune responses 
• 
No recent viral infections in past month: including colds and flu. 

 
 
 
 
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• 
No nasal anatomical abnormalities, sinus infections, polyps, nasal mucosal 
abnormalities 
• 
No prescribed anti-inflammatory drug therapy  
A. Healthy non-atopic volunteers 
• 
No clinical history of allergic rhinitis, allergic asthma or eczema. 
• 
Negative skin prick tests to a range of 6 common aeroallergens: cat, dog, grass 
pollen, tree pollen, house dust mite, fungal spores 
• 
Normal blood eosinophil count 
• 
Normal baseline forced expiratory volume (FEV1) i.e. ≥80% 
• 
Negative methacholine challenge (PC20> 8mg/ml) 
 
B.  Atopic subjects with Timothy Grass Pollen sensitivity  
• 
A clinical history of seasonal grass pollen allergic rhinitis: sneezing, running and 
itching nose, nasal drip in the UK grass pollen summer season (May-July).  
• 
Specific allergy confirmed by positive intra-epidermal skin prick test to Timothy grass 
pollen extract (Soluprick, Phleum pratense; ALK, Horsholm, Denmark), a positive 
reaction being a raised wheal of diameter >3mm larger than a negative saline 
control. 
• 
Normal baseline forced expiratory volume (FEV1) i.e. ≥80% 
 
 
C. Asthmatic subjects with Timothy Grass Pollen sensitivity 
• 
Physician diagnosed asthma up to stage 2 BTS/GINA guidelines 
• 
Seasonal grass pollen allergic rhinitis: sneezing, running and itching nose, nasal drip 
in the UK grass pollen summer season (May-July). 
• 
Specific allergy confirmed by positive intra-epidermal skin prick test to Timothy grass 
pollen extract (Soluprick, Phleum pratense; ALK, Horsholm, Denmark), a positive 
reaction being a raised wheal of diameter >3mm larger than a negative saline 
control. 
• 
Baseline forced expiratory volume (FEV1) ≥75% 
• 
Positive methacholine challenge (PC20< 8mg/ml) 
 
 
D. Asthmatic subjects with no allergies 
• 
Physician diagnosed asthma up to stage 2 BTS/GINA guidelines 
• 
No clinical history of allergic rhinitis or eczema 
• 
Negative skin prick tests to a range of 6 common aeroallergens: cat, dog, grass 
pollen, tree pollen, house dust mite, fungal spores 
• 
Normal blood eosinophil count 
• 
Baseline forced expiratory volume (FEV1) ≥75% 
• 
Positive methacholine challenge (PC20< 8mg/ml) 
 
 
EXCLUSION CRITERIA 
 
Nasal and Respiratory 

 
 
 
 
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• 
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 
• 
Signs or symptoms of significant nasal anatomical defects, hypertrophy of turbinates, 
major septum deviation, nasal polyposis or recurrent sinusitis 
• 
Nasal mucosal defects, injury, ulceration 
• 
Previous nasal or sinus surgery 
Therapy  
• 
Medical therapy other than that permitted for contraception. 
• 
Treatment with local or systemic corticosteroids during the previous 2 months (other than 
low dose inhaled corticosteroids for asthmatic subjects) 
• 
Anti-inflammatory therapy: including non-steroidal anti-inflammatory drugs (NSAIDs)  
Concomitant diseases 
Clinically significant diseases are not permitted 
• 
tuberculosis at any stage in life  
• 
active infectious disease 
• 
cardiovascular diseases 
• 
respiratory (other than hay fever or asthma where specified) 
• 
hepatic, gastrointestinal, renal, endocrine, infective, haematological, autoimmune, 
rheumatological, neurological, dermatological,  
• 
neoplastic conditions 
• 
metabolic diseases and extreme obesity 
• 
depression and psychiatric disorders 
Miscellaneous 
• 
Non-smokers: up to 10 cigarettes a year is permitted 
• 
Participation in a therapeutic drug trial in the prior 30 days. 
• 
Inability or unwillingness to use contraception if the patient is a female of child-bearing 
age. 
• 
Pregnant or breast feeding women  
• 
Inability to provide informed consent 
 
4.4 MIXED 
INCLUSION 
& 
EXCLUSION 
CRITERIA 
FOR 
TUBERCULIN 
CHALLENGE   ARM ONLY 
 
 
VOLUNTEERS WITH LATENT TB INFECTION 
 
INCLUSION CRITERIA 
• 
Males and females aged 18 to 60 years 
• 
Body mass index in the range 18-39  
• 
Positive blood Interferon-γ release assay (IGRA): Quantiferon TB Gold-in-Tube (QFT-
it), >0.35 IU/ml IFN-γ versus control 

 
 
 
 
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• 
Tuberculin skin test (TST), using RT23 tuberculin purified protein derivative (PPD), 
from Statens Serum Institut (SSI) of Copenhagen.  
• 
2 tuberculin units (TU) in 0.1ml injected intradermally (id) : >6mm to <25mm of 
induration at 48-120h OR >15mm to <25mm in subjects with BCG vaccination 
• 
Normal chest X-ray (CXR) or CT scan if performed routinely for clinical reasons 
• 
Previous chemoprophylaxis for TB is permitted.  
 
 
HEALTHY INTERFERON-Γ RELEASE ASSAY (IGRA) NEGATIVE VOLUNTEERS 
 
INCLUSION CRITERIA 
• 
Age and sex matched to latent TB subjects 
• 
Body mass index in the range 18-39  
• 
No clinical history of TB infection 
• 
Healthy with no lung nor systemic symptoms  
• 
No active nasal allergy 
• 
No BCG vaccination 
• 
Negative blood Interferon-γ release assay (IGRA): Quantiferon TB Gold-in-Tube 
(QFT-it), <0.35 IU/ml IFN-γ versus control 
• 
Tuberculin skin test (TST), using RT23 tuberculin purified protein derivative (PPD), 
from Statens Serum Institut (SSI) of Copenhagen. 
• 
2 tuberculin units (TU) in 1ml injected intradermally (id): <6mm of induration at 48-
120h. 
• 
Chest X-ray is not required 
 
 
EXCLUSION CRITERIA (FOR ALL VOLUNTEERS)  
 
 
Nasal and Respiratory 
• 
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 
• 
Signs or symptoms of significant nasal anatomical defects, hypertrophy of turbinates, 
major septum deviation, nasal polyposis or recurrent sinusitis 
• 
Nasal mucosal defects, injury, ulceration 
• 
Previous nasal or sinus surgery  
• 
No clinical history of allergic rhinitis, allergic asthma or eczema. 
 
 
 
Concomitant diseases  
• 
Active tuberculosis at any stage in life  
• 
ENT disease 
• 
Active infectious disease including hepatitis 
• 
Respiratory disease 
• 
Renal disease  
• 
Autoimmune disease 
• 
Rheumatological disease & vasculitis 
• 
Dermatological disease 
• 
Neoplastic conditions 

 
 
 
 
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• 
Metabolic diseases and extreme obesity 
• 
Severe depression and psychiatric disorders 
 
Therapy  
• 
Medical therapy other than that permitted for contraception or for chronic conditions 
unlikely to affect the results of the study as determined by the study physician. 
• 
Treatment with local or systemic corticosteroids during the previous 2 months 
• 
Anti-inflammatory therapy: including non-steroidal anti-inflammatory drugs (NSAIDs)  
 
Miscellaneous  
• 
Participation in a therapeutic drug trial in the prior 30 days. 
• 
Inability or unwillingness to use contraception if the patient is female of child-bearing 
age. 
• 
Pregnant or breast feeding women  
• 
Inability to provide informed consent  
 
NB: Smokers permitted – but must be willing to stop smoking for duration of the study 
 
 
4.5 
WITHDRAWAL CRITERIA 
 
In the case of the ascending dose tolerability studies, all clinical activities will discontinue if 
there is a significant level of adverse events that is judged to be related to the nasal 
challenge as judged by the patient, and in the opinion of the Study Physician and/or the 
Principal Investigator. 
 
 
5. 
ADVERSE EVENTS 
 
5.1 
DEFINITIONS   
 
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study subject.   
 
Serious Adverse Event (SAE): any untoward and unexpected medical occurrence or effect 
that: 
• 
Results in death 
• 
Is life-threatening – refers to an event in which the subject 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 
• 
Requires 
hospitalisation, 
or 
prolongation 
of 
existing 
inpatients’ 
hospitalisation 
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE is serious in other 
situations.  Important AEs that are not immediately life-threatening or do not result in death 
or hospitalisation but may jeopardise the subject or may require intervention to prevent one 
of the other outcomes listed in the definition above, should also be considered serious. 
 
5.2 
REPORTING PROCEDURES 
All adverse events should be reported.  Depending on the nature of the event the reporting 
procedures below should be followed.  Any questions concerning adverse event reporting 
should be directed to the Chief Investigator in the first instance.   

 
 
 
 
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5.2.1 Non serious AEs 
All such events, whether expected or not, should be recorded.   
5.2.2 Serious AEs 
An SAE form should be completed and faxed to the Chief Investigator and the Sponsor 
within 24 hours. However, relapse and death due to an independent condition, and 
hospitalisations for elective treatment of a pre-existing condition do not need reporting as 
SAEs. 
 
All SAEs should be reported to the NRES Committee London-Harrow where the Chief 
Investigator considers the event was: 
• 
‘related’, i.e. resulted from the administration of any of the research procedures; 
and 
• 
‘unexpected’, i.e. an event that is not listed in the protocol as an expected 
occurrence 
 
Reports of related and unexpected SAEs should be submitted within 15 days of the Chief 
Investigator becoming aware of the event, using the NRES SAE form for non-IMP studies.  
The Chief Investigator must also notify the Sponsor of all SAEs. 
Local investigators should report any SAEs as required by their Local Research Ethics 
Committee, Sponsor and/or Research & Development Office. 
 
Contact details for reporting SAEs 
Fax: 020 331 25750, attention Dr Trevor Hansel and/or Dr Akhilesh Jha 
Please send SAE forms to: Imperial Clinical Respiratory Research Unit (ICRRU) 
St, Mary’s Hospital 
Mint Wing, First Floor, Entrance C 
Paddington 
London 
W2 1NY 
Tel: 020 331 25744 (Mon to Fri 09.00 – 17.00) 
 
6. 
ASSESSMENT AND FOLLOW-UP:  
DOSAGE JUSTIFICATION 
SAFETY MEASURES 
SAMPLING SCHEDULE 
 
MPLA 
We are proposing nasal administration of MPLA at incremental ascending single doses from 
10 to 100 to 500µg/100µl/nostril. 
• We have recently completed a study in 15 subjects with nasal highly purified LPS at 
doses of up to 100µg (in 100 µl), in which there were no symptoms or adverse events 
caused by the nasal LPS. 
• MPLA is at least 100 fold less toxic than LPS, and has been administered to more than 
300,000 human subjects. 

 
 
 
 
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• MPLA has been used as an allergen immunotherapy adjuvant at doses of 50μg sub-
lingually and by subcutaneous injection (36;46;47).  
• MPLA has been given to rodents at doses of 2500μg/kg by injection (81). 
 
Poly-IC 
Poly-IC is a synthetic viral poly-inosine/cytosine molecule that stimulates TLR-3. Unmodified 
poly-IC given systemically for cancer caused toxic side effects and this was probably caused 
by IL-12 production (82). High molecular weight poly-IC is employed for in vitro stimulation of 
human cells at doses of 5-100 g/ml (83) (84) (85) (86) (87).  
 
• 
A pilot study of Poly-IC nasal challenge was published recently by a group from Lund 
University, Sweden (53). Seven healthy atopic volunteers were challenged with a 
total of 800μg Poly-IC, administered as 8 separate doses of 100µl into one nostril. 
Four doses were given over an hour on day 1 and four doses 24h later on day 2. 
Each volunteer was challenged twice - once outside the allergen season and once 
during the Swedish birch pollen allergy season. The challenge was tolerated well, 
causing no changes in total nasal symptom scores at this dose. Nasal lining cells 
(epithelial cells) were taken using an interdental brush, and the RNA levels analysed.  
 
• 
Our study similarly will recruit healthy volunteers, both atopic and non-atopic. We will 
not recruit atopic volunteers during allergy season, to avoid any confounding effects 
of allergy on results. Our study will have additional scientific merit to that published 
as we repeatedly sample the mucosal lining fluid and look directly at the proteins 
secreted, as well as the RNA. Alongside total nasal symptom scores, we also assess 
volunteer responses using peak nasal inspiratory flow measurements. This will allow 
both subjective and objective clinical measurements that we can correlate with the 
immune data generated. 
 
• 
Based on the successfully tolerated dosage with measurable mucosal immune 
responses reported by Brandelius et al. (53), we therefore propose omitting Part A 
(Poly-IC incremental dose escalation from 10µg to 500µg) from the study, and move 
directly onto Part B with Poly-IC top dose 500µg.  We intend to administer a total of 
500g Poly-IC into each nostril over 1 hour, in 5 equal doses.  
 
Poly-ICLC 
Poly-ICLC is poly-IC stabilised with poly-L-lysine and carboxymethylcellulose, and this 
molecule has more RNAse resistance, and is better able to stimulate intracellular 
endosomal TLR3. Poly-ICLC has been developed by a Pharmaceutical Company 
(Oncovir of Washington DC, USA) and has been administered to man in a range of 
clinical studies, including as a vaccine adjuvant (48;54).  
We propose to administer poly-ICLC at incremental ascending single nasal spray doses 
from 10 to 100 to 500g to 1000µg/in up to 500µl, topically onto the nasal mucosa. We 
have obtained poly-ICLC from Oncovir and will use doses previously well tolerated in 
man. 
• 
Nasal poly-ICLC. John Beigel of Baltimore, MD, USA has performed a human phase 
I dose escalation clinical study with nasally administered poly-ICLC. The study has 
not been published, but data has been sent to the FDA. Poly-ICLC was well tolerated 
at all doses between 0.25mg and 2mg (2000μg) per nostril. This was a simple nasal 
instillation, not a spray.   

 
 
 
 
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• 
Intramuscular poly-ICLC at 20μg/kg has been safely given to man, being a 2mg dose 
in a 100kg man (54)  
 
Subcutaneous administration of 1.6mg  (1600μg) of poly-ICLC in man caused some 
redness and induration at the site of injection, and some mild-to-moderate transient 
flu-like symptoms (48). In this study there was upregulation of blood innate 
transcriptional signals (48). 
 
Resiquimod (R848) 
We will employ resiquimod as a solution, proceeding in careful incremental steps up to a 
maximum dose of 500µg in 100µl saline as a nasal spray, to each nostril. 
• 
Resiquimod was well tolerated in mice and rats in extensive tolerability studies 
(57;89). 
• 
Topical dermal resiquimod is safe and effective as a skin cream for the treatment of 
anogenital warts, actinic keratosis and skin cancer (55). Human studies have been 
carried out giving topical resiquimod to inflamed skin with actinic keratosis at doses 
up to 0.25%. 
0.25g in 100ml= 250,000µg/100ml = 2500µg/ml = 250µg/100μl 
• 
Oral resiquimod has been given to patients with chronic hepatitis C virus (HCV)  
infection (90). Resiquimod was well tolerated orally at doses of 0.01mg/kg, equating  
to 1mg (1000μg) for a 100kg man. 
 
Tuberculin 
 
Table. TUBERCULIN DOSE JUSTIFICATION:  
Relative Skin, Inhaled and Nasal Doses 
 
 
Skin 
Nasal 
Inhaled 
Tuberculin PPD 
supplied as  
20 TU/ml 
100 TU/ml 
Intradermal 
injection of 
100l 
2, 5, 10 TU 
Proposal for  
0.1, 1, 2, 5, 10 TU per 
100µl nasal spray in 
separate cohorts of 
subjects with latent TB 
Bronchoscopic 
administration of  0.5 TU 
in 10 ml to one broncho-
pulmonary segment in 
patients with latent TB 
 
We propose nasal tuberculin challenge in a cautious incremental ascending dose regimen. 
We shall begin at 0.1TU since this is below the Minimum Anticipated Biological Effect Level 
(MABEL).  In the next cohort we shall give 1.0TU, followed by a cohort at 2.0TU, 5.0TU and 
finally 10.0TU. 
 
The nasal dose is delivered by a nasal spray and will be administered as a fine mist diffusely 
to the upper respiratory tract nasal passages. 
 
• 
For tuberculin injected intradermally at up to 10TU, there is a high local concentration 
within a bleb.  
• 
On the basis of skin and nasal tuberculin doses being in the same range, we shall 
give tuberculin at a top dose of 10TU/100μl spray.  
• 
Bronchial installation: Tuberculin has been administered safely to subjects with latent 
TB by bronchosegmental challenge at 0.5TU per 10ml.  

 
 
 
 
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In the studies of Richard Silver and colleagues in Ohio, 0.5TU tuberculin PPD was 
administered into a subsegment of the lingula in 10ml, covering an area of alveoli and small 
airways estimated at 100cm2 (58) (59) (60). 
We estimate our nasal spray is delivered over each nostril to an equivalent area of 
approximately 100cm2, that corresponds to the area of the nasal mucosa in a nostril. Hence 
we begin at a lower dose of 0.1TU per nostril spray. 
 
Allergen 
The dose of Timothy grass pollen allergen has previously been safely employed in a range 
of clinical studies in patients with allergic rhinitis and asthma (91).  
 
Vitamin D3 
This is a widely used nutritional supplement with an excellent safety profile with no adverse 
events reported at doses of 2000-4000 IU/day given orally. To study the actions of vitamin 
D3 we propose looking at the effects of NAC with Timothy grass pollen in people with grass 
pollen allergy outside the summer grass pollen season (May-July).  
 
We will use Vitamin D3 at a dose of 4000IU/tablet taken orally. This dose has been widely 
used in research studies and was used on a daily basis for 28 days in the VIDA Study: 
Vitamin D Supplementation in Asthma (65). There is no additional effect of increasing the 
dose further and very few studies go above 4000IU/day.  
 
The supplement used will be sourced from Lamberts Healthcare Ltd and is certified to be 
• 
Manufactured according to good medical practice guidelines (GMP)  
• 
Verified for quality (vitamin D levels) to British Pharmacopoiea (BP) standards  
• 
Licensed as a food supplement 
 
Serum calcium levels will be monitored as rarely hypercalcaemia can be an adverse event. 
 
Placebo 
This will be prepared by an NHS pharmacy in a similar format to vitamin D3 and will be taken 
by subjects in an identical manner to the vitamin D3 supplementation. 
 
 
 
DOSING SCHEDULE: SAFETY PRECAUTIONS 
 
 
A. Nasal Challenge with TLR-Agonists 
 
For each dosage cohort, a single subject will first be assessed for safety over 24h. We shall 
monitor all subjects for local and systemic symptoms, and record vital signs, for at least 6 
hours following each nasal challenge. For asthmatic subjects we will additionally monitor 
their forced expiratory volume (FEV1) and should respiratory symptoms or spirometry 
deteriorate significantly, this will be promptly treated by clinical staff, e.g. by administration of 
inhaled B2 agonists via inhaler or nebulizer. For the dose escalation phase of the study (Part 
A), we will additionally make a telephone call at 24 hours to ensure there are no persistent 
symptoms. We shall then proceed to further subjects if there are no adverse events. 
 

 
 
 
 
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Then the second and third subjects will be observed over 24h, again only proceeding if there 
are no adverse events. 
 
Then the 4 to 8th subjects can be given nasal challenge simultaneously. 
 
There will be at least 4 to 7 days interval between each challenge agent in any one volunteer 
subject. 
 
Dosage Regimen for poly-ICLC 
o First dose 10µg per 100μl per nostril 
o Second dose 100µg per 100μl per nostril 
o Third dose 500µg per 300μl per nostril 
o Fourth dose of 1000µg in 500µl per nostril administered over 1 hour 
Dosage Regimen for poly-IC 
o Single dose of 500µg per 500μl per nostril administered over 1 hour 
Dosage Regimen for MPLA and Resiquimod 
o First dose 10µg per 100μl per nostril 
o Second dose 100µg per 100μl per nostril 
o Third dose 500µg per 500μl per nostril administered over 1 hour 
o Jan 2016 update: due to the development of pyrexia in a volunteer given the 
10μg/nostril dose – we are moving to a weight based dosing regimen. This 
will involve using a dose between 0.02μg/kg to 0.05μg/kg per nostril dose in 
remaining subjects. 
 
 
B. Nasal Challenge with Tuberculin 
 
Visits to ICRRU 
 
Each subject shall be observed for up to 7 days following nasal administration of tuberculin 
 
Visits to ICRRU will be made at: 
• 
Screening 
• 
challenge day (with observation up to 8h after administration) 
• 
24h, 48h (2d), 3d, 4d, 7d 
 
Intervals between Patients 
 
In the ascending dose part of the study we study groups of 4 people given a single dose, 
before moving incrementally to the next group of 4 given a higher dose. 
 
For each dosage cohort, a single subject will first be observed for >96h. We shall then 
proceed to further subjects at the same dose, if there are no adverse events 
 
Then the second subject will be observed over >48h, again only proceeding if there are no 
adverse events in the first 2 subjects 
 
Then the 3rd and 4th subjects can be given nasal challenge simultaneously. 

 
 
 
 
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There will be follow-up for all subjects given nasal challenge at 7d after challenge. 
 
 
Ascending Dose Protocol in Subjects with Latent TB (LTB) 
 
Single dose of tuberculin PPD per subject (spray each nostril),  
4 subjects per dose without placebo initially, 4 groups of 4: 
 
• 
0.1TU/nostril, from tuberculin 20U/ml, n=4           
clinical observations: nasal examination and systemic features (BP, pulse, 
temperature) every 30min for 2h 
 
• 
1.0TU/nostril, from tuberculin 20U/ml, n=4           
clinical observations, nasal examination and systemic features (BP, pulse, 
temperature) 
 
• 
2.0TU/nostril, from tuberculin 20U/ml, n=4           
clinical observations, nasal examination and systemic features (BP, pulse, 
temperature) 
 
• 
5.0TU/nostril, from tuberculin 100U/ml, n=4        
clinical observations nasal examination and systemic features (BP, pulse, 
temperature) 
  
 
After 5.0TU has been administered clinical data and MSD analyses of nasosorption eluates 
from all doses will be made. 
Then the decision will be made on whether to continue 5.0TUnostril or move up to 
10.0TU/nostril, from tuberculin 100U/ml 
 
Highest dose nasal tuberculin cohort 
 
1. Decision on top dose. 
N=8 on top dose with latent TB, 6 versus 2 placebo 
N=8 healthy IGRA test negative, 6 versus 2 placebo 
 
2. Nasosorption: 
 
• cytokines and chempokines: - elute with buffer 
• lipidomics, prostanoids, resolvins, metabolomics – spin off neat 
 
3. Nasal scrapes for: 
• 
flow cytometry on T cells and DCs 
• 
transcriptomics 
 
 
7. 
STATISTICS AND DATA ANALYSIS 
 
Data and all appropriate documentation will be stored for a minimum of 10 years after the 
completion of the study, including the follow-up period.   
 
The major data will be levels of cytokines and chemokines in nasal SAM eluates. 

 
 
 
 
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We shall perform a test of distribution of data (Shapiro-Wilks test), although nasal levels of 
chemokines and cytokines are generally non parametric in distribution. 
 
Raw Data:  
We shall make plots for individual cytokines with raw data for individuals connected by lines. 
These “spaghetti plots” generally have different colours for different individuals. 
 
Summary Statistics 
Normal data with graphs with means and standard error of means (SEM) 
Non-normal data with graphs with medians, quartiles and ranges (box-whisker) 
 
Non-parametric tests are used to compare challenge effects with placebo at different times: 
 Willcoxon signed-rank test for paired and Mann-Whitney for unpaired data. 
 
The differences from baseline and difference compared with that from placebo (active-
placebo) at each time point and the differences from placebo (active-placebo) will be 
calculated, and the significance determined.  
 
We shall also analyse area under the curve (AUC), by selecting specified times in the early 
or late phase or for the entire response. 
e.g. PGD2 from 10min to 2h 
e.g. for IL-4 at 4 to 8h.  
 
A P value of <0.05 is considered significant. 
Randomization 
For the ViDAR study randomization will be supervised by the pharmacy at St Mary’s 
Hospital. The pharmacy will organise the packaging and have sole access to the 
randomization codes until the completion of all the clinical aspects of the study. 
8. 
REGULATORY ISSUES 
 
Topical Nasal Challenge Agents 
All our challenge agents are used to elicit an inflammatory response, and not employed for 
therapeutic benefit. 
Hence these agents are regarded by the UK regulatory authority (the Medicines and 
Healthcare Regulatory Authority, MHRA) as Non-Investigative Medicinal Products (non-IMP) 
(92). 
For this reason, the challenge agents do not have to be manufactured according to Good 
Manufacturing Practice (GMP). 
A proposal to employ these nasal challenges does not require supervision by the MHRA, 
where we have contacted the Clinical Trials Dept and Dr Elaine Godfrey. 
 
Vitamin D3 
We have obtained confirmation from the MHRA and the manufacturers of Vitamin D3 
(Lamberts Healthcare Ltd) that the vitamin D3 is classified as a food supplement and not an 
Investigative Medicinal Product (IMP). 
 
8.1 
ETHICS APPROVAL 
The Chief Investigator has obtained approval from the Research Ethics Committee.  The 
study must be submitted for Site Specific Assessment (SSA) at each participating NHS 
Trust.  The Chief Investigator will require a copy of the Trust R&D approval letter before 

 
 
 
 
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accepting participants into the study.  The study will be conducted in accordance with the 
recommendations for physicians involved in research on human subjects adopted by the 
18th World Medical Assembly, Helsinki 1964 and later revisions. 
 
Only for subjects receiving tuberculin we shall propose a limited range of genetic tests. This 
is because the immune response to TB is strongly influenced by a limited number of genes 
(61;93). These samples will be handled strictly under the Human Tissue Act, and data 
handled in a confidential manner, with no reporting back to the subject. 
 
 
8.2 
CONSENT  
• 
Anonymised samples will be aliquoted and stored prior to analysis in all cases. 
• 
An option for the participant to provide blood samples for DNA analysis has been 
included for Part B 
• 
An option for the participant to take part in a reproducibility element has been 
included for Part B  
• 
All DNA blood samples will be stored as per Human Tissue Act regulations. 
• 
All publications will be anonymised. 
 
Consent to enter the study must be sought from each participant only after a full explanation 
has been given, an information leaflet offered and time allowed for consideration.  Signed 
participant consent should be obtained.  If potential volunteers are identified from sites other 
than Imperial College Healthcare NHS trust, they will be asked to sign a pre-screening 
consent form that seeks permission for their identifiable demographic and medical data to be 
transferred from their base hospital to Imperial College Healthcare NHS trust. This data will 
be transferred on a password protected spread sheet via secure NHS mail and will 
subsequently be stored on a secure trust network that is restricted to researchers from the 
Imperial Clinical Respiratory Research Unit (ICRRU) based at St Mary’s Hospital, Paddington, 
London. The right of the participant to refuse to participate without giving reasons must be 
respected.  After the participant has entered the study the clinician remains free to give 
alternative treatment to that specified in the protocol at any stage if he/she feels it is in the 
participant’s best interest, but the reasons for doing so should be recorded.  In these cases 
the participants remain within the study for the purposes of follow-up and data analysis.  All 
participants are free to withdraw at any time from the protocol treatment without giving 
reasons and without prejudicing further treatment. 
 
8.3 
CONFIDENTIALITY 
The Chief Investigator will preserve the confidentiality of participants taking part in the study 
and is registered under the Data Protection Act. 
 
8.4 
INDEMNITY 
Imperial College London holds negligent harm and non-negligent harm insurance policies 
which apply to this study. 
The College holds two insurance policies which apply to healthcare research.  
 
1. Public Liability (negligent harm) 
 
The College holds Zurich Municipal Public Liability insurance which provides cover in the 
event that Imperial College is held legally liable for negligent harm caused to a research 
subject. 
 
2. Clinical Trial (non-negligent harm) 
 

 
 
 
 
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Cover for non-negligent harm arising from an Imperial project is provided by the College's 
Zurich Municipal Clinical Trials insurance policy. The policy document defines a "clinical trial" 
very broadly as: "Any investigation or series of investigations conducted on any person for a 
medicinal purpose". 
 
Consequently, this policy will be relevant if your study involves direct interventions with a 
research subject. 
 
The non-negligent harm policy specifically excludes cover for claims arising from children 
aged under five and pregnant women.  It has a number of other important exclusions, 
including, but not limited to, claims arising directly from HIV/AIDS, Hepatitis, Creutzfeldt-
Jakob disease, or the use of products or devices designed by Imperial College. 
 
It is usually possible to have the pregnant women and children aged under five exclusions 
waived on a study-by-study basis following liaison with the Insurer. 
 
http://www3.imperial.ac.uk/clinicalresearchgovernanceoffice/projectplanning/clinicalresearchoffice
approval/insurance 
 
8.5 
SPONSOR 
Imperial College London will act as the main Sponsor for this study. Delegated 
responsibilities will be assigned to the NHS Trust taking part in this study.   
 
8.6 
FUNDING 
Imperial NIHR BRC are funding this study 
 
 
8.7 
AUDITS  
The study may be subject to inspection and audit by Imperial College London under their 
remit as sponsor and other regulatory bodies to ensure adherence to GCP and the NHS 
Research Governance Framework for Health and Social Care (2nd edition).  
 
 
9. 
STUDY MANAGEMENT 
 
The day-to-day management of the study will be co-ordinated through Tanushree Tunstall 
(unit coordinator), Dr Akhilesh Jha (Clinical Research Fellow) and Jennifer Brimley 
(Research Nurse) with close support from Dr Trevor T. Hansel of ICRRU at St Mary’s 
Hospital.   
 
10. 
PUBLICATION POLICY 
 
Our aim is to ensure the full dissemination of all study results in medical publications of the 
highest impact and citation factor. Individual subjects will be strictly anonymised. 
 
 
 

 
 
 
 
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11. 
REFERENCES 
Reference List 
 
 
(1)  Hansel TT, Johnston SL, Openshaw PJ. Microbes and mucosal immune responses 
in asthma. Lancet 2013 February 18. 
 
(2)  Mirzakhani H, Al-Garawi A, Weiss ST, Litonjua AA. Vitamin D and the development 
of allergic disease: how important is it? Clin Exp Allergy 2014 October 13. 
 
(3)  Mann EH, Chambers ES, Pfeffer PE, Hawrylowicz CM. Immunoregulatory 
mechanisms of vitamin D relevant to respiratory health and asthma. Ann N Y Acad 
Sci 2014 May;1317:57-69. 
 
(4)  Kupfer SS, Maranville JC, Baxter SS, Huang Y, Di RA. Comparison of cellular and 
transcriptional responses to 1,25-dihydroxyvitamin d3 and glucocorticoids in 
peripheral blood mononuclear cells. PLoS ONE 2013;8(10):e76643. 
 
(5)  Boulet LP, Gauvreau G, Boulay ME, O'Byrne P, Cockcroft DW. The allergen 
bronchoprovocation model: an important tool for the investigation of new asthma 
anti-inflammatory therapies. Allergy 2007 October;62(10):1101-10. 
 
(6)  O'Byrne PM, Gauvreau GM, Brannan JD. Provoked models of asthma: what have 
we learnt? Clin Exp Allergy 2009 February;39(2):181-92. 
 
(7)  Devillier P, Le GM, Horak F. The allergen challenge chamber: a valuable tool for 
optimizing the clinical development of pollen immunotherapy. Allergy 2011 
February;66(2):163-9. 
 
(8)  Barnes PJ. Severe asthma: advances in current management and future therapy. J 
Allergy Clin Immunol 2012 January;129(1):48-59. 
 
(9)  Wechsler ME, Fulkerson PC, Bochner BS, Gauvreau GM, Gleich GJ, Henkel T et 
al. Novel targeted therapies for eosinophilic disorders. J Allergy Clin Immunol 2012 
September;130(3):563-71. 
 
(10)  Wenzel SE. Asthma phenotypes: the evolution from clinical to molecular 
approaches. Nat Med 2012 May;18(5):716-25. 
 
(11)  Kraft M. Asthma phenotypes and interleukin-13--moving closer to personalized 
medicine. N Engl J Med 2011 September 22;365(12):1141-4. 
 
(12)  Ingram JL, Kraft M. IL-13 in asthma and allergic disease: Asthma phenotypes and 
targeted therapies. J Allergy Clin Immunol 2012 October;130(4):829-42. 
 
(13)  Howarth PH, Persson CG, Meltzer EO, Jacobson MR, Durham SR, Silkoff PE. 
Objective monitoring of nasal airway inflammation in rhinitis. J Allergy Clin Immunol 
2005 March;115(3 Pt 2):S414-S441. 
 
(14)  Alam R, Sim TC, Hilsmeier K, Grant JA. Development of a new technique for 
recovery of cytokines from inflammatory sites in situ. J Immunol Methods 1992 
October 19;155(1):25-9. 

 
 
 
 
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(15)  Weido AJ, Reece LM, Alam R, Cook CK, Sim TC. Intranasal fluticasone propionate 
inhibits recovery of chemokines and other cytokines in nasal secretions in allergen-
induced rhinitis. Ann Allergy Asthma Immunol 1996 November;77(5):407-15. 
 
(16)  Sim TC, Reece LM, Hilsmeier KA, Grant JA, Alam R. Secretion of chemokines and 
other cytokines in allergen-induced nasal responses: inhibition by topical steroid 
treatment. Am J Respir Crit Care Med 1995 September;152(3):927-33. 
 
(17)  Sim TC, Grant JA, Hilsmeier KA, Fukuda Y, Alam R. Proinflammatory cytokines in 
nasal secretions of allergic subjects after antigen challenge. Am J Respir Crit Care 
Med 1994 February;149(2 Pt 1):339-44. 
 
(18)  Linden M, Svensson C, Andersson E, Andersson M, Greiff L, Persson CG. 
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APPENDICES 
Appendix 1: Schedule of Events: Summary of Screening Procedures 
 
Depending on the part of the study, a range of these procedures will be performed.  
 
Pre-challenge 
Telephone screening 
X 
Clinical history 
X 
Physical exam 
X 
Skin allergen test 
X 
Tuberculin skin test 
X 
 
Interferon-gamma release assay (IGRA) 
X 
FBC, U&E, LFT, IgE 
X 
Chest X-ray and ECG 
X 
Informed Consent 
X 
Nasal and Pharyngeal Swab Sample 
 
Calcium, 25-OH Vitamin D levels, melatonin 
X 
Pregnancy test (if applicable) 
X 
Methacholine bronchoprovocation  
X 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
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Appendix 2a: Summary of Procedures for Nasal Toll-Like Receptor (TLR) Agonist Challenge 
 
PART A: Incremental Ascending Dose Study based on Tolerability and SAM 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
• 
LEFT nostril: Nasosorption neat:  to include from lipidomics, mass spectrometry, prostanoids, PGD2, tryptase 
• 
RIGHT nostril: Nasosorption in assay buffer: for chemokines and cytokines, complement C3a & 5a  
Time 2a 
Total duration = 30 min 
Nasal sampling before NC 
 
Nasal Challenge (NC) 
 
Total duration=9h 
Hourly nasal sampling post NC 
 
-30m 
-20m 
-10m 
15m 
30m 
45m 
1h 
2h – 8h 
Nasal challenge 
Saline/TLR agonist (variable doses)  
 
 
 
X 
 
 
 
 
 
Nasal lavage (Washing only) 
 
L/R 
 
 
 
 
 
 
 
Nasal mucosal examination 
X 
 
 
 
X 
X 
X 
X 
X 
Total nasal symptom scores (TNSS) & PNIF 
X 
 
 
 
X 
X 
X 
X 
X 
Nasosorption (L and R) 
 
L/R 
 
L/R 
 
L/R 
L/R 
L/R 
L/R 
L/R 

 
 
 
 
 
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Appendix 2b: Summary of Procedures for Nasal Toll-Like Receptor (TLR) Agonist Challenge 
PART B: Highest Dose (1000µg over 1h, or highest single dose)  
• LEFT nostril: Nasosorption neat:  to include from lipidomics, mass spectrometry, prostanoids, PGD2, tryptase 
• RIGHT nostril: Nasosorption in assay buffer: for chemokines and cytokines, complement C3a & 5a  
Time 2b 
 
Screening 
Total duration = 30 min 
Nasal sampling before NC 
 
 
Total duration = 1h 
Nasal Challenge (NC) 
200µg/nostril on 5 occasions over 1h 
Total duration=8h 
Hourly nasal sampling post NC 
 
 
Total 
duration = 
2h 
D -7 
-30m 
-20m 
-10m 
0m 
15m 
30m 
45
m 
60m 
75m 
90m 
105m 
120m 
3h – 9h 
24h 
Nasal challenge 
Saline / TLR agonist 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Nasal lavage (Washing only) 
X 
 
L /R 
 
 
 
 
 
 
 
 
 
 
 
 
Nasal mucosal examination 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Total nasal symptom scores 
(TNSS) & PNIF 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Nasosorption (NS) 
 
L /R 
 
L /R 
 
 
 
 
 
L /R 
L /R 
L /R 
L /R 
L /R 
X 
Nasal curettage 
X 
Up to 4 
samples 
 
 
 
 
 
 
 
 
 
 
 
 
(9h) 
4L 
X 
4R 
Blood (20ml) 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
OPTIONAL: Nasal SSP (Real time MS) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

 
 
 
 
 
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Appendix 2c: Summary of Procedures for Nasal Resiquimod Challenge 
PART B: Highest Dose (10µg/100μl)[Jan 2016: move to a weight based dosing regimen between 0.02μg/kg to 0.05μg/kg]   
 
•  
Time 2c 
 
Screening 
 
Nasal sampling before NC 
Total duration = 30 min 
 
 
Nasal 
Challenge 
(NC) 
 
Hourly nasal sampling post NC 
Total duration=8h 
 
 
Final 
Sample 
D -7 
-30m 
-20m 
-10m 
0m 
15m 
30m 
45m 
60  
m 
1-10h 
24h 
Nasal challenge 
Saline / Resiquimod 
 
 
 
 
X 
X 
X 
X 
X 
 
 
Nasal lavage (Washing only) 
X 
 
L /R 
 
 
 
 
 
 
 
 
Nasal mucosal examination 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
Total nasal symptom scores 
(TNSS) & PNIF 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
Nasosorption (NS) 
 
L /R 
 
L /R 
 
L /R 
L /R 
L /R 
L /R 
L /R 
X 
Nasal curettage 
X 
Up to 4 
samples 
 
 
 
 
 
 
 
 
(6-8h) 
4L 
X 
4R 
Blood (up to 20ml) 
X 
 
X 
 
 
 
 
 
 
X (4h) 
X (8h) 
X 
Spirometry (for asthmatics) 
X 
X 
 
 
 
 
 
 
 
1,2,4,8h 
x 

 
 
 
 
 
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Appendix 3a: Part A Summary of Procedures for Tuberculin Nasal Challenge 
 
PART A: Nasal ascending dose (0.1TU, 1.0TU, 2.0TU, 5.0TU) tolerability and SAM 
 
• 
LEFT nostril: Nasosorption neat:  to include from lipidomics, mass spectrometry, prostanoids, PGD2, tryptase 
• 
RIGHT nostril: Nasosorption in assay buffer: for chemokines and cytokines 
Time 3a 
 
S 
D -7 
-30m 
-20m 
-10m 
Nasal 
Challenge 
(NC) 
1h – 8h 
d1 
d2 
d3 
d4 
d7 
Nasal challenge 
Saline/Tuberculin (variable 
doses) 
 
 
 
 
X 
X 
 
 
 
 
 
Bilateral nasal lavage for 
washing purposes  
X 
 
X 
 
 
X 
 
 
 
 
 
Nasal mucosal examination 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
Total nasal symptom scores 
(TNSS) & PNIF 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
Nasosorption 
 
 
X 
2L 
2R 
 
X 
2L 
2R 
 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
4L 
X 
4R 
X 
4R 
OPTIONAL  
Nasal curettage 
 
X 
 
 
 
 
 
 
X 
4R 
 
X 
4L 
X 
4R 

 
 
 
 
 
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Appendix 3b: Part B Summary of Procedures for Tuberculin Nasal Challenge 
 
PART B: Top dose (10.0TU) with Additional Sampling 
 
• 
LEFT nostril: Nasosorption neat:  to include from lipidomics, mass spectrometry, prostanoids, PGD2, tryptase 
• 
RIGHT nostril: Nasosorption in assay buffer: for chemokines and cytokines 
Time 3b 
S  
D -7 
-30m 
-20m 
-10m 
Nasal 
Challenge 
1h – 8h 
d1 
d2 
d3 
d4 
d7 
Nasal challenge 
Placebo/Tuberculin (Top dose)  
 
 
 
 
X 
 
 
 
 
 
 
Bilateral nasal lavage for washing 
purposes  
X 
 
X 
 
 
 
 
 
 
 
 
Nasal mucosal examination 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
Total nasal symptom scores 
(TNSS) & PNIF 
 
X 
 
 
 
X 
X 
X 
X 
X 
X 
Nasosorption 
 
 
X 
2L 
2R 
 
X 
2L 
2R 
 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
4L 
X 
4L 
X 
4R 
Nasal curettage 
 
X 
 
 
 
 
 
 
X 
4R 
 
X 
4L 
X 
4R 
Blood (20ml) 
 
X 
 
 
 
 
 
X 
 
X 
X 
OPTIONAL 
Nasal SSP 
 
 
 
 
 
 
X 
 
 
 
 

 
 
 
 
 
Page 51 of 54 
Protocol  
Version No: 7.0 
Date: 20th January 2016  
 
 
Appendix 4A:  Vitamin D3 in Nasal Allergic Rhinitis (ViDAR) 
Summary of Procedures for Nasal Allergen Challenge (NAC) Days 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Blood for full blood count (5ml); peripheral blood mononuclear cells (PBMC) (50ml);  serum vit D, calcium, cortisol, melatonin (10ml);  whole blood 
transcriptomics (10ml) 
Procedure 
-30m 
  
-20m 
-10m 
Nasal 
Challenge 
5m 
15m 
30m 
45m 
1h 
Hourly  
 2-8h 
Nasal allergen challenge 
Timothy grass pollen 
Phleum P5  
 
 
 
X 
~ 9AM 
 
 
 
 
 
 
Bilateral nasal lavage for 
washing purposes (discard) 
 
 
X 
 
 
 
 
 
 
 
 
Oral capsule:  
Day 28: vitamin D or placebo 
x 
 
 
 
 
 
 
 
 
 
Total nasal symptom scores 
(TNSS) and peak nasal 
inspiratory flow (PNIF) 
 
 
X 
 
X 
X 
X 
X 
X 
X 
Nasosorption (SAM) 
L/R 
 
L/R 
 
L/R 
L/R 
L/R 
L/R 
L/R 
L/R 
Blood (75ml)* 
X 
 
 
 
 
 
 
 
 
 
Nasal curettage (rhinoprobe)  
 
 
 
 
 
 
 
 
 
2L/2R 
At 8 hours 
(~5PM) 

 
 
 
 
 
Page 52 of 54 
Protocol  
Version No: 7.0 
Date: 20th January 2016  
 
 
 
Appendix 4B: Summary of Procedures for Nasal Allergen Challenge (NAC) 
 
 
• 
LEFT nostril: Nasosorption neat:  to include from lipidomics, mass spectrometry, prostanoids, PGD2, tryptase 
• 
RIGHT nostril: Nasosorption in assay buffer: for chemokines and cytokines 
Time 4 
-30m 
-20m 
-10m 
NC 
5m 
10m 
15m 
30m 
45m 
1h 
2h 
3h 
4h 
5h 
6h 
Nasal challenge 
Saline/Allergen 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Bilateral nasal 
lavage for washing 
purposes 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nasal mucosal 
examination 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Total nasal symptom 
scores (TNSS) & 
PNIF 
X 
 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Nasosorption 
 
X 
2L 
2R 
 
 
 
X 
2L 
2R 
 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
X 
2L 
2R 
Blood 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Nasal curettage 
X 
4L 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
4R 
OPTIONAL 
Nasal SSP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

 
 
 
 
Page 53 of 54 
Protocol  
Version No: 7.0 
Date: 20th January 2016  
 
 
Appendix 5: Pharmaceutical Quality of Agents delivered by Nasal Spray as 
Nasal Challenges or taken orally 
Challenge/Oral Agent 
Manufacturer 
Contact person 
Catalogue  
Dose 
Production 
Purity 
Timothy grass pollen 
allergen licensed for  
intraepidermal and 
subcutaneous skin 
testing 
 
Aquagen SQ Timothy 
grass Pollen 
Phleum pratense 
number 4 
Manufacturer: ALK-Abello: 
TEL 01488 686 016 
Andrew Young  
TEL 01488 688 337 MOB 07917 
191 
468 
andrew.young@alk-
abello.com  
 
Supplier: 
Healthcare 
Logistics 
TEL 01234 248 736, Mandy 
Meinertzhagen.  
Aquagen SQ 
Timothy grass 
Pollen, 
 internal code 
1001862  
product code  
225 156 
(ALK 225)  
 
ALK Abello freeze 
dried extract. 
Tuberculin purified 
protein derivative (PPD) 
licensed internationally 
for intradermal skin 
testing 
In the Mantoux test 
 
• 
20 
units/ml, 
1.5ml vial 
• 
100 
units/ml, 
1.5ml vial 
Heat-treated products 
of growth and lysis of  
appropriate 
Mycobacterium spp. 
Poly-ICLC 
(poly inosine-cytosine 
stabilised with poly-L-
lysine) 
 
Hiltonol 
Vaccine component 
(HTN code 3002200) 
Registered with FDA 
under IND 43984, held 
by Oncovir Inc. 
Lot PJ215-10-01 
Dalton Chemical Laboratories for 
Oncovir 
 
Andres M. Salazar, MD 
CEO & Scientific Director 
Oncovir, Inc.,  
3203 Cleveland Ave, NW 
Washington, DC 
200008-3450, USA 
asalazar@oncovir.com 
TEL 001 202 342 1726 
2mg/ml 
1ml/vial 
Chemical synthesis. 
Non-biological origin: 
does not contain any 
animal nor human 
components 
Poly-IC (inosine-
cytosine), high 
molecular weight, 
ultrapure 
Synthetic analogue of 
double stranded RNA 
Invivogen, California 
3950 Sorrento Valley Boulevard, 
Suite 100, San Diego, CA 92121, 
USA 
www.invivogen.com 
Tlrl-pic 
10mg and 50mg  
Lyophilised 
powder 
 
Reconstitute in 
endotoxin-free 
physiological water 
Chemical synthesis. 
Non-biological origin: 
does not contain any 
animal nor human 
componentsEndotoxin 
level 
<1.25 EU/mg 
MPLA (monophosphoryl 
lipid A) 
Synthetic vaccine grade 
Invivogen, California 
3950 Sorrento Valley Boulevard, 
Suite 100, San Diego, CA 92121, 
USA 
 
www.invivogen.com 
I mg MPLA 
VacciGrade 
supplied as a clear 
lipidic film. 
CAS 1246298-63-
4 
Reconstitute in 
endotoxin-free 
physiological water 
Chemical synthesis. 
Non-biological origin: 
Comprises   
lipid A from E.coli 
serotype R515, with 6 
fatty acyl groups 
Resiquimod 
R848 VacciGrade 
TLR 7/8 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 

 
 
 
 
Page 54 of 54 
Protocol  
Version No: 7.0 
Date: 20th January 2016  
 
 
 
Vitamin D3 
Vitamin D3 from Lamberts 
Healthcare Ltd  
http://www.lambertshealthcare.co
.uk 
4000IU 
Manufactured 
according to GMP & 
meets British 
Pharmacopoiea (BP) 
standards for Vitamin 
D levels