INFECTION AND IMMUNITY, May 2008, p. 2240–2248
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 5
Breadth and Magnitude of Antibody Responses to Multiple
Plasmodium falciparum Merozoite Antigens Are
Associated with Protection from Clinical Malaria?†
Faith H. A. Osier,1,2* Gregory Fegan,1,2Spencer D. Polley,2Linda Murungi,1Federica Verra,2,4
Kevin K. A. Tetteh,2Brett Lowe,1Tabitha Mwangi,1Peter C. Bull,1Alan W. Thomas,5
David R. Cavanagh,6Jana S. McBride,6David E. Lanar,7Margaret J. Mackinnon,1,3
David J. Conway,2,8and Kevin Marsh1
KEMRI Centre for Geographic Medicine Research, Coast, P.O. Box 230-80108, Kilifi, Kenya1; London School of Hygiene and
Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom2; Department of Pathology, University of Cambridge,
Tennis Court Road, Cambridge CB2 1QP, United Kingdom3; Dipartimento di Scienze di Sanita’ Pubblica, Sezione di
Parassitologia, University of Rome “La Sapienza,” 00185 Rome, Italy4; BPRC, Department of Parasitology,
P.O. Box 3306, 2280, GH Rijswijk, The Netherlands5; Institute of Immunology and Infection Research,
School of Biological Sciences, University of Edinburgh, EH9 3JT Edinburgh, United Kingdom6;
Department of Immunology, Walter Reed Army Institute of Research, Forest Glen Annex,
Silver Spring, Maryland 209107; and Medical Research Council Laboratories,
Fajara, P.O. Box 273, Banjul, The Gambia8
Received 1 December 2007/Returned for modification 9 February 2008/Accepted 23 February 2008
Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria
parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely
focused on the presence of responses to individual antigens and their associations with decreased morbidity.
We hypothesized that the breadth (number of important targets to which antibodies were made) and magni-
tude (antibody level measured in a random serum sample) of the antibody response were important predictors
of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium
falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for
uncomplicated malaria for 6 months (n ? 119). Serum antibody levels to apical membrane antigen 1 (AMA1)
and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the
probability of developing malaria, but levels to MSP-119and erythrocyte binding antigen (EBA-175) were not.
The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none
of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical
episode (17/119; 15%; P ? 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were
more strongly predictive of protection than others. The results were validated in a larger, separate case-control
study whose end point was malaria severe enough to warrant hospital admission (n ? 387). These findings
suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple
antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar
While large populations of the world are at risk of malaria
(30, 62), the brunt of mortality caused by Plasmodium falcip-
arum continues to be borne by children in sub-Saharan Africa.
It is estimated that in this region alone, nearly 1 million chil-
dren under the age of 5 years died as a direct consequence of
malaria in the year 2000 (59). An effective vaccine is urgently
needed but has proved challenging to obtain. In endemic areas,
older children and adults develop naturally acquired immunity
to severe and life-threatening malaria but remain susceptible
to infection (37). Classical experiments in which passively
transferred antibodies from immune adults were successfully
used to treat children with severe P. falciparum malaria (14, 40)
provide the strongest evidence that antibodies are important
mediators of naturally acquired immunity. Clinical symptoms
of malaria result from the asexual blood stage of the infection,
in which potential antibody targets include merozoite antigens
involved in invasion (18) and parasite-derived surface antigens
on infected erythrocytes (8).
Studies on protective immunity to malaria involve monitor-
ing subjects in endemic communities for variable durations of
time to measure the incidence of infection or clinical disease.
Associations between the presence of a specific immune re-
sponse to a target antigen and an outcome determine whether
an immune response to the specific antigen appears to be
“protective.” These immuno-epidemiological studies have of-
ten provided conflicting data, with responses to the same an-
tigen appearing to be protective in some studies but not in
others (2, 5, 10, 15, 21, 24, 31, 52, 57). Most antibody-based
analyses of protection are tethered on seropositivity (usually
* Corresponding author. Mailing address: KEMRI-CGMRC, P.O.
Box 230-80108, Kilifi, Kenya. Phone: 254 721 450307. Fax: 254 41
522390. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 3 March 2008.
defined as the mean plus 3 standard deviations of non-malaria-
exposed sera) and do not take into account the continuous,
quantitative nature of antibody concentrations. Furthermore,
the majority of studies have concentrated on associations be-
tween responses to a single or a limited number of antigens
and protection from clinical malaria, despite the fact that in-
dividuals living in endemic areas are simultaneously and re-
peatedly challenged with numerous malaria antigens. Few
studies have examined the interactions between specific anti-
body responses against multiple malaria antigens (32, 41) and
whether these might be synergistic, antagonistic, or neither
with regards to protection.
To test whether either the number of important target an-
tigens to which antibodies are made or the levels of such
antibodies in serum are associated with protection from ma-
laria, we analyzed naturally acquired antibodies to five leading
P. falciparum merozoite-stage vaccine candidate antigens (api-
cal membrane antigen 1 [AMA1], merozoite surface proteins
1, 2, and 3 [MSP-1, MSP-2, and MSP-3], and erythrocyte bind-
ing antigen [EBA-175]), as well as P. falciparum schizont ex-
tract, in a cohort of Kenyan children who were monitored
longitudinally for mild (uncomplicated) clinical malaria (Cho-
nyi cohort). We also examined combinations of, and interac-
tions between, antigen-specific antibodies to determine the
combination(s) that predicted the strongest protection from
clinical malaria. These antigens were selected for study be-
cause of the cumulative evidence that the presence of antibod-
ies to these antigens may be associated with protection (10, 15,
42, 50, 53–56, 65, 66), backed by evidence that polymorphisms
in their sequences are maintained by natural selection (16),
and their biological plausibility (3, 13, 20, 25, 36, 46, 60). The
analytical approaches were developed using data from the
Chonyi cohort, and the methods subsequently were validated
in an independent case-control study whose end point was
malaria severe enough to require admission to hospital.
MATERIALS AND METHODS
Cohort study. This study was conducted in Kilifi, a rural district along the
Kenyan coast. Details of the study area and population have been published
elsewhere (44), along with a description of a cohort comprising both adults and
children from Chonyi village in Kilifi. This area typically experiences two sea-
sonal peaks in malaria transmission (June to August and November to Decem-
ber) and has an average annual entomological inoculation rate of approximately
20 to 100 infective bites/person/year (38). Following a cross-sectional bleed at the
start of a malaria transmission season in October 2000, the cohort was followed
for clinical episodes of malaria in the ensuing 6 months. Asymptomatic para-
sitemias were not subjected to drug clearance at the beginning of the study or
during follow-up. Active detection of mild clinical malaria (outcome) was
achieved through weekly visits to participants’ homes, where a morbidity ques-
tionnaire (investigating symptoms occurring in the preceding week) was admin-
istered and the presence or absence of fever was ascertained (axillary tempera-
ture of ?37.5°C). Participants found to be unwell were referred for free
treatment to a dedicated outpatient clinic at the local district hospital, where
they also had open access as required at any time during the study (passive
case detection). Clinical episodes of malaria were treated with sulfadoxine-
pyrimethamine, as per the Kenyan national treatment guidelines at the time the
study was conducted. Age-specific criteria for defining clinical episodes of ma-
laria were developed previously for this area as follows: children under 1 year,
fever plus any parasitemia; children older than 1 year, fever plus a parasitemia of
greater than 2,500/?l (44). Participants were only included in the study if they
were present for all but three of the weekly visits during the 6 months of
follow-up. For analytical purposes, only the first clinical episode was counted,
although all children continued to be monitored until the close of the study.
Within the cohort, children aged 10 years and less (n ? 280) accounted for nearly
90% of all the clinical episodes. The current analysis focused on children who
were slide positive (they were all asymptomatic) at the time the cross-sectional
blood sample was collected (n ? 119; details are provided below in the descrip-
tion of the statistical analysis). Data on antibody responses to AMA1, MSP-2,
and MSP-3 were previously published (50, 53, 54), while data on the remaining
antigens (EBA-175, MSP-119, and MSP-1 block 2) are reported here (see Fig. S1
in the supplemental material).
Case-control study. The findings in the Chonyi cohort were validated in a
separate, spatially and temporally distinct group of children investigated in an
analysis of parasite antigens on the infected red blood cell surface that was
previously conducted in Kilifi (7). Children were recruited from an area imme-
diately surrounding the administrative town of Kilifi, with an entomological
inoculation rate of approximately 1.5 to 8 bites/person/year (39). In May 1995, a
cross-sectional survey was carried out during which finger prick blood samples
were collected from 4,783 children (aged 1 to 5 years), from which sera were
separated (stored at ?70°C) and thick blood smears were prepared and exam-
ined for parasites. Over the following 8 months, children who were part of this
survey and presented to hospital with malaria that was severe enough to warrant
pediatric ward admission were identified (passive case detection). A total of 89
children from the survey were admitted during that period and were frequency
matched, allowing for age and location of children (n ? 298) who took part in the
cross-sectional survey but did not present to hospital with malaria. Sera from the
cross-sectional survey for cases and controls (n ? 387) were analyzed for immu-
noglobulin G (IgG) antibodies to AMA1, MSP-2, MSP-3, EBA-175, MSP-1
block 2, MSP-119, and P. falciparum schizont extract. Ethical approval was
granted by the Kenya National Research Ethics Committee.
Recombinant antigens. The recombinant antigens were expressed in Esch-
erichia coli as glutathione S-transferase–fusion proteins (MSP-2_Dd2 and
MSP-2_CH150/9) (66), MSP-1 block 2 (RO33, Palo Alto, 3D7, MAD20, and
Wellcome) (12), and MSP-119(9), His-tagged (AMA1_3D7) (23) and EBA-
175_F2_CAMP (51), or as maltose-binding protein–fusion proteins (MSP-3_K1
and MSP-3_3D7 (56). Recombinant AMA1_FVO (35) was expressed in Pichia
pastoris, while EBA-175_F2_3D7 (19) is a baculovirus-expressed product. Details
on these antigens are provided in Table S1 of the supplemental material.
Antibody assays. Enzyme-linked immunosorbent assays (ELISAs) against
each recombinant antigen and against parasite schizont extract were performed
according to a standard protocol as previously described (50, 53, 54). Individual
wells of Dynex Immunolon 4HBX ELISA plates (Dynex Technologies Inc.) were
coated with 50 ng of antigen per 100 ?l of carbonate coating buffer (15 mM
Na2CO3, 35 mM NaHCO3, pH 9.3). Wells were coated with P. falciparum
schizont extract (the A4 strain for the Chonyi cohort and Wellcome strain for the
hospital cohort) in phosphate-buffered saline (PBS) according to the method of
Ndungu et al. (45). Plates were incubated overnight at 4°C before washing four
times in PBS-Tween (PBS–0.05% Tween 20) and blocking for 5 h at room
temperature with 1% skimmed milk in PBS-Tween (blocking buffer). Following
this, wells were washed again and incubated overnight at 4°C with 100 ?l of test
serum (1/1,000 dilution in blocking buffer). Plates were then washed four times
and incubated for 3 h at room temperature with 100 ?l of horseradish peroxi-
dase-conjugated rabbit anti-human IgG (Dako Ltd.) at a 1/5,000 dilution in
blocking buffer before final washing and detection with H2O2and o-phenylene-
diamine (Sigma). The reaction was stopped with 25 ?l of 2 M H2SO4per well,
and absorbance was read at 492 nm. The same positive controls (hyperimmune
sera) were run in duplicate on each day of the experiment, on each plate, to allow
for standardization of day-to-day and plate-to-plate variations. Single-dilution
serum ELISA optical density (OD) values were used as proxies for antibody
titers, as they correlate closely with full endpoint antibody titrations when used
at appropriate dilutions (22, 67).
Statistical analysis. All data analyses were performed with STATA version 9.2
(StataCorp, College Station, TX). Models were first developed using data from
the Chonyi cohort and subsequently validated in the case-control study with
some modifications (below). The primary analysis was on the subgroup of 119
children from the Chonyi cohort (n ? 280) who were asymptomatically parasit-
ized at the time of serum collection in October 2000, because in previous
analyses, P. falciparum parasitemia at the time of serum collection modified the
effects of antibodies to both variant red blood cell surface (6) and merozoite (50,
53, 54) antigens on the risk of disease. The confounding effects of exposure on
antibody responses were controlled for by adjusting both for age as well as
antibody reactivity to parasite schizont extract in multifactorial analyses.
The probability of a clinical episode for each antigen (and each allelic form)
for given antibody levels was estimated by logistic regression, fitting ELISA OD
values for the antigen as a linear covariate and adjusting for age (in 2-year
categories). The logits from these models were converted into probabilities (43)
to give estimates of risk (see Fig. 1, below). These analyses established that for
VOL. 76, 2008 BREADTH AND MAGNITUDE OF ANTIBODY RESPONSES2241
most antigens and antibodies, higher antibody levels were associated with a lower
risk of disease and that allelic versions of the same antigen (or the same allelic
family for MSP-1 block 2) generally gave similar patterns of protection. The
probability plots were used to define a threshold (cutoff) for high versus low/
undetectable antibodies as the OD level above which the risk of disease was
lower than the population’s average risk of 33.6% (i.e., the risk of disease
assuming no role for any antibodies) (see Fig. 1, below; see also Table S2 in the
supplemental material). The suitability of the logistic model was confirmed by
examining the residuals when the OD data were fitted in quintiles (data not
shown). The individual effects of high levels of each antibody on the risk of
disease were then reanalyzed, fitting antibody level as a factor rather than as a
linear covariate (Table 1) for ease of interpretation and to facilitate analyses of
the breadth and the interactions between antibodies. To avoid the lack of con-
vergence commonly encountered in conventional binomial regression analyses,
data were fitted to a modified Poisson regression model with robust error vari-
ance, which tends to provide conservative results (69).
Antibodies to different allelic forms of most antigens (AMA1, MSP-2, MSP-3,
and the F2 subdomain of EBA-175) and to the main allelic types of MSP-1 block
2 (K1 and MAD20 types) were highly correlated (see Table S3 in the supple-
mental material). Consequently, high levels of antibodies to only one allelic form
of each antigen were considered for the analysis of antibodies to multiple anti-
gens. Antibodies to MSP-1 block 2 (MSP-1_B2) were highly correlated only
within the main allelic families, and so for this antigen, antibodies to one antigen
from each of the three main allelic families (MAD20-like, K1-like, and RO33-
like) were included to give an overall MSP-1 block 2 response (any of MSP-
1_B2_Wellcome, MSP-1_B2_3D7, or MSP-1_B2_RO33). The criteria used to
select antibodies to a single allelic form of each antigen have been outlined (see
text in the supplemental material). The antigens retained for further analyses
(breadth and combined responses) were AMA1_3D7, MSP-2_Dd2, MSP-3_K1,
EBA-175_3D7, MSP-119, and MSP-1 block 2 (overall response).
Breadth was analyzed in an age- and schizont extract-adjusted modified Pois-
son regression model that compared the risk of disease among children who had
high levels of antibodies (fitted as a fixed level factor) to between one and six
antigens to those who had low/undetectable antibodies to all six antigens. The
combination of antibodies that was associated with the lowest risk of clinical
malaria was determined by analyzing all pair-wise combinations, investigating
interactions between antigens by fitting a model with two main effects and an
interaction term. Interaction as presented here refers to statistical interaction
where the estimate of risk obtained for antibodies to two antigens is significantly
lower than expected (i.e., lower than the product of the individual risk ratios). It
does not exclude biological interaction. To make certain that we were not simply
measuring correlated antibodies arising from shared exposure, we separately
included antibodies to all antigens in a single regression model, together with age
and reactivity to schizont extract, dropping each out sequentially in decreasing
order of their P values. Antigens that remained significant in this model at the
P ? 0.10 level were MSP-2, MSP-3, and AMA1.
Data on children from the hospital cohort were analyzed essentially as de-
scribed above with minor modifications. Models were fitted to data from the
entire hospital cohort (not only the subgroup that were parasitemic at the time
of serum collection) because it appeared that frequency matching of cases and
controls for location (and therefore exposure) successfully eliminated the inter-
action between the antibody’s protective effect and parasite infection status.
Magnitude of antibody response and protection. The prob-
ability of developing an episode of clinical malaria for a given
value of measured antibody level (OD) was estimated for each
antigen. We found that the levels of serum antibodies to some,
but not all, vaccine candidate antigens were inversely related to
the probability of developing malaria (Fig. 1). Increasing OD
levels to MSP-2, MSP-3, AMA1, and the MAD20-like antigens
of MSP-1 block 2 (denoted Wellcome and MAD20) were as-
sociated with reduced probability of malaria morbidity, while
those to MSP-119, EBA-175, and the K1- and RO33-like anti-
gens of MSP-1 block 2 had little effect. Within these loci (and
within the main allelic families for MSP-1 block 2), the pat-
terns were similar for the different allelic forms. Increased
antibody titers to whole parasite schizont extract were also
associated with a reduced probability of clinical malaria.
Breadth and protective efficacy of antibody response. The
probability plots (Fig. 1) were used to define a threshold (cut-
off) for high versus low/undetectable antibodies for each anti-
gen. This threshold varied both by antigen and by population
(the Chonyi cohort and the case-control study), ranging from
relatively low OD values for the MSP-1 block 2 antigens to
high values for MSP-2 (see Table S2 in the supplemental ma-
terial). Children who had high levels of antibodies to one, two,
three, four, five, or six unrelated (nonallelic) antigens were
compared with those who did not have high levels to any
antigen to test the hypothesis that the breadth of specificities
for unrelated antigens in the antibody response is important
for protection. The risk of malaria was inversely associated
with increasing breadth of antibody specificities in both study
groups (Fig. 2). None of the children in the Chonyi cohort who
had high-titer antibody responses to five or more antigens
TABLE 1. Protective effectsaof high levels of antibodies to individual antigens
% of children with
Age and schizont adjustedb
Risk ratio (95% CI)P valueRisk ratio (95% CI)P value Risk ratio (95% CI)P value
aRisk of developing clinical malaria associated with high titers compared to low/undetectable titers of antibodies to individual antigens in a subset of the Chonyi
cohort (n ? 119). Antigens are designated by their locus name and P. falciparum strain (?locus?_?strain?).
bRisk ratios (with 95% confidence intervals ?CI?) are presented for univariate and multivariate analyses (adjusted initially for age and subsequently for both age and
reactivity to P. falciparum parasite schizont extract as a proxy for exposure). *, P ? 0.05.
2242 OSIER ET AL.INFECT. IMMUN.
(17/119; 15%) experienced a clinical episode (P ? 0.0006 by
Fisher’s exact two-tailed test). Similarly, in the case-control
study, none of the children who had high-titer responses to five
or more antigens (23/298; 7.7%) was admitted to hospital with
severe malaria (P ? 0.004, Fisher’s exact two-tailed test).
Breadth of antibody specificity increases with age and con-
current parasitemia. The breadth of high-titer antigen-specific
responses increased with age in both groups of children (Fig.
3). Parasite positivity at the time of serum collection signifi-
cantly increased the breadth of the response. In the Chonyi
cohort, nearly three times as many children who were para-
sitemic at the time of serum collection had high antibody titers
to three or more antigens, compared to those who were apara-
sitemic (47% [56/119] versus 17.3% [28/161]; Pearson’s chi-
square, 28.67; P ? 0.001). This difference was more marked in
the case-control study, with over five times as many children
who were parasitemic at serum sampling having high-titer re-
sponses to three or more antigens compared to those who were
not parasitemic (57% [102/176] versus 10.4% [30/287]; Pear-
son’s chi-square, 120.77; P ? 0.001).
Combinations of antibodies and protection. Interactions
between antibodies were investigated to determine which com-
bination(s) was associated with the lowest risk of clinical epi-
sodes in the Chonyi cohort. High levels of antibodies to com-
binations that included MSP-2, MSP-3, and AMA1 were
associated with a lower risk of disease compared to their indi-
vidual effects (Table 2). While the combined effects of anti-
bodies were always greater than each of the individual effects,
there was no statistical evidence of synergism or antagonism,
i.e., more or less protection, respectively, than expected from
the combination of the two antigens acting additively. The
strongest protection was associated with high levels of antibod-
ies to both MSP-2 and MSP-3. Thirty-three children (of 119)
had high antibody levels to both MSP-2 and MSP-3, and none
of them experienced any episodes of disease (P ? 0.001 by
Fisher’s exact two-tailed test; still highly significant after a
Bonferroni correction , allowing for multiple comparisons,
P ? 0.003). This finding was validated in the case-control study,
where admission to hospital with malaria was the end point.
Children who had high levels of antibodies to both MSP-2 and
MSP-3 were significantly less likely to be admitted to hospital
with malaria (odds ratio, 0.26; 95% confidence interval, 0.08 to
0.81; P ? 0.020) (see Table S5 in the supplemental material).
FIG. 1. The predicted probability of an episode of malaria in children decreases with increasing antibody titer for most antigens (n ? 119). Each
panel represents the allelic antigens tested at each locus, as well as parasite schizont extract. The red horizontal line represents the risk of an
episode without taking antibody responses to any antigen into account. The final panel combines antibodies to one allelic form of each antigen
(and one antigen from each of the three main allelic families of MSP-1 block 2). The lines, from top to bottom, represent MSP-1_B2_Wellcome,
MSP-3_K1, MSP-2_Dd2, schizont extract, AMA1_3D7, EBA-175_F2_3D7, MSP-1_B2_3D7, MSP-1_B2_RO33, and MSP-119.
FIG. 2. Protective efficacy increased with increasing breadth of re-
sponse in children from the Chonyi (parasitemic children, n ? 119) (a)
and hospital cohorts (all children, n ? 387) (b). Each bar represents
the comparison between individuals making high-titer responses to n
number of antigens with those who make no responses to any antigen.
Proportions above each bar are the percentage of individuals making
high-titer responses to n antigens. The effect of high-titer responses to
P. falciparum schizont extract is also shown.
VOL. 76, 2008 BREADTH AND MAGNITUDE OF ANTIBODY RESPONSES2243
We found that in two independent studies conducted in both
high-transmission (Chonyi cohort) and low-transmission (case-
control study) settings at different times, both the breadth of
specificity for distinct merozoite antigens and the magnitude
of antibody responses to these antigens provide robust pre-
dictors of the immune status of children. High titers of
antibodies to combinations of three merozoite antigens in
particular (AMA1, MSP-2, and MSP-3) were more strongly
predictive of protection from clinical episodes of malaria
compared to other putative “protective” merozoite antigens
(MSP-1 or EBA-175).
Out of the panel of malaria vaccine candidate antigens stud-
ied here, high levels of antibodies to combinations that in-
cluded AMA1, MSP-2, and MSP-3 were the most strongly
associated with protection. This is consistent with other studies
in which naturally acquired antibodies to each of the three
antigens individually have been associated with protection
from clinical malaria in this and other populations (1, 41, 42,
53, 54, 61, 63, 65, 66). Recently, long-term clinical protection
was associated with IgG3 isotype antibodies to MSP-3 in Sen-
egalese children (58). In contrast, antibodies to MSP-1 block 2,
which have been associated with protection in two cohorts in
West Africa (10, 15, 55), were not similarly protective in the
two cohorts we studied from Kilifi, Kenya. Antibodies to MSP-
119have been associated with protection from clinical malaria
in some studies, but not in others (5, 10, 15, 21, 24, 31, 52, 57).
This may be explained in part by the finding that the fine
specificity of anti-MSP-119antibodies appears to be more im-
portant with regards to protection (17, 48). A separate study
found that individuals with high-titer anti-MSP-119-specific in-
vasion-inhibitory antibodies were protected from infection (33)
and underscored the importance of developing robust func-
tional assays for malaria. Antibodies to the F2 subdomain of
EBA-175 were not associated with protection from clinical
disease in our studies, as has been found in other parts of
Africa where this (49) and other subdomains of EBA-175 (32,
47, 49) have been studied. To date, only one study has reported
significantly higher antibody levels to EBA-175 peptide 4 (res-
idues 1062 to 1103, within region V) in children protected from
clinical attacks of malaria compared to susceptible children
The importance of allele-specific immunity was highlighted
in the Combination B malaria vaccine trial in Papua New
Guinea. Children who received this vaccine (containing a com-
bination of P. falciparum ring-infected erythrocyte surface an-
tigen, MSP-1, and the 3D7 allele of MSP-2) were less likely to
be infected with parasites bearing the homologous allele of
MSP-2 (28), suggesting (as was later confirmed) that the vac-
cine had induced primarily allele-specific MSP-2 antibodies
(26). In the context of naturally acquired infections, while
some data suggest that parasites bearing specific genotypes
induce allele-specific antibodies (11, 34, 53), to our knowledge
no studies have examined the protective effects of preexisting
allele-specific antibodies on subsequent disease caused by par-
asites bearing homologous alleles. We found that for most
antigens tested, responses to allelic forms of each antigen had
similar effects on the probability of mild or more severe ma-
laria, suggesting the possibility that there may be significant
cross-allele protection to clinical episodes.
In a study conducted in The Gambia, Gray et al. (29) found
that while antibodies to a similar panel of individual antigens
were only weakly correlated with protection, those to the com-
binations of AMA1 and MSP-2 were significantly associated
with protection from clinical malaria. There are two important
differences between this Gambian study and the results re-
ported here from Kenya. First, k-means clustering and phylo-
genetic networks were used to investigate associations between
antibody reactivity profiles and clinical status in the Gambian
cohort. These methods independently identified the group of
children who were asymptomatic (asymptomatic parasitemia,
splenomegaly, or both) at the end of the study and who had not
apparently experienced clinical disease. That end point differs
from that of the studies reported here, in which outcome was
simply defined as mild (Chonyi cohort) or severe (case-control
study) malaria during the period of observation. Second, the
magnitude of responses was not taken into account, mainly
because this generates increased individual differences, impair-
ing cluster analysis. One other longitudinal study, carried out
among children in Burkina Faso, examined antibodies to a
different set of blood-stage malaria antigens (glutamate-rich
protein, P. falciparum exported protein 1, and MSP-3) and, like
our studies, they found that the simultaneous presence of an-
tibodies to more than one antigen was associated with a lower
frequency of malaria episodes (41). However, in a separate
study on protection from malaria infection as opposed to clin-
FIG. 3. The breadth of antibody specificity increased with age in both the Chonyi cohort (n ? 119) (a) and the case-control study (n ?
2244 OSIER ET AL.INFECT. IMMUN.
TABLE 2. Protective effectsaof combinations of high-titer antibody responses
Main effects and interactionb
Risk ratio (95% CI)d
P value Risk ratio (95% CI)d
AMA1_3D7 ? MSP-3_K1
0.21 (0.05–0.88) 0.033*
AMA1_3D7 ? MSP-2_Dd2
0.24 (0.09–0.64) 0.004*
AMA1_3D7 ? EBA-175_F2_3D7
AMA1_3D7 ? MSP-119
MSP-2_Dd2 ? MSP-3_K1
Total protection (n ? 33)
Total protection (n ? 33)
MSP-2_Dd2 ? EBA-175_F2_3D7
MSP-2_Dd2 ? MSP-119
0.52 (0.21–1.30) 0.166
MSP-3_K1 ? EBA-175_F2_3D7
MSP-3_K1 ? MSP-119
EBA-175_F2_3D7 ? MSP-119
0.69 (0.26–1.78) 0.445
AMA1_3D7 ? MSP-1_B2
MSP-2_Dd2 ? MSP-1_B2
MSP-3_K1 ? MSP-1_B2
0.16 (0.02–1.13) 0.067
0.98 (0.48–2.01) 0.972
EBA-175_F2_3D7 ? MSP-1_B2
0.85 (0.36–2.00) 0.720
aRisk of developing clinical malaria associated with combinations of high titers compared to low/undetectable titers of antibodies to individual antigens in a subset
of the Chonyi cohort (n ? 119). In the majority of cases, significantly more protection was obtained with high-level antibody responses to pairs of antigens, compared
to single antigens (Table 1). No strong evidence of statistical interaction between pairs of antibodies was observed.
bThe main effects of antibodies to each antigen were adjusted for each other. Interaction effects (combinations of two antigens) are those over and above the main
cEffects of combinations of high-titer responses (combines the main effects and interaction effects).
dRisk ratios (with 95% confidence intervals ?CI?) are from multivariate analyses (adjusted for both age and reactivity to P. falciparum schizont extract). *, P ? 0.05.
VOL. 76, 2008BREADTH AND MAGNITUDE OF ANTIBODY RESPONSES2245
ical episodes in Kenyan adults, John et al. (32) found that high
antibody titers to multiple blood-stage antigens were not pro-
tective (though there was evidence of protection for responses
to preerythrocytic antigens). Our data suggest that the combi-
nation of blood-stage antigens analyzed in these Kenyan adults
(AMA1, EBA-175, and MSP-119) may not have been optimal.
While these studies are difficult to compare directly due to
differences in study design, study populations and end points,
antigens tested, and analytical methodologies, the picture that
nevertheless emerges clearly is that antibodies to key combi-
nations of multiple parasite targets are more strongly associ-
ated with protection from clinical malaria than are antibodies
to individual antigens.
With the completion of the P. falciparum genome, numerous
new (and old) antigens of the parasite have been identified and
are being characterized. High-throughput assays employing
suspension array technology (27) or microarrays (29, 64) now
allow for simultaneous analysis of antibodies to multiple anti-
gens using minimal amounts of sera. This technology has not
been matched with equivalently efficient tools for identifying
protective immune responses. Robust concurrent analyses of
numerous responses in relatively small studies, where children
have been monitored longitudinally over a limited time period
for disease episodes, remain challenging. The pair-wise analy-
ses of combinations of high-titer antibody responses as pre-
sented here have obvious limitations when numerous antibod-
ies are to be analyzed. Other analytical techniques, such as
clustering and the use of phylogenetic networks (29), while
attractive for screening of potential vaccine candidates, simi-
larly become more complex when increasing numbers of re-
sponses are analyzed and may well obscure genuinely “protec-
tive” responses. New strategies to identify protective responses
in humans are urgently needed.
Studies of associations between immune responses and clin-
ical malaria need to take into account the possibility that any
given response is merely a marker of cumulative exposure
(which is itself necessary to induce immunity) or of a response
to an as-yet-unidentified antigen(s) that elicits strongly protec-
tive immunity. In our study, the fact that antibodies to specific
antigens were more strongly predictive of protection than
those to whole-schizont extract (containing all the specific an-
tigens and many other blood-stage antigens) (Fig. 1) suggests
that specific responses do not merely reflect exposure. The
finding that protective efficacy increased with increasing
breadth of antibody specificity indicates that the effect of any
one apparently protective response does not simply result from
correlation with responses to other antigens (Fig. 2) and argues
for the interpretation that these are truly protective responses.
Ultimately, the critical test of any such hypotheses will be to
achieve equivalent protection through vaccination. Our dem-
onstration of strong protection against malaria associated with
high antibody levels to AMA1, MSP-2, and MSP-3 lends sup-
port to the development of vaccines based on combinations of
these key malaria antigens.
This paper is published with the permission of the director of
We thank Chetan Chitnis for provision of the recombinant antigen
representing the F2 subdomain of EBA-175. We also thank Moses
Mosobo for his invaluable assistance in the laboratory and Britta
Urban for helpful comments on the manuscript.
This work was supported by a research training fellowship for F.O.
from the Wellcome Trust, grant no. 073591.
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