Rationale and methods of a randomized controlled trial of immunogenicity, safety and impact on carriage of pneumococcal conjugate and polysaccharide vaccines in infants in Papua New Guinea
Lehmann et al. Pneumonia
Rationale and methods of a randomized controlled trial of immunogenicity, safety and impact on carriage of pneumococcal conjugate and polysaccharide vaccines in infants in Papua New Guinea
Deborah Lehmann 0
Wendy Kirarock 2
Anita H. J. van den Biggelaar 0
Megan Passey 1
Peter Jacoby 0
Gerard Saleu 2
Geraldine Masiria 2
Birunu Nivio 2
Andrew Greenhill 0 2 4
Tilda Orami 2
Jacinta Francis 2
Rebecca Ford 2
Lea-Ann Kirkham 0 3
Vela Solomon 2
Peter C. Richmond 0 3
William S. Pomat 0 2
v PCV trial team
0 Telethon Kids Institute, University of Western Australia , 100 Roberts Road, Subiaco, WA 6008 , Australia
1 The University of Sydney, University Centre for Rural Health, School of Public Health , 61 Uralba Street, Lismore, NSW 2480 , Australia
2 Papua New Guinea Institute of Medical Research , Homate Street, Goroka, Eastern Highlands Province 441 , Papua New Guinea
3 School of Paediatrics and Child Health, University of Western Australia , Roberts Road, Subiaco, WA 6008 , Australia
4 School of Applied and Biomedical Sciences, Federation University , Northways Road, Churchill, VIC 3842 , Australia
Background: Children in third-world settings including Papua New Guinea (PNG) experience early onset of carriage with a broad range of pneumococcal serotypes, resulting in a high incidence of severe pneumococcal disease and deaths in the first 2 years of life. Vaccination trials in high endemicity settings are needed to provide evidence and guidance on optimal strategies to protect children in these settings against pneumococcal infections. Methods: This report describes the rationale, objectives, methods, study population, follow-up and specimen collection for a vaccination trial conducted in an endemic and logistically challenging setting in PNG. The trial aimed to determine whether currently available pneumococcal conjugate vaccines (PCV) are suitable for use under PNG's accelerated immunization schedule, and that a schedule including pneumococcal polysaccharide vaccine (PPV) in later infancy is safe and immunogenic in this high-risk population. Results: This open randomized-controlled trial was conducted between November 2011 and March 2016, enrolling 262 children aged 1 month between November 2011 and April 2014. The participants were randomly allocated (1:1) to receive 10-valent PCV (10vPCV) or 13-valent PCV (13vPCV) in a 1-2-3-month schedule, with further randomization to receive PPV or no PPV at age 9 months, followed by a 1/5th PPV challenge at age 23 months. A total of 1229 blood samples were collected to measure humoral and cellular immune responses and 1238 nasopharyngeal swabs to assess upper respiratory tract colonization and carriage load. Serious adverse events were monitored throughout the study. Of the 262 children enrolled, 87% received 3 doses of PCV, 79% were randomized to receive PPV or no PPV at age 9 months, and 67% completed the study at 24 months of age with appropriate immunization and challenge. (Continued on next page)
(Continued from previous page)
Conclusion: Laboratory testing of the many samples collected during this trial will determine the impact
of the different vaccine schedules and formulations on nasopharyngeal carriage, antibody production and
function, and immune memory. The final data will inform policy on pneumococcal vaccine schedules in
countries with children at high risk of pneumococcal disease by providing direct comparison of an accelerated
schedule of 10vPCV and 13vPCV and the potential advantages of PPV following PCV immunization.
Trial registration: ClinicalTrials.gov CTN NCT01619462, retrospectively registered on May 28, 2012
An estimated 921,000 children die annually from
pneumonia, with the majority of deaths occurring in
thirdworld countries [
]. Streptococcus pneumoniae
(pneumococcus) is the leading cause of pneumonia deaths in
children under 5 years, being responsible for approximately
400,000 of these deaths annually [
]. The pneumococcus
is also a leading cause of meningitis, septicemia and
otitis media. Infants are at greatest risk of dying from
pneumococcal disease [
In Papua New Guinea (PNG), acute lower respiratory
infection (ALRI) is the most common reason for
hospitalization and cause of death in children [
the burden of invasive pneumococcal disease (IPD)
remains high. In a 7-valent pneumococcal conjugate
vaccine (7vPCV) trial that we conducted between
2005–2009 in the Asaro Valley, Eastern Highlands
Province (the site of this study), the overall incidence
rate of ALRI before age 18 months in 109 controls
was 1175/1000 person-years; there were 62 episodes
of moderate or severe ALRI (incidence rate 439/1000
person-years), 27% of which occurred between 10 and
18 months of age . In the same trial, the estimated
incidence rate of IPD in the first year of life was
4762/100,000 per annum (95% CI 1936–9665),
approximately 100 times the rates reported in European infants
]. Upper respiratory tract (URT) pneumococcal carriage
starts within weeks of birth in PNG (median age of
acquisition is 19 days) [
] and is persistent throughout early
childhood, with average pneumococcal carriage rates of 70–80%
from age 3 months to 5 years [
]. Moreover, the
distribution of pneumococcal serotypes in PNG is broad: in our
7vPCV trial more than 60 different pneumococcal
serotypes were identified in the URT of infants followed from
birth until 18 months of age, of which 53 were identified in
the first month of life [
]. Only 43% of pneumococcal
carriage isolates among controls who had not received
PCV would have been covered by 13vPCV [
Vaccines for pneumococcal disease
Protection against IPD is by circulating
opsonophagocytic IgG antibodies binding to capsular polysaccharides,
which determine serotype. More than 90 different
pneumococcal serotypes have been identified to date.
The recognition that immunity correlated with
anticapsular antibodies led to the development of
multivalent polysaccharide vaccines [
polysaccharides are T-cell independent antigens and therefore
the pneumococcal polysaccharide vaccine (PPV) is
poorly immunogenic in infants and does not induce
immune memory [
]. Pneumococcal conjugate
vaccines (PCV) containing polysaccharide antigens of
individual serotypes covalently bound to protein carriers,
hence transforming polysaccharides into T-cell
dependent antigens, became available in 2000. In
contrast to PPV, PCVs induce immunological memory and
serum antibodies with a strong opsonophagocytic
capacity in infants [
] and they are effective in
preventing vaccine serotype-associated IPD, pneumonia and
mortality in high-risk infants [
PCVs reduce URT carriage of vaccine serotypes in
vaccinated children, thus interrupting circulation of vaccine
serotypes in the community, which in turn reduces IPD
incidence in unvaccinated individuals through herd
The Global Alliance for Vaccines and Immunization
(Gavi) and the World Health Organization (WHO)
committed to the introduction of PCVs for infants in
Gavieligible countries (including PNG). Two PCVs are
currently available: i) 10-valent PCV (10vPCV, Synflorix®,
GlaxoSmith Kline [GSK], Belgium) containing serotypes
1, 4, 5, 6B, 7F, 9 V, 14, 18C, 19F & 23F; and ii) 13-valent
PCV (13vPCV, Prevenar 13®, Pfizer, United States of
America [USA]) containing serotypes 3, 6A and 19A in
addition to the 10 serotypes in 10vPCV. Both vaccines
have been shown to prevent vaccine serotype-associated
IPD in high-risk populations [
10vPCV also contains non-typeable Haemophilus
influenzae (NTHi) Protein D as a carrier, which may
offer protection against NTHi disease [
]. NTHi was
frequently isolated in lung aspirates from children with
pneumonia in PNG [
], is a predominant cause of otitis
media worldwide [
], and has also been reported as a
cause of invasive disease [
Policy on choice of PCV in low- and middle-income
countries has been hampered by the lack of data
comparing the two vaccines in high-risk infants. While the
three additional serotypes included in 13vPCV improve
coverage for prevention of pneumococcal disease, the
potential benefits of protection against NTHi-related
pneumonia and otitis media by 10vPCV need to be
19, 21, 28–30
The broad range of serotypes causing IPD in PNG and
other high-risk populations means that even 13vPCV
provides limited coverage (50–70%) against IPD [
There are also concerns that serotype replacement is
more likely to occur in high-risk populations, similar to
what was reported for native Alaskan infants after
7vPCV introduction .
Despite limited immunogenicity in infants, PPV given
to PNG children aged 6 months to 5 years of age has
been shown to reduce mortality and severe morbidity
due to ALRI [
], with an age-related maturation of
IgG responses correlating with efficacy data .
Therefore, in high-risk infants, priming with PCV followed by
a single dose of PPV in later infancy could potentially
increase protection against IPD, particularly for serotypes
such as type 2 that is an important cause of meningitis
but is not covered by either PCV [
]. This strategy
also has the potential to improve duration of protection
and reduce the risk of serotype replacement disease
compared to PCV alone.
A study in Fiji raised concerns on PPV immunization
in young children by reporting that children primed with
7vPCV in infancy followed by a dose of PPV at
12 months of age failed to further boost the already high
antibody titres following challenge with 1/5th of a
normal PPV dose 5 months later. However,
serotypespecific IgG responses in these children did not decline
and were robust with high avidity and opsonic functional
activity suggesting protective responses [
Followup of the Fijian study participants at pre-school age
showed that serotype-specific antibody levels were
similar in those who had and those who had not received
PPV at age 12 months, implying that there was no
evidence of long-term hyporesponsiveness at pre-school
In the 7vPCV trial PPV was found to be immunogenic
when given at age 9 months to PNG infants previously
primed with 3 doses of PCV [
], and antibody titres to
PCV and non-PCV serotypes rose in response to a 1/5th
PPV challenge dose at age 3–4 years. These data further
contribute to the evidence that long-lasting deleterious
effects of PPV immunization in infancy are unlikely [
Limitations of the 7vPCV trial were the relatively long time
interval between receipt of PPV at 9 months and
assessment of response to challenge at 3–4 years of age, and that
the study did not include a group of children who had
received PCV with no PPV. To determine whether priming
of high-risk infants with PCV followed by a single dose of
PPV in later infancy could be a preferred strategy to
increase serotype coverage for IPD, without compromising
both short- and long-term protective immune responses, a
randomized controlled trial including a control group not
receiving PPV is required.
Rationale for the present study
This head-to-head randomized trial aims to demonstrate
that 10vPCV and 13vPCV are suitable vaccines for use
under PNG’s accelerated immunization schedule by
comparing the safety and immunogenicity of 10vPCV
with that of 13vPCV in a 1-2-3-month schedule with
and without a PPV booster at 9 months of age. This is
important for the following reasons:
With support from Gavi, an increasing number of
low-resource countries are introducing PCV into
routine immunization schedules, with a comparable
number of 10vPCV and 13vPCV doses being used.
At the time of the primary schedule of this trial,
PCV had not been introduced into PNG and no
data were available directly comparing the safety and
immunogenicity of these two vaccines in a high-risk
population such as PNG. While PNG infants have
been receiving 13vPCV since 2014 (after completion
of the primary schedule for this trial), it remains
important to determine the safety and immunogenicity
of 10vPCV should there be a shortage in the supply of
13vPCV or if future research shows that 10vPCV
protects against NTHi disease.
There are limited data on the immunogenicity of
PCVs in children with very early onset of dense
URT pneumococcal and NTHi carriage.
The Expanded Program on Immunization (EPI)
schedule is accelerated in PNG (ages 1, 2 and
3 months); neither 10vPCV nor 13vPCV13 have
been evaluated under this schedule.
Given the broad range of serotypes causing disease
in PNG and in other high-risk populations, it is
important to investigate the safety and immunogenicity
of a booster dose of PPV at age 9 months following
priming with PCV.
Data are lacking on PCV-induced functional antibody
responses and 10vPCV-induced NTHi Protein D
antibody responses in populations with high URT
carriage rates from a young age.
While there is evidence that PCV impacts on vaccine
serotype-specific URT pneumococcal carriage in
high-risk settings [
], there are limited data on
the potential impact on carriage density in these
The overarching aim of the study is to ensure that the
PCVs under investigation are suitable for use under
PNG’s accelerated immunization schedule, and that a
schedule including PPV in later infancy is safe and
immunogenic in this high-risk population.
The primary objectives of this study are to:
1. Determine the safety and reactogenicity up to
24 months of age of 10vPCV and 13vPCV given in
the accelerated 1-2-3-month PNG schedule;
2. Evaluate serotype-specific serum IgG and functional
antibody responses at 4 and 9 months of age
following completion of 3 doses of 10vPCV or 13vPCV,
and antibody persistence and IgG avidity maturation
at 23 months of age;
3. Compare serotype-specific serum IgG titres,
functional antibody responses and circulating memory
B-cells at 10 months of age and the persistence of
these responses at 23 months of age in children
primed with 10vPCV or 13vPCV who did or did not
receive PPV at 9 months of age;
4. Assess the persistence of B-cell memory by
measuring circulating vaccine serotype-specific memory
B-cells, IgG and opsonophagocytic antibody responses
and IgG avidity maturation 1 month after challenge at
23 months with 1/5th of a normal PPV dose in
children who did or did not receive PPV at 9 months
of age; and
5. Compare the relative impact of 10vPCV and
13vPCV on pneumococcal (total and vaccine type)
and NTHi carriage rates and bacterial load up to
2 years of age and the effect of a PPV booster.
Secondary objectives of this study are to:
1. Compare development of IgG responses to NTHi
Protein D between infants receiving 10vPCV and
those receiving 13vPCV; and
2. Describe the frequency of pneumonia and otitis
media with perforation associated with
pneumococcal vaccine serotypes and/or NTHi
isolated from the nasopharynx and/or middle ear
The following paragraphs describe the methods used for
enrolment, clinical follow-up and specimen collection
Study design and location
Between November 2011 and March 2016, we
conducted an open randomized controlled trial of safety
and immunogenicity of 13vPCV and 10vPCV given in
an accelerated 1-2-3-month schedule, with further
randomization of children in each PCV group to
receive PPV or no PPV at age 9 months, followed by a
1/5th challenge dose of PPV to all children at age
23 months. The study was conducted in Goroka
Town and villages located within an hour’s drive from
Goroka in the Eastern Highlands Province of PNG.
Goroka is the provincial capital (population ~25,000;
altitude 1600 m) and the PNG Institute of Medical
Research (PNGIMR) laboratories and clinic are adjacent
to Eastern Highlands Provincial Hospital (EHPH), the only
tertiary hospital in the area. Outside the urban area,
people live in villages often accessible only by 4WD
vehicles. Living standards vary widely between urban,
peri-urban and rural settings.
Ethical approval was obtained from the PNG Medical
Research Advisory Committee (#11.03) and PNGIMR
Institutional Review Board (#1028). The study was
conducted according to Declaration of Helsinki International
Conference on Harmonisation Good Clinical Practice
(ICH-GCP) and local ethical guidelines. The study is
registered with ClinicalTrials.gov (CTN NCT01619462).
Recruitment, assent and consent process
Prior to starting the study, field staff visited communities
around Goroka Town and surrounding villages to
explain the study to community members and seek their
willingness to participate in the trial. Pregnant women
were invited by research nurses and health extension
officers (HEOs) to take part in the study during these field
visits or when women attended the PNGIMR clinic.
Local drivers who were familiar to and respected by the
local communities played an important role in
introducing the study team to the communities. Assent was
sought from pregnant women and their husbands,
contact details were recorded and information sheets were
provided. Women who had assented were seen again
close to their expected delivery date or when they
presented of their own accord at the PNGIMR clinic after
delivery. Informed consent was sought at time of
enrolment of the child.
Inclusion criteria for infants to be enrolled in the study
Aged 28–35 days;
Resident within 1 h drive from Goroka town; and
Intending to remain in the study area for at least
Exclusion criteria were:
Birth weight < 2000 g;
Severe congenital abnormality;
Mother or child known to be positive for Human
Immunodeficiency Virus (HIV); or
Unable to or did not provide consent.
A single dose (0.5 mL) of 10vPCV (Synflorix®, GSK,
Belgium, batches ASPNA0099AB, ASPNA267DD)
contains 1 μg of each pneumococcal polysaccharide for
serotypes 1, 5, 6B, 7F, 9 V, 14 and 23F and 3 μg of
serotype 4, each conjugated to Protein D of NTHi and
3 μg of serotypes 18C and 19F conjugated to tetanus
and diphtheria toxoids, respectively. One dose (0.5 ml)
of 13vPCV (Prevenar 13®, Pfizer, USA, batch numbers
F36226, G71540) contains 2.2 μg of pneumococcal
purified capsular polysaccharides for serotypes 1, 3, 4,
5, 6A, 7F, 9 V, 14, 18C, 19A, 19F and 23F, and 4.4 μg of
pneumococcal purified capsular polysaccharides for
serotype 6B and 0.125 mg aluminium adjuvant. Each
serotype is individually conjugated to non-toxic
diphtheria CRM197 protein.
Each 0.5 mL dose of PPV (Pneumovax®23 Merck &
Co, USA, batch numbers T0861, V1200, K006913)
contains 25 μg of purified capsular polysaccharides of each
of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9 N, 9 V, 10A, 11A,
12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F
and 0.25% phenol preservative.
Enrolment, randomization, vaccination and follow-up
Children who met the eligibility criteria were
randomized at 1 month of age to receive 10vPCV or 13vPCV
using a computer-generated random number list. This
assignment was specified inside a sealed envelope with
the next sequential number and kept in the participant’s
personal folder. Throughout the study laboratory staff
was blinded to the group allocation. The vaccine and
specimen collection schedules are shown in Table 1.
Prior to any vaccination (and PPV challenge at age
23 months), study nurses/HEOs obtained a medical
history and examined children to exclude contraindications
for vaccination (including temperature ≥ 38 °C,
anaphylaxis to previous vaccination, serious neurological illness
within 48 h of previous pentavalent vaccine) and
specimens were collected. Routine childhood vaccines
(including pentavalent whole-cell pertussis, diphtheria,
tetanus, Hepatitis B and Haemophilus influenzae type B
BCG Bacillus Calmette-Guérin vaccine; HepB Hepatitis B vaccine; Pentavalent is a diphtheria, tetanus, whole cell pertussis, Haemophilus influenzae type B, Hepatitis
B combination vaccine; PCV 10-valent or 13-valent pneumococcal conjugate vaccine; OPV oral polio vaccine; PPV 23-valent pneumococcal polysaccharide vaccine;
NPS nasopharyngeal swab; PBMC peripheral blood mononuclear cells
aBirth doses of BCG and Hepatitis B were given by hospital nurses before enrolment of infants into the study
bPCV and PPV are study vaccines while the other vaccines were routine EPI vaccines in PNG at the time of the trial
cMorbidity surveillance was conducted throughout the duration of the trial. Vitamin A was given at 6 and 9 months of age
(Hib) vaccine and oral polio vaccine; see Table 1) were
administered by the study HEOs/nurses at 1, 2, and
3 months of age, together with the 10vPCV or 13vPCV
study vaccines. Information on birth doses of Bacillus
Calmette-Guérin (BCG) vaccine and Hepatitis B
vaccines administered by hospital staff was collected from
hospital records, as were birth weights. At the initial visit
mothers were given a follow-up card on which dates for
future visits were recorded. Children living in rural areas
were generally transported to and from the clinic while
urban residents were either brought in or came of their
own accord to the clinic following a reminder by mobile
phone. At age 4 months children attended the clinic for
specimen collection only. At age 6 months, children
received their first dose of measles vaccine and a dose of
Vitamin A either at the PNGIMR clinic or at other child
health clinics. At age 9 months children received a
second dose of measles vaccine and Vitamin A, and were
randomized within each PCV group to receive or not
receive PPV. The PPV group allocation was assigned at
enrolment, at the same time as PCV randomization, but
kept in an unopened envelope until the 9-month visit.
Specimens were collected before vaccination and again
4 weeks later.
All participants were given a 1/5th challenge dose of
PPV at age 23 months with specimens collected
immediately before the challenge and 4 weeks later. All
vaccinations were documented in the child’s health book held
by the mother/guardian and also written on the child’s
follow-up card, which was stored electronically.
Photocopies of records in the parent-held books (which
include information on any illness episode) were stored in
the child’s personal folder.
Every effort was made to locate participants for
followup in a timely manner. Between 10 and 23 months, nurses
and field staff attempted to contact participants’ caregivers
at least twice to find out about any intervening illness and
changes in address. Children were withdrawn from the
study if the child could not be located on 3 home visits or
information had been obtained from relatives that the
family had moved out of the study area permanently, or if
parents withdrew their consent for continuation in the
study. Children were also withdrawn by the investigators
following a protocol violation.
Monitoring for adverse reactions
Following each vaccination, children were observed at
the clinic for 1 h for immediate local and systemic
reactions and generally taken home by the study team. This
helped locate participants for subsequent visits. Study
nurses/HEOs visited children at home 24–48 h
postvaccination to assess local or systemic side effects and
they provided treatment as required.
A nasopharyngeal swab (NPS) to assess pneumococcal
and NTHi carriage and bacterial load was collected at ages
1, 4, 9, 10, 23 and 24 months (Table 1) as previously
] except that a flocked swab (Copan Diagnostics,
USA) was used. NPS samples were placed in
skim-milktryptone-glucose-glycerol broth (STGGB) and stored at
−80 °C within 2 h of collection. Culture and
pneumococcal serotyping were conducted at PNGIMR as previously
]. Aliquots from the stored NPS were sent in
liquid nitrogen to the University of Western Australia to
measure bacterial load. If middle ear discharge was seen, a
flocked swab was used to collect and store the purulent
discharge in STGGB for culture, identification and
characterisation of bacterial pathogens [
Venous blood samples (up to 3 mL) for serum
collection were collected at ages 1, 4, 9, 10, 23 and 24 months
(Table 1) to measure pneumococcal serotype-specific
IgG antibody titres [
], IgG avidity [
opsonophagocytic antibody titres [
], and NTHi Protein D-specific IgG
antibody levels [
]. No more than 3 attempts to collect
blood were undertaken at a single time point. On
occasions when parents agreed, a further attempt at blood
collection was undertaken a few days later. Serum
samples were aliquoted and stored at −80 °C.
Serotypespecific IgG antibody responses were measured at
PNGIMR using a WHO standardized pneumococcal
enzyme-linked immunosorbent assay (ELISA) [
Serum aliquots were sent in liquid nitrogen to the
University of Western Australia (UWA) to conduct
functional pneumococcal antibody assays and NTHi protein
D antibody assays.
From age 4 months onwards, at each routine
followup visit, we aimed to collect 2–7 mL of heparinized
venous blood samples to isolate peripheral blood
mononuclear cells (PBMCs) to measure pneumococcal
serotype-specific B-cell responses and pneumococcal
protein-specific T-cell responses as shown in Table 1
]. After immediate processing at PNGIMR, PBMCs
were cryopreserved and sent in batches in liquid
nitrogen vapour phase to UWA.
Detailed laboratory methods will be reported
elsewhere with laboratory results.
Passive morbidity surveillance was conducted. Parents
were encouraged to attend the PNGIMR clinic if their
child was unwell. Generally parents liked to bring their
children directly to the PNGIMR clinic as they were seen
faster than at routine outpatient clinics, they knew the staff,
and were assisted with transport if required. In addition,
family members other than the study child could be seen
at the PNGIMR clinic for the duration of the study.
Children were referred to pediatricians at EHPH as required.
The EHPH pediatric ward was also monitored for any
hospitalizations of study participants. Information on any
intervening illnesses was documented on a morbidity
report form and in the parent-held child’s health book.
Children were treated according to standard management
protocols in PNG [
ALRI was defined as mild (cough and raised
respiratory rate, namely >60/min in infants less than 2 months
of age, or >40/min in infants aged 2 months and older),
moderate (as for mild with chest wall in-drawing), or
severe (as for moderate with cyanosis or inability to feed
or restlessness or heart failure). This is a slight
modification from that used generally in PNG. Standard
treatment protocols in PNG recommend hospitalization for
all moderate or severe ALRIs.
A serious adverse event (SAE) was defined as any
event requiring hospitalization or resulting in death. In
2015 there were disruptions of EHPH services. During
this period an SAE was also documented for illnesses
(mainly moderate ALRI cases) deemed serious enough
to require hospitalization, but the children had to be
treated as outpatients and were monitored closely. Blood
cultures and NPS were collected when children were
seen with a temperature > 38 °C or a provisional
diagnosis of moderate or severe ALRI, or meningitis.
PCV outcome measures are:
(1)Safety and reactogenicity of 10vPCV and 13vPCV in
(2)Serum PCV serotype-specific IgG and opsonizing
antibody geometric mean titres (GMTs), and
proportions of children with serum serotype-specific
IgG antibody concentrations ≥0.35 μg/mL and
opsonizing titres ≥8 for PCV serotypes at 4 and
9 months of age;
(3)Serum H. influenzae Protein D IgG GMTs at 4 and
9 months of age;
(4)Number of circulating memory B-cells specific for
PCV serotypes and PCV carrier proteins (NTHi
Protein D, Tetanus Toxoid and CRM197) and PCV
serotype-specific IgG avidity maturation at 4 and
9 months of age;
(5)T-cell responses to PCV carrier proteins (NTHi
Protein D, Tetanus Toxoid and CRM197) at 4 and
9 months of age;
(6)Nasopharyngeal carriage rates of S. pneumoniae
(vaccine and non-vaccine serotypes) and serotypeable
and non-serotypeable H. influenzae at 1, 4 and
9 months of age;
(7)Pneumococcal and H. influenzae nasopharyngeal
density at 1, 4 and 9 months of age; and
(8)Rates for hospitalization with ALRI or record of
otitis media with perforation between 4 and
9 months of age.
PPV outcome measures are:
(1)Serum PCV and PPV serotype-specific IgG and
opsonizing antibody GMTs, and proportions of
children with PCV and PPV serotype-specific IgG
concentrations ≥0.35 μg/mL and opsonizing titres
≥8 at 10 and 23 months of age;
(2)Number of circulating memory B-cells and IgG
avidity maturation for PCV and non-PCV serotypes
included in PPV at 10, 23 and 24 months of age;
(3)Nasopharyngeal carriage rates and density of
serotype-specific S. pneumoniae, and serotypeable
and non-serotypeable H. influenzae at 10, 23 and
24 months of age; and
(4)Rates for hospitalization with ALRI or record of
otitis media with perforation between 10 and
24 months of age.
Study and data management
To ensure any problems or queries were addressed as
soon as possible, investigators in PNG and Australia
attended monthly teleconferences.All data collection
forms were checked manually before entry into a
password-protected computer file using FileMaker Pro
version 11 (Santa Clara, CA, USA).
Data safety monitoring board
A data safety monitoring board (DSMB) was established,
which included independent clinicians in PNG and
Australia. All SAEs were reported within 24 h to an
appointed Papua New Guinean Safety Monitor and the
Chair of the DSMB for review, followed by a final report
with outcome. The Chair of the DSMB was informed
immediately when a death occurred. A quarterly report was
prepared by the investigators and sent to the Chair of the
DSMB and distributed to other DSMB members. This
included a summary of all serious adverse events, all
morbidities, protocol violations, and status of enrolment and
follow-up. A teleconference with DSMB members and the
study investigators was held annually.
Sample size calculations
Sample size calculations were based on an anticipated
80% follow-up rate with evaluable samples to the final visit
based on results from our 7vPCV trial (87% at age
18 months) [
]. Based on the study’s primary outcome, it
was postulated that 90% of children would achieve
serotype-specific antibody concentrations ≥0.35 μg/ml at
4 months of age for serotypes included in the PCV
children were given. Based on this assumption, a sample size
of 100 children (receiving 10vPCV or 13vPCV) would give
us 95% confidence that the true proportion is within 6%
of this level of 90%. For bacterial load analysis, previous
data for H. influenzae bacterial loads [
] indicate that a
within-group standard deviation in log10 bacterial load
(copies/ml) of 2.0 can be expected. With a sample size of
50 in each group (after randomization into PCV and PPV
group), this would give 80% power to detect a
betweengroup difference in mean log10 bacterial load of 0.9. In
view of the higher loss to follow-up than anticipated
during the first year of the study, the total number of children
to be enrolled was increased from 200 to 260.
Statistical analysis is by intention to treat. All serum
antibody concentrations and opsonophagocytic titres
were log-transformed and geometric mean
concentrations (GMC) and geometric mean titres (GMT)
calculated respectively with 95% confidence intervals. To
compare continuous and categorical variables between
groups, Mann-Whitney tests and Pearson chi-square
tests were used respectively. Spearman rank correlation
analysis was used to investigate correlations between
serotype-specific opsonophagocytic and IgG antibody
concentrations. Linear and logistic regression models
were used for further analyses examining associations
between specific outcomes and different factors
incorporating generalised estimating equations to take into
account multiple samples from individual children. For all
analyses, test outcomes were considered to be significant
if the p-value is smaller or equal to 0.05.
Assent and enrolment
A total of 431 women assented to having their child
participate in the study, of which 265 (61%) gave their
informed consent. Three children did not meet inclusion
criteria (2 were overage and 1 child’s mother was
HIVpositive). Hence 262 children were formally enrolled into
the study between 14 November 2011 and 16 April 2014,
including 131 children randomized to receive 10vPCV
and 131 randomized to receive 13vPCV. A flow chart
(Fig. 1) shows the number of women who assented,
number of infants randomized to receive one or other PCV at
1 month of age, number of children seen at subsequent
visits, number of withdrawals and reasons for withdrawal
according to randomization schedule.
A total of 86 (33%) children were withdrawn from the
study (including deaths and protocol violations), the
majority before age 9 months (41% before age 3-month
visit, 63% before 9-month visits) (Fig. 1). Parents
withdrew their consent for 20 children (8%) (8 in 10vPCV
group and 12 in 13vPCV group); 25 children (9.5%)
migrated with their parents to another location (12 in
10vPCV group and 13 in 13vPCV group); and 25
children (9.5%) were recorded as lost to follow-up after at
least 3 unsuccessful home visits by field staff (13 in
10vPCV group and 12 in 13vPCV group). Of the
children lost to follow up, two-thirds could not be located
within 2 months of enrolment, while approximately
onethird of those who migrated out of the study area did so
before age 4 months. Approximately two-thirds of
parents who withdrew consent did so before the 4-month
visit. None of the withdrawals were related to an adverse
There were 7 deaths, none of which were related to
any vaccination (4 in the 10vPCV group and 3 in the
13vPCV group). Documented causes of death, age at
death and PCV/PPV status were as follows:
– Severe pneumonia and presumed pulmonary
tuberculosis; 9 months; 13vPCV
– Blood dyscrasia, anaemia; 9 months; 10vPCV
– Moderate pneumonia, malnutrition, gastroenteritis;
15 months; 10vPCV, no PPV
– Malnutrition, anaemia, presumed pulmonary
tuberculosis; 17 months; 10vPCV, no PPV
– Gastroenteritis with dehydration; 17 month;
– Gastroenteritis with severe dehydration; 18 months;
– Severe malnutrition, dysentery, anaemia; 19 months;
13vPCV, no PPV
One other child received the first dose of 13vPCV
but soon after was diagnosed with a retroperitoneal
tumour, at which time this child was withdrawn from
the study and died at age 2 months.
There were 8 protocol violations: 3 were related to
the PCV schedule (2 in the 10vPCV group, 1 in the
13vPCV group), 1 was related to PPV immunization
(within the 13vPCV group) and 4 were related to the
low-dose challenge at 23 months of age (1 in the
10vPCV/no PPV group, 2 in the 13vPCV/no PPV
group and 1 in the 13vPCV/PPV group).
Follow-up of study participants
Of the 262 children enrolled in the study who
received PCV at age 1 month, 93% received 2 doses of
PCV and 87% received 3 doses of PCV. At age
9 months 79% of enrolled children were randomized
to receive PPV or no PPV; 77% of the 262 children
were seen at the 10 month visit; 69% at 23 months;
and 67% completed the study at 24 months of age
with appropriate immunization and challenge.
Followup was similar for the different PCV/PPV groups.
Fig. 1 flow chart
Table 2 shows that children received PCV and PPV
in a timely manner with no difference in age at
vaccination and follow-up between groups. The table also
shows the gender distribution and average body
weight according to PCV/PPV group at different time
points. There were no differences between the groups.
NPS and venous blood samples for serum collection
were successfully obtained from more than 96% of
attendees at different ages, while PBMCs were
successfully isolated from 64 to 69% of blood samples (Table 3).
On 4 occasions parents declined blood collection from
To our knowledge, our study is the only trial directly
comparing safety and immunogenicity of two different
PCVs followed by a PPV booster dose. A trial in The
Male, number (%) at enrolment
Age (days) at study visits
Visit 1 m/PCV1, mean ± sd
Gambia directly compared safety and immunogenicity of
10vPCV and 13vPCV as part of a larger trial of new
experimental pneumococcal protein-based vaccines, but
did not include a PPV booster [
]. Two other trials
comparing different schedules of the two PCVs are
underway, one in Vietnam (NCT01953510) and another in
Australian Aboriginal children (ACTRN12610000544077
Study strengths and limitations
Despite logistical difficulties in conducting the study in
urban and rural areas of the Eastern Highlands Province
of PNG, a large enough number of samples were
successfully collected to address study outcomes. Two-thirds of
those enrolled in this study were fully followed up to
2 years of age, with sera and NPS collected at >1200
routine visits and high numbers of PBMCs (Table 3) isolated
aPeripheral blood mononuclear cells bNumbers exclude those who did not meet inclusion criteria and those who were lost to follow-up or withdrawn
from 658 blood samples from age 4 months onwards. This
is a reflection of the long-established relationship and
trust between the community and PNGIMR staff, in
addition to the perseverance of field staff in locating
The two-stage process of assent followed by formal
consent was important, allowing parents to discuss their
participation with family members. Providing medical
treatment to participants and their families and close
follow-up in the event of illness was appreciated and a
motivation to take part in the study. In addition, all
study children received hand-knitted sweaters donated
by Australian volunteers, and at the end of the study
parents received a certificate of participation with a
photograph of their child.
Every vaccination entailed at least three trips to
communities (pick-up and drop-off, and 24–48 h follow-up
visit), with additional visits needed if not located the first
time. Roads were at times impassable and locals
repairing roads sometimes charged a ‘toll’. In 2012 there were
national elections and some families returned to their
home villages outside the study area to vote and did not
return. Furthermore, disputes between supporters of
rival candidates at times restricted movement in the
Despite every effort, approximately one-third of
participants did not complete the study. This is in part the
result of increasing mobility of the population: 10% of
participants migrated out of the area during this study
compared with 5% during the earlier 7vPCV study
conducted between 2005 and 2009 [
]. Loss to follow-up
and withdrawal of consent tended to occur at a young
age, similar to the 7vPCV study [
]. In some cases,
husbands had not been part of the assent and consent
processes and subsequently indicated they did not want
their children to participate in the study. While every
effort was made to provide full information about what
the study entailed, mothers may on further reflection
have had doubts about taking part in the study. Some
parents were concerned about the volume of blood that
was being collected from their child. Collecting blood
samples from young children was sometimes difficult,
though the nurses/HEOs did not persevere if the child
or parent was distressed.
Having a Papua New Guinean doctor to oversee the
clinical activities and train nurses and HEOs was a great
advantage and much appreciated by parents. When the
study doctor left a year and a half into the study to
pursue pediatric training, a succession of three competent
HEOs oversaw the field and clinical activities. However,
these staff changes reduced continuity over the course of
In contrast to laboratory staff who were blinded to the
vaccine randomization, study nurses were not blinded
due to the limited number of nurses/HEOs available to
administer vaccines and conduct follow-ups. However,
whenever possible, different nurse/HEOs administered
vaccines to those assessing reactogenicity. It is
acknowledged that this could potentially impact on the
interpretation of clinical outcomes, but cannot affect the primary
Despite the launch of the 13vPCV program in PNG in
2014, vaccine was not available in the EHP until 2015
and has been introduced slowly since: coverage with one
dose of 13vPCV was only around 25% and with three
doses around 5% in late 2015 in the study area [
Herd protection following launch of the PNG 13vPCV
program in PNG in late 2014 is therefore unlikely to
have impacted on the results of the present study.
Notably, there were no deaths among study participants
due to uncomplicated pneumonia in a country where
pneumonia remains the most common cause of childhood
]. This is attributed to all study children receiving
both pneumococcal and Hib vaccines and close
monitoring of study participants when they were sick. While
morbidity among study participants will be reported fully
elsewhere, 2 of the 3 cases of culture-proven
pneumococcal bacteraemia were due to non-PCV serotypes and the
third child with a PCV serotype-related bacteraemia had
only received one dose of PCV at the time of death [
The mean age at death was older than previously recorded
], with 5 of the 8 deaths occurring in the second year
of life. While a diagnosis of pulmonary tuberculosis was
assigned for two children who died, these were not
laboratory-confirmed. The emergence of malnutrition as
an important cause of death in PNG has been highlighted
]. This may be related to undiagnosed
tuberculosis, which was previously rare in the highlands of
PNG, overall poverty, or a reflection of societal challenges
including unwanted pregnancies [
] and associated
neglect of children . Finally, infants with a birth weight
of less than 2000 g were excluded as low birth weight was
used as a surrogate marker for prematurity, which may
affect immune responses to vaccines including PCVs
], as well as that prematurity is hard to assess in
this setting. As is true for randomized clinical trials in
general, field studies conducted post-implementation
may find different results in individuals due to the
influence of various external factors. However, the
authors believe that the results for 10vPCV and 13vPCV
immunogenicity and safety are largely generalizable to
newborns in PNG and other settings where
pneumococcal infections are highly endemic.
The introduction of PCV in high-risk settings facilitated
by Gavi is likely to have a significant impact on the burden
of IPD, although the high proportion of pneumococcal
disease due to non-vaccine serotypes raises concerns with
regard to serotype replacement. The lack of data
comparing PCVs has made it difficult for countries to choose the
PCV likely to provide the most benefit. This study
provides direct comparison of safety, immunogenicity
(including functional antibody responses) and impact on
carriage rates and bacterial load in a randomized controlled
trial of an accelerated 3-dose schedule of 10vPCV and
13vPCV in a low-income setting. Following completion of
the primary schedule in this trial without immediate
concerns regarding reactogenicity and safety of either PCV,
13vPCV was introduced into the PNG EPI Program in
2014. In addition to comparison of currently available
PCVs, this study will provide evidence on the potential
advantages of PPV following PCV immunization in infancy
in high-risk settings, including immune protection against
important IPD-causing non-PCV serotypes [
boosting of immune responses against PCV serotypes to
enhance long-term immunity, and will directly investigate
any effects on B-cell memory. Samples collected in this
cohort are currently undergoing laboratory testing to
determine the impact of the different vaccine schedules and
formulations on nasopharyngeal carriage, antibody
production and function, and immune memory response.
Assessment of antibody and T-cell responses to
pneumococcal and NTHi proteins will aid an understanding of the
development of natural immunity to these antigens
relevant to development of protein-based vaccines. These
studies will provide data to inform policy on
pneumococcal vaccine schedules in countries with children at high
risk of invasive pneumococcal disease.
10vPCV: 10-valent pneumococcal conjugate vaccine; 13vPCV: 13-valent
pneumococcal conjugate vaccine; AE: Adverse event; ALRI: Acute lower
respiratory infection; BCG: Bacillus Calmette-Guérin; CSL: Commonwealth
Serum Laboratories Ltd.; DSMB: Data safety monitoring board; EHPH: Eastern
Highlands Provincial Hospital; ELISA: Enzyme-linked immunosorbent assay;
EPI: Expanded Program on Immunization; Gavi: Global Alliance for Vaccines
and Immunization; GMC: Geometric mean concentration; GMT: Geometric
mean titre; HEO: Health extension officer; Hib: Haemophilus influenzae type B;
HIV: Human Immunodeficiency Virus; ICH-GCP: International Conference on
Harmonisation Good Clinical Practice; IPD: Invasive pneumococcal disease;
NHMRC: National Health and Medical Research Council; NPS: Nasopharyngeal
swab; NTHi: Non-typeable Haemophilus influenzae; PBMC: Peripheral blood
mononuclear cells; PCV: Pneumococcal conjugate vaccine; PNG: Papua New
Guinea; PNGIMR: PNG Institute of Medical Research; PPV: Pneumococcal
polysaccharide vaccine; SAE: Serious adverse event; STGGB: Skim milk
tryptone-glucose-glycerol broth; URT: Upper respiratory tract; UWA: University
of Western Australia; WHO: Word Health Organization
This study was funded through the Papua New Guinea Institute of Medical
Research Internal Competitive Research Award Scheme (ICRAS); Papua New
Guinea Liquefied Natural Gas Project (a joint venture of Exxon Mobil
Corporation, Oil Search, Santos, Nippon Oil, Mineral Resource Development
Company and Eda Oil) and the Australian National Health and Medical
Research Council (NHMRC) (APP 1087200); the Telethon Kids Institute’s
Wesfarmers Centre of Vaccines and Infectious Diseases provided salary support
to DL and AVDB; LAK is supported by a NHMRC Career Development Fellowship
(APP 1061428); MP was supported by fellowships from the NHMRC
(GNT1072213) and the Cancer Institute NSW (13/ECF/1–11); Seqirus (formerly
bioCSL) donated Pneumovax for the study. None of the funding bodies were
involved in the design of the study and collection, analysis, and interpretation of
data and in writing the manuscript.
Availability of data and materials
DL, PR, and WP conceived and designed the study, acquired funding, and
were responsible for overall oversight of the study; VS provided clinical
supervision and was responsible for recruitment and follow up of study
participants; WK provided program management support and was involved
in participant recruitment and follow-up; GS, GM and BN conducted the
recruitment and follow-up of the study participants; WP, AVDB and PR were
responsible for design, oversight and interpretation of immunological
experiments; JF was responsible for the processing of immunological
samples and assay conduct in PNG; AG and RF were responsible for all
aspects of bacterial analysis in PNG; LAK is responsible for design, oversight and
interpretation of microbiological experiments in Australia; TO was responsible
for processing microbiological samples and conducting analysis in PNG; PJ is
responsible for statistical methods and analysis; MP contributed to design and
implementation of the study. WK wrote the first draft of the manuscript and DL
and AVDB wrote the final version; PR, LAK, and MP critically reviewed and
edited the final version. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Ethical approval was obtained from the PNG Medical Research Advisory
Committee (#11.03) and PNGIMR Institutional Review Board (#1028). The
study was conducted according to Declaration of Helsinki International
Conference on Harmonization Good Clinical Practice (ICH-GCP) and local
ethical guidelines. Assent was sought from pregnant women and their
husbands, and informed consent was obtained from the mother at the time
of enrolment of the child.
Consent for publication
DL has received support from Pfizer Australia to attend conferences, an
honorarium from Merck Vaccines to give a seminar at their offices in
Pennsylvania and support from Merck Vaccines to attend a conference; and
is an investigator on an investigator-initiated research grant that was funded
by Pfizer Australia. PR has received non-financial support from Pfizer, grants
from GlaxoSmithKline, grants from Pfizer, and non-financial support from
GlaxoSmithKline for work outside the submitted work. WP has received
funding from Pfizer Australia to attend a conference. AVDB was previously an
employee of Janssen Pharmaceuticals, Johnson and Johnson. LAK has received
educational grants and travel support from Pfizer and GSK to attend
conferences, is an investigator on an investigator-initiated research grant
funded by Pfizer Australia, and is an inventor on patents for a pneumococcal
protein vaccine antigen. All other authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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