Safety and immunogenicity of a tetravalent dengue vaccine in healthy children aged 2-11 years in Malaysia: A randomized, placebo-controlled, Phase III study

Article (PDF Available)inVaccine 31(49) · October 2013with88 Reads
DOI: 10.1016/j.vaccine.2013.10.013 · Source: PubMed
Abstract
Dengue disease is a major public health problem across the Asia-Pacific region for which there is no licensed vaccine or treatment. We evaluated the safety and immunogenicity of Phase III lots of a candidate vaccine (CYD-TDV) in children in Malaysia. In this observer-blind, placebo-controlled, Phase III study, children aged 2-11 years were randomized (4:1) to receive CYD-TDV or placebo at 0, 6 and 12 months. Primary endpoints included assessment of reactogenicity following each dose, adverse events (AEs) and serious AEs (SAEs) reported throughout the study, and immunogenicity expressed as geometric mean titres (GMTs) and distribution of dengue virus (DENV) neutralizing antibody titres. 250 participants enrolled in the study (CYD-TDV: n=199; placebo: n=51). There was a trend for reactogenicity to be higher with CYD-TDV than with placebo post-dose 1 (75.4% versus 68.6%) and post-dose 2 (71.6% versus 62.0%) and slightly lower post-dose 3 (57.9% versus 64.0%). Unsolicited AEs declined in frequency with each subsequent dose and were similar overall between groups (CYD-TDV: 53.8%; placebo: 49.0%). Most AEs were of Grade 1 intensity and were transient. SAEs were reported by 5.5% and 11.8% of participants in the CYD-TDV and placebo groups, respectively. No deaths were reported. Baseline seropositivity against each of the four DENV serotypes was similar between groups, ranging from 24.0% (DENV-4) to 36.7% (DENV-3). In the CYD-TDV group, GMTs increased post-dose 2 for all serotypes compared with baseline, ranging from 4.8 (DENV-1) to 8.1-fold (DENV-3). GMTs further increased post-dose 3 for DENV-1 and DENV-2. Compared with baseline, individual titre increases ranged from 6.1-fold (DENV-1) to 7.96-fold (DENV-3). This study demonstrated a satisfactory safety profile and a balanced humoral immune response against all four DENV serotypes for CYD-TDV administered via a three-dose regimen to children in Malaysia.
Vaccine
31 (2013) 5814–
5821
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at
ScienceDirect
Vaccine
j
our
nal
homep
ag
e:
www.elsevier.com/locate/vaccine
Safety
and
immunogenicity
of
a
tetravalent
dengue
vaccine
in
healthy
children
aged
2–11
years
in
Malaysia:
A
randomized,
placebo-controlled,
Phase
III
study
,夽夽
Amar-Singh
HSS
a
,
Mia-Tuang
Koh
b
,
Kah
Kee
Tan
c
,
Lee
Gaik
Chan
d
,
Lynn
Zhou
e
,
Alain
Bouckenooghe
f
,
Denis
Crevat
g
,
Yanee
Hutagalung
f,
a
Department
of
Pediatrics,
Hospital
Raja
Permaisuri
Bainun,
Jalan
Hospital,
Ipoh,
Perak,
Malaysia
b
Department
of
Pediatrics,
University
of
Malaya
Medical
Centre,
Jalan
Universiti,
59100
Kuala
Lumpur,
Malaysia
c
Department
of
Pediatrics,
Hospital
Tuanku
Jaafar,
Jalan
Rasah,
70300
Seramban,
Negeri
Sembilan,
Malaysia
d
Department
of
Pediatrics,
Sarawak
General
Hospital,
Jln
Tun
Ahmad
Zaidi
Adruce,
93586
Kuching,
Sarawak,
Malaysia
e
Biometry
China,
Sanofi
Pasteur,
Beijing,
PR
China
f
Clinical
R&D,
Sanofi
Pasteur,
C/o
Sanofi-Aventis
Singapore
Pte
Ltd.,
6
Raffles
Quay
#18-00,
Singapore
048580,
Singapore
g
Clinical
R&D,
Sanofi
Pasteur,
Marcy
l’Etoile
69280,
France
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
17
May
2013
Received
in
revised
form
8
August
2013
Accepted
2
October
2013
Available online 14 October 2013
Keywords:
Suggestions
dengue
Paediatric
population
Vaccine
Malaysia
Safety
Immunogenicity
a
b
s
t
r
a
c
t
Background:
Dengue
disease
is
a
major
public
health
problem
across
the
Asia-Pacific
region
for
which
there
is
no
licensed
vaccine
or
treatment.
We
evaluated
the
safety
and
immunogenicity
of
Phase
III
lots
of
a
candidate
vaccine
(CYD-TDV)
in
children
in
Malaysia.
Methods:
In
this
observer-blind,
placebo-controlled,
Phase
III
study,
children
aged
2–11
years
were
randomized
(4:1)
to
receive
CYD-TDV
or
placebo
at
0,
6
and
12
months.
Primary
endpoints
included
assessment
of
reactogenicity
following
each
dose,
adverse
events
(AEs)
and
serious
AEs
(SAEs)
reported
throughout
the
study,
and
immunogenicity
expressed
as
geometric
mean
titres
(GMTs)
and
distribution
of
dengue
virus
(DENV)
neutralizing
antibody
titres.
Results:
250
participants
enrolled
in
the
study
(CYD-TDV:
n
=
199;
placebo:
n
=
51).
There
was
a
trend
for
reactogenicity
to
be
higher
with
CYD-TDV
than
with
placebo
post-dose
1
(75.4%
versus
68.6%)
and
post-dose
2
(71.6%
versus
62.0%)
and
slightly
lower
post-dose
3
(57.9%
versus
64.0%).
Unsolicited
AEs
declined
in
frequency
with
each
subsequent
dose
and
were
similar
overall
between
groups
(CYD-TDV:
53.8%;
placebo:
49.0%).
Most
AEs
were
of
Grade
1
intensity
and
were
transient.
SAEs
were
reported
by
5.5%
and
11.8%
of
participants
in
the
CYD-TDV
and
placebo
groups,
respectively.
No
deaths
were
reported.
Baseline
seropositivity
against
each
of
the
four
DENV
serotypes
was
similar
between
groups,
ranging
from
24.0%
(DENV-4)
to
36.7%
(DENV-3).
In
the
CYD-TDV
group,
GMTs
increased
post-dose
2
for
all
serotypes
compared
with
baseline,
ranging
from
4.8
(DENV-1)
to
8.1-fold
(DENV-3).
GMTs
further
increased
post-
dose
3
for
DENV-1
and
DENV-2.
Compared
with
baseline,
individual
titre
increases
ranged
from
6.1-fold
(DENV-1)
to
7.96-fold
(DENV-3).
Conclusions:
This
study
demonstrated
a
satisfactory
safety
profile
and
a
balanced
humoral
immune
response
against
all
four
DENV
serotypes
for
CYD-TDV
administered
via
a
three-dose
regimen
to
children
in
Malaysia.
© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
This
is
an
open-access
article
distributed
under
the
terms
of
the
Creative
Commons
Attribution
License,
which
permits
unrestricted
use,
distribution,
and
reproduction
in
any
medium,
provided
the
original
author
and
source
are
credited.
夽夽
Trial
registration:
The
trial
is
registered
on
ClinicalTrials.gov.:
National
Clinical
Trials
Identifier
(NCT
ID):
NCT01254422.
Corresponding
author.
Tel.:
+65
6225
3836/6431
2396;
fax:
+65
6431
2445.
E-mail
addresses:
amarhss@gmail.com
(A.-S.
HSS),
kohmt@um.edu.my
(M.-T.
Koh),
kktalk@gmail.com
(K.K.
Tan),
dr
chanlg@hotmail.com
(L.G.
Chan),
Linghua.Zhou@sanofipasteur.com
1.
Introduction
Dengue
disease
is
caused
by
dengue
virus
(DENV)
serotypes
(1–4)
and
is
transmitted
mainly
by
Aedes
aegypti
mosquitoes
[1].
Dengue
disease
is
classified
as
with
or
without
warning
signs,
or
(L.
Zhou),
Alain.Bouckenooghe@sanofipasteur.com
(A.
Bouckenooghe),
Denis.Crevat@sanofipasteur.com
(D.
Crevat),
Yanee.Hutagalung@sanofipasteur.com
(Y.
Hutagalung).
0264-410X/$
see
front
matter ©
2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.10.013
A.-S.
HSS
et
al.
/
Vaccine
31 (2013) 5814–
5821 5815
severe
dengue
disease
[2].
Infection
with
one
DENV
serotype
usu-
ally
imparts
immunity
to
that
serotype
[3].
No
licensed
vaccine
or
treatment
for
dengue
disease
currently
exists.
Prevention
relies
on
individual
protection
and
vector
control
measures,
which
have
limited
effectiveness
[1].
The
Asia-Pacific
region
is
considered
the
global
epicentre
of
the
disease,
with
1.8
billion
people
at
risk
[4].
Dengue
disease
is
endemic
in
Malaysia
[5–7]
and
its
incidence
has
increased
dramat-
ically,
from
<20/100,000
in
the
1970s
[7]
to
>150/100,000
in
2010
[8],
with
an
increased
prevalence
in
adults
relative
to
children
(the
main
target
population
for
potential
dengue
disease
vaccines)
and
several
changes
in
serotype
distributions
and
increases
in
disease
severity
[7–13].
Increasing
disease
burden
has
spurred
the
development
of
potential
dengue
vaccines
[14].
One
live-attenuated
tetravalent
dengue
vaccine
(CYD-TDV,
Sanofi
Pasteur,
Lyon,
France),
con-
tains
four
recombinant
viruses
engineered
with
DENV1–4.
Each
CYD-TDV
virus
expresses
dengue
pre-membrane
and
envelope
proteins
of
the
associated
serotype,
and
the
non-structural
and
capsid
proteins
of
the
attenuated
Yellow
Fever
(YF-17D)
vaccine
virus
[15–19].
In
Phase
I
and
II
studies,
CYD-TDV
elicited
balanced
neutralizing
antibody
responses
against
DENV1–4
and
was
well
tol-
erated
[20–28].
In
a
large
Phase
IIb
study
in
Thai
schoolchildren,
CYD-TDV
appeared
to
be
efficacious
against
three
of
the
four
DENV
serotypes
and
was
well
tolerated
over
2
years
of
follow-up
with
no
safety
concerns
[29].
The
primary
objectives
of
our
study
were
to
evaluate
the
safety
and
immunogenicity
of
CYD-TDV
in
children
aged
2–11
years
in
Malaysia.
This
is
the
first
Phase
III
CYD-TDV
study
to
be
reported
that
uses
Phase
III
lots
(manufactured
by
large-scale
production
processes)
in
an
area
where
dengue
disease
is
endemic.
Addition-
ally,
one
of
the
study
sites
was
located
in
the
Sarawak
state
of
Malaysia,
where
Japanese
Encephalitis
(JE)
is
endemic
and
JE
vac-
cination
is
routine.
2.
Methods
2.1.
Study
design
and
participants
This
multicentre,
randomized,
observer-blind,
placebo-
controlled,
Phase
III
study
was
conducted
between
December
2010
and
August
2012
at
four
sites
in
Malaysia:
Kuala
Lumpur,
Ipoh
(Perak
state),
Seremban
(Negeri
Sembilan
state)
and
Kuching
(Sarawak
state).
The
methodology
was
similar
to
that
of
previous
CYD-TDV
Phase
II
studies
[22,26]
but
used
Phase
III
vaccine
lots.
Healthy
children
(aged
2–11
years)
were
assigned
randomly
to
two
groups
(4
CYD-TDV:
1
placebo).
All
participants
received
three
injections
at
0,
6
and
12
months
and
were
followed
up
for
6
months
post-dose
3
to
assess
safety.
Participants
were
recruited
by
the
study
investigators,
sub-
investigators
and
research
nurses.
Randomization
was
performed
by
study-site
personnel
via
an
Interactive
Voice
Recognition
System
(IVRS),
using
a
permuted
block
method
with
stratification
by
cen-
tre
and
age.
A
double
randomization
system
separated
treatment
allocation
from
doses
dispensed.
The
IVRS
was
used
in
a
blind
man-
ner
to
ensure
a
balanced
distribution
of
the
number
of
participants
with
a
history
of
JE
vaccination,
known
JE
infection
or
dengue
dis-
ease
infection
in
each
age
group
(2–5
and
6–11
years)
of
the
study
groups,
based
on
medical
records
or
parents’
recollection.
Girls
of
childbearing
age
were
checked
for
pregnancy
(urine
test)
and
required
to
abstain
from
sexual
intercourse
or
use
contraception
from
four
weeks
before
the
first
dose
until
four
weeks
after
the
last
dose.
Exclusion
criteria
included
prior
or
current
participation
in
another
clinical
study;
receipt
of
blood
or
blood-derived
products
in
the
previous
3
months
that
could
interfere
with
immunogenicity
assessments;
hypersensitivity
to
the
vaccine
components;
and
vaccination
with
any
other
vaccine
(except
for
pandemic
influenza)
in
the
4
weeks
prior
to
enrolment.
Contraindications
to
receiving
subsequent
vaccinations
included
significant
allergic
reaction
(AR),
serious
adverse
event
(SAE)
or
ongoing
adverse
event
(AE)
related
to
the
previous
vaccination.
2.2.
Ethical
considerations
The
study
was
conducted
in
accordance
with
the
Declaration
of
Helsinki
(Seoul
revision),
with
Good
Clinical
Practice
(defined
by
the
International
Conference
on
Harmonisation),
and
with
appli-
cable
national
and
local
requirements.
The
Medical
Research
and
Ethics
Committee
of
the
Ministry
of
Health
of
Malaysia
approved
the
protocol.
Each
investigator
obtained
approval
from
an
indepen-
dent
ethics
committee
or
institutional
review
board.
Participants’
parents
or
legal
representatives
provided
written
informed
con-
sent
and
children
aged
7
years
signed
an
assent
form.
The
trial
is
registered
on
ClinicalTrials.gov.
(NCT01254422).
2.3.
Study
procedures
Blood
samples
were
taken
prior
to
vaccination,
to
assess
flavivirus
(FV)
serostatus
(dengue
disease
or
JE)
using
plaque
reduc-
tion
neutralization
tests
(PRNT
50
)
[30,31],
and
28
days
post-dose
2
and
3
to
assess
immunogenicity.
2.4.
Vaccine
CYD-TDV
Phase
III
lots
were
manufactured
using
a
large-scale
production
process.
CYD-TDV
vaccine
details
have
been
published
[17–19].
CYD-TDV
comprises
a
powder
and
solvent
for
suspension
and
must
be
stored
at
2–8
C.
Each
0.5
ml
dose
of
reconstituted
vaccine
contained
approximately
5
±
1
log
10
cell-culture
infec-
tious
dose
50%
(CCID50)
of
each
live,
attenuated,
recombinant
CYD
serotypes
1–4
(batch
numbers:
dose
1:
S4316F02
and
D0822;
dose
2:
S4316F08
and
D1148;
dose
3:
S4316F14
and
D1148).
Placebo
was
0.9%
saline
solution
(batch
numbers:
dose
1:
E5594F04;
dose
2:
E5594F08;
dose
3:
E5594F06).
Vaccinations
were
by
subcutaneous
injection
in
the
deltoid
region
of
the
upper
arm
using
a
25G
×
5/8
in.
(0.5
mm
×
16
mm)
needle.
2.5.
Primary
endpoints
2.5.1.
Safety
and
reactogenicity
Safety
and
reactogenicity
data
(graded
in
severity
from
1
to
3)
were
collected
after
each
dose:
immediate
AEs
within
30
min;
solicited
injection-site
reactions
within
7
days;
solicited
systemic
reactions
within
14
days;
and
unsolicited
AEs
within
28
days
of
vaccination.
SAEs
and
suspected
dengue
disease
cases
requiring
hospitalization
were
monitored
throughout
the
study.
An
indepen-
dent
data
monitoring
committee
reviewed
all
SAEs.
2.5.2.
Immunogenicity
Neutralizing
antibody
levels
against
DENV1–4
were
assessed
28
days
post-dose
2
and
3
using
a
PRNT
50
compliant
with
WHO
guidelines
[30,31]
and
expressed
as
geometric
mean
titres
(GMTs)
and
seropositivity
rates
(percentage
of
participants
with
titres
10
1/dil).
Distribution
of
individual
titres
at
enrolment
and
post-
dose
2
and
3
of
CYD-TDV
or
placebo
was
analyzed
using
reverse
cumulative
distribution
curves.
2.6.
Secondary
endpoints
CYD-TDV
safety
and
immunogenicity
assessments
were
stratified
according
to
baseline
FV-serostatus
(seropositive
or
5816 A.-S.
HSS
et
al.
/
Vaccine
31 (2013) 5814–
5821
seronegative
for
dengue
disease
or
JE)
and
by
age
(2–5
and
6–11
years).
Pre-vaccination,
post-dose
2
and
post-dose
3
GMTs
were
also
analyzed
according
to
FV-serostatus
at
baseline
(seropositive
or
seronegative
for
dengue
disease
and
JE).
2.7.
Statistical
methods
2.7.1.
Sample
size
and
study
populations
With
a
planned
sample
size
of
250
participants
(CYD-TDV:
n
=
200;
placebo:
n
=
50)
and
assuming
a
drop-out
rate
of
15%,
the
probability
of
observing
a
common
AE
after
three
vaccina-
tions
was
100%
and
82%
if
the
incidence
of
the
AE
was
5%
and
1%,
respectively.
Of
the
200
CYD-TDV
recipients,
60–140
were
expected
to
be
FV-seropositive
at
baseline.
Based
on
a
variability
of
0.7
log
10
for
the
PRNT
50
assay,
the
power
to
detect
differences
of
0.4
in
log
10
GMTs
between
FV-seropositive
and
-seronegative
participants
and
between
age
groups
was
>90%.
The
popula-
tions
analyzed
included
a
Safety
Analysis
Set
(SAS,
participants
receiving
at
least
one
dose
of
study
vaccine)
and
a
Full
Anal-
ysis
Set
(FAS,
participants
receiving
at
least
one
dose
of
study
vaccine,
with
a
valid
post-vaccination
serology
result).
Further
analyses
were
conducted
stratified
by
FV-serostatus
and
by
age
group.
2.7.2.
Statistical
analyses
Analyses
were
descriptive
with
no
hypothesis
tested.
For
the
main
parameters,
95%
confidence
intervals
of
point
estimates
were
calculated
using
a
normal
approximation
for
quantitative
data
and
exact
binomial
distribution
(Clopper–Pearson
method)
for
propor-
tions
[32,33].
Analyses
were
conducted
with
SAS
software,
version
9.1
or
above
(SAS
Institute,
Cary,
NC,
USA).
3.
Results
The
first
participant
was
enrolled
on
2
December
2010
and
the
last
participant’s
6
month
follow-up
visit
was
14
August
2012.
We
enrolled
250
participants
in
the
study
(CYD-TDV:
n
=
199;
placebo:
n
=
51),
of
whom
196
(98.5%)
and
50
(98%)
in
the
CYD-TDV
and
placebo
group,
respectively,
completed
the
vaccination
phase
and
were
included
in
the
FAS
(n
=
246,
Fig.
1).
In
the
SAS
(n
=
250),
55.8%
of
participants
in
the
CYD-TDV
group
and
60.8%
in
the
placebo
group
were
FV-seropositive
(seropositive
for
DENV
and/or
JE).
In
the
CYD-TDV
and
placebo
groups,
49.7%
and
52.0%
of
participants
were
aged
2–5
years,
respectively.
In
the
CYD-
TDV
group,
57.3%
of
children
aged
2–5
years
were
FV-seropositive
and
42.7%
were
FV-seronegative
and
in
children
aged
6–11
years,
54.0%
were
FV-seropositive
and
46.0%
were
FV-seronegative.
Enrolled: n=250
Randomized to
CYD-TDV group
n=199
Randomized to
placebo group
n=51
Received CYD-TDV
at visit 01:
n=199 (100%)
Received placebo
at visit 01:
n=51 (100%)
Received CYD-TDV
at visit 03:
n=197 (99%)
Received placebo
at visit 03:
n=50 (98%)
Received CYD-TDV
at visit 05:
n=196 (98.5%)
Received placebo
at visit 05:
n=50 (98%)
Completed vaccination
phase, visit 06
n=196 (98.5%)
Did not complete vaccination phase: n=3 (1.5%)
Adverse event: n=1 (0.5%);
Lost to follow-up: n=1 (0.5%);
Non-compliance with protocol: n=1 (0.5%)
Did not complete vaccination phase: n=1 (2%)
Serious adverse event: n= 1 (2%)
Discontinued
n=1 (2%)
Discontinued
n=2 (1%)
Discontinued
n=1 (0.5%)
Completed vaccination
phase, visit 06
n=50 (98%)
Fig.
1.
Study
flow
chart:
progress
of
participants
through
the
study.
Reasons
for
withdrawal:
In
the
CYD-TDV
group,
one
participant
withdrew
due
to
an
AE
(allergic
conjunctivitis),
one
was
lost
to
follow-up
after
the
first
vaccination,
and
one
did
not
comply
with
the
protocol
by
refusal
of
a
blood
sample
before
injection
3.
In
the
placebo
group,
one
participant
experienced
a
SAE
(right
VII
nerve
paralysis)
after
the
first
vaccination,
which
was
assessed
by
the
investigator
to
be
related
to
treatment.
A.-S.
HSS
et
al.
/
Vaccine
31 (2013) 5814–
5821 5817
Table
1
Demographic
and
baseline
characteristics
of
participants:
Safety
Analysis
Set
(all
participants
and
categorized
according
to
flavivirus
serostatus
at
baseline
and
stratified
by
age
[2–5
and
6–11
years]).
CYD-TDV
Placebo
All
FV-seropositive
at
baseline
FV-seronegative
at
baseline
Age
2–5
years
Age
6–11
years
N
199
111
88
99
100
51
Sex,
n
(%)
Male
96
(48.2) 51
(45.9) 45
(51.1)
51
(51.5)
45
(45.0)
32
(62.7)
Female
103
(51.8)
60
(54.1)
43
(48.9)
48
(48.5)
55
(55.0)
19
(37.3)
Age
(years)
Mean
(±SD)
6.4
(2.8)
6.5
(2.9)
6.2
(2.7)
4.0
(1.2)
8.7
(1.7)
6.5
(3.0)
Range
2.0–11.9
2.1–11.9
2.0–11.6
2.0–5.9
6.0–11.9
2.1–11.8
Height
(cm)
Mean
(±SD) 113.7
(18.1) 114.4
(19.1) 112.8
(16.9) 99.5
(10.6) 127.8
(11.9) 115.7
(19.4)
Range
78.0–157.0 78.0–153.0
81.0–157.0
78.0–121.0
104.0–157.0
84.0–158.0
Weight
(kg)
Mean
(±SD)
21.9
(10.6)
22.9
(11.5)
20.7
(9.2)
15.6
(4.7)
28.2
(11.0)
22.7
(10.8)
Range
8.9–77.0 9.0–77.0
8.9–61.5
8.9–37.6
15.4–77.0
11.0–54.9
Body
mass
index
(kg/m
2
)
Mean
(±SD)
16.2
(3.6)
16.6
(3.6)
15.7
(3.5)
15.6
(2.9)
16.8
(4.0)
16.2
(3.8)
Range
11.7–34.8
12.1–34.7
11.7–34.8
11.7–34.8
12.1–34.7
12.0–32.5
FV,
flavivirus;
n,
number
of
participants;
SD,
standard
deviation.
Baseline
demographic
characteristics
were
similar
between
groups
and
in
the
FV-serostatus
subsets
(Table
1).
However,
base-
line
GMTs
against
DENV1–4
were
slightly
higher
in
those
aged
6–11
years
than
in
those
aged
2–5
years
(Table
2).
The
majority
of
par-
ticipants
were
of
Asian
origin
(n
=
248)
and
two
participants
were
of
Asian/Caucasian
origin.
3.1.
Safety
and
reactogenicity
The
proportion
of
participants
reporting
solicited
reactions
(total
and
injection
site
and
systemic
reactions)
was
similar
in
the
CYD-TDV
(89.4%)
and
placebo
(94.1%)
groups
(Table
3).
However,
there
was
a
trend
for
slightly
higher
reactogenicity
with
CYD-TDV
compared
with
placebo
post-dose
1
and
2
(75.4%
versus
68.6%
and
71.6%
versus
62.0%,
respectively)
and
slightly
lower
reactogenicity
in
the
CYD-TDV
group
compared
with
placebo
post-dose
3
(57.9%
versus
64.0%,
Fig.
2).
The
number
of
solicited
systemic
reactions
decreased
after
each
injection
in
both
groups.
Solicited
injection
site
reactions
in
the
CYD-TDV
group
tended
to
be
more
frequent
post-dose
2
compared
with
post-dose
1
and
less
frequent
post-dose
3.
In
the
placebo
group,
solicited
injection
site
reactions
were
more
frequent
post-
dose
2
and
3
than
post-dose
1
(Fig.
2).
Most
solicited
injection
site
reactions
and
systemic
reactions
were
Grade
1
and
lasted
<3
days.
The
most
frequently
reported
injection
site
reactions
were
pain
(CYD-TDV:
69.3%;
placebo:
56.9%),
erythema
(46.7%
and
49.0%)
and
swelling
(38.7%
and
35.3%).
In
both
study
groups,
malaise
(CYD-TDV:
54.3%;
placebo:
41.2%)
and
headache
(52.3%
and
39.2%)
were
the
most
frequently
reported
solicited
systemic
reactions,
followed
by
asthenia,
myalgia
and
fever.
Fever
was
the
most
commonly
reported
Grade
3
reaction
in
both
groups
(CYD-TDV:
6.6%;
placebo:
3.9%),
and
approximately
half
of
these
reported
concomitantly
with
intercurrent
infections
(Supplementary
table
S1).
Table
2
Geometric
mean
titres
(GMTs)
for
dengue
serotype-specific
plaque
reduction
neutralization
tests
(PRNT
50
)
antibodies
before
vaccination
and
28
days
after
two
and
three
injections
of
CYD-TDV
or
placebo:
Full
Analysis
Set
for
all
participants
and
stratified
according
to
flavivirus
serostatus
at
baseline
and
age
(2–5
years
and
6–11
years).
CYD-TDV
group
GMT
1/dil
(95%
CI)
Placebo
group
GMT
1/dil
(95%
CI)
All
(n
=
196)
FV-seropositive
at
baseline
(n
=
109)
FV-seronegative
at
baseline
(n
=
87)
Age
2–5
years
(n
=
96)
Age
6–11
years
(n
=
100)
All
(n
=
50)
DENV-1
Pre-vacc
15.3
(11.5;
20.4)
37.5
(24.0;
58.6)
5.0
(NC)
10.4
(7.51;
14.5)
22.2
(14.1;
34.8)
18.6
(9.69;
35.8)
Post-dose
2
119
(90.7;
155)
248(171;
361)
47.1
(35.3;
62.9)
95.9
(69.0;
133)
146
(95.4;
222)
21.0
(10.6;
41.9)
Post-dose
3
151(121;
188)
247(178;
343)
81.6
(66.3;
101)
117
(91.2;
151)
192(136;
272)
18.9
(9.94;
35.8)
DENV-2
Pre-vacc
15.9
(11.8;
21.3)
39.9
(25.2;
63.1)
5.0
(NC)
11.1
(7.79;
15.7)
22.5
(14.2;
35.6)
18.6
(10.0;
34.5)
Post-dose
2
160(127;
203)
306(221;
424)
71.4
(55.4;
92.1)
138(106;
181)
185(126;
272)
17.9
(9.91;
32.2)
Post-dose
3
180(146;
221)
292(217;
395)
97.5
(77.5;
123)
158(125;
201)
203(144;
285)
16.3
(9.59;
27.7)
DENV-3
Pre-vacc
15.6
(12.3;
19.9)
38.8
(27.3;
55.4)
5.0
(NC)
12.1
(8.97;
16.3)
20.0
(13.7;
29.2)
15.9
(9.57;
26.5)
Post-dose
2
196(163;
235)
296(230;
381)
116
(92.6;
146)
167(134;
207)
228(170;
306)
15.9
(9.27;
27.4)
Post-dose
3
193(161;
231)
287(219;
376)
117
(97.4;
141)
168(136;
208)
220(164;
295)
16.3
(9.81;
27.0)
DENV-4
Pre-vacc
9.92
(8.17;
12.0)
17.1
(12.5;
23.5)
5.0
(NC)
8.28
(6.44:
10.6)
11.8
(8.79:
15.8)
12.3
(7.96;
19.0)
Post-dose
2
110
(88.9;
136)
152(115;
201)
73.3
(53.7;
100)
101
(75.8:
134)
119
(87.0:
164)
13.3
(7.94;
22.1)
Post-dose
3
114
(97.0;
134)
155(125;
191)
78.0
(62.0;
98.1)
105
(86.0:
129)
123
(95.7:
159)
10.9
(7.34;
16.2)
CI,
confidence
interval;
FV,
flavivirus;
GMT,
geometric
mean
titre;
n,
number
of
participants;
NC,
not
calculated.
5818 A.-S.
HSS
et
al.
/
Vaccine
31 (2013) 5814–
5821
Table
3
Overview
of
safety
data
up
to
28
days
after
any
injection:
Safety
Analysis
Set.
Participants
experiencing
at
least
one
of:
CYD-TDV
group
(n
=
199)
Placebo
group
(n
=
51)
n
%
95%
CI
n
%
95%
CI
Solicited
reaction
178/199
89.4
(84.3;
93.3)
48/51
94.1
(83.8;
98.8)
Solicited
injection
site
reaction
163/199
81.9
(75.8;
87.0)
40/51
78.4
(64.7;
88.7)
Solicited
systemic
reaction 141/199
70.9 (64.0;
77.1) 37/51
72.5
(58.3;
84.1)
Unsolicited
AE
107/199
53.8
(46.6;
60.8)
25/51
49.0
(34.8;
63.4)
Unsolicited
non-serious
AE
105/199
52.8
(45.6;
59.9)
20/51
39.2
(25.8;
53.9)
Unsolicited
AR
22/199
11.1
(7.1;
16.3)
5/51
9.8
(3.3;
21.4)
Unsolicited
non-serious
AR
22/199
11.1
(7.1;
16.3)
4/51
7.8
(2.2;
18.9)
AE
leading
to
study
discontinuation
a
1/199
0.5
(0.0;
2.8)
1/51
2.0
(0.0;
10.4)
SAE
b
11/199
5.5 (2.8;
9.7) 6/51
11.8 (4.4;
23.9)
AE,
adverse
event;
AR,
allergic
reaction;
CI,
confidence
interval;
n,
number
of
participants;
SAE,
serious
adverse
event.
a
Identified
in
the
termination
form
as
SAE
or
other
AE.
b
Includes
SAEs
collected
up
to
after
the
third
injection
analysis.
The
proportion
of
participants
experiencing
unsolicited
AEs
was
similar
between
groups
overall
(CYD-TDV:
53.8%;
placebo:
49.0%)
and
post-dose
1
and
2
(30.7%
and
27.5%;
and
26.9%
and
24.0%,
respectively,
Table
3
and
Fig.
2),
and
slightly
higher
with
CYD-TDV
than
with
placebo
post-dose
3
(CYD-TDV:
22.4%;
placebo:
14.0%).
Infections
and
infestations
were
most
frequently
reported,
with
upper
respiratory
tract
infections
predominant
(CYD-TDV:
13.8%;
placebo:
9.8%).
The
proportion
of
participants
experiencing
unso-
licited
ARs
was
similar
between
groups
(CYD-TDV:
11.1%;
placebo:
9.8%).
Injection
site
induration
was
the
most
frequent
AR.
The
pro-
portions
of
both
unsolicited
AEs
(Fig.
2)
and
unsolicited
ARs
were
generally
lower
after
each
injection
for
both
groups
(unsolicited
ARs:
CYD-TDV:
7.5%,
2%,
2.6%;
placebo:
7.8%,
2%,
0%).
Five
partic-
ipants
experienced
Grade
1
rash
episodes
post-dose
1
(CYD-TDV:
n
=
3;
placebo:
n
=
1),
or
post-dose
2
(CYD-TDV:
n
=
1)
lasting
6
days.
Most
unsolicited
AEs
and
ARs
were
non-serious
and
did
not
lead
to
study
discontinuation
(Table
3).
SAEs
were
reported
by
5.5%
of
participants
in
the
CYD-TDV
group
and
11.8%
in
the
placebo
group
(
Table
3).
All
SAEs
were
assessed
as
unrelated
to
study
vaccine,
except
for
an
SAE
of
VII
nerve
paralysis
in
the
placebo
group.
This
participant
did
not
receive
any
further
vaccinations
and
recovered
4
months
later.
No
deaths
were
reported.
3.1.1.
Safety
and
reactogenicity
by
baseline
FV-serostatus
and
age
An
analysis
by
subset
showed
that
safety
and
reactogenicity
data
were
not
markedly
affected
by
FV-serostatus
or
age
(Fig.
2).
The
decrease
in
the
incidence
of
systemic
reactions
post-dose
3
appeared
more
marked
in
the
FV-seropositive
group
than
the
FV-
seronegative
group.
3.2.
Immunogenicity
Baseline
seropositivity
against
DENV1–4
was
similar
in
both
treatment
groups
(CYD-TDV:
31.1%,
27.6%,
36.7%
and
24.0%;
placebo:
32.0%,
30.0%,
36.7%
and
30.0%,
for
DENV1–4,
respec-
tively).
A
large
proportion
of
participants
were
seropositive
at
baseline
for
at
least
one
DENV
serotype
(CYD-TDV:
44.9%;
placebo:
48%).
A
small
proportion
of
participants
were
seropositive
at
baseline
for
JE
only
(CYD-TDV:
10.7%;
placebo:
13.7%).
In
the
CYD-
TDV
group,
pre-vaccination
GMTs
against
DENV1–4
ranged
from
9.92
1/dil
(DENV-4)
to
15.9
1/dil
(DENV-2).
Baseline
seropositivity
rates
against
DENV1–4
differed
between
age
groups
in
both
treat-
ment
groups,
being
higher
in
children
aged
6–11
years
compared
with
those
aged
2–5
years
(Supplementary
table
S2).
Similarly
in
the
CYD-TDV
group,
GMTs
were
higher
in
children
aged
6–11
years
compared
with
children
aged
2–5
years
(Table
2).
When
stratified
by
FV
serostatus,
GMTs
were
higher
in
those
aged
6–11
years
than
in
those
aged
2–5
years
in
participants
who
were
FV-seropositive
at
baseline
(Supplementary
table
S3).
Robust
immune
responses
were
reported
after
vaccination
with
CYD-TDV
regardless
of
FV-serostatus
(Table
2).
The
geometric
mean
fold
rise
of
individual
antibody
titres
ranged
from
4.8-fold
(DENV-
1)
to
8.1-fold
(DENV-3)
post-dose
2
and
from
6.1-fold
(DENV-1)
to
8.0-fold
(DENV-3)
post-dose
3.
Post-dose
2
and
3
GMTs
were
higher
in
the
FV-seropositive
group.
Post-dose
3
GMTs
showed
a
greater
increase
in
the
FV-seronegative
group
than
in
the
FV-seropositive
group.
Pre-vaccination
and
post-dose
3
GMTs
were
higher
in
par-
ticipants
who
were
seropositive
for
dengue
disease
at
baseline
than
in
participants
who
were
seronegative,
regardless
of
JE
serostatus.
Post-dose
3
GMTs
were
slightly
higher
in
JE
seropositive
partici-
pants
versus
JE
seronegative
participants.
However,
this
difference
was
not
significant.
The
highest
post-dose
3
GMTs
were
observed
in
participants
who
were
seropositive
for
both
dengue
disease
and
JE
at
baseline.
Robust
immune
responses
were
also
reported
regard-
less
of
age,
although
post-dose
2
and
3
GMTs
were
higher
in
6–11
year-olds
compared
with
2–5
year-olds
(Table
2).
The
post
dose
2
and
3
antibody
reverse
cumulative
distribu-
tion
curves
demonstrated
good
similarity
of
responses
(curves
close
together
and
parallel,
Fig.
3).In
FV-seronegative
children,
the
dose
3
vaccination
increased
the
overall
neutralizing
titres
with
a
left
shift
of
the
curves
for
serotypes
1
and
2.
4.
Discussion
CYD-TDV
is
in
advanced
stages
of
clinical
development
and
large
Phase
III
efficacy
studies
are
underway
in
Latin
America
(NCT01374516)
and
Asia
(NCT01373281);
no
other
dengue
disease
candidate
vaccine
has
reached
this
stage
of
development
to
date.
As
the
first
paediatric
trial
to
use
Phase
III
CYD-TDV
lots
manufactured
using
large-scale
processes,
the
present
study
is
a
key
component
of
the
global
clinical
trial
programme
for
CYD-TDV
[17–19].
Results
showed
that
CYD-TDV
had
a
satisfactory
safety
profile
and
gen-
erated
a
balanced
humoral
immune
response
against
DENV1–4
in
this
paediatric
population.
All
four
DENV
serotypes
circulate
in
Malaysia
and
their
relative
geographic
distributions
vary
over
time
[11].
Baseline
seroposi-
tivity
against
DENV1–4
was
similar
in
both
groups,
which
was
expected,
given
that
dengue
disease
is
endemic
in
Malaysia.
The
safety
profile
of
CYD-TDV
was
satisfactory
for
the
total
pop-
ulation
and
in
the
subsets
analyzed.
Reactogenicity
decreased
with
subsequent
doses
of
CYD-TDV,
consistent
with
observations
from
previous
phase
I
and
II
studies
[11].
Injection
site
erythema
and
swelling
were
reported
frequently,
but
at
Grade
1
intensity
and
at
similar
rates
in
both
treatment
groups.
Reactogenicity
was
similar
in
the
subsets
of
FV-serostatus
and
age
ranges
and
in
the
whole
A.-S.
HSS
et
al.
/
Vaccine
31 (2013) 5814–
5821 5819
A
90
80
70
60
50
Participants (%)
40
30
20
10
0
Post-dose 1
CYD-TDV Placebo CYD-TDV Placebo CYD-TDV Placebo
Post-dose 2 Post-dose 3
Any solicited reaction
Solicited injection site reactions
Solicited systemic reactions
Unsolicited AEs
B
90
80
70
60
50
Participants (%)
40
30
20
10
0
Post-dose 1
FV-seropositive
FV-seronegative
FV-seropositive
FV-seronegative
FV-seropositive
FV-seronegative
Post-dose 2 Post-dose 3
C
90
80
70
60
50
Participants (%)
40
30
20
10
0
Post-dose 1
2–5 years
6–11 years
2–5 years
6–11 years
2–5 years
6–11 years
Post-dose 2 Post-dose 3
Fig.
2.
Proportion
of
participants
with
different
categories
of
adverse
events
and
adverse
reactions
after
each
vaccination:
Safety
Analysis
Set
for
(A)
all
participants
and
(B)
in
the
CYD-TDV
Group
by
FV-serostatus
at
baseline
and
(C)
in
children
stratified
by
age
(2–5
and
6–11
years).
AE,
adverse
event;
FV,
flavivirus.
population,
without
increased
reactogenicity
in
2–5
year-olds,
or
baseline
FV-seropositive
children
compared
with
6–11-year-olds
or
FV-seronegative
children.
The
favourable
safety
and
reactogen-
icity
results
observed
are
similar
to
those
from
previous
Phase
I
and
II
studies
[20–29].
Our
data
from
Malaysia
are
consistent
with
those
reported
from
2
to
11-year
olds
in
Peru
[22]
and
Singapore
[26],
from
4
to
11-year
olds
in
Thailand
[29]
and
from
9
to
16-year
olds
in
Latin
America
[28].
The
three-dose
regimen
of
CYD-TDV
elicited
a
good
immune
response
in
terms
of
GMTs,
regardless
of
FV-serostatus
or
age
group.
GMTs
increased
both
post-dose
2
and
3
in
the
overall
population.
The
robust
and
balanced
antibody
responses
against
DENV1–4
were
similar
to
those
reported
in
other
studies
of
CYD-
TDV
in
children
[22,26,28].
However,
the
results
of
the
Phase
IIb
proof-of-concept
efficacy
study
by
Sabchereon
et
al.
challenged
the
hypothesis
that
such
a
robust,
balanced
antibody
response
profile,
as
assessed
by
PRNT
50
,
translates
to
similar
levels
of
protection
against
all
viruses
of
each
DENV
serotype
[29].
The
higher
GMTs
reported
after
vaccination
in
the
FV-seropositive
group
compared
with
the
FV-seronegative
group
were
also
observed
in
children
vac-
cinated
in
Latin
America
[28].
The
third
dose
had
little
impact
in
the
FV-seropositive
group
but
was
more
marked
in
the
FV-seronegative
group.
Therefore,
in
the
context
of
a
mixed
population
with
both
FV-seropositive
and
FV-seronegative
children,
a
three-dose
regi-
men
is
beneficial
to
induce
a
balanced
immune
response.
Baseline
serostatus
for
dengue
disease
but
not
for
JE
had
an
impact
on
post-
dose
3
GMTs.
However,
it
should
be
noted
that
the
sample
size
of
this
subset
was
limited-only
67
participants
in
the
CYD-TDV
5820 A.-S.
HSS
et
al.
/
Vaccine
31 (2013) 5814–
5821
DENV-1
≥20
≥40
≥60
≥80
≥160
≥320
≥640
≥1280
≥2560
≥5120
Pre-vaccination FV-seropositive
100
Participants (%)
80
60
40
20
Antibody titre (l/dil)
0
≥10
100
Participants (%)
80
60
40
20
Antibody titre (l/dil)
DENV
-2
0
≥10
≥20
≥40
≥60
≥80
≥160
≥320
≥640
≥1280
≥2560
≥5120
100
Participants (%)
80
60
40
20
Antibody titre (l/dil)
DENV-3
0
≥10
≥20
≥40
≥60
≥80
≥160
≥320
≥640
≥1280
≥2560
≥5120
100
Participants (%)
80
60
40
20
Antibody titre (l/dil)
DENV-4
0
≥10
≥20
≥40
≥60
≥80
≥160
≥320
≥640
≥1280
≥2560
≥5120
Post-dose 2 FV-seropositive
Post-dose 3 FV-seropositive
Pre-vaccination FV-seronegative
Post-dose 2 FV-seronegative
Post-dose 3 FV-seronegative
Fig.
3.
Reverse
cumulative
distribution
of
serotype-specific
PRNT
50
antibody
titres
curves
for
DENV
serotypes
1–4
by
baseline
FV-serostatus,
pre-vaccination
and
after
two
and
three
doses
of
CYD-TDV
(Full
Analysis
Set).
DENV,
dengue
virus;
FV,
flavivirus;
PRNT,
plaque
reduction
neutralization
test.
group
and
17
participants
in
the
placebo
group
were
seropositive
for
JE
antibodies
(PRNT
50
titre
10
l/dil).
The
immune
response
in
the
current
study
was
satisfactory
in
both
age
groups,
although
higher
GMTs
were
reported
in
those
aged
6–11-years
olds
than
2–5-year
olds
post-dose
2
and
3.
This
observation
was
probably
the
result
of
the
difference
in
baseline
DENV
serostatus
between
the
two
age
groups
(difference
in
pre-vaccination
GMTs
for
DENV1–4)
as
the
main
driver
for
vaccine
immunogenicity.
The
link
between
baseline
FV-serostatus
and
age
range
on
the
immune
response
is
presumed
to
be
the
result
of
a
longer
period
of
exposure
to
nat-
ural
dengue
infection
in
the
6–11
years
group
compared
with
the
younger
cohort.
Although
the
highest
attack
rate
for
dengue
infection
is
com-
monly
observed
in
young
adults
aged
20–24
years
old
[34],
the
large
proportion
of
children
who
were
seropositive
at
baseline
for
at
least
one
DENV
serotype
indicates
that
dengue
disease
is
endemic
and
infects
children
in
Malaysia
at
a
young
age.
However,
no
suspected
cases
of
dengue
disease
requiring
hospitalization
were
reported
by
the
investigators
or
participants’
parents
in
this
study
and
antibody
titres
in
the
placebo
group
did
not
increase,
suggesting
the
absence
or
very
limited
circulation
of
wild-type
DENV
strains
in
this
pop-
ulation
during
the
study
period.
Therefore,
the
results
of
vaccine
immunogenicity
were
probably
not
biased
by
natural
dengue
infec-
tions.
However,
the
placebo
group
was
small
and
conclusions
based
on
these
results
need
to
be
kept
in
context.
This
study
was
not
designed
to
assess
CYD-TDV
efficacy
and
did
not
address
long-term
vaccine
safety
or
immune
persistence,
with
follow-up
limited
to
6
months.
However,
large
Phase
III
efficacy
studies
are
underway
and
long-term
follow-up
is
ongoing
in
these
studies,
as
well
as
in
other
Phase
I
[20]
and
Phase
II
studies
[29].
Furthermore,
the
sample
size
of
this
study
was
too
small
to
accu-
rately
analyze
any
potential
priming
effect
of
previous
exposure
to
individual
FV
subtypes
(e.g.
JE).
5.
Conclusion
In
conclusion,
this
Phase
III
study
demonstrated
a
satisfactory
safety
profile
and
a
balanced
humoral
immune
response
against
DENV1–4
for
CYD-TDV
administered
via
a
three-dose
regimen
to
children
in
Malaysia
living
in
an
area
where
dengue
disease
is
endemic.
Acknowledgements
The
authors
thank
the
Director
General
of
Health,
Min-
istry
of
Health
Malaysia,
for
supporting
their
involvement
in
this
publication,
Mark
Boaz
from
Sanofi
Pasteur
Global
Clini-
cal
Immunology
(GCI,
Swiftwater,
PA,
USA)
for
conducting
the
immunology
assessments,
Sophia
Gailhardou
from
Sanofi
Pasteur
A.-S.
HSS
et
al.
/
Vaccine
31 (2013) 5814–
5821 5821
Global
Pharmacovigilance
and
Epidemiology,
Marcy
l’Etoile,
Lyon,
France
and
Mandy
Khoo
from
Sanofi
Pasteur
Regional
Study
Man-
agement
and
Logistic,
Sanofi
Pasteur
Singapore,
for
co-ordinating
study
activities.
The
authors
also
thank
all
of
the
children
who
participated
in
the
study
and
their
parents/guardians,
and
the
study-site
personnel
for
their
contributions.
The
authors
take
full
responsibility
for
the
content
of
this
contri-
bution
and
thank
Communigen
Ltd.
(supported
by
Sanofi
Pasteur)
for
editorial
assistance
with
the
preparation
of
this
manuscript
and
inputting
author
comments.
The
authors
also
thank
Grenville
Marsh
for
providing
critical
comments
and
suggestions
on
the
draft.
Contributions:
The
authors
made
the
following
contributions
to
the
study:
study
conception
and
design
(AB,
DC,
YH,
KKT),
laboratory
data
acquisition
(MTK),
patient
enrolment/patient
data
acquisition
(AB,
DC,
LGC,
MTK,
YH,
ASHSS,
KKT)
and
study
data
analysis
and
interpretation
(AB,
DC,
MTK,
YH,
ASHSS,
LZ).
All
authors
contributed
to
the
conceptualization
and
drafting
of
the
article,
participated
in
the
critical
review
of
the
article
and
approved
the
final
ver-
sion
submitted
for
publication.
All
authors
were
involved
in
the
decision
to
submit
to
Vaccine.
Conflict
of
interest:
Lynn
Zhou,
Alain
Bouckenooghe,
Denis
Crevat
and
Yanee
Hutagalung
are
employ-
ees
of
Sanofi
Pasteur.
Alain
Bouckenooghe
and
Yanee
Hutagalung
own
stock
options
in
Sanofi
Pasteur.
Mia-Tuang
Koh
has
received
financial
support
from
Sanofi
Pasteur
to
travel
to
meetings.
Tan
Kah
Kee
has
received
financial
support
from
Sanofi
Pasteur
to
travel
to
meetings
and
has
received
study
equipment
and
study
drugs.
Amar-Singh
HSS
and
Lee
Gaik
Chan
declare
no
conflicting
inter-
ests.
Role
of
the
funding
source:
This
study
was
funded
by
Sanofi
Pasteur
who
also
contributed
to
the
study
design,
data
collection,
analysis
and
interpretation,
review
of
the
manuscript
and
decision
to
publish.
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/
j.vaccine.2013.10.013
.
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