Maternal antibodies to pneumolysin but not to pneumococcal surface protein A delay early pneumococcal carriage in high-risk Papua New Guinean infants.
ABSTRACT Immunization of pregnant women can be an efficient strategy to induce early protection in infants in developing countries. Pneumococcal protein-based vaccines may have the capacity to induce pneumococcal serotype-independent protection. To understand the potential of maternal pneumococcal protein-specific antibodies in infants in high-risk areas, we studied the placental transfer of naturally acquired antibodies to pneumolysin (Ply) and pneumococcal surface protein A family 1 and 2 (PspA1 and PspA2) in relation to onset of pneumococcal nasopharyngeal carriage in infants in Papua New Guinea (PNG). In this study, 76% of the infants carried Streptococcus pneumoniae in the upper respiratory tract within the first month of life, at a median age of 19 days. Maternal and cord blood antibody titers to Ply (rho = 0.824, P < 0.001), PspA1 (rho = 0.746, P < 0.001), and PspA2 (rho = 0.631, P < 0.001) were strongly correlated. Maternal pneumococcal carriage (hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.25 to 5.39) and younger maternal age (HR, 0.74; 95% CI, 0.54 to 1.00) were independent risk factors for early carriage, while higher cord Ply-specific antibody titers predicted a significantly delayed onset (HR, 0.71; 95% CI, 0.52 to 1.00) and cord PspA1-specific antibodies a significantly younger onset of carriage in PNG infants (HR, 1.57; 95% CI, 1.03 to 2.40). Maternal vaccination with a pneumococcal protein-based vaccine should be considered as a strategy to protect high-risk infants against pneumococcal disease by reducing carriage risks in both mothers and infants.
- SourceAvailable from: Geert Leroux-Roels[Show abstract] [Hide abstract]
ABSTRACT: New vaccines containing highly conserved Streptococcus pneumoniae proteins such as pneumolysin toxoid (dPly) and histidine-triad protein D (PhtD) are being developed to provide broader protection against pneumococcal disease. This study evaluated the safety, reactogenicity and immunogenicity of different pneumococcal protein-containing formulations in adults. In a phase I double-blind study (www.clinicaltrials.gov: NCT00707798), healthy adults (18-40 years) were randomized (1:2:2:2:2:2:2) to receive two doses of one of six investigational vaccine formulations 2 months apart, or a single dose of the control 23-valent pneumococcal polysaccharide vaccine (23PPV; Pneumovax23™, Sanofi Pasteur MSD) followed by placebo. The investigational formulations contained dPly alone (10 or 30μg), or both dPly and PhtD (10 or 30μg each) alone or combined with the polysaccharide conjugates of the 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV; Synflorix™, GlaxoSmithKline Vaccines). Two groups primed with a formulation containing dPly and PhtD (10 or 30μg each) continued to the follow-up phase II study (NCT00896064), in which they received a booster dose at 5-9 months after primary vaccination. Of 156 enrolled and vaccinated adults, 146 completed the primary immunization and 43 adults received a booster dose. During primary and booster vaccination, for any formulation, ≤8.9% of doses were followed by grade 3 solicited local or general adverse events. No fever >39.5°C (oral temperature) was reported. Unsolicited adverse events considered causally related to vaccination were reported following ≤33.3% of investigational vaccine doses. No serious adverse events were reported for adults receiving investigational vaccine formulations. Formulations containing dPly with or without PhtD were immunogenic for these antigens; polysaccharide conjugate-containing formulations were also immunogenic for those 10 polysaccharides. Investigational vaccine formulations containing dPly and PhtD were well tolerated and immunogenic when administered to healthy adults as standalone protein vaccine or combined with PHiD-CV conjugates.Vaccine 03/2014; · 3.77 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Influenza-like-illness can be caused by a wide range of respiratory viruses. The etiology of influenza-like-illness in developing countries such as Papua New Guinea is poorly understood. The etiological agents associated with influenza-like-illness were investigated retrospectively for 300 nasopharyngeal swabs received by the Papua New Guinea National Influenza Centre in 2010. Real-time PCR/RT-PCR methods were used for the detection of 13 respiratory viruses. Patients with influenza-like-illness were identified according to the World Health Organization case definition: sudden onset of fever (>38°C), with cough and/or sore throat, in the absence of other diagnoses. At least one viral respiratory pathogen was detected in 66.3% of the samples tested. Rhinoviruses (17.0%), influenza A (16.7%), and influenza B (12.7%) were the pathogens detected most frequently. Children <5 years of age presented with a significantly higher rate of at least one viral pathogen and a significantly higher rate of co-infections with multiple viruses, when compared to all other patients >5 years of age. Influenza B, adenovirus, and respiratory syncytial virus were all detected at significantly higher rates in children <5 years of age. This study confirmed that multiple respiratory viruses are circulating and contributing to the presentation of influenza-like-illness in Papua New Guinea. J. Med. Virol. © 2013 Wiley Periodicals, Inc.Journal of Medical Virology 10/2013; · 2.37 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Assessment of antibody responses to pneumococcal colonisation in early childhood may aid our understanding of protection and inform vaccine antigen selection. Serum samples were collected from mother-infant pairs during a longitudinal pneumococcal colonisation study in Burmese refugees. Maternal and cord sera were collected at birth and infants were bled monthly (1-24 months of age). Nasopharyngeal swabs were taken monthly to detect colonisation. Serum IgG titres to 27 pneumococcal protein antigens were measured in 2,624 sera and IgG to dominant serotypes (6B,14,19F,19A,23F) were quantified in 864 infant sera. Antibodies to all protein antigens were detectable in maternal sera. Titres to four proteins (LytB,PcpA,PhtD,PhtE) were significantly higher in mothers colonised by pneumococci at delivery. Maternally-derived antibodies to PiuA and Spr0096 were associated with delayed pneumococcal acquisition in infants in univariate, but not multivariate models. Controlling for infant age and previous homologous serotype exposure, nasopharyngeal acquisition of serotypes 19A, 23F, 14, or 19F were associated significantly with a ≥2-fold antibody response to the homologous capsule (OR 12.84, 7.52, 6.52, 5.33; p<0.05). Acquisition of pneumococcal serotypes in the nasopharynx of infants was not significantly associated with a ≥2-fold rise in antibodies to any of the protein antigens studied. In conclusion, nasopharyngeal colonisation in young children resulted in demonstrable serum IgG responses to pneumococcal capsules and surface/virulence proteins. However, the relationship between serum IgG and the prevention of, or response to, pneumococcal nasopharyngeal colonisation remains complex. Mechanisms other than serum IgG are likely to have a role but are currently poorly understood. This article is protected by copyright. All rights reserved.Clinical Microbiology and Infection 05/2013; · 4.58 Impact Factor
CLINICAL AND VACCINE IMMUNOLOGY, Nov. 2009, p. 1633–1638
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 16, No. 11
Maternal Antibodies to Pneumolysin but Not to Pneumococcal Surface
Protein A Delay Early Pneumococcal Carriage in High-Risk
Papua New Guinean Infants?
Jacinta P. Francis,1,2,3Peter C. Richmond,2William S. Pomat,1Audrey Michael,1Helen Keno,1
Suparat Phuanukoonnon,1Jan B. Nelson,2Melissa Whinnen,2Tatjana Heinrich,3
Wendy-Anne Smith,3Susan L. Prescott,2Patrick G. Holt,3Peter M. Siba,1
Deborah Lehmann,3and Anita H. J. van den Biggelaar3*
Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea1; School of Paediatrics and Child Health, University of
Western Australia, Perth, Australia2; and Telethon Institute for Child Health Research, Centre for Child Health Research,
University of Western Australia, Perth, Australia3
Received 29 May 2009/Returned for modification 1 September 2009/Accepted 14 September 2009
Immunization of pregnant women can be an efficient strategy to induce early protection in infants in
developing countries. Pneumococcal protein-based vaccines may have the capacity to induce pneumococcal
serotype-independent protection. To understand the potential of maternal pneumococcal protein-specific
antibodies in infants in high-risk areas, we studied the placental transfer of naturally acquired antibodies to
pneumolysin (Ply) and pneumococcal surface protein A family 1 and 2 (PspA1 and PspA2) in relation to onset
of pneumococcal nasopharyngeal carriage in infants in Papua New Guinea (PNG). In this study, 76% of the
infants carried Streptococcus pneumoniae in the upper respiratory tract within the first month of life, at a
median age of 19 days. Maternal and cord blood antibody titers to Ply (? ? 0.824, P < 0.001), PspA1 (? ? 0.746,
P < 0.001), and PspA2 (? ? 0.631, P < 0.001) were strongly correlated. Maternal pneumococcal carriage
(hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.25 to 5.39) and younger maternal age (HR, 0.74; 95%
CI, 0.54 to 1.00) were independent risk factors for early carriage, while higher cord Ply-specific antibody titers
predicted a significantly delayed onset (HR, 0.71; 95% CI, 0.52 to 1.00) and cord PspA1-specific antibodies a
significantly younger onset of carriage in PNG infants (HR, 1.57; 95% CI, 1.03 to 2.40). Maternal vaccination
with a pneumococcal protein-based vaccine should be considered as a strategy to protect high-risk infants
against pneumococcal disease by reducing carriage risks in both mothers and infants.
Every year approximately 1 million children under 5 years
of age die of pneumococcal pneumonia, meningitis, or sepsis,
mostly in developing countries (4). Despite the efficacy of
pneumococcal conjugate vaccines, Streptococcus pneumoniae
remains an important cause of serious morbidity and mortality
in young infants in developing countries (5, 8, 44), where the
age of onset of disease is often younger than the recommended
vaccination age of 6 weeks old and many of the serotypes
causing serious disease are not included in currently available
conjugate vaccines. Alternative vaccines and vaccine strategies
are therefore needed to induce the earliest protection possible
in high-risk infants.
Early onset of pneumococcal colonization and prolonged
carriage in the upper respiratory tract are believed to play
important roles in the high incidence and early onset of pneu-
mococcal diseases in children in developing countries (10, 25).
In the highlands of Papua New Guinea (PNG), where this
study was performed, all infants carry pneumococci in the
upper respiratory tract by the age of 3 months old, and 60% of
them are already carriers during the neonatal period at a
median age of 17 days old (14). This is in contrast to high-
income countries, where less than half of the children experi-
ence pneumococcal colonization within the first year of life (3,
10, 43). Besides children, pneumococcal carriage rates remain
higher in adulthood in developing countries, including PNG,
where approximately half of the adults carry pneumococci in
the upper respiratory tract (17, 39), compared to 1 to 13% of
adults in low-risk countries (12, 15, 20). Consequently, mater-
nal pneumococcal carriage may be an important risk factor for
early colonization in infants in high-risk areas, in particular
considering the frequent and close contact between mother
and child in the critical early period of life when infants are
In the first few months of life, when the human immune
system is still highly immature (27), infants largely depend on
passively acquired maternal immunoglobulin G (IgG) antibod-
ies to protect themselves against invading pathogens. Immuni-
zation of pregnant women is a strategy that has been proven to
reduce infection risks in both mothers and infants (9, 13, 45).
This includes the potential to reduce acute lower respiratory
illnesses in infants in high-risk areas, as shown with maternal
immunization with the 23-valent pneumococcal polysaccharide
vaccine (36, 37). However, the efficacy of pneumococcal poly-
saccharide vaccines on reducing nasopharyngeal colonization
is limited, whereas the protective effect of pneumococcal con-
jugate vaccines is restricted by the number of pneumococcal
serotypes that can be included. On the other hand, novel vac-
cines based on conserved pneumococcal proteins may offer
* Corresponding author. Mailing address: Telethon Institute for
Child Health Research, P.O. Box 855, West Perth, WA 6872, Austra-
lia. Phone: 61 8 9489 7919. Fax: 61 8 9489 7700. E-mail: Anitav@ichr
?Published ahead of print on 23 September 2009.
better, serotype-independent protection against pneumococcal
carriage and disease. This may include maternal immunization
strategies, as supported by findings in mice (21).
Pneumolysin (Ply) and pneumococcal surface protein A
(PspA) are two conserved proteins that are expressed by vir-
tually all S. pneumoniae isolates and that are being considered
as vaccine candidates. Pneumolysin is the thiol-activated cyto-
lysin produced by S. pneumoniae that enables the bacterium to
penetrate the host’s physical defenses through its cytotoxic
effect on epithelial cells, thus facilitating carriage and disease
(28). PspA is a cell wall-associated protein that plays a role in
inhibiting complement-mediated opsonization (7, 33) and can
prevent lactoferrin-mediated clearance (19). In contrast to Ply,
PspA shows structural diversity between pneumococcal strains
and has been classified into three families based on the se-
quence variability of the most C-terminal 100 amino acids of
the N-terminal domain of PspA. Although S. pneumoniae
strains expressing family 1 or 2 PspA proteins account for 98%
of clinical isolates, protective PspA-specific IgG antibodies
binding to this highly variable region are family dependent (7).
Both Ply and PspA have been shown to be highly immuno-
genic and to protect mice against disease and colonization
following pneumococcal challenge (2, 6, 7, 11, 34). There is
evidence that in humans naturally acquired IgA and IgG an-
tibodies to PspA and Ply can mediate protection against sub-
sequent pneumococcal carriage and disease (22, 24, 30, 31, 38,
46). Moreover, naturally acquired antibodies to Ply and PspA
have been shown to be transferred from mother to child and to
protect against early pneumococcal carriage and infection, at
least in populations in low-risk areas (18, 38). It is not known
whether these findings hold true for areas of high endemicity,
where infants are at a considerably higher risk for early car-
riage and disease.
In order to understand the role of maternal antibodies to Ply
and PspA in protecting high-risk infants against early carriage,
we studied antibody titers in paired maternal and cord blood
samples in relation to the infant’s age of first pneumococcal
nasopharyngeal carriage. We hypothesized that, compared to
lower-risk settings, maternal Ply- and PspA-specific antibody
titers would be higher and would be associated with a delay in
the age of first pneumococcal carriage in the offspring.
MATERIALS AND METHODS
Study population. Study mothers and newborns were participants in a neona-
tal pneumococcal conjugate vaccination trial performed in the Asaro Valley in
the Eastern Highlands Province of PNG (42). Pregnant women were recruited in
villages located within an hour’s drive from Goroka town, the provincial capital,
or the antenatal clinic of Goroka General Hospital, the only tertiary hospital in
the province. Inclusion criteria for newborns were the intention of the family to
remain in the study area for at least 2 years, a birth weight of at least 2,000 g, no
acute neonatal infection, and no severe congenital abnormality. While there was
no routine human immunodeficiency virus testing, antenatal testing is recom-
mended and children of mothers known to be human immunodeficiency virus
positive were excluded.
After birth, umbilical cord blood samples (25 to 50 ml) were collected in sterile
tubes containing an equal volume of RPMI 1640 (Invitrogen-Life Technologies,
Melbourne, Australia) and preservative-free heparin (20 IU/ml). The total vol-
ume of cord blood collected was recorded to correct for the plasma dilution
factor. Venous blood samples (10 ml) were collected from mothers 1 month
postpartum in 100 IU/ml preservative-free heparin. Since cord blood collections
were started only later in the trial, paired maternal and cord blood plasma
samples were available for only 89 of the 313 newborns enrolled in the vaccina-
tion study. Pernasal swabs (PNS) were collected from the infants at 1, 2, 3, and
4 weeks of age and from the mothers at the time of delivery.
Apart from two sets of twins that were born by caesarean section, all 89 babies
(58% boys) were born by natural delivery at an overall mean gestation age of 39.5
weeks (standard deviation [SD], 1.4) with a mean birth weight of 3,300 g (SD,
540). Thirty-eight percent of the pregnancies were primigravidae. At the time of
delivery, the 87 PNG study mothers had a mean age of 26 years (SD, 6). As part
of the vaccination trial, one-third of the newborns had been randomized to
receive one dose of a 7-valent pneumococcal conjugate vaccine (Prevnar; Wyeth)
at birth (34 of the 89 newborns with paired maternal samples). All study children
received bacillus Calmette-Gue ´rin vaccine, a dose of oral polio vaccine, and a
dose of hepatitis B vaccine at birth.
As a maternal control group from a low-risk area, serum samples collected 1
month postpartum from 50 women participating in a neonatal allergy study in
Perth, Western Australia, were included.
In PNG, assent was sought from women and their partners at the time of
recruitment, and written informed consent was obtained shortly after delivery. In
Australia, written informed consent was obtained from the pregnant women at
the time of recruitment. Ethical approval for this study was obtained from the
PNG Medical Research Advisory Committee and the Princess Margaret Hospi-
tal Ethics Committee in Perth, Australia.
Pneumococcal protein antigens. The pneumolysin toxoid used in this study
(PdB) was genetically engineered from a serotype 2 pneumococcus and kindly
provided by James Paton (School of Molecular and Biomedical Science, The
University of Adelaide, Adelaide, Australia) (2). The toxoid, which carries a
Trp-433 3Phe substitution, was purified from Escherichia coli JM109(pJCP202)
and stored in 50% glycerol.
PspA1 (family 1, clade 2) was derived from the recombinant PspA/
Rx1AA1.0.302protein, which comprised 302 N-terminal amino acids of the pneu-
mococcal strain Rx1 PspA, and PspA2 (family 2, clade 3) from PspA/
V-24AA1.0.410, consisting of amino acids 1 to 410 of the wild-type mature PspA of
S. pneumoniae Taiwan19F-14. Both recombinant PspA expression constructs
were transformed into competent E. coli BL21 Star(DE3)pLysS cells in the
presence of 100 ?g/ml ampicillin and 34 ?g/ml chloramphenicol (Invitrogen
Corp., Carlsbad, CA). Recombinant protein was expressed with a C-terminal
hexahistidine fusion tag, and soluble recombinant proteins were purified using
Ni2?-nitrilotriacetic acid agarose chromatography in the presence of 600 mM
NaCl (Qiagen GmbH, Germany). Fractions containing the relevant proteins
were pooled, dialyzed into 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM
EDTA, and applied to a Bio-Rad Macro-prep High Q anion exchange support
(Bio-Rad, Hercules, CA). Elution was achieved with a linear gradient of 100 to
500 mM NaCl in Tris-HCl, pH 7.4, 2 mM EDTA. Fractions containing the
relevant protein were pooled and further purified using size exclusion chroma-
tography by applying the samples to a HiPrep HR S200 26/60 column. A single
peak was obtained for each of the PspA proteins. Finally, the proteins were
sterilized and endotoxin removed using 0.2-?m Mustang E filters (Pall Life
Sciences, Portsmouth, United Kingdom). The purities of both PspA1 (?40 kDa)
and PspAs (?55 kDa) were checked on a 12.5% sodium dodecyl sulfate-poly-
acrylamide gel by the method of Laemmli (23), and the concentrations were
determined using the optical density at 280 nm (OD280) measurements and
extinction coefficients (PspA1 ε, 10,430; PspA2 ε, 13,410).
Pneumococcal protein enzyme-linked immunosorbent assay (ELISA). Plasma
was separated from umbilical cord blood samples within 18 h of collection by
spinning for 30 min at 400 ? g, while maternal venous blood samples were spun
for 10 min at 700 ? g within 2 h of collection. Samples were stored at ?20°C until
shipment of aliquots (1 to 1.5 ml) to Perth for further analysis.
Microtiter ELISA plates (96-well flat-bottomed Polysorp; Nunc, Denmark)
were coated with Ply (1.25 ?g/ml), PspA1 (2.5 ?g/ml), or PspA2 (2.5 ?g/ml) in
coating buffer (sodium carbonate buffer, pH 9.6) and incubated overnight at 4°C
for Ply and at 37°C in 5% CO2for PspA. Plates were washed four times with
phosphate-buffered saline (PBS)–0.05% Tween before blocking with PBS–0.05%
Tween plus 5% skim milk powder at 37°C, 5% CO2for 1 h to prevent nonspecific
binding. In-house reference and quality control (high and low) sera standards
(obtained from healthy laboratory volunteers) and test samples (maternal and
cord blood plasma) were diluted in assay buffer solution (1? PBS–0.05% Tween
plus 5% skim milk powder) at predetermined starting dilutions (for cord blood,
1/50; PNG maternal, 1/400; Australian maternal, 1/25) and serially diluted (two-
fold) before being added to the plates. After discarding the block buffer, in-house
reference sera, high and low quality control and test sera were added and
incubated for 2 h at room temperature on a plate shaker. After four washes, an
alkaline phosphatase-conjugated goat anti-human IgG diluted 1/2,000 (Bio-
source) was added and plates were incubated for 1.5 h at room temperature on
a plate shaker before washing five times (three times with wash buffer and two
1634 FRANCIS ET AL.CLIN. VACCINE IMMUNOL.
times with distilled water) and incubating with p-nitrophenyl phosphate (tablets;
Sigma) substrate buffer for 1.5 h at 37°C and 5% CO2. Substrate color develop-
ment was stopped by adding a solution of 3 M sodium hydroxide. Absorbance
levels were measured as ODs by using an automated microtiter plate reader
(Sunrise; Tecan Austria GmbH, Austria) at a reading wavelength of 405 nm and
reference wavelength of 620 nm. The OD readings were then converted into
arbitrary ELISA units (AEU).
Bacteriology. PNS samples were stored in 1 ml of skim milk-glucose-glycerine
broth at ?70°C until further processing at the Papua New Guinea Institute for
Medical Research to determine pneumococcal carriage, using standard bacteri-
ological culturing, isolation, and pneumococcal identification methods (25, 32).
In summary, after thawing and vortexing, 10 ?l of the PNS sample suspension
(primary inocula) was transferred onto culture plates containing either plain
horse blood agar, chocolate agar, gentamicin (5 ?g/ml) blood agar, or bacitracin
(300 ?g/ml) chocolate agar (Oxoid, Australia), using sterile disposable inoculat-
ing loops (3 mm), and incubated at 36°C in 5% CO2for 24 h and a further 24 h
if extra bacterial growth was required. Presumptive pneumococcal colonies were
then cultured with an optochin disc and confirmed to be S. pneumoniae based on
Statistical analyses. All statistical analyses were performed using the statistical
package SPSS 15.0 (SPSS Inc.). IgG antibody levels were log transformed into
geometric mean titers (GMT). Differences between groups were tested using the
nonparametric Mann-Whitney U test, and correlations were studied using the
Spearman’s rank correlation method. Logistic regression was used to analyze
associations between antibody responses and risk for infant carriage within the
first month of life, whereas the Cox regression was used to study associations
between antibody titers and age of first pneumococcal carriage of the infants
within the first month of life. In the regression models, antibody responses were
studied as Z-scores, which were calculated based on the following equation: (log
antibody titer ? mean log antibody titer)/standard deviation of log antibody titer.
Where indicated, regression models were adjusted for confounding by maternal
pneumococcal carriage, maternal age, and pneumococcal conjugate vaccination.
An association was considered to be significant at a P level of ?0.05.
Pneumococcal carriage in PNG mothers and infants. At the
time of delivery, 30% of the PNG study mothers carried pneu-
mococci in the upper respiratory tract, with higher carriage
rates being observed in younger compared to older mothers
(Fig. 1). Of the newborns that had completed all four weekly
visits, 76% (55/72) carried S. pneumoniae at least once during
the first month of life, with a median age of onset of 19 days
(interquartile range, 12 to 28). Maternal pneumococcal car-
riage at the time of delivery (hazard ratio [HR], 1.97; 95%
confidence interval [CI], 1.01 to 3.85; P ? 0.046) (Fig. 2) and
younger maternal age (HR, 0.76; 95% CI, 0.57 to 1.01, for
every 5 years of age; P ? 0.062) were found to be independent
risk factors for earlier onset of pneumococcal carriage in in-
Antibody responses to Ply and PspA in mothers and new-
borns. Compared to plasma samples from Australian (AUS)
mothers, geometric mean IgG antibody titers to Ply, PspA1,
and PspA2 were significantly higher in plasma samples of PNG
mothers (Fig. 3). Antibody titers in paired PNG mothers and
newborn cord blood samples were strongly correlated for all
three studied pneumococcal proteins (Spearman correlation
[?] for Ply ? 0.824, P ? 0.001; for PspA1, ? ? 0.746, P ? 0.001;
for PspA2, ? ? 0.631, P ? 0.001), but cord antibody titers to
Ply were on average 7.2-fold (median) lower compared to titers
in paired maternal samples, whereas PspA1 and Pspa2 titers
were, respectively, 1.8-fold and 1.7-fold lower (Fig. 3). IgG
antibody titers specific for PspA1 and PspA2 were strongly
correlated in sera of PNG mothers (? ? 0.652, P ? 0.001),
PNG newborns (? ? 0.677, P ? 0.001), and AUS mothers (? ?
0.620, P ? 0.001), whereas such a correlation was not found
between antibody titers to PspA1 and Ply (PNG mothers, ? ?
0.261, P ? 0.015; PNG newborns, ? ? 0.178, P ? 0.096; AUS
mothers, ? ? 0.160, P ? 0.266), or PspA2 and Ply (PNG
FIG. 1. Pneumococcal nasopharyngeal carriage in PNG mothers
according to their age.
FIG. 2. Maternal pneumococcal carriage as a predictor of age of
first pneumococcal carriage in PNG infants. The solid line represents
the group of PNG infants whose mothers carried pneumococci at the
time of delivery (n ? 20), and the broken line represents infants whose
mothers were noncarriers (n ? 47).
FIG. 3. Antibody titers to pneumococcal proteins in PNG mothers
and newborns and AUS mothers. Data shown are GMTs (and 95%
confidence intervals) of antibodies to Ply, PspA1, and PspA2 in plasma
samples of Australian mothers (light gray bars; n ? 50) and of Papua
New Guinean mothers (white bars; n ? 87) and their newborns (dark
gray bars; n ? 89), with*indicating significant differences (P ? 0.05)
VOL. 16, 2009 Ply AND PspA MATERNAL ANTIBODIES IN HIGH-RISK INFANTS1635
mothers, ? ? 0.137, P ? 0.207; PNG newborns, ? ? 0.142, P ?
0.184; AUS mothers, ? ? 0.193, P ? 0.178).
Antibody titers to Ply decreased significantly with increasing
age of PNG mothers in maternal sera (linear regression coef-
ficient [?], ?0.30 standard deviation (SD)/5 years of age; 95%
CI, ?0.48 to ?0.12; P ? 0.002) as well as in cord blood samples
(?, ?0.23 SD/5 years of age; 95% CI, ?0.43 to ?0.05; P ?
0.015). In contrast, antibody titers to Ply increased with age in
sera of AUS mothers (?, 0.34 SD/5 years of age; 95% CI,
?0.14 to 0.69; P ? 0.060). No age-related changes were found
for antibodies to PspA1 and PspA2 in either PNG or AUS
mothers (data not shown). No associations were found be-
tween pneumococcal carriage in PNG mothers at the time of
delivery and antibody titers to Ply, PspA1, or PspA2 (Table 1).
Protective effects of maternally derived Ply- and PspA-spe-
cific antibodies on early carriage. Cord IgG antibody titers to
Ply tended to be lower in children that carried pneumococci
within the first 2 weeks of life than in those that carried for the
first time between 2 and 4 weeks of age or that did not carry
within the first month, but this association was not significant
(P ? 0.321) (Fig. 4). In contrast, antibody titers for PspA1 or
PspA2 tended to be the lowest in children that remained free
of pneumococcal carriage within the first month of life (PspA1,
P ? 0.166; PspA2, P ? 0.677). To study associations in relation
to age of first pneumococcal carriage, cord and maternal an-
tibody titers to Ply, PspA1, and PspA2 were studied in three
different Cox regression models (Table 2). No significant as-
sociations were found when antibody responses were studied
in univariate models (model I) or together in a multivariate
Cox regression model (model II). However, IgG antibody ti-
ters to Ply were associated with a significantly delayed onset of
first pneumococcal carriage and higher antibody titers to
PspA1 with a significantly earlier onset of carriage in a multi-
variate regression model (model III) adjusting for maternal
age (HRadjusted, 0.74; 95% CI, 0.54 to 1.00 for every 5 years of
age; P ? 0.048) and maternal pneumococcal carriage at the
time of delivery (HRadjusted, 2.60; 95% CI, 1.25 to 5.39; P ?
0.010). Findings were similar whether cord blood or maternal
antibody titers to Ply and PspA1 were studied. Associations
remained unchanged when models were coadjusted for 7-va-
lent pneumococcal conjugate vaccine immunization at birth
(data not shown).
IgG antibodies to Ply that are transferred from mother to child
were associated with delaying the age of first pneumococcal car-
riage in high-risk infants in PNG. This is in line with earlier
findings for infants living in a relatively lower-risk area in the
Philippines (18). Maternal antibodies to PspA family 1 did not
protect, but in fact were associated with a significantly younger
age of first carriage in the PNG infants. This is in contrast with an
experimental human adult carriage study that reported a protec-
in line with a study in the Philippines that found no association
between maternally derived PspA1-specific antibodies and pro-
tection against early carriage in infants (18). No associations were
found between maternal antibodies to PspA family 2 and risk for
early carriage in PNG infants. To our knowledge no other studies
in humans have previously reported a possible protective effect of
In contrast to Ply, which is homogeneously expressed by
virtually all pneumococcal strains, PspA has been classified
into three families, with limited cross-protection between an-
tibodies recognizing different PspA families (7). Low-affinity
binding of antibodies from different PspA families may, how-
ever, hinder recognition and clearance by specific antibodies.
This could explain the positive association between PspA an-
tibodies and risk for early carriage observed in the PNG in-
fants. To test this hypothesis and confirm a potential cross-
inhibiting effect of PspA family-specific antibodies in young
infants, functional antibody assays will have to be developed.
In addition, more information will be needed regarding the
geographical distribution and possible preferential nasopha-
ryngeal colonization of young infants compared to adults by
FIG. 4. Cord antibody titers to Ply, PspA1, and PspA2 in relation
to early pneumococcal carriage. Data shown are GMTs (and 95%
confidence intervals) of antibodies to Ply, PspA1, and PspA2 in cord
samples of PNG newborns that carried pneumococci within the first
2 weeks of life (white bars; n ? 27), within 2 to 4 weeks of life (gray
bars; n ? 28), or did not carry within the first 4 weeks of life (black
bars; n ? 17).
TABLE 1. IgG antibody titers in maternal and cord samples in relation to maternal pneumococcal carriage
Geometric mean IgG titera(95% CI)
Maternal venous blood from mother who was: Cord blood from mother who was:
NoncarrierCarrierP valueNoncarrier CarrierP value
aValues are geometric mean concentrations (and 95% confidence intervals) of IgG antibodies to Ply, PspA1, and Pspa2 for venous blood samples of PNG mothers
1 month postpartum (n ? 87) and paired cord blood samples (n ? 89) in relation to nasopharyngeal pneumococcal carriage of the mothers at the time of delivery.
1636FRANCIS ET AL.CLIN. VACCINE IMMUNOL.
different PspA families to understand why findings can vary for
different study populations.
In contrast to the Philippines study, we found that antibody
titers were not equivalent in mother-newborn pairs (18) but
were sevenfold and twofold lower in PNG newborns compared
to their mothers for Ply-specific and PspA-specific antibodies,
respectively. A difference between the two studies is that in the
Philippines study, mothers were bled during the second to
third trimester of pregnancy, when IgG antibodies are known
to be decreased (1), whereas in our study maternal blood
samples were collected 1 month postpartum, when titers have
already started to increase. Alternatively, a reduced transfer
efficiency of higher maternal IgG titers due to competition for
receptor binding (16) may explain the lower antibody titers in
PNG newborns compared to their mothers, although this does
not offer an explanation for the relative lower transfer of Ply-
specific compared with PspA-specific antibodies. Nevertheless,
maternal and cord blood antibody titers gave very similar results
in relation to early carriage risk, which indicates that the transfer
of maternal antibodies was optimal in our study children. More-
over, this observation implies that maternal blood samples, even
when collected 1 month postpartum, can be used as a surrogate
for cord blood antibodies when cord samples are not available.
Maternal pneumococcal carriage and younger maternal age
at the time of delivery were independent risk factors for early
onset of pneumococcal carriage in PNG infants. It is not un-
expected that mothers, in addition to siblings and other family
members (34, 39), play an important role in transmission of S.
pneumoniae in a high-risk area such as PNG, considering the
high rates of pneumococcal carriage reported for adults com-
pared with that of adults in low-risk areas (12, 15, 17, 20, 39).
Serotyping of pneumococci isolated from nasopharyngeal
swabs collected from mother-child pairs could be applied to
further confirm the role of mother-to-child transmission, but
due to lack of power this was not feasible for the current study.
We suggest that behavioral changes may explain the protective
role of older maternal age on early pneumococcal carriage, but
as yet we have no insight into what these changes may be.
Importantly, our observation reconfirms that prevention of
maternal pneumococcal carriage through maternal immuniza-
tion and/or reducing transmission risks through introducing
measures such as improved hand washing practices (35, 40) will
contribute to reducing early carriage risks in infants in high-
risk areas such as in PNG.
In addition to antibodies transferred in utero, antibodies
transmitted through breast milk can mediate protection
against acute lower respiratory infections in young infants (26).
Although breastfeeding is practiced by most Papua New
Guinea women (41), this was not recorded on an individual
basis for the mother-child pairs in our study. We acknowledge
that the potential protective effect of breastfeeding is a rele-
vant issue to take into account in future studies, as will be
studying correlations between Ply and PspA antibody titers in
breast milk compared to serum.
Finally, it is important to recognize that the protective effect
of maternal Ply-specific antibodies was limited, since young
infants in the highlands of PNG remain at high risk of early
pneumococcal carriage. Comparable to findings from a study
in the same area more than 20 years ago (14), nearly 80% of
the study infants were found to carry S. pneumoniae in the
upper respiratory tract within the first month of life, at a
median age of 19 days. It remains to be shown that new vac-
cines such as pneumococcal protein-based vaccines can over-
come early pneumococcal carriage and disease in young infants
in developing countries, but vaccination strategies involving
maternal immunization to enhance passive immunity and po-
tentially reduce maternal pneumococcal carriage, in combina-
tion with neonatal vaccination to induce early memory T-cell
responses (42), should be considered.
This work was supported by International Collaborative Research
Grant Scheme, 071613/Z/03/Z, from the Wellcome Trust and Austra-
lian National Health and Medical Research Council (NHMRC). D.
Lehmann is supported by an NHMRC program grant (353514), and A.
van den Biggelaar is supported by an NHMRC R. Douglas Wright
Biomedical Career Development grant (458780).
We thank J. Paton for providing pneumolysin antigen, the parents
and guardians of the study children for their participation and ongoing
support, and all staff of the Papua New Guinea Neonatal Pneumococ-
cal Conjugate Vaccine Trial Team (in particular, G. Saleu, C. Opa, T.
Orami, P. Namuigi, A. Javati, A. Sie, B. Nivio, J. Totave, R. Sehuko, L.
Pui, N. Fufu, M. Dreyum, G. Inapero, and J. Reeder) and village
reporters in the Asaro Valley for their contributions to this work.
TABLE 2. Cox regression for risk of early pneumococcal carriage in relation to cord and maternal IgG antibody titera
Model IModel II Model III
HR (95% CI)P value HR (95% CI)P valueHR (95% CI)P value
aCox regression analysis was used to study associations between age at onset of pneumococcal carriage and Z-scores of cord and maternal antibody titers to Ply,
PspA1, and PspA2, using a univariate model (model I), a multivariate model (model II), and a multivariate model adjusting for maternal pneumococcal carriage and
maternal age at the time of delivery in cord blood and maternal venous blood.
VOL. 16, 2009Ply AND PspA MATERNAL ANTIBODIES IN HIGH-RISK INFANTS1637
1. Ailus, K. T. 1994. A follow-up study of immunoglobulin levels and autoan-
tibodies in an unselected pregnant population. Am. J. Reprod. Immunol.
2. Alexander, J. E., R. A. Lock, C. C. Peeters, J. T. Poolman, P. W. Andrew, T. J.
Mitchell, D. Hansman, and J. C. Paton. 1994. Immunization of mice with
pneumolysin toxoid confers a significant degree of protection against at least
nine serotypes of Streptococcus pneumoniae. Infect. Immun. 62:5683–5688.
3. Aniansson, G., B. Alm, B. Andersson, P. Larsson, O. Nylen, H. Peterson, P.
Rigner, M. Svanborg, and C. Svanborg. 1992. Nasopharyngeal colonization
during the first year of life. J. Infect. Dis. 165(Suppl. 1):S38–S42.
4. Anonymous. 2003. The world’s forgotten children. Lancet 361:1.
5. Black, R. E., S. S. Morris, and J. Bryce. 2003. Where and why are 10 million
children dying every year? Lancet 361:2226–2234.
6. Campos, I. B., M. Darrieux, D. M. Ferreira, E. N. Miyaji, D. A. Silva, A. P.
Areas, K. A. Aires, L. C. Leite, P. L. Ho, and M. L. Oliveira. 2008. Nasal
immunization of mice with Lactobacillus casei expressing the pneumococcal
surface protein A: induction of antibodies, complement deposition and par-
tial protection against Streptococcus pneumoniae challenge. Microbes In-
7. Darrieux, M., E. N. Miyaji, D. M. Ferreira, L. M. Lopes, A. P. Lopes, B. Ren,
D. E. Briles, S. K. Hollingshead, and L. C. Leite. 2007. Fusion proteins
containing family 1 and family 2 PspA fragments elicit protection against
Streptococcus pneumoniae that correlates with antibody-mediated enhance-
ment of complement deposition. Infect. Immun. 75:5930–5938.
8. Duke, T. 2005. Neonatal pneumonia in developing countries. Arch. Dis.
Child. Fetal Neonatal 90:F211–F219.
9. Englund, J. A. 2007. The influence of maternal immunization on infant
immune responses. J. Comp. Pathol. 137(Suppl. 1):S16–S19.
10. Faden, H., L. Duffy, R. Wasielewski, J. Wolf, D. Krystofik, Y. Tung, et al.
1997. Relationship between nasopharyngeal colonization and the develop-
ment of otitis media in children. J. Infect. Dis. 175:1440–1445.
11. Ferreira, D. M., M. Darrieux, D. A. Silva, L. C. Leite, J. M. Ferreira, Jr.,
P. L. Ho, E. N. Miyaji, and M. L. Oliveira. 2009. Characterization of pro-
tective mucosal and systemic immune responses elicited by pneumococcal
surface proteins PspA and PspC nasal vaccines against a respiratory pneu-
mococcal challenge in mice. Clin. Vaccine Immunol. 16:636–645.
12. Givon-Lavi, N., R. Dagan, D. Fraser, P. Yagupsky, and N. Porat. 1999. Marked
differences in pneumococcal carriage and resistance patterns between day care
centers located within a small area. Clin. Infect. Dis. 29:1274–1280.
13. Glezen, W. P., and M. Alpers. 1999. Maternal immunization. Clin. Infect.
14. Gratten, M., H. Gratten, A. Poli, E. Carrad, M. Raymer, and G. Koki. 1986.
Colonisation of Haemophilus influenzae and Streptococcus pneumoniae in
the upper respiratory tract of neonates in Papua New Guinea: primary
acquisition, duration of carriage, and relationship to carriage in mothers.
Biol. Neonate 50:114–120.
15. Gunnarsson, R. K., S. E. Holm, and M. Soderstrom. 1998. The prevalence of
potential pathogenic bacteria in nasopharyngeal samples from healthy chil-
dren and adults. Scand. J. Prim. Health Care 16:13–17.
16. Hartter, H. K., O. I. Oyedele, K. Dietz, S. Kreis, J. P. Hoffman, and C. P.
Muller. 2000. Placental transfer and decay of maternally acquired anti-
measles antibodies in Nigerian children. Pediatr. Infect. Dis. J. 19:635–641.
17. Hill, P. C., A. Akisanya, K. Sankareh, Y. B. Cheung, M. Saaka, G. Lahai, B. M.
Greenwood, and R. A. Adegbola. 2006. Nasopharyngeal carriage of Streptococ-
cus pneumoniae in Gambian villagers. Clin. Infect. Dis. 43:673–679.
18. Holmlund, E., B. Quiambao, J. Ollgren, H. Nohynek, and H. Kayhty. 2006.
Development of natural antibodies to pneumococcal surface protein A,
pneumococcal surface adhesin A and pneumolysin in Filipino pregnant
women and their infants in relation to pneumococcal carriage. Vaccine
19. Jedrzejas, M. J. 2007. Unveiling molecular mechanisms of bacterial surface
proteins: Streptococcus pneumoniae as a model organism for structural
studies. Cell. Mol. Life Sci. 64:2799–2822.
20. Jousimies-Somer, H. R., S. Savolainen, and J. S. Ylikoski. 1989. Comparison
of the nasal bacterial floras in two groups of healthy subjects and in patients
with acute maxillary sinusitis. J. Clin. Microbiol. 27:2736–2743.
21. Katsurahara, T., M. Hotomi, K. Yamauchi, D. S. Billal, and N. Yamanaka.
2008. Protection against systemic fatal pneumococcal infection by maternal
intranasal immunization with pneumococcal surface protein A (PspA). J. In-
fect. Chemother. 14:393–398.
22. Kohler, C., A. A. Adegnika, R. Van der Linden, S. T. Agnandji, S. K. Chai,
A. J. Luty, Z. Szepfalusi, P. G. Kremsner, and M. Yazdanbakhsh. 2008.
Comparison of immunological status of African and European cord blood
mononuclear cells. Pediatr. Res. 64:631–636.
23. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
24. Laine, C., T. Mwangi, C. M. Thompson, J. Obiero, M. Lipsitch, and J. A. Scott.
2004. Age-specific immunoglobulin g (IgG) and IgA to pneumococcal protein
antigens in a population in coastal Kenya. Infect. Immun. 72:3331–3335.
25. Leach, A. J., J. B. Boswell, V. Asche, T. G. Nienhuys, and J. D. Mathews.
1994. Bacterial colonization of the nasopharynx predicts very early onset and
persistence of otitis media in Australian aboriginal infants. Pediatr. Infect.
Dis. J. 13:983–989.
26. Lehmann, D., W. S. Pomat, I. D. Riley, and M. P. Alpers. 2003. Studies of
maternal immunisation with pneumococcal polysaccharide vaccine in Papua
New Guinea. Vaccine 21:3446–3450.
27. Levy, O. 2007. Innate immunity of the newborn: basic mechanisms and
clinical correlates. Nat. Rev. Immunol. 7:379–390.
28. Marriott, H. M., T. J. Mitchell, and D. H. Dockrell. 2008. Pneumolysin: a
double-edged sword during the host-pathogen interaction. Curr. Mol. Med.
29. McCool, T. L., T. R. Cate, G. Moy, and J. N. Weiser. 2002. The immune
response to pneumococcal proteins during experimental human carriage. J.
Exp. Med. 195:359–365.
30. McCool, T. L., T. R. Cate, E. I. Tuomanen, P. Adrian, T. J. Mitchell, and
J. N. Weiser. 2003. Serum immunoglobulin G response to candidate vaccine
antigens during experimental human pneumococcal colonization. Infect. Im-
31. Melin, M. M., S. K. Hollingshead, D. E. Briles, M. I. Lahdenkari, T. M.
Kilpi, and H. M. Kayhty. 2008. Development of antibodies to PspA families
1 and 2 in children after exposure to Streptococcus pneumoniae. Clin. Vac-
cine Immunol. 15:1529–1535.
32. Montgomery, J. M., D. Lehmann, T. Smith, A. Michael, B. Joseph, T.
Lupiwa, C. Coakley, V. Spooner, B. Best, I. D. Riley, et al. 1990. Bacterial
colonization of the upper respiratory tract and its association with acute
lower respiratory tract infections in Highland children of Papua New
Guinea. Rev. Infect. Dis. 12(Suppl. 8):S1006–S1016.
33. Ochs, M. M., W. Bartlett, D. E. Briles, B. Hicks, A. Jurkuvenas, P. Lau, B.
Ren, and A. Millar. 2008. Vaccine-induced human antibodies to PspA aug-
ment complement C3 deposition on Streptococcus pneumoniae. Microb.
34. Ogunniyi, A. D., M. Grabowicz, D. E. Briles, J. Cook, and J. C. Paton. 2007.
Development of a vaccine against invasive pneumococcal disease based on
combinations of virulence proteins of Streptococcus pneumoniae. Infect. Im-
35. Pickering, H., and G. Rose. 1988. Nasal and hand carriage of Streptococcus
pneumoniae in children and mothers in the Tari Basin of Papua New
Guinea. Trans. R. Soc. Trop. Med. Hyg. 82:911–913.
36. Quiambao, B. P., H. Nohynek, H. Kayhty, J. Ollgren, L. Gozum, C. P.
Gepanayao, V. Soriano, and P. H. Makela. 2003. Maternal immunization
with pneumococcal polysaccharide vaccine in the Philippines. Vaccine 21:
37. Quiambao, B. P., H. M. Nohynek, H. Kayhty, J. P. Ollgren, L. S. Gozum,
C. P. Gepanayao, V. C. Soriano, and P. H. Makela. 2007. Immunogenicity
and reactogenicity of 23-valent pneumococcal polysaccharide vaccine among
pregnant Filipino women and placental transfer of antibodies. Vaccine 25:
38. Rapola, S., V. Jantti, R. Haikala, R. Syrjanen, G. M. Carlone, J. S. Sampson,
D. E. Briles, J. C. Paton, A. K. Takala, T. M. Kilpi, and H. Kayhty. 2000.
Natural development of antibodies to pneumococcal surface protein A,
pneumococcal surface adhesin A, and pneumolysin in relation to pneumo-
coccal carriage and acute otitis media. J. Infect. Dis. 182:1146–1152.
39. Riley, I. D. 1979. Pneumonia in Papua New Guinea: a study of the effects of
Western medicine upon disease in a developing country. University of Syd-
ney, Sydney, Australia.
40. Stubbs, E., K. Hare, C. Wilson, P. Morris, and A. J. Leach. 2005. Strepto-
coccus pneumoniae and noncapsular Haemophilus influenzae nasal carriage
and hand contamination in children: a comparison of two populations at risk
of otitis media. Pediatr. Infect. Dis. J. 24:423–428.
41. Tracer, D. P. 2009. Breastfeeding structure as a test of parental investment
theory in Papua New Guinea. Am. J. Hum Biol. 21:635–642.
42. van den Biggelaar, A. H., P. C. Richmond, W. S. Pomat, S. Phuanukoonnon,
M. A. Nadal-Sims, C. J. Devitt, P. M. Siba, D. Lehmann, and P. G. Holt.
2009. Neonatal pneumococcal conjugate vaccine immunization primes T
cells for preferential Th2 cytokine expression: A randomized controlled trial
in Papua New Guinea. Vaccine 27:1340–1347.
43. Watson, K., K. Carville, J. Bowman, P. Jacoby, T. V. Riley, A. J. Leach, and
D. Lehmann. 2006. Upper respiratory tract bacterial carriage in Aboriginal
and non-Aboriginal children in a semi-arid area of Western Australia. Pe-
diatr. Infect. Dis. J. 25:782–790.
44. WHO Young Infants Study Group. 1999. Bacterial etiology of serious infec-
tions in young infants in developing countries: results of a multicenter study.
Pediatr. Infect. Dis. J. 18:S17–S22.
45. Zaman, K., E. Roy, S. E. Arifeen, M. Rahman, R. Raqib, E. Wilson, S. B.
Omer, N. S. Shahid, R. F. Breiman, and M. C. Steinhoff. 2008. Effectiveness
of maternal influenza immunization in mothers and infants. N. Engl. J. Med.
46. Zhang, Q., J. Bernatoniene, L. Bagrade, A. J. Pollard, T. J. Mitchell, J. C.
Paton, and A. Finn. 2006. Serum and mucosal antibody responses to pneu-
mococcal protein antigens in children: relationships with carriage status.
Eur. J. Immunol. 36:46–57.
1638FRANCIS ET AL.CLIN. VACCINE IMMUNOL.