Cell Mediated Immunity Against RSV and Influenza in a Human Experimental Challenge
Phase: NA
Conditions: Respiratory Syncytial Virus Infections, Influenza, Human
Sponsor: Imperial College London

 
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Cell mediated immunity against 
RSV and influenza in a human 
experimental challenge model 
 
 
 
 
 
 
 
 
RESEARCH PROTOCOL 
 
 
 
Version Fourteen 
12th July 2018 
 
 
 
 
 
 
 
 
MAIN SPONSOR: Imperial College London  
FUNDER: MRC & European Union Framework 7 
STUDY COORDINATION CENTRE: Respiratory Infections, NHLI, 
St Mary’s Campus 
 
 
NRES/REC reference: 11/LO/1826 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Table of Contents
TABLE OF CONTENTS ........................................................................................................... 2 
STUDY SUMMARY .................................................................................................................. 5 
GLOSSARY OF ABBREVIATIONS ......................................................................................... 5 
INTRODUCTION ...................................................................................................................... 6 
1.1. 
BACKGROUND TO RESEARCH ............................................................................................................................ 6 
1.2. 
RESEARCH HYPOTHESES .................................................................................................................................. 8 
STUDY OBJECTIVES .............................................................................................................. 8 
1.3. 
PRIMARY OBJECTIVE: ....................................................................................................................................... 8 
1.4. 
SECONDARY OBJECTIVE: .................................................................................................................................. 8 
PARTICIPANT ENTRY ............................................................................................................ 9 
1.5. 
RECRUITMENT .................................................................................................................................................. 9 
1.6. 
PRE-REGISTRATION EVALUATIONS..................................................................................................................... 9 
1.6.1. 
Screening Visit .........................................................................................................................................................9 
1.7. 
INCLUSION CRITERIA ......................................................................................................................................... 9 
1.8. 
EXCLUSION CRITERIA ........................................................................................................................................ 9 
1.9. 
WITHDRAWAL CRITERIA ..................................................................................................................................10 
STUDY DESIGN ..................................................................................................................... 11 
1.10. 
STUDY VISITS FOR ALL SUBJECTS ..................................................................................................................12 
1.10.1. 
Baseline visit (Day -14) ....................................................................................................................................... 13 
1.10.2. 
Confinement period for RSV and influenza arms (confinement, day 0 – day 8-10 and day 168 to 177-179) ..... 13 
1.10.3. 
Follow-up period (outpatient, day 11 – day 196) ................................................................................................. 13 
STUDY PROCEDURES ......................................................................................................... 14 
1.11. 
VIRUS INOCULATION .....................................................................................................................................14 
1.12. 
SWABS FOR MICROBIAL ANALYSIS .................................................................................................................14 
1.12.1. 
Throat swab.......................................................................................................................................................... 14 
1.12.2. 
Stool swab ............................................................................................................................................................ 14 
1.13. 
NASAL SAMPLING PROCEDURES ....................................................................................................................14 
1.13.1. 
Nasal scrape using Rhinopro®.............................................................................................................................. 15 
1.13.2. 
Nasosorption ........................................................................................................................................................ 15 
1.13.3. 
Nasal Lavage........................................................................................................................................................ 15 
1.14. 
BRONCHOSCOPY ..........................................................................................................................................15 
1.14.1. 
Bronchosorption: ................................................................................................................................................. 16 
1.14.2. 
Bronchial Biopsies: .............................................................................................................................................. 17 
1.14.3. 
Bronchial Brushings: ........................................................................................................................................... 17 
1.14.4. 
Bronchoalveolar Lavage (BAL): ......................................................................................................................... 17 
1.15. 
BLOOD SAMPLING .........................................................................................................................................17 
1.16. 
PHYSICAL EXAMINATION ...............................................................................................................................17 
1.17. 
LUNG FUNCTION TESTS ................................................................................................................................17 
1.17.1. 
Measuring FEV1 and FVC by Spirometry 
1.17.2. 
Collection and analysis of exhaled breath 
 
1.18. 
CLINICAL SYMPTOM SCORES .........................................................................................................................18 
Upper Respiratory Tract Symptoms ..................................................................................................................... 19 
Systemic, Lower Respiratory Tract and Other Symptoms ................................................................................... 20 

 
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ADVERSE EVENTS ............................................................................................................... 20 
1.19. 
DEFINITIONS .................................................................................................................................................20 
1.20. 
EXPECTED ADVERSE EVENTS ........................................................................................................................21 
1.20.1. 
Potential adverse events related to RSV and influenza infection ......................................................................... 21 
1.20.2. 
Potential adverse events of bronchosorption ........................................................................................................ 21 
1.20.3. 
Potential adverse events of chest X-ray ............................................................... Error! Bookmark not defined. 
1.21. 
REPORTING PROCEDURES .............................................................................................................................21 
1.21.1. 
Non serious AEs .................................................................................................................................................. 21 
1.21.2. 
Serious AEs.......................................................................................................................................................... 21 
ASSESSMENT AND FOLLOW-UP ....................................................................................... 22 
STATISTICS AND DATA ANALYSIS .................................................................................... 22 
REGULATORY ISSUES ........................................................................................................ 23 
1.22. 
ETHICS APPROVAL ........................................................................................................................................23 
1.23. 
CONSENT .....................................................................................................................................................23 
1.24. 
CONFIDENTIALITY .........................................................................................................................................23 
1.25. 
INDEMNITY....................................................................................................................................................23 
1.26. 
SPONSOR .....................................................................................................................................................23 
1.27. 
FUNDING ......................................................................................................................................................23 
1.28. 
AUDITS AND INSPECTIONS .............................................................................................................................23 
STUDY MANAGEMENT ........................................................................................................ 24 
PUBLICATION POLICY ......................................................................................................... 24 
REFERENCES ....................................................................................................................... 25 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Study Management Group 
Chief Investigator:  
Professor Peter Openshaw 
 
Co-investigators:  
Professor Sebastian Johnston 
 
 
 
Dr Christopher Chiu  
 
 
 
 
Study Site 
The study will take place at Imperial College Healthcare NHS Trust or Surrey Clinical Research Centre 
in Guildford. 
 
Study Coordination Centre 
For general queries, supply of study documentation, and collection of data, please contact: 
 
Study Coordinator: Dr Christopher Chiu 
Address: Department of Respiratory Medicine,Commonwealth Building, Hammersmith Campus, 
Imperial College London.  
 
                             
 
Position: Clinician Scientist & Honorary Consultant in Infectious Diseases  
 
Tel: +44-20-8383 2301 
E-mail:  c.chiu@imperial.ac.uk 
 
 
 
 
Clinical Queries 
Clinical queries should be directed to Dr Christopher Chiu who will answer or direct the query to the 
appropriate person. 
 
Sponsor 
Imperial College London is the research sponsor for this study.  For further information regarding the 
sponsorship conditions, please contact the Research Governance Manager (Ruth Nicholson) at: 
 
 
 
Joint Research Office, Room 221, 
 Level 2, Medical School Building 
Norfolk Place, 
London W2 1PG 
020759 41862 
r.nicholson@imperial.ac.uk 
Funder 
The MRC has provided Clinician Scientist Fellowship funding for this study.  This protocol describes the 
above study and provides information about procedures for entering participants.  Every care was taken 
in its drafting, but corrections or amendments may be necessary.  These will be circulated to 
investigators in the study.  Problems relating to this 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 (3rd  edition).  It will be conducted in compliance with the protocol, the Data Protection 
Act and other regulatory requirements as appropriate including Good Clinical Practice. 
 
 
 
 
 
 
 

 
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STUDY SUMMARY 
TITLE Cell mediated immunity against RSV and influenza in a human experimental 
challenge model 
DESIGN Human viral challenge study in healthy volunteers 
AIMS 1. To test the hypothesis that RSV and influenza lead to quantitative and 
functional differences in T-cell responses. 
2. To identify the mechanisms underlying poor antigen-specific cell mediated 
immunity (CMI) to RSV by comparing the transcriptional changes in T-cells 
following experimental challenge with RSV and influenza. 
POPULATION Healthy persons aged 18 to 55 years 
ELIGIBILITY Healthy persons aged 18 to 55 years that fit the inclusion and exclusion criteria 
DURATION 
3 Years 
GLOSSARY OF ABBREVIATIONS 
AE 
Adverse Event 
BAL 
Bronchoalveolar Lavage 
CMI 
Cell Mediated Immunity 
CRI 
Centre for Respiratory Infection 
CTL 
Cytotoxic T cell 
DC 
Dendritic Cell 
DNA 
Deoxyribonucleic Acid 
ELF 
Epithelial Lining Fluid 
FEV1 
Forced Expiratory Volume in One Second 
FI-RSV 
Formalin-inactivated RSV vaccine 
GC-MS 
Gas Chromatography with Mass Spectrometry 
GCP 
Good Clinical Practice 
HA 
Haemagglutinin 
ICRRU 
Imperial Clinical Respiratory Research Unit 
LAIV 
Live Attenuated Influenza Vaccine 
NA 
Neuraminidase 
NHLI 
National Heart and Lung Institute 
NP 
Nucleoprotein 
PBMC 
Peripheral Blood Mononuclear Cell 
PCR 
Polymerase Chain Reaction 
PEF 
Peak Expiratory Flow 
PIS 
Participant Information Sheet 
RSV 
Respiratory Syncytial Virus 
RV 
Rhinovirus 
SAE 
Serious Adverse Event 
SAM 
Synthetic Absorptive Matrix 
URT 
Upper Respiratory Tract 
VOC 
Volatile Organic Compound 
WHO 
World Health Organisation 

 
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KEYWORDS 
Respiratory Syncytial Virus, RSV, immune, virus, bronchiolitis, viral challenge, viral lung disease, 
influenza 
 
INTRODUCTION 
1.1. 
Background to research 
Influenza and Respiratory Syncytial Virus (RSV) are the two most common causes of severe viral 
respiratory tract infection.  Seasonal influenza has an overall incidence of 10-20% per annum with 
frequent complications, and the annual mortality in the USA has been estimated at up to 9.9 deaths per 
100,0001. During the 2009 H1N1 influenza pandemic, the fatality rate has been 0.02% with many deaths 
occurring in young adults, and asthmatics at particularly high risk with up to 50% of asthma 
exacerbations thought to be due to influenza.  According to WHO estimates, RSV causes around 64 
million infections per annum and 160,000 deaths. It is the leading cause of severe respiratory illness in 
young children (associated with severe infant wheezing illness) and is also a significant problem in 
susceptible adults (including the elderly and those with airways disease) in whom RSV is responsible 
for around 22% of winter respiratory illnesses with a case fatality rate of 2-8%2. 
 
Vaccine development remains an urgent priority.  While CMI is important in viral clearance and may 
enhance vaccine efficacy, its relative contribution in humans has not been fully determined. We aim to 
elucidate its role by comparing the human T cell responses to influenza and RSV.  These differ in that 
re-infection with antigenically identical strains of influenza does not occur, while within 2 months of RSV 
challenge even subjects with high antibody levels may be re-infected with the same strain.  The reasons 
for this are unclear but may involve immune modulation by viral products or impaired antigen 
presentation by RSV-infected dendritic cells leading to impaired T cell numbers function. 
 
Immune targets in influenza and RSV The major surface antigenic determinants of influenza, 
haemaglutinin (HA) and neuraminidase (NA), both elicit protective humoral responses.  However rapid 
mutation and reassortment of the segmented genome leads to immune escape. Internal viral 
components, such as the nucleoprotein (NP), which are relatively conserved, do stimulate cross-reactive 
T cell responses3 but these do not appear to prevent infection with novel strains in the long term.  The 
genome of RSV encodes 3 transmembrane proteins (the attachment glycoprotein G, fusion F and small 
hydrophobic SH proteins) and both the G and F proteins stimulate antibody production.  Like HA and 
NA, the G protein undergoes sequence mutation but the F and SH proteins exhibit limited variability4.  
Cytotoxic T cell (CTL) responses have been demonstrated against the F and SH proteins as well as 
several internal proteins but not the G protein5. 
 
Correlates of protection against influenza and RSV infection The immune factors that determine 
whether an individual will develop symptomatic infection following exposure to a respiratory pathogen 
are poorly understood. Traditionally, serum antibody has been used as a correlate of protection against 
influenza and, indeed, high neutralising antibody titres do correlate somewhat with a decreased risk of 
influenza infection. However, it is clear that antibodies do not explain all aspects of immune protection 
as some individuals with no detectable neutralising antibody are still protected. With RSV, this is 
additionally complicated as there is no antibody level at which protection is assured. Increasing evidence 
indicates that protective immunity is mediated by a number of factors including early innate immune 
mechanisms and cell mediated immunity. Furthermore, these arms of the immune system are 
interlinked, with innate immunity critically influencing later adaptive responses. Thus a comprehensive 
understanding of both the early innate responses and cell mediate immunity will be essential for the 
development of effective vaccines. 
 
Vaccines against influenza and RSV There are few treatments available for influenza and RSV 
infection, and those that exist cannot be used in the developing world, where a large proportion of 
disease burden lies, due to cost.  For these reasons, effective vaccines remain of utmost importance.  
Influenza vaccination is currently achieved by subunit or live attenuated influenza vaccines (LAIV) which 
are re-formulated each year to maintain their antigenicity.  Production of these vaccines each year is 
time-consuming and slow to respond to emerging viruses such as the current H1N1 pandemic strain.  

 
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No influenza vaccine has yet been shown to elicit broad protection against all virus strains.  Meanwhile, 
an effective vaccine against RSV still does not exist.  Studies using formalin-inactivated RSV carried out 
in the 1960s led to unexpected results as not only was there no protection but also 80% of vaccinees 
required hospitalisation with vaccine-enhanced disease6.  It has therefore been essential to balance 
vaccine-induced immunopathology with immunogenicity, and few vaccine candidates have fulfilled both 
criteria. 
 
The importance of cell mediated immunity There is evidence that cell mediated immunity (CMI) is 
important for protection against and clearance of viral infections.  Vaccines that induce both humoral 
immunity and CMI may be more effective and protect against a wider range of virus serotypes.  In animal 
models, CTL responses have been correlated with reductions in influenza viral load and some protection 
has been demonstrated using prior inoculation with a serologically distinct strain7.  The protective role 
of CMI in humans has been more difficult to determine, and the contribution of reduced CMI in infective 
exacerbations of airways disease is unclear.  T cells have been identified in humans that have cross-
reactive potential and one study has directly demonstrated correlation between high CTL levels and 
reduced virus shedding8, while in vaccinated individuals, levels of serum antibody correlate poorly with 
vaccine efficacy9.  LAIV stimulates more vigorous CMI than subunit vaccines and there is an association 
between the number of interferon-γ (IFN-γ) producing peripheral blood mononuclear cells (PBMCs) and 
the protective efficacy of the vaccine10.  These may explain why LAIV is more effective than subunit 
vaccines.  In mice, depletion of CD4+ and CD8+ T cells suggests that both make significant contributions 
to the inhibition of RSV replication.  Athymic nude mice infected with RSV exhibit persistent shedding of 
virus which can be terminated by adoptive transfer of T cells from previously primed animals11.  In the 
clinical setting, children with defects in CMI suffer more severe RSV disease and shed the virus for 
several months compared with normal children who shed for only 7-21 days12.  This is also true of bone 
marrow transplant recipients. 
 
Contrasting immune responses to influenza and RSV In a mouse model of influenza infection, viral 
load falls as soon as CTLs appear in the lung13 and effector memory CTLs act rapidly on re-challenge 
to recruit T cells to then expand and form a new effector pool.  Along with the humoral response against 
HA and NA, this leads to protective immunity and re-infection with the same strain of influenza does not 
occur14.  In contrast, infection with RSV does not confer full protective immunity.  Almost all children will 
have seroconverted to RSV by 24 months of age, but re-infection is common throughout life.  This occurs 
despite persistent antibodies against the relatively conserved F protein which are cross-reactive against 
the different serogroups of RSV15.  In human experimental challenge studies, where adults naturally 
infected with RSV were later challenged 6 times with a similar laboratory strain, 73% could be re-infected 
and 50% re-infected twice or more16.  Re-infection took place at a rate of 25% even in those subjects 
with the highest levels of IgG and IgA against F and G proteins within as little as 2 months.  These 
differences are likely to be due to differential immune responses against influenza and RSV and may 
be quantitative or functional.  In adult blood and lung, influenza-specific memory T cells are 2-4 times 
more frequent than those specific for RSV17,18, while analysis of cytokine responses in the 
nasopharyngeal aspirates of infected children show higher levels of IFN-γ and decreased IL-4 and IL-5 
in influenza compared with RSV19. 
 
Mechanisms of immune modulation by RSV The reasons for these contrasting immune responses 
and incomplete protection against RSV infection are unclear.  In vitro studies have suggested many 
RSV proteins possess immunomodulatory activity.  NS1 and NS3 proteins have been shown to inhibit 
response to interferons20, while replication of RSV within dendritic cells (DCs) has also been shown to 
interfere with IFN-α and IL-12 responses, as well as inducing IFN-λ production that suppresses CD4+ T 
cells21.  Furthermore, animal studies have shown RSV infection depleting DC precursors and leading to 
the inhibition of DC expansion on re-infection22.  RSV may also interfere with the normal interaction 
between DCs and T cells.  In mice, RSV-specific CTLs are short-lived and have impaired cytolytic ability 
and reduced production of IFN-γ23.  These deficiencies can be reversed by incubation with exogenous 
IL-2, and are believed to be due to a defect in the TCR signalling pathway, which may be due to the 
effects of direct contact with cells expressing F protein on their surface and/or inhibition of formation of 
the immunological synapse24. 
 
 

 
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Human challenge model In the early 1990s, Peter Openshaw’s group carried out human volunteer 
challenge studies showing that individuals could be repeatedly infected with the same strain of RSV 
although the mechanism remained unclear (A.H. Cherrie, PhD thesis, University of London 1992).  
Although human challenge experiments with RSV were discontinued, Sebastian Johnston’s group has 
since performed many volunteer challenge studies to investigate immune responses to rhinovirus.  Their 
recent studies on exacerbations of asthma by rhinovirus have demonstrated the altered cytokine profiles 
of subjects, and their experience in this area will form the basis of this study25. 
 
Participants in the influenza arm of this study will be challenged with either a fully virulent influenza strain 
live attenuated influenza vaccine (LAIV). Challenge experiments with virulent influenza overcomes the 
many disadvantages associated with studying a patient group infected with naturally-acquired influenza: 
diagnosis is often delayed, the timing and dose of inoculating virus is extremely variable, many patients 
have underlying medical conditions influencing their immune responses, and they may be subject to 
therapeutic measures that alter those responses. Finally, it would not be possible to re-challenge these 
individuals in order to examine a recall response. Using a well-characterised, fully virulent GMP-certified 
challenge strain of influenza will allow us to closely replicate natural infection but also provide us with 
the control required to answer important questions about the essential immune responses required for 
protection against re-infection. Since, LAIV replicates and undergoes intracellular processing in a similar 
way to virulent influenza, it is also able to elicit similar immune responses including mucosal immunity 
and IFN-γ producing T cells in the blood10. In children it is highly effective in preventing subsequent 
infection with antigenically similar strains of seasonal influenza. However, its efficacy in adults 
diminishes with age and duration of protection is poor. By comparing the immune responses of LAIV to 
virulent influenza, we will be able to define the differences in phenotype and function that are responsible 
for the more robust immunity seen with natural infection. 
 
This study will for the first time systematically investigate the mechanisms behind the differential 
responses to RSV and influenza.  The data obtained will be essential for further understanding of the 
natural history of human antiviral responses, and will have direct bearing on future studies into the effect 
of these infections on the elderly and asthmatics as well as novel approaches to vaccine development. 
 
1.2. 
Research Hypotheses 
1. RSV specifically interferes with T cell activation, causing impairment of cell-mediated immunity 
and allowing recurrent infection with antigenically similar strains. 
 
2. By contrast, repeated infection with the same strain of influenza is prevented by antibody and T 
cell dependent immune responses that show quantitative and/or qualitative differences to the 
responses elicited by RSV infection. 
 
STUDY OBJECTIVES 
1.3. 
Primary Objective: 
 
 
To test the hypothesis that RSV and influenza lead to quantitative and functional differences in 
T-cell responses. 
 
1.4. 
Secondary Objective: 
 
 
To identify the mechanisms underlying poor antigen-specific CMI to RSV by comparing the 
transcriptional changes in T-cells following experimental challenge with RSV and virulent 
influenza. 
 
 

 
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PARTICIPANT ENTRY  
1.5. 
Recruitment 
Subjects will be recruited by advertisement in local newspapers, around College sites, and online: the 
Imperial Trust website, Gumtree, Student Union websites, ICRF website and on Social Media such as 
the Imperial CRF twitter page.  Additionally, respondents to adverts for prior research projects in the 
department that were not subsequently enrolled, but were otherwise eligible for this study, will be 
contacted and invited to take part in our study (they have previously given their consent to be contacted).  
If interested, they will be invited for screening.   
 
1.6. 
Pre-registration evaluations  
1.6.1. Screening Visit 
The screening visit will involve each participant attending the Imperial Clinical Respiratory Research 
Unit (ICRRU) at St Mary’s Hospital, Paddington or Imperial Clinical Research Facility (ICRF) at 
Hammersmith Hospital for a brief interview and medical examination to find out if they are suitable for 
the study.  Potential subjects will be given a participant information sheet (PIS) detailing the study and 
experimental procedures.  When the subject has had enough time to consider their participation in this 
study, ask any questions they may have, and only when they have agreed to take part will they be asked 
to read, sign and date a consent form in the presence of the study doctor who will also sign the consent 
form.  Consent will be obtained prior to any history-taking, examination or tests are carried out. A copy 
will be kept in the research file, a copy given to the patient and a copy put into their medical notes. 
 
A medical history will then be taken and clinical examination, lung function tests, chest X-ray, ECG and 
blood tests performed by the study doctor.  Women of childbearing potential will be asked about current 
contraceptive use, and be required to use effective contraception (barrier, oral contraceptive pill, depot 
injection, implant, or total abstinence) throughout the study.  Blood tests include general screening for 
underlying illness, particularly full blood count, urea and electrolytes, liver function tests, coagulation C-
reactive protein, lymphocyte subsets, HIV serology and baseline titres of neutralising antibodies against 
RSV and influenza.  A urine drug screen for illicit drugs will also be performed.  
 
These will all be done in the ICRRU at St Mary’s Hospital or ICRF at Hammersmith Hospital.  If the 
evaluation and the results of these tests show no evidence of infection or any other problems with the 
participants’ health, and matches the inclusion / exclusion criteria then they will continue to the main 
part of the study. 
 
1.7. 
Inclusion criteria 
Healthy persons aged 18 to 55 years, able to give informed consent 
 
1.8. 
Exclusion criteria 
Chronic respiratory disease (asthma, COPD, rhinitis, sinusitis) in adulthood 
Inhaled bronchodilator or steroid use within the last 12 months 
Use of any medication or other product (prescription or over-the-counter) for symptoms of rhinitis or 
nasal congestion within the last 3 months 
Acute upper respiratory infection (URI or sinusitis) in the past 6 weeks 
Smoking in the past 6 months OR >5 pack-year lifetime history 
Subjects with allergic symptoms present at baseline 
Clinically relevant abnormality on chest X-ray 
Any ECG abnormality 

 
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Those in close domestic contact (i.e. sharing a household with, caring for, or daily face to face contact) 
with children under 3 years, the elderly (>65 years), immunosuppressed persons, or those with 
chronic respiratory disease 
Subjects with known or suspected immune deficiency  
Receipt of systemic glucocorticoids (in a dose ≥ 5 mg prednisone daily or equivalent) within one month, 
or any other cytotoxic or immunosuppressive drug within 6 months prior to challenge 
Known IgA deficiency, immotile cilia syndrome, or Kartagener’s syndrome 
History of frequent nose bleeds 
Any significant medical condition or prescribed drug deemed by the study doctor to make the participant 
unsuitable for the study 
Pregnant or breastfeeding women 
Positive urine drug screen 
Detectable baseline antibody titres against influenza challenge strains 
Influenza arm only: history of hypersensitivity to eggs, egg proteins, gentamicin, gelatin or arginine, or 
with life-threatening reactions to previous influenza vaccinations. 
 
All women of childbearing age will have a pregnancy test performed prior to virus inoculation to 
exclude pregnancy and be required to use contraception throughout the study. 
 
1.9. 
Withdrawal criteria 
Any subjects can withdraw from the study at any time if they wish to. Subjects can also be removed from 
the study if an investigator feels this is necessary or appropriate.  Subjects will be closely monitored 
throughout by the study doctor.  This is defined further in the section on ‘Serious Adverse Events’. 
 

 
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Day 0 
48 
subjects 
enrolled 
33 subjects 
challenged with 
4 log PFU RSV & 
40 subjects with 
virulent 
influenza 
Comparison of T cell 
response 
Day 168 
Sampling 
of nasal 
secretions, 
throat, and 
blood
33 subjects 
challenged with 
4 log PFU RSV 
Sampling 
of nasal 
secretions, 
throat, and 
blood
STUDY DESIGN 
Figure 1: Subject allocation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 1: Study procedures (Screening to day 9) 
Procedures 
DAY (relative to viral inoculation) 
 
Screen 
-14 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination 
X 
X 
X 
 
 
 
 
 
 
X 
 
 
Lung Function 
Testing – FEV1 , 
peak flow & breath 
tests 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Screening blood 
tests 
40 
 
 
 
 
 
 
 
 
 
 
 
Blood - serum 
 
 
5 
 
 
 
 
 
 
 
 
 
Blood for HLA typing 
5 
 
 
 
 
 
 
 
 
 
 
 
Blood - PBMCs (mls) 
 
 
40 
 
 
 
 
 
 
 
 
 
Blood – plasma (mls) 
 
X 
6 x 2 
6 x 2 
6 x 2 
6 x 2 
6 x 2 
6 x 2 
6 x 2 
6 x 2 
6 
6 
Blood – RNA (mls) 
 
 
7.5 x 2 7.5 x 2 7.5 x 2 7.5 x 2 7.5 x 2 7.5 x 2 7.5 x 2 7.5 x 2 
7.5 
7.5 
Throat & stool swabs 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Nasal lavage (daily) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Nasosorption using 
SAM (right nostril) 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Nasal scrape 
(Rhinopro®) 
X 
X 
 
X 
X 
X 
 
 
 
X 
 
 
Bronchoscopy: 
Bronchosorption 
using SAM (4) 
Bronchial biopsy (6) 
Bronchial brush (4) 
Bronchoalveolar 
lavage (BAL) 
 
X 
 
 
 
 
 
 
 
 
 
 
Virus inoculation 
 
 
X 
 
 
 
 
 
 
 
 
 
Review symptom 
diaries daily 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Collect symptom 
diaries 
 
 
 
 
 
 
 
 
 
 
 
 
Urine pregnancy test 
X 
 
X 
 
 
 
 
 
 
 
 
 
 

 
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1.10. Study visits for all subjects 
RSV and Influenza Study Arm 
The RSV and influenza arms of the study are divided into outpatient and confinement phases.  Subjects 
will stay overnight for a period of 8-10 nights in total, from the day of viral challenge, to the 8-10th day 
after viral challenge and, for RSV only, a second subsequent 8-10-night stay after a second viral 
inoculation.  This period of confinement has been chosen to eliminate the possibility of subjects in the 
study transmitting the virus to anyone not involved in the study (i.e. family, household contacts, and the 
wider community).  Confinement is not used to enable closer monitoring or to enhance safety for study 
subjects, although this may be an additional benefit in some circumstances.  During the confinement 
period, all study procedures will take place in the confinement facility (except bronchoscopy – see 
sections 1.10.2 and 7)(Figure 1 & Tables 1 & 2). 
Table 2: Study procedures (day 10 to day 196) 
Procedures 
DAY (relative to viral inoculation) 
 
10 
14 
28 
161 
168 
178 
182 
196 
Consent 
 
 
 
X 
 
 
 
 
Physical Examination 
X 
X 
X 
X 
X 
X 
X 
X 
Lung Function 
Testing – FEV1, peak 
flow & breath tests 
X 
X 
X 
X 
X 
X 
 
 
Screening blood 
tests 
 
 
 
 
 
 
 
 
Blood - serum 
 
 
5 
 
5 
 
5 
5 
Blood for HLA typing 
 
 
 
 
 
 
 
 
Blood - PBMCs (mls) 
60 
 
40 
20 
40 
60 
60 
60 
Blood – plasma (mls) 
6 
6 
6 
 
 
 
 
 
Blood – RNA (mls) 
7.5 
7.5 
7.5 
7.5 
7.5 
7.5 
7.5 
7.5 
Throat & stool swabs 
X 
X 
X 
 
X 
X 
X 
X 
Nasal lavage (daily) 
X 
X 
X 
X 
 
X 
 
 
Nasosorption using 
SAM (right nostril) 
X 
X 
X 
 
X 
X 
X 
X 
Nasal scrape 
(Rhinopro®) 
X 
 
 
 
X 
X 
X 
X 
Bronchoscopy: 
Bronchosorption 
using SAM (4) 
Bronchial biopsy (6) 
Bronchial brush (4) 
Bronchoalveolar 
lavage (BAL) 
X 
 
X 
 
 
 
X 
 
Virus inoculation 
 
 
 
 
X 
 
 
 
Review symptom 
diaries daily 
X 
X 
 
 
X 
X 
X 
X 
Collect symptom 
diaries 
 
X 
 
 
 
 
X 
 
Urine pregnancy test 
 
 
X 
X 
 
 
 
 
 
SAM = synthetic absorptive matrix 
 

 
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1.10.1. Baseline visit (, day -14) 
 
On the first visit (baseline – around one week before infection with the virus) we will perform lung 
function, blood tests (serology and PBMCs), a throat swab, take washings from the participant’s nose 
(nasal lavage), a nasal scrape, a nasal SAM and bronchoscopy.  At this visit the participant will also be 
given a symptom diary card and asked to complete it (before any nasal procedures), detailing any 
respiratory symptoms they experience in their upper and lower airways.  They will be asked to complete 
the symptom diary on a daily basis for one week following the first bronchoscopy and then daily for two 
weeks after infection with the virus. The first bronchoscopy will be performed on this visit  
  
On the next visit, a week later, subject to a satisfactory assessment by the study doctor (brief interview 
and medical examination) and the taking of blood and nasal lavage samples, we will infect volunteers 
with RSV or virulent influenza.  Those challenged with RSV and virulent influenza will then be confined 
as described (Section 1.10.2).  
1.10.2. Confinement period for RSV and influenza arms (confinement, day 0 – day 8-10 and day 
168 to 177-179) 
On the morning of viral challenge, participants will attend the ICRRU or ICRF. A single nasal lavage 
sample will be taken to exclude coincident infection and blood will be taken for PBMCs.  Following this, 
subject to a satisfactory assessment by the study doctor (brief interview and medical examination), we 
will infect the volunteers with RSV using a dose of 4 log PFU or with influenza at a dose of 3.5x106 
TCID50.  After infection, participants will be observed for a period of 30 minutes to ensure no adverse 
reactions have occurred. They will enter a residential research facility (confinement facility), where they 
will reside for the next 8-10 nights so we can monitor the development of cold symptoms and collect 
samples to evaluate immune responses and test the hypotheses.  These will include daily blood 
samples, throat swabs, nasal lavage, nasal SAM, nasal scrapes, lung function tests, as well as 
bronchoscopy on day 7 or 10 post-infection. Participants will be seen daily by the study team.  On the 
8th day of residence, assuming all significant symptoms have resolved and at the discretion of the 
principle investigator, participants will leave the confinement facility and asked to return daily for 
assessment and sampling. If symptoms continue or the study doctors deems it necessary, the volunteer 
will be asked to remain until the 9th or 10th day of residence at which time they will be discharged, subject 
to a satisfactory assessment by the study doctor. If a subject is discharged before the 10th day or wishes 
to withdraw after virus inoculation but before the 10th day, we will strongly advise them to confine 
themselves in their own homes, and to strictly avoid any contact with young children, the elderly or other 
high-risk individuals for the remainder of the period during which viral shedding may occur. Additionally, 
subjects infected with influenza withdrawing before the 8th day post-inoculation will be treated with 
oseltamivir to prevent reduce the risk of viral shedding having left confinement. 
1.10.3. Follow-up period (outpatient, day 11 – day 196) 
Participants will return to the ICRRU or ICRF on days 14 and 28 post infection for assessment and 
sampling, including bronchoscopy on day 28. During the 6 months after challenge, subjects will be asked 
to report all episodes of upper respiratory tract symptoms.  If an episode of natural infection occurs, the 
subject will be asked to re-attend for blood and upper respiratory tract samples to be collected.  After 6 
months, subjects will be asked to return for further samples to be taken, including bronchoscopy if 
subject has been included for this. A second dose of RSV administered a week later in the RSV arm 
only. Further samples will be collected at the peak of the secondary response 7 days post-infection. We 
will not do every test on every visit as can be seen from Table 2. The total study period for each subject 
is 210 in the RSV arm and 42 in the influenza arm.  
 
 

 
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STUDY PROCEDURES  
A variety of procedures are carried out during the study period.  The frequency and timing of these 
procedures are shown in  above. For the screening, and days -14, 14, 28 and 196 visits, procedures will 
take place in the procedures room on ICRRU or ICRF; for days 0 to 10 and 168 to 179 subjects in the 
RSV and influenza arms of the study will reside in the confinement facility and procedures will take place 
there (with the possible exception of virus inoculation and bronchoscopy). 
 
1.11. Virus Inoculation 
GMP influenza A/California/04/2009-like (H1N1); influenza A/Belgium/4217/2015 (H3N2); or RSV 
Memphis 37 virus strains will be used for experimental infection of volunteers. On the day of inoculation 
(day 0 and day 168), RSV or influenza virus stock will be defrosted from its storage in the freezer.  
Subjects will be inoculated with intra-nasal drops on a single occasion with diluted inoculum at a given 
dose divided equally between the two nostrils.  This will be done slowly with sufficient interval between 
each inoculation (2-3 minutes) to ensure maximum contact time between with the nasal and pharyngeal 
mucosa.  Subjects will be asked not to swallow during the procedure to ensure maximal pharyngeal 
contact.  The inoculation procedure will be performed using a ventilated body box, allocated isolation 
bay or negative pressure room in the ICRRU or ICRF. Inoculations using intranasal drops will be done 
using a 1mL pipette with subjects supine. Following inoculation, advice regarding hand hygiene will be 
given and subjects will be provided with alcohol hand gel and face-masks to reduce spread of virus in 
the home environment. 
 
1.12. Swabs for microbial analysis 
1.12.1. Throat swab 
A sterile dry cotton-headed swab is used to obtain samples from the pharynx for bacterial 16S gene 
analysis.  This is performed with the subject sitting.  Ensure adequate lighting and use a tongue 
depressor if required.  Remove the swab from the container carefully to ensure the tip is not 
contaminated, and swab the dorsal aspect of the pharynx and soft palate, avoiding the tongue.  Some 
subjects may experience a strong gag reflex.  Obtain two samples; place one used swab into a dry 
container and freeze at -80°C prior to analysis and another into bacteriology culture medium. 
1.12.2. Stool swab 
Sterile dry cotton-headed swabs will be used to obtain stool samples for bacterial 16S gene analysis. 
These will be collected by the subject from the toilet paper after opening their bowels.  
 
Procedure: 
- 
Remove the swab from the collection tube by holding it firmly by the red cap. DO NOT TOUCH 
THE COTTON PART WITH YOUR BARE HANDS! 
- 
Collect a small amount of fecal material by rubbing the cotton tip of the swab on a faecal sample: 
a piece of used bathroom tissue is the best material possible. A small amount is enough: it should 
cover half of the cotton tip. Do not try to collect too much biomass. 
- 
Replace the swab in the collection tube and close it by pushing firmly on the cap. 
- 
Store the swab at -80°C within 48h. If it is not possible to store at -80°C, store the sample at 4°C 
until transfer into a cryogenic environment. 
 
1.13. Nasal sampling procedures 
All nasal procedures will be performed in the order below prior to the bronchoscopy to avoid 
contamination by the local anaesthetic gel and bronchoscope introduced into the nose. 

 
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1.13.1. Nasal scrape using Rhinopro® 
Rhinopro® curettes will be used to obtain a sample of nasal epithelial cells from each nostril.  This is a 
painless procedure and will not require local anaesthetic.  The following technique is used: 
 
The subject should be sat comfortably, ideally with their head fixed, looking forward, while their chin 
rests on a support (if available) 
Tear bag and remove the flexible plastic Rhinopro® without contaminating the scoop end 
Place a speculum in the nose to keep the cavity open and employ good lighting 
Under direct visual inspection, insert the cupped probe onto the surface of the mid-inferior portion of the 
inferior turbinate.  Note: Avoid the anterior bulb. 
The Rhinopro® should be 3cm up the nose; the floor of the nostril can be used to rest on 
Have the cup of the Rhinopro® at the correct angle 
Gently press the cupped tip on mucosal surface and move out and in of nostril 3mm up to 3 times 
Note that this area has limited sensitivity and the subject should not find this procedure painful, although 
a nasolacrimal reaction usually occurs 
 
The cell harvest is epithelial cells, goblet cells and mast cells.  It does not contain deeper layers of the 
mucosa.  The sample obtained should be placed immediately into a tube containing RNA Cell Protect® 
(Qiagen) or Trizol and frozen at -80°C for storage prior to analysis. 
 
1.13.2. Nasosorption 
Up to four strips of SAM will be used (2 per nostril, one after the other) for 2 minutes to obtain repeated 
samples of neat nasal ELF.  This is a painless minimally invasive procedure that will not require any 
local anaesthetic.  Following sampling, SAM will be placed in a 1mL microfuge spin filter tube containing 
250μL of elution buffer (PBS/1% bovine serum albumin/0.05% azide/0.05% Triton®).  Further details are 
given in the SOP Human Sampling Procedures for Challenge Study. 
 
The SAM will be transported on ice to the laboratory. 
1.13.3. Nasal Lavage 
Nasal lavage is performed using the following technique: 
 
 
5mL of 0.9% saline is introduced into each nostril using a syringe attached to a nasal olive with the 
subject sitting with the head tilted forward 
 
The saline is then washed in and out of the nose approximately 10 times by alternately withdrawing 
and advancing the plunger of the syringe while the subject maintains a tight seal between the nasal 
olive and the nostril; the aim is to recover ~80% of the saline from the nose 
 
The fluid is then aliquotted into sterile microfuge tubes and centrifuged for analysis of cells 
Lavage fluid will later be analysed to quantify the degree of RSV or influenza shedding.  Multiplex PCR 
will be performed on the pre-inoculation lavage and post-inoculation lavage collected during the study 
to exclude the presence of other respiratory viruses.  Supernatants will be frozen and stored at -80°C.  
Further details are given in the SOP Human Sampling Procedures for Challenge Study. 
 
1.14. Bronchoscopy 
Bronchoscopies will be performed in the endoscopy suite at St Mary’s Hospital, or at Hammersmith 
hospital, in accordance with BTS guidelines.  During the confinement period, private transport from the 
confinement facility to the endoscopy suite will be mandatory. Subjects will be instructed to wear face 
masks during this visit, and staff will use appropriate personal protective equipment (PPE). 
 

 
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 
Subjects will sign a consent form prior to being screened for the study and will sign a separate 
consent form for each bronchoscopy. The procedure will be explained during the assessment 
stage and subjects will be given written information specifically regarding bronchoscopies in 
addition to the Participant Information Sheet 
 
 
Subjects will fast for four hours prior to the procedure 
 
 
Resuscitation equipment (for intubation, ECG monitoring and defibrillation) and necessary drugs 
(salbutamol, theophylline, adrenaline, hydrocortisone) will be available in the bronchoscopy room 
 
 
Premedication may be given including: 
 
 
Nebulised salbutamol 2.5mg 10-15 minutes prior to bronchoscopy 
 
Sedation – midazolam (2-10mg) or fentanyl (50-100μg) as necessary 
 
Lignocaine – gel and solutions (1-4%) for topical anaesthesia.  The total dose will not exceed 
400mg 
 
 
Supplemental oxygen at a rate of 2Lmin-1 is given via a nasal cannula and oxygen saturations and 
heart rate are monitored with a pulse oximeter continuously.  Intravenous access will be mandatory 
in all cases 
 
 
The subject will be monitored during the bronchoscopy by adequately trained clinical staff, according 
to Research Bronchoscopy SOP (V3). 
 
 
The following samples are collected in this order: (i) Bronchosorption using Synthetic Absorptive 
Matrix (SAM) (ii) Bronchial Brushings (iii) Bronchial Biopsy (iv) Bronchoalveolar lavage (BAL) 
 
 
All adverse events – pain, bleeding, hypoxia etc will be recorded and reported according to Section 
8 of this protocol 
 
 
Subjects will be observed for a minimum of 2 hours and nil by mouth until safe 
 
 
Transportation will be arranged as subjects should not drive on the day of the procedure 
 
 
All subjects will have a contact telephone number on discharge 
1.14.1. Bronchosorption: 
 
‘Bronchosorption™ FX·i is a non-sterile, single-use device consisting of a synthetic absorptive matrix 
(SAM™) strip enclosed within a catheter for bronchial sampling. Bronchosorption™ FX·i is designed to 
operate through a flexible video bronchoscope with a maximum working length of 815mm. The SAM™ 
strip is 1.0mm wide, designed to work with a minimum instrument channel diameter of 2.0mm.’ Hunt 
Developments UK (Ltd).  
 
 
The device will be passed down the bronchoscope. 
 
The probe will be deployed for up to 120 seconds in segmental and larger bronchi to allow the SAM 
to absorb local epithelial lining fluid (ELF). 
 
The probe will then be resheathed and removed via the operating port of the bronchoscope.  
 
This will be repeated up to 4 times with a new device. 
 
The SAM will be transferred to polypropylene tubes for transport on ice to the laboratory and stored 
at -80 degrees C until analysis. 
 

 
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1.14.2. BronchoBronchial Biopsies: 
Six bronchial biopsies will be taken from the segmental and sub-segmental bronchi of the right lower 
and middle lobe (RLL, ML).  This is performed using Keymed 2mm biopsy channel cupped and 
fenestrated biopsy forceps [FB-19C-1 (1111065)]; four biopsies will be placed in 4% paraformaldehyde 
and stored in paraffin blocks and two placed immediately into RNAlater® (Qiagen) stabilisation solution 
and refrigerated for 24 hours prior to freezing at -80°C.  
1.14.3. Bronchial Brushings: 
Four bronchial brushings will be taken from the left lower lobe (LLL) sub-segmental bronchi with a 
standard cytology brush.  The brush is washed in a tube containing RNA Cell Protect® (Qiagen) to 
preserve cells prior to freezing at -80°C.  A new brush is used each time the brushes are washed and 
discarded. 
1.14.4. Bronchoalveolar Lavage (BAL): 
 
BAL is performed by instillation of sterile physiological (0.9%) saline at room temperature into the 
left upper lobe (LUL) bronchus in 30ml aliquots to a total of 180-240ml 
 
Aiming for 80% volume recovery and aspirating after each instillation 
 
The BAL fluid is collected into a plastic chamber and transferred to polypropylene tubes for transport 
on ice to the laboratory 
 
BAL processing - Keep BAL collected at bronchoscopy on ice at all times 
 
1.15. Blood sampling 
Screening visit blood will be taken for full blood count, renal function, liver function tests, glucose, 
clotting, CRP, lymphocytes, immunoglobulins, HIV, hepatitis B and C.  These will be processed in the 
Haematology and Chemical Pathology Laboratories of Imperial College Healthcare NHS Trust.  
Serology will be performed at screening and day -14 by IgG microneutralisation assay. 
 
The total amount of blood taken at screening would amount to 40mL.  Blood for peripheral blood 
mononuclear cells (PBMCs) will also be taken at day -14, 0, 3, 7, 14, 28, 161, 168, 175 and 196. On 
these occasions, 20-60mls of blood will be taken (see Table 1 & 2). In addition, during the first challenge, 
blood for gene expression profiling, serum and plasma will be taken from day 0 to day 10 plus day 14 
and 28. Blood for gene expression profiling will be taken once or twice day. A maximum of 72 mls of 
blood will be taken on any single day for a total of 478.5 mls over the 28-day challenge period (see 
Tables 1 & 2). 
 
1.16. Physical examination 
Physical examination, including ENT, respiratory and cardiac assessment will be performed by the 
study doctor at screening and days -14, 0 (prior to inoculation),8, 9,10 14, 28 and 175 days post 
inoculation. 
 
1.17. Lung Function Tests 
Predicted Values for Lung Function Measurements: the predicted or reference values for lung function 
measurements are those recommended by the Report Working Party for the European Community for 
Coal and Steel.  Also incorporated are the recommendations of the British Thoracic Society and the 
Association of Respiratory Technicians and Physiologists. 
 
 

 
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1.17.1. Measuring FEV1 and FVC by Spirometry 
Performing the measurement 
 
 
Posture must be consistent during a study, either standing or sitting, with no breathing limitation 
 
The subject should breathe in to total lung capacity.  A good tight seal by the lips round the 
mouthpiece is essential.  The subject should then exhale forcibly into the spirometer, blowing as 
hard as possible and continue to residual volume 
 
The best value of 3 attempts will be recorded 
Calibration:  Diagnostic spirometers will be serviced and calibrated by the relevant company for the 
brand.  However, the calibration will be checked at least once a month using a calibrated one litre 
syringe. 
 
At the screening visit and at other visits as described in the table in, measurements of FEV1 and FVC 
will be made as outlined above. 
 
1.17.2. Collection and analysis of exhaled breath 
 
Exhaled breath is believed to contain infectious virus during natural influenza infections. To collect 
potentially infectious particles in breath exhaled during infection, we will use an altered resuscitation 
mask with Teflon filter mouthpiece into which the participant will be asked to breath for 20 minutes. 
 
1. 
The test will take a total of thirty minutes including twenty minutes of quiet breathing.  
2. 
A study team member will explain what the device consists of and why the test is being 
performed. 
3. 
The participant will hold the mask by the cardboard tubing and not touch any plastic surface. 
4. 
They will gently press the mask against their face, covering the mouth and nose. 
5. 
Then they test breathing through their nose and mouth while study staff check for leaks 
around the face. 
6. 
If the participant experiences pinching of their nose, difficulty in nasal breathing or a leak is 
detected air can be removed with a 20 ml catheter tip syringe from the inflatable cuff on the 
facemask and the mask re-tested. 
7. 
Once steps 4 and 5 have been optimised the participant will be asked to breath in through 
their mouth and out through their nose (normal tidal volumes and rate). 
8. 
If their nose is blocked due to symptomatic infection mouth breathing will be performed and 
this will be documented in the Breath Collection Log. 
9. 
The participant will be asked not to speak, unless it is necessary. 
10. 
They will be asked not to remove the mask from their face, unless absolutely necessary. 
11. 
It will be explained that if the seal around their face is broken, e.g. if they remove the mask, 
the test will be restarted. 
 
1.18. Clinical symptom scores 
A self-completed diary card of both upper and lower respiratory tract clinical symptoms will be made at 
baseline 14 days prior to inoculation (prior to nasal washing and / or bronchoscopy), on day 0, and daily 
for 14 days.  
 
Additionally, the symptom diary will be completed daily for 7 days after the 1st bronchoscopy (i.e. day -
14 to day -7).  This is to allow the effect of bronchoscopy on symptoms to be measured and adjusted. 
 
Individual symptom scores will be accumulated over the six-day period of maximal illness (days 2-7) 
after inoculation and the baseline recording (including any effect measured from the 1st bronchoscopy 
where appropriate) subtracted from the post inoculation recordings. Thus, for a patient who has a score 
of zero on day 0 prior to inoculation, the maximum cumulative score for the following six days is 144. 

 
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Upper Respiratory Tract Symptoms 
A total ‘upper respiratory clinical symptom score’ will be derived using a four-point scale (0-3 for absent, 
mild, moderate and severe) for each of the following eight respiratory symptoms: sneezing, headache, 
malaise, fever/chills, nasal discharge, nasal obstruction, sore throat and cough according to established 
methods, giving a maximum clinical severity score of 24.  This is an established method for studies of 
common cold illnesses44. Symptoms will be recorded at the same time of day and before any procedures 
such as bronchoscopy or nasal lavage are performed. 
 
An example is shown below: 
 
Symptom 
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
Day 6 
Day 7 
Sneezing 
 
 
 
 
 
 
 
Headache 
 
 
 
 
 
 
 
Malaise 
 
 
 
 
 
 
 
Fever / chills 
 
 
 
 
 
 
 
Nasal discharge 
 
 
 
 
 
 
 
Nasal obstruction 
 
 
 
 
 
 
 
Sore throat 
 
 
 
 
 
 
 
Cough 
 
 
 
 
 
 
 
Total score 
 
 
 
 
 
 
 
 
0 = absent, 1 = mild, 2 = moderate, 3 = severe 
Definition of a clinical cold 
 
A clinical cold is diagnosed if two or more of the following are present: 
 
A cumulative clinical symptom score of 14 or greater over a 6 day period 
 
Nasal discharge is present on three or more days over the six-day period post viral inoculation 
 
A subjective impression of a cold developing. This latter criterion is used because there are a few 
subjects who have had a very strong subjective impression of a clinical cold but the cumulative 
clinical score does not reach the arbitrary cut-off level 
 
 

 
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 
 
System, Lower Respiratory Tract and Other Symptoms 
A diary card of lower respiratory tract symptoms will be completed with a scoring system outlined below. 
SYMPTOM 
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
Day 6 
Day 7 
Cough 
 
 
 
 
 
 
 
Difficulty breathing 
 
 
 
 
 
 
 
Hoarseness 
 
 
 
 
 
 
 
Chest discomfort 
 
 
 
 
 
 
 
Chills 
 
 
 
 
 
 
 
Diarrhoea 
 
 
 
 
 
 
 
Muscle aches 
 
 
 
 
 
 
 
Feeling/being sick 
 
 
 
 
 
 
 
Red/watery eyes 
 
 
 
 
 
 
 
Wheezy chest 
 
 
 
 
 
 
 
TOTAL SCORE 
 
 
 
 
 
 
 
Absent = 0    Mild = 1   Moderate = 2    Severe = 3 
 
ADVERSE EVENTS 
1.19. 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 
 
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. 
 

 
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1.20. Expected adverse events 
1.20.1. Potential adverse events related to RSV and influenza infection 
We would expect subjects to experience typical symptoms of a common cold (including, but not limited 
to: fever, headache, malaise, rhinorrhoea, nasal congestion, sneezing, sore throat, and cough).  These 
would not be deemed adverse events, unless in the opinion of the study doctor. However, fever greater 
than 38ºC for more than three consecutive days or withdrawal from the study due to intolerable 
symptoms in more than two subjects in any arm will lead to a suspension of the study. The safety 
monitoring committee will be convened to determine any systematic cause for unexpectedly severe 
symptoms.  
 
Any influenza-like illness resulting in 
 
 
Sustained elevated heart rate >120bpm AND 
 
Sustained low blood pressure SBP<100 
 
Sustained elevated respiratory rate >30/min AND 
 
Sustained low blood oxygen SaO2<94% 
 
Evidence of pneumonia on clinical examination 
 
New ECG abnormalities 
 
will lead to referral for assessment in Accident and Emergency. Hospitalisation of any subject will lead 
to immediate suspension of the trial. The safety monitoring committee will be convened to assess the 
clinical evidence in order to determine whether the study may proceed. 
1.20.2. Potential adverse events of bronchosorption 
Bronchosorption is likely to have less adverse effects than bronchoalveolar lavage, brushing and biopsy 
(the standard bronchoscopy tools) as it should not cause bleeding, infection or a reactive pyrexia.  The 
only additional adverse event relating specifically to bronchosorption is dislodgement of the SAM from 
the forceps.  If this does occur the SAM can be retrieved using standard endobronchial forceps and 
snares available routinely in the bronchoscopy suite. Emergency procedures SOP is in place.   
 
1.20.3 Potential adverse effects of chest X-ray 
Volunteers participating in this study will receive 1 chest Xray, which is entirely for research purposes. 
The estimated dose will be 0.02mSv (national Diagnostic Reference Level), which is approximately 
equivalent to 3 days natural background radiation and carries risk of inducing a cancer of approximately 
1:1000,000 based on risk factors for a healthy adult. This is classified as a trivial risk level (ICRP 62). 
 
1.21. 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.   
1.21.1. Non serious AEs 
All such events, whether expected or not, should be recorded.  These will be discussed by the safety 
monitoring committee (see Section 12) 
1.21.2. Serious AEs 
An SAE form should be completed and faxed to the Chief Investigator and the Sponsor within 24 hours.  
The safety monitoring committee (see Section 12) will also be informed and a meeting convened as 
soon as possible. 
 
All SAEs should be reported to the West London REC 2 Research Ethics Committee where in the 
opinion of the Chief Investigator, the event was: 

 
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 
‘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 to ethics, the sponsor and the R&D office 
within 15 days of the Chief Investigator becoming aware of the event, using the NRES SAE form for 
non-IMP studies.   
 
Contact details for reporting SAEs 
Fax: 020 7262 8913 for the attention of Professor Peter Openshaw and/or Dr Christopher Chiu 
Please send SAE forms to: Respiratory Infections, Wright Fleming Institute, NHLI, Imperial 
College, St Mary’s Campus, London, W2 1PG 
Tel: 020 7594 3854 (Mon to Fri 09.00 – 17.00) 
 
ASSESSMENT AND FOLLOW-UP  
Study participants will be seen frequently during the study period, and daily for 10 days following 
infection.  They have details to contact the study doctor and research nurses and will be offered daily 
telephone contact.  In this way, participants will be assessed regularly by the investigating team and any 
adverse events detected rapidly; subjects meeting the criteria for a serious adverse event will be offered 
prompt treatment as appropriate. 
 
Subjects will have completed the study when they have had final convalescence investigations, 
expected to be 28 days after the initial inoculation with the influenza virus and 196 days after initial 
inoculation with the RSV virus.  The overall study will be completed when sufficient numbers of subjects 
have been recruited.  The end of the study is defined as the last visit of the last participant. 
 
When the study is completed they will not be routinely followed-up.  Subjects will return to the care of 
their GP following completion of the study. 
 
STATISTICS AND DATA ANALYSIS 
Based on pilot data from de Bree et al., we calculate that a sample size of 16 in each arm will be sufficient 
to find a difference of 1.4 between epitope-specific T cell counts in blood and lung, with 80% power 
using a 2-sided unpaired t-test with 5% significance level (where the variability in each group is 1.3).  T 
cell numbers will be compared between RSV and influenza using an unpaired t-test (if normality of the 
T cell distribution, or its transformation, is satisfactory as assessed by histogram with q-qplot and 
Shapiro-Wilks test) or a Mann-Whitney test (if normality is not satisfactory).  We will perform microarray 
analysis on each of 6 patients prior to inoculation and at 3 post-baseline timepoints.  Our main analysis 
will identify differentially expressed genes between influenza and RSV at each post-baseline timepoint.  
The sample size, calculated using the size package from bioconductor which provides pilot data of gene 
expression variability from the U95 package, predicts that 6 individuals in each group will give us 80% 
power to detect a potential fold change of 3 in up to 67% of the genes with a Bonferroni multiple testing 
correction with a genome-wide 5% significance level. 
 
Quantitative assessments of symptom scores, lung function, virus load, leukocyte numbers and 
inflammatory markers will be compared within subjects to determine differences between baseline and 
during infection.  Intra-subject differences will be analysed using ANOVA and 2-tailed paired Student’s 
t-tests or Wilcoxon signed rank test as appropriate.  Correlations between inflammatory cell, illness 
severity, viral load and leukocyte counts will be examined using Spearman’s rank correlations to 
investigate possible causal relationships. 
 

 
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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 according to Imperial College London policy. 
REGULATORY ISSUES 
1.22. Ethics approval 
The Chief Investigator has obtained approval from the Fulham Research Ethics Committee for this 
study.  The study will be submitted for Site Specific Assessment (SSA) at Imperial College Healthcare 
NHS Trust.  The Chief Investigator will require a copy of the R&D approval letter before 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, 
Declaration of Helsinki 1964 and later revisions. 
 
1.23. Consent  
Consent to enter the study will be sought from each participant only after a full explanation has been 
given, an information leaflet offered, time allowed for consideration, and any questions participants may 
have answered.  Signed participant consent will be obtained prior to any screening tests being carried 
out.  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. 
 
1.24. Confidentiality 
The Chief Investigator and all of the research team will preserve the confidentiality of participants taking 
part in the study and abide by the Data Protection Act. 
 
1.25. Indemnity 
Imperial College, London as sponsor of this study holds negligent and non-negligent harm insurance 
policies which apply to this study.  These have been arranged through the Joint Research Office. 
 
1.26. Sponsor 
Imperial College London will act as the main sponsor for this study.  Delegated responsibilities will be 
assigned to the NHS trusts taking part in this study. 
 
1.27. Funding 
Following a successful Clinician Scientist Fellowship application the MRC is funding this study.  They 
are acting as sole funders and this agreement is in place.  The investigators will not receive any 
additional payment above their normal salaries.  Participants in the study will have their travel costs 
refunded.  They will also be given a donation of up to £3000 to compensate for the time and 
inconvenience of taking part in the study. 
 
1.28. Audits and inspections  
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 (3rd  edition). 
 
 

 
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1.29. Sample storage and usage 
 
Samples of tissue, cells and fluids will be stored at Imperial College London. Samples will be fully 
anonymised. These may be used for further assays or in other ethically approved studies. Samples may 
be shared with UK and international collaborators in studies that have been approved by local ethics 
committee and subject to a valid Materials Transfer Agreement. 
STUDY MANAGEMENT 
The day-to-day management of the study will be co-ordinated through Dr Christopher Chiu, Clinical 
Senior Lecturer & Honorary Consultant in Infectious Diseases, with close support from Professor Peter 
Openshaw.  In addition, a safety monitoring committee will convene monthly during the study to discuss 
all adverse events, protocol deviations, and other safety issues. 
PUBLICATION POLICY 
Our expectation is that after analysis the data from this study will be widely distributed in the medical 
and scientific community.  Facilitated with presentations at local, national and international meetings, 
we hope to publish widely in the medical literature.  In addition we have an excellent media department 
at Imperial College and publicise research that has public interest when it is published.  No identifying 
participant information will be published. 
 
 
 

 
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