A Study in Adolescent Females to Explore Cytomegalovirus Infection
Phase: NA
Conditions: Infections, Cytomegalovirus
Sponsor: GlaxoSmithKline

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Study Protocol
Sponsor:
GlaxoSmithKline Biologicals
Rue de l’Institut, 89
1330 Rixensart, Belgium
eTrack study number and 
Abbreviated Title
115639 (CMV-014 EXPLO)
Date of protocol
Final: 06 April 2012
Date of protocol amendment
Protocol Amendment 1 Final: 31 July 2013
Protocol Amendment 2 Final: 19 February 2015
Title
A study in adolescent females to explore 
cytomegalovirus infection.
Detailed Title
A study to explore cytomegalovirus primary 
infection, re-activation and re-infection in an 
adolescent female population.
Co-ordinating author
(Keyrus Biopharma contractor for 
GSK Biologicals), 
, Scientific 
Writers
Contributing authors
(Amended 19 February 2015)

,
, Clinical 
Research and Development Leads

,
, Study 
Delivery Lead

, Study Delivery Manager

,
, 
Project Statistician

, 
, 
 (Business & Decision 
Life Sciences for GSK Biologcals), GVCL
Representatives

,
,
 
(Keyrus Biopharma contractor for 
GSK Biologicals),
(TCS 
Consultant for GSK Biologicals),
 
, Study Data Manager

, Project Data Manager

,
 
, 
, Safety Representatives

, Epidemiologist
GSK Biologicals’ protocol template for observational studies and interventional studies 
without administration of medicinal products as described in a research protocol based 
on the Protocol Document Standard version 13.2
Copyright 2012-2015 of the GlaxoSmithKline group of companies. All rights reserved. 
Unauthorised copying or use of this information is prohibited.
 
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Protocol Amendment 2 Sponsor Signatory Approval
eTrack study number and 
Abbreviated Title
115639 (CMV-014 EXPLO)
Date of protocol amendment
Protocol Amendment 2 Final: 19 February 2015
Detailed Title 
A study to explore cytomegalovirus primary 
infection, re-activation and re-infection in an 
adolescent female population.
Sponsor signatory
(Amended 19 February 
2015)
Jeanne-Marie Devaster
Director Clinical Research and Translational 
Science
Signature
Date
 
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Protocol Amendment 2 Rationale
Amendment number:
Amendment 2 Final
Rationale/background for changes: 
Protocol amendment 2 was prepared:

To cancel the planned interim analyses and to keep the final analysis only: The 
need to validate and develop some assays has led to a delay in the release of data 
on time. Since no vaccine is administered in the CMV-014 EXPLO study and as 
the interim analyses were not linked to subjects’ safety or efficacy, but rather to 
exploring the natural history of CMV disease, it was decided to group the planned 
interim analysis after Year 1 and Year 2 and to have a single final analysis of the 
total follow-up at the end of the study.

To clarify that the tertiary endpoints (i.e. exploratory tests) and the subcohort 
selected for testing will be based on the evaluations of primary and secondary 
endpoints.

To stop collection of blood samples for gene expression signature (qPCR or RNA 
microarray) as of protocol amendment 2 approval: A Year 1 dataset will be used 
for analysis of gene expression in a subset of subjects.
In addition, the list of contributing authors together with the Sponsor Signatory 
Approval page have been updated following changes in the team members/ function 
names. The notation of the terms “ATP cohort” and “Total cohort” were adapted to 
maintain consistency throughout the protocol.
The summary of the amendment will be provided in APPENDIX C.
 
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Protocol Amendment 2 Investigator Agreement
I agree:

To conduct the study in compliance with this protocol, any mutually agreed 
future protocol amendments or protocol administrative changes, and with 
any other study conduct procedures and/or study conduct documents 
provided by GlaxoSmithKline Biologicals (GSK Biologicals).

To assume responsibility for the proper conduct of the study at this site.

That I am aware of, and will comply with, ‘Good Clinical Practice’ (GCP)
and all applicable regulatory requirements.

To ensure that all persons assisting me with the study are adequately 
informed about study-related duties and functions as described in the 
protocol.

To acquire the reference ranges for laboratory tests performed locally and, if 
required by local regulations, obtain the laboratory’s current certification or 
Quality Assurance procedure manual.

To ensure that no samples (including serum samples) are retained onsite or 
elsewhere without the approval of GSK Biologicals and the express written 
informed consent of the subject and/or the subject’s legally authorised 
representative.

To perform no other biological assays on the samples except those described 
in the protocol or its amendment(s).

To co-operate with a representative of GSK Biologicals in the monitoring 
process of the study and in resolution of queries about the data.

That I have been informed that certain regulatory authorities require the 
sponsor to obtain and supply, as necessary, details about the investigator’s 
ownership interest in the sponsor, and more generally about his/her financial 
ties with the sponsor. GSK Biologicals will use and disclose the information 
solely for the purpose of complying with regulatory requirements.
Hence I:

Agree to supply GSK Biologicals with any necessary information regarding 
ownership interest and financial ties (including those of my spouse and 
dependent children).

Agree to promptly update this information if any relevant changes occur 
during the course of the study and for one year following completion of the 
study. 

Agree that GSK Biologicals may disclose any information it has about such 
ownership interests and financial ties to regulatory authorities.

Agree to provide GSK Biologicals with an updated Curriculum Vitae and 
other documents required by regulatory agencies for this study.
 
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eTrack study number and 
Abbreviated Title
115639 (CMV-014 EXPLO)
Date of protocol amendment
Protocol Amendment 2 Final: 19 February 2015
Detailed Title
A study to explore cytomegalovirus primary 
infection, re-activation and re-infection in an 
adolescent female population.
Investigator name
Signature
Date
 
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Sponsor Information
1.
Sponsor
GlaxoSmithKline Biologicals
Rue de l’Institut, 89
1330 Rixensart, Belgium
2.
Sponsor Medical Expert for the Study
Refer to the local study contact information document.
3.
Sponsor Study Monitor
Refer to the local study contact information document.
4.
Sponsor Study Contact for Reporting of a Serious Adverse Event
GSK Biologicals Central Back-up Study Contact for Reporting SAEs: refer to
protocol Section 6.3.2.
 
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SYNOPSIS
Detailed Title
A study to explore cytomegalovirus primary infection, re-
activation and re-infection in an adolescent female population.
Rationale for the 
study 
GSK Biologicals anticipates developing a prophylactic 
cytomegalovirus (CMV) vaccine indicated for the use in 
women and adolescents to protect their offspring against 
congenital cytomegalovirus (cCMV) infection and disease. 
This study is therefore designed:

To estimate the incidence of CMV secondary 
infections (re-infections / re-activations) in adolescent 
females.

To estimate the incidence of CMV primary infection 
in adolescent females.

To develop assays able to distinguish and detect re-
infections versus re-activations.
Objectives
Primary

To estimate the incidence of CMV secondary 
infections in seropositive adolescent females.
Secondary

To estimate the incidence of CMV primary infections 
in seronegative adolescent females.
Tertiary

To develop assays that allow the differentiation of 
CMV secondary infections caused either by re-
infection or by re-activation, in CMV seropositive 
adolescent females.

To assess the diagnostic value of different assays to 
evaluate seroconversion in blood and the detection of 
CMV DNA in different body fluids.

To describe the proportion of secondary infection 
caused either by re-infection or re-activation.

To collect samples for immunological and biological 
disease-related exploratory assays.

To explore socio-demographic or behavioral factors 
associated with CMV infection.

In case of pregnancies, to document the birth 
prevalence of CMV congenital infections.
 
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
To evaluate the concordance between screening testing 
data at local laboratories (or alternatively, central 
laboratories) and the GSK Biologicals or designated 
laboratory testing data.
Study design

Prospective, multi-center, multi-country study.

Visit schedule:

10 scheduled Site Visits approximately every 
4 months.

9 Sample Collection Visits approximately at mid-
term between 2 site visits (i.e. approximately 
2 months after a Site Visit) where samples will be 
collected either through self-collection by the 
subjects, through collection by a home-visiting nurse 
or through a new visit at the study center.

Study population: Adolescent females aged between 
10 and 17 years regardless of CMV status.

Type of study: Self-contained.

Data collection: Electronic Case Report Form (eCRF).

Duration of the study:
Participant: Approximately 36 months.
Newborn (in case of pregnancy): Approximately 10 days.

Epoch 001: Prospective data collection starting at 
Visit 1 (Month 0) and ending at Visit 10 (Month 36).
Synopsis Table 1
Study groups foreseen in the study
Study Groups
Number of subjects
Age (Min/Max)
S+
+/- 240 seropositive subjects
10 – 17 years
S-
Estimated approximate number of seronegative subjects(1): 160
10 – 17 years
(1) As subjects will be enrolled consecutively without previous CMV screening with a target number of 
240 seropositive subjects, the number of seronegative subjects enrolled will depend on the seroprevalence of the 
participating countries.
 
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Discussion of study 
design
The study sites will be selected in multiple countries, 
including countries where the CMV prevalence is assumed to 
be high in order to enroll primarily CMV seropositive 
adolescent females to allow the evaluation of CMV secondary 
infections. Since enrolment will proceed without previous 
CMV screening, some of the enrolled subjects will be 
seronegative. The enrolled seronegative subjects will allow 
the estimation of the incidence of CMV primary infections in 
the adolescent population.
To increase the probability to capture all CMV infections 
(primary and secondary infections), regular sample collection 
time-points have been arranged:

Site Visits: Subjects will be asked to perform a Site 
Visit approximately every 4 months for 3 years 
(10 Site Visits in total). At Site Visits, blood, urine and 
saliva, will be collected.

Sample Collection Visits: Subjects will be contacted 
to perform urine and saliva sample collections at home
either by self-collection or by a home-visiting nurse. 
Alternatively subjects may be asked to come back to 
the study center where the investigator or designate 
will collect the samples. These Sample Collection 
Visits will occur approximately every 4 months for 
3 years between 2 Site Visits (9 Sample Collection 
Visits in total).
Number of subjects
Subjects will be enrolled consecutively without previous 
CMV screening. Enrolment will be terminated once 
approximately 240 seropositive subjects are included in the 
trial. The number of seronegative subjects enrolled will 
depend on the seroprevalence of the participating countries. It 
is estimated that approximately 400 subjects will be enrolled 
in total.
Endpoints
Primary

Occurrence of CMV secondary infections determined in 
all seropositive subjects on samples collected during the 
4-month Site Visits until study conclusion:

Anti-CMV tegument protein IgG antibody
concentration in serum (ELISA).

Number of CMV DNA copies (pp65 or other genes) 
in urine (qPCR).
 
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Secondary

Occurrence of CMV primary infection determined in all 
seronegative subjects on samples collected during the 4-
month Site Visits until study conclusion:

Anti-CMV tegument protein IgG antibody 
concentration in serum (ELISA).
Tertiary (amended 19 February 2015)

Further characterization of primary infection in initially
seronegative subjects during the 4-month Site Visits until 
study conclusion (testing done upon seroconversion)
might be done:

Number of CMV DNA copies (pp65 or other genes) 
in urine (qPCR).

Further characterization of primary and /or secondary 
CMV infections in a subset of subjects on selected 
samples of the Sample Collection Visits* might be done.

Number of CMV DNA copies (pp65 or other genes) 
in urine (qPCR).

Number of CMV DNA copies (pp65 or other genes) 
in saliva (qPCR).
* In case of a positive sample at a 4-month Site Visit time point, 
testing might be done on selected samples collected during the 
Sample Collection Visit. The selection of samples to be tested will 
be based on the qPCR results obtained following the Site visits.

Further characterization of primary and /or secondary 
CMV infections in a subset of subjects on all available 
samples of the 4-month Site Visits might be done:

Anti-CMV tegument protein IgG antibody avidity 
index in serum (ELISA).

Anti-gB IgG antibody avidity index in serum 
(ELISA).

Anti-gB IgG antibody concentration in serum 
(ELISA).

Anti-CMV IgM antibody concentration in serum 
(ELISA).

Number of CMV DNA copies (pp65 or other genes) 
in saliva and blood (qPCR).

Assessment of anti-CMV neutralizing antibodies on 
different target cells and/or other virus strains.
 
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
Development of assays that will allow differentiating 
re-infection from re-activation from primary infection 
in a subset of subjects might be done.

Characterization of CMV strains by 
genotyping/sequencing.

Exploring the CMV strain specific antibody profile 
using peptide microarrays.

Assessment of the expression of host genes* using 
techniques such as qPCR or mRNA microarray in a 
subset of subjects might be performed.
* excluding genes related to hereditary characteristics of 
the subject.

Evaluation of the impact of demographic, social or 
behavioral factors upon CMV infection might be 
performed.

Occurrence of congenital CMV infection in newborns 
of subjects who become pregnant during the study
might be evaluated:

Evidence of CMV DNA in urine and/or saliva of 
newborns within 10 days of delivery by using qPCR 
(pp65 or other genes) might be tested.
 
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TABLE OF CONTENTS
PAGE
SPONSOR INFORMATION ............................................................................................6
SYNOPSIS......................................................................................................................7
LIST OF ABBREVIATIONS (AMENDED 19 FEBRUARY 2015) ....................................17
GLOSSARY OF TERMS ...............................................................................................19
1.
INTRODUCTION....................................................................................................22
1.1.
Background ................................................................................................22
1.2.
Rationale for the study................................................................................23
2.
OBJECTIVES.........................................................................................................23
2.1.
Primary objective ........................................................................................23
2.2.
Secondary objective ...................................................................................24
2.3.
Tertiary objectives.......................................................................................24
3.
STUDY DESIGN OVERVIEW ................................................................................25
3.1.
Discussion of study design .........................................................................26
4.
STUDY POPULATION...........................................................................................27
4.1.
Number of subjects/ centers .......................................................................27
4.2.
Inclusion criteria for enrolment....................................................................27
4.3.
Exclusion criteria for enrolment...................................................................28
5.
CONDUCT OF THE STUDY ..................................................................................29
5.1.
Regulatory and ethical considerations, including the informed 
consent process..........................................................................................29
5.2.
Subject identification...................................................................................30
5.3.
General study aspects ................................................................................30
5.4.
Outline of study procedures ........................................................................31
5.5.
Detailed description of study procedures ....................................................32
5.5.1.
Procedures prior to study participation.........................................32
5.5.1.1.
Informed consent........................................................32
5.5.1.2.
Check inclusion and exclusion criteria ........................33
5.5.2.
Procedures during study participation..........................................33
5.5.2.1.
Collect demographic data...........................................33
5.5.2.2.
Collect social and behavioral data ..............................33
5.5.2.3.
Medical history ...........................................................33
5.5.2.4.
CMV serostatus..........................................................33
5.5.2.5.
Blood sampling...........................................................33
5.5.2.6.
Urine sampling ...........................................................34
5.5.2.7.
Saliva sampling ..........................................................34
5.5.2.8.
Distribution of subject sample collection 
booklet........................................................................34
5.5.2.9.
Check Elimination criteria ...........................................35
5.5.2.10.
Record any concomitant 
medication/vaccination ...............................................35
 
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5.5.2.11.
Recording of Serious Adverse Events related 
to study participation...................................................35
5.5.2.12.
Reporting of pregnancy and pregnancy 
outcome .....................................................................35
5.5.2.13.
Study conclusion ........................................................35
5.5.3.
Procedure during study participation for the newborns (in 
case of pregnancy and delivery during the study period) .............35
5.5.3.1.
Urine/saliva collection.................................................36
5.5.3.2.
Record CMV infection status ......................................36
5.5.3.3.
Assessment of CMV disease at birth..........................36
5.6.
Biological Sample handling and analysis ....................................................36
5.6.1.
Use of specified study materials ..................................................37
5.6.2.
Biological samples.......................................................................37
5.6.3.
Laboratory assays .......................................................................37
5.6.4.
Biological samples evaluation......................................................40
5.6.4.1.
Read-outs...................................................................40
6.
SERIOUS ADVERSE EVENTS..............................................................................40
6.1.
Safety definitions ........................................................................................41
6.1.1.
Definition of an adverse event......................................................41
6.1.2.
Definition of a serious adverse event ...........................................42
6.1.3.
Clinical laboratory parameters and other abnormal 
assessments qualifying as serious adverse events......................42
6.2.
Detecting and recording serious adverse events.........................................43
6.2.1.
Time period for detecting and recording serious adverse 
events..........................................................................................43
6.2.2.
Evaluation of serious adverse events...........................................43
6.2.2.1.
Active questioning to detect serious adverse 
events.........................................................................43
6.2.2.2.
Assessment of intensity..............................................44
6.2.2.3.
Assessment of causality .............................................44
6.2.2.4.
Assessment of outcomes............................................45
6.3.
Reporting of serious adverse events...........................................................45
6.3.1.
Prompt reporting of serious adverse events related to a 
study procedure to GSK Biologicals.............................................45
6.3.2.
Contact information for reporting serious adverse events 
and other events to GSK Biologicals............................................45
6.3.3.
Completion and transmission of SAE reports related to a 
study procedure to GSK Biologicals.............................................45
6.3.3.1.
Back-up system in case the electronic SAE 
reporting system does not work..................................46
6.3.3.2.
Updating of SAE information after freezing of 
the subject’s eCRF .....................................................46
6.4.
Follow-up of serious adverse events...........................................................46
7.
SUBJECT COMPLETION AND WITHDRAWAL.....................................................47
7.1.
Subject completion .....................................................................................47
7.2.
Subject withdrawal......................................................................................47
8.
DATA EVALUATION: CRITERIA FOR EVALUATION OF OBJECTIVES...............48
8.1.
Endpoints....................................................................................................48
8.1.1.
Primary endpoints........................................................................48
 
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8.1.2.
Secondary endpoints ...................................................................48
8.1.3.
Tertiary endpoints (amended 19 February 2015) .........................48
8.2.
Sample size consideration ..........................................................................49
8.3.
Classification of cases (amended 19 February 2015)..................................49
8.4.
Study cohorts to be evaluated.....................................................................50
8.4.1.
Total cohort..................................................................................50
8.4.2.
According-to-protocol cohort (amended 19 February 2015).........50
8.5.
Derived and transformed data.....................................................................51
8.6.
Conduct of analyses ...................................................................................51
8.6.1.
Sequence of analyses (amended 19 February 2015)...................51
8.7.
Statistical methods......................................................................................51
8.7.1.
Analysis of demographics/baseline characteristics ......................52
8.7.2.
Analysis of primary objective........................................................52
8.7.3.
Analysis of secondary objective...................................................52
8.7.4.
Tertiary objectives (amended 19 February 2015).........................52
9.
ADMINISTRATIVE MATTERS ...............................................................................53
9.1.
Remote Data Entry instructions ..................................................................53
9.2.
Monitoring by GSK Biologicals....................................................................53
9.3.
Archiving of data at study sites ...................................................................54
9.4.
Audits .........................................................................................................55
9.5.
Posting of information on public registers....................................................55
9.6.
Ownership, confidentiality and publication ..................................................55
9.6.1.
Ownership ...................................................................................55
9.6.2.
Confidentiality ..............................................................................55
9.6.3.
Publication...................................................................................56
9.6.4.
Provision of study results to investigators and publication............56
10. COUNTRY SPECIFIC REQUIREMENTS...............................................................56
11. REFERENCES.......................................................................................................57
 
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LIST OF TABLES
PAGE
Table 1
Study groups foreseen in the study ........................................................26
Table 2
List of study procedures (amended 19 February 2015) ..........................31
Table 3
List of study procedures for newborns/mothers (in case of 
pregnancy).............................................................................................32
Table 4
Intervals between study Site Visits/Sample Collection Visits ..................32
Table 5
Biological samples (amended 19 February 2015)...................................37
Table 6
Humoral Immunity (Antibody determination) (amended 19 
February 2015) ......................................................................................38
Table 7
Molecular Biology (amended 19 February 2015)....................................39
Table 8
Read-outs (amended 19 February 2015) ...............................................40
Table 9
Timeframes for submitting SAEs............................................................45
Table 10
GSK Biologicals’ laboratories .................................................................62
Table 11
Outsourced laboratories.........................................................................62
 
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LIST OF APPENDICES
PAGE
APPENDIX A LABORATORY ASSAYS .......................................................................59
APPENDIX B CLINICAL LABORATORIES ..................................................................62
APPENDIX C AMENDMENTS TO THE PROTOCOL...................................................63
 
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LIST OF ABBREVIATIONS (amended 19 February 2015)
AE
Adverse Event
Ag
Antigen
ATP
according-to-protocol
CI
Confidence Interval
cCMV
congenital Cytomegalovirus
CIOMS
Council for International Organizations of Medical 
Sciences
CMV
Cytomegalovirus
DNA
Deoxyribonucleic acid
eCRF
electronic Case Report Form
ED50
Effective dose 50
ELISA
Enzyme-linked immunosorbant assay
EU
ELISA unit
gB
Glycoprotein B
GCP
Good Clinical Practice
gH
Glycoprotein H
GSK
GlaxoSmithKline
GMT
Geometric mean titer
HIV
Human Immunodeficiency Virus
HRP
Horseradish peroxidase
ICF
Informed Consent Form
ICH
International Conference on Harmonization
IEC
Independent Ethics Committee
Ig
Immunoglobulin
IgG
Immunoglobulin class G
 
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IgM
Immunoglobulin class M
IRB
Institutional Review Board
LAR
Legally Acceptable Representative
NA
Not applicable
mRNA
Messenger Ribonucleic acid
OD
Optical density
PCR
Polymerase Chain Reaction
qPCR
Quantitative Polymerase Chain Reaction
pp65
Phosphoprotein 65
RDE
Remote Data Entry
RNA
Ribonucleic acid
S-
CMV Seronegative group
S+
CMV Seropositive group
SAE
Serious Adverse Event
SAP
Statistical Analysis Plan
SDV
Source Document Verification
SPM
Study Procedures Manual
TBD
To be determined
 
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GLOSSARY OF TERMS
Adverse event:
Any untoward medical occurrence in a subject,
temporally associated with the use of a medicinal product, 
whether or not considered related to the medicinal 
product, or temporally associated with a study procedure.
An AE can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding), 
symptom, or disease (new or exacerbated) temporally 
associated with the use of a medicinal product. For 
marketed medicinal products, this also includes failure to 
produce expected benefits (i.e. lack of efficacy), abuse or 
misuse.
Anonymisation:
Information that identifies a specific individual 
(including, but not limited to name, address and national 
identification number such as social security number, date 
of birth) has been removed and no link to the donor, 
through a code number for example, is maintained.
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.
CMV secondary 
infection:
Recurrent CMV infection, defined as either a viral re-
activation or a re-infection with a new strain of CMV.
Coded:
Information is associated with a subject number i.e. a 
code number. Coded information can only be linked back 
to the individual via a key code i.e. a listing of the 
research participants and their code. Within the 
pharmaceutical industry coding data is the usual
mechanism used for protecting an individual’s research 
data. The key code is kept secure, usually by the 
investigator, and GSK researchers cannot identify the 
research individual other than in exceptional and 
controlled circumstances.
 
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Cohort study:
A form of epidemiology 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). 
Eligible:
Qualified for enrolment into the study based upon strict 
adherence to inclusion/exclusion criteria.
Epidemiology study:
An observational study or an interventional study without 
administration of medicinal product(s) as described in a 
research protocol.
Epoch:
An epoch is a well defined part of a protocol that covers a 
set of consecutive time points. Generally, an epoch is 
self-contained and allows to perform a data analysis to 
address some of the trial objectives (e.g. primary, booster, 
yearly follow-ups, retrospective data collection, 
prospective data collection).
eTrack:
GSK’s tracking tool for clinical/epidemiology trials.
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 8.4 for details on criteria for evaluability).
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.
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 endpoints.
Research protocol:
A document that describes the objective(s), design, 
methodology, statistical considerations, and organization 
of a trial. The protocol usually also gives the background 
and rationale for the trial, but these could be provided in 
other protocol referenced documents.
Seronegative subject:
Subject for whom anti-CMV immunoglobulin G (IgG) 
antibodies are not detected in serum sample collected at 
Visit 1.
Seropositive subject:
Subject for whom anti-CMV IgG antibodies are detected 
in serum sample collected at Visit 1.
 
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Site Monitor:
An individual assigned by the sponsor who is responsible 
for assuring proper conduct of clinical/ epidemiology
studies at one or more investigational sites.
Study population:
Sample of population of interest.
Subject:
Term used throughout the protocol to denote an 
individual who has been contacted in order to participate 
or participates in the clinical/ epidemiology 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.
 
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1.
INTRODUCTION
1.1.
Background
Cytomegalovirus (CMV), a member of the herpes virus family, is a double-stranded 
deoxyribonucleic acid (DNA) virus. CMV infects a large proportion of the world’s 
population, with a seropositivity ranging from 30% to 100% in adults, depending on the 
countries, populations, density and socioeconomic status [Cannon, 2010].
CMV is generally passed from infected people to others through direct contact with body 
fluids (such as saliva, tears, blood, breast milk, semen and urine). It can also be 
transmitted from a pregnant woman to her fetus during pregnancy. The virus in the 
mother’s blood crosses over the placenta and infects the fetus’s blood. A primary 
infection with CMV is likely to be asymptomatic in healthy individuals, although it may 
cause a mild mononucleosis-like syndrome. However, CMV infection can be serious or 
even life-threatening in subjects with deficient or immature immune systems, such as 
congenitally infected fetuses, organ-transplant patients and human immunodefiency virus 
(HIV)-infected patients. The most frequent complications from congenital CMV (cCMV) 
infection are neonatal neurological sequelae (mainly sensorineural hearing loss) 
following infection of the mother during pregnancy [Mussi-Pinhata, 2009; Fowler, 2006].
Congenital CMV infection results from in utero transmission from the mother to her 
fetus, either because she undergoes a primary (i.e. first) CMV infection during pregnancy 
or because she experiences a recurrent CMV infection, defined as either viral re-
activation or viral re-infection with a different strain of CMV [Hyde, 2010]. Recent 
studies have shown that maternal re-infection by new strains of CMV is a major source of 
congenital infection in seropositive population [Yamamoto, 2010; Ross, 2006].
The overall prevalence of cCMV infection in industrialized countries, regardless of the 
serostatus of the mother, is estimated to be 0.6-0.7% [Dollard, 2007]. From these, it is 
estimated that 12.7% of the CMV infected children present CMV-specific symptoms 
(such as mental retardation, deafness) at birth. From the remaining 87.3% of children 
without symptoms at birth an additional 13.5% may develop permanent sequelae (such as 
primarily deafness) [Dollard, 2007].
Upon primary infection of the mother during pregnancy (between 1.38% and 3.85% per 
year [Colugnati, 2007]), the transmission rate of the virus to the fetus is approximately 
30% in contrast to seropositive women in whom vertical transmission has been estimated 
to be 1%. In countries or populations where high seropositivity rates are observed 
actually more cCMV infected babies will be born from secondary infections (re-
infection/ re-activation) than from primary infections. 
As pre-existing maternal immunity to CMV provide some protection against vertical 
transmission of the virus [Nyholm, 2010], primary maternal infection may place the child 
at higher risk of permanent and serious disabilities [Hyde, 2010]. It is generally believed 
that primary infections cause more severe disease than re-infections or re-activations 
[Fowler, 2003]. However, several investigators have also reported severe cCMV disease 
in babies born from seropositive mothers resulting in lifelong disabilities, mainly leading 
 
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to hearing loss [Boppana, 1999; Ross, 2006; Nyholm, 2010; Yamamoto, 2011; Mussi-
i-Pinhata, 2009]. 
Some attempts have been made to evaluate the relative contribution of re-activation of 
latent virus or re-infection with a different CMV strain to intrauterine transmission of the 
virus to the infants born to women with pre-existing immunity. An enzyme-linked 
immunosorbant assay (ELISA) was developed to distinguish serological responses 
against infection with different CMV strains [Novak, 2008; Boppana, 2001]. The 
principle of this assay was based on the defined heterogeneity of antibody binding
epitopes of glycoprotein H (gH) and/or glycoprotein B (gB) from two laboratory strains 
of CMV (AD169 and Towne). The detection of new antibody reactivity to either epitope 
on gH or gB in serum samples of seropositive women was considered as seroconversion 
and infection with a new virus strain (re-infection). One limitation of this assay is that not 
all CMV-specific IgG-positive individuals can be identified using these antigens. 
Specimens from more than a third of the seropositive individuals did not contain 
antibodies against any of the four antigenic determinants tested. This suggests the 
presence of additional polymorphic epitopes on gH and gB as well as on other envelope 
glycoproteins of CMV, such as glycoprotein N.
Providing protection against cCMV infection in babies born to mothers with pre-existing 
immunity will be a major challenge for any vaccine in development. The feasibility of 
running vaccine trials to demonstrate vaccine efficacy in this seropositive population 
needs to be carefully explored.
1.2.
Rationale for the study
GSK Biologicals anticipates developing a prophylactic CMV vaccine indicated for the 
use in women and adolescents to protect their offspring against cCMV infection and 
disease. This study is therefore designed:

To estimate the incidence of CMV secondary infections (re-infections / re-
activations) in adolescent females.

To estimate the incidence of CMV primary infection in adolescent females.

To develop assays able to distinguish and detect re-infections versus re-
activations.
2.
OBJECTIVES
2.1.
Primary objective

To estimate the incidence of CMV secondary infections in seropositive adolescent 
females.
Refer to Section 8.1 for the definition of the primary endpoint.
 
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2.2.
Secondary objective

To estimate the incidence of CMV primary infections in seronegative adolescent 
females.
Refer to Section 8.1.2 for the definition of the secondary endpoint.
2.3.
Tertiary objectives

To develop assays that allow the differentiation of CMV secondary infections 
caused either by re-infection or by re-activation, in CMV seropositive adolescent 
females.

To assess the diagnostic value of different assays to evaluate seroconversion in 
blood and the detection of CMV DNA in different body fluids.

To describe the proportion of secondary infection caused either by re-infection or 
re-activation.

To collect samples for immunological and biological disease-related exploratory 
assays.

To explore socio-demographic or behavioral factors associated with CMV 
infection.

In case of pregnancies, to document the birth prevalence of CMV congenital 
infections.

To evaluate the concordance between screening testing data at local laboratories 
(or alternatively, central laboratories) and the GSK Biologicals or designated 
laboratory testing data.
Refer to 8.1.3 for the definition of the tertiary endpoints.
 
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3.
STUDY DESIGN OVERVIEW
Time point
Year 1
Month
M0
M2
M4
M6
M8
M10
M12
Site Visits
V1
V2
V3
V4
Sample Collection Visits
SCV1
SCV2
SCV3
Year 2
Month
M14
M16
M18
M20
M22
M24
Site Visits
V5
V6
V7
Sample Collection Visits
SCV4
SCV5
SCV6
Year 3
Month
M26
M28
M30
M32
M34
M36
Site Visits
V8
V9
V10
Sample Collection Visits
SCV7
SCV8
SCV9
Samples:
Blood




Saliva







Urine







In case of pregnancy resulting in live birth: Urine and or saliva from the newborn (please refer to Table 3) will be 
collected (if possible) as per standard of care in the center where the subject has delivered or through a home 
visiting nurse or alternatively, the subject may be asked to come back to the study center within 10 days of 
delivery.

Type of design: Prospective, multi-center, multi-country study.

Study Design:

10 scheduled Site Visits approximately every 4 months.

9 Sample Collection Visits, approximately at mid-term between 2 Site Visits 
(i.e. approximately 2 months after a Site Visit) where samples will be collected 
either through self-collection by the subjects, by a home-visiting nurse or 
through a new visit at the study center.

Study population: Adolescent females aged between 10 and 17 years regardless 
of CMV status.

Number of subjects and study groups: 
At Visit 1, subjects will be enrolled regardless of their seropositivity status. Subjects 
will be allocated to the CMV seropositive (S+) or the CMV seronegative (S-) groups
based on the local laboratory results (alternatively, a GSK designated and validated
central laboratory can perform the analyses). Enrolment will be terminated when 
approximately 240 CMV seropositive females have been enrolled.

CMV Seropositive Group (S+): Subjects defined as CMV seropositive based on 
the local laboratory results (or alternatively based on central laboratory results) 
obtained from the sample collected at Visit 1.

CMV Seronegative Group (S-): Subjects defined as CMV seronegative based on 
the local laboratory results (or alternatively based on central laboratory results) 
obtained from the sample collected at Visit 1.
For statistical analysis, the serostatus will be based on the laboratory results obtained 
at the GSK Biologicals or designated laboratory.
 
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
Controls: Uncontrolled.

Type of study: Self-contained.

Data collection: Electronic Case Report Form (eCRF).

Duration of the study:

Participant: Approximately 36 months.

Newborn (in case of pregnancy): Approximately 10 days.
Epoch 001: Prospective data collection starting at Visit 1 (Month 0) and ending at 
Visit 10 (Month 36).
Table 1
Study groups foreseen in the study
Study Groups
Number of subjects
Age (Min/Max)
S+
+/- 240 seropositive subjects
10 – 17 years
S-
Estimated approximate number of seronegative subjects(1): 160
10 – 17 years
(1)As subjects will be enrolled consecutively without previous CMV screening with a target number of 240
seropositive subjects, the number of seronegative subjects enrolled will depend on the seroprevalence of the 
participating countries.

Time points and biological samples

Blood: approximately every 4 months;

Saliva and urine: approximately every 2 months;

Urine and /or saliva of newborn (in case of pregnancy): within 10 days post-
delivery.
3.1.
Discussion of study design
The study sites will be selected in multiple countries, including countries where the CMV 
prevalence is assumed to be high in order to enroll primarily CMV seropositive 
adolescent females to allow the evaluation of CMV secondary infections. Since 
enrolment will proceed without previous CMV screening, some of the enrolled subjects 
will be seronegative. The enrolled seronegative subjects will allow the estimation of the 
incidence of CMV primary infections in the adolescent population.
To increase the probability to capture all CMV infections (primary and secondary 
infections), regular sample collection time-points have been arranged:

Site Visits: Subjects will be asked to perform a Site Visit approximately every 
4 months for 3 years (10 Site Visits in total). At Site Visits, blood, urine and 
saliva will be collected.

Sample Collection Visits: Subjects will be contacted to perform urine and 
saliva sample collections at home either by self-collection or by a home visiting 
nurse. Alternatively subjects may be asked to come back to the study center 
where the investigator or designate will collect the samples. These Sample 
 
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Collection Visits will occur approximately every 4 months for 3 years between 
2 Site Visits (9 Sample Collection Visits in total).
4.
STUDY POPULATION
4.1.
Number of subjects/ centers
The study will be conducted world-wide in countries with a high seroprevalence. 
Considering a 15-20% drop-out rate over the study period it is estimated that 
approximately 240 seropositive subjects should be enrolled to obtain approximately 
200 evaluable seropositive subjects. 
Overview of the recruitment plan
Subjects will be enrolled consecutively without previous CMV screening. Enrolment will 
be terminated once approximately 240 seropositive subjects are included in the trial, 
based on the local laboratory results (or alternatively, based on the GSK designated and 
validated central laboratory results) obtained from the sample collected at Visit 1. The 
number of seronegative subjects enrolled will depend on the seroprevalence of the 
participating countries. It is estimated that approximately 400 subjects will be enrolled in 
total.
4.2.
Inclusion criteria for enrolment
All subjects must satisfy ALL the following criteria at study entry:

A female adolescent between, and including 10 and 17 years at the time of 
enrolment (subjects become ineligible for enrolment on their 18th birthday) 
regardless of pregnancy status and contraception method used or not used.

Subjects who the investigator believes that the subject and/or the subject’s 
parent(s)/Legally Acceptable Representative(s) (LAR[s]) can and will comply 
with the requirements of the protocol (e.g.: sample collection either through self –
collection or through a home-visiting nurse, return for follow-up visits).

Written informed assent and/or consent obtained from the subject and/or the 
parent(s)/LAR(s) of the subject.

Subject is likely to remain in the area and/or return for required study Site Visits 
and complete Sample Collection Visits.
 
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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:

Child in care
Please refer to the glossary of terms for the definition of child in care.

Use or planned use of any investigational or non-registered antiviral drug or 
vaccine during the study period.

Known medical history of any recurrent clinical herpes episodes requiring 
episodic or chronic suppressive treatment with oral or parenteral antiviral 
treatment such as acyclovir, famciclovir, valacyclovir or any other anti-herpes 
virus anti-viral during the year preceding enrolment (no laboratory testing 
required). Topical anti-viral are allowed.

Subjects with history of previous vaccination against CMV.

Chronic administration (defined as more than 14 days in total) of 
immunosuppressants or other immune-modifying drugs within 6 months prior to 
Visit 1 or planned administration during the study (for corticosteroids, this will 
mean prednisone, 0.5 mg/kg/day, or equivalent). Inhaled and topical steroids are 
allowed.

Administration of immunoglobulins and/or any blood products within 3 months 
prior to Visit 1 or planned administration during the study.

Any confirmed or suspected immunosuppressive or immunodeficient condition
including HIV-infection, based on medical history and physical examination (no 
laboratory testing required).

Any major congenital defects, serious chronic illness or organ transplantation.
The criterion that may eliminate subjects from ATP analyses can be found in Section 
5.5.2.9.
 
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5.
CONDUCT OF THE STUDY
5.1.
Regulatory and ethical considerations, including the 
informed consent process
The study will be conducted in accordance with all applicable regulatory requirements.
The study will be conducted in accordance with the ICH Guideline for Good Clinical 
Practice (GCP), all applicable subject privacy requirements and the guiding principles of 
the Declaration of Helsinki. 
The study has been designed and will be conducted in accordance with the ICH 
Harmonized Tripartite Guideline for clinical investigation of medicinal products in the 
pediatric population (ICH E11) and all other applicable ethical guidelines.
GSK will obtain favorable opinion/approval to conduct the study from the appropriate 
regulatory agency, in accordance with applicable regulatory requirements, prior to a site 
initiating the study in that country or will document that neither a favorable opinion nor 
an approval to conduct the study is needed.
Conduct of the study includes, but is not limited to, the following:

Institutional Review Board (IRB)/Independent Ethics Committee (IEC) review and 
favorable opinion/approval of study protocol and any subsequent amendments.

Subject/ subject’s parent(s)/LAR(s) informed consent and subject informed assent, as 
appropriate.

Investigator reporting requirements as stated in the protocol.
GSK will provide full details of the above procedures to the investigator, either verbally, 
in writing, or both.
Freely given and written informed consent must be obtained from each subject and/or
each subject’s parent(s)/LAR(s) and subject informed assent, as appropriate, prior to 
participation in the study. 
GSK Biologicals will prepare a model Informed Consent Form (ICF) which will embody 
the applicable ICH GCP and GSK Biologicals required elements. While it is strongly 
recommended that this model ICF be followed as closely as possible, the informed 
consent requirements given in this document are not intended to pre-empt any local 
regulations which require additional information to be disclosed for informed consent to 
be legally effective. Clinical judgment, local regulations and requirements should guide 
the final structure and content of the local version of the ICF. 
In accordance with the ICH Harmonized Tripartite Guidelines for GCP, those subjects 
who can only be enrolled in the study with the consent of the subject’s parent(s) or 
legally acceptable representative (e.g. minors), should be informed about the study to the 
extent compatible with the subject’s understanding and, if capable, the subject should 
sign and personally date a written informed assent. It is required that the assent be signed 
 
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by each subject, if capable, in addition to the informed consent that is to be signed by
his/her parent or legal representative. It should be assessed whether an assent is required 
depending on the age of the study population and the local requirements.
GSK Biologicals strongly recommends that if the subject reaches the age of consent 
during the study they will be asked to provide consent at the next study visit (if 
applicable). This procedure should be applied according to local laws and regulations.
The investigator has the final responsibility for the final presentation of the ICF, 
respecting the mandatory requirements of local regulations. The ICF generated by the 
investigator with the assistance of the sponsor’s representative must be acceptable to 
GSK Biologicals and be approved (along with the protocol, and any other necessary 
documentation) by the IRB/IEC.
5.2.
Subject identification
Subject numbers will be assigned sequentially to subjects consenting to participate in the 
study, according to the range of subject numbers allocated to each study center.
5.3.
General study aspects
Supplementary study conduct information not mandated to be present in this protocol is 
provided in the accompanying Study Procedures Manual (SPM). The SPM provides the 
investigator and the site personnel with administrative and detailed technical information 
that does not impact the safety of the subjects.
 
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5.4.
Outline of study procedures
Table 2
List of study procedures (amended 19 February 2015)
Age
10-17 years
Epoch
001
Year 1
Time-point (Months)
M0
M2
M4
M6
M8
M10
M12
Visit/Sample Collection Visit
V1
SCV1
V2
SCV2
V3
SCV3
V4
Year 2
Time-point (Months)
M14
M16
M18
M20
M22
M24
Visit/Sample Collection Visit
SCV4
V5
SCV5
V6
SCV6
V7
Year 3
Time-point (Months)
M26
M28
M30
M32
M34
M36
Visit/Sample Collection Visit
SCV7
V8
SCV8
V9
SCV9
V10
Informed consent
●
Check inclusion/exclusion criteria
●
Record demographic data
●
Record social and behavioral data(1)
●
●
●
Medical history
●
CMV serostatus(2) (~3.5 to 5 mL)
●
Blood sample(3)
- for humoral immunity (10 mL)
- for molecular biology (4 mL)
●
●
●
●
●
Blood sample for gene expression signature 
(qPCR or RNA microarray; 2.5 mL)(4)
●
●
Urine sample(5) (~10 mL)
●
●
●
●
●
●
●
●
Saliva 
●
●
●
●
●
●
●
●
Subject sample collection booklet distribution(6)
○
Check elimination criteria
●
●
●
●
●
Record any concomitant 
medication/vaccination(7)
●
●
●
●
●
Reporting of SAEs related to study participation 
●
●
●
●
●
Reporting of pregnancy and outcome
●
●
●
●
●
Study conclusion 
●
● is used to indicate a study procedure that requires documentation in the individual eCRF. 
○ is used to indicate a study procedure that does not require documentation in the individual eCRF
(1)Social and behavioral data will be recorded at inclusion (Visit 1 [Month 0]), at Visit 4 (Month 12), Visit 7 (Month 24) 
and Visit 10 (Month 36).
(2) CMV serostatus, for allocation to a study group (S+ or S-), will be determined by the local laboratory as per local 
practices or by a GSK designated and validated central laboratory.
(3) CMV serostatus for endpoints analysis will be determined by GSK Biologicals or designated laboratory at Visit 1 
(Month 0).
(4) These blood samples will be collected until approval of protocol amendment 2.
(5) Prepared from the 20 mL urine samples collected from the subject.
(6) The sample collection booklet will instruct the subjects of the study procedures to be performed at the Sample 
Collection Visits. The sampling can be done through self-collection, through a home-visiting nurse or the subject 
may be asked to come back to the study center. 
(7) Only concomitant medication/vaccination related to inclusion/exclusion and elimination criteria will be recorded.
 
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Table 3
List of study procedures for newborns/mothers (in case of 
pregnancy)
Age
0-10 days old
Visit/ time-point
Visit 1/Day 0-10
Informed consent(1)
●
Collect urine (5 mL) and/or saliva from newborn
●
Record CMV infection status, if available
●
Assessment of CMV disease from newborn, if applicable(2)
●
● is used to indicate a study procedure that requires documentation in the individual eCRF.
(1) For practical reasons the informed consent may be obtained during pregnancy.
(2) Symptomatic or asymptomatic.
Table 4
Intervals between study Site Visits/Sample Collection Visits
Interval
Optimal length of interval(1)
(Days)
Allowed interval(2)
(Days)
Visit 1 (M0) Visit 2 (M4)
122
112 - 132
Visit 1 (M0) Visit 3 (M8)
244
234 - 254
Visit 1 (M0) Visit 4 (M12)
365
355 - 375
Visit 1 (M0) Visit 5 (M16)
488
478 - 498
Visit 1 (M0) Visit 6 (M20)
610
600 - 620
Visit 1 (M0) Visit 7 (M24)
730
720 - 740
Visit 1 (M0) Visit 8 (M28)
854
844 - 864
Visit 1 (M0) Visit 9 (M32)
976
966 - 986
Visit 1 (M0) Visit 10 (M36)
1095
1085 - 1105
(1) Whenever possible the investigator should arrange study visits within this interval.
(2) Subjects will not be eligible for inclusion in the according-to-protocol (ATP) cohort if they make the study visit 
outside this interval (See Section 8.4.2 for definition of the ATP cohort).
(Amended 19 February 2015)
Between study visits, Sample Collection Visits should occur at approximately 2 months 
interval of the Site Visits (i.e. mid-term between 2 Site Visits). Every effort should be 
made to collect samples at 61-day (2-month) intervals with approximately a 10-day
range.
5.5.
Detailed description of study procedures
5.5.1.
Procedures prior to study participation
5.5.1.1.
Informed consent
Before performing any other study procedure, the signed informed consent of the subject 
or subject’s parent(s)/LAR(s) needs to be obtained. If capable, before performing any 
other study procedure, the signed informed assent of the subject below the age of consent 
should be obtained in addition to the signed informed consent by her parent(s)/LAR(s) 
according to local rules and regulations. 
In case of pregnancy and before performing any study procedure for the newborns, the
subject /subject’s parent(s)/LAR(s) informed consent must be obtained. If capable, before 
performing any other study procedure, the signed informed assent of the subject below 
 
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the age of consent should be obtained in addition to the signed informed consent by her 
parent(s)/LAR(s) according to local rules and regulations.
Refer to Section 5.1 for the requirements on how to obtain informed consent and assent, 
as appropriate.
5.5.1.2.
Check inclusion and exclusion criteria
Check all applicable inclusion and exclusion criteria as described in Sections 4.2 and 4.3
before enrolment.
5.5.2.
Procedures during study participation
5.5.2.1.
Collect demographic data
Record demographic data such as age and race in the subject’s eCRF.
5.5.2.2.
Collect social and behavioral data
Record the social and behavioral data in the subject’s eCRF, based on the questionnaire 
completed by the subject (a blank questionnaire is provided in the SPM).
5.5.2.3.
Medical history
Perform a history-directed medical examination and record any pre-existing conditions or 
signs and/or symptoms present in a subject prior to the start of the study in the eCRF.
Treatment of any abnormality observed during this examination has to be performed 
according to local medical practice outside this study or by referral to an appropriate 
health care provider.
5.5.2.4.
CMV serostatus
Collect a blood sample (approximately 3.5 to 5 mL) at Site Visit 1 to determine the 
subject’s serostatus by the local laboratory (or alternatively, by a GSK designated and 
validated central laboratory). Record results of the test in the eCRF.
5.5.2.5.
Blood sampling
As specified in the List of Study Procedures in Section 5.4 (Table 2), blood samples will 
be collected at each Site Visit (approximately 14 to 16.5 mL). Refer to the module on 
Biospecimen Management in the SPM for general handling and storage condition of 
blood samples.

A volume of approximately 10 mL of whole blood should be drawn from all 
subjects for analysis of humoral immune response at each Site Visit. This blood 
sample might also be used for exploratory testing.

A volume of approximately 4 mL of whole blood should be drawn from all 
subjects for molecular biology tests at each Site Visit. This blood sample might 
also be used for exploratory testing.
 
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
A volume of approximately 2.5 mL of whole blood should be drawn from all 
subjects for ribonucleic acid (RNA) assays using qPCR or microarray at Site 
Visits 1 (Month 0) and 4 (Month 12). This blood sample might also be used for 
exploratory testing.
(Amended 19 February 2015)
5.5.2.6.
Urine sampling
A volume of approximately 10 mL of urine will be collected from all subjects at each Site 
Visit by the investigator or designate.
A volume of approximately 10 mL of urine will also be collected at each Sample 
Collection Visit either by the subject herself or a home-visiting nurse. Alternatively, the 
subject can be asked to come back to the study center and the sample will be collected by 
the investigator or designate.
If samples are self-collected by the subject, the investigator or designated is responsible 
for instructing the subjects on how to take the sample appropriately.
Refer to the module on Biospecimen Management in the SPM and the sample collection 
booklet for the general handling of urine samples.
5.5.2.7.
Saliva sampling
Saliva will be collected from all subjects using a specific device at each Site Visit by the 
investigator or designate.
Saliva will also be collected at each Sample Collection Visit either by the subject herself 
or a home visiting nurse. Alternatively, the subject can be asked to come back to the 
study center and the sample will be collected by the investigator or designate.
If samples are self-collected by the subject, the investigator or designate is responsible for 
instructing the subjects on how to take the sample appropriately. 
Refer to the module on Biospecimen Management in the SPM and the sample collection
booklet for general handling of saliva samples.
5.5.2.8.
Distribution of subject sample collection booklet
If it’s foreseen that the subject herself or a home-visiting study nurse will collected urine 
and saliva at the Sample Collection Visit, a sample collection booklet will be given to the 
subject at the first Study Visit (SV1, Month 0). This sample collection booklet will detail 
the study procedures to be performed during Sample Collection Visit.
 
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5.5.2.9.
Check Elimination criteria
Check the following elimination criteria:

Known medical history of any recurrent clinical herpes episodes requiring 
episodic or chronic suppressive treatment with oral or parenteral antiviral 
treatment such as acyclovir, famciclovir, valacyclovir or any other anti-herpes 
virus anti-viral since previous visit (no laboratory testing required). Topical anti-
viral are allowed.
5.5.2.10.
Record any concomitant medication/vaccination
Concomitant medication/vaccination related to inclusion/exclusion and elimination 
criteria (i.e. administration of immunosuppressants, other immune-modifying drugs, 
immunoglobulins, any blood products and/or investigational/non-registered vaccine, any 
antiviral drug including anti-herpes) must be recorded in the eCRF.
5.5.2.11.
Recording of Serious Adverse Events related to study participation
The subject/subjects’ parent(s)/LAR(s) will be instructed to contact the investigator 
immediately should they/the subjects manifest any signs or symptoms she/they perceive 
as serious.
Refer to Section 6.2 for procedures for the Investigator to record serious adverse events 
(SAEs) that are related to study participation or GSK concomitant medication/vaccination 
and to Section 6.3 for guidelines on how to report these SAEs to GSK Biologicals.
5.5.2.12.
Reporting of pregnancy and pregnancy outcome
If the investigator becomes aware that a subject is pregnant, the pregnancy should be 
reported in the eCRF of the subject. 
Pregnancy will be followed-up only during the study period. If the delivery occurs during 
the study period, the pregnancy outcome should be recorded.
5.5.2.13.
Study conclusion
The investigator will review safety data collected to ensure accuracy and completeness 
and will complete the Study Conclusion screen in the eCRF.
5.5.3.
Procedure during study participation for the newborns (in case of 
pregnancy and delivery during the study period)
If the delivery occurs during the study period, the pregnancy and, if the subject and/or 
subject’s parent(s)/LAR(s) has given her/his/their consent, information regarding CMV 
infection of the newborn will be documented (see Section 5.5.3) in the newborn’s eCRF.
 
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5.5.3.1.
Urine/saliva collection
A urine (approximately 5 mL) and/or saliva sample will be collected from newborns for 
virology analysis. Samples will be collected (if possible):

As per standard of care in the center where the subject has delivered.

OR through a home visiting nurse within 10 days of delivery.

OR the subject may be asked to come back to the study center within 10 days of 
delivery.
Refer to the module on Biospecimen Management in the SPM for general handling of 
urine and/or saliva samples.
5.5.3.2.
Record CMV infection status
The CMV infection status (positive or negative) of the newborns will be recorded in the 
newborn’s eCRF.
5.5.3.3.
Assessment of CMV disease at birth
Assessment of CMV disease (if applicable, symptomatic or asymptomatic) will be 
performed for newborns diagnosed as CMV-positive and record in the newborn’s eCRF.
The investigator or designate (such as a pediatrician) will perform the assessment 
him/herself during the Site Visit which should occur within 10 days of delivery or will 
contact the center where the subject has delivered to obtain a copy of the medical dossier.
5.6.
Biological Sample handling and analysis
Please refer to the SPM and sample collection booklet for details of biospecimen 
management (handling, storage and shipment).
Samples will not be labeled with information that directly identifies the subjects but will 
be coded with the identification number for the subject (subject number). 
Collected samples may be used in other assays, for test improvement or test development 
of analytical methods related to a study vaccine(s)/product(s) and its constituents or the 
disease under study to allow to achieve a more reliable measurement of the vaccine
response. Under these circumstances, additional testing on the samples may be performed 
by GSK Biologicals outside the scope of this protocol.
Information on further investigations and their rationale can be obtained from GSK 
Biologicals.
Any sample testing will be done in line with the consent of the individual 
subject/subject’s parent(s)/LAR(s).
 
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Any human pharmacogenetic testing will require specific consent from the individual 
subjects/subject’s parent(s)/LAR(s) and the ethics committee approval. Any HIV testing 
will also require specific consent and ethics committee approval.
Refer also to the Investigator Agreement, where it is noted that the Investigator cannot 
perform any other biological assays except those described in the protocol or its 
amendment(s).
Collected samples will be stored for up to 15 years (counting from when the last subject 
performed the last study visit), unless local rules, regulations or guidelines require 
different timeframes or different procedures, which will then be in line with the 
subjects/subject’s parent(s)/LAR(s) consent. These extra requirements need to be 
communicated formally to and discussed and agreed with GSK Biologicals.
5.6.1.
Use of specified study materials
When materials are provided by GSK Biologicals, it is MANDATORY that all samples 
(including serum samples) be collected and stored exclusively using those materials in 
the appropriate manner. The use of other materials could result in the exclusion of the 
subject from the ATP analysis (See Section 8.4 for the definition of study cohorts to be 
evaluated). The investigator must ensure that his/her personnel and the laboratory(ies) 
under his/her supervision comply with this requirement. However, when GSK 
Biologicals does not provide material for collecting and storing samples, appropriate 
materials from the investigator’s site must be used. Refer to the Module on Clinical Trial 
Supplies in the SPM.
5.6.2.
Biological samples
Each sample will be collected from all subjects.
Table 5
Biological samples (amended 19 February 2015)
Sample type
Quantity 
Time point
Priority 
rank
Blood for humoral immunity 
determination
Approximately 10 mL
Months 0, 4, 8, 12, 16, 20, 24, 28, 32 
and 36
1
Blood for molecular biology
Approximately 4 mL
Months 0, 4, 8, 12, 16, 20, 24, 28, 32 
and 36
1
Blood for gene signature expression
(RNA transcript)
Approximately 2.5 mL
Months 0, and 12*
2
Urine
Approximately 10 mL
Months 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 
20, 22, 24, 26, 28, 30, 32, 34 and 36
1
Saliva
NA
Months 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 
20, 22, 24, 26, 28, 30, 32, 34 and 36
1
* Blood samples will be collected until approval of protocol amendment 2.
5.6.3.
Laboratory assays
Please refer to APPENDIX A for a detailed description of the assays performed in the 
study and to APPENDIX B for the address of the clinical laboratories used for sample 
analysis.
 
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Table 6
Humoral Immunity (Antibody determination) (amended 19 February 
2015)
System
Component
Method
Kit / 
Manufacturer
Unit
Cut-off
Number 
of 
subject
Laboratory
(3)
Serum(1)
Anti-CMV 
tegument protein 
IgG 
ELISA
Biotest like 
assay
EU/mL
TBD
All 
subjects
TBD
Serum(1)
Anti-gB IgG
ELISA
In house
EU/mL
54
Subset(2)
TBD
Serum(1)
Anti-CMV 
tegument protein 
avidity index
ELISA
Biotest like 
assay
NA
NA
Subset(2)
TBD
Serum(1)
Anti-gB IgG 
avidity index
ELISA
In house
NA
NA
Subset(2)
TBD
Serum(1)
Anti-CMV IgM
ELISA
Diasorin or 
equivalent 
NA
NA 
Subset(2)
TBD
Serum
Anti-CMV 
neutralizing 
antibodies 
(multiple cell 
lines) and /or 
other virus strains
Seroneutralisation
In house
ED50
TBD
Subset(2)
TBD
Serum(1)
Antibody 
recognition profile
Peptide 
microarray
TBD
NA
NA
Subset(2)
TBD
Note: Most of the read-outs described in this table might be done if deemed necessary.
(1) Coating Ag will be defined based on available commercial kits or in house developed assays (such as whole 
virus proteins or tegument proteins or others to be defined).
(2) Subset will be defined based on the outcome of primary/secondary endpoints or the development of the assay. 
The subset is the minimum number of subjects that will be tested. If applicable, all subjects could be tested.
(3) Refer to APPENDIX B for the laboratory addresses.
TBD = To be determined, gB = glycoprotein B; Ig = immunoglobulin, ELISA = Enzyme-linked immunosorbant 
assay, EU = ELISA unit, NA = not applicable; ED50 = effective dose 50, Ag = antigen.
The laboratories that will perform antibody determination are not yet identified and will 
be defined as soon as available.
 
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Table 7
Molecular Biology (amended 19 February 2015)
System
Test
Component
Method
Unit
Number of 
subject
Laboratory (4)
Urine
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
All subjects(1)
DDL
Urine
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
All Newborns
BARC
Saliva
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
Subset(3)
DDL
Saliva
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
All Newborns
BARC
Plasma/
buffy coat
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
Subset(3)
DDL
TBD(2)
Multi strain 
typing
CMV DNA
CMV strain 
typing
NA
Subset(3)
TBD
Whole blood RNA transcript
TBD
mRNA 
microarray
or qPCR
NA
Subset(3)
TBD
Note: Most of the read-outs described in this table might be done if deemed necessary.
(1) In seronegative subjects, testing of viral load will only be done upon seroconversion. 
(2) The matrix will be determined based on the results obtained with the PCR and the availability of the assay.
(3) Subset will be defined based on the outcome of primary/secondary endpoints or the development of the assay. 
The subset is the minimum number of subjects that will be tested. If applicable, all subjects could be tested.
(4) Refer to APPENDIX B for the laboratory addresses.
TBD = To be determined, mRNA = messenger Ribonucleic acid; PCR = Polymerase Chain reaction; DNA = 
deoxyribonucleic acid; NA = not applicable.
In case the results from a 4-month Site Visit suggests primary or secondary (re-
infection/re-activation) infection, testing related to the tertiary endpoints will be done on 
selected samples collected during the Sample Collection Visit. The selection of samples 
to be tested will be based on the qPCR results obtained following the Site visits.
If the results from a 4-month Site Visit suggest no primary or secondary (re-infection/re-
activation) infection, the samples collected during the Sample Collection Visit period will 
be stored and used if applicable (e.g. negative control for laboratory validation).
Collected samples will be used for purposes related to the quality assurance of data 
generated within the scope of this protocol, such as for maintenance of assays described 
in this protocol and comparison between analytical methods and/or laboratories.
The laboratories that will perform antibody recognition profile, multi strain typing and 
RNA transcript assays are not yet identified and will be defined as soon as available.
 
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5.6.4.
Biological samples evaluation
5.6.4.1.
Read-outs
Table 8
Read-outs (amended 19 February 2015)
Sampling time point
Subset Name
No. 
subjects
Component
Type of 
contact and 
time point 
Sampling 
time point
Site visits:
(V1 to V10)
Months 0, 4, 8, 
12, 16, 20, 24, 
28, 32, and 36
All subjects
All
Anti-CMV tegument protein IgG (serum)
Subset(2)
TBD
Anti-gB IgG (serum)
All subjects(1)
TBD
CMV DNA (urine)
Subset(2)
TBD
Anti-CMV tegument protein IgG antibody avidity index
Anti-gB IgG antibody avidity index
Subset(2)
TBD
CMV DNA (saliva, blood)
Subset(2)
TBD
Anti-CMV IgM (serum)
Subset(2)
TBD
Anti-CMV neutralizing antibodies (multiple cell lines
and/or other viral strains)
Subset(2)
TBD
Antibody recognition profile
Subset(2)
TBD
Multi strain typing
Site visit 
(V1 and V4*)
Months 0 and
12*
Subset(2)
TBD
RNA transcript
Sample 
Collection Visits 
(SCV 1 to SCV 
9)
Month 2, 6, 
10, 14, 18, 22, 
26, 30, 34(3)
Subset(2)
TBD
CMV DNA (urine + saliva)
Upon delivery
Newborn
TBD
CMV DNA urine and/or saliva
(1) In seronegative subjects, testing of viral load will only be done upon seroconversion.
(2) Subset will be defined based on the outcome of primary/secondary endpoints or the development of the assay. 
The subset is the minimum number of subjects that will be tested. If applicable, all subjects could be tested.
(3) Testing will be done on selected samples only if the samples at the following 4-month Site Visit time-point is 
positive. If the 4-month sample is negative for CMV infection/re-infection, the samples self-collected during the inter-
visit period will be stored and be used if applicable. The selection of samples to be tested will be based on the qPCR 
results obtained following the Site visits.
* Blood sample will be collected until approval of protocol amendment 2.
DNA = deoxyribonucleic acid; RNA = Ribonucleic acid
IgG = immunoglobulin G; IgM = immunoglobulin M; gB = glycoprotein B; TBD = To be determined.
6.
SERIOUS ADVERSE EVENTS
Only SAEs related to study participation will be collected and reported during the entire 
study period.
The investigator or site staff is/are responsible during the study for the detection and 
documentation of events meeting the criteria and definition of a SAE as provided in this 
protocol.
Each subject/subject’s parent(s)/LAR(s) will be instructed to contact the investigator 
immediately should they/the subject manifest any signs or symptoms they perceive as 
serious.
 
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6.1.
Safety definitions
6.1.1.
Definition of an adverse event
An adverse event (AE) is any untoward medical occurrence in a subject, temporally 
associated with the use of a medicinal product, whether or not considered related to the 
medicinal product, or temporally associated with a study procedure.
For an AE to be reportable, there is a minimum set of criteria (as recommended by the 
Council for International Organizations of Medical Sciences (CIOMS) and ICH) to be 
considered:

an identifiable reporter 

an identifiable patient,

suspect medicinal product (immunization history)

detailed description of the event

suspected causal relationship (event is explicitely labelled as "related" by the 
reporter)
An AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) temporally associated with 
the use of a medicinal product. For marketed medicinal products, this also includes 
failure to produce expected benefits (i.e. lack of efficacy), abuse or misuse. 
Examples of an AE include:

Significant or unexpected worsening or exacerbation of the condition/indication 
under study. 

Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition.

Signs, symptoms, or the clinical sequelae of a suspected overdose of a concurrent 
medication (overdose per se should not be reported as an AE/SAE).
Examples of an AE DO NOT include:

Medical or surgical procedures (e.g. endoscopy, appendectomy); the condition that 
leads to the procedure is an AE.

Situations where an untoward medical occurrence did not occur (e.g. social and/or 
convenience admission to a hospital, admission for routine examination).

Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected at the start of the study that do not worsen. 
Example of events to be recorded in the medical history section of the CRF/eCRF:

Pre-existing conditions or signs and/or symptoms present in a subject prior to the 
start of the study (i.e. prior to enrolment in an observational study).
 
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6.1.2.
Definition of a serious adverse event
A SAE is any AE that:
a.
Results in death,
b.
Is life-threatening,
NB: The term ‘life-threatening’ in the definition of ‘serious’ refers to an event in 
which the subject was at risk of death at the time of the event. It does not refer to an 
event, which hypothetically might have caused death, had it been more severe.
c.
Requires hospitalization or prolongation of existing hospitalization,
NB: In general, hospitalization signifies that the subject has been admitted at the 
hospital or emergency ward for observation and/or treatment that would not have 
been appropriate in the physician’s office or in an out-patient setting. 
Complications that occur during hospitalization are also considered adverse events. 
If a complication prolongs hospitalization or fulfills any other serious criteria, the 
event will also be considered serious. When in doubt as to whether ‘hospitalization’ 
occurred or was necessary, the adverse event should be considered serious.
Hospitalization for elective treatment of a pre-existing condition (known or 
diagnosed prior to informed consent signature) that did not worsen from baseline is 
NOT considered an SAE.
d.
Results in disability/incapacity, OR
NB: The term disability means a substantial disruption of a person’s ability to 
conduct normal life functions. This definition is not intended to include experiences 
of relatively minor medical significance such as uncomplicated headache, nausea, 
vomiting, diarrhea, influenza like illness, and accidental trauma (e.g. sprained ankle) 
which may interfere or prevent everyday life functions but do not constitute a 
substantial disruption.
e.
Is a congenital anomaly/birth defect in the offspring of a study subject.
Medical or scientific judgment should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize the 
subject or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition. These should also be considered serious. 
Examples of such events are invasive or malignant cancers, intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions 
that do not result in hospitalization.
6.1.3.
Clinical laboratory parameters and other abnormal assessments 
qualifying as serious adverse events
Abnormal laboratory findings (e.g. clinical chemistry, hematology, urinalysis) or other 
abnormal assessments that are judged by the investigator to be clinically significant will 
be recorded as SAEs if they meet the definition of an SAE, as defined in Section 6.1.2. 
 
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Clinically significant abnormal laboratory findings or other abnormal assessments that 
are detected during the study or are present at baseline and significantly worsen following 
the start of the study will be reported as SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant.
6.2.
Detecting and recording serious adverse events
6.2.1.
Time period for detecting and recording serious adverse events
In order to fulfill international reporting obligations, SAEs that are related to study 
participation (i.e. protocol-mandated procedures, invasive tests, a change from existing 
therapy) will be collected and recorded from the time the subject consents to participate 
in the study until she is discharged.
6.2.2.
Evaluation of serious adverse events
6.2.2.1.
Active questioning to detect serious adverse events
Each subject/subject’s parent(s)/LAR(s) will be instructed to contact the investigator 
immediately should the subject manifest any signs and symptoms she perceives/they
perceive as serious.
All SAEs either observed by the investigator or his/her staff or reported by the 
subject/subject’s parent(s)/LAR(s) spontaneously or in response to a direct question will 
be evaluated by the investigator. The nature of each event, data and time (where 
appropriate) of onset, outcome, and relationship to the study procedures should be 
established.
When an SAE occurs, it is the responsibility of the investigator to review all 
documentation (e.g. hospital progress notes, laboratory, and diagnostics reports) relative 
to the event. The investigator will then record all relevant information regarding an SAE 
on the eCRF or SAE Report screens, as applicable. It is not acceptable for the 
investigator to send photocopies of the subject’s medical records to GSK Biologicals 
instead of the appropriate completed SAE pages on the SAE screens in the eCRF. 
However, there may be instances when copies of medical records for certain cases are 
requested by GSK Biologicals. In this instance, all subject identifiers will be blinded on 
the copies of the medical records prior to submission to GSK Biologicals.
The investigator will attempt to establish a diagnosis pertaining to the event based on 
signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be 
documented as the SAE and not the individual signs/symptoms.
 
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6.2.2.2.
Assessment of intensity
The investigator will assess the maximum intensity that occurred over the duration of the 
event for all SAEs reported during the study. The assessment will be based on the 
investigator’s clinical judgement.
The intensity of each SAE recorded in the eCRF or SAE Report screens, as applicable, 
should be assigned to one of the following categories:
1 (mild)
=
An SAE which is easily tolerated by the subject, causing minimal 
discomfort and not interfering with everyday activities.
2 (moderate)
=
An SAE which is sufficiently discomforting to interfere with 
normal everyday activities.
3 (severe)
=
An SAE which prevents normal, everyday activities 
In adults/adolescents, such an SAE would, for example, prevent 
attendance at work/school and would necessitate the 
administration of corrective therapy.
6.2.2.3.
Assessment of causality 
The investigator should assess the causality of each SAE. The investigator will use 
clinical judgment to determine the relationship of SAEs to study procedures. Alternative 
causes, such as natural history of the underlying diseases, concomitant therapy and other 
risk factors will be considered and investigated.
There may be situations when a SAE has occurred and the investigator has minimal 
information to include in the initial report to GSK Biologicals. However it is very 
important that the investigator always makes an assessment of causality for every event 
prior to submission of the SAE to GSK Biologicals. The investigator may change his/her 
opinion of causality in light of follow-up information, amending the SAE information 
accordingly. 
If an event meets the criteria to be determined ‘serious’ (refer to Section 6.1.2), it will be 
examined by the investigator to the extent to enable determination of all contributing 
factors applicable to each SAE. 
Possible contributing factors include:

Medical history.

Concomitant medication.

Protocol required procedure.

Other procedure not required by the protocol.

Other cause (specify).
 
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6.2.2.4.
Assessment of outcomes
Outcome of any SAE reported during the entire study will be assessed as:

Recovered/resolved.

Recovering/resolving.

Not recovered/not resolved.

Recovered with sequelae/resolved with sequelae.

Fatal.
6.3.
Reporting of serious adverse events
6.3.1.
Prompt reporting of serious adverse events related to a study 
procedure to GSK Biologicals
SAEs that occur in the time period defined in Section 6.2.1 will be reported promptly to 
GSK within the timeframes described in Table 9 once the investigator determines that the 
event meets the protocol definition of a SAE.
Table 9
Timeframes for submitting SAEs 
Type of Event
Initial Reports
Follow-up of Relevant Information
on a Previous Report
Timeframe
Documents
Timeframe
Documents
SAEs related to a study procedure
24 hours(1)
SAE screen
24 hours(1)
SAE screen
(1) Timeframe allowed after receipt or awareness of the information.
6.3.2.
Contact information for reporting serious adverse events and 
other events to GSK Biologicals
Please see the Sponsor Information Sheet for contact details. 
Back-up Study Contact for Reporting SAEs
GSK Biologicals Clinical Safety & Pharmacovigilance 
Fax: 
 or 
24/24 hour and 7/7 day availability
6.3.3.
Completion and transmission of SAE reports related to a study 
procedure to GSK Biologicals
Once an investigator becomes aware that a SAE has occurred in a study subject, the 
investigator (or designate) must complete the information in the SAE screens of the 
eCRF WITHIN 24 HOURS. The SAE screens will always be completed as thoroughly as 
possible with all available details of the event. Even if the investigator does not have all 
information regarding a SAE, the SAE screens should still be completed within 24 hours. 
Once additional information is received, the SAE screens in the eCRF should be updated
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The investigator will always provide an assessment of causality at the time of the initial 
report.
6.3.3.1.
Back-up system in case the electronic SAE reporting system does not 
work
If the electronic SAE reporting system does not work, the investigator (or designate) must 
complete, then date and sign a SAE Report Form and fax it to the GSK Biologicals 
Clinical Safety and Pharmacovigilance department within 24 hours. 
This back-up system should only be used if the electronic SAE reporting system is not 
working and NOT if the system is slow. As soon as the electronic SAE reporting system 
is working again, the investigator (or designate) must complete the SAE screens in the 
eCRF within 24 hours. The final valid information for regulatory reporting will be the 
information reported through the electronic SAE reporting system.
6.3.3.2.
Updating of SAE information after freezing of the subject’s eCRF
When additional information is received on a SAE after freezing of the subject’s eCRF, 
new or updated information is to be recorded on a SAE Report Form, with all changes 
signed and dated by the investigator. The updated form should be faxed to the GSK 
Biologicals Clinical Safety and Pharmacovigilance department or to the Study Contact 
for Reporting SAEs (refer to the Sponsor Information Sheet) WITHIN 24 HOURS of 
receipt of the follow-up information.
6.4.
Follow-up of serious adverse events
After the initial SAE report, the investigator is required to proactively follow each subject 
and provide further information on the subject’s condition to GSK Biologicals.
All SAEs documented at a previous visit/contact and designated as not recovered/not 
resolved or recovering/resolving will be reviewed at subsequent visits/contacts until the 
end of the study.
Investigators will follow-up subjects with SAEs until the event has resolved, subsided, 
stabilized, disappeared, or until the event is otherwise explained, or the subject is lost to 
follow-up. 
Clinically significant laboratory abnormalities will be followed up until they have 
returned to normal, or a satisfactory explanation has been provided. Additional 
information (including but not limited to laboratory results) relative to the subsequent 
course of such abnormalities noted for any subject must be made available to the Site 
Monitor.
GSK Biologicals may request that the investigator performs or arranges for the conduct 
of additional clinical examinations/tests and/or evaluations to elucidate as fully as 
possible the nature and/or causality of the SAE. The investigator is obliged to assist. If a 
subject dies during participation in the study or during a recognized follow-up period, 
 
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GSK Biologicals will be provided with a copy of any available post-mortem findings, 
including histopathology.
7.
SUBJECT COMPLETION AND WITHDRAWAL
7.1.
Subject completion
A subject who returns for the concluding visit foreseen in the protocol is considered to 
have completed the study.
7.2.
Subject withdrawal
Subjects who are withdrawn because of SAE must be clearly distinguished from subjects 
who are withdrawn for other reasons. Investigators will follow subjects who are 
withdrawn as result of a SAE until resolution of the event (see Section 6.3).
From an analysis perspective, a ‘withdrawal’ from the study refers to any subject who did 
not come back for the concluding visit foreseen in the protocol.
All data collected until the date of withdrawal/last contact of the subject will be used for 
the analysis.
A subject is considered a ‘withdrawal’ from the study when no study procedure has 
occurred, no follow-up has been performed and no further information has been collected 
for this subject from the date of withdrawal/last contact.
Investigators will make an attempt to contact those subjects who do not return for 
scheduled visits or follow-up. 
Information relative to the withdrawal will be documented in the eCRF. The investigator 
will document whether the decision to withdraw a subject from the study was made by 
the subject herself, by the subject’s parent(s) or LAR(s), or by the investigator, as well as 
which of the following possible reasons was responsible for withdrawal:

SAE.

Protocol violation (specify).

Consent withdrawal, not due to an adverse event.

Moved from the study area.

Lost to follow-up.

Death.

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8.
DATA EVALUATION: CRITERIA FOR EVALUATION OF 
OBJECTIVES
8.1.
Endpoints
8.1.1.
Primary endpoints

Occurrence of CMV secondary infections determined in all seropositive subjects 
on samples collected during the 4-month Site Visits until study conclusion:

Anti-CMV tegument protein IgG antibody concentration in serum (ELISA).

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).
8.1.2.
Secondary endpoints

Occurrence of CMV primary infection determined in all seronegative subjects on 
samples collected during the 4-month Site Visits until study conclusion:

Anti-CMV tegument protein IgG antibody concentration in serum (ELISA).
8.1.3.
Tertiary endpoints (amended 19 February 2015)

Further characterization of primary infection in initially seronegative subjects 
during the 4-month Site Visits until study conclusion (testing done upon 
seroconversion) might be done:

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Further characterization of primary and /or secondary CMV infections in a subset 
of subjects on selected samples of the Sample Collection Visits* might be done.

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Number of CMV DNA copies (pp65 or other genes) in saliva (qPCR).
* In case of a positive sample at a 4-month Site Visit time point, testing might be done on 
selected samples collected during the Sample Collection Visit. The selection of samples 
to be tested will be based on the qPCR results obtained following the Site visits.

Further characterization of primary and /or secondary CMV infections in a subset 
of subjects on all available samples of the 4-month Site Visits might be done:

Anti-CMV tegument protein IgG antibody avidity index in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-gB IgG antibody avidity index in serum (ELISA).

Anti-CMV IgM antibody concentration in serum (ELISA).

Number of CMV DNA copies (pp65 or other genes) in saliva and blood (qPCR).

Assessment of anti-CMV neutralizing antibodies on different target cells and/or 
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
Development of assays that will allow differentiating re-infection from re-
activation from primary infection in a subset of subjects might be done.

Characterization of CMV strains by genotyping/sequencing.

Exploring the CMV strain specific antibody profile using peptide microarrays.

Assessment of the expression of host genes* using techniques such as qPCR or 
mRNA microarray in a subset of subjects might be performed.
* excluding genes related to hereditary characteristics of the subject.

Evaluation of the impact of demographic, social or behavioral factors upon CMV 
infection might be performed.

Occurrence of congenital CMV infection in newborns of subjects who become 
pregnant during the study might be evaluated:

Evidence of CMV DNA in urine and/or saliva of newborns within 10 days of 
delivery by using qPCR (pp65 or other genes) might be tested.
8.2.
Sample size consideration
The sample size for this study is not based on formal statistical considerations. The target
is to recruit approximately 200 evaluable seropositive adolescent females. Considering a 
conservative incidence rate of CMV infection of 2% per year, it is expected that 
approximately 12 seropositive subjects will be re-infected during the study period.
A target number of approximately 200 evaluable seropositive adolescent females will be 
analyzed. Considering a 15-20% drop out rate during the 3-year study period, 
approximately 240 seropositive subjects will be enrolled.
Some laboratory assays will be performed on a subset. The subset will be defined based 
on the outcome of the primary/secondary endpoints or the development of the assay. The 
subset is the minimum number of subjects that will be tested. If applicable, all subjects 
could be tested.
8.3.
Classification of cases (amended 19 February 2015)

A primary infection is defined as the first infection with CMV in subjects who were 
seronegative at enrolment and will be evaluated by:

Appearance of anti-CMV tegument protein IgG antibodies in serum
AND/OR

Appearance of anti-gB IgG antibodies in serum
WITH or WITHOUT

Presence of CMV DNA in urine.
 
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
CMV re-infection* with a different CMV strain in subjects who were seropositive at 
the time of enrolment will be evaluated by:

Appearance or increase of anti-CMV tegument protein IgG antibodies in serum
AND/OR

Appearance or increase of anti-gB IgG antibodies in serum
AND/OR

Appearance or increase of CMV DNA in urine
AND (depending on assay availability) in the presence of 

Change in polymorphism in the antibody recognition profile in follow-up serum 
samples 
AND/OR

Genotyping data characteristic of a re-infection 

CMV re-activation* of latent CMV in subjects who were seropositive at the time of 
enrolment will be evaluated by:

Appearance or increase of anti-CMV tegument protein IgG antibodies in serum
AND/OR

Appearance or increase of anti-gB IgG antibodies in serum
AND/OR

Appearance or increase of CMV DNA in urine
AND (depending on assay availability) in absence of 

Change in polymorphism in the antibody recognition profile in follow-up serum 
samples 
AND/OR

Genotyping data characteristic of a re-infection 
* Depending on the future scientific knowledge and the data generated in this study 
multiple case definition might be explored.
8.4.
Study cohorts to be evaluated
8.4.1.
Total cohort
The Total cohort will include all subjects enrolled in the study. 
8.4.2.
According-to-protocol cohort (amended 19 February 2015)
The according-to-protocol (ATP) cohort will include all subjects who meet all 
inclusion criteria and no exclusion criteria for the study, who do not have any elimination 
criteria during the study (Section 5.5.2.9) and who comply with the study procedures.
 
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A detailed, comprehensive list of reasons for elimination from ATP analyses will be 
established at the time of data cleaning.
8.5.
Derived and transformed data
Immunogenicity
The cut-off value is defined by the laboratory before the analysis and is described in 
Table 6.

A seronegative subject is a subject whose titer is below the cut-off value. 

A seropositive subject is a subject whose titer is greater than or equal to the cut-
off value. 
For seronegative subjects, seroconversion is defined as the appearance of antibodies (i.e. 
titer greater than or equal to the cut-off value). 
The geometric mean titers (GMTs) calculations will be performed by taking the anti-log 
of the mean of log titer transformations. Antibody titers below the cut-off of the assay 
will be given an arbitrary value of half the cut-off for the purpose of GMT calculation.
Virology
For seronegative subjects, CMV DNAeamia is defined as the detection of CMV DNA 
(i.e. number of CMV DNA copies/mL is greater than or equal to the cut-off value).
8.6.
Conduct of analyses
Any deviation(s) or change(s) from the original statistical plan outlined in this protocol 
will be described and justified in the final study report.
8.6.1.
Sequence of analyses (amended 19 February 2015)

The final analyses on all endpoints will be done when all data are available for 
Visit 10 (Month 36), and provided in a full study report.

All analyses based on tertiary endpoints not available at the time of the writing of the 
full report, will be provided in annex reports.
All analyses will be performed on clean data.
8.7.
Statistical methods
Statistical analyses will be described in detail in a separate document, the statistical 
analysis plan (SAP).
The primary analysis will be performed on the ATP cohort(s). Analysis on the Total 
cohort may be performed to complement the primary analysis. (Amended 19 February 
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8.7.1.
Analysis of demographics/baseline characteristics
Demographic (e.g. age at study entry in years, race) and baseline characteristics will be 
summarized using descriptive statistics.

Frequency tables will be generated for categorical variable such as race.

Mean, median, standard error will be provided for continuous data such as age.
8.7.2.
Analysis of primary objective
Descriptive statistics will be provided for each primary endpoint at all available time-
points and will include (not exhaustive) the following tabulations (with 95% confidence 
interval [CI]):

The percentage of CMV seropositive subjects with appearance or increase of anti-
CMV tegument protein IgG antibodies in serum.

The percentage of CMV seropositive subjects with appearance or increase of anti-gB 
IgG antibodies in serum when applicable.

The percentage of CMV seropositive subjects with appearance or increase of CMV 
DNA in urine.
8.7.3.
Analysis of secondary objective
Descriptive statistics will be provided for each secondary endpoint at each timepoint for 
which a sample is available time-points and will include (not exhaustive) the following 
tabulations (with 95% CI):

The percentage of CMV seronegative subjects with appearance of anti-CMV 
tegument protein IgG antibodies in serum.

The percentage of CMV seronegative subjects with appearance of anti-gB IgG 
antibodies in serum when applicable.
8.7.4.
Tertiary objectives (amended 19 February 2015)
Based on the results of primary and secondary endpoints, following statistical analyses 
on tertiary endpoints might be done:
Descriptive statistics will be provided for the number of CMV DNA copies in urine at
each timepoint for which a sample is available time-points and the percentage of CMV 
seronegative subjects with presence of CMV DNA in urine will be tabulated (with 95% 
CI).
For all results of assays performed for the further characterization of primary and/or 
secondary CMV infections, descriptive statistics will be provided for each assay at each 
timepoint for which a sample is available time-points.
Analysis performed for the evaluation of the impact of demographic, social or behavioral 
factors upon CMV infection will be described in a separate document (SAP).
 
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Analysis of results following genotyping/sequencing, mRNA microarray and peptide 
microarrays will also be described in a separate document (SAP).
The number and percentage of CMV infected newborns will be tabulated and descriptive 
statistics will be provided for the number of CMV DNA copies.
9.
ADMINISTRATIVE MATTERS
To comply with ICH GCP administrative obligations relating to data collection, 
monitoring, archiving data, audits, confidentiality and publications must be fulfilled. 
9.1.
Remote Data Entry instructions
Remote Data Entry (RDE), a validated computer application, will be used as the method 
for data collection. 
In all cases, subject initials will not be collected nor transmitted to GSK. Subject data 
necessary for analysis and reporting will be entered/transmitted into a validated database 
or data system. Clinical data management will be performed in accordance with 
applicable GSK standards and data cleaning procedures.
While completed eCRFs are reviewed by a GSK Biologicals’ Site Monitor at the study 
site, omissions or inconsistencies detected by subsequent eCRF review may necessitate 
clarification or correction of omissions or inconsistencies with documentation and 
approval by the investigator or appropriately qualified designate. In all cases, the 
investigator remains accountable for the study data. 
The investigator will be provided with a CD-ROM of the final version of the data 
generated at the investigational site once the database is archived and the clinical study 
report is complete and approved by all parties.
9.2.
Monitoring by GSK Biologicals
Monitoring visits by a GSK Site Monitor are for the purpose of confirming that GSK 
Biologicals’ sponsored studies are being conducted in accordance with the ethical 
principles that have their origins in the Declaration of Helsinki and that are consistent 
with GCP and the applicable regulatory requirement(s) (verifying continuing compliance 
with the protocol, amendment(s), verifying that the site staff and facilities continue to be 
adequate to conduct the study). 
The investigator must ensure provision of reasonable time, space and qualified personnel 
for monitoring visits.
Direct access to all study-site related and source data is mandatory for the purpose of 
monitoring review. The monitor will perform a RDE review and a Source Document 
Verification (SDV). By SDV we understand verifying CRF/ RDE entries by comparing 
them with the source data that will be made available by the investigator for this purpose. 
 
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The Source Documentation Agreement Form describes the source data for the different 
data in the RDE. This document should be completed and signed by the site monitor and 
investigator and should be filed in the monitor’s and investigator’s study file. Any data 
item for which the RDE will serve as the source must be identified, agreed and 
documented in the source documentation agreement form. 
For RDE, the monitor will mark completed and approved screens at each visit 
In accordance with applicable regulations, GCP, and GSK procedures, GSK monitors 
will contact the site prior to the start of the study to review with the site staff the protocol, 
study requirements, and their responsibilities to satisfy regulatory, ethical, and GSK 
requirements. When reviewing data collection procedures, the discussion will also 
include identification, agreement and documentation of data items for which the CRF
entries will serve as the source document.
GSK will monitor the study to verify that, amongst others, the: 

Data are authentic, accurate, and complete.

Safety and rights of subjects are being protected.

Study is conducted in accordance with the currently approved protocol and any 
amendments, any other study agreements, GCP and all applicable regulatory 
requirements. 
The investigator and the head of the medical institution (where applicable) agrees to 
allow the monitor direct access to all relevant documents.
Upon completion or premature discontinuation of the study, the monitor will conduct site 
closure activities with the investigator or site staff, as appropriate, in accordance with 
applicable regulations, GCP, and GSK procedures.
9.3.
Archiving of data at study sites
Following closure of the study, the investigator must maintain all site study records in a 
safe and secure location. The records must be maintained to allow easy and timely 
retrieval, when needed (e.g. audit or inspection), and, whenever feasible, to allow any 
subsequent review of data in conjunction with assessment of the facility, supporting 
systems, and staff. Where permitted by applicable laws/regulations or institutional policy, 
some or all of these records can be maintained in a validated format other than hard copy 
(e.g. microfiche, scanned, electronic for studies with an eCRF); however, caution needs 
to be exercised before such action is taken. The investigator must assure that all 
reproductions are legible and are a true and accurate copy of the original and meet 
accessibility and retrieval standards, including re-generating a hard copy, if required. 
Furthermore, the investigator must ensure there is an acceptable back-up of these 
reproductions and that an acceptable quality control process exists for making these 
reproductions.
GSK will inform the investigator/institution of the time period for retaining these records 
to comply with all applicable regulatory requirements. However, the 
 
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investigator/institution should seek the written approval of the sponsor before proceeding 
with the disposal of these records. The minimum retention time will meet the strictest 
standard applicable to that site for the study, as dictated by ICH GCP any institutional 
requirements or applicable laws or regulations, or GSK standards/procedures; otherwise, 
the minimum retention period will default to 15 years. 
The investigator/institution must notify GSK of any changes in the archival 
arrangements, including, but not limited to, the following: archival at an off-site facility, 
transfer of ownership of the records in the event the investigator leaves the site.
9.4.
Audits
To ensure compliance with GCP and all applicable regulatory requirements, GSK may 
conduct a quality assurance audit. Regulatory agencies may also conduct a regulatory 
inspection of this study. Such audits/inspections can occur at any time during or after 
completion of the study. If an audit or inspection occurs, the investigator and institution 
agree to allow the auditor/inspector direct access to all relevant documents and to allocate 
his/her time and the time of his/her staff to the auditor/inspector to discuss findings and 
any relevant issues.
9.5.
Posting of information on public registers
Study information from this protocol will be posted on public registers (e.g. GSK Clinical 
Study Register, clinicaltrials.gov) before enrolment of subjects begins as applicable.
9.6.
Ownership, confidentiality and publication
9.6.1.
Ownership
All information provided by GSK and all data and information generated by the site as 
part of the study (other than a subject’s medical records) are the sole property of GSK.
All rights, title, and interests in any inventions, know-how or other intellectual or 
industrial property rights which are conceived or reduced to practice by site staff during 
the course of or as a result of the study are the sole property of GSK, and are hereby 
assigned to GSK.
If a written contract for the conduct of the study which includes ownership provisions 
inconsistent with this statement is executed between GSK and the study site, that 
contract’s ownership provisions shall apply rather than this statement.
9.6.2.
Confidentiality
Documented evidence that a potential investigator is aware and agrees to the confidential 
nature of the information related to the study must be obtained by means of a 
confidentiality agreement.
All information provided by GSK and all data and information generated by the site as 
part of the study (other than a subject’s medical records) will be kept confidential by the 
 
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investigator and other site staff. This information and data will not be used by the 
investigator or other site personnel for any purpose other than conducting the study. 
These restrictions do not apply to: (i) information which becomes publicly available 
through no fault of the investigator or site staff; (ii) information which it is necessary to 
disclose in confidence to an IEC or IRB solely for the evaluation of the study; (iii) 
information which it is necessary to disclose in order to provide appropriate medical care 
to a study subject; or (iv) study results which may be published as described in the next 
paragraph. If a written contract for the conduct of the study which includes 
confidentiality provisions inconsistent with this statement is executed, that contract’s 
confidentiality provisions shall apply rather than this statement.
9.6.3.
Publication
For multi-center studies, the first publication or disclosure of study results shall be a 
complete, joint multi-center publication or disclosure coordinated by GSK. Thereafter, 
any secondary publications will reference the original publication(s).
Prior to submitting for publication, presentation, use for instructional purposes, or 
otherwise disclosing the study results generated by the site (collectively, a ‘Publication’), 
the investigator shall provide GSK with a copy of the proposed Publication and allow 
GSK a period to review the proposed Publication (at least twenty-one working days, or at 
least fifteen working days for abstracts/posters/presentations). Proposed Publications 
shall not include either GSK confidential information other than the study results or 
personal data on any subject, such as name or initials.
At GSK’s request, the submission or other disclosure of a proposed Publication will be 
delayed a sufficient time to allow GSK to seek patent or similar protection of any 
inventions, know-how or other intellectual or industrial property rights disclosed in the 
proposed Publication.
If a written contract for the conduct of the study, which includes publication provisions 
inconsistent with this statement is executed, that contract’s publication provisions shall 
apply rather than this statement.
9.6.4.
Provision of study results to investigators and publication
Where required by applicable regulatory requirements, an investigator signatory will be 
identified for the approval of the study report. The investigator will be provided 
reasonable access to statistical tables, figures, and relevant reports and will have the 
opportunity to review the complete study results at a GSK site or other mutually-
agreeable location.
A manuscript will be progressed for publication in the scientific literature if the results 
provide important scientific or medical knowledge.
10.
COUNTRY SPECIFIC REQUIREMENTS
Not applicable
 
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11.
REFERENCES
Boppana SB, Fowler KB, Britt WJ, Stagno S, Pass RF. Symptomatic congenital 
cytomegalovirus infection in infants born to mothers with preexisting immunity to 
cytomegalovirus. Pediatrics. 1999;104:55-60. 
Boppana SB, Rivera LB, Fowler KB, Mach M, Britt WJ. Intrauterine transmission of 
cytomegalovirus to infants of women with preconceptional immunity. N Engl J Med.
2001;344(18):1344:1366-71.
Cannon MJ, Schmid DS, Hyde TB. Review of cytomegalovirus seroprevalence and 
demographic characteristics associated with infection. Rev Med Virol. 2010;20(4):202-
13. 
Colugnati FA, Staras SA, Dollard SC, Cannon MJ. Incidence of cytomegalovirus 
infection among the general population and pregnant women in the United States. BMC 
Infect Dis. 2007;7:71.
De Souza S, Bonon SH, Costa SC, Rossi CL. Evaluation of an in-house specific 
immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from longterm 
human cytomegalovirus (HCMV) infection. Rev Inst Med Trop Sao Paulo. 2003; 45:323-
326.
Dollard SC, Grosse SD, Ross DS. New estimates of the prevalence of neurological and 
sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev 
Med Virol. 2007;17(5):355-63.
Fowler KB, Stagno S, Pass RF. Maternal immunity and prevention of congenital 
cytomegalovirus infection. JAMA. 2003;289:1008-1011.
Fowler KB, Boppana SB. Congenital cytomegalovirus (CMV) infection and hearing 
deficit. J Clin Virol. 2006:35(2):226-31.
Hyde TB, Schmid DS, Cannon MJ. Cytomegalovirus seroconversion rates and risk 
factors: implications for congenital CMV. Rev Med Virol. 2010;20(5):311-26.
Mussi-Pinhata MM, Yamamoto AY, Moura Brito RM, de Lima Isaac M, de Carvalho e 
Oliveira PF, Boppana S, Britt WJ. Birth prevalence and natural history of congenital 
cytomegalovirus infection in a highly seroimmune population. Clin Infect Dis. 
2009;49(4):522-8.
Novak Z, Ross SA, Patro RK, Pati SK, Kumbla RA, Brice S and Boppana SA. 
Cytomegalovirus Strain Diversity in Seropositive Women. Journal of 
ClinicalMicrobiology 2008, 46(3):882–886. 
Nyholm JL, Schleiss MR. Prevention of maternal cytomegalovirus infection: current 
status and future prospects. Int J Women Health. 2010;2:23-35.
 
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Ross SA, Fowler KB, Ashrith G, Stagno S, Britt WJ, Pass RF, Boppana SB. Hearing loss 
in children with congenital cytomegalovirus infection born to mothers with preexisting 
immunity. J Pediatr. 2006;148(3):332-6. 
Yamamoto AY, Mussi-Pinhata MM, Boppana SB, Novak Z, Wagatsuma VM, Oliveira 
PdeF, Duarte G, Britt WJ. Human cytomegalovirus reinfection is associated with 
intrauterine transmission in a highly cytomegalovirus-immune maternal population. Am J 
Obstet Gynecol. 2010;202(3):297.e1-8
Yamamoto AY, Mussi-Pinhata MM, Isaac MD, Amaral FR, Carvalheiro CG, Aragon 
DC, da Silva Manfredi AK, Boppana SB, Britt WJ. Congenital Cytomegalovirus 
Infection as a Cause of Sensorineural Hearing Loss in a Highly Immune Population. 
Pediatr Infect Dis J. 2011;30(12):1043-1046.
 
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APPENDIX A
LABORATORY ASSAYS
Note that some assays, the general principles of which are described below, are still in 
development or planned for development at GSK laboratories or delegate and that it 
cannot be assumed that all of them will be successfully developed due to possible 
technical constraints. It is also assumed that some already available assays can be slightly 
modified in order to improve robustness and quality of these assays.
1.
Anti-CMV tegument proteins IgG ELISA (biotest like assay)
The assay is an indirect solid-phase ELISA for the detection of specific IgG antibodies 
using two highly purified, autologous fusion proteins, CG1 and CG2, each combining 
two immunodominant fragments from HCMV tegument (pp150; CG1:UL32, amino acids 
(aa) 495-691 and 862-1048; CG2:UL32, aa 695-854) and the delayed-early DNA binding 
protein (pp52; UL44, aa 297-433). If the sample contains specific antibodies, these bind 
to the recombinant antigens in the microtiter wells. Non-specific antibodies are removed 
by washing steps. The antibody-antigen complex is detected by peroxidase-labeled anti-
human IgG antibodies. The presence of bound antibodies is demonstrated by adding the 
chromogen-substrate solution.
2.
Anti-gB IgG ELISA
Anti-HCMV gB antibody concentrations are measured by ELISA using recombinant gB 
as coating antigen on 96-well microplates. After washing, the wells are blocked with a 
saturating reagent. Two dilutions of sera, in duplicate, as well as controls are incubated 
onto the coated wells to allow the specific antibodies, present in the sample, to react with 
gB. Non-specific antibodies are removed by washing steps. The antibody-antigen 
complex is detected by peroxidase-labeled anti-human IgG antibodies. The presence of 
bound antibodies is demonstrated by adding the chromogen-substrate solution.
3.
Anti-CMV IgM ELISA
The method used for qualitative determination of specific IgM antibodies to 
cytomegalovirus in human serum is an immunoenzymatic assay based on the antibody 
capture ELISA technique. The removable polystyrene wells of the 96 well microplates 
are precoated with IgG mouse monoclonal to human IgM. During the first step of the 
immunological reaction, diluted serum samples and controls (negative, positive, cut-off 
control and diluent) are incubated into the pre-coated plate. The plates are washed to 
remove non-specific reactants, and a mix of enzyme-labeled detection antibody (IgG to 
CMV conjugated to horseradish peroxidase) and CMV antigen is added. The presence of 
specific IgM to CMV allows the tracer to bind to the solid phase through the presence of 
CMV antigen. After a washing step to remove excess of conjugate, the chromogen-
enzyme substrate is added to measure the enzyme activity. After incubation at room 
temperature away from direct light the enzymatic reaction is stopped by the addition of 
Stop Reagent. The intensity of the resultant color change is measured by a 
spectrophotometer. The optical density (OD) is proportional to the concentration of the 
anti-CMV IgM present in the sample. The IgM concentration in the cut-off control is 
calibrated in order to provide a cut-off, unknown samples with an OD above the cut-off 
OD + 10% cut-off OD are positive, those with an OD below the cut-off OD – 10% cut-
 
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off OD are negative. In the greyzone around the cut-off OD (cut-off OD +/- 10% cut-off 
OD) samples are retested.
4.
Anti-CMV tegument protein IgG avidity index
This assay will be performed using an elution step with a chaotropic agent to remove 
low-avidity antibodies bound to CG1 and CG2 fusion proteins. Briefly, each diluted 
serum is first added to wells of plates coated with CG1 and CG2 fusion proteins. After 
incubation, the unbound antibodies are removed by washing, and a chaotropic agent is 
then added in the coated wells to dissociate low-avidity antibodies. The microplate is then 
washed, and horseradish peroxidase (HRP)-conjugated anti-human IgG antibodies is 
added. After incubation, unbound antibodies are removed by washing and the 
chromogen-substrate solution is added to reveal the enzyme activity. The color reaction is 
stopped by addition of a stop reagent and the resulting color is measured 
spectrophotometrically. An avidity index is then calculated.
5.
Anti-gB IgG avidity index
This assay will be performed according to [De Souza, 2003] using an elution step with 
urea to remove low-avidity antibodies from gB antigen. Briefly, each diluted serum is 
first added to wells of plates coated with gB antigen. After incubation, the unbound 
antibodies are removed by washing, and 8M urea in phosphate buffered saline is then 
added in the coated wells to dissociate low-avidity antibodies. The microplate is then 
washed, and HRP-conjugated anti-human IgG antibodies are added. After incubation, 
unbound antibodies are removed by washing and the chromogen-substrate solution is
added to reveal the enzyme activity. The color reaction is stopped by addition of a stop 
reagent and the resulting yellow color is measured spectrophotometrically.
The avidity index is calculated as the mean absorbance of reactions in which the immune 
complexes are exposed to urea divided by the mean absorbance of reactions in which the 
immune complexes are not exposed to urea, expressed as a percentage.
6.
Anti-CMV neutralisation assay
The microneutralization test uses MRC-5 human lung embryo fibroblast cells as the 
infection target. The different serum sample dilutions are mixed with a fixed amount of 
virus at different dilutions and the serum-virus mixture is incubated at 37°C with MRC-5 
cells. Infected cells are then fixed with acetone. The degree to which neutralizing
antibodies decrease CMV infection of cells in the assay is determined by counting the 
number of nuclei expressing the CMV 72 kDa IE1 protein. Nuclear IE1 is detected using 
highly sensitive immunoperoxidase staining. The stained nuclei are counted. The 
neutralization titer is expressed as the reciprocal of the serum dilution that reduced by 
50% the number of infected cells as compared to a control without serum.
Additional microneutralization assays using other cell types such as epithelial, 
trophoblast cells as the infection targets and other virus strains may also be performed.
 
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7.
Antibody recognition profile
The exact methodology to perform antibody recognition profiling is not yet decided but 
the type of assay may be derived from classical high or low density peptide arrays. The 
peptide –microarray is a technology allowing the simultaneous measurement of serum 
reactivity (IgG, IgM, IgA or IgE) to hundreds or thousands of peptides in a single 
biological sample. Briefly, solid support spotted/coated with CMV peptides are incubated 
with serum dilutions. Slides are then washed to remove unbound antibodies and 
incubated with labeled anti-human Immunoglobulin. After a washing step, antibody 
recognition profile against different peptides is revealed by fluorescence or other means 
following the technology platform selected.
8.
Detection of CMV-DNA (qPCR)
Quantitative PCR provides a rapid and sensitive method for the diagnosis of CMV 
infection. The CMV detection test used by GSK Biologicals is designed to detect the 
pp65 gene.
Real-time PCR technology allows the determination of the copy number of sequence-
specific nucleic acids target molecules. The amplification of the target sequence is 
detected using the 5’ nuclease assay based on the Taqman chemistry. In those PCR 
experiments, the formation of a PCR product is monitored in real-time during 
amplification by means of a fluorogenic probe that binds specifically to the amplified 
product. The reporter fluorophore is at the 5’ end of the Taqman probe and the quencher 
is at the 3’ end. As long as the probe is intact, no fluorescence is observed from the 
fluorophore. During the polymerization step, the Taq DNA polymerase displaces the 
Taqman probe by 3-4 nucleotides, and the 5’ nuclease activity of the DNA polymerase 
separates the fluorophore from the quencher. The fractional cycle number at which the 
generated fluorescence passes a fixed threshold above the baseline defines a “Threshold 
Cycle” (Ct value). A standard curve can be generated from the log of the starting copy 
number of DNA references against the measured Ct value.
In the context of this study, the qPCR technology is used to detect CMV DNA in 
different matrix (blood, urine and saliva) following adapted purification methodology.
9.
CMV strain typing
In addition to direct detection of CMV through PCR, the company is seeking to develop, 
evaluate and validate next generation sequencing based assay and/or other technologies
aiming to characterize CMV genomic diversity that allow discriminating re-infection or 
re-activation to primary infection.
10. mRNA microarray /qPCR
Transcriptional profiling in the blood consists of measuring RNA abundance in 
circulating nucleated cells. Changes in transcript abundance can result from exposure to 
host or pathogen-derived immunogenic factors and/or changes in relative cellular 
composition. Techniques such as mRNA microarrays of qPCR may be used to evaluate 
these changes, if available.
 
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APPENDIX B
CLINICAL LABORATORIES
Table 10
GSK Biologicals’ laboratories
Laboratory
Address
GSK Biologicals Global Vaccine 
Clinical Laboratory, Rixensart
Biospecimen Reception - B7/44
Rue de l'Institut, 89 - B-1330 Rixensart - Belgium
GSK Biologicals Global Vaccine 
Clinical Laboratory, North America-
Laval
Biospecimen Reception - Clinical Serology
525 Cartier blvd West - Laval - Quebec - Canada -
H7V 3S8
GSK Biologicals Global Vaccine 
Clinical Laboratory, Wavre-Nord 
Noir Epine
Avenue Fleming, 20 - B-1300 Wavre - Belgium
Table 11
Outsourced laboratories
Laboratory
Address
DDL Diagnostic Laboratory B.V.
Fonteijnenburghlaan 7
Voorburg
Netherland
BARC USA Inc
5, Delaware Drive
Lake Success
NY 11042-1114
USA
CERBA European labs
7/11 Rue de l’Equerre, Parc d’activités ‘les Béthunes’
95310 Saint Ouent l’Aumone
France
 
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APPENDIX C
AMENDMENTS TO THE PROTOCOL
GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 1
eTrack study number 
and Abbreviated Title
115639 (CMV-014 EXPLO)
Amendment number:
Amendment 1
Amendment date:
31-JUL-2013
Co-ordinating author:
, GSK Biologicals’
Rationale/background for changes: 
Initially, the study was set-up, as a preparation for large phase III trials, to assess with 
good precision the rates of CMV re-infections and reactivations in CMV seropositive 
adolescents from different countries or areas with presumably high CMV 
seroprevalence. Additionally, the study aimed at collecting human biological samples 
to support the development of new laboratory assays that would be both specific and 
sensitive for the detection of these biological events but also that would facilitate the 
conduct of large efficacy trials using non/less-invasive sampling methods. 
Since the initial discussions on the CMV-014 study, the company has progressed on its 
reflection of a clinical program needed for the development of a CMV vaccine. Over 
time it appeared clearer that the success of a phase III trial necessitated a better 
understanding of the complex virology and immune parameters in CMV seropositive 
subjects. Consequently, the Company has decided to switch the CMV program from a 
classical development strategy to a more experimental, clinical research status. 
The CMV-014 EXPLO study has been redesigned to better align with this new scope 
and will serve the design of future vaccine research studies in terms of scientific 
questions to be addressed, like assays to be used and developed. The protocol 
amendment of the study CMV-014 EXPLO is reflecting these strategic changes : the 
incidence of CMV primary and secondary infection in adolescent females will be 
evaluated using less stringent assumptions on CMV re-infection and reactivation attack 
rates . Assays primarily intended to evaluate the feasibility of sample collection and 
testing in the context of large phase III trials were removed from the present study that 
will focus more on the development of assays used to better decipher the complex 
virological and immune parameters associated to CMV infection.
 
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In consequence:

The planned number of seropositive subjects was decreased from 475 to 240, 
with an estimated total enrolled subjects of 400 instead of 800 which is still 
considered sufficient to evaluate the incidence of CMV primary and secondary 
infection.

The objectives and endpoints of the study were revised:

The anti-tegument IgG ELISA testing in saliva was removed, as it was more 
aimed to address operational questions in anticipation of Phase III clinical 
trials rather than to address the actual evaluation of the incidence of CMV 
primary and secondary infections. Indeed, in case of good correlation between 
the anti-tegument IgG ELISA in serum and in saliva, the anti-tegument IgG 
ELISA in saliva was intended to be used as a more practical diagnostic 
readout to evaluate CMV infection in large Phase III trials. 

Similarly, the qPCR on dry saliva and dry urine samples were removed. They 
were initially included to assess the feasibility to collect some samples using 
more practical sampling devices in large Phase III trials. 

The analysis of anti-gB IgG ELISA in serum was moved to tertiary endpoint 
because the anti-tegument IgG ELISA in serum (which should provide 
equivalent results) is already tested as per primary and secondary endpoints. 
The anti-gB ELISA might only be used in a subset of subjects to confirm 
results generated with the anti-tegument IgG ELISA and/or to confirm the 
case definition. 

The occurrence of congenital CMV infections of newborns from subjects who 
became pregnant during the study will still be evaluated. Nevertheless deep 
genotypic characterization activities will be reserved to CMV strains infecting 
the mothers and not the babies. 

The evaluation of T-cell proliferation (mentioned as a tertiary endpoint) was 
removed as it was finally decided not to collect PBMC in this study. 
Note that all samples will still be used, except the dry urine and dry saliva samples 
which are prepared from the collected urine and saliva samples collected from the 
subjects (no change of procedure for the subjects). Some dry urine samples were 
already prepared for the enrolled subjects and will not be used. No dry saliva samples 
were already prepared. Dry saliva and dry urine samples will not be prepared from 
upcoming samples. The section on the laboratory assays was updated accordingly.
 
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
The list of laboratories to perform the tests was updated, as the collaboration 
with ImmuneHealth will be stopped before the first testing of CMV-014 
samples. A service provider was selected for the development of the qPCR in 
saliva, urine and blood and for the associated clinical testing. Some other 
laboratories are still to be identified.

Appendix A was slightly reworded to give a more general description of the 
principle of the assays. Indeed, some of these assays are still in development or 
are susceptible to improvement for robustness and quality purposes.

Some minor updated were made

The list of contributing authors was updated following changes in the team 
members/ function names.
Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
Co-ordinating author: 
(Keyrus Biopharma contractor for GSK Biologicals), 
 
, Scientific Writers
Contributing authors:

,
, Clinical Development Manager

, 
, Study Delivery Lead Global Study Manager

, 
, Project Statistician

, 
, 
 (Keyrus Biopharma 
contractor for GSK Biologicals), Study Data Manager Clinical Data Coordinator 

, 
, Safety Representative (Valesta 
contractor for GSK Biologicals)
Synopsis
Rationale for the study design

To evaluate the operational feasibility for future studies.
Tertiary objectives

To evaluate the degree of matching of the detected CMV strain(s) in newborn(s) 
and the mother.
 
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Synopsis Table 1: study groups foreseen in the study
Study Groups
Number of subjects
Age (Min/Max)
S+
+/- 475 240 seropositive subjects
10 – 17 years
S-
Estimated approximate number of seronegative subjects(1): 
325160
10 – 17 years
(1) As subjects will be enrolled consecutively without previous CMV screening with a target number of 
475 240 seropositive subjects, the number of seronegative subjects enrolled will depend on the seroprevalence of 
the participating countries.
Discussion of study design
The study sites will be selected in multiple countries, including countries where the 
high CMV prevalence is assumed to be high in order to enroll primarily CMV 
seropositive adolescent females to allow the evaluation of CMV secondary infections. 
Since enrolment will proceed without previous CMV screening, some of the enrolled 
subjects will be seronegative. The enrolled seronegative subjects will allow the 
estimation of the incidence of CMV primary infections in the adolescent population.
To increase the probability to capture all CMV infections (primary and secondary 
infections), regular sample collection time-points have been arranged:

Site Visits: Subjects will be asked to perform a Site Visit approximately every 
4 months for 3 years (10 Site Visits in total). At Site Visits, blood, urine, dry 
urine, and saliva, and dry saliva will be collected.
Number of subjects
Subjects will be enrolled consecutively without previous CMV screening. Enrolment will 
be terminated once approximately 475 240 seropositive subjects are included in the trial. 
The number of seronegative subjects enrolled will depend on the seroprevalence of the 
participating countries. It is estimated that approximately 800 400 subjects will be 
enrolled in total.
Primary endpoint

Occurrence of CMV secondary infections determined in all seropositive subjects on 
samples collected during the 4-month Site Visits until study conclusion:

Anti-CMV tegument protein IgG antibody concentration in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-CMV tegument protein IgG antibody concentration in saliva (ELISA).

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).
Secondary endpoints

Occurrence of CMV primary infection determined in all seronegative subjects on 
samples collected during the 4-month Site Visits until study conclusion:

Anti-CMV tegument protein IgG antibody concentration in serum (ELISA).
 
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
Anti-gB IgG antibody concentration in serum (ELISA).

Anti-CMV tegument protein IgG antibody concentration in saliva (ELISA).
Tertiary endpoints

Further characterization of primary and /or secondary CMV infections in a subset of 
subjects on all available selected samples of the Sample Collection Visits*.

Anti-CMV tegument protein IgG antibody concentration in saliva (ELISA).

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Number of CMV DNA copies (pp65 or other genes) in saliva (qPCR).
* In case of a positive sample at a 4-month Site Visit time point, testing will be done on 
all available selected samples collected during the Sample Collection Visit. The selection 
of samples to be tested will be based on the qPCR results obtained following the Site 
visits.

Further characterization of primary and /or secondary CMV infections in a subset of 
subjects on all available samples of the 4-month Site Visits:

Anti-CMV tegument protein IgG antibody avidity index in serum (ELISA).

Anti-gB IgG antibody avidity index in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-CMV IgM antibody concentration in serum (ELISA).

Number of CMV DNA copies (pp65 or other genes) in saliva, dry saliva, dry 
urine, blood (qPCR).

Assessment of anti-CMV neutralizing antibodies on different target cells and/or 
other virus strains.

Development of assays that will allow differentiating re-infection from re-
activation from primary infection in a subset of subjects

Characterization of CMV strains by genotyping/sequencing.

Exploring the CMV strain specific antibody profile using peptide microarrays.

Assessment of the expression of host genes* related to immunity using 
techniques such as qPCR or mRNA microarray in a subset of subjects, and/or 
other functions such as T-cell proliferation.
* excluding genes related to hereditary characteristics of the subject.

Evaluation of the impact of demographic, social or behavioral factors upon CMV 
infection.

Occurrence and characterization of congenital CMV infection in newborns of 
subjects who become pregnant during the study:

Evidence of CMV DNA in urine and/or saliva of newborns within 10 days of 
delivery by using qPCR (pp65 or other genes).
 
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
Characterization of strains by genotyping/sequencing in newborns from re-
infected mothers.
1.2. Rationale for the study

To evaluate the operational feasibility for future studies.
2.3. Tertiary objectives

To evaluate the degree of matching of the detected CMV strain(s) in newborn(s) 
and the mother.
3. Study design overview
Time point
Year 1
Month
M0
M2
M4
M6
M8
M10
M12
Site Visits
V1
V2
V3
V4
Sample Collection Visits
SCV1
SCV2
SCV3
Year 2
Month
M14
M16
M18
M20
M22
M24
Site Visits
V5
V6
V7
Sample Collection Visits
SCV4
SCV5
SCV6
Year 3
Month
M26
M28
M30
M32
M34
M36
Site Visits
V8
V9
V10
Sample Collection Visits
SCV7
SCV8
SCV9
Samples:
Blood




Saliva







Dry Saliva




Urine







Dry Urine




In case of pregnancy resulting in live birth: Urine and or saliva from the newborn (please refer to Table 3) will be 
collected (if possible) as per standard of care in the center where the subject has delivered or through a home 
visiting nurse or alternatively, the subject may be asked to come back to the study center within 10 days of 
delivery.

Number of subjects and study groups: 
At Visit 1, subjects will be enrolled regardless of their seropositivity status. Subjects 
will be allocated to the CMV seropositive (S+) or the CMV seronegative (S-) groups 
based on the local laboratory results (alternatively, a GSK designated and validated
central laboratory can perform the analyses). Enrolment will be terminated when 
approximately 475 240 CMV seropositive females have been enrolled.
 
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Table 1: Study groups foreseen in the study
Study Groups
Number of subjects
Age (Min/Max)
S+
+/- 240 240 seropositive subjects
10 – 17 years
S-
Estimated approximate number of seronegative subjects(1): 160 160
10 – 17 years
(1) As subjects will be enrolled consecutively without previous CMV screening with a target number of 475 240
seropositive subjects, the number of seronegative subjects enrolled will depend on the seroprevalence of the 
participating countries.

Time points and biological samples

Blood, dry saliva and dry urine: approximately every 4 months;

Saliva and urine: approximately every 2 months;

Urine and /or saliva of newborn (in case of pregnancy): within 10 days post-
delivery.
3.1. Discussion of the study design
The study sites will be selected in multiple countries, including countries where the
high CMV prevalence is assumed to be high in order to enroll primarily 
CMV seropositive adolescent females to allow the evaluation of CMV secondary 
infections. Since enrolment will proceed without previous CMV screening, some of the 
enrolled subjects will be seronegative. The enrolled seronegative subjects will allow the 
estimation of the incidence of CMV primary infections in the adolescent population.

Site Visits: Subjects will be asked to perform a Site Visit approximately every 
4 months for 3 years (10 Site Visits in total). At Site Visits, blood, urine, dry 
urine, and saliva and dry saliva will be collected.
4.1. Number of subjects/ centers
The study will be conducted world-wide in countries with a high seroprevalence. 
Considering a 15-20% drop-out rate over the study period it is estimated that 
approximately 475 240 seropositive subjects should be enrolled to obtain approximately 
400200 evaluable seropositive subjects. 
Overview of the recruitment plan
Subjects will be enrolled consecutively without previous CMV screening. Enrolment will 
be terminated once approximately 475 240  seropositive subjects are included in the trial, 
based on the local laboratory results (or alternatively, based on the GSK designated and 
validated central laboratory results) obtained from the sample collected at Visit 1. The 
number of seronegative subjects enrolled will depend on the seroprevalence of the 
participating countries. It is estimated that approximately 800 400 subjects will be 
enrolled in total.
 
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5.4. Outline of study procedures
Table 2: List of study procedures
Age
10-17 years
Epoch
001
Year 1
Time-point (Months)
M0
M2
M4
M6
M8
M10
M12
Visit/Sample Collection Visit
V1
SCV1
V2
SCV2
V3
SCV3
V4
Year 2
Time-point (Months)
M14
M16
M18
M20
M22
M24
Visit/Sample Collection Visit
SCV4
V5
SCV5
V6
SCV6
V7
Year 3
Time-point (Months)
M26
M28
M30
M32
M34
M36
Visit/Sample Collection Visit
SCV7
V8
SCV8
V9
SCV9
V10
CMV serostatus(2) ( 2 ~3.5 to 5 mL)
●
Blood sample for gene expression signature 
(qPCR or RNA microarray; 2.5 mL)
●
●
●
Dry Urine
●
●
●
●
●
Saliva 
●
●
●
●
●
●
●
●
Dry saliva
●
●
●
●
●
5.5.2.4. CMV serostatus
Collect a blood sample (approximately 2 3.5 to 5 mL) at Site Visit 1 to determine the 
subject’s serostatus by the local laboratory (or alternatively, by a GSK designated and 
validated central laboratory). Record results of the test in the eCRF.
5.5.2.5. Blood sampling

A volume of approximately 2.5 mL of whole blood should be drawn from all 
subjects for ribonucleic acid (RNA) assays using qPCR or microarray and 
exploratory testing at Site Visits 1 (Month 0), 4 (Month 12), 7 (Month 24), and 10 
(Month 36).
5.5.2.6. Urine and dry urine sampling
Urine and dry urine will be collected for virology analysis and exploratory testing at each 
time-point specified in the list of study procedure (Table 2).
Refer to the module on Biospecimen Management in the SPM for general handling of dry 
urine samples and to the module on Biospecimen Management in the SPM and the 
sample collection booklet for the general handling of urine samples.
5.5.2.7. Saliva and dry saliva sampling
Saliva and dry saliva will be collected for virology analysis and exploratory testing at 
each time-point specified in the list of study procedure (Table 2).
Saliva will be collected from all subjects using a specific device at each Site Visit by the 
investigator or designate. A dry saliva sample will also be collected at each visit site.
 
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Refer to the module on Biospecimen Management in the SPM for general handling of dry 
saliva samples and to the module on Biospecimen Management in the SPM and the 
sample collection booklet for general handling of saliva samples.
5.5.3. Procedure during study participation for the newborns (in case of pregnancy 
and delivery during the study period)
If the delivery occurs during the study period, the pregnancy and, if the subject and/or 
subject’s parent(s)/LAR(s) has given her/his/their consent, information regarding CMV 
infection of the newborn will be documented (see Section 5.5.3) in the subject newborn’s
eCRF.
5.5.3.2. Record CMV infection status
If available, The CMV infection status (positive or negative) of the newborns will be 
recorded in the newborn’s eCRF of his/her mother.
5.5.3.3. Assessment of CMV disease at birth
Assessment of CMV disease (if applicable, symptomatic or asymptomatic) will be 
performed for newborns diagnosed as CMV-positive and record in the newborn’s eCRF 
of his/her mother. The investigator or designate (such as a pediatrician) will perform the 
assessment him/herself during the Site Visit which should occur within 10 days of 
delivery or will contact the center where the subject has delivered to obtain a copy of the 
medical dossier.
5.6.2. Biological samples
Table 5: Biological samples
Sample type
Quantity 
Time point
Priority 
rank
Dry urine
NA
Months 0, 4, 8, 12, 16, 20, 24, 28, 32 
and 36
2
Dry saliva
NA
Months 0, 4, 8, 12, 16, 20, 24, 28, 32 
and 36
2
 
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5.6.3. Laboratory assays
Table 6: Humoral immunity (antibody determination
System
Component
Method
Kit / 
Manufacturer
Unit
Cut-off
Number 
of 
subject
Laboratory 
(3)
Serum(1)
Anti-CMV 
tegument protein 
IgG 
ELISA
Biotest like 
assay
EU/mL
TBD
All 
subjects
Immune 
Health
TBD
Serum(1)
Anti-gB IgG
ELISA
In house
EU/mL
54
All 
subjects
Subset(2)
Immune 
Health
TBD
Saliva(1)
Anti-CMV 
tegument proteins 
IgG
ELISA
TBD
TBD
TBD
All 
subjects
Immune 
Health
Serum(1)
Anti-CMV 
tegument protein 
avidity index
ELISA
Biotest like 
assay
NA
NA
Subset(2)
Immune 
Health
TBD
Serum(1)
Anti-gB IgG 
avidity index
ELISA
In house
NA
NA
Subset(2)
Immune 
Health
TBD
Serum(1)
Anti-CMV IgM
ELISA
Diasorin or 
equivalent 
NA
NA 
Subset(2)
Immune 
Health
TBD
Serum
Anti-CMV 
neutralizing 
antibodies 
(multiple cell 
lines) and /or 
other virus strains
Seroneutralisation
In house
ED50
TBD
Subset(2)
Immune 
Health
TBD
Serum(1)
Antibody 
recognition profile
Peptide 
microarray
TBD
NA
NA
Subset(2)
TBD
(1) Coating Ag will be defined based on available commercial kits or in house developed assays (such as whole 
virus proteins or tegument proteins or others to be defined).
(2) Subset will be defined based on the outcome of primary/secondary endpoints or the development of the assay. 
The subset is the minimum number of subjects that will be tested. If applicable, all subjects could be tested.
(3) Refer to APPENDIX B for the laboratory addresses.
TBD = To be determined, gB = glycoprotein B; Ig = immunoglobulin, ELISA = Enzyme-linked immunosorbant 
assay, EU = ELISA unit, NA = not applicable; ED50 = effective dose 50, Ag = antigen.
The laboratories that will perform antibody determination are not yet identified and 
will be defined as soon as available.
 
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Table 7: Molecular Biology
System
Test
Component
Method
Unit
Number of 
subject
Laboratory (4)
Urine
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
All subjects(1)
Newborns
TBD
DDL
Urine
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
All Newborns
BARC
Saliva
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
Subset(3)
+ Newborns
TBD
DDL
Saliva
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
All Newborns
BARC
Dry Urine
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
Subset(3)
TBD
Dry 
Saliva
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
Subset(3)
TBD
Plasma/
buffy 
coat
Viral load 
detection
CMV DNA
quantitative 
PCR
Copy/mL
Subset(3)
TBD
DDL
TBD(2)
Multi strain 
typing
CMV DNA
CMV strain 
typing
NA
Subset(3)
+ Newborns (urine 
only)
TBD
Whole 
blood 
RNA transcript
TBD
mRNA 
microarray 
or qPCR
NA
Subset(3)
TBD
(1) In seronegative subjects, testing of viral load will only be done upon seroconversion. 
(2) The matrix will be determined based on the results obtained with the PCR and the availability of the assay.
(3) Subset will be defined based on the outcome of primary/secondary endpoints or the development of the assay. 
The subset is the minimum number of subjects that will be tested. If applicable, all subjects could be tested.
(4) Refer to APPENDIX B for the laboratory addresses.
(TBD = To be determined, mRNA = messenger Ribonucleic acid; PCR = Polymerase Chain reaction; DNA = 
deoxyribonucleic acid; NA = not applicable.
In case the results from a 4-month Site Visit suggests primary or secondary (re-
infection/re-activation) infection, testing related to the tertiary endpoints will be done on 
all available selected samples collected during the Sample Collection Visit. The selection 
of samples to be tested will be based on the qPCR results obtained following the Site visits.
The laboratories that will perform antibody recognition profile, multi strain typing and 
RNA transcript assays are not yet identified and will be defined before study start as soon 
as available.
 
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5.6.4.1. Read-outs
Table 8: Read-outs
Sampling time point
Subset Name
No. 
subjects
Component
Type of 
contact and 
time point 
Sampling 
time point
Site visits:
(V1 to V10)
Months 0, 4, 8, 
12, 16, 20, 24, 
28, 32, and 36
All subjects
All
Anti-CMV tegument protein IgG (serum + saliva)
Subset(2)
TBD
Anti-gB IgG (serum)
All subjects(1)
All TBD
CMV DNA (urine)
Subset(2)
TBD
Anti-CMV tegument protein IgG antibody avidity 
index
Anti-gB IgG antibody avidity index
Subset(2)
TBD
CMV DNA (saliva, dry saliva, dry urine, blood)
Subset(2)
TBD
Anti-CMV IgM (serum)
Subset(2)
TBD
Anti-CMV neutralizing antibodies (multiple cell lines 
and/or other viral strains)
Subset(2)
TBD
Antibody recognition profile
Subset(2)
TBD
Multi strain typing
Site visit 
(V1, V4, V7, 
V10)
Months 0, 12, 
24, and 36
Subset(2)
TBD
RNA transcript
Sample 
Collection Visits 
(SCV 1 to SCV 
9)
Month 2, 6, 
10, 14, 18, 22, 
26, 30, 34(3)
Subset(2)
TBD
Anti-CMV tegument proteins IgG (saliva)
Subset(2)
TBD
CMV DNA (urine + saliva)
Upon delivery
Newborn(4)
TBD
CMV DNA urine and/or saliva
Multi-strain typing
(1) In seronegative subjects, testing of viral load will only be done upon seroconversion.
(2) Subset will be defined based on the outcome of primary/secondary endpoints or the development of the assay. 
The subset is the minimum number of subjects that will be tested. If applicable, all subjects could be tested.
(3) Testing will be done on selected samples only if the samples at the following 4-month Site Visit time-point is 
positive. If the 4-month sample is negative for CMV infection/re-infection, the samples self-collected during the 
inter-visit period will be stored and be used if applicable. The selection of samples to be tested will be based on 
the qPCR results obtained following the Site visits.
(4) Not done in seronegative mothers who remain seronegative during pregnancy
DNA = deoxyribonucleic acid; RNA = Ribonucleic acid
IgG = immunoglobulin G; IgM = immunoglobulin M; gB = glycoprotein B; TBD = To be determined.
8.1.1. Primary endpoint

Occurrence of CMV secondary infections determined in all seropositive subjects 
on samples collected during the 4-month Site Visits until study conclusion: 

Anti-CMV tegument protein IgG antibody concentration in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-CMV tegument protein IgG antibody concentration in saliva (ELISA).

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).
 
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8.1.2. Secondary endpoints

Occurrence of CMV primary infection determined in all seronegative subjects on 
samples collected during the 4-month Site Visits until study conclusion: 

Anti-CMV tegument protein IgG antibody concentration in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-CMV tegument protein IgG antibody concentration in saliva (ELISA).
Tertiary endpoints

Further characterization of primary infection in initially seronegative subjects 
during the 4-month Site Visits until study conclusion (testing done upon 
seroconversion): 

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Further characterization of primary and /or secondary CMV infections in a subset 
of subjects on all available selected samples of the Sample Collection Visits*. 

Anti-CMV tegument protein IgG antibody concentration in saliva (ELISA).

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Number of CMV DNA copies (pp65 or other genes) in saliva (qPCR
* In case of a positive sample at a 4-month Site Visit time point, testing will be done on 
all available selected samples collected during the Sample Collection Visit. The selection 
of samples to be tested will be based on the qPCR results obtained following the Site 
visits.

Further characterization of primary and /or secondary CMV infections in a subset 
of subjects on all available samples of the 4-month Site Visits: 

Anti-CMV tegument protein IgG antibody avidity index in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-gB IgG antibody avidity index in serum (ELISA).

Anti-CMV IgM antibody concentration in serum (ELISA).

Number of CMV DNA copies (pp65 or other genes) in saliva, dry saliva, dry 
urine, blood (qPCR).

Assessment of anti-CMV neutralizing antibodies on different target cells and/or 
other virus strains.

Development of assays that will allow differentiating re-infection from re-
activation from primary infection in a subset of subjects

Characterization of CMV strains by genotyping/sequencing.

Exploring the CMV strain specific antibody profile using peptide microarrays.
 
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
Assessment of the expression of host genes* related to immunity using 
techniques such as qPCR or mRNA microarray in a subset of subjects, and/or 
other functions such as T-cell proliferation. 
* excluding genes related to hereditary characteristics of the subject.

Evaluation of the impact of demographic, social or behavioral factors upon CMV 
infection. 

Occurrence and characterization of congenital CMV infection in newborns of 
subjects who become pregnant during the study:

Evidence of CMV DNA in urine and/or saliva of newborns within 10 days of 
delivery by using qPCR (pp65 or other genes).

Characterization of strains by genotyping/sequencing in newborns from re-
infected mothers.
8.2. Sample size consideration
The sample size for this study is not based on formal statistical considerations. A The
target is to recruit number of approximately 400 200 evaluable seropositive adolescent 
females is considered sufficient to obtain a reasonable number of CMV infections during 
the study. Considering a conservative incidence rate of CMV infection of 1 2% per year, 
it is expected that approximately 12 seropositive subjects will be re-infected during the 
study period.
A target number of approximately 400 200 evaluable seropositive adolescent females will 
be analyzed. Considering a 15-20% drop out rate during the 3-year study period, 
approximately 475 240 seropositive subjects will be enrolled.
8.3. Classification of cases

A primary infection is defined as the first infection with CMV in subjects who were 
seronegative at enrolment and will be evaluated by:

Appearance of anti-CMV tegument protein IgG antibodies in serum and/or 
saliva
AND/OR

Appearance of anti-gB IgG antibodies in serum
WITH or WITHOUT

Presence of CMV DNA in urine.
 
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
CMV Re-infection* with a different CMV strain in subjects who were seropositive 
at the time of enrolment will be evaluated by:

Appearance or increase of anti-CMV tegument protein IgG antibodies in serum 
and/or saliva
AND/OR

Appearance or increase of anti-gB IgG antibodies in serum
AND/OR

Appearance or increase of CMV DNA in urine
AND in the presence of 

Change in polymorphism in the antibody recognition profile in follow-up serum 
samples (assay to be further developed)
AND/OR

Genotyping data characteristic of a re-infection (assay to be further developed)

CMV re-activation* of latent CMV in subjects who were seropositive at the time of 
enrolment will be evaluated by:

Appearance or increase of anti-CMV tegument protein IgG antibodies in serum 
and /or saliva
AND/OR

Appearance or increase of anti-gB IgG antibodies in serum
AND/OR

Appearance or increase of CMV DNA in urine
AND in absence of 

Change in polymorphism in the antibody recognition profile in follow-up serum 
samples (assay to be further developed)
AND/OR

Genotyping data characteristic of a re-infection (assay to be further developed)
* Depending on the future scientific knowledge and the data generated in this study 
multiple case definition might be explored.
8.7. Statistical methods
The primary analysis will be performed on the Total Cohort. Analyses on the ATP 
cohort(s). Analysis on the Total Cohort may be performed to complement the primary 
analysis.
 
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8.7.2. Analysis of primary objective
Descriptive statistics will be provided for each primary endpoint at all available time-
points and will include (not exhaustive) the following tabulations (with 95% confidence
interval [CI]):

The percentage of CMV seropositive subjects with appearance or increase of anti-gB 
IgG antibodies in serum when applicable.

The percentage of CMV seropositive subjects with appearance or increase of anti-
CMV tegument protein IgG antibodies in saliva.
8.7.3. Analysis of secondary objectives
Descriptive statistics will be provided for each secondary endpoint at all each timepoint 
for which a sample is available time-points and will include (not exhaustive) the 
following tabulations (with 95% CI):

The percentage of CMV seronegative subjects with appearance of anti-CMV 
tegument protein IgG antibodies in serum.

The percentage of CMV seronegative subjects with appearance of anti-gB IgG 
antibodies in serum when applicable.

The percentage of CMV seronegative subjects with appearance of anti-CMV 
tegument protein IgG antibodies in saliva.
8.7.4. Tertiary objectives
Descriptive statistics will be provided for the number of CMV DNA copies in urine at all
each timepoint for which a sample is available time-points and the percentage of CMV 
seronegative subjects with presence of CMV DNA in urine will be tabulated (with 95% 
CI).
For all results of assays performed for the further characterization of primary and/or 
secondary CMV infections, descriptive statistics will be provided for each assay at all
each timepoint for which a sample is available time-points.
The number and percentage of CMV infected newborns will be tabulated and descriptive 
statistics will be provided for the number of CMV DNA copies. Characterization of CMV 
strains in newborns will be described.
 
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Appendix A: Laboratory assays
Note that some assays, the general principles of which are described below, are still in 
development or planned for development at GSK laboratories or delegate and that it 
cannot be assumed that all of them will be successfully developed due to possible 
technical constraints. It is also assumed that some already available assays can be 
slightly modified in order to improve robustness and quality of these assays.
1.
Anti-CMV tegument proteins IgG ELISA (biotest like assay)
The assay is an indirect solid-phase ELISA for the detection of specific IgG antibodies 
using two highly purified, autologous fusion proteins, CG1 and CG2, each combining 
two immunodominant fragments from HCMV tegument (pp150; CG1:UL32, amino acids 
(aa) 495-691 and 862-1048; CG2:UL32, aa 695-854) and the delayed-early DNA binding 
protein (pp52; UL44, aa 297-433). If the sample contains specific antibodies, these bind 
to the recombinant antigens in the microtiter wells. Non-specific antibodies are removed 
by washing steps. The antibody-antigen complex is detected by peroxidase-labeled anti-
human IgG antibodies. The presence of bound antibodies is demonstrated by adding the 
chromogen-substrate solution (TMB and H202).
2.
Anti-gB IgG ELISA
Anti-HCMV gB antibody concentrations are measured by ELISA using recombinant gB 
as coating antigen on 96-well microplates. After washing, the wells are blocked with a 
saturating reagent bovine serum albumin. Two dilutions of sera, in duplicate, as well as 
controls are incubated onto the coated wells to allow the specific antibodies, present in 
the sample, to react with gB. Non-specific antibodies are removed by washing steps. The 
antibody-antigen complex is detected by peroxidase-labeled anti-human IgG 
antibodies. The presence of bound antibodies is demonstrated by adding the 
chromogen-substrate solution. 
Non-specific reactants are removed by washing, and peroxidase-conjugated anti-human 
IgG polyclonal antibodies are added to bind with anti-gB IgG antibodies. Excess 
conjugate is removed by washing. Enzyme substrate solution (TMB) is added, and the 
blue color is allowed to develop. The reaction is stopped by addition of sulphuric acid, 
resulting in a color change to yellow. The intensity of the color is quantified and 
expressed in EU/mL. The optical density (OD) is proportional to the concentration of anti 
gB antibodies present in the sample. Titers of each serum dilution are calculated by 
reference to a standard serum using the 4-parameters equation (mean OD), and the final 
titer of a serum is the mean of the titers falling in the proportional part of the reference 
curve.
3.
Anti-CMV IgM ELISA
The method used for qualitative determination of specific IgM antibodies to 
cytomegalovirus in human serum is an immunoenzymatic assay based on the antibody 
capture ELISA technique. The removable polystyrene wells of the 96 well microplates 
are precoated with IgG mouse monoclonal to human IgM. During the first step of the 
immunological reaction, diluted (1:101) serum samples and controls (negative, positive, 
 
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cut-off control and diluent) are incubated into the pre-coated plate. The plates are washed 
to remove non-specific reactants, and a mix of enzyme-labeled detection antibody (IgG to 
CMV conjugated to horseradish peroxidase) and CMV antigen is added. The presence of 
specific IgM to CMV allows the tracer to bind to the solid phase through the presence of 
CMV antigen. After a washing step to remove excess of conjugate, the chromogen-
enzyme substrate solution containing 3, 3’, 5, 5’-tetramethyl benzidine and hydrogen 
peroxide is added to measure the enzyme activity. After incubation at room temperature 
away from direct light the enzymatic reaction is stopped by the addition of Stop Reagent 
(sulphuric acid solution). The intensity of the resultant color change is measured by a 
spectrophotometer at a defined wavelength (450 nm) against a reference wavelength (630 
nm). The optical density (OD) is proportional to the concentration of the anti-CMV IgM 
present in the sample. The IgM concentration in the cut-off control is calibrated in order 
to provide a clinical cut-off, unknown samples with an OD above the cut-off OD + 10% 
cut-off OD are positive, those with an OD below the cut-off OD – 10% cut-off OD are 
negative. In the greyzone around the cut-off OD (cut-off OD +/- 10% cut-off OD) 
samples are retested.
4.
Anti-CMV tegument protein IgG avidity index
This assay will be performed using an elution step with a chaotropic agent to remove 
low-avidity antibodies bound to CG1 and CG2 fusion proteins. Briefly, each diluted 
serum is first added to wells of plates coated with CG1 and CG2 fusion proteins. After 
incubation, the unbound antibodies are removed by washing, and a chaotropic agent is 
then added in the coated wells to dissociate low-avidity antibodies. The microplate is then 
washed, and horseradish peroxidase (HRP)-conjugated anti-human IgG antibodies is 
added. After incubation, unbound antibodies are removed by washing and the 
chromogen-substrate solution is TMB and oxygen peroxide are added to reveal the 
enzyme activity. The color reaction is stopped by addition of a stop reagent sulphuric 
acid and the resulting yellow color is measured spectrophotometrically. An avidity index 
is then calculated.
5.
Anti-gB IgG avidity index
This assay will be performed according to [De Souza, 2003] using an elution step with 
urea to remove low-avidity antibodies from gB antigen. Briefly, each diluted serum is 
first added to wells of plates coated with gB antigen. After incubation, the unbound
antibodies are removed by washing, and 8M urea in phosphate buffered saline is then 
added in the coated wells to dissociate low-avidity antibodies. The microplate is then 
washed, and HRP-conjugated anti-human IgG antibodies are added. After incubation, 
unbound antibodies are removed by washing and the chromogen-substrate solution is
TMB and oxygen peroxide are added to reveal the enzyme activity. The color reaction is 
stopped by addition of sulphuric acid a stop reagent and the resulting yellow color is 
measured spectrophotometrically. 
The avidity index is calculated as the mean absorbance of reactions in which the immune 
complexes are exposed to urea divided by the mean absorbance of reactions in which the 
immune complexes are not exposed to urea, expressed as a percentage.
 
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6.
Anti-CMV neutralisation assay
The microneutralization test uses MRC-5 human lung embryo fibroblast cells as the 
infection target. The different serum sample dilutions are mixed with a fixed amount of 
virus at different dilutions and the serum-virus mixture is incubated overnight at 37°C 
with MRC-5 cells. Infected cells are then fixed with acetone. The degree to which 
neutralizing antibodies decrease CMV infection of cells in the assay is determined by 
counting the number of nuclei expressing the CMV 72 kDa IE1 protein. Nuclear IE1 is 
detected using highly sensitive immunoperoxidase staining. The stained nuclei are 
counted using a manual reading method. The neutralization titer is expressed as the 
reciprocal of the serum dilution that reduced by 50% the number of infected cells as 
compared to a control without serum. Other target cells and clinical virus isolates 
obtained from the urine of pregnant women may be used to explore more deeply the 
neutralization function of the anti-CMV antibodies and its possible association with the 
virus genotype as determined by DNA sequencing.
Additional microneutralization assays using other cell types such as epithelial, 
trophoblast cells as the infection targets and other virus strains may also be performed.
7.
Antibody recognition profile
The exact methodology to perform antibody recognition profiling is not yet decided but 
the type of assay may be derived from classical high or low density peptide arrays. 
Whatever the technology used, The peptide –microarray is a technology allowing the 
simultaneous measurement of serum reactivity (IgG, IgM, IgA or IgE) to hundreds or 
thousands of peptides in a single biological sample. Briefly, solid support spotted/coated 
with CMV peptides selected based on their capacity to discriminate the antibody profiles 
induced either by the vaccine or by reinfection with a different CMV strains, are 
incubated with serum dilutions. Slides are then washed to remove unbound antibodies 
and incubated with labeled anti-human Immunoglobulin. After a washing step, antibody 
recognition profile against different peptides is revealed by fluorescence or other means 
following the technology platform selected.
8.
Detection of CMV-DNA (qPCR)
Quantitative PCR provides a rapid and sensitive method for the diagnosis of CMV 
infection. The CMV detection test used by GSK Biologicals is designed to detect the 
pp65 gene.
Real-time PCR technology allows the determination of the copy number of sequence-
specific nucleic acids target molecules. The amplification of the target sequence is 
detected using the 5’ nuclease assay based on the Taqman chemistry. In those PCR 
experiments, the formation of a PCR product is monitored in real-time during 
amplification by means of a fluorogenic probe that binds specifically to the amplified 
product. The reporter fluorophore is at the 5’ end of the Taqman probe and the quencher 
is at the 3’ end. As long as the probe is intact, no fluorescence is observed from the 
fluorophore. During the polymerization step, the Taq DNA polymerase displaces the 
Taqman probe by 3-4 nucleotides, and the 5’ nuclease activity of the DNA polymerase 
separates the fluorophore from the quencher. The fractional cycle number at which the 
 
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generated fluorescence passes a fixed threshold above the baseline defines a “Threshold 
Cycle” (Ct value). A standard curve can be generated from the log of the starting copy 
number of DNA references against the measured Ct value.
In the context of this study, the qPCR technology is used to detect CMV DNA in 
different matrix (blood, urine, dry urine, and saliva, and dry saliva) following adapted 
purification methodology.
9.
CMV strain typing
In addition to direct detection of CMV through PCR, the company is seeking to develop, 
evaluate and validate next generation sequencing based assay and/or other technologies 
aiming to characterize CMV genomic diversity that to allow discriminating re-infection 
or re-activation to primary infection.
10. mRNA microarray /qPCR
Transcriptional profiling in the blood consists of measuring RNA abundance in 
circulating nucleated cells. Changes in transcript abundance can result from exposure to 
host or pathogen-derived immunogenic factors and/or changes in relative cellular 
composition. Techniques such as mRNA microarrays of qPCR may be used to evaluate 
these changes, if available.
The study of transcriptomics may be explored to examine the expression level of mRNAs 
related to immune genes by using high-throughput techniques based on DNA microarray 
technology.
Appendix B: Clinical laboratories
Table 11: Outsourced laboratories
Laboratory
Address
Immune Health
Rue A. Bolland
B-6041 Charleroi
Belgium
DDL Diagnostic Laboratory B.V.
Fonteijnenburghlaan 7
Voorburg
Netherland
BARC USA Inc
5, Delaware Drive
Lake Success
NY 11042-1114
USA
CERBA European labs
7/11 Rue de l’Equerre, Parc d’activités ‘les Béthunes’
95310 Saint Ouent l’Aumone
France
 
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GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 2
eTrack study number 
and Abbreviated Title
115639 (CMV-014 EXPLO)
Amendment number:
Amendment 2
Amendment date:
19-FEB-2015
Co-ordinating author:
 (XPE Pharma & Science for GSK 
Biologicals)
Rationale/background for changes: 
Protocol amendment 2 was prepared:

To cancel the planned interim analyses and to keep the final analysis only: The need 
to validate and develop some assays has led to a delay in the release of data on time. 
Since no vaccine is administered in the CMV-014 EXPLO study and as the interim 
analyses were not linked to subjects’ safety or efficacy, but rather to exploring the 
natural history of CMV disease, it was decided to group the planned interim 
analysis after Year 1 and Year 2 and to have a single final analysis of the total 
follow-up at the end of the study.

To clarify that the tertiary endpoints (i.e. exploratory tests) and the subcohort 
selected for testing will be based on the evaluations of primary and secondary 
endpoints.

To stop collection of blood samples for gene expression signature (qPCR or RNA 
microarray) as of protocol amendment 2 approval: A Year 1 dataset will be used for 
analysis of gene expression in a subset of subjects.
In addition, the list of contributing authors and the Sponsor Signatory Approval page 
have been updated following changes in the team members/ function names. The 
notation of the terms “ATP cohort” and “Total cohort” were adapted to maintain 
consistency throughout the protocol.
 
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Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
Contributing authors:

,
, Clinical Research and Development Leads
Manager

, Study Delivery Manager

, 
, Project Statistician

, 
, 
(Business & 
Decision Life Sciences for GSK Biologicals), Clinical Immunology GVCL 
Representatives

, 
, 
 (Keyrus Biopharma 
contractor for GSK Biologicals),
(TCS Consultant for GSK 
Biologicals),
, Study Data Manager

, Project Data Manager

,
, 
, Safety 
Representatives (Valesta contractor for GSK Biologicals)

, Epidemiologist
Sponsor Signatory Approval:
Sponsor signatory
Jeanne-Marie Devaster
Director Clinical Research and Translational 
Science Development Director
List of abbreviations
ATP
Aaccording-Tto-Pprotocol
 
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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
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Synopsis
Tertiary endpoints

Further characterization of primary infection in initially seronegative subjects during 
the 4-month Site Visits until study conclusion (testing done upon seroconversion)
might be done:

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Further characterization of primary and /or secondary CMV infections in a subset of 
subjects on selected samples of the Sample Collection Visits* might be done.

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Number of CMV DNA copies (pp65 or other genes) in saliva (qPCR).
* In case of a positive sample at a 4-month Site Visit time point, testing will might be done on 
selected samples collected during the Sample Collection Visit. The selection of samples to be 
tested will be based on the qPCR results obtained following the Site visits.

Further characterization of primary and /or secondary CMV infections in a subset of 
subjects on all available samples of the 4-month Site Visits might be done:

Anti-CMV tegument protein IgG antibody avidity index in serum (ELISA).

Anti-gB IgG antibody avidity index in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-CMV IgM antibody concentration in serum (ELISA).

Number of CMV DNA copies (pp65 or other genes) in saliva and blood (qPCR).

Assessment of anti-CMV neutralizing antibodies on different target cells and/or 
other virus strains.

Development of assays that will allow differentiating re-infection from re-
activation from primary infection in a subset of subjects might be done.

Characterization of CMV strains by genotyping/sequencing.

Exploring the CMV strain specific antibody profile using peptide microarrays.

Assessment of the expression of host genes* using techniques such as qPCR or 
mRNA microarray in a subset of subjects might be performed. 
* excluding genes related to hereditary characteristics of the subject.

Evaluation of the impact of demographic, social or behavioral factors upon CMV 
infection might be performed.

Occurrence of congenital CMV infection in newborns of subjects who become 
pregnant during the study might be evaluated:
Evidence of CMV DNA in urine and/or saliva of newborns within 10 days of delivery by 
using qPCR (pp65 or other genes) might be tested.
 
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Section 5.4
Outline of study procedures
Table 2
List of study procedures
Age
10-17 years
Epoch
001
Year 1
Time-point (Months)
M0
M2
M4
M6
M8
M10
M12
Visit/Sample Collection Visit
V1
SCV1
V2
SCV2
V3
SCV3
V4
Year 2
Time-point (Months)
M14
M16
M18
M20
M22
M24
Visit/Sample Collection Visit
SCV4
V5
SCV5
V6
SCV6
V7
Year 3
Time-point (Months)
M26
M28
M30
M32
M34
M36
Visit/Sample Collection Visit
SCV7
V8
SCV8
V9
SCV9
V10
Informed consent
●
Check inclusion/exclusion criteria
●
Record demographic data
●
Record social and behavioral data(1)
●
●
●
Medical history
●
CMV serostatus(2) (~3.5 to 5 mL)
●
Blood sample(3)
- for humoral immunity (10 mL)
- for molecular biology (4 mL)
●
●
●
●
●
Blood sample for gene expression signature 
(qPCR or RNA microarray; 2.5 mL)(4)
●
●
●
Urine sample (4) (5) (~10 mL)
●
●
●
●
●
●
●
●
Saliva 
●
●
●
●
●
●
●
●
Subject sample collection booklet distribution(5) (6)
○
Check elimination criteria
●
●
●
●
●
Record any concomitant 
medication/vaccination(6) (7)
●
●
●
●
●
Reporting of SAEs related to study participation 
●
●
●
●
●
Reporting of pregnancy and outcome
●
●
●
●
●
Study conclusion 
●
● is used to indicate a study procedure that requires documentation in the individual eCRF. 
○ is used to indicate a study procedure that does not require documentation in the individual eCRF
The double line border indicates that an interim analysis will be performed on clean data each year (Month 12 and 
Month 24).
(1) Social and behavioral data will be recorded at inclusion (Visit 1 [Month 0]), at Visit 4 (Month 12), Visit 7 (Month 24) 
and Visit 10 (Month 36).
(2) CMV serostatus, for allocation to a study group (S+ or S-), will be determined by the local laboratory as per local 
practices or by a GSK designated and validated central laboratory.
(3) CMV serostatus for endpoints analysis will be determined by GSK Biologicals or designated laboratory at Visit 1 
(Month 0).
(4) These blood samples will be collected until approval of protocol amendment 2.
(4) (5) Prepared from the 20 mL urine samples collected from the subject.
(5) (6) The sample collection booklet will instruct the subjects of the study procedures to be performed at the Sample 
Collection Visits. The sampling can be done through self-collection, through a home-visiting nurse or the subject 
may be asked to come back to the study center. 
(6) (7) Only concomitant medication/vaccination related to inclusion/exclusion and elimination criteria will be recorded.
 
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Footnote of Table 4 Interval between study Site Visits/Sample Collection Visits
(2) Subjects will not be eligible for inclusion in the according-to-protocol (ATP) cohort if they make the study visit outside 
this interval (See Section 8.4.2 for definition of the ATP cohort).
Section 5.5.2.5.
Blood sampling

A volume of approximately 10 mL of whole blood should be drawn from all 
subjects for analysis of humoral immune response at each Site Visit. This blood 
sample might also be used for exploratory testing.and exploratory testing at each 
Site Visit. 

A volume of approximately 4 mL of whole blood should be drawn from all 
subjects for molecular biology tests at each Site Visit. This blood sample might 
also be used for exploratory testing. and exploratory testing at each Site Visit.

A volume of approximately 2.5 mL of whole blood should be drawn from all 
subjects for ribonucleic acid (RNA) assays using qPCR or microarray at Site 
Visits 1 (Month 0) and 4 (Month 12). This blood sample might also be used for 
exploratory testing. and exploratory testing at Site Visits 1 (Month 0), 4 
(Month 12), 7 (Month 24), and 10 (Month 36). 
Section 5.6.2. Biological samples
Table 5
Biological samples
Sample type
Quantity 
Time point
Priority 
rank
Blood for humoral immunity 
determination 
Approximately 10 mL
Months 0, 4, 8, 12, 16, 20, 24, 28, 32 
and 36
1
Blood for molecular biology 
Approximately 4 mL
Months 0, 4, 8, 12, 16, 20, 24, 28, 32 
and 36
1
Blood for gene signature expression 
(RNA transcript)
Approximately 2.5 mL
Months 0 and 12, 24* , and 36
2
Urine
Approximately 10 mL
Months 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 
20, 22, 24, 26, 28, 30, 32, 34 and 36
1
Saliva
NA
Months 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 
20, 22, 24, 26, 28, 30, 32, 34 and 36
1
* Blood samples will be collected until approval of protocol amendment 2.
 
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Section 5.6.3 Laboratory assays

Footnote of Table 6 Humoral Immunity (Antibody determination)
Note: Most of the read-outs described in this table might be done if deemed necessary.

Footnote of Table 7 Molecular Biology
Note: Most of the read-outs described in this table might be done if deemed necessary.
Section 5.6.4.1.
Read-outs
Table 8
Read-outs 
Sampling time point
Subset Name
No. 
subjects
Component
Type of 
contact and 
time point 
Sampling 
time point
Site visits:
(V1 to V10)
Months 0, 4, 8, 
12, 16, 20, 24, 
28, 32, and 36
All subjects
All
Anti-CMV tegument protein IgG (serum)
Subset(2)
TBD
Anti-gB IgG (serum)
All subjects(1)
TBD
CMV DNA (urine)
Subset(2)
TBD
Anti-CMV tegument protein IgG antibody avidity index
Anti-gB IgG antibody avidity index
Subset(2)
TBD
CMV DNA (saliva, blood)
Subset(2)
TBD
Anti-CMV IgM (serum)
Subset(2)
TBD
Anti-CMV neutralizing antibodies (multiple cell lines 
and/or other viral strains)
Subset(2)
TBD
Antibody recognition profile
Subset(2)
TBD
Multi strain typing
Site visit 
(V1 and, V4, 
V7, V10)*
Months 0 and
12*, 24, and 
36
Subset(2)
TBD
RNA transcript
Sample 
Collection Visits 
(SCV 1 to SCV 
9)
Month 2, 6, 
10, 14, 18, 22, 
26, 30, 34(3)
Subset(2)
TBD
CMV DNA (urine + saliva)
Upon delivery
Newborn
TBD
CMV DNA urine and/or saliva
(1) In seronegative subjects, testing of viral load will only be done upon seroconversion.
(2) Subset will be defined based on the outcome of primary/secondary endpoints or the development of the assay. 
The subset is the minimum number of subjects that will be tested. If applicable, all subjects could be tested.
(3) Testing will be done on selected samples only if the samples at the following 4-month Site Visit time-point is 
positive. If the 4-month sample is negative for CMV infection/re-infection, the samples self-collected during the inter-
visit period will be stored and be used if applicable. The selection of samples to be tested will be based on the qPCR 
results obtained following the Site visits.
* Blood sample will be collected until approval of protocol amendment 2.
DNA = deoxyribonucleic acid; RNA = Ribonucleic acid
IgG = immunoglobulin G; IgM = immunoglobulin M; gB = glycoprotein B; TBD = To be determined.
Section 8.1.3 Tertiary endpoints 

Further characterization of primary infection in initially seronegative subjects 
during the 4-month Site Visits until study conclusion (testing done upon 
seroconversion) might be done: 

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).
 
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
Further characterization of primary and /or secondary CMV infections in a subset 
of subjects on selected samples of the Sample Collection Visits* might be done. 

Number of CMV DNA copies (pp65 or other genes) in urine (qPCR).

Number of CMV DNA copies (pp65 or other genes) in saliva (qPCR).
* In case of a positive sample at a 4-month Site Visit time point, testing will might be 
done on selected samples collected during the Sample Collection Visit. The selection of 
samples to be tested will be based on the qPCR results obtained following the Site visits.

Further characterization of primary and /or secondary CMV infections in a subset 
of subjects on all available samples of the 4-month Site Visits might be done: 

Anti-CMV tegument protein IgG antibody avidity index in serum (ELISA).

Anti-gB IgG antibody concentration in serum (ELISA).

Anti-gB IgG antibody avidity index in serum (ELISA).

Anti-CMV IgM antibody concentration in serum (ELISA).

Number of CMV DNA copies (pp65 or other genes) in saliva and blood (qPCR).

Assessment of anti-CMV neutralizing antibodies on different target cells and/or 
other virus strains.

Development of assays that will allow differentiating re-infection from re-
activation from primary infection in a subset of subjects might be done.

Characterization of CMV strains by genotyping/sequencing.

Exploring the CMV strain specific antibody profile using peptide microarrays.

Assessment of the expression of host genes* using techniques such as qPCR or 
mRNA microarray in a subset of subjects might be performed. 
* excluding genes related to hereditary characteristics of the subject.

Evaluation of the impact of demographic, social or behavioral factors upon CMV 
infection might be performed. 

Occurrence of congenital CMV infection in newborns of subjects who become 
pregnant during the study might be evaluated:

Evidence of CMV DNA in urine and/or saliva of newborns within 10 days of 
delivery by using qPCR (pp65 or other genes) might be tested.
 
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Section 8.3
Classification of cases 

A primary infection is defined as the first infection with CMV in subjects who were 
seronegative at enrolment and will be evaluated by:

Appearance of anti-CMV tegument protein IgG antibodies in serum
AND/OR

Appearance of anti-gB IgG antibodies in serum
WITH or WITHOUT

Presence of CMV DNA in urine.

CMV re-infection* with a different CMV strain in subjects who were seropositive at 
the time of enrolment will be evaluated by:

Appearance or increase of anti-CMV tegument protein IgG antibodies in serum
AND/OR

Appearance or increase of anti-gB IgG antibodies in serum
AND/OR

Appearance or increase of CMV DNA in urine
AND (depending on assay availability) in the presence of 

Change in polymorphism in the antibody recognition profile in follow-up serum 
samples (assay to be further developed)
AND/OR

Genotyping data characteristic of a re-infection (assay to be further developed)

CMV re-activation* of latent CMV in subjects who were seropositive at the time of 
enrolment will be evaluated by:

Appearance or increase of anti-CMV tegument protein IgG antibodies in serum
AND/OR

Appearance or increase of anti-gB IgG antibodies in serum
AND/OR

Appearance or increase of CMV DNA in urine
AND (depending on assay availability) in absence of 

Change in polymorphism in the antibody recognition profile in follow-up serum 
samples (assay to be further developed)
AND/OR

Genotyping data characteristic of a re-infection (assay to be further developed)
 
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115639 (CMV-014 EXPLO)
Protocol Amendment 2 Final
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19-FEB-2015

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115639 (CMV-014 EXPLO)
Protocol Amendment 2 Final
19-FEB-2015
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Section 8.4.2 According-tTo-pProtocol cohort 
The according-to-protocol (ATP) Ccohort will include all subjects who meet all 
inclusion criteria and no exclusion criteria for the study, who do not have any elimination 
criteria during the study (Section 5.5.2.9) and who comply with the study procedures 
indicated below.
The ATP Cohort Year 1 will include all subjects who attend the 4 Site Visits (Visit 1 to 
Visit 4) and have samples collected from at least 2 Sample Collection Visits during 
Year 1.
The ATP Cohort Year 2 will include all subjects who attend the 3 Site Visits (Visit 5 to 
Visit 7) and have samples collected from at least 2 Sample Collection Visits during 
Year 2.
The ATP Cohort Year 3 will include all subjects who attend the 3 Site Visits (Visit 8 to 
Visit 10) and have samples collected from at least 2 Sample Collection Visits during 
Year 3.
A detailed, comprehensive list of reasons for elimination from ATP analyses will be 
established at the time of data cleaning.
Section 8.6.1 Sequence of analyses 
The analyses will be performed stepwise: 

An interim analysis will be done every 12 months (Month 12 and Month 24). No 
study report will be written.
Section 8.6.2 Statistical considerations for interim analyses
In this study no treatment is given to the subjects. An interim analysis can therefore be 
performed every 12 months (Month 12 and Month 24) on all available results without 
adjustment of the sample size.
Section 8.7
Statistical methods
Statistical analyses will be described in detail in a separate document, the statistical 
analysis plan (SAP).
The primary analysis will be performed on the ATP cohort(s). Analysis on the Total 
Ccohort may be performed to complement the primary analysis.
 
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Section 8.7.4 Tertiary objectives
Based on the results of primary and secondary endpoints, following statistical analyses 
on tertiary endpoints might be done:
Descriptive statistics will be provided for the number of CMV DNA copies in urine at 
each timepoint for which a sample is available time-points and the percentage of CMV 
seronegative subjects with presence of CMV DNA in urine will be tabulated (with 95% 
CI).
 
CONFIDENTIAL
115639 (CMV-014 EXPLO)
Protocol Amendment 2 Final
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19-FEB-2015

PPD