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

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0019-9567/95/$04.0010
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

Downloaded from iai.asm.org at Princeton University Library on January 17, 2010

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

4034
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

Downloaded from iai.asm.org at Princeton University Library on January 17, 2010

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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the highest risk of an episode of clinical malaria. The risk of an
episode of clinical malaria was substantially higher in the rainy
season (P 5 0.008), and we found a borderline significant effect
of the high levels of anti-MSP119 antibodies at birth (P 5
0.047); i.e., the risk of an episode of clinical malaria decreased
with increasing values of MSP119. No other explanatory vari-
able (see Materials and Methods) had a significant effect on
the risk of an episode of clinical malaria. Furthermore, we
found that the effect of anti-MSP119 antibodies was the same
for all ages of the infants and for both the rainy and dry
seasons; i.e., there was no correlation between age and anti-
MSP119 antibodies or between season and anti-MSP119 anti-
bodies. Thus, the final model for the probability of an episode
of clinical malaria is as follows:
e a 1 b1x1 1 b2x2 1 b3x3 1 b4x4 1 b5x5
P5
1 1 e a 1 b1x1 1 b2x2 1 b3x3 1 b4x4 1 b5x5
where x1, x2, x3, and x4 are indicator variables for age 3, 6, and
9 months and rainy season, respectively, and x5 is ln OD of
MSP119. The estimated coefficients a and b are shown in Table
3. This final model is illustrated in Fig. 1 for two infants, one
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
response (OD 5 0.1) (■) and for an infant who has a positive anti-MSP119
antibody response (OD 5 0.5) (F).
in infants who had the highest levels of anti-MSP119 antibodies
at birth, when age and season were entered in the model.
Interestingly, we found that the potentially protective effect of
a high level of anti-MSP119 antibodies at birth extended be-
yond the period at which maternally derived antibodies would
be expected to play a role. Passively acquired antibodies would
be expected to be present mainly during the first few months of
life, with only a minor amount after 6 months of age. IgG has
a half-life of 23 days and a breakdown of 3%/day; this leaves
only 10% 2.5 months after birth. An explanation for the ex-
tended effect could be that a high level of anti-MSP119 at birth
did not interfere with the natural boosting but rather protected
the infant until active naturally acquired immunity was
achieved.
Antibodies against MSP1 inhibit invasion of erythrocytes by
merozoites in vitro (4). However, there is considerable uncer-
tainty as to whether anti-MSP1 antibodies block invasion, and
protective activity may require a cellular component (10). A
longitudinal immunoepidemiological study in The Gambia
suggested that antibodies to MSP142 were associated with pro-
tection in young children (31), and data from a longitudinal
study of antimalarial antibody levels and malaria morbidity
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
confirmed in an extensive review (5).
The likelihood of clinical attacks was inversely related to
levels of MSP119 antibodies in infants at birth but did correlate
VOL. 63, 1995 MATERNALLY DERIVED ANTI-P. FALCIPARUM ANTIBODIES
with maternal anti-MSP119. Clearly, maternal and infant levels
of anti-MSP119 did not correlate with each other. It is there-
fore likely that there has been some selective transplacental
transfer of antibodies, perhaps related to differences in anti-
body subclass.
Splenomegaly in infants in The Gambia was associated with
increased concentrations of IgG and IgM (26). In the present
study, the hematocrit was lowest and the spleen rate was high-
est at an age when the probability of an episode of clinical
malaria was highest, i.e., 6 to 9 months. Natural boosting of the
immune response seemed to have occurred by 6 months of age,
in accordance with studies that found no evidence of serocon-
version before the fifth month (3) and other studies in which
most infants lost all detectable malaria-specific maternal IgG
between 4 and 7 months of age (32).
The ideal malaria vaccine will probably contain antigens
from different malaria strains and developmental stages
(preerythrocytic, asexual, and gamete) so as to reduce mortal-
ity and morbidity and block transmission. Vaccines, however,
must be more effective in stimulating the host’s immune de-
fenses than the parasite under natural conditions. We do not
know the best age at which to vaccinate to achieve the maxi-
mum impact on the incidences of infection related to morbidity
and mortality.
The gap window problem, i.e., the combination of high
transmission rates and low efficacy of vaccination in children
with measurable titers of maternally derived antibodies, makes
it difficult to decide at which age the majority of children are
susceptible to infection and hence susceptible to vaccination.
Vaccination at too young an age could waste too much vaccine
on children with maternally derived protection that could po-
tentially lower vaccine efficacy. Studies using mice have shown
that the progeny of immune mothers respond poorly to active
immunization, and it was concluded that maternal IgG anti-
bodies interfered with both priming and helper T-cell function
(18), and neonatal T-cell tolerance was induced by peptides
causing clonal inactivation in mice (15). In other studies, active
immunization with radiation-attenuated Plasmodium berghei
sporozoites of infant mice gave a protection of 71 to 100%
against challenge with infective sporozoites, and offspring of
immunized adult female mice received passive transfer of im-
munity which seemed to be largely through the milk (29).
However, contrasting observations have been made in rats; i.e.,
enhanced responsiveness to vaccination in offspring of P.
berghei-infected female rats was demonstrated (12). Mice and
rats therefore offer two disparate models for the role of con-
genital immune status in malaria vaccination. The immune
responsiveness to malaria vaccination in infants in areas where
malaria is endemic therefore needs careful consideration.
The prevalence of naturally acquired antimalarial IgG is
high among adult populations in our study area in the Nimba
region, Liberia, reflecting the high exposure to malaria. We
have demonstrated that infants born of immune mothers re-
ceive antimalarial IgG, but this IgG is not sufficient to prevent
P. falciparum infection and disease. Therefore, safety and im-
munogenicity studies of future malaria vaccines should exam-
ine the possibility of immunizing infants as soon as possible
after birth, or even the possibility of immunizing the pregnant
mother, thus inducing a relative protection of the infant during
the first year of life.
ACKNOWLEDGMENTS
This study was supported by the Science and Technology for Devel-
opment Programme of the Commission of the European Community
contract TS3p-CT92p138 and by the UNDP/World Bank/WHO Spe-
cial Programme for Research and Training in Tropical Diseases. The
4037
field work was supported by the Liberian American-Swedish Minerals
Company. The activities of the Danish Epidemiology Science Centre
are financed by a grant from the Danish National Research Founda-
tion.
We gratefully acknowledge the work of the staff at Malaria Research
Unit, Yekepa, Liberia, and the collaboration during the field work of
A. Björkman, Department of Infectious Diseases, Karolinska Institute,
Stockholm, Sweden, and A. P. Hanson, Liberian Institute of Biomed-
ical Research, Robertsfield, Liberia. We thank Lis Wassmann and
Nanna Aaby Kruse Laboratory of Parasitology, Statens Seruminstitut,
for excellent technical assistance.
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