d4.1 first study subject approvals package all clinical...

Reproduction of this document or part of this document without RESCEU consortium permission is forbidden. Any use of any part must acknowledge the RESCEU consortium as “RESCEU REspiratory Syncytial virus Consortium in EUrope” n°116019 (Innovative Medicines Initiative Joint Undertaking)”. This document is shared in the RESCEU Consortium under

the conditions described in the RESCEU Consortium Agreement, Clause 9.

D4.1 First study subject approvals package – all clinical studies

116019 - RESCEU

REspiratory Syncytial virus Consortium in EUrope

WP4 – Prospective data collection

Lead contributor Louis Bont (3 – UMC Utrecht (UMCU))

Email: [email protected]

Other contributors Clinical study sites:

UEDIN, UA, UMCU, UOXF, SERGAS, TUCH, UMCG, Imperial

Pharmaceutical partners: GSK, AZ, JPNV, SP, Pfizer, Novavax

Due date 30/04/2017

Delivery date 28/04/2017

Deliverable type R

Dissemination level PU

Description of Work Version Date

V2.1 28/04/2017

116019 – RESCEU – D4.1

© Copyright 2017 RESCEU Consortium 2

Table of contents

Document History ........................................................................................................................................................................... 3

Definitions ........................................................................................................................................................................................... 4

Publishable Summary ................................................................................................................................................................... 6

1. Introduction ............................................................................................................................................................................... 7

2. Methods...................................................................................................................................................................................... 8

3. Results ..................................................................................................................................................................................... 10

4. Discussion .............................................................................................................................................................................. 11

5. Conclusion and next steps ............................................................................................................................................ 12

ANNEX I. Clinical study 1: Birth cohort study protocol.............................................................................................. 13

ANNEX II. Clinical study 2: Case-control study protocol ......................................................................................... 41

ANNEX III. Clinical study 3: Older adults cohort study protocol ........................................................................... 65

ANNEX IV. Clinical study 4: COPD cohort study protocol ...................................................................................... 94

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Document History

Version Date Description

V1.0 03/04/2017 First Draft

V1.1 07/04/2017 PMO review

V2.0 20/04/2017 Second draft

SC review

V2.1 28/04/2017 Final version for IMI submission

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Definitions

Participants of the RESCEU Consortium are referred to herein according to the following codes:

▪ UEDIN. University of Edinburgh (United Kingdom)

▪ UA. Universiteit Antwerpen (Belgium)

▪ UMCU. University Medical Centre Utrecht (Netherlands)

▪ UOXF. The Chancellor, Masters and Scholars of the University of Oxford (United Kingdom)

▪ SYNAPSE. Synapse Research Management Partners S.L. (Spain)

▪ Imperial. Imperial College of Science, Technology and Medicine (United Kingdom)

▪ SERGAS. Servicio Galego de Saúde (Spain)

▪ TUCH. Varsinais-Suomen sairaanhoitopiirin kuntayhtymä (Finland)

▪ RIVM. Rijksinstituut voor Volksgezondheid en Milieu - National Institute for Public Health and the Environment (Netherlands)

▪ SSI. Statens Serum Institut (Denmark)

▪ UMCG. Academisch Ziekenhuis Groningenand (Netherlands)

▪ PENTA. Fondazione PENTA for the treatment and care of children with HIV-ONLUS (Italy)

▪ AZ. Astrazeneca AB (Sweden)

▪ Pfizer. Pfizer Limited (United Kingdom)

▪ GSK Bio. GlaxoSmithKline Biologicals S.A. (Belgium)

▪ SP. Sanofi Pasteur (France)

▪ JPNV. Janssen Pharmaceutica, N.V (Belgium)

▪ Novavax. Novavax Inc. (United States of America)

▪ Grant Agreement. The agreement signed between the beneficiaries and the IMI JU for the

undertaking of the RESCEU project (116019).

▪ Project. The sum of all activities carried out in the framework of the Grant Agreement.

▪ Work plan. Schedule of tasks, deliverables, efforts, dates and responsibilities corresponding to

the work to be carried out, as specified in Annex I to the Grant Agreement.

▪ Consortium. The RESCEU Consortium, comprising the above-mentioned legal entities.

▪ Consortium Agreement. Agreement concluded amongst RESCEU participants for the

implementation of the Grant Agreement. Such an agreement shall not affect the parties’

obligations to the Community and/or to one another arising from the Grant Agreement.

▪ SC. Steering committee.

▪ PAB. Patient Advisory Board.

▪ ISAG. International Scientific Advisory Group.

▪ EAC. Ethics Advisory Committee.

▪ IRB. Institutional Review Board.

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▪ COPD. Chronic Obstructive Pulmonary Disease

▪ RSV. Respiratory Syncytial Virus

▪ ARTI. Acute Respiratory Tract Infection

▪ RT-PCR. Reverse Transcriptase Polymerase Chain Reaction

▪ HRQoL. Health Related Quality of Life

116019 – RESCEU – D4.1


Publishable Summary

The birth cohort study protocol (n=10,000) has been approved by the Steering committee (SC), Patient Advisory Board (PAB), International Scientific Advisory Group (ISAG) and Ethics Advisory Committee (EAC). The protocol has been submitted to the Institutional Review Board (IRB) at UMCU, the other centers will submit to the IRB in April. The Chronic Obstructive Pulmonary Disease (COPD) study protocol (N=500) has been approved by the SC, PAB, ISAG and EAC. The protocol has been submitted to the IRB at UMCG. The older adults study protocol (n=1,000) has been approved by the SC, PAB, ISAG and EAC. All centers plan to submit to the IRB in the next months. The infant case-control study protocol (N=630) has been approved by the SC, PAB, ISAG and EAC. All centers plan to submit to the IRB in the next months.

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1. Introduction

We will prospectively collect Respiratory Syncytial Virus (RSV) incidence data in healthy infants, older

adults (aged ≥60 years) and adults (aged ≥40 years) with COPD. This will include data both on RSV

infection leading to medical care (GP visits, hospital outpatient visits, hospital and ICU admission)

and non-medically-attended RSV infections. In addition, the clinical studies will provide high quality

data on direct and indirect costs and health related quality of life (HRQoL) (WP3) and clinical samples

for biomarker research (WP5).

The four clinical studies are:

▪ Clinical study 1: Birth cohort study (N=10,000) consisting of an active (N=1,000) and a passive

cohort (N=9,000)

▪ Clinical study 2: Case-control study (N=630)

▪ Clinical study 3: Adult cohort study (N=1,000)

▪ Clinical study 4: COPD cohort study (N=500)

116019 – RESCEU – D4.1


2. Methods

Clinical study 1: Birth cohort study (N=10,000)

Participating centers: UMCU, UOXF, UEDIN, SERGAS, TUCH

A healthy birth cohort (n=10,000) will be studied. Babies will be recruited at birth. To determine the

incidence of RSV hospitalization in the first year of life participants will be screened for hospital

admission for respiratory symptoms by parental questionnaire at age 1 year. In those who screen

positive, ascertainment of RSV admission will be achieved by patient record assessment, including

viral diagnosis.

Within the healthy birth cohort a nested cohort (n=1,000) will be used to measure the incidence of

medically attended (MA) RSV acute respiratory tract infection (ARTI). In the nested cohort active

surveillance through weekly contact, alternating by telephone and telephone app, will ensure that all

(including non-medically attended) RSV-related respiratory infections (acute upper and lower

respiratory tract infections) and associated medical consumption episodes are captured by the study.

Throughout the first year of life samples will be collected from the participants of the nested cohort for

analysis. Nasal swabs will be collected at the time of an ARTI episode for diagnosis of RSV by RT-

PCR. A subset of the nested cohort (n=600) will deliver samples for the biomarker studies (WP5).

Premorbid samples will be collected at time of neonatal heel prick. In this subset, blood will be

collected at time of RSV positive ARTI episode and at convalescence from three study sites (UOXF,

UMCU, SERGAS) to validate biomarkers discovered in the case control studies.

From 1 to 3 years of age, questionnaire-based follow-up will be limited to children in the nested cohort

and all children hospitalized for RSV ARTI and will be censored at one year after the last patient has

entered the study. Follow-up will be used to estimate how RSV infections of different severity relate

to wheeze up to age 3, and will include associated costs and health care consumption.

For health economic studies, cost, resource use and HRQoL data will be collected using

questionnaires for caregivers, with a follow-up diary from the time of diagnosis as established through

the active surveillance.

Clinical study 2: Case-control study (N=630)

Participating centers: UMCU, UOXF, SERGAS, Imperial

We will undertake a case-control study to provide samples to the RESCEU biobank (for analysis in

WP5) which has the aim of identifying biomarkers of severe disease and of sequelae in infants after

RSV infection. Cases (N=250) will be drawn from RSV infected hospitalized, previously healthy infants

and controls (N=250) will be mildly affected RSV positive, previously healthy infants who do not

require hospital admission. In addition, 50 infants (25 cases and 25 controls) with comorbidities as an

exploratory study and 80 healthy control infants without RSV infection will be recruited. Blood, stool,

urine and nasopharyngeal samples will be obtained from all infants at presentation and in

convalescence 6 weeks later for biomarker studies in WP5. Families will be followed up yearly till

maximum 3 years to document the onset of respiratory sequelae.

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Clinical study 3: Adult cohort study (N=1,000)

Participating centers: UMCU, UOXF, UA

A prospective observational study with data and sample collection will be performed to better

determine the incidence rate of RSV infection, including MA RSV infection in older adults. We will

recruit adults aged ≥60 years (n=1,000, including 500 elderly, ≥75 years) randomly selected from a

population-based patient registry from local general practitioner networks. We will determine the

incidence and severity of RSV MA-ALRI in adults ≥ 60 years of age. We will follow the participants by

active surveillance through weekly contacts during one RSV season. Cost, resource use and HRQoL

data will be collected using questionnaires, with a follow-up diary from the time of diagnosis as

established through the active surveillance.

RSV diagnosis will be based on respiratory samples at the time of symptoms and serology pre- and

post-RSV season. To this end, nasal swabs and, if possible, sputum will be collected at the time of

any respiratory episode with or without medical attention, during home visits for respiratory pathogen

detection. Serum will be collected at the beginning (October) and end (May) to measure a relevant

increase in antibody titer during the RSV season. For biomarker studies, samples will be collected for

analysis in WP5.

Clinical study 4: COPD cohort study (N=500)

Participating centers: Imperial, UMCG

An observational, prospective study with data and sample collection will be performed. Five hundred

subjects with COPD (GOLD I-IV) will be followed for 3 RSV seasons to determine the incidence and

severity of RSV medically attended respiratory illness in adults with COPD. At the start of the study

and the end of the study patients will be characterized, including by FEV1 measurement. We will

follow the participants by active surveillance through weekly contacts. Cost, resource use and HRQoL

data will be collected using questionnaires, with a follow-up diary from the time of diagnosis, as

established through the active surveillance. RSV diagnosis will be based on respiratory samples at

time of symptoms and serology pre- and post-RSV season. To this end, nasal swabs and sputum will

be collected at the time of exacerbations, with or without medical attention, for respiratory pathogen

detection. Serum will be collected at the beginning (October) and end (May) to measure a relevant

increase in antibody titer during the RSV season. For biomarker studies, samples will be collected for

analysis in WP5.

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3. Results

D4.1. First study subject approvals package – all clinical studies (M4) Report on the approval of the clinical study protocols on healthy infants, older adults > 60 years and

adults with COPD by the Patient Advisory Board (PAB) and Ethics Advisory Committee (EAC)

Submission to the local IRB’s is conducted at the moment. Update on this deliverable:

• The birth cohort study protocol (n=10,000) has been approved by the SC, PAB, ISAG and EAC. The protocol has been submitted to the IRB at UMCU, the other centers will submit to their IRB in April.

• The COPD study protocol (N=500) has been approved by the SC, PAB, ISAG and EAC. The protocol has been submitted to the IRB at UMCG.

• The older adults study protocol (n=1000) has been approved by the SC, PAB, ISAG and EAC. All centers plan to submit to their IRB in the next months.

• The infant case-control study protocol (N=630) has been approved by the SC, PAB, ISAG and EAC. All centers plan to submit to their IRB in the next months.

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4. Discussion

Not applicable

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5. Conclusion and next steps

The clinical study protocols on healthy infants, older adults > 60 years and adults with COPD have

been approved by the Patient Advisory Board (PAB) and Ethics Advisory Committee (EAC).

Submission to the IRB of each participating center is conducted for the four clinical studies at the

moment in order to obtain IRB approval in the coming months.

13

ANNEX I. Clinical study 1: Birth cohort study protocol

NLxxxxx.xxx.xx Burden of disease of RSV

14

REspiratory Syncytial virus Consortium in EUrope (RESCEU) study:

Defining the burden of disease of Respiratory Syncytial Virus in

Europe.

Protocol ID <include protocol ID given by sponsor or

investigator>

Short title Burden of disease of RSV

EudraCT number Not applicable

Version 1

Date January 2nd 2017

Coordinating

investigator/project leader

<Name, function and contact details>

Principal investigator(s) (in

Dutch: hoofdonderzoeker/

uitvoerder)


Sponsor (in Dutch:

verrichter/opdrachtgever)

[Comment: Add site specific

information]

Subsidising party Innovative Medicines Initiative (IMI)

EU-H2020

Independent expert (s) <please include name and contact data>


15

Laboratory sites UMCU

[Comment: Add site specific information]

Pharmacy Not applicable


16

PROTOCOL SIGNATURE SHEET [Comment: Add site specific information]

Name Signature Date

Sponsor or legal representative:

Head of Department:

[Coordinating Investigator/Project

leader/Principal Investigator]:


17

TABLE OF CONTENTS

1. INTRODUCTION AND RATIONALE .............................................................................21

2. OBJECTIVES ................................................................................................................23

3. STUDY DESIGN ...........................................................................................................23

4. STUDY POPULATION ..................................................................................................25

4.1 Population (base) ...................................................................................................25

4.2 Inclusion criteria .....................................................................................................25

4.3 Exclusion criteria ....................................................................................................25

4.4 Sample size calculation ..........................................................................................25

5. METHODS ....................................................................................................................26

5.1 Study parameters/endpoints ...................................................................................26

5.1.1 Main study parameter/endpoint .......................................................................26

5.1.2 Secondary study parameters/endpoints ..........................................................26

5.2 Randomisation, blinding and treatment allocation ..................................................27

5.3 Study procedures ...................................................................................................27

5.4 Withdrawal of individual subjects ............................................................................29

5.5 Replacement of individual subjects after withdrawal ...............................................29

5.6 Premature termination of the study .........................................................................29

6. SAFETY REPORTING ..................................................................................................30

6.1 Section 10 WMO event ..........................................................................................30

6.1.1 Adverse events (AEs) and Serious adverse events (SAEs) .............................30

6.1.2 Suspected unexpected serious adverse reactions (SUSARs) .........................30

6.1.3 Annual safety report ........................................................................................30

6.2 Follow-up of adverse events ...................................................................................30

6.3 Data Safety Monitoring Board (DSMB) ...................................................................30

7. STATISTICAL ANALYSIS ..........................................................................................31

8. ETHICAL CONSIDERATIONS ...................................................................................31

8.1 Regulation statement .............................................................................................31

8.2 Recruitment and consent........................................................................................31

8.3 Objection by minors or incapacitated subjects ........................................................32

8.4 Benefits and risks assessment, group relatedness .................................................32

8.5 Compensation for injury .........................................................................................33

9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .........................33

9.1 Handling and storage of data and documents ........................................................33

9.2 Monitoring and Quality Assurance ..........................................................................34

9.3 Amendments ..........................................................................................................34

9.4 Annual progress report ...........................................................................................34

9.5 End of study report .................................................................................................34

9.6 Public disclosure and publication policy ..................................................................34

10. REFERENCES ..........................................................................................................35

11. APPENDIX A Governance structure of RESCEU………………………………………..36

12. APPENDIX B Glossary of terms…………………………………………………………...39

18

LIST OF ABBREVIATIONS (FOR RELEVANT DEFINITIONS, SEE APPENDIX 2)

ABR ABR form, General Assessment and Registration form, is the application form that

is required for submission to the accredited Ethics Committee (In Dutch, ABR =

Algemene Beoordeling en Registratie)

AE Adverse Event

AR Adverse Reaction

ARTI Acute Respiratory Tract Infection

CA Competent Authority

CCMO Central Committee on Research Involving Human Subjects; in Dutch: Centrale

Commissie Mensgebonden Onderzoek

COPD Chronic obstructive pulmonary disease

CV Curriculum Vitae

DSMB Data Safety Monitoring Board

EDC system Electronic Data Capture system

EU European Union

GCP Good Clinical Practice

IC Informed Consent

MA Medically Attended

METC Medical Research Ethics Committee (MREC); in Dutch: Medisch Ethische

Toetsing Commissie (METC)

POC test Point of Care test

RSV Respiratory Syncytial Virus

RT-PCR Reverse Transcriptase Polymerase Chain Reaction

(S)AE (Serious) Adverse Event

SUSAR Suspected Unexpected Serious Adverse Reaction

Wbp Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens)

WMO Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-

wetenschappelijk Onderzoek met Mensen)

19

SUMMARY

Rationale: The REspiratory Syncytial virus Consortium in EUrope (RESCEU) is an Innovative

Medicine Initiative (IMI) effort funded by the EU under the H2020 framework to define and

understand the burden of disease caused by human respiratory syncytial virus (RSV) infection.

RSV causes severe disease in individuals at the extremes of the age spectrum and in high risk

groups. It was estimated that RSV was associated with 34 million cases of acute respiratory

tract infection (ARTI), 3.4 million ARTI hospitalizations and 55,000 to 199,000 deaths in

children <5 years in 2005 worldwide. These estimates were based on limited data and there is

a substantial gap in knowledge on morbidity and associated healthcare and social costs in

Europe. New vaccines and therapeutics against RSV are in development and will soon be

available on the European market. RESCEU will deliver knowledge of the incidence and

burden of disease RSV in young children and older adults in Europe, which is essential for

stakeholders (governments, etc) to take decisions about prophylaxis and treatment.

Objective: To determine the burden of disease due to RSV in young children.

Study design: Prospective epidemiological, observational, multi-country, multicenter, cohort

study.

Study population: Birth cohort of healthy infants (follow-up from birth until the age of 3 years

maximum):

- Passive birth cohort (n=9,000).

- Active birth cohort (n=1,000).

Main study parameters/endpoints:

The primary endpoint of the study is the incidence of RSV infection-associated ARTI, RSV

associated medically attended (MA) ARTI (active birth cohort) and RSV related hospitalization

(passive birth cohort) in infants (< 1 year) during 3 RSV seasons. In addition, a major

secondary endpoint is RSV attributable burden of wheezing.

Nature and extent of the burden and risks associated with participation, benefit and

group relatedness:

1a Passive birth cohort

Parents who agree with participation in the study will be asked to fill out a questionnaire at

inclusion in the first week(s) after birth and at age one year. Only children who were admitted

to the hospital for ARTI during the first year of life will be followed up to the age of maximum 3

years by yearly questionnaires.

1b Active birth cohort (nested cohort)

Parents who agree with participation in the study will be asked to fill in a baseline questionnaire

at inclusion in the first week after birth and yearly questionnaires until the age of 3 years or

end of study. In addition, a blood sample, a nasopharyngeal swab, a buccal swab, and stool

and urine samples will be collected at time of heel prick around the fifth day of life as baseline

samples. By collecting capillary blood (1 ml) at the moment of the heel prick an extra moment

of discomfort/pain by the procedure will be avoided. Taking a nasopharyngeal swab and a

buccal swab can give a brief moment of discomfort at the moment of sampling.

20

During the RSV season within the first year of life parents will be contacted weekly (by

telephone or email and/or fill in a (daily) telephone app) to monitor respiratory symptoms of

their child. Parents are asked to contact the study team if the infant experiences a new episode

of ARTI. The study team will visit the infant within 3 days and take 2 nasopharyngeal swabs,

one to perform a point of care (POC) test for RSV and one for RT-PCR (and additional analyses

if RSV is positive). Taking a nasopharyngeal swab can give a brief moment of discomfort at

the moment of sampling. If RSV is positive, informed consent will be asked for participation in

the biomarker sub-study (n=480 maximum). If included in the biomarker sub-study, the

following additional sampling procedures will be performed by the study team at the moment

of infection and 6-8 weeks after infection: A venous puncture will be drawn (max 4 ml blood).

This is moderately painful. However, discomfort will be minimalized by the use of a local

anaesthetic cream. In addition, stool and urine samples and a nasopharyngeal swab will be

collected (if not already collected as part of the study).

Possible benefit: There is no clear clinical benefit for the subjects participating in this

proposed study. However, the results of this study aim to support the understanding of the

burden of RSV disease which is important for the implication of future preventive and

therapeutic interventions.

None of the study procedures is associated with any risk for serious complications. However,

there is a minimal risk of minor complications due to study procedures (for example a nose

bleed after a nose swab or bruise after blood sampling).

21

1. INTRODUCTION AND RATIONALE

The RESCEU clinicalstudies - summary

The IMI-funded REspiratory Syncytial virus Consortium in EUrope (RESCEU) effort includes

an observational study to define the burden of disease caused by human respiratory syncytial

virus (RSV) infection. A total of 4 clinical studies in specific risk groups will be performed in

several European countries as part of the RESCEU study. The sites of these studies were

selected because of their experience in ARTI and/or RSV research in specific risk groups.

The clinical studies in young children (clinical study 1 and 2) will be conducted at the UMC

Utrecht (UMCU, Netherlands), University of Turku (TUH, Finland), Servicio Galego de Saúde

(SERGAS, Spain), University of Oxford (UOXF), University of Edinburgh (UEDIN) and Imperial

College London (United Kingdom).

The study in older adults (elderly) will be conducted at the UMC Utrecht (Netherlands),

University of Antwerp (Belgium) and University of Oxford (United Kingdom).

The study in chronic obstructive pulmonary disease (COPD) patients will be conducted at the

UMC Groningen (Netherlands) and Imperial College London (United Kingdom).

This protocol is restricted to Clinical Study 1.

Figure 1. Overview of clinical studies embedded in the RESCEU effort and follow up periods

for included (individual) patients.

22

Background

Human respiratory syncytial virus (RSV) causes severe disease in individuals at the extremes

of the age spectrum and in high risk groups. It was estimated that RSV was associated with

34 million cases of acute respiratory tract infection (ARTI), 3.4 million ARTI hospitalizations

and 55,000 to 199,000 deaths in children <5 years in 2005 worldwide 1. These estimates were

based on limited data and there is a substantial gap in knowledge on morbidity and associated

healthcare and social costs in Europe. Although prematurity and cardiorespiratory comorbidity

are well-known risk factors for severe disease in young children, a substantial number of

children admitted to pediatric intensive care units because of severe RSV ARTI are previously

healthy infants. Data about RSV incidence and burden of disease in healthy children are

scarce, since most studies are performed only in high risk groups. Moreover, RSV infection in

childhood is associated with subsequent wheezing and asthma 2-4. These long‐term sequelae

pose a substantial additional burden on the healthcare system. In addition, RSV is a significant

cause of ARTI morbidity in elderly and COPD patients 5,6.

Treatment and prophylaxis options are very limited. Mostly only supportive care is available for

infants with severe RSV ARTI. Ribavirin has been used as treatment, but with limited evidence

of benefit and is therefore not routineously recommended 7. Passive prophylaxis with RSV

specific antibodies (palivizumab) is available for high risk groups for severe disease

(prematurely born infants and infants with significant cardiac and/or respiratory comorbidity).

However, palivizumab has to be given intramuscular monthly during the RSV season and

protection varies. Palivizumab is also expensive and therefore not readily available for lower

income countries.

Various new RSV vaccines and therapeutics are expected to be available in the near future 8.

To use these new vaccines and therapeutics in the best possible way, it is necessary to

determine the burden of RSV disease in Europe to gain better insight into disease severity in

young children and the associated societal and healthcare costs.

There is a parallel need to assemble clinical resources to identify the correlates of severe RSV

disease for clinical management, classification of disease severity in clinical trials and

identification of biomarkers for severe disease, which are currently lacking 9.

For this purpose, the RESCEU (Respiratory Syncytial virus Consortium in Europe) consortium

has been set up. RESCEU will perform the first prospective multi‐center study in healthy

children to provide accurate data on RSV disease incidence and sequelae (long‐term airway

morbidity, including asthma) and economic consequences of RSV infection. We will

prospectively follow-up a birth cohort of 10,000 healthy children (1,000 active and 9,000

passive) during at least one year to obtain incidence data on RSV infection, medically attended

(MA) RSV infection and hospitalization due to RSV.

23

2. OBJECTIVES

Primary objective

• To determine the incidence of RSV infection-associated ARTI, RSV associated

medically attended ARTI (MA-ARTI) (active cohort) and RSV-related hospitalization

(all) during the first year of life.

Secondary objectives

• To estimate how RSV infection of different severity relates to wheeze up to age 3

years. The incidence and severity of wheeze will be determined by annual

questionnaires at age 1 year, 2 years and 3 years (active cohort and all children

hospitalized for ARTI) maximum or till end of study.

• To determine the rate of all-cause medically attended (inpatient or outpatient) ARTI

(active cohort).To determine mortality (RSV associated and all-cause) through all

RSV seasons of follow up (all).

• To determine health care costs, health care resource use, interruption of normal

activities, and Health Related Quality of Life (HRQoL) in RSV-associated and all-

cause medically attended (inpatient or outpatient) ARTI patients and their families

(active cohort).

• To determine the incidence of RSV-related secondary bacterial respiratory tract

infections within 21 days after onset of RSV infection and their association with

antibiotic use in hospitalized RSV ARTI patients (all children) and non-hospitalized

RSV ARTI patients (active cohort).

• To collect clinical samples (blood, nasopharygeal, stool and urine) for biomarker

analysis from a subset of infants in the active cohort.

• To determine the incidence rate of other respiratory pathogens (influenza,

rhinovirus, human metapneumovirus, parainfluenzavirus, etc.) associated with all

medically attended (inpatient or outpatient) ARTI (active cohort).

• To determine the proportion of viral ARTI attributable to RSV (active cohort).

• To determine important risk factors for RSV infection (by severity and healthcare

utilization) (all).

3. STUDY DESIGN

This will be a multi-country, multicenter, prospective, observational cohort study conducted

across 3 consecutive years to determine the incidence of RSV infection, RSV associated MA-

ARTI and RSV related hospitalization in a birth cohort of healthy subjects, recruited from the

general population.

At birth parents will be asked by a member of the study team to participate in the active cohort.

If enrolled, a nasopharyngeal sample, a blood sample, a buccal sample, and urine and stool

samples will be collected from the baby at the time of the heel prick around the fifth day of life.

Respiratory tract symptoms will be assessed weekly during the RSV season by telephone or

email or (daily) telephone app. If a child experiences a new episode of ARTI according to the

24

parents, the study team will visit the child to collect two nasopharyngeal samples, one to

perform a point of care (POC) test for RSV, and one for additional viral testing by RT-PCR. If

RSV is positive, parents will be asked informed consent [Comment: If preferred by participating

center informed consent for this part can also be asked at birth] to obtain additional blood,

nasopharyngeal, urine and stool samples at the time of RSV infection and 6-8 weeks after RSV

infection. Parents of all children in the active cohort will be asked yearly to fill in a questionnaire

until age 3 years maximum or till end of study (defined as the moment that the last included

subject has been followed up for 12 months).

If parents decline to participate in the active birth cohort, informed consent will be asked for

passive follow up. Parents will be asked to fill in a questionnaire at birth and after one year.

If their child was admitted to the hospital because of an ARTI, clinical data will be collected

retrospectively from the hospital. Participating hospitals will perform RSV tests as part of

standard diagnostic care in children <1 year of age who are admitted with ARTI. Only children

with hospitalization due to ARTI will be followed up by a yearly questionnaire until age 3 years

maximum or till end of study (defined as the moment that the last included subject has been

followed up for 12 months).

An overview of the study design and main procedures is given below (Figure 2).

Figure 2. Overview of study design and main procedures of the birth cohort study.

25

4. STUDY POPULATION

4.1 Population (base)

Birth cohort of 10,000 healthy term infants of the general population including a nested (active)

cohort of 1,000 healthy term infants. Subjects will be recruited from maternity wards during the

first days after birth in the following countries: the Netherlands (UMCU), United Kingdom

(UEDIN, UOXF), Spain (SERGAS) and Finland (TUH).

4.2 Inclusion criteria for enrolment

All subjects must satisfy ALL the following criteria at study entry:

- Healthy* children, gestation age at least 37+0, born at participating centers.

- Written informed consent obtained from parents.

- Parents ability and willingness to adhere to protocol-specified procedures (active

cohort).

* children with transition problems, including mild to moderate asphyxia, respiratory distress or

suspected early onset neonatal infection will be included and are distinguished and analysed

separately at end of study.

4.3 Exclusion criteria for enrolment

The following criteria should be checked at the time of study entry. If ANY

exclusion criterion applies, the subject must not be included in the study:

- Major congenital defects or serious chronic illness (i.e. severe congenital heart and/or

lung disease, genetic, immunologic and/or metabolic disorder).

- Gestational age of less than 37+0 weeks.

- Acute severe medical condition at moment of heel prick (e.g. sepsis, severe asphyxia,

for which the child is admitted to the hospital).

- Child in care.

- Parents not able to understand and communicate in the local language.

- Living outside catchment area of study sites.

- Mother vaccinated against RSV during pregnancy

4.4 Sample size calculation

For the primary analysis the ratio between cases of RSV-related hospitalizations and number

of children in the total population will be calculated (full birth cohort). In addition, the ratio

between the cases of MA RSV infection and the number of children undergoing active

surveillance will be calculated (active cohort).

To estimate the incidence of MA RSV infection and RSV-related hospitalization during the first

year of life a birth cohort is followed for 1 year. For sample size calculations a statistic expert of

the Julius Support Center was consulted. The following assumptions were made: α=0.05; power

≥ 0.95 and loss to follow-up = 10%.

26

Patient populations

Sites Outcome

Persons

RSV seasons

Expected

Incidence (%)/y

CI Half-Width (%)

Healthy baby,

at least AD

37+0

(full cohort)

NL,

UK,

SP, FI

RSV-

hospitalization 10,000 1 0,8 10,11 0,5-1,3

Healthy baby,

at least AD

37+0

(active cohort)

NL,

UK,

SP, FI MA-RSV 1,000 1 10 10-12 8,0-12,0

5. METHODS

5.1 Study parameters/endpoints

5.1.1 Main study parameter/endpoint

The primary outcome is the incidence rate of RSV-associated ARTI leading to hospitalization in

the first year of life. To determine the incidence of RSV hospitalization in the first year of life

participants will be screened for hospital admission for respiratory symptoms by parental

questionnaire at age 1 year. Second, in those who screen positive, ascertainment of RSV

admission will be achieved by patient record assessment, including viral diagnosis.

In the active cohort the primary outcome is RSV associated ARTI and MA RSV infection defined

as any medical care for RSV infection. Nasopharyngeal swabs will be collected during home

visits for all ARTI episodes during the RSV season, and will be used for a RSV POC test and

reverse transcription-polymerase chain reaction (RT-PCR) detection of RSV.

5.1.2 Secondary study parameters/endpoints

1 Mortality through all RSV seasons of follow up including RSV-associated deaths and all

cause deaths.

2 Health care utilization for RSV-associated and all-cause medically attended (inpatient

or outpatient) ARTI or respiratory events (active birth cohort).

3 The incidence of RSV-associated secondary bacterial pneumonia and associated

antibiotic consumption events within 21 days after onset of RSV-related symptoms.

4 Parent reported wheeze and doctor’s diagnosis of wheeze by routine care (active birth

cohort and children admitted for ARTI of passive birth cohort).

5 The incidence of nosocomial RSV infection.

6 Sample collection for biomarker analysis.

27

5.2 Randomisation, blinding and treatment allocation

No investigational product is being administered in this study.

5.3 Study procedures

Passive birth cohort

• Informed consent will be obtained from parents prior to any study procedure.

• At birth (within 7 days) a questionnaire about pregnancy, perinatal course and potential

risk factors for RSV-hospitalization will be filled in (amongst others use of vaccines

during pregnancy, gestational age, birth weight, transition problems, including mild to

moderate asphyxia, respiratory distress or suspected early onset neonatal infection).

• At age 1 year participants will be screened for hospital admission for respiratory

symptoms by parental questionnaire.

• In those who screen positive, ascertainment of RSV admission will be achieved by

patient record assessment, including viral diagnosis. Children hospitalized for ARTI will

be followed up from age one to three years maximum by means of a yearly

questionnaire on RSV-related sequelae.

Active birth cohort (nested cohort of 1,000 healthy term infants of the birth cohort)

• Informed consent will be obtained from parents prior to any study procedure.

• At birth (within 7 days) a questionnaire about pregnancy, perinatal course and potential

risk factors for RSV-hospitalization will be filled in (amongst others use of vaccines

during pregnancy, gestational age, birth weight, transition problems, including mild to

moderate asphyxia, respiratory distress or suspected early onset neonatal infection).

• Around the fifth day of life (together with heel prick) a blood sample (max 1 ml), a

nasopharyngeal sample, a buccal sample, and urine and stool samples will be collected

and stored at -70 to -80°C.

• During the RSV season (between October 1st and May 1st, or longer if RSV is still

circulating) within the first year of life, parents/caregivers will be asked about respiratory

symptoms of their infant through weekly contact, alternating by telephone and email or

(daily) telephone app.

• If their child experiences an ARTI, parents are instructed to contact the study team.

Two nasopharyngeal samples will be collected by home visits within 3 days after

contact with the study team. One nasopharyngeal sample will be used for direct POC

RSV testing. The other nasopharyngeal sample will be diluted and aliquoted in 200

microliter samples and stored at -70 to -80°C.

• If the POC RSV test is positive, informed consent will be asked for the biomarker sub-

study and a blood sample (max 4 ml) will be collected by the study team. Parents will

be asked to collect a urine sample and a stool sample which can be stored in the freezer

at home till collection by the study team. All samples will be stored at -70 to -80°C.

• If included in the biomarker sub-study, the study team will visit the child again six to

eight weeks after the RSV positive ARTI for collection of nasopharyngeal and blood

samples (max 4 ml) and urine and stool samples collected by the parents. Samples will

be stored at -70 to -80°C.

28

• From age 1 to 3 years maximum children will be followed up by means of a yearly

questionnaire on RSV-related sequelae.

Table 1. Overview of sampling in active birth cohort.

Moment of sampling Sample Volume Analysis (minimum

amount)

At moment of heel

prick (around 5th day)

Serum (capillary) Max 0,5 ml RSV serology (350-400

µl)

Proteome (100 µl)*

Paxgene (capillary) Max 0,5 ml Transcriptome (200 µl)*

Nasopharyngeal swab

(aliquoted)

n/a Airway microbiome

Airway transcriptome

Buccal swab 3 swabs DNA/GWAS

Stool 5-10 ml (min 2 ml) Microbiome

Urine 3 ml Metabolomics

RTI Nasopharyngeal swab

(1 for POC, 1 for PCR)

RSV POC (qualitative)

RSV RT-PCR#

(quantitative)

RSV RTI (if informed

consent for biomarker

sub-study)

Serum (venous) 1-2 ml RSV serology (350-400

µl)

Proteome (100 µl)*

Paxgene (venous) 1-2 ml Transcriptome (200 µl)*

Whole blood$ 1-2 ml Cellular immunology$*


Nasopharyngeal swab

(aliquoted)

3-4 aliquots of 200

microliter

Airway microbiome


(RSV viral (deep)

sequence analysis)


6 weeks after RSV

RTI (if informed

consent for biomarker

sub-study)

Serum (venous) 1-2 ml RSV serology (350-400

µl)

Proteome (100 µl)*

Paxgene (venous) 1-2 ml Transcriptome (200 µl)*

Whole blood$ 1-2 ml Cellular immunology$*


Nasopharyngeal swab

(aliquoted)

3-4 aliquots of 200

microliter

Airway microbiome


Multiplex RT-PCR

respiratory viruses

(quantitative)


29

* and additional RSV-related biomarkers # and multiplex RT-PCR respiratory viruses if RSV POC is positive $ in subset of infants

Handling and storage of samples

All samples will be stored at the site where they are collected:

• Blood samples will be collected in appropriate tubes as described in table 1 and will be

stored at -70 to-80°C for later analysis.

• Nasopharyngeal swab: a nasopharyngeal swab will be collected and aliquoted bed-site in

3-4 samples of 200 microliter. All aliquots will be stored at -70 to -80°C for later analysis.

• Buccal swabs: 3 buccal swabs will be collected and stored at -70 to -80°C for later analysis.

• Urine: 3x1 ml of urine will be collected and stored at -70 to -80°C for later analysis.

• Stool: at least 2 ml of stool will be collected and stored at -70 to -80°C for later analysis.

5.4 Withdrawal of individual subjects

Subjects can leave the study at any time for any reason if they wish to do so without any

consequences. The investigator can decide to withdraw a subject from the study for urgent

medical reasons.

Withdrawal of consent: If consent is withdrawn, the subject will not have any further study

procedures or study observations. Samples and data collected to that point will continue to

form part of the study.

Lost to follow-up: Subjects will be considered lost-to-follow-up only if no contact has been

established by the time the study is completed such that there is insufficient information to

determine the subject’s RSV status at approximately 1 year.

5.5 Replacement of individual subjects after withdrawal

After withdrawal of an individual subject he or she will be replaced depending on the moment

of withdrawal.

5.6 Premature termination of the study

Not applicable.

6. SAFETY REPORTING

6.1 Section 10 WMO event

In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects

and the reviewing accredited METC if anything occurs, on the basis of which it appears that

the disadvantages of participation may be significantly greater than was foreseen in the

research proposal. The study will be suspended pending further review by the accredited

30

METC, except insofar as suspension would jeopardise the subjects’ health. The investigator

will take care that all subjects are kept informed.

6.1.1 Adverse events (AEs) and Serious adverse events (SAEs)

Every SAE directly related to any of the sampling interventions (vene or capillary puncture,

buccal swab, nasal swab) will be registered. Only SAEs will be registered as this is a non-

interventional, low risk, observational study. Expected AEs directly related to one of the

interventions (for example a nose bleed after a nose swab or bruise after a blood test) will not

be registered.

6.1.2 Suspected unexpected serious adverse reactions (SUSAR)

NA

6.1.3 Annual safety report

NA

6.2 Follow-up of adverse events

All AEs will be followed until they have abated, or until a stable situation has been reached.

Depending on the event, follow up may require additional tests or medical procedures as

indicated, and/or referral to the general physician or a medical specialist.

SAEs need to be reported to the local METC till end of study within the Netherlands, as defined

in the protocol [Comment: Adapt according to local regulations].

6.3 Data Safety Monitoring Board (DSMB)

No Data Safety Monitoring Board is needed. However, there will be 3 Advisory Boards which

will act as consultative bodies for ethical, scientific and technical matters.

The following advisory boards will be formed by external experts:

• International Scientific Advisory Group (ISAG)

• RESCEU Ethics Advisory Committee (EAC)

• RESCEU Patient Advisory Board (PAB).

See also appendix 1 for a detailed description of the governance structure of RESCEU.

7. STATISTICAL ANALYSIS

Descriptive statistics will be used to describe the incidence rate of hospitalization for RSV, MA

RSV infection and RSV associated ARTI in the birth cohort. Demographic parameters, clinical

parameters and outcome and laboratory test results will be displayed as categorical data with

percentages or continuous variables with mean (+/-SD) and/or median (interquartile range).

Comparisons between groups will be performed using chi-square for categorical variables,

Student-t-test for normally distributed continuous variables or Mann-Whitney U test for not

normally distributed continuous variables.

31

Statistical analyses will be performed using SPSS version 20 or a more recent version or with

R statistical software version 3.0.1 or higher.

8. ETHICAL CONSIDERATIONS

8.1 Regulation statement

The study will be conducted according to the principles of the Declaration of Helsinki

(www.wma.net) and in accordance with the Medical Research Involving Human Subjects Act

(WMO) and other guidelines, regulations and Acts.

The investigator will explain the nature of the study and will inform the parents/legal

representative of the subject that participation is voluntary and that the subject can withdraw

or be withdrawn from the study at any time. Written informed consent will be obtained from

parents/legal representative of each subject prior to any study procedure. A copy of the signed

consent form will be given to the parents /legal representative of the subject and the original

will be maintained by the research team.

8.2 Recruitment and consent

[Comment: Recruitment procedures can be adapted to local preferences and regulations]

Recruitment will take place around birth at maternity wards of participating hospitals All parents

will be given an information letter. If they stay in hospital long enough after birth, information

will also be given by the investigator and/or research nurses directly or if parents already left

the maternity ward, they will be contacted to discuss the study further.

Parents will be asked informed consent to participate in the active birth cohort. If they do not

want to participate in the active birth cohort, they will be asked informed consent to participate

in the passive birth cohort. All parents of participating subjects will be asked permission to

contact them in the future.

If parents decide to participate in the active birth cohort and their child gets a RSV associated

ARTI, informed consent will be asked for the biomarker sub-study and additional samples

during that episode and 6-8 weeks later will be obtained.

Continuous recruitment will take place over a period of 26 months (between July 1st 2017 and

September 1st 2019) to include three RSV seasons, the inclusion rate will be evenly distributed

over the whole inclusion period.

8.3 Objection by minors or incapacitated subjects

Subjects or their legal guardians can object to any procedure related to the study at any time

for any reason and participation will be terminated. According to the behavioural code

guidelines of the Dutch Society of Pediatrics (NVK 2001) [Comment: Change according to local

regulations], in case of minors or incapacitated subjects, any physical signs interpreted by the

investigator or legal guardian as an objection to a procedure will terminate participation.

32

8.4 Benefits and risks assessment, group relatedness

Active birth cohort

• Capillary puncture: This will be done together with/instead of the heel prick if possible,

so there will be no additional painful procedure. If it is not possible to combine with the

heel prick, an additional blood sample will be taken with verbal consent. With a capillary

puncture device a puncture will be done on the heel after which the blood drops will be

collected in tubes and routine heel prick sampling could be collected at the same time

• Nasopharyngeal swab: A small swab will be introduced into the nose towards the

nasopharynx and some mucus will be collected. This is a non-invasive technique. The

procedure can cause a brief moment of discomfort, however, the duration of this

procedure is less than 10 seconds and the swab is very soft. This procedure will be

performed by trained personnel. Minor complications (like a nose bleed) have been

described, but are rare.

• Buccal swab: a swab will be introduced in the mouth and will be gently rubbed and

rotated against the inside of the cheek for ~30 seconds. This is a non-invasive

technique. The procedure can cause a brief moment of discomfort, however, the

duration of this procedure is short. It will be performed by trained personnel. No

complications have been described.

• Stool sample: a stool sample will be collected from diapers. This will cause no

discomfort.

• Urine sample: a urine sample will be collected by putting a gauze in the diaper. If the

child has urinated the wet gauze will be squeezed into a tube. This will cause no

discomfort.

Extra procedures in active birth cohort with biomarker sampling if RSV positive (at moment of

infection and 6-8 weeks later)

• Venepuncture: Drawing venous blood is moderately painful. This will be performed by

trained personnel. If the child is aged >4 weeks anaesthetic cream will be offered. The

risk of complications is negligible. Occasionally bruising has been seen.

• Stool sample: a stool sample will be collected from diapers. This will cause no

discomfort.

• Urine sample: a urine sample will be collected by putting a gauze in the diaper. If the

child has urinated the wet gauze will be squeezed into a tube. This will cause no

discomfort.

The study is group related because the primary aim is to determine the burden of RSV disease

in a specific risk for RSV disease, namely infants. It is therefore necessary to perform this in

subjects belonging to these groups.

There is no clear clinical benefit for the subjects participating in this proposed study. However,

the results of this study aim to support the understanding of the burden of RSV disease which

is important for the implication of future preventive and therapeutic interventions.

33

8.5 Compensation for injury

Due to the type of study, observational with non-invasive diagnostic procedures without

complications (as previously described), no adverse or serious adverse events are to be

expected and participating in the study is with minimal risks. Therefore, we request

dispensation from the statutory obligation to provide insurance [Comment: The obligation to

provide insurance falls under the responsibility of each participating center/country].

9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION

9.1 Handling and storage of data and documents

Full data management will be performed by Julius Center. Data will be stored in a cloud-based

database. Data will be anonymized before they enter the database. Each subject will receive

a unique identification number, which cannot be traced back directly to the subject. The study

team will keep a subject identification code list to trace data to an individual subject, if

necessary. Data will be kept 15 years. The handling of personal data will be in compliance with

the Dutch Personal Data Protection Act [Comment: Change according to local regulations].

Data management of this study will be performed by a professional and experienced data

management team. This team will coordinate and implement a high quality IT-infrastructure

which will be necessary for the collection, controlling and reporting of the research data of this

study.

A GCP compliant electronic data capture (EDC) system will be used to guarantee a correct,

complete and consistent data collection. Web-based case report forms will be developed and

implemented on the EDC system. By using comprehensive data validation checks within these

forms, only data of high quality can be submitted to the study database. The forms, integrated

into the EDC system, can easily be accessed by a standard web browser.

The data management system facilitates the collection of data, supports the monitoring

processes and provides real time progress reports for management of the study. After last

patient out, the database can rapidly be closed and data made available for further analysis

and publication purposes.

The system meets all GCP guidelines for electronic data collection in terms of protecting data

integrity and securing the information collected. This means, among other things, that users

will get a role based access to the system after they have logged-in using their own username

and password. The system will log all data entry steps with timestamps, update reasons and

user information. The role based access to the system will avoid unauthorised data access

and prevents that users perform actions that they are not allowed to do. Data from the EDC

system will be transferred over the internet using secured data communication protocols. Data

will be stored automatically and regularly back-ups will make sure that data never will be lost.

Databases and web servers will be hosted in data centers that meet the highest possible

security requirements.

34

9.2 Monitoring and Quality Assurance

Monitoring of the conduct of the study will be performed according to GCP guidelines at

initiation and once yearly for the duration of the study.

9.3 Amendments

Amendments are changes made to the research after a favourable opinion by the accredited

METC has been given. All amendments will be notified to the METC that gave a favourable

opinion. All substantial amendments will be notified to the METC and to the competent

authority. Non-substantial amendments will not be notified to the accredited METC and the

competent authority, but will be recorded and filed by the sponsor.

9.4 Annual progress report

The sponsor/investigator will submit a summary of the progress of the trial to the accredited

METC once a year. Information will be provided on the date of inclusion of the first subject,

numbers of subjects included and numbers of subjects that have completed the trial, serious

adverse events/ serious adverse reactions, other problems, and amendments.

9.5 Temporary halt and (prematurely) end of study report

The investigator/sponsor will notify the accredited METC of the end of the study within a period

of 8 weeks. The end of the study is defined as the moment that the last included subject has

been followed up for 12 months.

The sponsor will notify the METC immediately of a temporary halt of the study, including the

reason of such an action.

In case the study is ended prematurely, the sponsor will notify the accredited METC within 15

days, including the reasons for the premature termination.

Within one year after the end of the study, the investigator/sponsor will submit a final study

report with the results of the study, including any publications/abstracts of the study, to the

accredited METC.

9.6 Public disclosure and publication policy

Results of this research are disclosed unreservedly.

35

10. REFERENCES

1. Nair H, Nokes DJ, Gessner BD, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet. 2010;375(9725):1545-1555.

2. Blanken MO, Rovers MM, Molenaar JM, et al. Respiratory syncytial virus and recurrent wheeze in healthy preterm infants. N Engl J Med. 2013;368(19):1791-1799.

3. Lotz MT, Moore ML, Peebles RS, Jr. Respiratory syncytial virus and reactive airway disease. Curr Top Microbiol Immunol. 2013;372:105-118.

4. Stein RT, Sherrill D, Morgan WJ, et al. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet. 1999;354(9178):541-545.

5. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med. 2005;352(17):1749-1759.

6. Zwaans WA, Mallia P, van Winden ME, Rohde GG. The relevance of respiratory viral infections in the exacerbations of chronic obstructive pulmonary disease-a systematic review. J Clin Virol. 2014;61(2):181-188.

7. Ventre K, Randolph AG. Ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children. Cochrane Database Syst Rev. 2007(1):CD000181.

8. Mazur NI, Martinon-Torres F, Baraldi E, et al. Lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics. Lancet Respir Med. 2015;3(11):888-900.

9. Brown PM, Schneeberger DL, Piedimonte G. Biomarkers of respiratory syncytial virus (RSV) infection: specific neutrophil and cytokine levels provide increased accuracy in predicting disease severity. Paediatr Respir Rev. 2015;16(4):232-240.

10. Zomer-Kooijker K, Uiterwaal CS, van der Gugten AC, Wilbrink B, Bont LJ, van der Ent CK. Decreased lung function precedes severe respiratory syncytial virus infection and post-respiratory syncytial virus wheeze in term infants. Eur Respir J. 2014;44(3):666-674.

11. Hall CB, Weinberg GA, Iwane MK, et al. The burden of respiratory syncytial virus infection in young children. N Engl J Med. 2009;360(6):588-598.

12. Houben ML, Bont L, Wilbrink B, et al. Clinical prediction rule for RSV bronchiolitis in healthy newborns: prognostic birth cohort study. Pediatrics. 2011;127(1):35-41.

13. Modjarrad K, Giersing B, Kaslow DC, Smith PG, Moorthy VS, Group WRVCE. WHO consultation on Respiratory Syncytial Virus Vaccine Development Report from a World Health Organization Meeting held on 23-24 March 2015. Vaccine. 2016;34(2):190-197.

36

11. APPENDIX A: Governance structure of RESCEU

RESCEU will adopt a governance model that will promote the active participation of national

public health agencies, academia and pharmaceutical companies (EFPIA) in order to achieve

maximum collaboration and data sharing. The management structure of RESCEU has been

developed to respond to the needs of an international large-scale multi-stakeholder project. It

is based on a traditional management structure adapted to the particular attributes of RESCEU.

Managing an organization like RESCEU can be challenging due to the size of the project, its

ambition, the variety of activities and their interdependencies. The project aims to harmonize

the interests of the public and EFPIA partners. Therefore, a strong internal trust and

communication interface is crucial to setting the project up for success.

The project is composed of complementary, as well as parallel activities, with strong inter-

dependencies between critical work packages outputs. This will require the need for a detailed

time schedule for many of the tasks, which will need close monitoring and communication

between team members to avoid bottlenecks and to allow effective progress of deliverables.

The management structure needs to be a balance between a simplistic standard scheme

(which will not be able to address the needs of a project of this level of complexity) and an

excessive super-structure (that would impose a cumbersome bureaucracy to the project and

thus impede its scientific and technical progress).

Taking into account these project characteristics, the management structure proposed for

RESCEU is based on a multi-level organisation that balances:

• The fulfilment of the work plan per se.

• The management of trade-offs affecting scope, quality, time and cost.

• The primary focus needed on critical activities that aim to ensure the achievement of

milestones and that contribute to strategic objectives.

• The relationships and trust amongst partners, including conflict resolution.

• The quality and efficiency with which the project activities are carried out.

• The appropriate implementation of the Consortium Agreement, with careful attention to

the governance procedures, intellectual property policy and the related use of results.

• The implementation of the Grant Agreement, including administrative and financial

elements.

• The creation and management of a wider scientific forum encompassing interested

organisations beyond the project partnership (Associate Partners).

Taking into account the above, a management structure has been designed with the following

components included:

• RESCEU Forum (RF): forum for discussion, dissemination and scientific community-

building within the project. The RF will consist of the project partners (Beneficiaries)

and the Associate Partners and may be convened by electronic means or face-to-face

with the purpose of stimulating discussion and promoting dialogue on scientific issues.

The RF will not have decision-making powers.

37

• General Assembly (GA): body composed of all Beneficiaries participating in the

project, with the ultimate decision-making responsibility in matters affecting the overall

project strategy and composition of the consortium. The GA will meet annually and will

adopt decisions by majority – each partner having a vote-, except in cases were

unanimity is required according to IMI rules.

• Steering Committee (SC): leadership team with 50/50 vote allocation between

EFPIA/non-EFPIA members, composed of WP (Co-) Leads (from academia and

EFPIA) or their designated representatives. The SC is responsible for decision making

on most issues related to project execution, technical development decisions, work

plan updates, and effort/budget re-assignment in order to pursue optimal efficiency.

Meetings will take place regularly, typically every two months. The attendance of one

representative from each WP will be required for quorum. Decisions will be determined

by majority vote of attendees.

• Operations Team (OT): executive group composed of the Coordinator, the Project

Leader and the Project Manager (but not restricted to those), responsible for the day-

to-day operational and technical aspects of the project. The OT will meet frequently

(i.e. bi-weekly by teleconference) to monitor the project progress and to address any

issues that may arise.

• Project Management Office (PMO): team dealing with the day-to-day management of

the project. Regular meetings, mostly by teleconference, will be established to

appropriately follow up on management matters.

• Task Forces: Result-oriented ad-hoc teams will be created as needed, with a clear

and exclusive mission of studying/resolving any issues between WPs.

• Advisory Boards: consultative bodies for ethical, scientific and technical matters.

RESCEU intends to establish three advisory boards formed by external experts: the

International Scientific Advisory Group (ISAG), the RESCEU Ethics Advisory

Committee (EAC) and the RESCEU Patient Advisory Board (PAB).

38

Figure 3. Governance structures in RESCEU

39

12. APPENDIX B: Glossary of Terms

According-To-Protocol (ATP) cohort: This cohort will include all cases enrolled in the study

who meet the criteria defined in the protocol for the considered analysis.

Acute respiratory tract infection (ARTI): symptoms of an upper and/or lower respiratory tract

infection, such as runny or blocked nose, coughing, fast breathing, chest indrawing, shortness

of breath, low oxygen saturation.

Child in care: A child who has been placed under the control or protection of an agency,

organization, institution or entity by the courts, the government or a government body, acting

in accordance with powers conferred on them by law or regulation. The definition of a child in

care can include a child cared for by foster parents or living in a care home or institution,

provided that the arrangement falls within the definition above. The definition of a child in care

does not include a child who is adopted or has an appointed legal guardian.

Cohort study: A form of epidemiological study where subjects in a study population are

classified according to their exposure status/disease and followed over time (prospective/

retrospective) to ascertain the outcome(s).

Epidemiological study: An observational or interventional study without administration of

medicinal product(s) as described in a research protocol.

Evaluable: Meeting all eligibility criteria, complying with the procedures defined in the protocol,

and, therefore, included in the According-To-Protocol (ATP) analysis (see Section 9.3 for

details on criteria for evaluability).

Health Burden: Burden of the disease imposed on the study population in terms of incidence

of the disease and associated healthcare utilization in any healthcare setting.

Healthcare settings (Healthcare Utilization):

Primary, secondary and tertiary care settings such as selfcare with over-the-counter [OTC]

drugs, general practitioner (GP) visits, emergency room (ER) visits, hospital visits, etc

Interventional Human Subject Research:

Studies in which participants are administered medical care, medicinal products and/or

medical/scientific procedures as described in a research protocol.

Lost-to-Follow-up is defined as no contact by the subject’s parent(s)/LAR(s) over the period

of 3 planned contacts and/or 1 month and after a final attempt has been made by mail. Once

this has been reached, the subject is censored at the time of last contact.

Severe and very severe lower respiratory tract infection:

WHO candidate case definition (Modjarrat et al.13):

40

Sponsor: The sponsor is the party that commissions the organisation or performance of the

research, for example a pharmaceutical company, academic hospital, scientific organisation

or investigator. A party that provides funding for a study but does not commission it is not

regarded as the sponsor, but referred to as a subsidising party.

Prospective study: A study in which the subjects/cases are identified and then followed

forward in time in order to address one or more study objectives.

Research protocol: A document that describes the objective(s), design, methodology,

statistical considerations, and organization of a study. The protocol usually also gives the

background and rationale for the study, but these could be provided in other protocol

referenced documents.

Seroprevalence: The total number of cases within the study population at a specific time that

test positive for the disease based on blood serum specimens.

Study population: Sample of population of interest.

Sub-cohort: A subgroup of the total cohort of study subjects for whom the planned study

procedures are different from those planned for the other study subjects.

Subject: Term used throughout the protocol to denote an individual who has been contacted

in order to participate or participates in the epidemiological study or a person about whom

some medical information has been recorded in a database.

Subject number: A unique number identifying a subject, assigned to each subject consenting

to participate in the study.

Surveillance: The ongoing systematic collection, collation, analysis, and interpretation of

descriptive epidemiological health data on a specific disease. Surveillance can monitor

incidence and/or prevalence, and/or inform about when and where health problems are

occurring and who is affected.

41

ANNEX II. Clinical study 2: Case-control study protocol

Near final version

Date and version No: 21th December 2016 version 1.4

42

• Study Title: REspiratory Syncytial virus Consortium in EUrope (RESCEU) Work Package

5- Presumed risk factors and biomarkers for RSV-related severe disease and related

sequelae

Internal Reference Number / Short title: Understanding RSV: Severe disease and the long

term consequences

Ethics Ref: TBC

Date and Version No: 30th March 2017 version 1.7

Chief Investigator: Professor Andrew J Pollard Oxford Vaccine Group (OVG) Department of Paediatrics University of Oxford Centre for Clinical Vaccinology & Tropical Medicine (CCVTM) Churchill Hospital Oxford OX3 7LE United Kingdom Tel/Fax: 00 44(0)1865 857420 [email protected]

Investigators: Simon Drysdale, University of Oxford Matthew Snape, University of Oxford Christoph Blohmke, University of Oxford Simon Nadel, Imperial College London Peter Openshaw, Imperial College London Louis Bont, University Medical Centre, Utrecht Joanne Wildenbeest, University Medical Centre, Utrecht Debby Bogaerts, University Medical Centre, Utrecht Federico Martinon-Torres, Servicio Galego de Saúde (SERGAS) Irene Rivero Calle, Servicio Galego de Saúde Pablo Obando Pacheco, Servicio Galego de Saúde Antonio Justicia Grance, Servicio Galego de Saúde Carmen Rodriguez Tenreiro, Servicio Galego de Saúde Jeroen Aerssens, Janssen Pharmaceutica nv

Insert names of key collaborators, including institutional affiliations

Sponsor: University of Oxford

Funder: The Innovative Medicines Initiative (IMI)

Chief Investigator

Signature:

The approved protocol should be signed by author(s) and/or

person(s) authorised to sign the protocol

Andrew Pollard has previously conducted vaccine clinical trials on behalf of Oxford University

funded by vaccine manufacturers but he no longer does so and did not receive any personal

reimbursement from them. He is chair of the Department of Health’s (DH) Joint Committee on

Vaccination and Immunisation (JCVI) but the reviews expressed herein do not necessarily

represent those of DH or JCVI.


43

Others conflicts of interest….

Confidentiality Statement

This document contains confidential information that must not be disclosed to anyone other than

the Sponsor, the Investigator Team, HRA, host organisation, and members of the Research Ethics

Committee, unless authorised to do so.


44

TABLE OF CONTENTS

To update table of contents (TOC), hover cursor over the table and ‘right click’. Choose ‘update field’,

then ‘update entire table’.

1. SYNOPSIS .................................................................................................................................... …. 46

2. ABBREVIATIONS ............................................................................................................................. 47

3. BACKGROUND AND RATIONALE ............................................................................................... 48

3.1 Background information………………………………………………………………………………………………………48

3.2 Main research questions……………………………………………………………………………………………………..48

3.3 Potential risks and benefits……………………………………………………………………………..............................48

3.4 Study Participants and generalisability………………………………………………………………………………..48

4. OBJECTIVES AND OUTCOME MEASURES .............................................................................. 49

5. STUDY DESIGN ............................................................................................................................... 50

6. PARTICIPANT IDENTIFICATION .................................................................................................. 51

6.1 Study Participants……………………………………………………………………………………………………………….51

6.2 Inclusion Criteria…………………………………………………………………………………………………………………51

6.3 Exclusion Criteria………………………………………………………………………………………………………………..51

7. STUDY PROCEDURES .................................................................................................................. 52

7.1 Recruitment…………………………………………………………………………………………………………………………52

7.2 Screening and Eligibility Assessment………………………………………………………………………………….52

7.3 Informed Consent……………………………………………………………………………………………………………….53

7.4 Baseline Assessments…………………………………………………………………………………...............................53

7.5Subsequent Visits……………………………………………………………………………………………………………….54

7.6 Sample Handling……………………………………………………………………………………………………………….54

7.7 Discontinuation/Withdrawal of Participants from Study…………………………………………..................55

7.8 Definition of End of Study…………………………………………………………………………………………………..55

8. SAFETY REPORTING……………………………………………………………………………………56

8.1 Definition of Serious Adverse Events………………………………………………………………………………...56

8.2 Reporting Procedures for Serious Adverse Events……………………………………………………………56

9. STATISTICS AND ANALYSIS………………………………………………………………………….56

9.1 Description of Statistical Methods……………………………………………………………………………………..56

9.2 The Number of Participants……………………………………………………………………………………………...57

10. DATA MANAGEMENT………………………………………………………………………………..57

10.1 Access to Data……………………………………………………………………………………………………………….57

10.2 Data Recording and Record Keeping……………………………………………………………………………..57

11. QUALITY ASSURANCE PROCEDURES…………………………………………………..58

12. ETHICAL AND REGULATORY CONSIDERATIONS……………………………………..58

121 Declaration of Helsinki…………………………………………………………………………………………………….58


45

12.2 Guidelines for Good Clinical Practice………………………………………………………………………………58

12.3 Approvals………………………………………………………………………………………...............................................58

12.4 Reporting…………………………………………………………………………………………………………………………58

12.5 Participant Confidentiality…………………………………………………………………………………………………58

12.6 Expenses and Benefits…………………………………………………………………………………………………….59

12.7 Other Ethical Considerations…………………………………………………………………………………………….59

13. FINANCE AND INSURANCE ......................................................................................... 59

13.1 Funding……………………………………………………………………………………………………………………………..59

13.2 Insurance……………………………………………………………………………………….................................................59

14. PUBLICATION POLICY................................................................................................. .59

15. REFERENCES ............................................................................................................... 60

17. APPENDIX B: SCHEDULE OF STUDY PROCEDURES ............................................... 62

18. APPENDIX B: SCHEDULE OF STUDY PROCEDURES ............................................... 63

19. APPENDIX C: AMENDMENT HISTORY ....................................................................... 64


46

1. SYNOPSIS

Study Title REspiratory Syncytial virus Consortium in EUrope (RESCEU): Work Package 5- Presumed risk factors and biomarkers for RSV-related severe disease and related sequelae

Internal ref. no. / short title

Understanding RSV: Severe disease and the long-term consequences

Study Design Case-control study

Study Participants Infants with RSV infection and healthy controls

Planned Sample Size 630

Planned Study Period 1st September 2017 – 31st December 2021

Objectives Outcome Measures

Primary

• To establish biomarkers predictive of, or associated with, lower respiratory tract RSV infection and disease severity in infants

• Discover biomarkers that are associated with RSV lower respiratory tract infection or severe RSV disease in infants.

Secondary

• To establish biomarkers associated with sequelae following RSV infection in infants

• To characterise the viral load and genetic sequence of RSV associated with mild and severe disease

• To characterise the immune response to mild and severe RSV disease

• To describe the transcriptomic, proteomic, metabolomic and epigenetic signatures associated with RSV disease severity and/or respiratory sequelae

•

• To biobank specimens for later analysis of respiratory/stool microbiome, genetic associations with severe RSV disease and/or respiratory sequelae

• Discover biomarkers that are associated with respiratory sequelae after RSV infection

• RSV viral load (copies/ml) and genetic sequence in infants with mild and severe disease.

• Immune responses (flow cytometric cell phenotyping, intracellular cytokine staining, systems serology, neutralising antibody) of infants with mild and severe RSV disease.

• Transcriptomic, proteomic, metabolomic and epigenetic signatures of infants with RSV disease severity and/or respiratory sequelae

• Biobanking of specimens (respiratory, blood, stool and urine) collected in WP4 and WP5.


47

2. ABBREVIATIONS

ALRI Acute lower respiratory tract infection

BAL Bronchoalveolar lavage

CI Chief Investigator

CRF Case Report Form

CTRG Clinical Trials & Research Governance, University of Oxford


GP General Practitioner

HDU High dependency unit

HRA Health Research Authority

HRQoL Health related quality of life

ICF Informed Consent Form

LRTI Lower respiratory tract infection

NHS National Health Service

NRES National Research Ethics Service

PI Principal Investigator

PICU Paediatric intensive care unit

PIL Participant/ Patient Information Leaflet

R&D NHS Trust R&D Department

REC Research Ethics Committee

RSV Respiratory syncytial virus

SOP Standard Operating Procedure

URTI Upper respiratory tract infection

WP Work package


48

3. BACKGROUND AND RATIONALE

3.1 Background information

Human respiratory syncytial virus (RSV) causes severe disease in the very young, elderly and in

high risk groups. Worldwide in 2005 there were an estimated 34 million cases of acute lower

respiratory tract infection (ALRI), 3.4 million ALRI hospitalisations and 55,000 to 199,000 deaths

associated with RSV in children <5 years old [1]. RSV infection in childhood is associated with

subsequent wheezing and asthma [2, 3]. These long-term sequelae pose a substantial additional

burden on healthcare systems. There is a parallel need to assemble clinical resources to identify

the correlates of severe RSV disease for clinical management, classification of disease severity

in clinical trials and identification of biomarkers for severe disease, which are currently lacking [4].

We have prioritised biomarker investigation based on key knowledge gaps that will facilitate

improved understanding of the biology of RSV infection and its sequelae as well as better control

and treatment of RSV infections. There is an urgent need to better define correlates of protection

and we therefore prioritise analysis of potential serological biomarkers of protection in infants

(using functional and total antibody assays). We will use virological sequencing and host

transcriptomics [5] to investigate phenotypic differences in each of our populations that may

account for severity, susceptibility and sequelae in view of the considerable power of these

approaches and the expertise we have developed in analysis [6]. Findings from these analyses

may identify mechanistic pathways involved in protection or the development of sequelae and

provide targets for therapeutic intervention and/or monitoring in interventional treatment trials. We

will also examine other key “omics” approaches to biomarker discovery including proteomics,

microbiome studies, metabolomics, and epigenetic studies [7].

3.2 Main research questions

We will address the following questions:

• Are there serological or mucosal biomarkers (type, level or function of antibody; soluble

immune mediators) that predict, correlate with protection from, or susceptibility to, severe

RSV disease?

• Are there virological factors (viral load, sequence, coinfection) associated with severe RSV

disease?

• Can a transcriptomic, proteomic, metabolomic or epigenetic signature be found that is

associated with susceptibility to RSV disease, severe disease or wheezing sequelae?

• Are there RSV-specific T cell biomarkers (phenotype or function) or markers of innate

immunity that correlate with susceptibility, recovery, or protection from infection?

• Are alterations in the respiratory mucosa, or stool present in RSV infection?

• Are alterations in microbiome associated with susceptibility to, or severity of RSV disease

or sequelae?

• By analysing stored host DNA, can we find (epi)genetic associations with susceptibility to,

or severity of RSV disease or wheezing sequelae?

3.3 Potential risks and benefits

There are few risks of participating in the study. Blood and respiratory sampling can be associated with minor local effects, for example, discomfort, bruising or nose bleeds. There are no risks associated with collection of urine or stool samples. Benefits of participating in the study: There are no particular benefits to participating in this study, apart from knowing knowledge obtained from it may benefit other patients in the future.

3.4 Study Participants and generalisability


49

226 infants without comorbidities less than 12 months old with severe proven RSV disease

requiring hospitalisation (“cases”) and 226 age- and sex-matched community controls without

comorbidities with RSV infection (“controls”) but not requiring hospitalisation will be selected along

with 50 infants (25 cases and 25 controls) with RSV infection less than 12 months old with any

comorbidities (i.e. any child who is not “healthy” and term born) (“exploratory group”) and 80

healthy controls with no acute RSV infection (“healthy controls”). Blood, stool, urine and

nasopharyngeal samples will be obtained from all infants at presentation, and in convalescence

6-8 weeks later. In ventilated infants, bronchoalveolar lavage (BAL) and/or tracheal aspirate

samples will be obtained at the participating sites where study procedures and ethical approvals

are already in place. We plan to recruit 10% over these numbers to account for anticipated drop

out. The results from this study should be generalizable to almost all other infants with RSV

infection.

4. OBJECTIVES AND OUTCOME MEASURES

Objectives Outcome Measures Timepoint(s) of

evaluation of this

outcome measure (if

applicable)

Primary Objective To establish biomarkers predictive of, or associated with, lower respiratory tract RSV infection and disease severity in infants

Discovery of biomarkers that are associated with lower respiratory tract RSV infection and disease severity in infants

Blood, respiratory and urine sampling at day 0 and day 49 (+/- 7 days) and stool sampling at day 0.

Secondary Objectives 1. To characterise the viral load

and sequence of RSV

associated with mild and

severe disease

2. To characterise the immune

response to mild and severe

RSV disease

3. To describe the

transcriptomic, proteomic,

metabolomic and epigenetic

signatures associated with

RSV disease severity and/or

respiratory sequelae

4. To biobank specimens for

later analysis of

respiratory/stool microbiome,

1. RSV viral load and

sequence associated with

mild and severe disease

2. Immune responses to mild

and severe RSV disease

3. Transcriptomic, proteomic,

metabolomic and

epigenetic signatures

associated with RSV

disease severity and/or


4. Biobanking of specimens

1. Respiratory sampling

at day 0 and day 49

(+/- 7 days) (and daily

while in hospital for

“cases”)

2. Blood sampling at

day 0 and day 49 (+/-

7 days)

3. Blood, urine and

respiratory sampling

at day 0 and at day

49 (+/- 7 days) and

stool sampling at day

0. Follow up

(telephone, internet,

questionnaire,

medical records

review) at 1,2, 3

years of age.


50

genetic associations with

severe RSV disease and/or


4. Blood, urine and

respiratory sampling

at day 0 and at day

49 (+/- 7 days) and

stool sampling at day

0

5. STUDY DESIGN

The study design is a case-control study. Cases, infants <12 months old hospitalised with RSV

infection, will be recruited from paediatrics emergency departments, clinical decision

units/observation bays and paediatric wards (including high dependency units [HDU] and

paediatric intensive care units [PICU]). Controls, infants <12 months old with RSV infection not

requiring hospitalisation (discharged from hospital within 12 hours from registration in emergency

department), will be recruited from paediatrics emergency departments and General Practitioner

(GP) surgeries. An exploratory cohort of 50 infants with comorbidities (25 case and 25 controls)

and 80 healthy controls will also be recruited. Infants with respiratory symptoms will be identified

and pre-consent will be taken to take a respiratory sample and test for RSV with a bedside RSV

test and/or by PCR. If either test is positive for RSV they will be eligible to be enrolled in the study.

If the patient is already known to be RSV positive a bedside test will not be required. Participants

will be seen at enrolment and in convalescence 7 weeks (+/- 1 week) later (after hospital discharge

in “cases”). At these time points clinical and health economic and quality of life data and blood,

respiratory, urine and stool samples will be collected. In ventilated infants, bronchoalveolar lavage

(BAL) or tracheal/bronchial aspirate samples will be obtained at enrolment at the participating sites

where study procedures and ethical approvals are feasible. There will be further contact via

telephone/telephone app/email when the participant is one, two and three years of age. In total,

participants will be followed up for up to three years after enrolment. At these time points the

participant’s parent/guardian/legally authorised representative will be asked to complete a

questionnaire to gather data on healthcare resource use and associated costs and respiratory

health and health related quality of life (HRQoL).

At enrolment, sampling will be done as follows (for all groups):

• Nasopharyngeal swab or nasopharyngeal aspirate [NPA] for bedside RSV test (one

sample)

• Nasopharyngeal bacterial swab (one swab aliquoted into several samples)

• Respiratory viral sample (flocked swab or synthetic absorption matrix [SAM] swab) (one

sample)

• Respiratory viral sample (flocked swab or synthetic absorption matrix [SAM] swab) (one

sample) daily until discharge for those infants admitted to hospital

• If the child is intubated and ventilated and has a bronchoalveolar lavage or tracheal

aspirate taken as part of routine clinical care a sample will be collected (one sample)

• Venous or capillary blood sample (maximum of 0.8ml/kg)

• Bag or clean catch urine sample (3-5ml)

• Stool sample (from a “dirty” nappy) (2ml)

Ideally, at least one hour should elapse between initial nasal sampling (for bedside RSV test or

diagnostic RSV PCR) and any further respiratory samples being taken.

See Appendix A for study flow chart.


51

6. PARTICIPANT IDENTIFICATION

6.1 Study Participants

Participants include infants less than 12 months old with a proven RSV respiratory tract infection.

However, recruitment is stratified to include 500 otherwise well infants, born at more than 36+6

weeks of gestation at birth, 50 infants (25 cases and 25 controls) with comorbidities as an

exploratory study. In addition, 80 healthy control infants without RSV infection will also be

recruited.

Cases are defined as infants with an RSV infection and requiring hospitalisation for at least 12

hours.

Controls are defined as infants with an RSV infection not requiring hospitalisation (discharged

home within 12 hours of registration in emergency department)

The exploratory group are defined as infants with an RSV infection with any co-morbidity that

would exclude them from being a case or control.

Healthy controls are defined as infants without comorbidities and without acute RSV infection.

Participants may initially be a recruited as a “control” and later become a “case” either within the

same respiratory illness or with a separate respiratory illness. In this scenario repeat samples

(blood/urine/stool/respiratory) will be taken as per the “case” arm of the flow diagram if at least 24

hours have elapsed since previous sampling and blood sampling will not exceed the maximum

allowable blood volume. In addition, healthy controls may later become “RSV” cases or controls.

6.2 Inclusion Criteria

The participant may enter the study if ALL of the following apply:

• Parent/carer of infant is willing and able to give informed consent for participation in the

study.

• Male or female, and less than 12 months of age at enrolment.

• Hospitalised for <48 hours at enrolment.

• Live near enough to a participating study centre for the 6-8 week home visit/hospital

appointment to be feasible.

• Parent has a telephone.

6.3 Exclusion Criteria

The participant may not enter the study if ANY of the following apply:

• History of receipt of medication to treat RSV infection (e.g. ribavirin).

• Prior exposure to an RSV investigational vaccine or medication.

Infants should not be enrolled as cases, controls or healthy controls if they have any of the

following criteria. Infants recruited to the “exploratory” group may have these criteria:


52

• History of concurrent clinically significant medical illness (not directly attributable to RSV

infection) including but not limited to, cardiovascular, respiratory, renal, gastrointestinal,

haematology, neurology, endocrinology, immunology, musculoskeletal, oncological or

congenital disorders, as judged by the investigator. Specifically excluded examples

include, but are not limited to:

o Immunosuppressed states

o Bronchopulmonary dysplasia/chronic lung disease of infancy

o Congenital heart disease

o Down’s syndrome

• Prematurity, as defined as gestational age <37 weeks at birth.

• History of receipt of immunoglobulin or monoclonal antibodies (including palivizumab).

• Use of steroids or montelukast within 7 days of enrolment in the study

7. STUDY PROCEDURES

See Appendix B for schedule of procedures.

7.1 Recruitment

Potential participants for the study will be identified by any of the following routes:

• Clinicians reviewing medical handover lists and clinical records of new admissions and

emergency department attendances

• Site study team contacting relevant wards in the hospital where potential participants could

be attending/admitted (e.g. GP surgeries, outpatient clinics, emergency department,

general paediatric wards, paediatric high dependency unit or paediatric intensive care unit)

to enquire about any new attendances/admissions.

• Microbiologists and/or virologists identifying children with a positive test for RSV

• Participants in this study will be given information about a separate, ethically approved,

study called Oxford Vaccine Centre Biobank (LREC 10/H0504/25) and asked if they would

like to consent to this study on a separate consent form. If they give consent for Biobank,

samples taken will be stored for future infection and immunity studies.

7.2 Screening and Eligibility Assessment

If a potential participant has been identified by any of the above methods, the relevant clinical

team will first be informed. A member of the clinical team would then approach the

parent/guardian/legally authorised representative to seek their interest in knowing more about the

study. Verbal consent will be sought from the parent/ guardian/legally authorised representative

for a member of the clinical team to pass their details on to the study team. Where such consent

is obtained, this will be documented in the child’s medical notes. Only then would the study team

contact the family and subsequently give them the participant information sheet (PIS). A member

of the study team will check the patient’s eligibility by asking the parent/ guardian/legally authorised

representative questions, in line with the inclusion and exclusion criteria (see section 6, participant

identification) before obtaining consent, if the parent/guardian/legally authorised representative

agrees for their child to participate. If the delegated party is unsure if the patient can participate in

the study they should first speak with the PI at site or contact the Department of Paediatrics,

University of Oxford coordinating centre to clarify eligibility.


53

7.3 Informed Consent

Pre-consent will be obtained from the parent/guardian/legally authorised representative of the

potential participant to obtain a respiratory sample for a bedside RSV test to assess eligibility for

entry into the study. If this is positive, or if the infant has already tested positive for RSV, informed

written consent will be requested for the full study. If the bedside test is negative the potential

participant will not be eligible for entry into the full study. Healthy controls will be recruited from

outpatient clinics or inpatient wards (children undergoing elective surgery) and informed written

consent will be requested for the full study from the parent/guardian/legally authorised

representative of the potential participant.

The parent/guardian/legally authorised representative must personally sign and date the latest

approved version of the Informed Consent form before any study specific procedures are

performed.

Written and verbal versions of the Participant Information and Informed Consent will be presented

to the parent/guardian/legally authorised representative detailing no less than: the exact nature of

the study; what it will involve for the participant; the implications and constraints of the protocol;

the known side effects and any risks involved in taking part. It will be clearly stated that the

parent/guardian/legally authorised representative is free to withdraw the infant from the study at

any time for any reason without prejudice to future care, without affecting their legal rights, and

with no obligation to give the reason for withdrawal.

The parent/guardian/legally authorised representative will be allowed as much time as wished to

consider the information, and the opportunity to question the Investigator, their GP or other

independent parties to decide whether they will participate in the study. Written Informed Consent

will then be obtained by means of parent/guardian/legally authorised representative dated

signature and dated signature of the person who presented and obtained the Informed Consent.

The person who obtained the consent must be suitably qualified and experienced, and have been

authorised to do so by the Chief/Principal Investigator. A copy of the signed Informed Consent will

be given to the parent/guardian/legally authorised representative. The original signed form will be

retained at the study site.

7.4 Baseline Assessments

Information will be collected on participant’s medical history, clinical examination and any

laboratory and radiological investigations that are undertaken as part of routine medical care. A

standardised respiratory clinical severity score grading the severity of the RSV disease will be

calculated at enrolment for “cases” and “controls”.

The following samples will be obtained:

• Nasopharyngeal bacterial swab for microbiome analysis (one swab)

• Respiratory viral sample for PCR/qPCR analysis(swab) (one sample at enrolment and

daily samples on infants admitted to hospital with RSV infection)

• If the child in intubated and ventilated and has a bronchoalveolar lavage/tracheal aspirate

taken as part of routine clinical care, a sample will be taken for PCR/qPCR analysis (one

sample)

• Venous or capillary blood sample for transcriptomic/proteomic etc analysis (maximum of

~0.8ml/kg)


54

• Bag or clean catch urine sample for metabolomics analysis (3ml)

• Stool sample for microbiome analysis (from a nappy) (2ml)

7.5 Subsequent Visits

Six weeks (convalescent time point)

At 7 weeks (+/- 1 week) after discharge from hospital/emergency department (when well)

participants (excluding healthy controls) will have one follow up visit. This will either be a home

visit by the study staff or a clinic visit depending on parent/guardian/legally authorised

representative preference. At this time the following assessments will be made:

• Questionnaire of recent respiratory health

• Clinical examination

• Nasopharyngeal bacterial swab (one swab)

• Respiratory viral sample (swab) (one sample)

• Venous or capillary blood sample (maximum of ~0.8ml/kg)

• Bag or clean catch urine sample (3-5ml)

• Stool sample for microbiome analysis (from a nappy) (2ml)

One year of age

• Assessment of respiratory symptoms by parental questionnaire at age 1 year through

telephone contact, email & telephone app

• Cost, resource use and HRQoL data will be collected using questionnaires for caregivers

Two years of age

• Assessment of respiratory symptoms by parental questionnaire at age 2 years through



Three years of age

• Assessment of respiratory symptoms by parental questionnaire at age 3 years through



If a child is initially enrolled as a “control” but during the same illness or a subsequent illness

becomes a “case” (after 24 hours) they will start with the baseline assessment and sampling again.

We would ensure the maximum amount of blood taken remains within that allowable in a research

study.

7.6 Sample Handling

See sections 7.4 and 7.5 above for samples to be taken, volume and frequency of sampling.

Samples collected will be biobanked and stored for use in future ethically approved studies.


55

Sample handling and processing

Serum: Blood will be drawn into serum collection tubes containing a clot activator and placed on

ice. Tubes will be centrifuged at 3000 rpm, 4oC for 10min, serum extracted and stored at -80oC for

later analysis.

Whole blood: Whole blood will be collected in Lithium Heparin tubes, have FACS lysis buffer

added and then be spun down and stored at -80C for later analysis.

Functional Genomics: Whole blood will be added to Paxgen tubes containing a RNA stabilization

reagent and stored at -80oC. Subsequently, RNA will be extracted and gene transcription

determined using next generation sequencing approaches.

Respiratory samples: A bedside RSV test (nasopharyngeal swab) will be carried out prior to

enrolment. After enrolment nasopharyngeal swabs will be collected and aliquoted directly and

stored at -80oC for later analysis.

Urine: 3mL of urine will be collected and placed on ice. The sample will be centrifuged at 3000

rpm and 4C for 10 minutes, filtered (0.2 micron filter) and stored at -80oC for later analysis.

Stool: 2ml of stool will be collected and stored at -80oC for later analysis.

Prioritisation may be required for biomarkers in the blood (but not other samples) as a result of limited sample volume. We will prioritise serological and transcriptomic analysis but will store whole blood whenever sample volume permits. Appendix B shows the sample collection chart.

7.7 Discontinuation/Withdrawal of Participants from Study

Each participant has the right to withdraw from the study at any time. In addition, the Investigator

may discontinue a participant from the study at any time if the Investigator considers it necessary

for any reason including:

• Ineligibility (either arising during the study or retrospectively having been overlooked at

screening)

• Significant protocol deviation

• Withdrawal of Consent

• Loss to follow up

If the parent/guardian/legally authorised representative withdraws consent, we will analyse any

previously collected sample and include their data in further analyses unless they state otherwise.

Withdrawn participants will not be replaced but we have factored a 10% drop out rate in our sample

size.

The reason for withdrawal will be recorded in the CRF.

7.8 Definition of End of Study

The end of study is the date of the last telephone/email follow up of the last participant.


56

8. SAFETY REPORTING

8.1 Definition of Serious Adverse Events

A serious adverse event is any untoward medical occurrence that:

• results in death

• is life-threatening

• requires inpatient hospitalisation or prolongation of existing hospitalisation

• results in persistent or significant disability/incapacity

• consists of a congenital anomaly or birth defect.

Other ‘important medical events’ may also be considered serious if they jeopardise the participant

or require an intervention to prevent one of the above consequences.

NOTE: The term "life-threatening" in the definition of "serious" refers to an event in which

the participant was at risk of death at the time of the event; it does not refer to an event

which hypothetically might have caused death if it were more severe.

8.2 Reporting Procedures for Serious Adverse Events

In this observational study, only serious adverse event (SAEs) related to sampling, or death

regardless of cause, occurring to a participant will be reported to the REC that gave a favourable

opinion of the study where in the opinion of the Chief Investigator the event was ‘related’ (resulted

from administration of any of the research procedures) and ‘unexpected’ in relation to those

procedures. Reports of related and unexpected SAEs will be submitted within 15 working days of

the Chief Investigator becoming aware of the event, using the HRA report of serious adverse event

form (see HRA website).

9. STATISTICS AND ANALYSIS

9.1 Description of Statistical Methods

We will generate large data sets for integrative computational analyses in order to identify profiles

associated with disease. In the first instance our biomarker data, including immunological,

transcriptional, proteomic and metabolomic data will be used to identify acute cases within each

data type and subsequently also all data types combined. Specificity of the results will be tested

against our validation datasets and other publicly available data by computational meta-analysis

using a variety of classification analyses approaches. With the availability of nasal microbiome

data, we will analyse the interaction between bacterial species colonizing the nasopharynx and

disease severity and outcome. For prediction of disease, transcriptomics, metabolomics and

proteomics data will be used for predictive modelling. The different data types (immunological,

virological, transcriptional, metabolomics and proteomics) will be scaled in order to facilitate

feature selection for subsequent training of well-established algorithms including support vector

machines, random forest or nearest shrunken centroid classifiers with extensive inner cross-

validation approaches (x-fold or LOOCV). For increased depth of the analysis, cases and controls

maybe stratified by nasal microbiome data in order to assess potential interaction of predictive

signatures and the nasal microbiota. Data will be used from all participants including those that

were lost to follow up or who withdrew consent.

http://www.nres.npsa.nhs.uk/docs/forms/Safety_Report_Form_(non-CTIMPs).doc


57

9.2 The Number of Participants

Using RSV PCR, 226 cases without comorbidities with severe disease requiring hospitalisation

and 226 age- and sex-matched without comorbidities community controls not requiring

hospitalisation will be selected to reveal 90% power (SD 0.4), with a true biomarker rate of 0.5%

and log odds ratio of 0.18. We plan to recruit 10% over these numbers to account for anticipated

drop out for a sample size of 500 infants without comorbidities. An additional 50 participants with

comorbidities (25 case and 25 controls) will be recruited as an exploratory study as well as 80

healthy controls giving a total sample size of 630 infants.

10. DATA MANAGEMENT

10.1 Access to Data

Direct access will be granted to authorised representatives from the Sponsor and host institution

for monitoring and/or audit of the study to ensure compliance with regulations. Partners in the

study will have access to de-identified data.

10.2 Data Recording and Record Keeping

Source documents are where data are first recorded, and from which participants’ CRF data are

obtained (except where CRF is the source). These include, but are not limited to, patient medical

notes (from which medical history, investigation results, previous and concurrent medication may

be summarised into OpenClinica), research notes, clinical charts, laboratory results, pharmacy

records and drug charts, radiology images, questionnaires, and any correspondences relating to

the participant’s involvement in the study. All documents will be stored safely in confidential

conditions. On all study-specific documents, other than the signed consent form and participant

identification log, the participant will be referred to by the trial participant number/code, not by

name. OpenClinica is the primary data collection instrument for the study and will be a password

protected, central web based database, based at Oxford. This database is stored on a secure

server within the UK with accountability records and will include validation processes to encourage

high quality data entry. All data requested in OpenClinica must be recorded. All missing data must

be explained. All entries made to the research notes should be printed legibly. If any entry error

has been made, to correct such an error, a single straight line should be drawn through the

incorrect entry and the correct data entered above it. All such changes must be initialled and dated.

DO NOT ERASE OR WHITE OUT ERRORS. For clarification of illegible or uncertain entries, the

clarification should be printed above the item, and this should also be initialled and dated.

Information entered into the research notes must be subsequently transferred onto OpenClinica.

The participants will be identified by a unique study specific number and/or code in any database.

The name and any other identifying detail will NOT be included in any study data file. The study

admission record should be completed within 2 weeks of the patient’s admission and once

discharged the all other required data should be entered onto OpenClinica within 4 weeks. If any

relevant information has not been recorded in the hospital notes or for situations where a

participant is transferred to a non-participating hospital, this will be obtained from either the

participant’s parent/guardian/legally authorised representative, GP or the clinician involved in the

participant’s ongoing care. The University of Oxford UK coordinating centre will retain a sponsor

file of all non-patient identifiable information relating to the study from all participating sites.

The investigator at each investigational site must make arrangements to store the essential study

documents, (as defined in Essential Documents for the Conduct of a Clinical Trial (International


58

Conference on Harmonisation (ICH) E6, Guideline for Good Clinical Practice) including the

Investigator Site File. All study documents will be retained after the completion or discontinuation

of the study for 3 years after the youngest participant turns 18 years. In addition, the investigator

is responsible for archiving of all relevant source documents so that the study data can be

compared against source data after completion of the study (e.g. in case of inspection from

authorities). The investigator is required to ensure the continued storage of the documents, even

if the investigator, for example, leaves the clinic/practice or retires before the end of required

storage period. Delegation must be documented in writing.

The University of Oxford UK coordinating centre undertakes to store any of the above documents

including returned questionnaires for the same period. The University of Oxford UK coordinating

centre will archive the documents in compliance with GCP utilising the Records Management

Service of the University of Oxford. All electronic CRFs and study data will be archived onto an

appropriate media for long term accessible storage. Hard copies of data will be boxed and

transferred to specially renovated, secure, premises where unique reference numbers are applied

to enable confidentiality, tracking and retrieval.

11. QUALITY ASSURANCE PROCEDURES

The study may be monitored, or audited in accordance with the current approved protocol, GCP,

relevant regulations and standard operating procedures.

12. ETHICAL AND REGULATORY CONSIDERATIONS

121 Declaration of Helsinki

The Investigator will ensure that this study is conducted in accordance with the principles of the

Declaration of Helsinki.

12.2 Guidelines for Good Clinical Practice

The Investigator will ensure that this study is conducted in accordance with relevant regulations

and with Good Clinical Practice.

12.3 Approvals

The protocol, informed consent form, participant information sheet and any proposed advertising

material will be submitted to an appropriate Research Ethics Committee (REC), and HRA for

written approval. The Investigator will submit and, where necessary, obtain approval from the

above parties for all substantial amendments to the original approved documents.

12.4 Reporting

The CI shall submit once a year throughout the study, or on request, an Annual Progress report

to the REC Committee, HRA (where required), host organisation and Sponsor. In addition, an End

of Study notification and final report will be submitted to the same parties.

12.5 Participant Confidentiality

The study staff will ensure that the participants’ anonymity is maintained. The participants will be

identified only by a participant ID number on all study documents and any electronic database,

with the exception of the CRF, where participant initials may be added. All documents will be

stored securely and only accessible by study staff and authorised personnel. The study will comply

with the Data Protection Act, which requires data to be anonymised as soon as it is practical to do

so.


59

12.6 Expenses and Benefits

Reasonable travel expenses for any visits additional to normal care will be reimbursed on

production of receipts, or a mileage allowance provided as appropriate.

12.7 Other Ethical Considerations

The testing of samples is intended solely for research and not diagnostic purposes and therefore

is not a substitute for a clinical appointment. Analysis of samples may not be done in a timely

fashion to be useful clinically. In the case of an incidental finding of a possible abnormality, the

results will be discussed with the clinical team at the site where the participant was recruited.

Where the participant’s ongoing care is in a local hospital not participating in the study the PI will

inform the appropriate clinical team. The clinical team will discuss implications with the

parent/guardian/legally authorised representative and further investigations will be arranged as

necessary.

13. FINANCE AND INSURANCE

13.1 Funding

The study is funded by the Innovative Medicines Initiative (IMI). The IMI is a joint undertaking

between the European Union and the European Federation of Pharmaceutical Industries and

Associations (EFPIA).

13.2 Insurance

The University has a specialist insurance policy in place which would operate in the event of any

participant suffering harm as a result of their involvement in the research (Newline Underwriting

Management Ltd, at Lloyd’s of London). NHS indemnity operates in respect of the clinical

treatment that is provided.

14. PUBLICATION POLICY

The Investigators will be involved in reviewing drafts of the manuscripts, abstracts, press releases

and any other publications arising from the study. Authors will acknowledge that the study was

funded by the IMI. Authorship will be determined in accordance with the ICMJE guidelines and

other contributors will be acknowledged.


60

15. REFERENCES

1. Harish Nair, D James Nokes, Bradford D Gessner, Mukesh Dherani, Shabir A Madhi, Rosalyn J Singleton, Katherine L O’Brien, Anna Roca, Peter F Wright, Nigel Bruce, Aruna Chandran, Evropi Theodoratou, Agustinus Sutanto, Endang R Sedyaningsih, Mwanajuma Ngama HC (2010) Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 375:1173–1181. doi: 10.1016/S0140-6736(09)62100-0

2. Blanken MO, Rovers MM, Molenaar JM, et al. (2013) Respiratory syncytial virus and recurrent wheeze in healthy preterm infants. N Engl J Med 368:1791–9. doi: 10.1056/NEJMoa1211917

3. Stein RT, Sherrill D, Morgan WJ, et al. (1999) Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 354:541–5. doi: 10.1016/S0140-6736(98)10321-5

4. Brown PM, Schneeberger DL, Piedimonte G (2015) Biomarkers of respiratory syncytial virus (RSV) infection: Specific neutrophil and cytokine levels provide increased accuracy in predicting disease severity. Paediatr Respir Rev 16:232–240. doi: 10.1016/j.prrv.2015.05.005

5. Mejias A, Dimo B, Suarez NM, et al. (2013) Whole Blood Gene Expression Profiles to Assess Pathogenesis and Disease Severity in Infants with Respiratory Syncytial Virus Infection. PLoS Med 10:e1001549. doi: 10.1371/journal.pmed.1001549

6. Blohmke CJ, Darton TC, Jones C, et al. (2016) Interferon-driven alterations of the host’s amino acid metabolism in the pathogenesis of typhoid fever. J Exp Med 213:1061–1077. doi: 10.1084/jem.20151025

7. Wouter A.A. de Steenhuijsen Piters, Santtu Heinonen, Raiza Hasrat, Eleonora Bunsow, Bennett Smith, Maria-Carmen Suarez-Arrabal, Damien Chaussabel, Daniel M. Cohen, Elisabeth A.M. Sanders, Octavio Ramilo DB and AM (2016) Nasopharyngeal microbiota, host transcriptome and disease severity in children with respiratory syncytial virus infection. Am J Respir Crit Care Med 1–61. doi: http://dx.doi.org/10.1094/MPMI-03-13-0062-R


61

16. APPENDIX A: STUDY FLOW CHART

Potential eligibility notified

Approach by study team

Confirm eligibility

Obtain pre-consent to undertake RSV bedside test

Confirm RSV test positive (if negitive infants not eligible)

Obtain full consent

Collect demographic and clinical data and assess as "case" or "control"

Obtain biological samples: Blood/respiratory/urine/stool/buccal swabs

Daily swab to test for RSV in hospitalised cases

At discharge (from ward/ED/clinic/GP):

Complete research notes and CRF

At 7 weeks (+/- 1 week) home/clinic visit:

Complete questionnaire and collect biological samples (blood/urine/respiratory/stool)

At one year of age:

Telephone/telephone app/email contact and parent to complete questionnaire

At two years of age:

Telephone/telephone app/email contact and parent to complete questionnaire

At three year of age:

-Telephone/telephone app/email contact and parent to complete questionnaire

-Complete research notes and CRF including termination page

Fo

llo

w u

p

Du

rin

g h

osp

ital

/ e

mer

gen

cy d

epar

tmen

t /G

P/c

lin

ic s

tay


62

17. APPENDIX B: SCHEDULE OF STUDY PROCEDURES

Day 0 Day 0 7 weeks 1 year 2 years 3 years

Procedures Screening Baseline

Demographics X

Medical history X

Pre-consent X

RSV bedside test X

Eligibility assessment X

Informed consent X

Physical examination X X

Assessment 1 (Blood test) X X

Assessment 2 (Respiratory sample- bacterial swab)

X X

Assessment 3 (respiratory sample- viral swab)

X* X

Assessment 4 (urine sample) X X

Assessment 5 (stool sample) X X

Questionnaire X X X X

Adverse event assessments X X

*Infants hospitalised with RSV will have a daily swab taken to test for RSV and a clinical score calculated

while they remain in hospital.


63

18. APPENDIX B: SCHEDULE OF STUDY PROCEDURES

1WB = whole blood; 2follow up after discharge; 3Gold Top Tube;4Antibody mediated immune response.

Sto

ol S

amp

le

Uri

ne

Met

abo

lom

ics

Res

pir

ato

ry s

amp

le (

mu

ltip

lex

PC

R &

RSV

seq

uen

cin

g)

Res

pir

ato

ry s

amp

le (

mic

rob

iom

e)

Cel

lula

r im

mu

no

logy

(fl

ow

cyto

met

ric

cell

ph

eno

typ

ing

&

intr

acel

lula

r cy

toki

ne

stai

nin

g)

Epig

enet

ics

DN

A

GW

AS

DN

A

Blo

od

tra

nsc

rip

tom

e

Seru

m P

rote

om

e

Seru

m M

etab

olo

mic

s

Mu

ltip

lex

RSV

F, G

a, G

b a

nd

N

sero

logy

Syst

ems

Sero

logy

4 Pre

-F/P

ost

-F n

eutr

alis

ing

anti

bo

die

s

TOTAL blood

volume all

samples

Sample Tube Stool pot

urine pot

swab swab 1WB Gold Top Clot

Gold Top Clot

PAX Gene

Seru

m3

Seru

m3

Seru

m3

Seru

m3

Seru

m3

Sample Vol (mL) 2-5mL

3mL 1

swab 1

swab 0.35/Ag (3 Ag)

0 0 0.2 0.1 0.5 0.05 0.1 0.1

Total vol/sample (mL)

NA NA NA NA 1.05 0 0 0.2 0.1 0.5 0.05 0.1 0.1 2.1mL / visit

N Infant Case-Control Study RSV Diagnosis 670 x x x x x x x x x x x x x 6-8w follow up2 590 x x x x x x x x x x x x x


64

19. APPENDIX C: AMENDMENT HISTORY

Amendment No.

Protocol Version No.

Date issued

Author(s) of changes Details of Changes made

List details of all protocol amendments here whenever a new version of the protocol is

produced. This is not necessary prior to initial REC submission.

65

ANNEX III. Clinical study 3: Older adults cohort study protocol

XXxxxxx.xxx.xx Burden of disease of RSV

66

REspiratory Syncytial virus Consortium in EUrope

(RESCEU) study: Defining the burden of disease of

Respiratory Syncytial Virus in older adults in Europe.

Protocol ID <include protocol ID given by sponsor or

investigator>

Short title Burden of disease of RSV in older adults

EudraCT number Not applicable

Version 1

Date January 23rd 2017

Coordinating





uitvoerder)


Sponsor (in Dutch:


[Comment: Add site-specific

information]

Subsidising party Innovative Medicines Initiative (IMI)

EU-H2020

Independent expert (s) <please include name and contact data>


67

Laboratory sites UMCU

[Comment: Add site-specific information]

Pharmacy Not applicable


68

PROTOCOL SIGNATURE SHEET [Comment: Add site-specific information]

Name Signature Date


Head of Department:

[Coordinating Investigator/Project

leader/Principal Investigator]:


69

TABLE OF CONTENTS

1. INTRODUCTION AND RATIONALE ............................................................................ 74

2. OBJECTIVES (see also Table 1) ................................................................................. 75

3. STUDY DESIGN .......................................................................................................... 77

4. STUDY POPULATION ................................................................................................. 78

4.1 Population (base) ................................................................................................... 78

4.2 Inclusion criteria for enrolment ................................................................................ 78

4.3 Exclusion criteria for enrolment .............................................................................. 78

4.4 Sample size calculation .......................................................................................... 78

5.1 Study parameters/endpoints (see also Table 1)...................................................... 79

5.1.1 Main study parameter/endpoint ........................................................................ 79

5.1.2 Secondary study parameters/endpoints ........................................................... 79

5.2 Randomisation, blinding and treatment allocation ................................................... 80

5.3 Study procedures ................................................................................................... 80

5.4 Withdrawal of individual participants ....................................................................... 82

5.5 Replacement of individual participants after withdrawal .......................................... 82

5.6 Premature termination of the study ......................................................................... 82

6. SAFETY REPORTING ................................................................................................. 82

6.1 Section 10 WMO event ........................................................................................... 82

6.1.1 Adverse events (AEs) and Serious adverse events (SAEs) ............................. 83

6.1.2 Suspected unexpected serious adverse reactions (SUSAR) ............................ 83

6.1.3 Annual safety report ......................................................................................... 83

6.2 Follow-up of adverse events ................................................................................... 83

6.3 Data Safety Monitoring Board (DSMB) ................................................................... 83

7. STATISTICAL ANALYSIS ............................................................................................ 83

8. ETHICAL CONSIDERATIONS ..................................................................................... 20

8.1 Regulation statement.............................................................................................. 20

8.2 Recruitment and consent ........................................................................................ 84

8.3 Benefits and risks assessment, group relatedness ................................................. 84

8.4 Compensation for injury .......................................................................................... 85

9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION............................ 21

9.1 Handling and storage of data and documents ........................................................ 21

9.2 Monitoring and Quality Assurance .......................................................................... 86

9.3 Amendments .......................................................................................................... 86

9.4 Annual progress report ........................................................................................... 86

9.5 Temporary halt and prematurely end of study report .............................................. 22

9.6 End of the study ..................................................................................................... 86

9.7 Public disclosure and publication policy .................................................................. 86

10. REFERENCES .......................................................................................................... 87

11. APPENDIX 1 .............................................................................................................. 88

12. APPENDIX 2 .............................................................................................................. 88

13. APPENDIX 3 .............................................................................................................. 88


70

LIST OF ABBREVIATIONS (FOR RELEVANT DEFINITIONS, SEE APPENDIX 2)

ABR ABR form, General Assessment and Registration form, is the application

form that is required for submission to the accredited Ethics Committee

(In Dutch, ABR = Algemene Beoordeling en Registratie)

AE Adverse Event

AR Adverse Reaction

ARTI Acute Respiratory Tract Infection

CA Competent Authority

CCMO Central Committee on Research Involving Human Subjects; in Dutch:

Centrale Commissie Mensgebonden Onderzoek

COPD Chronic Obstructive Pulmonary Disease

CV Curriculum Vitae

DSMB Data Safety Monitoring Board

EDC

system

Electronic Data Capture system

EU European Union


GP General Practitioner

HRQoL Health related Quality of life

IC Informed Consent

MA Medically Attended

METC Medical Research Ethics Committee (MREC); in Dutch: Medisch

Ethische Toetsing Commissie (METC)

NA Not applicable

POC test Point of Care test

QoL Quality of Life

RSV Respiratory Syncytial Virus

RT-PCR Reverse Transcriptase Polymerase Chain Reaction


SD Standard deviation

SUSAR Suspected Unexpected Serious Adverse Reaction

Wbp Personal Data Protection Act (in Dutch: Wet Bescherming

Persoonsgevens)

WMO Medical Research Involving Human Subjects Act (in Dutch: Wet

Medisch-wetenschappelijk Onderzoek met Mensen)


71

SUMMARY

Rationale: The REspiratory Syncytial virus Consortium in EUrope (RESCEU) is an

Innovative Medicine Initiative (IMI) funded by the EU under the H2020 framework to define

and understand the burden of disease caused by human respiratory syncytial virus (RSV)

infection. RSV causes severe disease in individuals at the extremes of the age spectrum

and in high risk groups. It was estimated that RSV was associated with 34 million cases of

acute respiratory tract infection (ARTI), 3.4 million ARTI hospitalizations and 55,000 to

199,000 deaths in children <5 years in 2005 worldwide. The estimated burden of disease

in older adults is comparable with non-pandemic influenza A (for which a vaccine is

available). These estimates were based on limited data and there is a substantial gap in

knowledge on morbidity and associated healthcare and social costs in Europe. New

vaccines and therapeutics against RSV are in development and could soon be available on

the European market. RESCEU will deliver knowledge of the incidence and burden of RSV

disease in young children and older adults in Europe, which is essential for stakeholders

(governments, etc.) to take decisions about prophylaxis and treatment.

Objective: To determine the burden of disease due to RSV in older adults.

Study design: Prospective epidemiological, observational, multi-country, multicenter

cohort study.

Study population: Adults aged 60 years and up (n= approximately 1,000).

Main study parameters/endpoints:

The primary endpoints of the study are;

• The incidence of RSV infection-associated ARTI.

• RSV associated medically attended (MA) ARTI.

• RSV related hospitalization.


group relatedness:

A blood sample (60 ml) and both a nasopharyngeal and an oropharyngeal swab will be

collected at the beginning (August/September) and end (May/June) of the RSV season. The

drawing of blood can be moderately painful. The collection of both a nasopharyngeal and

an oropharyngeal swab can cause a brief moment of discomfort. Participants will be asked

weekly by telephone (verbal or text message), email or telephone app about any signs of

respiratory tract infections. In the event of an ARTI two nasopharyngeal and one

oropharyngeal swab will be collected to perform a direct reverse transcriptase polymerase

chain reaction (RT-PCR) for RSV and additional analyses if RT-PCR is positive for RSV. If

a participant experiences a RSV positive ARTI, blood will be drawn (60 ml) at the time of

the infection and an additional respiratory sample will be collected 1-2 weeks after onset of

symptoms. During the course of each ARTI, independent of RSV status, participants will be

asked to complete a short daily diary in order to score respiratory symptoms and quality of

life. At inclusion and after approximately one year (+/- 2 months, at least after the RSV

season) participants are asked to fill in a short questionnaire.


72

None of the RESCEU study procedures is associated with any risk for serious

complications. However, there is a minimal risk of minor complications due to study

procedures (for example a nose bleed after a nasopharyngeal swab or bruise after a blood

test).

Possible benefit: There is no clear clinical benefit for the participants taking part in this

proposed study. However, the results of this study aim to support the understanding of the

burden of RSV disease, which is important for the implication of future preventive and

therapeutic interventions.


73


The RESCEU clinical cohort studies - summary

The IMI-funded REspiratory Syncytial virus Consortium in EUrope (RESCEU) programme

includes an observational study to define the burden of disease caused by human

respiratory syncytial virus (RSV) infection. A total of 4 clinical studies in specific risk groups

will be performed in several European countries as part of the RESCEU study. The sites of

these studies were selected because of their experience in acute respiratory tract infection

(ARTI) and/or RSV research in specific risk groups.

The clinical cohort studies in young children (clinical study 1 and 2, fig. 1) will be conducted

at the UMC Utrecht (Netherlands), University of Turku (Finland), Servicio Galego de Saúde

(Spain), University of Oxford, University of Edinburg and Imperial College London (United

Kingdom).

The clinical cohort study in older adults (elderly) (clinical study 3, fig.1) will be conducted at

the UMC Utrecht (Netherlands), University of Antwerp (Belgium) and University of Oxford

(United Kingdom).

The clinical cohort study in chronic obstructive pulmonary disease (COPD) patients (clinical

study 4, fig. 1) will be conducted at the UMC Groningen (Netherlands) and Imperial College

London (United Kingdom).

This protocol is restricted to Clinical Study 3.

Figure 1. Overview of clinical studies embedded in the RESCEU effort and follow up

periods for included (individual) patients.

0 1 2 3

Passive surveillance

n=9,000

Clinical Study 3

Elderly

n=1,000

Clinical Study 1

Birth cohort

Active surveillance,

n=1,000*

Clinical Study 4

COPD

n=500

RSV season

Sampling during respiratory episodes

Frequent questioning to assess acute

airway symptomsSerology

Lung function

Annual questionnaire

at birth

at birth

* Blood, respiratory, urine and stool sampling during acute and convalescent phase of RSV ARTI Follow-up

Clinical Study 2

Infant case-control

n=500*

Sampling during RSV infection


74

Background

Human respiratory syncytial virus (RSV) causes severe disease in individuals at the

extremes of the age spectrum and in high risk groups. It was estimated that RSV was

associated with 34 million cases of acute respiratory tract infection (ARTI), 3.4 million ARTI

hospitalizations and 55,000 to 199,000 deaths in children <5 years in 2005 worldwide.1

These estimates were based on limited data and there is a substantial gap in knowledge on

morbidity and associated healthcare and social costs in Europe. RSV infection in childhood

is associated with subsequent wheezing and asthma.2-4 These long‐term sequelae pose a

substantial additional burden on the healthcare system. In addition, RSV is a significant

cause of ARTI morbidity in elderly and COPD patients.5,6 Most published data on RSV

disease burden in the elderly (aged >65 years) are from the United States and from hospital

settings and describe a disease burden similar to non-pandemic influenza A.5

Treatment and prophylaxis options are limited. Mostly only supportive care is available for

patients with severe RSV ARTI. Ribavirin has been used as treatment, but with limited

evidence of benefit and is therefore not routinely recommended. Various new RSV vaccines

and therapeutics could be available in the near future.7 To use these new vaccines and

therapeutics in the best possible way and to guide their development and implementation,

it is necessary to determine the burden of RSV disease in Europe to gain better insight in

disease severity in young children and older adults and the associated societal and

healthcare costs.

There is a parallel need to assemble clinical resources to identify the correlates of severe

RSV disease for clinical management, classification of disease severity in clinical trials and

identification of biomarkers for severe disease, which are currently lacking.8

For this purpose RESCEU (REspiratory Syncytial virus Consortium in EUrope) has been

set up. RESCEU will perform the first prospective multi‐center study in both older adults

and children to provide accurate data on RSV disease incidence and sequelae (long‐term

airway morbidity, including asthma) and economic consequences of RSV infection.

The following document will describe only the protocol for the adult cohort study. The other

prospective cohort studies are presented in separate protocols for these specific cohorts.

We will prospectively follow-up a cohort of approximately 1,000 older adults (≥60 years, of

whom approximately 500 will be ≥75 years) living in the community during one year to obtain

incidence data on RSV infection, medically attended (MA) RSV infection and hospitalization

due to RSV.


75

2. OBJECTIVES

Table 1. Primary and secondary objectives and associated endpoints.

Objectives Endpoints

Primary • To estimate the incidence of

RSV infection-associated ARTI,

RSV MA-ARTI and RSV

hospitalization in older patients.

• Incidence rate of RT-PCR

confirmed RSV infection-

associated ARTI, MA-ARTI and

hospitalization in older patients.

Data collected by using samples,

medical data from the hospital

and questionnaires.

Secondary • To estimate the rate of all-

cause MA (inpatient or

outpatient) ARTI and related

medical complications

(exacerbations of chronic

conditions, acute cardiovascular

events).

• Incidence rate of all-cause

MA ARTI or events leading to

worsening of cardiorespiratory

status. Data collected by using

diary, questionnaires.

• To estimate the RSV-

associated and all-cause

mortality.

• Mortality through the RSV

season of follow up for RSV-

associated deaths and all cause

deaths. Data collected by using

medical data from the hospital

and questionnaires.

• To estimate health care

costs, health care resource use,

interruption of normal activities,

and HRQoL in RSV-associated

and all-cause MA (inpatient or

outpatient) ARTI patients.

• Health care costs and

resource use, interruption of

normal activities, and HRQoL in

RSV-associated and all-cause

MA (inpatient or outpatient) ARTI

patients. Data collected by using

diary, questionnaires.

• To estimate the incidence of

RSV-related secondary

bacterial pneumonia events and

their association with antibiotic

use within 21 days after onset

of RSV infection.

• Incidence rate of RSV-

associated secondary bacterial

pneumonia events (defined as

pneumonia within 21 days after

RSV infection) and associated

antibiotic use. Data collected by

using medical data from the

hospital, diary and

questionnaires.

• To collect clinical samples

for biomarker analysis.

• Sample collection for

biomarker analysis.


76

• To examine the incidence of

other respiratory pathogens

associated with all MA-ARTI

• Incidence rate of other

respiratory pathogens

associated with all MA-ARTI.

Data collected by using samples.

• To estimate the proportion of

viral ARTI attributable to RSV.

• Proportion of viral ARTI

attributable to RSV. Data

collected by using samples.

• To estimate important risk

factors for RSV infections (by

severity and healthcare

utilizations).

• Important risk factors of RSV

infection. Data collected by

using baseline questionnaires.

• To determine change in

frailty over the course of the

study

• Change in frailty over the

course of the study. Data

collected by using diary,

questionnaires.


77

3. STUDY DESIGN

This will be a multi-country, multicenter, prospective, observational cohort study conducted

across 2 consecutive winter seasons to determine the incidence of RSV infection, RSV

associated MA-ARTI and RSV related hospitalization in participants ≥60 years of age,

recruited from the general population.

These older adults will be recruited from general practitioner (GP) networks between May

and October of each year for 2 consecutive years based on pre-specified selection criteria

(see section 4.2 and 4.3). Practice lists will be screened for potentially eligible participants.

They will be informed by their GP about the study. If they consent that their contact details

can be given to the study team, interested potential participants will be contacted by a

member of the study team who will answer any questions they may have about the study

[Comment: The method of recruitment older adults can be adapted according to local

circumstances and regulations].

When participants are willing to be enrolled in the study, an inclusion visit is booked at the

beginning of the RSV season (August/September). During the same visit a blood sample,

a nasopharyngeal and an oropharyngeal sample will be collected and a baseline

questionnaire about demographic data, medical history, smoking habits and quality of life

will be completed.

During the RSV season (October 1st to May 1st, or longer if RSV is still circulating, based on

country specific surveillance reports), respiratory tract symptoms will be assessed weekly

by telephone contact (verbal or text message), email, (daily) telephone app or online

questionnaire. If the participant experiences an ARTI, the study team will visit the participant

to collect two nasopharyngeal and one oropharyngeal sample for direct RSV RT-PCR and

additional analyses. If RSV is positive a blood sample will be obtained at the time of infection

and an additional nasopharyngeal sample will be collected 1-2 weeks after the onset of

symptoms. At the end of the RSV season (May/June) a blood, nasopharyngeal and

oropharyngeal sample will be collected again. Individual participants will be followed up for

one RSV season. After approximately one year (+/-2 months) participants will be asked to

fill in a questionnaire to finalize follow-up. This questionnaire is about, but not limited to,

respiratory disease in the past year, changes in living conditions, health status, frailty and

quality of life.

An overview of the study design and main procedures is given below (Figure 2).

Figure 2. Overview of study design and main procedures of cohort of older adults.


78

4. STUDY POPULATION

4.1 Population (base)

Cohort of approximately 1,000 older adults (≥60 years, including approximately 500 ≥75

years). Participants will be randomly recruited from the database of general practitioners in

the following countries: the Netherlands (UMCU), Belgium (UA) and United Kingdom

(UOXF).

4.2 Inclusion criteria for enrolment

All participants must satisfy ALL the following criteria at study entry:

- Male and female adults ≥60 years of age (comorbidity, including chronic heart

disease is not an exclusion criterion)

- Willing and able to give written informed consent

- Willing and able to adhere to protocol-specified procedures

4.3 Exclusion criteria for enrolment

The following criteria should be checked at the time of study entry. If ANY exclusion criterion applies, the subject must not be included in the study:

- Current alcohol or drug abuse or history of unsuccessfully treated alcohol or drug

abuse within the past year

- Unable to perform the study procedures

- Dementia

- Life expectancy less than 1 year

- Any known or suspected immunosuppressive condition, acquired or congenital, as

determined by history and/or physical examination (a more detailed description/list

can be found in appendix 3).

- Chronic administration (defined as more than 14 continuous days) of

immunosuppressants or other immune-modifying drugs within 6 months prior to

study participation. The use of topical, inhaled, and nasal glucocorticoids will be

permitted (a more detailed description/list can be found in appendix 3).

- Previous participation in this study or in a RSV interventional trial (vaccine,

antivirals).

4.4 Sample size calculation

For the primary analysis the ratio between cases of RSV-related hospitalizations and number

of older adults in the total population will be calculated. In addition, the ratio between the

cases of MA-RSV infection and the number of older adults undergoing active surveillance

will be calculated.

To estimate the incidence of MA-RSV infection and RSV-related hospitalization at an older

age, a prospective cohort of approximately 1,000 older adults ≥60 is followed for 1 year. For

sample size calculations a statistic expert of the Julius Support Center was consulted.

Assuming a yearly MA-RSV incidence of 3,0% based on literature5,9, a sample size of 800

will produce a two-sided >95% confidence interval with a symmetric half width of 0.01


79

(Confidence interval formula: Exact, Clopper-Pearson). Accounting for a 10% loss to follow

up, approximately 1000 elderly will be included in the cohort.

Patient

population

Sites Outcome

Persons

RSV

seasons

Expected

Incidence

per year (%)

95% Confidence Interval

Half-Width (%)

Older Adults

(≥ 60 years)

NL,

UK,

BE

Incidence

rate of

MA-RSV

1,000 1 3,0 5,9 1,9 – 5,0

5. METHODS

5.1 Study parameters/endpoints (see also Table 1)

5.1.1 Main study parameter/endpoint

The primary endpoint is the incidence rate of RT-PCR confirmed RSV infection-associated

ARTI, MA-ARTI and hospitalization in older patients. The incidence rate will also be

summarized separately for outpatient events and for both inpatient and outpatient events

combined through the RSV season of follow up. Nasopharyngeal and oropharyngeal swabs

collected during ARTI episodes during the RSV season will be used for reverse transcriptase

polymerase chain reaction (RT-PCR) detection of RSV. Pre- and post RSV-season RSV

serology will be performed in order to capture RSV infected individuals which will be missed

by active surveillance. In order to analyze the microbiome and transcriptome a

nasopharyngeal and oropharyngeal swab will be collected pre- and post RSV-season.


1. The incidence rate of all-cause MA-ARTI or events leading to worsening of

cardiorespiratory status. The incidence rate will also be summarized separately for

outpatient events and for both inpatient and outpatient events combined through the

RSV season of follow up. Subgroup analyses will summarize these endpoints by

RSV season.

2. Mortality through the RSV season of follow up for RSV-associated deaths and all

cause deaths.

3. Health care costs and resource use for RSV-associated and all-cause MA-ARTI

(inpatient or outpatient) or events leading to worsening cardiorespiratory status with

regard to hospital duration, incidence and duration of intensive care unit stay,

supplemental oxygen use, antibiotic and antiviral use and number of outpatient visits

(e.g., ER visit, physician office/outpatient visits) and HRQoL.

4. The incidence rate of RSV-associated secondary bacterial pneumonia events

(defined as pneumonia within 21 days after RSV infection) and associated antibiotic

use will be summarized.

5. Change in frailty over the course of study.

6. Sample collection for biomarker analysis.


80

5.2 Randomisation, blinding and treatment allocation

There is no randomisation, blinding or treatment allocation, because no investigational

product is being administered in this study.


1. Inclusion Visit (August/September)

At the inclusion visit, after eligibility has been confirmed and fully informed consent

completed, the following procedures will take place:

• A baseline questionnaire about, but not limited to, demographic data, medical

history, risk factors for RSV disease and quality of life will be completed.

• A blood sample (60 ml) and both a nasopharyngeal and an oropharyngeal sample

will be collected.

2. Throughout the RSV season (October 1st to May 1st, or longer if RSV is still circulating

based on national viral surveillance programs):

Participants will be asked about respiratory symptoms during the RSV winter season by

weekly contact by telephone (verbally or text message), email, telephone app or online

questionnaire. If they experience an ARTI, participants are instructed to contact the study

team. Two nasopharyngeal and one oropharyngeal sample will be collected by home visits

(or in the doctor’s office when preferred by participant) within 3 days after contact with the

study team. One nasopharyngeal sample will be used for direct RSV RT-PCR testing. The

other samples will be stored at -80°C. If RSV is positive a blood sample will be drawn (60

ml) and stored at -80°C. In addition, in case of a RSV ARTI, 1-2 weeks after onset of

symptoms another nasopharyngeal sample will be collected. During the episode of

respiratory disease, participants are asked to complete a diary on respiratory symptoms

and HRQoL. At the end of the episode, participants are asked to complete a questionnaire

on medical resource use, interruption of daily activities and HRQoL. If participants have

been admitted to the hospital, a questionnaire about the reason for hospitalization,

diagnosis and treatment will be completed by the study team using medical data from the

admitted hospital.

3. At the end of the RSV Season (May/June)

• A blood sample (60 ml) and both a nasopharyngeal and an oropharyngeal sample

will be collected.

4. Approximately one year after inclusion (+/- 2 months).

• A questionnaire about changes in living conditions, health status and quality of life

will be completed.

Table 2. Overview of sampling of older adults study.

Moment of

sampling

Sample Volume Analysis

(minimum amount)

At beginning of

RSV season (Oct.)

Serum (venous) 20 ml RSV serology (350-

400 µl)


81

Proteome (100 µl)*

Paxgene (venous) 10 ml Transcriptome (200

µl)*

Whole blood

(venous)

30 ml DNA/GWAS (200 µl)

Epigenetics*

Nasopharyngeal

swab and

oropharyngeal swab


Airway

transcriptome*

ARTI Nasopharyngeal

swab (2x) and

oropharyngeal swab

n/a RSV RT-PCR

(qualitative)

Multiplex RT-PCR

respiratory viruses

(quantitative)

(pending funding)

RSV ARTI Serum (venous) 20 ml RSV serology (350-

400 µl)

Proteome (100 µl)*

Paxgene (venous) 10 ml Transcriptome (200

µl)*

Whole blood

(venous)

30 ml Epigenetics*

Cellular

immunology#

Nasopharyngeal

and oropharyngeal

swab (aliquoted)


Airway

transcriptome*

(RSV viral (deep)

sequence analysis)

1-2 weeks after

RSV ARTI

Nasopharyngeal

swab

n/a Airway microbiome*

At end of RSV

season (May)

Serum (venous) 20 ml RSV serology (350-

400 µl)

Proteome (100 µl)*

Paxgene (venous) 10 ml Transcriptome*

Whole blood

(venous)

30 ml Epigenetics*

Nasopharyngeal

and oropharyngeal

swab

Airway microbiome

Airway

transcriptome*

* and additional RSV related biomarkers # in subset of subjects


82

Handling and storage of samples

All samples will be stored at the site where they are collected (medical center or local

laboratory):

• Blood samples will be collected in appropriate tubes as described in table 2 and will be

stored at -80°C for later analysis.

• Nasopharyngeal swab: a nasopharyngeal swab will be collected and aliquoted directly

in 3-4 samples of 200 microliter. All aliquots will be stored at -80°C for later analysis

• Oropharyngeal swab: an oropharyngeal swab will be collected and stored at -80°C for

later analysis.

5.4 Withdrawal of individual participants

Participants can withdraw from the study at any time without having to provide a reason if

they wish to do so and without any consequences for their health care. The investigator can

also decide to withdraw a subject from the study if they meet the pre-defined exclusion

criteria (see section 4.3).

Withdrawal of consent: If consent is withdrawn, the participant will not have any further study

procedures or study observations. All previously collected samples and data will be retained

and used as planned, unless consent is specifically withdrawn for this.

Lost to follow-up: Participants will be considered lost-to-follow-up only if no contact has been

established by the time the study is completed such that there is insufficient information to

determine the subject’s RSV status at approximately 1 year.

5.5 Replacement of individual participants after withdrawal

After withdrawal of an individual participant he or she will be replaced depending on the

moment of withdrawal.


Not applicable.

6. SAFETY REPORTING

6.1 Section 10 WMO event

In accordance to section 10, subsection 1, of the WMO, the investigator will inform the

participants and the reviewing research ethics committee if anything occurs, on the basis of

which it appears that the disadvantages of participation may be significantly greater than

was foreseen in the research proposal. The study will be suspended pending further review


83

by the research ethics committee, except insofar as suspension would jeopardise the

participants’ health. The investigator will take care that all participants are kept informed.

6.1.1 Adverse events (AEs) and Serious adverse events (SAEs)

SAEs directly related to one of the interventions (venepuncture, nasopharyngeal or

oropharyngeal swab) will be registered. Only these SAEs will be registered, as this is a non-

interventional, low risk, observational study. AEs directly related to one of the interventions

(for example a nose bleed after a nose swab or bruise after a blood test) will not be

registered.

6.1.2 Suspected unexpected serious adverse reactions (SUSAR)

NA

6.1.3 Annual safety report

NA


All AEs and SAEs will be followed until they have abated, or until a stable situation has been

reached. Depending on the event, follow up may require additional tests or medical

procedures as indicated, and/or referral to the general physician or a medical specialist.

SAEs need to be reported till end of study within the Netherlands, as defined in the protocol

[Comment: Adapt according to local regulations].

6.3 Data Safety Monitoring Board (DSMB)

No Data Safety Monitoring Board is needed. However, there will be 3 Advisory Boards,

which will act as consultative bodies for ethical, scientific and technical matters.

The following advisory boards will be formed by external experts:

• International Scientific Advisory Group (ISAG)

• RESCEU Ethics Advisory Committee (EAC)

• RESCEU Patient Advisory Board (PAB).

See also appendix 1 for a detailed description of the governance structure of RESCEU.


Descriptive statistics will be used to describe the incidence rate of hospitalization for RSV

and MA-RSV infection in the cohort of older adults. Demographic parameters, clinical

parameters and outcome and laboratory test results will be displayed as categorical data

with percentages or continuous variables with mean (+/-SD) and/or median (interquartile

range). Comparisons between groups will be performed using chi-square for categorical

variables, Student-t-test for normally distributed continuous variables or Mann-Whitney U

test for not normally distributed continuous variables. Multivariate regression analysis will

be performed to analyse multiple risk factors for RSV disease.


84

Statistical analyses will be performed using SPSS version 20 or a more recent version or

with R statistical software version 3.0.1 or higher.



The study will be conducted according to the principles of the Declaration of Helsinki

(www.wma.net) and in accordance with the Medical Research Involving Human Subjects

Act (WMO) and other guidelines, regulations and Acts.

The recruiter will explain the nature of the study and will inform the participant that

participation is voluntary and that the participant can withdraw from the study at any time.

Written informed consent will be obtained from each participant prior to any study

procedure. A copy of the signed consent form will be given to every participant and the

original will be maintained by the research team.


General practice patient lists will be screened for potentially eligible participants (see section

4.2 and 4.3). The latter will be informed by their GP about the study. If they are interested

in taking part their contact details will be given to the study team. Interested potential

participants will be contacted by a member of the study team who will answer any questions

they may have about the study and if they are still interested booked in for an inclusion visit.

Recruitment procedures can be adapted to local circumstances and regulations


• Nasopharyngeal swab: A small swab will be introduced deep into the nose and some

mucus will be collected. The procedure can cause a brief moment of discomfort,

however, the duration of this procedure is less than 10 seconds and the swab is very

soft. Trained personnel will perform this. Minor complications (for example nose

bleed) have been described, but are rare.

• Oropharyngeal swab: A small swab will be introduced into the mouth towards the

oropharynx and some mucus of the oropharynx will be collected. This is a non-

invasive technique. The procedure can cause a brief moment of discomfort,

however, the duration of this procedure is less than 10 seconds and the swab is very

soft. Trained personnel will perform this.

• Venipuncture: Drawing venous blood is moderately painful. Trained personnel will

perform this. Minor complications like bruising have been described.

There is no clear clinical benefit for the participants taking part in this proposed study.

However, the results of this study aim to support the understanding of the burden of RSV

disease, which is important for the implication of future preventive and therapeutic

interventions.


85


Due to the type of study, observational with non-invasive diagnostic procedures without

major complications, as previously described, no serious adverse events are to be expected

and participating in the study is with minimal risks. Therefore we request dispensation from

the statutory obligation to provide insurance [Comment: The obligation to provide insurance

falls under the responsibility of each participating center/country].



Full data management will be performed by Julius Center. Data will be stored in a cloud-

based database. Data will be anonymized before they enter the database. Each participant

will receive a unique identification number, which cannot directly be traced back to the

participant. The study team will keep a participant identification code list to trace data to an

individual participant, if necessary. Data will be kept for 15 years. The handling of personal

data will be in compliance to local regulations.

Data management of this study will be performed by a professional and experienced data

management team. This team will coordinate and implement a high quality IT-infrastructure

that will be necessary for the collection, controlling and reporting of the research data of this

study.

A GCP compliant electronic data capture (EDC) system will be used to guarantee a correct,

complete and consistent data collection. Web-based case report forms will be developed

and implemented on the EDC system. By using comprehensive data validation checks

within these forms, only data of high quality can be submitted to the study database. The

forms, integrated into the EDC system, can easily be accessed by a standard web browser.

The data management system facilitates the collection of data, supports the monitoring

processes and provides real time progress reports for management of the study. After last

patient out, the database can rapidly be closed and data made available for further analysis

and publication purposes.

The system meets all GCP guidelines for electronic data collection in terms of protecting

data integrity and securing the information collected. This means, among other things, that

users will get a role based access to the system after they have logged-in using their own

username and password. The system will log all data entry steps with timestamps, update

reasons and user information. The role based access to the system will avoid unauthorised

data access and prevents that users perform actions that they are not allowed to do. Data

from the EDC system will be transferred over the internet using secured data

communication protocols. Data will be stored automatically and regularly back-ups will

make sure that data never will be lost. Databases and web servers will be hosted in data

centers that meet the highest possible security requirements.


86


Monitoring of the conduct of the study will be performed according to GCP guidelines at

initiation and once yearly for the duration of the study.

9.3 Amendments

Amendments are changes made to the research protocol after a favourable opinion by the

accredited METC has been given. All amendments will be notified to the METC that gave a

favourable opinion. All substantial amendments will be notified to the METC. Non-

substantial amendments will not be notified to the accredited METC, but will be recorded

and filed by the sponsor.





adverse events, other problems, and amendments.

9.5 Temporary halt and prematurely end of study report



In case the study is ended prematurely, the sponsor will notify the accredited METC within

15 days, including the reasons for the premature termination.



accredited METC.

9.6 End of the study

The investigator/sponsor will notify the accredited METC of the end of the study within a

period of 8 weeks. The end of the study is defined as the moment that the last included

participant has been followed up for 12 months.


Results of this research are disclosed unreservedly.


87

10. REFERENCES

1. Nair H, Nokes DJ, Gessner BD, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet. 2010;375(9725):1545-1555.

2. Blanken MO, Rovers MM, Molenaar JM, et al. Respiratory syncytial virus and recurrent wheeze in healthy preterm infants. N Engl J Med. 2013;368(19):1791-1799.

3. Lotz MT, Moore ML, Peebles RS, Jr. Respiratory syncytial virus and reactive airway disease. Curr Top Microbiol Immunol. 2013;372:105-118.

4. Stein RT, Sherrill D, Morgan WJ, et al. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet. 1999;354(9178):541-545.

5. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med. 2005;352(17):1749-1759.

6. Zwaans WA, Mallia P, van Winden ME, Rohde GG. The relevance of respiratory viral infections in the exacerbations of chronic obstructive pulmonary disease-a systematic review. J Clin Virol. 2014;61(2):181-188.

7. Mazur NI, Martinon-Torres F, Baraldi E, et al. Lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics. Lancet Respir Med. 2015;3(11):888-900.

8. Brown PM, Schneeberger DL, Piedimonte G. Biomarkers of respiratory syncytial virus (RSV) infection: specific neutrophil and cytokine levels provide increased accuracy in predicting disease severity. Paediatr Respir Rev. 2015;16(4):232-240.

9. Shinde V. Randomized Phase 2 Trial of an RSV F nanoparticle vaccine in the elderly: Epidemiology and Efficacy. RSV Vaccines for the World. 2015. La Jolla, California, USA. 2015.


88

10. APPENDIX 1: Governance structure of RESCEU

RESCEU will adopt a governance model that will promote the active participation of national

public health agencies, academia and pharmaceutical companies (EFPIA) in order to

achieve maximum collaboration and data sharing. The management structure of RESCEU

has been developed to respond to the needs of an international large-scale multi-

stakeholder project. It is based on a traditional management structure adapted to the

particular attributes of RESCEU.

Managing an organisation like RESCEU can be challenging due to the size of the project,

its ambition, the variety of activities and their interdependencies. The project aims to

harmonise the interests of the public and EFPIA partners. Therefore, a strong internal trust

and communication interface is crucial to setting the project up for success.

The project is composed of complementary, as well as parallel activities, with strong inter-

dependencies between critical work packages outputs. This will require the need for a

detailed time schedule for many of the tasks, which will need close monitoring and

communication between team members to avoid bottlenecks and to allow effective progress

of deliverables.

The management structure needs to be a balance between a simplistic standard scheme

(which will not be able to address the needs of a project of this level of complexity) and an

excessive super-structure (that would impose a cumbersome bureaucracy to the project

and thus impede its scientific and technical progress).

Taking into account these project characteristics, the management structure proposed for

RESCEU is based on a multi-level organisation that balances:

• The fulfilment of the work plan per se.

• The management of trade-offs affecting scope, quality, time and cost.

• The primary focus needed on critical activities that aim to ensure the achievement

of milestones and that contribute to strategic objectives.

• The relationships and trust amongst partners, including conflict resolution.

• The quality and efficiency with which the project activities are carried out.

• The appropriate implementation of the Consortium Agreement, with careful attention

to the governance procedures, intellectual property policy and the related use of

results.

• The implementation of the Grant Agreement, including administrative and financial

elements.

• The creation and management of a wider scientific forum encompassing interested

organisations beyond the project partnership (Associate Partners).

Taking into account the above, a management structure has been designed with the

following components included:

• RESCEU Forum (RF): forum for discussion, dissemination and scientific

community-building within the project. The RF will consist of the project partners

(Beneficiaries) and the Associate Partners and may be convened by electronic


89

means or face-to-face with the purpose of stimulating discussion and promoting

dialogue on scientific issues. The RF will not have decision-making powers.

• General Assembly (GA): body composed of all Beneficiaries participating in the

project, with the ultimate decision-making responsibility in matters affecting the

overall project strategy and composition of the consortium. The GA will meet

annually and will adopt decisions by majority – each partner having a vote-, except

in cases were unanimity is required according to IMI rules.

• Steering Committee (SC): leadership team with 50/50 vote allocation between

EFPIA/non-EFPIA members, composed of WP (Co-) Leads (from academia and

EFPIA) or their designated representatives. The SC is responsible for decision

making on most issues related to project execution, technical development

decisions, work plan updates, and effort/budget re-assignment in order to pursue

optimal efficiency. Meetings will take place regularly, typically every two months.

The attendance of one representative from each WP will be required for quorum.

Decisions will be determined by majority vote of attendees.

• Operations Team (OT): executive group composed of the Coordinator, the Project

Leader and the Project Manager (but not restricted to those), responsible for the

day-to-day operational and technical aspects of the project. The OT will meet

frequently (i.e. bi-weekly by teleconference) to monitor the project progress and to

address any issues that may arise.

• Project Management Office (PMO): team dealing with the day-to-day management

of the project. Regular meetings, mostly by teleconference, will be established to

appropriately follow up on management matters.

• Task Forces: Result-oriented ad-hoc teams will be created as needed, with a clear

and exclusive mission of studying/resolving any issues between WPs.

• Advisory Boards: consultative bodies for ethical, scientific and technical matters.

RESCEU intends to establish three advisory boards formed by external experts: the

International Scientific Advisory Group (ISAG), the RESCEU Ethics Advisory

Committee (EAC) and the RESCEU Patient Advisory Board (PAB).


90

Figure 3. Governance structures in RESCEU.


91

12. APPENDIX 2: Glossary of Terms

According-To-Protocol (ATP) cohort: This cohort will include all cases enrolled in the

study who meet the criteria defined in the protocol for the considered analysis.

Acute respiratory tract infection (ARTI): symptoms of an upper and/or lower respiratory

tract infection, such as runny or blocked nose, coughing, fast breathing, chest indrawing,

shortness of breath, low oxygen saturation.

Cohort study: A form of epidemiological study where subjects in a study population are

classified according to their exposure status/disease and followed over time (prospective/

retrospective) to ascertain the outcome(s).

Epidemiological study: An observational or interventional study without administration of

medicinal product(s) as described in a research protocol.

Evaluable: Meeting all eligibility criteria, complying with the procedures defined in the

protocol, and, therefore, included in the According-To-Protocol (ATP) analysis (see Section

9.3 for details on criteria for evaluability).

Health Burden: Burden of the disease imposed on the study population in terms of

incidence of the disease and associated healthcare utilization in any healthcare setting.

Healthcare settings (Healthcare Utilization): Primary, secondary and tertiary care

settings such as self-care with over-the-counter [OTC] drugs, general practitioner (GP)

visits, emergency room (ER) visits, hospital visits, etc.

Interventional Human Subject Research:

Studies in which participants are administered medical care, medicinal products and/or

medical/scientific procedures as described in a research protocol.

Lost-to-Follow-up is defined as no contact by the subject’s parent(s)/LAR(s) over the

period of 3 planned contacts and/or 2 months and after a final attempt has been made by

mail. Once this has been reached, the subject is censored at the time of last contact.

Sponsor: The sponsor is the party that commissions the organisation or performance of

the research, for example a pharmaceutical company, academic hospital, scientific

organisation or investigator. A party that provides funding for a study but does not

commission it is not regarded as the sponsor, but referred to as a subsidising party.

Prospective study: A study in which the subjects/cases are identified and then followed

forward in time in order to address one or more study objectives.

Research protocol: A document that describes the objective(s), design, methodology,

statistical considerations, and organization of a study. The protocol usually also gives the

background and rationale for the study, but these could be provided in other protocol

referenced documents.


92

Seroprevalence: The total number of cases within the study population at a specific time

that test positive for the disease based on blood serum specimens.

Study population: Sample of population of interest.

Sub-cohort: A subgroup of the total cohort of study participants for whom the planned study

procedures are different from those planned for the other study participants.

Participant: Term used throughout the protocol to denote an individual who has been

contacted in order to participate or participates in the epidemiological study or a person

about whom some medical information has been recorded in a database.

Participant number: A unique number identifying a subject, assigned to each participant

consenting to participate in the study.

Surveillance: The ongoing systematic collection, collation, analysis, and interpretation of

descriptive epidemiological health data on a specific disease. Surveillance can monitor

incidence and/or prevalence, and/or inform about when and where health problems are

occurring and who is affected.


93

13. APPENDIX 3: Clarification of exclusion criteria based on immunosuppression

Exclusion criteria based on immunosuppression related exclusion criteria are as follows:

• Exclusionary immunosuppressive conditions: “Any known or suspected

immunosuppressive condition, acquired or congenital, as determined by history

and/or physical examination.”

• Exclusionary immunosuppressive medications: “Chronic administration (defined as

more than 14 continuous days) of immunosuppressants or other immune-modifying

drugs within 6 months prior to study participation. The use of topical, inhaled, and

nasal glucocorticoids will be permitted.”

Below is a non-exhaustive list of immunosuppressive conditions and immunosuppressive

medications/therapies that are exclusionary according to above two exclusion criteria:

A) Potential subjects with HIV infection, regardless of whether the subject is

receiving anti-retroviral treatment.

B) Potential subjects with congenital immunodeficiencies.

C) Potential subjects with active leukemia, lymphoma, or other hematologic

malignancy according to following criteria:

i. Disease known to be present and active (previously treated patients with no

evidence of active disease in the previous 6 months are acceptable,

provided they meet the requirements for no ongoing cytotoxic drug therapy).

ii. With or without ongoing therapy.

D) Potential subjects with any prior history of hematopoietic stem cell

transplantation.

E) Potential subjects with certain medical conditions (for example, but not limited

to, solid tumors/malignancies, or solid organ transplant recipients) requiring any

of the following ongoing:

i. Cytotoxic drug therapy ongoing or within 6 months prior to study participation

ii. Systemic glucocorticoids in excess of the limits of the protocol (≥10mg of

prednisone per day or equivalent for more than 14 continuous days within 6

months prior to study participation)

iii. Chronic immunosuppressant therapies ongoing or within 1 month prior to

study participation to manage solid organ transplants: Calcineurin Inhibitors

(e.g. Tacrolimus and Cyclosporine), Antiproliferative agents (e.g.

Mycophenolate Mofetil, Mycophenolate Sodium and Azathioprine), mTOR

inhibitor (e.g. Sirolimus, Tacrolimus), and/or steroids (e.g. prednisone)

F) Potential subjects with end-stage renal failure or hepatic failure

The following potential subjects should NOT be excluded from participation:

1) Potential subjects with known or suspected "autoimmune diseases" who are

untreated or treated with immunomodulatory monoclonal antibodies/biologicals

and who have NOT had opportunistic infections.

94

ANNEX IV. Clinical study 4: COPD cohort study protocol

NL60190.042.16, RESCUE study, version 1.0, December 6st 2016

95

RESCUE STUDY: BURDEN OF RSV

An Observational Study to Assess Respiratory Syncytial Virus (RSV)-associated

Illness in Adults With Chronic Obstructive Pulmonary Disease (COPD)

Protocol ID NL60190.042.16

Short title RESCUE: Burden of RSV

EudraCT number Applicable

Version 1

Date 6-12-2016

Coordinating


Not applicable



uitvoerder

Dr. M. van den Berge, chest physician

Department of pulmonology, AA11

University Medical Center Groningen

Sponsor (in Dutch:


University Medical Center Groningen,


Head of department of Pulmonology: Prof.

Dr. H.A.M. Kerstjens

Subsidising party Part of this research is funded by an EU-H2020

grant. The remaining part is funded by the

department of Pulmonology of the UMCG by

research funds of dr. M van den Berge

Independent expert (s) Dr. O.W. Akkerman, chest physician



Laboratory sites <if applicable> Not applicable. Only the local laboratory in the

UMCG will be used

Pharmacy <if applicable> Not applicable


96

PROTOCOL SIGNATURE SHEET

Name Signature Date



Head of Department of Pulmonogy:

Prof. Dr. H.A.M. Kerstjens

Coordinating Investigator/Project

leader/Principal Investigator:


Department of Pulmonogy:

Dr. M. van den Berge


97

TABLE OF CONTENTS

LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS ................................... 99

INTRODUCTION AND RATIONALE ....................................................................... 102

OBJECTIVES ......................................................................................................... 104

STUDY DESIGN ..................................................................................................... 105

4.1 Definitions ........................................................................................................ 105

4.1.1 COPD Exacerbation Definition .................................................................. 105

4.1.2 Definition of a mild, moderate and severe COPD exacerbation:................ 105

4.1.3 Defining the end of an exacerbation ......................................................... 106

4.1.4 Defining a new exacerbation or the same prolonged exacerbation ........... 106

STUDY POPULATION ............................................................................................ 106

5.1 Inclusion criteria: .............................................................................................. 106

5.2 Exclusion criteria:............................................................................................. 106

SAMPLE SIZE CALCULATION .............................................................................. 106

METHODS .............................................................................................................. 107

7.1 Baseline visits and investigations ..................................................................... 107

7.2 Table 1 Time and Events Schedule ................................................................. 108

7.3 Exacerbation visits and investigations.............................................................. 109

7.4 Study parameters/endpoints ............................................................................ 109

7.4.1 Main study parameter / endpoint............................................................... 109

7.4.2 Secondary study parameters/endpoints .................................................... 109

7.5 Study procedures ............................................................................................ 110

7.5.1 A. Obtain informed consent ..................................................................... 110

7.5.2 B. Review inclusion / exclusion criteria ..................................................... 110

7.5.3 C. Medical / disease history ...................................................................... 110

7.5.4 D. Concomitant Medication Review .......................................................... 110

7.5.5 E. Review of adverse events/ exacerbations............................................. 110

7.5.6 F. Diary and PEF ..................................................................................... 110

7.5.7 G. Questionnaire ...................................................................................... 111

7.5.8 H. Pre- and post-bronchodilator spirometry / Body Box and diffusion

capacity 111

7.5.9 I. Post-bronchodilator IOS ........................................................................ 112

7.5.10 J. HRCT .................................................................................................... 112

7.5.11 K. MBNW .................................................................................................. 112

7.5.12 M. PExA measurements ........................................................................... 113

7.5.13 N. Nasal brush for mRNA ......................................................................... 113

7.5.14 O. Nasopharyngeal swab.......................................................................... 113


98

7.5.15 Q. Blood collection .................................................................................... 115

7.5.16 R. Sputum sample .................................................................................... 116

7.6 Withdrawal of individual subjects ..................................................................... 116

7.7 Specific criteria for withdrawal (if applicable) .................................................... 116

7.8 Replacement of individual subjects after withdrawal ........................................ 117

7.9 Premature termination of the study .................................................................. 117

SAFETY REPORTING ............................................................................................ 117

8.1 Temporary halt for reasons of subject safety ................................................... 117

8.2 AEs, SAEs and SUSARs ................................................................................. 117

8.2.1 Adverse events (AEs) ............................................................................... 117

8.2.2 Serious adverse events (SAEs) ................................................................ 117

8.3 Follow-up of adverse events ............................................................................ 118

STATISTICAL ANALYSIS ....................................................................................... 118

9.1 Analysis of demographic variables ................................................................... 118

9.2 Analysis of the primary and secondary endparameters .................................... 118

9.3 GWAS, microbiome, and genome-wide mRNA and microRNA expression

analyses. .................................................................................................................... 119

9.4 Interim analysis (if applicable) .......................................................................... 119

ETHICAL CONSIDERATIONS ............................................................................ 119

10.1 Regulation statement ....................................................................................... 119

10.2 Recruitment and consent ................................................................................. 119

10.3 Benefits and risks assessment, group relatedness .......................................... 120

10.4 Compensation for injury ................................................................................... 120

10.5 Incentives (if applicable) .................................................................................. 121

ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ................... 121

11.1 Handling and storage of data and documents .................................................. 121

11.2 Monitoring and Quality Assurance ................................................................... 121

11.3 Handling and storage of samples ..................................................................... 121

11.4 Amendments ................................................................................................... 122

11.5 Annual progress report .................................................................................... 122

11.6 Temporary halt and (prematurely) end of study report ..................................... 122

11.7 Public disclosure and publication policy ........................................................... 122

REFERENCES .................................................................................................... 123


99

1.LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS

ABR

ALRI

ABR form, General Assessment and Registration form, is the application

form that is required for submission to the accredited Ethics Committee (In

Dutch, ABR = Algemene Beoordeling en Registratie)

Acute Lower Respiratory Infection

AE Adverse Event

AR Adverse Reaction

CA

COPD

Competent Authority

Chronic Obstructive Pulmonary Disease

CCMO Central Committee on Research Involving Human Subjects; in Dutch:

Centrale Commissie Mensgebonden Onderzoek

CV Curriculum Vitae

EU European Union

GCP

GWAS

Good Clinical Practice

Genome Wide Association Studies

IC Informed Consent

IMP Investigational Medicinal Product

IMPD Investigational Medicinal Product Dossier

METC

RSV

Medical research ethics committee (MREC); in Dutch: medisch ethische

toetsing commissie (METC)

Respiratory Syncytial Virus


Sponsor The sponsor is the party that commissions the organisation or performance

of the research, for example a pharmaceutical

company, academic hospital, scientific organisation or investigator. A party

that provides funding for a study but does not commission it is not regarded

as the sponsor, but referred to as a subsidising party.

Wbp Personal Data Protection Act (in Dutch: Wet Bescherming

Persoonsgevens)

WMO Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-

wetenschappelijk Onderzoek met Mensen


100

SUMMARY

Rationale: RSV is a significant cause of ALRI morbidity in elderly and COPD patients. Most

published data on RSV disease burden in the elderly are from the United States and from

hospital settings. The knowledge gaps have an impact on Europe’s ability to make

evidence-based decisions nationally about novel vaccines and therapeutics. There is a

parallel need to assemble clinical resources to identify correlates of severe RSV disease

for clinical management, classification of disease severity in clinical trials and identification

of biomarkers for severe disease.

Objective: The primary objective of this prospective observational study is to determine the

incidence of RSV disease and document resource utilization in patients with COPD. In

addition, we will assess if and how the occurrence of an RSV virus infection affects the long-

term outcome of COPD as reflected by rate of lung function decline, course of symptoms,

and rate of COPD exacerbations during the follow-up of this study.

Study design: Prospective observational study

Study population: 500 COPD patients will be followed up for a period of three consecutive

years.

Intervention (if applicable): This is a non-interventional study.

Main study parameters/endpoints: The primary objective of this prospective

observational study is to determine the incidence of RSV disease and document resource

utilization in patients with COPD. In addition, we will assess if and how the occurrence of

an RSV virus infection affects the long-term outcome of COPD as reflected by rate of lung

function decline, course of symptoms, and rate of COPD exacerbations during the follow-

up of this study.


group relatedness: No investigational product will be used in this study. It is not expected

that subjects will receive any individual benefit from participation in this study; however, the

findings from this study may increase knowledge about the role RSV plays in the onset of

events leading to worsening cardiorespiratory status in the aforementioned patient

populations and in the pathological and biological changes that occur during such events.

This knowledge may help in understanding if there is a need for future RSV interventions

or prophylaxes in this patient population. In this study, subjects will be treated according to

usual standard of care as determined by the treating physician. Some procedures in this

study, such as nasopharyngeal swab collection and spontaneous sputum, may not be usual

practice for study sites. Nasopharyngeal swab collections have the potential to irritate the

intranasal cavity and lead to acute epistaxis; however, the risks associated with discomforts

from such events are minimal. Additional risks include obtaining blood that may sometimes


101

cause pain at the site where the blood is drawn, bruising, and occasional light-headedness

and, rarely, fainting; performing spirometry may cause mild chest tightening and coughing;

performing HRCT scan is accompanied with a low radiation dose that is within the maximum

dose allowed for research purposes. There are no other risks to subjects in this study above

that from the usual treatment of their disease.


102


Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide and

its morbidity and mortality are still rising. The WHO predicts that COPD will become the

fourth leading cause of death worldwide by 2030 [1].

COPD is defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) as

a preventable and treatable disease with some significant extra-pulmonary effects that may

contribute to the severity in individual patients [2]. Its pulmonary component is characterized

by airflow limitation that is not fully reversible. The airflow limitation is usually progressive

and associated with an abnormal inflammatory response of the lungs to noxious particles

or gases. The main pathologic features of COPD include tissue remodelling in the small

airways (fibrosis and smooth muscle hypertrophy) and tissue destruction in the lung leading

to emphysema [3,4]. The first signs of COPD are often chronic cough, increased sputum

production, and dyspnea. The presence and severity of COPD is generally documented by

a decrease in FEV1 compared to the predicted FEV1 and a decreased FEV1/FVC ratio that

is not or only little reversible by an inhaled bronchodilator. In addition, air trapping is present

in a considerable proportion of COPD patients, as reflected by an increased RV%TLC. This

trapped air, i.e. hyperinflation, contributes to the dyspnea intensity that is experienced by

COPD patients.

Exacerbations are regarded as important events for COPD prognosis, since an increased

frequency of these episodes may hasten disease progression by accelerated decline in lung

function and increased mortality rates, particularly if these require a hospital admission [5,6].

The mechanisms by which exacerbations lead to progressive loss of lung function are not

yet unknown. However, it is likely due to effects of acute inflammation and associated lung

tissue damage. Of interest, the Cosmic study showed that symptoms persist for several

weeks after an exacerbation, suggesting that underlying pathophysiology is not resolved

with a two-week course of oral corticosteroids or antibiotics [7]. It may thus be of importance

to attack the ongoing inflammation with appropriate treatment at the appropriate location in

the lungs.

Viruses that are commonly associated with acute exacerbations in COPD patients include

influenza viruses, picornaviruses, coronaviruses, and paramyxoviruses. Most of the

published studies to date have involved small study populations, and the percentage of

illnesses caused by RSV in persons with COPD ranges widely from 0% to 17.4% [3–10].


103

Though the percentage of illnesses caused by RSV shows a wide range, RSV has been

nevertheless recognized as an important cause of COPD exacerbations [11,12].

Human respiratory syncytial virus (RSV) causes severe disease in the very young, elderly

and in high-risk groups. It has been estimated that RSV was associated with 34 million

cases of acute lower respiratory tract infection (ALRI), 3.4 million ALRI hospitalisations and

55,000 to 199,000 deaths in children <5 years in 2005 [13]. These estimates are based on

limited data and there is a substantial gap in knowledge (on morbidity and associated

healthcare and social costs) across Europe. RSV infection in childhood is associated with

subsequent wheezing and asthma [14–16]. These long-term sequelae pose a substantial

additional burden on the healthcare system. In addition, RSV is a significant cause of ALRI

morbidity in elderly and COPD patients [11,17]. Most published data on RSV disease

burden in the elderly are from the United States and from hospital settings. The knowledge

gaps have an impact on Europe’s ability to make evidence-based decisions nationally about

novel vaccines and therapeutics. There is a parallel need to assemble clinical resources to

identify correlates of severe RSV disease for clinical management, classification of disease

severity in clinical trials and identification of biomarkers for severe disease.

COPD has been traditionally considered a self-inflicted disease induced by tobacco

smoking. In healthy subjects, lung function declines physiologically with age. By contrast,

the traditional pathophysiological paradigm of COPD proposed by Fletcher and Peto in the

late seventies states that, COPD develops in the so called “susceptible” individuals because

smoking enhances the physiological decline of lung function through life [18]. Recent

research, however, has challenged this traditional paradigm by showing that an enhanced

decline of lung function occurs only in half of the COPD patients whereas the other half

develop COPD because of poor lung function development early in life [19]. Potential

causes of poor lung development are multiple and include genetic and epigenetic factors,

associated with environmental exposures such as, poor diet, repeated lung infections in

infancy, passive smoking and/or prematurity. It is likely that the driving biological

mechanisms vary between these conditions, thereby making COPD the common clinical

endpoint of diverse molecular pathologies. In any case, this new concept has fundamental

implications for the understanding of COPD, since it allows a novel stratification of patients

based on their lung function trajectories that may be highly relevant for their individualized

management. It provides the opportunity to treat or even prevent COPD with tailored

interventions, targeting specific molecular networks operating in (innate and acquired)

immunity, inflammation and remodelling. This study will aim to identify patients who are


104

more susceptible to viral exacerbations with the potential to develop further treatments to

prevent RSV exacerbations.

3. OBJECTIVES

The primary objective of this prospective observational study is to determine the incidence

of RSV disease and document resource utilization in patients with COPD. In addition, we

will assess if and how the occurrence of an RSV virus infection affects the long-term

outcome of COPD as reflected by rate of lung function decline, course of symptoms, and

rate of COPD exacerbations during the follow-up of this study.

As a secondary objective, we will investigate whether it is possible to predict the long-term

outcome of COPD, occurrence of COPD exacerbations and susceptibility to respiratory viral

infections including RSV. To this end, we will analyse clinical data as well as GWAS in blood

and genome-wide gene expression in brushed nasal epithelium.


105

4. STUDY DESIGN

This is a prospective, observational study conducted across three consecutive RSV

seasons to determine the incidence of RSV disease and document the incident rate of

ALRI’s and COPD exacerbations in patients with COPD. Clinically stable subjects with

COPD (i.e. Global Initiative for Chronic Obstructive Lung Disease [GOLD] Stage I-IV will be

enrolled and followed for three years. At baseline, subjects will be more extensively

characterized with a full medical history, lung function, blood for transcriptomics, nasal

mucosal sampling, body plethysmography, multiple breath nitrogen washout (MBNW),

blood and sputum cell differential, HRCT, and particles in exhaled air (PExA). Subjects will

have twice yearly scheduled visits to obtain blood, nasopharyngeal swab, sputum, and

clinical data and perform a spirometry. One visit will be scheduled before the RSV season

(in the months between May and October), and one visit will be scheduled during the RSV

season (between October and April). In addition, unscheduled visits to collect blood,

nasopharyngeal swab, sputum, and clinical data will be conducted in cases where a subject

experiences an increase in symptoms consistent with a COPD exacerbation. Participants

will be seen within 7 days of symptom onset.

4.1 Definitions

4.1.1 COPD Exacerbation Definition

A COPD Exacerbation will be defined as the presence of two new symptoms (one of which

must be major) for two or more consecutive days.

- Major symptoms: increased breathlessness (A), increased sputum colour (B1) or

increased sputum amount (B2).

- Minor symptoms: a cold (C), increased wheeze or chest tightness (D), Sore throat

(E1), Increased cough (E2), Fever (F).

- Or symptoms suggesting a COPD exacerbation in the opinion of the attending

clinician.

4.1.2 Definition of a mild, moderate and severe COPD exacerbation:

- A mild COPD exacerbation is defined as: an exacerbation according to symptom

criteria that does not require any change in treatment.

- A moderate COPD exacerbation is defined as an exacerbation according to

symptom criteria which requires a change in treatment e.g. treatment with antibiotics

or oral corticosteroids, but does not require hospital admission

- A severe exacerbation is defined when admission to the hospital is required or when

the exacerbation results in death.


106

4.1.3 Defining the end of an exacerbation

The end of an exacerbation is defined when there are 2 consecutive symptom free days.

The last day of symptoms is defined as the end of exacerbation.

4.1.4 Defining a new exacerbation or the same prolonged exacerbation

If the participant has had five symptom free days following an exacerbation then 2 days of

2 new symptoms are defined as a new exacerbation. If a participant has had less than 5

symptom free days since his last exacerbation then this is defined as one exacerbation

(prolonged exacerbation).

5. STUDY POPULATION

This is a prospective observational study of 500 COPD patients followed-up for a period of

three consecutive years.

5.1 Inclusion criteria:

- Age ≥40 years at recruitment.

- Smoking history of >10 pack years.

- COPD patients with an FEV1/FVC <0.7.

5.2 Exclusion criteria:

- Patients with a history of asthma, significant bronchiectasis, carcinoma of the

bronchus, or other significant respiratory disease.

- Patients taking immunosuppressive medications.

- Active cancer diagnosis.

- Long-term steroid therapy (≥10 mg/day).

6. SAMPLE SIZE CALCULATION

- To estimate the incidence of medically attended RSV infection 500 COPD patients are

follow-up for 3 years. With an expected incidence of 5.4 events per year (confidence interval

(CI) 4.3-6.9) and an anticipated loss to follow-up of 1 year, we expect between 58 – 93 RSV

infections in our cohort and between 357 – 392 COPD patients who not affected by an RSV

virus infection. This way, we will have at least 80% power to detect a difference in the annual

rate of decline in FEV1 of 15 ml between COPD patients with and without an RSV infection

assuming a standard deviation of 40 ml and a two-tailed alpha of 0.05.


107

7. METHODS

7.1 Baseline visits and investigations

Patients will visit the outpatient or research clinic at recruitment and thereafter every 6

months (± 1month) to obtain blood, nasopharyngeal swab, sputum, mucosal sampling. In

addition, patients are asked to visit the outpatient clinic in case of an increase in symptoms

compatible with a COPD exacerbation. An overview of the investigations performed at each

baseline visit is presented in Table 1.


108

7.2 Table 1 Time and Events Schedule

- Vis

it 1

Baselin

e

Vis

it 2

½ y

ear

Vis

it 3

1 y

ear

Vis

it 4

1½

years

Vis

it 5

2 y

ears

Vis

it 6

2½

years

Vis

it 7

3 y

ears

Vis

it afte

r:

Exacerb

atio

n

A. Obtain informed consent

B. Review Inclusion / Exclusion criteria

C. Medical / Disease history

D. Concomitant Medication Review

E. Review of adverse events/

exacerbations

F. Diary and PEF

G. Questionnaires (CCQ, CAT, SGRQ)

H. Pre- and post-bronchodilator

spirometry

H. Post-bronchodilator Body Box and

diffusion capacity

H. Post-bronchodilator spirometry

I. Post-bronchodilator IOS

J. HRCT

K. MBNW

M. PExA measurement

N. Nasal brush for mRNA

O. Nasopharyngeal swab

P. Blood collection for serum and

inflammatory markers

P. Blood for transcriptomics

P. Blood PBMCs and frozen whole

blood

Q. Spontaneous sputum sample


109

7.3 Exacerbation visits and investigations

Participants will be instructed to report symptoms of an exacerbation to the study team

within seven days of symptom onset and before treatment with antibiotics or oral

corticosteroids. The study team will then arrange for the participant to attend the outpatient

clinic for an exacerbation visit. At an exacerbation visit clinical data, post-bronchodilator

spirometry, nasopharyngeal swab, spontaneous sputum and blood will be obtained.

Participants will be treated for the exacerbation according to the decision of the clinician.

7.4 Study parameters/endpoints

7.4.1 Main study parameter / endpoint

The primary objective of this prospective observational study is to determine the incidence

of RSV acute respiratory infection or events leading to worsening cardiorespiratory status

across multiple consecutive RSV. In addition, we will assess if and how the occurrence of

an RSV virus infection affects the long-term outcome of COPD as reflected by rate of lung

function decline, course of symptoms, and rate of COPD exacerbations during the follow-

up of this study.


As a secondary objective, we will investigate whether it is possible to predict the long-term

outcome of COPD, occurrence of COPD exacerbations and vulnerability to develop viral

infections including RSV. To this end, we will analyse clinical data as well as markers of

mucosal immunity and gene expression. The following parameters will be assessed:

- Lung function (PEF, FEV1, FEV1/FVC, FVC, FEF25-75%).

- Daily PEF monitoring and symptoms recordings.

- IOS measurements, R5, R10, R15, R20, R5-R20, Fres, X5, AX.

- Multiple Breath Nitrogen Washout.

- In- and expiratory HRCT scan will be performed at baseline.

- PExA measurement will be performed at baseline.

- Questionnaires (health status [CCQ and SGRQ] and CAT).

- Blood transcriptomics for genetic susceptibility.

- Blood RSV serology.

- Blood cell differential counts.

- Frozen whole blood.

- Peripheral blood mononuclear cells for innate immune response.


110

- Spontaneous sputum for viral RT-PCR, inflammatory cell counts, inflammatory cytokine

release and bacteriology.

- Nasopharyngeal swab for virus detection.


7.5.1 A. Obtain informed consent

A signed informed consent will be obtained from the subject prior to the start of the study.

7.5.2 B. Review inclusion / exclusion criteria

For verification of inclusion and exclusion criteria.

7.5.3 C. Medical / disease history

Demographic variables include: age, sex, smoking habits, education, work, other

exposures, height and weight. Medical history of the subjects will be assessed; comorbidity,

history of surgical procedures, recent and active medication use. Additionally, physical

examination will be performed.

7.5.4 D. Concomitant Medication Review

Medication use for COPD and comorbidity will be assessed.

7.5.5 E. Review of adverse events/ exacerbations

Subjects are instructed to fill in their diary and to report worsening of symptoms (see F.

Diary and PEF). Adverse events - excluding these addressed below - will be reviewed

during the half yearly visits.

7.5.6 F. Diary and PEF

Patients will be asked to record their symptoms on a daily basis throughout the study period

(see F2. RESCUE Diary Dutch, version 1.0). Participants will record symptoms using a daily

diary card to record day-to-day variations in symptoms. This has been used extensively in

the London COPD cohort to encourage early exacerbation reporting. Participants will be

asked to record and code symptoms as described below:

• Increase in breathless (A)

• Increase in sputum purulence (B1)

• Increase in sputum volume (B2)

• Increase in cold-like symptoms e.g. nasal congestion (C)

• Increase in chest tightness or wheeze (D)

• Increase in sore throat (E)

• Presence of a fever (F)


111

If participants experience two or more symptoms for two or more days they will be instructed

to contact the study team. Participants will also be asked to record their daily morning PEF

throughout the study period, at the same time each day. The highest of three values is used

for analysis.

7.5.7 G. Questionnaire

Clinical COPD Questionnaire (CCQ)

The CCQ consists of 10 items, divided into three domains (symptoms, functional and mental

state), which are scored on a 7-point Likert scale (0=best, 6=worst). It has a high Cronbach’s

alpha (0.91) and test-retest reliability in a 2-week interval. Significant correlations were

found between the CCQ total score and domains of the SF-36 (rho=0.48 to rho=0.69) and

the SGRQ (rho=67 to rho=0.72) [20].

St. George's Respiratory Questionnaire (SGRQ)

Disease-specific instrument designed to measure impact on overall health, daily life, and

perceived well-being in patients with obstructive airways disease.

COPD assessment test (CAT)

The COPD Assessment Test is a new questionnaire for people with COPD. It is designed

to measure the impact of COPD on a person's life, and how this changes over time.

7.5.8 H. Pre- and post-bronchodilator spirometry / Body Box and diffusion

capacity

Spirometry measurements will take place at the lung function department or research clinic

by trained personnel. Spirometry will be performed according to the international guidelines

[21]. A daily-calibrated pneumotachograph will be used throughout this study. The following

parameters will be assessed: FEV1, FVC and FEV1/FVC. Reversibility of airways

obstruction will be measured 15 minutes after administering 400mcg salbutamol per

metered dose-inhaler connected to a spacer following the same procedures.

Measurements of Body Box and diffusion capacity will be performed according to the

international guidelines with a constant volume bodyplethysmograph. The following

parameters will be assessed: TLC, RV and FRC. Specific airway conductance (sGaw),

airway resistance (Raw), FRC, VC and IVC will be measured. The TLC and RV can be

calculated. Reference values will be obtained from Quanjer et al. [22]. Furthermore, the


112

diffusion capacity (transfer factor) for carbon-monoxid (DLCO and KCO) will be measured

using the single breathholding method.

7.5.9 I. Post-bronchodilator IOS

IOS measurements will be performed (IOS Masterscreen, E. Jaeger, Wurzburg), Germany

according to the standard recommendations. The loudspeaker sends pressure impulses to

the respiratory system, during tidal breathing through a mouthpiece. The impulses come

with an interval of 0,2s for 30s. During the test the patient is breathing quietly and is sitting

upright on a char while supporting their cheeks with his hands. Parameters assessed with

this test are: R5, R20, X5, R5-R20.

7.5.10 J. HRCT

CT allows study of both emphysema and the small airways in COPD that will be used for

phenotyping the disease.

All scans will be obtained with in slices of 0.75-mm section thickness, and 26.8-mm table

feed per rotation (pitch of 1.4) in a caudocranial scan direction to minimize breathing

artefacts. Participants will be asked to take a deep breath and to hold their breath. Exposure

settings will be 20 mAs at 100 kVp for patients weighing less than 50 kg, 20 mAs at 120

kVp for patients weighing between 50 and 80 kg, and 30 mAs at 140 kVp for patients

weighing more than 140 kg without dose modulation. During expiration, the exposure

settings will be 20 mAs at 100 kVp for patients less than 80 kg and 20 mAs at 120 kVp for

those weighing more than 80 kg. Data will be transferred from the CT scanner to a digital

workstation.

In case of any unexpected findings, both the patient and the general practitioner will be

informed. If necessary, the patient will be referred to our outpatient clinic for regular medical

advice and/or treatment.

7.5.11 K. Multiple Breath Nitrogen Washout (MBNW)

– After inhaling 100% pure oxygen, multiple breath washout analysis will be performed

with the inert gas nitrogen at a fixed tidal volume. Curves are plotted with end-tidal inert gas

concentration (Cet) on the y-axis, and either breath number or turnover number (TO) on the

x-axis. Turnover number refers to the cumulative expired volume, expressed in terms of

multiples of functional residual capacity (FRC). Lung clearance index (LCI) is the most

commonly used index of VH, and is defined as the number of turnovers required for the Cet

to reach 1/40th of its initial value.


113

7.5.12 M. PExA measurements

With the PExA instrument, particles from a subject’s exhaled breath can be counts and

collected in a non-invasive way (www.pexa.se). Particles in exhaled air (PExA) are derived

from the small airways and are formed during airway closure and re-opening. Subjects are

instructed as follows:

a. First slowly breathe out as deep as possible and hold breath for 3-5 seconds. Then,

forcefully inhale to TLC.

b. Next, slowly exhale to RV during sample collection.

c. Repeat steps a-c until enough samples are collected or a maximum of 25 manoeuvres

are performed.

The particles collected consist mainly of phospholipids, proteins and several other well-

known mediators that control the development of inflammation. The sensitivity and

specificity of the PExA method combined with its non-invasive nature makes it a patient

friendly way to study the development, cause of action and treatment response of many

different respiratory diseases involving the peripheral airways.

7.5.13 N. Nasal brush for mRNA

A nasal swab will be collected at all visits and at the exacerbation visit to analyse the

presence of viruses during a stable phase of COPD and during an exacerbation.

Subjects are asked to blow their nose. The swabs are gently inserted into a nostril towards

the pharynx until resistance is felt and then rotate three times to obtain epithelial cells. The

swabs are then withdrawn and put into a tube containing universal transport medium. All of

the specimens are kept cool and are delivered to the laboratory within 3 hours of collection.

7.5.14 O. Nasopharyngeal swab

A nasopharyngeal swab will be collected at each visit to identify the presence of viruses

during a stable phase of COPD and during an exacerbation. Nasosorption may be

performed using devices produced by Hunt Developments (West Sussex, UK).

Respiratory virus diagnosis depends on the collection of high-quality specimens, their rapid

transport to the laboratory and appropriate storage before laboratory testing. Virus is best

detected in specimens containing infected cells and secretions. Specimens for the direct

detection of viral antigens or nucleic acids and virus isolation in cell cultures should be taken

preferably during the first 3 days after onset of clinical symptoms [23].

Subjects are then asked to blow their nose. The swabs are gently inserted into a nostril

towards the pharynx until resistance is felt and then rotate three times to obtain epithelial

cells [24]. The swabs are then withdrawn and put into a tube containing universal transport


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medium. All of the specimens are kept cool and are delivered to the laboratory within 3

hours of collection.

Nasal epithelium

We have previously found that the microarray technique is a useful tool to investigate the

mechanisms underlying relative corticosteroid sensitivity [25]. In the present study, we will

apply this technique to investigate genome-wide expression of mRNA, MiRNA and

methylation status in epithelial cells derived from nasal brushings. A major advantage of

nasal brushings above bronchial brushings is that they can be obtained in a non-invasive

way. In this context, it is important to mention that gene expression changes in bronchial

epithelium, e.g. after smoking are closely correlated to gene expression changes in the

nasal epithelium [26]. The present study aims to find predictors for a better ICS treatment

response e.g. fewer exacerbations, in patients with COPD. To this end, we will analyse

clinical data as well as nasal epithelial genome-wide gene expression data. We will perform

this in both a targeted way (i.e. the Th2 related gene expression signature associated with

a favourable treatment response in COPD) and unbiased (genome-wide) way.

Nasal epithelium collection will be performed as follows. The right nostril of the subjects is

examined and the inferior turbinate is located using a speculum and penlight. Subjects are

then asked to blow their nose and attempt to remove any mucous form the nose. 1mL of

lidocaine is collected in a 1mL or 2 mL syringe. Subjects are asked to make the sound of

the letter K repeatedly while .5mL of lidocaine is injected into the right nostril (aiming toward

the inferior turbinate) to numb the area to be brushed. The subjects are asked to lean

forward and any remaining lidocaine is collected onto a tissue. The steps are repeated with

another .5mL of lidocaine. After a few minutes, using the speculum to open the nostril, the

lateral area underneath the inferior turbinate is the brushed for 3 seconds and the brush is

placed in 2 mL screw-cap Eppendorf tube containing 1.5 mL of RNAProtect Cell solution.

The brush is then cut into the tube using a wire cutter cleaned with RNAse Zap and alcohol.

A second brush is collected in the same manner and placed in the same tube. Next, a third

brush is collected in the same manner and placed in new empty Eppendorf tube for DNA

collection. Both tubes are then labeled and stored at -80 oC until processing or shipping.

Finally, a fourth and fifth brush is collected and put in 10 mL PBS for cell cultures. As

performed previously in our lab, these cultured epithelial will be stored at -80 oC. This way

we have the unique opportunity to perform functional experiments to validate and further

investigate our findings of the genome-wide mRNA, miRNA, and methylation status

analyses described above.


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1. Clean wire cutter with RNAse Zap and alcohol

↓

2. Visualize inferior turbinate of right nostril with speculum and pen light

↓

3. Draw 1 mL of Lidocaine in 1 or 2 mL syringe

↓

4. Inject 0.5 mL of 1 or 2% Lidocaine into right nostril and ask to lean forward

↓

5. Repeat step 4 with leftover Lidocaine and wait 2 minutes

↓

6. Use speculum and brush lateral inferior turbinate for 3 seconds (or 3 rotations)

↓

7. Cut brush (below plastic) into Eppendorf cup filled with Cell Protect

↓

8. Repeat Step 6 and 7 with second brush

9. Repeat Step 6 with third brush and cut the third brush (below plastic)

in an empty Eppendorf tube.

↓

10. Repeat Step 6 with fourth and fifth and cut the fourth and fifth brushes

(below plastic) in an Eppendorf tube containing PBS.

↓

11. Label samples and store at -80o C

7.5.15 P. Blood collection

A total of 50 mL peripheral blood will be collected at visits 1-7 and at the exacerbation visit

for: Leucocytes and cell differentiation. This requires 10 mL peripheral blood collected in

EDTA tubes (purple cap). Two tubes for serum that will be stored to enable future analyses

of proteins depending on the GWAS and mRNA/microRNA analyses. Finally, genome-wide

genotyping will be performed to investigate genetic mechanisms underlying a favourable

course of FEV1 in COPD or a higher risk to have exacerbations. This requires 10 mL

peripheral blood collected in EDTA tubes (purple cap). From the same tubes plasma will be

obtained to be stored to enable future analyses of serum proteins depending on the GWAS

and mRNA/microRNA analyses. Finally, two PAXgene tubes for mRNA will be collected

(maximum 5 ml per tube).


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7.5.16 Q. Spontaneous sputum sample

– Sputum will be obtained at each visit, if the participant is able to produce spontaneous

sputum.

Sputum processing

The procedure for sputum processing is as follows:

1. Select sputum plugs from the saliva using fine forceps, with the aim that

approximately of 0.5-1g of plug weight sputum is subsequently processed (if only a

small amount of sputum is obtained, select as much as possible).

2. Transfer sputum plugs into an empty (pre-weighed) polypropylene centrifuge tube

with a screw top.

3. Calculate the weight of the sputum to be processed (weight of tube and sputum –

weight of the empty centrifuge tube).

4. Add 8 volumes x sputum weight (in grams) Dulbecco’s phosphate buffered saline

(D-PBS) to the tube.

5. Disperse the sputum by repeated gentle aspiration into a plastic pipette.

6. Add approximately 0.5mL of glass beads to the D-PBS tube.

7. Vortex tube for 15 seconds and then 15 minutes on a bench rocker. Re-vortex for

further 15 seconds.

8. Subaliquot 500 µl samples into 1.5ml Eppendorf tubes. Label with specimen type,

participant code, date of sample and store at -80oC for microbiology qPCR/16S and

any further virology studies.

9. Filter the remaining sputum suspension through a 48 µm nylon gauze placed in a

funnel, pre- wet filter with D-PBS and shake off the excess. Filter the suspension

into a pre-weighed, clean polypropylene centrifuge tube.

10. Centrifuge filtered sample at 2000g for 10 minutes (brake off).

11. Collect the supernatant into Eppendorf tubes and store at -80oC for any further

cytokine analysis.

7.6 Withdrawal of individual subjects

Subjects can leave the study at any time for any reason if they wish to do so without any

consequences. The investigator can decide to withdraw a subject from the study for urgent

medical reasons.

7.7 Specific criteria for withdrawal (if applicable)

Not applicable


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7.8 Replacement of individual subjects after withdrawal

No replacement of individual subjects will take place after withdrawal.


In case of premature ending of the study, the investigator will notify the METC including the

reasons for the premature termination.

8. SAFETY REPORTING

8.1 Temporary halt for reasons of subject safety

In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study

if there is sufficient ground that continuation of the study will jeopardise subject health or

safety. The sponsor will notify the accredited METC without undue delay of a temporary

halt including the reason for such an action. The study will be suspended pending a further

positive decision by the accredited METC. The investigator will take care that all subjects

are kept informed.

8.2 AEs, SAEs and SUSARs

8.2.1 Adverse events (AEs)

Adverse events are defined as any undesirable experience occurring to a subject during the

study, whether or not considered related to [the investigational product / trial procedure/ the

experimental intervention]. All adverse events reported spontaneously by the subject or

observed by the investigator or his staff will be recorded.

8.2.2 Serious adverse events (SAEs)

A serious adverse event is any untoward medical occurrence or effect that results in death;

- is life threatening (at the time of the event);

- requires hospitalisation or prolongation of existing inpatients’ hospitalisation;

- results in persistent or significant disability or incapacity;

- is a congenital anomaly or birth defect; or

- any other important medical event that did not result in any of the outcomes listed

above due to medical or surgical intervention but could have been based upon

appropriate judgement by the investigator.

An elective hospital admission will not be considered as a serious adverse event.


118

The investigator will report all SAEs to the sponsor without undue delay after obtaining

knowledge of the events, except for the following SAEs: mild, moderate and severe COPD

exacerbations.

The sponsor will report the SAEs through the web portal ToetsingOnline to the accredited

METC that approved the protocol, within 7 days of first knowledge for SAEs that result in

death or are life threatening followed by a period of maximum of 8 days to complete the

initial preliminary report. All other SAEs will be reported within a period of maximum 15 days

after the sponsor has first knowledge of the serious adverse events.


All AEs will be followed until they have abated, or until a stable situation has been reached.

Depending on the event, follow up may require additional tests or medical procedures as

indicated, and/or referral to the general physician or a medical specialist. SAEs need to be

reported till end of study within the Netherlands, as defined in the protocol


9.1 Analysis of demographic variables

Demographic variables as age, gender, BMI, smoking history, medication use, medical

history will be expressed as means with standard deviations or medians with interquartile

ranges as appropriate for continuous variables, and number (percentages) for dichotomous

variables. Spirometry data and (FEV1, IVC, FVC, FEF25, FEF50, FEF75, FEF25-75), MBNW

data (LCI, Sacin, Scond), PExA data (number of exhaled particles), HRCT scan data

(percentage of emphysema and functional small airways disease), body plethysmography

data (TLC, FRC, RV), IOS data (R5, R20, R5-R20, X5), and diffusion capacity data (TLCOc,

TLCOcVA) will be described likewise. Statistical analyses will be performed using either

SPSS version 18 or a more recent version or with R statistical software version 3.0.1 or

higher.

9.2 Analysis of the primary and secondary end-parameters

The primary end-parameter will be analysed using a linear mixed effects model, adjusting

for age, gender, smoking status and baseline FEV1, with the logarithm of time (in days)

during which patients were followed-up after the occurrence of an RSV infection as offset

variable. Other secondary variables will be analysed using a linear model adjusting for age,

gender, smoking status and baseline FEV1.


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9.3 GWAS, microbiome, and genome-wide mRNA and microRNA expression

analyses.

For the GWAS, microbiome, and genome-wide mRNA and microRNA expression, and DNA

methylation analyses, we will apply the following approach on base-2 log-transformed

expression data using the statistical software R version 3.0.1 or a later version. First, we

will analyse the baseline samples obtained at visit 1. In these samples, we will assess which

SNPs, microbiome characteristics, and mRNA and microRNA expression levels are

associated with severity of airflow obstruction in COPD and with the number of

exacerbations during the follow-up period of the study. To this end, a generalized linear

model will be applied with the logarithm of time during follow-up after the start of treatment

(in days) as offset variable. For all analyses, a loose false discovery rate (FDR) cut-off of

0.25 will be maintained. Once identified, we will validate our findings by PCR and or

measurement of protein levels in blood, plasma, nasal epithelium, or sputum supernatant.

For this purpose samples will be stored allowing future measurements. Second, functional

enrichment analysis will be performed using Gene Set Enrichment Analysis (GSEA) version

2.07 or a later version [27]. To this end, genes will be ranked according to the strength of

their t-statistic reflecting their association with parameters for large or small airway function

or their change with corticosteroid-treatment and a GSEA analysis applied.

9.4 Interim analysis (if applicable)

No interim analyses will be performed.



The study will be conducted in accordance with the Declaration of Helsinki (October 2013).

In addition, the study will be conducted in accordance with the Medical Research Involving

Human Subjects Act.


COPD patients will be recruited via the outpatient clinic of the department of pulmonary

diseases of the UMCG or other study site in the Netherlands. Applicable subjects who are

enrolled in the METC-approved SMART study (METc 2014/335) will be asked to participate

in this study after they finalized the SMART study participation; the characterisation of both

studies has a substantial overlap. Of course, this will only be done if the subjects indicated

on their informed consent form that they gave approval to be asked to participate in future

studies. In addition, they will be recruited via outpatient clinics of hospitals surrounding the


120

UMCG. To this end, patients will first be contacted by their treating physician. If patients are

willing to participate, they are referred by their treating physician to the investigator. In

addition, we will mail a letter to patients with known COPD asking if they would be interested

to participate in a study (see Appendix I). If patients choose to take part they will be able

send the reply-card back to the investigator (see Appendix I). Finally, patients will be

recruited via advertisements in local newspapers (see Appendix II). If patients respond

positively, a first visit will be planned. The investigator will explain the purpose and the detail

of the study to each patient. Their decision is voluntary and each should be competent to

understand what is involved. Written informed consent will be obtained from all patients.

This way, we expect to reach the target number of patients needed for this study within 12-

18 months.


No investigational product will be used in this study. It is not expected that subjects will

receive any individual benefit from participation in this study; however, the findings from this

study may increase knowledge about the role RSV plays in the onset of events leading to

worsening cardiorespiratory status in the aforementioned patient populations and in the

pathological and biological changes that occur during such events. This knowledge may

help in understanding if there is a need for future RSV interventions or prophylaxes in this

patient population. In this study, subjects will be treated according to usual standard of care

as determined by the treating physician. Some procedures in this study, such as nasal swab

collection and spontaneous sputum, may not be usual practice for study sites. Nasal swab

collections have the potential to irritate the intranasal cavity and lead to acute epistaxis;

however, the risks associated with discomforts from such events are minimal. Additional

risks include obtaining blood that may sometimes cause pain at the site where the blood is

drawn, bruising, and occasional light-headedness and, rarely, fainting; performing

spirometry may cause mild chest tightening and coughing. There are no other risks to

subjects in this study above that from the usual treatment of their disease.


The sponsor/investigator has a liability insurance, which is in accordance with article 7,

subsection 6 of the WMO.

The sponsor (also) has an insurance, which is in accordance with the legal requirements in

the Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for


121

Clinical Research in Humans of 23th June 2003). This insurance provides cover for

unexpected damage to research subjects through injury or death caused by the study.

1. € 450.000,-- (i.e. four hundred and fifty thousand Euro) for death or injury for each subject

who participates in the Research;

2. € 3.500.000,-- (i.e. three million five hundred thousand Euro) for death or injury for all

subjects who participate in the Research;

3. € 5.000.000,-- (i.e. five million Euro) for the total damage incurred by the organization for

all damage disclosed by scientific research for the Sponsor as ‘verrichter’ in the meaning of

said Act in each year of insurance coverage.

The insurance applies to the damage that becomes apparent during the study or within 4

years after the end of the study.

10.5 Incentives

Participants will receive compensation for travelling to and from the hospital. In addition,

subjects will receive a compensation for lost working hours. They will receive 25 euro for

each visit resulting in a maximum of 175 euro if they have completed the whole study, i.e.

attended 7 visits to the outpatient clinic. For exacerbation visits, patients will receive

compensation for travelling to and from the hospital, but no additional compensation for time

to avoid an ‘incentive bias’.



Handling of the personal data in this observational study will comply with the Dutch Personal

Data Protection Act. Data will be handled confidentially and anonymously. To trace the data

of individual patients, the study will use a subject identification code list that is linked to the

date of the participating patients. The code will not be based on the patient initials and birth

date, but on number of enrolment into the study.


The study will be monitored by an independent monitor with appropriate training according

to GCP guidelines.

11.3 Handling and storage of samples

Samples of serum, plasma, blood, sputum supernatant and epithelial RNA


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The samples mentioned here are optional samples collected for future analyses (plasma,

serum, nasal epithelium and sputum supernatant). Samples will be stored after the end of

the study for a maximum of 25 years.

With respect to the sponsor guarantees for storage of the samples, the following:

1. Future analyses of the samples will relate only or in relationship to the pathobiology

of COPD.

1. The samples are sent are encrypted and cannot be traced to the subject directly.

2. The facilities where these samples are sent to are designed for the safe storage of

samples.

3. Samples will be stored after the end of the study for a maximum of 25 years.

11.4 Amendments

Amendments are changes made to the research after a favourable opinion by the

accredited METC has been given. All amendments will be notified to the METC that gave a

favourable opinion.





adverse events/ serious adverse reactions, other problems, and amendments.

11.6 Temporary halt and (prematurely) end of study report

The investigator/sponsor will notify the accredited METC of the end of the study within a

period of 8 weeks. The end of the study is defined as the last patient’s last visit.



In case the study is ended prematurely, the sponsor will notify the accredited METC within

15 days, including the reasons for the premature termination.



accredited METC.


The study will be registered in a public trial registry and the results of this study will be

disclosed unreservedly.


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