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INFECTION AND IMMUNITY, Oct. 1995, p. 4034–4038 Vol. 63, No.

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Copyright q 1995, American Society for Microbiology

Relationship between Maternally Derived Anti-Plasmodium falciparum


Antibodies and Risk of Infection and Disease in Infants Living in an
Area of Liberia, West Africa, in Which Malaria Is Highly Endemic
BIRTHE HØGH,1* NUAHN T. MARBIAH,2 PETRA A. BURGHAUS,3 AND PER KRAGH ANDERSEN1,4
Epidemiology Research Unit and Danish Epidemiology Science Center, Statens Seruminstitut,1 and Department of
Biostatistics, University of Copenhagen,4 Copenhagen, Denmark, and London School of Hygiene and Tropical
Medicine2 and Division of Parasitology, National Institute for Medical Research,3 London, England
Received 15 June 1995/Returned for modification 11 July 1995/Accepted 25 July 1995

In areas where Plasmodium falciparum is endemic, immunoglobulin G is acquired by the fetus in utero,
mainly during the third trimester of pregnancy. The potential protective effect of transferred anti-P. falciparum
maternal antibodies was examined in a longitudinal study of 100 infants from birth to 1 year of age. The

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probability of acquiring a P. falciparum infection and developing an episode of clinical malaria was determined
in relation to the P. falciparum-specific antibody level of the infant at birth against P. falciparum schizont
antigen or recombinant merozoite surface protein MSP119 antigen. The risk of acquiring an episode of clinical
malaria increased from birth to 6 months of age, after which it decreased. The overall prevalence of P.
falciparum parasitemia was highest (48.9%) in the 6-month-old infants. The age-specific hematocrit value
showed the lowest mean value (30.2) from 6 to 9 months, and the spleen rate was the highest (69.8%) at the
same age. There was a lower risk of developing an episode of clinical malaria during the first year of life in the
infants with high levels of anti-MSP119 antibodies at birth. The level of maternally derived overall anti-schizont
antigen antibodies did not seem to play a role in the relative risk of developing malaria infection or disease
during the first year of life, though the level of specific anti-MSP119 antibodies may be associated with
protection.

The role of congenital immunity in the natural history of precursor of several surface proteins of the merozoite, and the
human malaria in areas where the disease is highly endemic is MSP1 C-terminal 19-kDa polypeptide (MSP119) remains on
unclear. A relative insusceptibility to malaria of young infants the surface during invasion and is the target of monoclonal
has been observed (16), and the most important factor in invasion-inhibiting antibodies (4); antibodies against this frag-
modifying the clinical symptoms has been ascribed to the pas- ment may therefore play an important role in protection.
sive transfer of maternal anti-malarial immunoglobulin G This paper describes the results of a longitudinal study of
(IgG) antibodies (6, 32). Other factors may include the inabil- 100 infants from birth to 1 year of age. The objective was to
ity of fetal hemoglobin to support plasmodial development determine whether transplacentally acquired schizont anti-P.
within the erythrocytes (30), the deficiency of p-aminobenzoic falciparum antibodies and/or antibodies to the defined recom-
acid in the exclusively milk diet of the breast-fed infant (19), binant protein representing the MSP119 polypeptide protect
and a relative aversion of Anopheles mosquitoes to feeding on against infection and disease by relating the antibody level at
infants (7). However, the splenomegaly observed in early in- birth to the risk of acquiring P. falciparum infection or disease
fancy suggests that passively acquired immunity does not pre- during the first year of life.
vent or adequately control infection (11).
The rate of decay of maternally derived antibodies and the
degree to which maternal antibodies protect against infection MATERIALS AND METHODS
are not clear for Plasmodium falciparum malaria (15). It has Study area and study population. The field study was carried out in 1987 to
been suggested that in low-endemicity areas, transplacentally 1988 in two villages, Beytonwee and Bonah, in a rural area of northwestern
acquired antibody may not be clinically relevant in protecting Liberia, in the Mount Nimba region close to the border of Guinea and Ivory
the infant (9). Age-specific parasite incidence rates show little Coast. The altitude is 600 m above sea level, the annual rainfall varies between
2,000 and 2,500 mm, and the temperature is generally between 21 and 328C. The
evidence that incidence is markedly reduced in the first climate is that of a tropical rain forest, with a rainy season from May to October
months. In the field study in Garki, Nigeria, the observed and a dry season from November to April. The Mount Nimba region is an area
cumulative prevalence of P. falciparum was compatible with of intense and perennial malaria transmission.
the hypothesis that the incidence rate is constant during the Study design. Pregnant women in the two villages were registered and moni-
tored during pregnancy. As soon as possible after delivery, usually within 4 to 14
first year of life, and this finding suggests that either the ma- days after birth, the mother and the newborn baby were examined clinically and
ternal antibodies have a protective effect during all of the first parasitologically. One hundred infants were included in an open cohort, and
year or the effect is negligible from birth (28). passive case detection was performed during the weekly clinics by the mobile
The major merozoite surface protein of P. falciparum research team; parasitological and active-case detection was carried out through
cross-sectional surveys every 3 months. Thick and thin blood films were made
(MSP1) is a candidate for a malaria vaccine (21). MSP1 is the from finger prick blood specimens whenever a child was unwell and/or had a
temperature of $37.58C. Infants found positive were treated with chloroquine
(25 mg/kg of body weight).
Morbidity measurements. A standardized clinical assessment was made, both
* Corresponding author. Mailing address: Epidemiology Research at the weekly clinics and at the 3-monthly surveys, by the medical doctor on the
Unit, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, basis of the history obtained from the child’s attendant, usually the mother. The
Denmark. Phone: 45 32 68 37 22. Fax: 45 32 68 31 65. mother or attendant was asked whether the child was (i) well, (ii) unwell but able

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VOL. 63, 1995 MATERNALLY DERIVED ANTI-P. FALCIPARUM ANTIBODIES 4035

to continue normal activities, or (iii) unwell and unable to continue normal TABLE 1. Age-specific P. falciparum parasite positivity and
activities and whether the child had had fever, had been vomiting, or had had geometric mean density per microliter in the cross-sectional surveys
diarrhea or cough during the previous week. Body temperature was measured
with an axillary thermometer (Terumo Corp., Tokyo, Japan). Asexual parasites Gametocytes
We compared our classification of an episode of clinical malaria (20) with the Age
criteria of different parasite densities from 1 to 5,000 ml and found that 1,000 Geometric Geometric
(mo) Positivity Positivity
parasites per ml and a body temperature of $37.58C gave a probability for an n mean density/ n mean density/
(%) (%)
episode of clinical malaria that was similar to our clinical stage classification. ml (CVa) ml (CV)
Individuals with a parasite density of $1,000/ml and a body temperature of
$37.58C were therefore considered to have an episode of clinical malaria.
0 100 5.0 1,114 (1.3) 100 0.0 000 (0.0)
Spleen size. The spleens were examined with the infant lying down and were 3 96 27.1 3,728 (1.5) 96 8.3 332 (0.9)
classified by the method described by Hackett (17). 6 88 48.9 5,431 (1.4) 88 17.1 290 (0.7)
Parasitological examinations. Thick and thin blood films were air dried and 9 99 43.4 9,260 (1.8) 99 9.1 455 (0.9)
stained with Giemsa stain. Two hundred fields were examined before a slide was 12 35 25.7 6,243 (1.2) 35 11.4 246 (1.0)
considered negative. Parasite density was calculated as the parasite-positive
a
geometric mean density, assuming that 100 fields were equal to 0.2 ml of blood. CV, coefficient of variation.
Hematological examinations. Plasma was obtained every 3 months from finger
prick blood samples collected in preheparinized 75-ml capillary tubes. Blood was
centrifuged, the hematocrit value was registered, and the plasma was stored at
falciparum sporozoites. The entomological inoculation rate was the product of
2208C until analysis.
the human-biting rate and the sporozoite rate.
Antigens and antigen preparation. (i) MSP119. The DNA sequence corre-
Statistical analysis. Log-transformed parasite densities and log-transformed
sponding to the natural MSP119 fragment lacking the membrane anchor (amino
OD values were used for the analyses and expressed as geometric means and

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acids Asn-1631 to Asn-1726; numbering according to Miller et al. [27]) was
coefficients of variation. To obtain a description of the probability of an episode
amplified by PCR using the Wellcome strain gene as a template. The PCR
of clinical malaria in relation to the level of antimalarial antibodies at birth, a
product was cloned into the expression vector pGEX-3X (Amrad Corp.). The
logistic regression model was used (23). The following candidate explanatory
MSP119 gene was fused to the carboxy terminus of the Schistosoma japonicum
glutathione S-transferase (GST) gene. Expression of the fusion protein (GST- variables for the probability of having clinical malaria at a given examination
MSP119) was induced with 1 mM isopropyl-b-D-thiogalactopyranoside in Esch- (number x) were included: information on clinical malaria at examination num-
erichia coli TOPP 1 (Stratagene). The cell lysate was applied to a glutathione- ber x 2 1, antibody levels at birth, season of examination number x (rainy or dry),
agarose column (Sigma) which bound the fusion protein. The column was age of child at examination number x, antibodies of mother, sex, and hemoglobin
washed, and MSP119 was cleaved from the GST with the protease factor Xa type (AA versus Ab or AS). The ages at examination were grouped into the
(Boehringer), leaving the fusion protein bound to the column. The eluate was intervals ,1.5, $1.5 and ,4.5, $4.5 and ,7.5, $7.5 and ,10.5, and $10.5 and
passed through a benzamidine-Sepharose 6B column (Pharmacia) to extract the #12 months. The results of the analysis are presented as estimated effects of the
protease. The eluted protein runs on a sodium dodecyl sulfate-polyacrylamide significant explanatory variables and as estimated probabilities based on the
gel as a single band with an apparent molecular mass of 20 kDa. The recombi- model.
nant protein was recognized by nine specific monoclonal antibodies. It appeared
to be correctly folded. Further characterization of the recombinant protein is RESULTS
described by Burghaus and Holder (8).
(ii) Purified schizont-stage P. falciparum antigen. The schizont antigen was Parasitological findings. A total of 100 infants were regis-
prepared from continuous cultures of P. falciparum isolate F32 in serum-free
RPMI 1640 culture medium supplemented with N-2-hydroxyethylpiperazine-N9-
tered in the open cohort and monitored longitudinally. The
2-ethanesulfonic acid (HEPES) buffer, 0.2% NaHCO3, and a growth-promoting age-specific P. falciparum parasite rates and densities of tro-
factor (1). Asexual parasitemias of 10 to 15% were achieved in 8 days, and the phozoites and gametocytes are shown in Table 1.
schizonts were harvested and enriched on a 20% discontinuous Nycodenz gra- Hematologic parameters and spleen rate and index. The
dient (Nycomed DAK A/S, Copenhagen, Denmark). The enriched schizont-
stage antigen preparation was obtained by freeze-thawing the purified, washed
age-specific hematocrit value was lowest (30.2) from 6 to 9
asexual parasites; this preparation contains merozoite products including MSP1. months, and the spleen rate was highest at the same age
The purified antigen preparation was sonicated for 30 s on ice and kept cold at (69.8%) (data not shown).
all times. The disrupted parasite suspension was centrifuged (10,000 3 g, 10 min, ELISA for recombinant MSP-119 and P. falciparum schizont
48C), and the protein concentration was determined in the supernatant, which
was then stored in aliquots at 2708C.
antigen. Anti-P. falciparum-specific antibodies against schizont
Enzyme-linked immunosorbent assay (ELISA). (i) Recombinant MSP119. antigen and MSP119 antigen were detected by ELISA in
Plasma samples diluted 1:1,000 were tested for reactivity against MSP119. The plasma samples of 100% of the mothers and 90.1% of their
saturating concentration of MSP119 protein, determined by titration, was 1 mg/ infants and in 96.2% of the mothers and 80.2% of their infants,
ml. Microtiter plates (Maxisorp; Tecknunc, Roskilde, Denmark) were coated
overnight at 48C with proteins diluted in coating buffer (0.05 M sodium carbon-
respectively. The geometric mean indices of response by age
ate-bicarbonate [pH 9.6]). Plates were washed four times in phosphate-buffered are summarized in Table 2.
saline (PBS; 0.5 M NaCl, 1 mM KH2PO4, 6.4 mM Na2HPO4, 2.7 mM KCl, 1% Entomological parameters. Anopheles gambiae and Anoph-
[vol/vol] Triton X-100 [pH 7.2]). Then 100 ml of plasma diluted 1:1,000 in eles funestus are the two main vectors. The mosquito densities
dilution buffer (PBS supplemented with 1% [wt/vol] bovine serum albumin and
0.5% phenol red [pH 7.2]) was added to duplicate wells, and the wells were
are seasonal, with 0.5 infectious bite per person per night
incubated at 378C for 1 h. After washing, horseradish peroxidase-conjugated during the rainy season and 0.02 infectious bite per person per
rabbit anti-human IgG (100 ml of a 1:5,000 dilution; Dako A/S, Glostrup, Den- night late in the dry season.
mark) was added to the plates, incubated for 20 min at room temperature, and
developed with H2O2 and o-phenyldiamine (Sigma, St. Louis, Mo.) as the sub-
strate. An antibody level greater than the mean plus 2 standard deviations of the
antibody levels among 100 nonimmune European blood donors was defined as TABLE 2. Age-specific seropositivity with two antigens (Schizont
the upper limit of the normal range; in this way, seropositivity was defined as an and MSP119) and geometric mean OD
optical density (OD) greater than 0.1. The relative absorbance of bound IgG was
determined from a standard curve obtained with a high-titer standard plasma run Schizont antigen MSP119
in parallel. Age
(ii) P. falciparum schizont-stage antigen. Plasma samples diluted 1:1,000 were Sero- Sero-
(mo) Geometric mean Geometric mean
tested for reactivity against the schizont-enriched antigen preparation. The sat- n positivity n positivity
OD (CVa) OD (CV)
urating concentration of the antigen preparation, determined by titration, was (%) (%)
1:800. Microtiter plates (Maxisorp) were coated overnight at 48C with proteins
diluted in coating buffer (0.1 M sodium carbonate-bicarbonate [pH 9.6]). Sub- 0 100 90.1 0.60 (0.6) 100 80.2 0.32 (1.2)
sequent procedures were as described above. 3 96 57.0 0.32 (0.5) 96 66.3 0.25 (1.0)
Entomological investigations. Every second month, pyrethrum spray collec- 6 88 61.9 0.36 (0.6) 88 86.9 0.40 (1.0)
tion of indoor-resting mosquitoes was performed in 10% of the houses in each 9 95 67.4 0.40 (0.6) 95 92.5 0.35 (0.9)
village. The human-biting rate was calculated from the number of human-fed 12 33 72.7 0.38 (0.8) 33 84.9 0.27 (1.2)
mosquitoes divided by the number of occupants of the room. The mosquitoes
a
were examined for sporozoites by ELISA, using monoclonal antibodies against P. CV, coefficient of variation.
4036 HØGH ET AL. INFECT. IMMUN.

TABLE 3. Estimated effects of the significant explanatory variables with negative and one with high levels of anti-MSP119 at birth.
in the logistic regression model for the probability of an episode of The estimated probabilities are based on the model for the two
clinical malaria arbitrarily chosen infants (in the rainy season) with anti-
Parameter MSP119 antibodies (OD 5 0.1, corresponding to the negative
Factor Symbol SE Wald x2 P cutoff OD value) and anti-MSP119 antibodies (OD 5 0.5, cor-
estimate
responding to an OD value above the mean), respectively.
Intercept a 23.511 0.734
Age (mo) compared 8.22 ,0.05
with 12 mo DISCUSSION
3 b1 20.209 0.756
6 b2 0.972 0.680 The relative insusceptibility to malaria of young infants born
9 b3 0.789 0.681 in areas where the disease is highly endemic is followed by a
Rainy season compared b4 0.993 0.374 7.05 ,0.01 period of increased risk. In Tanzania, the critical age at which
with dry season the first infection was observed was conditioned by date of
ln OD of MSP119 b5 20.365 0.184 3.94 ,0.05 birth in relation to season, with an average probability of first
infection at 2 months (2). We found an increase in the risk of
acquiring an episode of clinical malaria from birth to 6 months
of age. Logistic regression results suggest that there was a
Probability model for episodes of clinical malaria. Age had consistently lower probability of an episode of clinical malaria
a significant effect (P , 0.05); infants aged 6 to 9 months had

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in infants who had the highest levels of anti-MSP119 antibodies
the highest risk of an episode of clinical malaria. The risk of an at birth, when age and season were entered in the model.
episode of clinical malaria was substantially higher in the rainy Interestingly, we found that the potentially protective effect of
season (P 5 0.008), and we found a borderline significant effect a high level of anti-MSP119 antibodies at birth extended be-
of the high levels of anti-MSP119 antibodies at birth (P 5 yond the period at which maternally derived antibodies would
0.047); i.e., the risk of an episode of clinical malaria decreased be expected to play a role. Passively acquired antibodies would
with increasing values of MSP119. No other explanatory vari- be expected to be present mainly during the first few months of
able (see Materials and Methods) had a significant effect on life, with only a minor amount after 6 months of age. IgG has
the risk of an episode of clinical malaria. Furthermore, we a half-life of 23 days and a breakdown of 3%/day; this leaves
found that the effect of anti-MSP119 antibodies was the same only 10% 2.5 months after birth. An explanation for the ex-
for all ages of the infants and for both the rainy and dry tended effect could be that a high level of anti-MSP119 at birth
seasons; i.e., there was no correlation between age and anti- did not interfere with the natural boosting but rather protected
MSP119 antibodies or between season and anti-MSP119 anti- the infant until active naturally acquired immunity was
bodies. Thus, the final model for the probability of an episode achieved.
of clinical malaria is as follows: Antibodies against MSP1 inhibit invasion of erythrocytes by
merozoites in vitro (4). However, there is considerable uncer-
e a 1 b1x1 1 b2x2 1 b3x3 1 b4x4 1 b5x5 tainty as to whether anti-MSP1 antibodies block invasion, and
P5
1 1 e a 1 b1x1 1 b2x2 1 b3x3 1 b4x4 1 b5x5 protective activity may require a cellular component (10). A
longitudinal immunoepidemiological study in The Gambia
where x1, x2, x3, and x4 are indicator variables for age 3, 6, and
suggested that antibodies to MSP142 were associated with pro-
9 months and rainy season, respectively, and x5 is ln OD of
tection in young children (31), and data from a longitudinal
MSP119. The estimated coefficients a and b are shown in Table
study of antimalarial antibody levels and malaria morbidity
3. This final model is illustrated in Fig. 1 for two infants, one
suggest that antibodies to MSP119 are associated with protec-
tion (14). The structure and antigenicity of the recombinant
protein MSP119 resemble those of the natural P. falciparum
antigen, and therefore MSP119 is considered a good candidate
for inclusion in a subunit vaccine against malaria (8).
It has been suggested that passive immunity does not sup-
press parasitemia, though it can diminish clinical symptoms
(25). Our clinical criteria for an episode of clinical malaria,
based on the history of fever and clinical status of the infant,
resulted in a pattern of probability of acquiring clinical symp-
toms similar to that found when we applied the criteria of body
temperature of $37.58C and $1,000 asexual parasites per ml.
Average parasite density is not high in early infancy, and par-
asite density has been suggested as a surrogate measure for
malaria-associated morbidity and mortality (24).
Although IgG passes transplacentally, its ability to control
parasite infection in the infant could be limited if much of the
IgG is nonprotective, and we did not find any relationship
between high total anti-P. falciparum antibodies at birth and
protection during the first year of life. This finding is in accor-
dance with Macdonald’s original conclusion in 1950 that pas-
sively acquired maternal antibodies did not influence the par-
asite rate in infants (22), and this conclusion was recently
FIG. 1. Estimated probabilities of an episode of clinical malaria during the
rainy season for an infant who at birth has a negative anti-MSP119 antibody
confirmed in an extensive review (5).
response (OD 5 0.1) (■) and for an infant who has a positive anti-MSP119 The likelihood of clinical attacks was inversely related to
antibody response (OD 5 0.5) (F). levels of MSP119 antibodies in infants at birth but did correlate
VOL. 63, 1995 MATERNALLY DERIVED ANTI-P. FALCIPARUM ANTIBODIES 4037

with maternal anti-MSP119. Clearly, maternal and infant levels field work was supported by the Liberian American-Swedish Minerals
of anti-MSP119 did not correlate with each other. It is there- Company. The activities of the Danish Epidemiology Science Centre
fore likely that there has been some selective transplacental are financed by a grant from the Danish National Research Founda-
transfer of antibodies, perhaps related to differences in anti- tion.
We gratefully acknowledge the work of the staff at Malaria Research
body subclass. Unit, Yekepa, Liberia, and the collaboration during the field work of
Splenomegaly in infants in The Gambia was associated with A. Björkman, Department of Infectious Diseases, Karolinska Institute,
increased concentrations of IgG and IgM (26). In the present Stockholm, Sweden, and A. P. Hanson, Liberian Institute of Biomed-
study, the hematocrit was lowest and the spleen rate was high- ical Research, Robertsfield, Liberia. We thank Lis Wassmann and
est at an age when the probability of an episode of clinical Nanna Aaby Kruse Laboratory of Parasitology, Statens Seruminstitut,
malaria was highest, i.e., 6 to 9 months. Natural boosting of the for excellent technical assistance.
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