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Differences in Human Antibody Reactivity to Plasmodium falciparum
Variant Surface Antigens Are Dependent on Age and Malaria Transmission
Intensity in Northeastern Tanzania
Article  in  Infection and immunity · July 2008
DOI: 10.1128/IAI.01401-06 · Source: PubMed
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INFECTION AND IMMUNITY, June 2008, p. 2706–2714 Vol. 76, No. 6
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Differences in Human Antibody Reactivity to Plasmodium falciparum
Variant Surface Antigens Are Dependent on Age and Malaria
Transmission Intensity in Northeastern Tanzania

Lasse S. Vestergaard,1* John P. Lusingu,1,2 Morten A. Nielsen,1 Bruno P. Mmbando,2 Daniel Dodoo,3
Bartholomew D. Akanmori,3 Michael Alifrangis,1 Ib C. Bygbjerg,1 Martha M. Lemnge,2
Trine Staalsoe,1 Lars Hviid,1 and Thor G. Theander1
Centre for Medical Parasitology at Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), and
Institute of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark1;
National Institute for Medical Research, Tanga Medical Research Centre, Tanga, Tanzania2; and
Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana3
Received 31 August 2006/Returned for modification 7 October 2006/Accepted 24 January 2008
Plasmodium falciparum variant surface antigens (VSA) are involved in the pathogenesis of malaria. Immu-
noglobulin G (IgG) with specificity for VSA (anti-VSA IgG) is therefore considered important for acquired
immunity. To better understand the nature and dynamics of variant-specific IgG responses at population level,
we conducted an immunoepidemiological study in nearby communities in northeastern Tanzania, situated at
different altitudes and therefore exposed to different levels of P. falciparum transmission intensity. Samples of
plasma and infected red blood cells (IRBC) were collected from 759 individuals aged 0 to 19 years. Plasma
levels of IgG with specificity for VSA expressed by a panel of different parasite isolates were measured by flow
cytometry, while the ability of plasma to inhibit IRBC adhesion to CD36 was examined in cellular assays. The
level and repertoire of the heterologous anti-VSA IgG response developed dramatically in individuals at 1 to
2 years of age in the high-transmission area, reaching a maximum level at around 10 years of age; only a modest
further increase was observed among older children and adults. In contrast, at lower levels of malaria
transmission, anti-VSA IgG levels were lower and the repertoire was more narrow, and similar age- and
transmission-dependent differences were observed with regard to the ability of the plasma samples to inhibit
adhesion of IRBC to CD36. These differences indicate a strong and dynamic relationship between malaria
exposure and functional characteristics of the variant-specific antibody response, which is likely to be impor-
tant for protection against malaria.
In areas where malaria is endemic, the age-specific bur- brane protein 1 (PfEMP1), mediates the binding of IRBC to
den of Plasmodium falciparum infection and clinical disease endothelial receptors such as CD36 and ICAM-1 (13, 24, 36).
are closely related to the level of malaria transmission. In This IRBC adhesion enables the parasites to avoid splenic
high-transmission areas the youngest children suffer from clearance (2, 8, 29). The development of clinical immunity
high parasite loads and frequent episodes of disease, while coincides with the gradual acquisition of a broad repertoire of
older individuals are better able to control parasitemia and VSA-specific antibodies (6, 20). Each new parasite infection
in general only suffer from mild malaria episodes. In con- induces a variant-specific immunoglobulin G (IgG) response,
trast, in areas with low levels of malaria transmission, the with specificity for the VSA expressed by the infecting parasite
incidence and severity of clinical disease in adults remains (23, 33). This response appears to protect the host from future
similar to that of children (18, 25, 38, 39). This transmission- clinical episodes arising from parasites expressing antigenically
dependent difference is in agreement with the concept that similar VSA. VSA expressed by parasites isolated from chil-
immunity to malaria is acquired as a result of antigenic dren with severe disease have been found to be more com-
stimulation through repeated parasite infections from early monly recognized than VSA expressed by parasites isolated
childhood onwards (28). from children with nonsevere disease (4, 5, 31). It has been
Among immune responses associated with protection against suggested that, in high-transmission areas, infants and young
clinical malaria are immunoglobulin G antibodies with specific- children quickly acquire antibodies and protection against ma-
ity for variant surface antigens (VSA) expressed on the surface laria parasites expressing VSA types associated with severe
of P. falciparum-infected red blood cells (IRBC) (6, 7, 9, 16, 26, disease outcomes, while in the following years of life individ-
27). The best-studied VSA, P. falciparum erythrocyte mem- uals gradually expand their anti-VSA IgG repertoire toward
parasites expressing VSA associated with uncomplicated ma-
laria (20). According to this hypothesis, the rate of acquisition
* Corresponding author. Mailing address: Centre for Medical Par- of IgG repertoires to VSA would also be assumed to be lower
asitology, Institute of International Health, Immunology and Microbi- in low-transmission areas. To tests these assumptions in order
ology, University of Copenhagen, CSS Building 22, Øster Farimags-
gade 5, DK-1014 Copenhagen K, Denmark. Phone: 45 35 32 76 77. to better understand the dynamics of naturally acquired het-
Fax: 45 32 68 33 03. E-mail: l.vestergaard@cmp.dk. erologous anti-VSA IgG responses at the population level, we

 Published ahead of print on 4 February 2008. conducted an immunoepidemiological study among individuals
2706
VOL. 76, 2008 MALARIA TRANSMISSION INTENSITY AND ACQUIRED IMMUNITY 2707
FIG. 1. Levels of anti-VSA IgG in Ghanaian children against P. falciparum isolates of different origin. Plasma from 96 healthy children
(3 to 8 years old) living in an area of hyperendemic malaria transmission were tested against 13 parasite isolates from asymptomatically
infected children in Tanzania, 5 isolates from children suffering from malarial illness in Ghana (31), and 2 laboratory-based parasite isolates,
3D7 and the in vitro-selected 3D7 Dodowa isolate (41), respectively. The vertical bars represent geometric mean MFI values corrected for
assay-specific background reactivity defined as the mean reactivity of the negative controls plus two SD. Error bars represent 95% confidence
intervals.
living in areas of different altitudes and therefore exposed to Mgome, a point prevalence of 40.8% and a mean parasite density of 391/l in
different intensities of malaria transmission in northeastern Ubiri, and a point prevalence of 12.1% and a mean parasite density of 127/l in
Tanzania (3, 12). By flow cytometry we examined the level and Magamba. Parasite counts in Mgome showed the typical pattern of a high-transmission village with a particularly high parasite burden in children less than
repertoire of anti-VSA antibodies in different age groups, and 5 years old, while no such age-specific difference were observed in Ubiri and
we measured the adhesion-inhibitory effect of the donor Magamba, respectively. Thus, malaria transmission levels and levels of acquired
plasma in a CD36-specific adhesion inhibition assay. malarial immunity differed markedly between the three study villages. Informed
written consent was obtained from all study participants or from their parents or
guardians. The study protocol was approved by the Ethical Committee of the
MATERIALS AND METHODS National Institute for Medical Research and Ministry of Health, Dar es Salaam,
Study sites and populations. The study was conducted in the Tanga region in Tanzania.
northeastern Tanzania. This area is characterized by marked variations in inten- Blood samples. Blood samples were collected from all study individuals during
sity of P. falciparum transmission related to variations in altitude. Very intense cross-sectional surveys in the three study villages in April 2001 before the peak
perennial transmission, with reported entomological inoculation rates (EIRs) in transmission season. From children under the age of 2 to 3 years, 200- to 300-l
the range between 91 and 405 infective bites per person per year, is found in the samples of finger prick blood were collected in Eppendorf tubes with EDTA.
lowland areas toward the Indian Ocean, with peak seasons following the long From older individuals, 5-ml samples of venous blood were collected into Va-
rains in May and the short rains in November. Moderate but stable transmission cutainer tubes with citrate buffer. Hemoglobin concentrations were measured
is found at intermediate altitudes of around 1,000 to 1,200 meters above sea level with a HemoCue photometer (Ångelholm, Sweden), and thick and thin malaria
(EIRs in the range 1.8 to 34 infective bites per person per year reported), while blood smears were prepared and examined according to standard procedures.
very low and unstable transmission is found in highland areas at around 1,600 to After centrifugation, all blood samples were separated into plasma and red blood
1,800 meters above sea level, with an estimated EIR of only 0.03 infective bites cells (RBC); plasma samples were frozen and stored at minus 20°C, while IRBC
per person per year (3). Three study villages were selected; these were located mixed with sorbitol freezing solution were snap-frozen and stored in liquid
within short geographical distances but at different altitudes and thus had dif- nitrogen.
ferent malaria transmission intensities: Mgome village at low altitude (200 m), To test how well the parasite isolates obtained from Tanzanian children were
Ubiri village at intermediate altitude (1,100 to 1,200 m), and Magamba village at recognized by individuals living in another part of the African continent, we also
high altitude (1,600 to 1,700 m). In each village, cohorts of approximately 250 analyzed plasma from 96 asymptomatic children aged 3 to 8 years, obtained as part
healthy individuals between 0 and 19 years of age were randomly recruited for of previous studies in an area of moderate malaria transmission in Ghana (32).
cross-sectional and longitudinal malariometric studies from April to September Parasite isolates and in vitro cultivation. Cryopreserved parasite isolates col-
2001; these results are described in detail elsewhere (25). The villages were lected from 13 asymptomatically infected individuals in the high-transmission area
selected according to predefined criteria to minimize differences in socioeco- were randomly picked, thawed, and successfully adapted to in vitro cultures with O

nomic status, ethnicity, seasonal migration, and access to health care, as assessed blood and culture medium according to previously described procedures (15). PCR
by village-level socioeconomic surveys. In April and May 2001 the P. falciparum was used to genotype the parasite genes msp-1 block 2 (primers recognizing allelic
parasite prevalence and density in the 0- to 19-year-old age group as a whole was variants of MAD20, K1, and RO33) and msp-2 (primers recognizing allelic variants
as follows: a point prevalence of 81.1% and a mean parasite density of 771/l in of FC27 and IC1) of each parasite sample as previously described (37). To compare
2708 VESTERGAARD ET AL. INFECT. IMMUN.
TABLE 1. Proportion of positive anti-VSA IgG responders
Proportion (%) of positive anti-VSA IgG respondersb
Village and subject age in yr (n)a P. falciparum isolates
Mean
TA225 TA006 TA170 TA799 TA453 TA124
Low-transmission village
1 (25) 4.0 0.0 0.0 4.0 4.0 0.0 2.0
1–2 (46) 6.5 17.4 28.3 10.9 6.5 15.2 14.1
3–4 (52) 3.8 3.8 19.3 1.9 7.7 7.7 7.3
5–9 (50) 28.0 16.0 26.0 10.0 24.0 40.0 24.0
10–14 (42) 35.7 31.0 19.0 14.3 23.8 28.6 25.4
15–19 (40) 30.0 27.5 12.5 20.0 25.0 27.5 23.8
Total (255) 18.3 16.5 19.0 10.2 16.7 21.2 17.0
Moderate-transmission village
1 (23) 52.2 4.3 30.4 21.7 8.7 17.4 22.5
1–2 (46) 78.3 50.0 50.0 45.7 6.5 43.5 45.7
3–4 (30) 70.0 46.6 76.7 70.0 6.7 56.7 54.5
5–9 (67) 70.1 67.2 70.1 83.6 26.9 67.2 64.2
10–14 (48) 100.0 87.5 85.4 93.8 54.2 85.4 84.4
15–19 (36) 100.0 88.9 97.2 97.2 80.6 91.7 92.6
Total (250) 80.0 62.8 70.4 73.2 32.0 64.0 63.7
High-transmission village
1 (19) 73.7 10.5 73.6 57.9 15.8 52.6 47.4
1–2 (44) 97.7 90.9 93.2 97.7 52.3 90.9 87.1
3–4 (40) 100.0 100.0 100.0 100.0 90.0 97.5 97.9
5–9 (69) 100.0 100.0 100.0 100.0 97.1 100.0 99.5
10–14 (51) 100.0 100.0 100.0 100.0 96.1 100.0 99.4
15–19 (31) 100.0 100.0 100.0 100.0 100.0 100.0 100.0
Total (254) 97.6 91.7 96.9 96.5 82.3 94.5 93.3
a n, Number of individuals tested.
b Anti-VSA IgG above the antibody cutoff is defined as the mean of negative controls plus two SD.
the recognition of the parasites obtained from asymptomatic children in Tanzania plus two standard deviations (SD) of negative control samples. To calculate
with the recognition of parasites from children with strictly defined malaria, five cumulated antibody responses and to be able to compare anti-VSA IgG levels
parasite isolates obtained from children admitted to Korle-bu Hospital in Accra in between parasite isolates and plasma samples, we subtracted the mean plus two
Ghana as part of previous studies (31) were included for analysis. These isolates SD of log MFI values obtained with the six control samples from all test MFI
were selected on the basis of previous knowledge about their VSA expression to values. To further allow for direct comparisons of antibody recognition of the
represent a spectrum from poorly recognized to well-recognized parasites. Further- different parasite isolates, a standardized score was assigned to each parasite-
more, the two laboratory lines 3D7 and 3D7 Dodowa were tested, of which 3D7 plasma combination. Adjusted test sample MFI values above the MFI of the
Dodowa had been selected to express VSA shown to be associated with severe undiluted (1:1) hyperimmune control pool were assigned an anti-VSA IgG
malaria in young children (21, 41). score of 5. Values between the 1:1 and 1:2 dilutions of the positive control
Analysis of P. falciparum anti-VSA IgG by flow cytometry. The anti-VSA pool were assigned an antibody score of 4, values between the 1:2 and 1:4
plasma IgG levels were measured by flow cytometry as previously described (40). dilutions of the positive control pool were assigned an antibody score of 3,
In vitro cultures with the majority of parasites in the late trophozoite and and so on, until samples with MFI levels below 1:16 dilutions of the positive
schizont stages and parasitemias of 2 to 3% were enriched to 75% parasitemia control pool were assigned an antibody score of 0.
by exposure to a strong magnetic field (34, 40). Aliquots of 2  105 purified CD36-specific parasite adhesion inhibition assays. Inhibition of CD36-specific
IRBC labeled with ethidium bromide were sequentially incubated for 30 min cytoadhesion with the donor plasma samples were examined according to the
with 5 l of human plasma, 0.4 l of goat anti-human IgG (Dako, Glostrup, method described by Hasler et al. (19) with some modifications. A 3D7 parasite
Denmark), and 4 l of fluorescein isothiocyanate-conjugated rabbit anti-goat
isolate was selected for CD36 adhesion by panning the isolate in vitro on CD36-
IgG (Dako). Samples were washed twice in phosphate-buffered saline between
transfected Chinese ovarian hamster (CHO) cells (40). The CD36-specific IRBC
each incubation step. A titrated hyperimmune reference plasma pool and plasma
from six Danish individuals with no previous exposure to malaria served as were radiolabeled by incubating the cultures overnight in the presence of
3H-
positive and negative controls, respectively. A minimum of 5,000 events were labeled phenylalanin (1 MBq for a standard culture containing 200 l of packed
recorded for each parasite-plasma combination, measured on a Coulter EPICS IRBC). Prior to the assay, CD36-transfected CHO cells were grown to a mono-
XL-MCL flow cytometer (Coulter Electronics, Luton, United Kingdom) and layer in 96-well microtiter plates (Nunc, Roskilde, Denmark). Wild-type CHO
7
thereafter analyzed with WinList software (version 5.0; Verity Software, Maine). cells were used as negative controls. IRBC with late-stage parasites (100 l, 10
IRBC and RBC were gated according to the ethidium bromide fluorescence, and RBC/ml), enriched by gelatin sedimentation (22), were added to the CHO cell
for each sample the geometric mean fluorescence index (MFI) was recorded as monolayer and incubated for 1 h at 37°C before unbound RBC were washed
a measure of the amount of anti-VSA IgG present with specificity for that away from the cell monolayer. The number of IRBC remaining in the wells after
3
particular parasite isolate. Nonspecific labeling was evaluated by analysis of the washing step was quantified by measuring the H activity in the wells by liquid
ethidium bromide-negative RBC. Plasma samples obtained at a particular study scintillation. The ability of the plasma to inhibit this CD36-specific adhesion was
site and relating to a particular parasite isolate were processed and analyzed in tested in duplicates by the addition of 10 l of undiluted plasma to the microtiter
a single assay. Samples from the three study sites were tested within as few wells with the CHO-CD36 cell monolayer and IRBC before incubation. To
parasite growth cycles as possible (typically one or two) to minimize any inter- confirm the CD36 specificity of the assay, wells with human immune plasma
assay variations arising from antigenic variation of the isolate during its cultiva- samples were compared to wells with 5 l of monoclonal anti-CD36 antibodies
tion. or anti-ICAM antibodies, respectively (Dako), which were added in triplicates.
A positive anti-VSA IgG response was defined as a MFI value above the mean Levels of maximum CD36 adhesion were defined as the mean reactivity in wells
VOL. 76, 2008 MALARIA TRANSMISSION INTENSITY AND ACQUIRED IMMUNITY 2709
FIG. 2. Cumulative levels of anti-VSA IgG in Tanzanian plasma donors by age and intensity of malaria transmission. The symbols indicate
levels of total cumulative anti-VSA IgG against six Tanzanian P. falciparum isolates (TA225, TA006, TA170, TA799, TA453, and TA124), where
all plasma samples have been tested against each of the six isolates at a single dilution and where the MFI values for each isolate have been adjusted
for assay-specific background reactivity before the MFI values for all six isolates were totaled. Plasma samples from all study individuals from each
of the three study villages areas with high (n  254), moderate (n  250), and low (n  254) intensities of malaria transmission, respectively, were
included and stratified according to age. Error bars indicate 95% confidence intervals. y, years; mo, months.
coated with CHO-CD36 cells and incubated with IRBC and 10 l of normal antibodies at the different levels of transmission, plasma
human serum from individuals never exposed to malaria. samples from all study individuals (n  759) in each of the
Statistical analysis. All data were double entered into the Epi-Info database
(version 6.04; Centers for Disease Control and Prevention, Atlanta, GA), and three study villages were tested against six of the Tanzanian
statistical analyses were done with STATA version 8 (STATA Corp., Texas). parasite isolates (TA225, TA006, TA170, TA799, TA453,
Differences betweens means and proportions were compared by the Student t and TA124). These parasites were chosen on the basis of the
test and 2 test, respectively. The Pearson correlation test was used to examine initial screening (Fig. 1) to ensure the inclusion of both
pairwise correlations between cumulated MFI responses of individual plasma “well” and “poorly” recognized parasites in this analysis.
samples and the degree of CHO-CD36/IRBC adhesion inhibition. P values of
0.05 were considered significant. There were very marked differences between the three vil-
lages with respect to the proportion of individuals who had
a measurable anti-VSA IgG response to the different para-
RESULTS site isolates (Table 1, the proportions of positive antibody
Recognition of Tanzanian parasites by plasma antibodies responders are listed for each of the six parasite isolates and
from Ghanaian children. The tested field parasite isolates for each age group and are further summarized as the mean
from Tanzania (n  13) and Ghana (n  5) each had a unique proportions of responders for the combined collection of
genotype as determined by msp-1 and msp-2 PCR analysis. parasites across all age groups). In the low-transmission
Most parasite samples were polyclonal, with a median clone area, the average recognition of the six tested isolates were
number of three per sample (minimum, 1; maximum, 5). No 2% in children 1 year of age and peaked at 25% in the
correlations between the estimated clone number and the level children aged 10 to 14 years. In the moderate-transmission
of antibody recognition were found (data not shown). area, an average parasite was recognized by 92.6% in 15- to
The parasites obtained from the asymptomatic carriers in 19-year-old individuals, whereas in the high-transmission
Tanzania were all found to be recognized by plasma antibodies village this figure was 97.9% in children aged 3 to 4 years.
of the asymptomatic Ghanaian children (Fig. 1). The levels of Similar differences between the three transmission settings
anti-VSA IgG against these parasites were generally low. By were observed with respect to the mean plasma anti-VSA IgG
comparison, the Ghanaian children had higher plasma anti- levels at different ages (Fig. 2). In the high-transmission village,
body levels against the parasites that had been isolated from the amount of anti-VSA IgG remained constant or dropped
Ghanaian children admitted to hospital with malaria. The un- slightly during the first year of life but thereafter increased
selected 3D7 parasite line was poorly recognized by plasma rapidly and reached a plateau around 10 years of age. In the
antibodies from the asymptomatic Ghanaian children, while moderate-transmission village, the amounts of anti-VSA IgG
the 3D7 Dodowa isolate was more strongly recognized (Fig. 1). increased slowly and did not reach a plateau. In the low-
Level and repertoire of anti-VSA IgG in Tanzanian plasma transmission area, anti-VSA IgG levels remained low in all age
donors. To examine age-specific acquired levels of anti-VSA groups.
2710 VESTERGAARD ET AL. INFECT. IMMUN.
FIG. 3. Repertoires of anti-VSA IgG in Tanzanian plasma donors by age and intensity of malaria transmission. Each square represents one
plasma donor, tested against a panel of parasite isolates obtained in Tanzania (TA038, TA237, TA092, and TA124) and Ghana (L1018, L1106,
and L1093) and two laboratory isolates (3D7 and 3D7 Dodowa). All values have been adjusted for assay-specific background MFI reactivity defined
as the mean of six negative controls plus two SD. To allow for a direct comparisons between the different isolates, a standardized score 0 to 5 was
assigned to each parasite-plasma combination, signifying the antibody level of each sample relative to the geometric mean level of MFI values
against all of the testes parasite isolates (see the text for further details). Each score were then assigned a color according to its level from white
(lowest antibody score) to black (highest antibody score). (A) Antibody scores compared between children and young adults in each of the three
study areas with low, moderate, and high transmission of malaria, respectively. (B) Antibody scores compared between infants and young children
living in the high-transmission area. y, years; mo, months.
To further examine the age-specific repertoire of anti-VSA analyzed in the same assay to allow direct comparisons of
IgG acquired by the plasma donors in the three transmission anti-VSA IgG responses between transmission settings. The
settings, a broader panel of parasites were tested. This panel number of plasma donors was therefore limited to 30 from
included five Tanzanian parasite isolates (TA124, TA799, each village and comprised 10 randomly selected individuals of
TA092, TA237, and TA038), three Ghanaian isolates (L1093, three age groups: 3 to 4 years, 10 to 11 years, and 18 to 19
L1106, and L1018), and the 3D7 and 3D7 Dodowa lines. In years. As shown in Fig. 3A, the proportion of parasites recog-
these experiments, plasma samples from all three villages were nized and the anti-VSA IgG levels increased with the trans-
VOL. 76, 2008 MALARIA TRANSMISSION INTENSITY AND ACQUIRED IMMUNITY 2711
TABLE 2. Proportion of positive parasite-plasma combinations Antibodies to VSA mediate protection against severe malaria
Proportion (%) of positive parasite-plasma (6, 27, 43), uncomplicated malarial fevers (9, 16), and malaria
Age (yr) of combinationsa in pregnancy (11, 35, 42); however, our understanding of the
plasma Pb
Low Moderate High dynamics of naturally acquired anti-VSA IgG responses at thedonors
transmission transmission transmission population level remains limited. To elucidate how anti-VSA
3–4 10 27 95 0.001 responses are influenced and shaped by cumulative malaria
10–11 27 78 100 0.001 exposure, we examined the acquisition of anti-VSA IgG in
18–19 19 74 95 0.001 different age groups among populations residing in nearby
a Anti-VSA IgG tested in samples of plasma from 10 individuals against 10 communities at different altitude levels and therefore at mark-
parasite isolates for each age group. edly different levels of malaria transmission.
b Determined by Kruskal-Wallis one-way analysis of variance on ranks. Major differences in the acquisition of VSA-specific antibod-
ies were observed. In the high-transmission village, 3- to
mission intensity. In the high-transmission village, broad rep- 4-year-old children had already acquired antibodies to most
ertoires of VSA-specific IgG were already acquired by subjects parasites and the anti-VSA IgG levels increased rapidly with
at the age of 3 to 4 years. In the moderate-transmission village, age and reached a high-level plateau at about the age of 10
broad repertoires of antibodies were not acquired until around years. This suggests that the overall level of anti-VSA IgG
10 to 11 years of age, while in the low-transmission village the saturates and is maintained in an ongoing dynamic process
antibody repertoires remained narrow even in the oldest age with decay of old and acquisition of new VSA antibodies. In
group. Proportions of positive parasite-plasma combinations the high-transmission village, most if not all individuals were
by age and transmission level are listed in Table 2. infected with P. falciparum blood-stage parasites, and in chil-
Antibody responses to VSA among children younger than 3 dren aged 1 to 2 years these were carried at high densities.
years were examined in the high-transmission village (Fig. 3B). From age 3 to 10 years the carrier rate and the mean parasite
The antibody repertoire among 0- to 5-month-old infants ap- density declined, and this coincided with the drop in malaria
peared to be broader than among the 6- to 11-month-old fever incidence rate, which decreased markedly after the age of
children (34% versus 10% of tested parasites recognized), al- 4 years (25).
though this difference was not statistically significant (P  In the moderate-transmission setting most parasites were
0.08). From the age of 6 to 11 months, children rapidly devel- recognized by more than 90% of individuals aged 15 to 19
oped a broader repertoire of variant-specific antibodies, and in years, but the levels of antibodies were much lower than in the
the group of children aged 12 to 17 months the mean number plasma samples collected in the high-transmission village. In
of parasites recognized had increased to 60% (P  0.004 com- this village about half of the volunteers were infected, and
pared to 34% of parasites recognized by children aged 0 to 5 there were no marked differences in the parasite point preva-
months). Thus, in the high-transmission village, VSA-specific
IgG responses developed rapidly and antibody repertoires in lence between the ages of 1 and 19 years. We do not have
1- to 2-year-old children were comparable to the antibody age-specific morbidity data from this village, but in a village of
repertoires in the 10- to 11-year-old children living in the coastal Ghana with similar carrier rates, the incidence of fe-
moderate-transmission village (Fig. 3). brile malaria declined after 10 years of age (10). It is interest-
Antibody inhibition of parasite adhesion to CD36. The re- ing that the age at which children had acquired a broad anti-
lationship between malaria transmission and the ability of VSA response in the moderate- and high-transmission villages
plasma to inhibit adhesion to CD36 was investigated in an largely coincided with the age at which clinical immunity would
assay measuring the adhesion of a CD36-selected parasite line be predicted to occur. The levels of VSA-specific antibodies
to a monolayer of CD36-transfected CHO cells. In this assay were markedly higher in individuals living in the high-trans-
approximately half of the maximal adhesion can be inhibited mission village than in individuals from the moderate-trans-
by anti-CD36 monoclonal antibodies (Fig. 4). We first com- mission village, despite the fact that a high proportion of the
pared plasma samples from the 10 18- to 19-year-old individ- individuals in the moderate-transmission village were also in-
uals from each of the three study villages and found that the fected with blood-stage parasites. This finding suggests that it
adhesion-inhibitory effect was highest in plasma samples col- is the number of new infections, which is determined by the
lected in the high-transmission village (Fig. 4A). Acquisition of intensity of transmission, rather than the presence of parasites
the adhesion inhibition capacity by age in this village was per se, that determines the level of anti-VSA antibodies.
investigated by comparing samples obtained from different age In the low-transmission village, the anti-VSA repertoire was
groups. The inhibitory effect was higher in the children aged 10 very incomplete, and the level of antibodies to parasites rec-
to 11 years than in the group aged 3 to 4 years (P  0.004). In ognized was low. In this village, the carrier rate was ca. 10%. In
addition, a positive correlation was found between the cumu- areas of low and seasonal transmission anti-VSA antibody lev-
lated MFI level of the individual sample and its capacity to els show marked seasonal variations (17), and the measured
inhibit CD36-specific cytoadhesion (Pearson correlation test, levels of anti-VSA IgG might have been higher if samples had
R  0.63, P  0.001) (Fig. 4C). been collected just after the peak transmission season. In a
Sudanese village with a similar parasite point prevalence, we
DISCUSSION previously found that individuals of all age groups regularly
suffer from febrile malaria attacks. However, the malaria inci-
The importance of variant-specific antibodies against P. fal- dence rate is lower in adults than in children and adolescent
ciparum for protection against malaria is well documented. (14), and it is possible that some adults mount efficient and
2712 VESTERGAARD ET AL. INFECT. IMMUN.
FIG. 4. Inhibition of parasite adhesion to CD36. The adhesion of IRBC to CD36-transfected CHO cells and the adhesion-inhibitory effects of
monoclonal antibodies and human plasma antibodies, respectively, are shown. Binding levels are expressed as percentages of maximum binding,
measured in wells with IRBC, CHO-CD36 cells, and normal human plasma from negative controls. Vertical bars represent binding of IRBC to
CHO-CD36 cells in wells with monoclonal anti-CD36 and anti-ICAM1 antibodies, respectively (means of triplicates with error bars representing
standard errors of the mean). The box plots represent human plasma samples (n  10 per group, samples are identical to those shown in Fig. 3A),
showing medians, 25th and 75th percentiles, 5th and 95th percentiles, and outliers. (A) Comparison of the adhesion-inhibitory effect of plasma
anti-VSA IgG in samples from 18- to 19-year-old individuals living in areas with low, moderate, or high malaria transmission. (B) Comparison of
the adhesion-inhibitory effect of plasma anti-VSA IgG in samples from individuals aged 3 to 4 years, 10 to 11 years, and 18 to 19 years, living with
high malaria transmission. (C) Correlation between total cumulated MFI to six P. falciparum isolates (see the text) of the 30 plasma donors of the
high-transmission village also shown in panel B and the adhesion-inhibitory effect of the same samples. Statistical comparisons between groups
were performed with the Student t test, and the correlation coefficient was calculated with the Pearson correlation test.
VOL. 76, 2008 MALARIA TRANSMISSION INTENSITY AND ACQUIRED IMMUNITY 2713
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Editor: J. F. Urban, Jr.
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