Table of Contents
Association of naturally acquired IgG antibodies against Plasmodium falciparum serine
repeat antigen-5 with reduced placental parasitemia and normal birth weight in
pregnant Ugandan women: A pilot study
Tonny J. Owalla a, Nirianne Marie Q. Palacpac b, Hiroki Shirai c, Toshihiro Horii b,⁎, Thomas G. Egwang a,⁎⁎
a
b
c
Med Biotech Laboratories, P.O. Box 9364, Kampala, Uganda
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
The Research Foundation for Microbial Diseases of Osaka University, 2-9-41 Yahata-cho, Kanonji, Kagawa 768-0061, Japan
a r t i c l e
i n f o
Article history:
Received 7 December 2012
Received in revised form 10 January 2013
Accepted 30 January 2013
Available online 6 February 2013
Keywords:
Malaria
Serine repeat antigen
Pregnancy
Birth weight
a b s t r a c t
Plasmodium falciparum infection during pregnancy contributes substantially to malaria burden in both
mothers and offspring. Analysis of naturally acquired immune responses that confer protection against
parasitemia and clinical disease is important to guide vaccine evaluation as well as identify immune
correlates. Unfortunately, few studies have addressed the relationship between immune responses to malaria
vaccine candidate antigens and protection against adverse effects on pregnant women and newborn birth
weight. This study examines the relationship of maternal antibody responses to serine repeat antigen-5
(SE36) and merozoite surface protein-1 (MSP119 and MSP142) with placental parasitemia and birth weight.
In a peri-urban setting in Uganda, pregnant women without placental parasites have high median ODs for
antibodies against SE36 (P b 0.001). Naturally acquired anti-SE36 IgG was most prevalent in women without
placental parasitemia (P b 0.001). Furthermore, pregnant women with significantly high levels of anti-SE36
IgG delivered babies with normal birth weights (P b 0.001). That antibody to SE36 was associated with both
a reduced risk of placental parasitemia and resulting normal birth weight in newborns suggests some protective role. In contrast, although antibody to MSP142 was also associated with reduced placental parasitemia
and immune responses to both MSP119 and MSP142 may be of importance, there was no association between
anti-MSP119 antibodies and infant birth weight outcomes. This study highlights the need for conducting
further studies to investigate the association of antibodies against SE36 and outcomes of malaria infection
in pregnant women.
© 2013 Elsevier Ireland Ltd. All rights reserved.
In malaria endemic areas of Africa, pregnant women are a particular
risk group for Plasmodium falciparum infection [1,2]. Although, the impact of malaria during pregnancy varies with epidemiological setting
or pre-existing immunity [3], blood-stage parasites are significant contributors to maternal anemia and morbidity [4], low birth weight (LBW)
[5], and infant morbidity and mortality [6]. The molecular basis for
parasite adhesion and immune evasion in placental malaria has largely
been clarified but other factors may still be involved in parasite sequestration in the placenta [7]. Serological clues are crucial for vaccine
evaluation as well as for epidemiological studies to identify immune
correlates [8]. Antibodies to the erythrocyte surface ligand that binds
chondroitin sulfate A were associated with reduced placental malaria
[7,9–11]. Antibodies that inhibit blood-stage replication may also be
⁎ Corresponding author. Tel.: +81 6 6879 8280; fax: +81 6 6879 8281.
⁎⁎ Corresponding author. Tel.: +256 711998881.
E-mail addresses: [email protected] (T. Horii), [email protected]
(T.G. Egwang).
important in mediating immunity. However, the presence of antibodies
to ring-infected erythrocyte surface antigen (RESA) [9], glutamate-rich
protein (GLURP) [10], apical membrane antigen-1 (AMA-1), and merozoite surface protein-1(MSP-119) [11] were found not to correlate with
pregnancy or parity of mothers.
P. falciparum serine repeat antigen-5 (SE36) is a blood-stage vaccine
candidate currently in clinical trials [12]. SE36 antibody was found to correlate inversely with malaria symptoms and severe disease [13,14], inhibit cell proliferation by complement-mediated lysis of schizonts [15],
agglutinate schizonts and merozoites blocking re-invasion to red blood
cells [16,17], and exhibit antibody-dependent monocyte-mediated parasite growth inhibition [18]. Since a majority of asexual-stage parasites in
the placenta consist of mature forms [7], the presence of anti-SE36 antibody that blocks merozoite release/invasion could reduce the number of
infected erythrocytes both in the peripheral blood and in the intervillous
spaces of the placenta. The association of anti-SE36 antibody with
pregnancy-associated malaria, however, has not been investigated.
Here, we sought to determine if antibody levels against SE36 correlate
with placental parasitemia and pregnancy outcome in a peri-urban
Reproduced from Parasitology International 62: 237-239 (2013).
Nirianne Marie Q. Palacpac: Participant of the 21st UM, 1993-1994.
390
location in Kampala, a region characterized by low, seasonal malaria
transmission.
Serum samples were obtained from a cross-sectional study
designed to investigate the prevalence and effect of P. falciparum
infections during pregnancy at St. Francis Nsambya Hospital in
Makindye division, Kampala, during a 3-month dry spell from July
to Oct 1998. Details of the study and demographic characteristics of
the subjects have been published [19]. Pregnant women were
recruited when they presented at the labor ward for delivery. The
study was approved by the hospital ethical committee and the
Uganda National Council for Science and Technology. Each participant
donated 3 blood samples: peripheral blood (collected by venepuncture
on admission to the labor ward), placental and cord blood (collected
immediately upon uncomplicated delivery). Prevalence of malaria
was evaluated in each of the 3 samples using thick and thin smears
and parasitemia, relative to 200 white blood cells, was estimated
assuming 8000 white blood cells per microliter. All malaria infections
observed were exclusively of P. falciparum. Samples were classified
according to the following criteria for analysis: (a) placental malarianegative or positive, depending on the absence or presence of parasites
in the placenta; (b) peripheral malaria-negative or positive, depending
on the absence or presence of parasites in the venous blood. The classification was based on the characteristic feature of infection during
pregnancy which results in the accumulation of P. falciparum infected
erythrocytes in the placental intervillous blood spaces even in the
absence of detectable peripheral parasitemia [7].
Birth weights of live newborns were measured, with LBW defined
as birth weight below 2500 g. For the present study, information on
the pattern of infection and birth weights of live newborns were
obtained using anonymized data and samples that cannot be linked
to original personal identifiers.
For ELISA measurements, ELISA plates (Dynatech) were coated overnight at 4 °C with 100 μL of antigens MSP142, MSP119 (100 μg/mL=
10 μg/well) and SE36 (1 μg/mL =0.1 μg/well) in carbonate buffer.
MSP142 and MSP119, which represent the amino acid sequence derived
from the Wellcome strain of P. falciparum and expressed as GST fusion
proteins in E. coli, were a kind gift from Dr. A. Holder (Department of
Parasitology, National Institute of Medical Research, Mill Hill, London
UK). Recombinant SE36, which represents the N-terminal 47-kDa
domain without the serine repeat region of P. falciparum SERA-5 was
prepared as described [14]. After washing 3 times in phosphate buffered
saline (PBS) containing 0.05% v/v Tween 20 (PBS-T), plates were
blocked (1% non-fat dry milk in PBS-T) for an hour at 37 °C and washed
again before use/storage. Placental or peripheral serum samples were
diluted 1:1000 in blocking buffer, added to plates in duplicate (100 μL
per well), incubated for 1 h at 37 °C and washed prior to addition of
100 μL horseradish peroxidase-conjugated rabbit anti-human IgG
diluted 1:6000 in PBS-T (Dako Ltd, High Wycombe, UK). The optimal
dilutions of the antigens and rabbit anti-human IgG were previously determined by checkerboard titration. Following 1 h incubation at 37 °C,
plates were washed 4 times with PBS-T and then the substrate orthophenylenediamine (Sigma Chemical Industries, Inc, St. Louis, MO)
was added to wells and the plates kept in the dark for 25 min. The reaction was stopped by addition of 25 μL 2 M sulfuric acid and incubation
for 5 min before optical density (OD) readings at 405 nm (LabSystems
Multiscan352, Helsinki, Finland). For GST fusion proteins, a GST control
plate was processed in parallel. The OD value of the GST control was
subtracted from the OD of the GST fusion protein to obtain the corresponding OD for the antigens alone. When no fusion protein was
involved, as in the case of SE36, the original OD values are shown.
Median and interquartile range (IQR) of antibody ODs in women
with and without peripheral or placental malaria during pregnancy
and between women with normal or low newborn birth weight deliveries were compared using the Mann–Whitney U non-parametric
test. In order to investigate the effect of antigen specific IgG antibody
prevalence on peripheral or placental malaria we sub-categorized
antibody levels into seronegative or seropositive. A cut-off value for
positive ELISA was set as the mean plus 3 standard deviation (SD) of
OD readings from a pool of malaria naïve European sera. The Pearson's
chi-square test was used to compare antibody prevalence in malarianegative or malaria-positive women. Spearman's correlation analysis
was used for the relationship between antibody levels and parasitemia.
Out of 544 women enrolled 537 (98%) provided a complete set of
blood samples for analysis [19]. However, although 537 women is a
sizeable sample, the overall prevalence of parasitemia was low. Only
8.6% (n = 46) had peripheral blood parasitemia and 6.7% (n = 36)
had placental blood parasitemia at delivery. Thirty-six (78.2%) of
the 46 subjects with positive peripheral-blood smears also had positive placental-blood smears. Moreover, only 30 of the 36 samples
with placental blood parasitemia were available for the present
study. There were no congenital malaria infections (or positive
cord-blood smears). Primi-, secundi- and multi-gravid women were
generally comparable, although understandably, multigravidae were
significantly older than primigravid women [19]. Pregnant women
with or without peripheral blood or placental parasitemia at delivery
were matched for parity and geographical location of residence in
Kampala.
Women without placental parasitemia (malaria-negative) had
significantly high antibody levels for SE36 (P b 0.001) (Table 1). The
prevalence of antibodies to SE36 was 60% in women without placental
parasitemia as compared to 10% in women with placental parasitemia
(P b 0.001). Thus, despite the small sample size, anti-SE36 antibodies
were associated with decreased malaria infection and/or the absence
of placental parasitemia at delivery. Likewise, these women also have
significantly high antibody levels to both MSP142 and MSP119 (P =
0.005 and P = 0.026, respectively). High ODs for MSP142 did correlate
Table 1
Prevalence and levels of SE36 IgG antibodies in pregnant women.
Antigen
Placental parasitemia
SE36
MSP119
MSP142
Peripheral blood parasitemia
SE36
MSP119
MSP142
Median OD (IQR)
Antibody prevalence, % (n)
Malaria-positive
Malaria-negative
(n = 30)
0.111
(0.043–0.191)
0.079
(0.026–0.616)
0.484
(0.243–0.850)
(n = 46)
0.364
(0.202–0.599)
0.226
(0.151–0.626)
0.559
(0.370–0.934)
(n = 38)
0.292
(0.164–0.421)
0.291
(0.135–0.635)
0.729
(0.507–1.028)
(n = 61)
0.338
(0.187–0.447)
0.217
(0.124–0.317)
0.399
(0.264–0.691)
P
Malaria-positive
Malaria-negative
χ2
P
b0.001
(n = 30)
10.0 (3)
(n = 38)
60.5 (23)
18.123
b0.001
0.026
26.7 (8)
34.2 (13)
0.447
0.504
0.005
53.3 (16)
89.5 (34)
11.250
0.001
0.252
(n = 46)
63.0 (29)
(n = 61)
63.9 (39)
0.009
0.924
0.180
32.6 (15)
14.8 (9)
4.805
.028
0.012
80.4 (37)
59.6 (36)
5.549
0.018
391
Table of Contents
Acknowledgement
Table 2
Newborn birth weights and SE36 IgG antibody.
We acknowledge the contribution of Ruth Nambowa who carried
out data entry and statistical analyses. The study was supported by
grants from Grant-in-Aid for Scientific Research on Priority Areas
(13226058) (13225001) from the Japanese Ministry of Education,
Science, Sports, Culture and Technology to T.H. and from the UNDP/
WHO/Special Program for Training and Research in Tropical Diseases
(WHO/TDR) to T.G.E. The funding sources have no role in the study
design, collection, analysis, interpretation and writing, or in the decision to submit the paper for publication.
Median OD (IQR)
Antigen
Normal birth weight (n = 63)
Low birth weighta (n = 16)
P
SE36
0.240
(0.211–0.296)
0.764
(0.366–1.495)
0.056
(0.021–0.205)
1.046
(0.163–1.657)
b0.001
MSP119
a
0.760
Defined as birth weight b2500 g.
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Women with peripheral blood parasitemia at delivery had significantly high antibody levels (MSP142, P = 0.012) or antibody prevalence (MSP119, P = 0.028 and MSP142, P = 0.018) to MSP1 (Table 1).
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