INFECTION AND IMMUNITY, Mar. 2009, p. 1189–1196
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 3
Evidence for Multiple B- and T-Cell Epitopes in
Plasmodium falciparum Liver-Stage Antigen 3?
Aissatou Toure-Balde,1Blanca-Liliana Perlaza,2Jean-Pierre Sauzet,2Mouhamadou Ndiaye,1
Georgette Aribot,1Adama Tall,1Cheikh Sokhna,3Christophe Rogier,4Giampietro Corradin,5
Christian Roussilhon,2and Pierre Druilhe2*
Institut Pasteur de Dakar, Dakar, Senegal1; Bio-Medical Parasitology Unit, Institut Pasteur, Paris, France2; Institut de Recherche et de
Developpement, Dakar, Senegal3; Institut de Medecine Tropicale du Service de Sante des Armees, Marseille, France4; and
Institute of Biochemistry, University of Lausanne, Lausanne, Switzerland5
Received 8 June 2007/Returned for modification 8 September 2007/Accepted 1 January 2009
Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium
falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the
210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the
villages of Dielmo (n ? 143) and Ndiop (n ? 60) in Senegal (the level of malaria transmission differs in these
two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences
(up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of
responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were
detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria,
although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell
epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum
vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were
found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in
children between the ages of 7 and 15 years, but not in older individuals.
Preerythrocytic malaria antigens are critical targets of pro-
tective immune responses induced by irradiated sporozoites in
humans (9, 22). The demonstration of T-cell-mediated protec-
tion in mice immunized by this means (10, 16), the acquisition
of a significant level of protection against homologous Plasmo-
dium falciparum challenge in human volunteers (22), and the
induction by liver-stage antigens of a high level of protection
against P. falciparum infection in chimpanzees (19, 31) all
point to a major role for preerythrocytic stage antigens as
Liver-stage antigen 3 (LSA-3) is a novel antigen expressed at
the preerythrocytic stages (4). LSA-3 was selected by the dif-
ferential immune response found between protected and non-
protected volunteers, both similarly immunized with irradiated
sporozoites (4, 9). The gene encoding LSA-3 is unusually well-
conserved (4), in contrast with many other malaria vaccine
candidates (11, 19, 23). More than 10 dominant T-helper (Th),
cytotoxic T-lymphocyte, and B-cell epitopes have already been
characterized in LSA-3 (20), some of them displaying cross-
reactivity with an homologous antigen in Plasmodium yoelii (2).
The protective potential of LSA-3 was demonstrated by a se-
ries of experiments in chimpanzees and Aotus monkeys chal-
lenged with P. falciparum (4, 21) and in mice challenged by P.
yoelii following immunization either by recombinant proteins
with adjuvant or by formulations without adjuvant, such as
recombinant proteins adsorbed on microparticles or lipopep-
tides in phosphate-buffered saline (PBS) (4), or DNA-based
immunization (29). These convergent results stress the poten-
tial of LSA-3 as a prime vaccine candidate.
We therefore decided to further analyze the antigenicity of
LSA-3 and to investigate immune responses to discrete regions
of the protein in exposed individuals living in areas where
malaria is endemic.
T- and B-cell responses were evaluated in subjects living in
two villages in Senegal, West Africa, where malaria is endemic,
using three small synthetic peptides and a series of 17 over-
lapping long synthetic peptides (LSP) encompassing most of
the LSA-3 protein. In keeping with preliminary results (20), we
found a high prevalence of responses to most regions of this
preerythrocytic stage antigen in individuals of different age
groups. These results bring additional arguments in favor of
the potential of the LSA-3 protein for vaccine development.
MATERIALS AND METHODS
Population studied. A total of 294 inhabitants from Dielmo and Ndiop, two
villages located in a rural area of Senegal (West Africa), 270 km southwest of
Dakar were studied. In Dielmo, a group of 143 villagers and then a second group
of 91 villagers 3 to 87 years of age were enrolled in this study; in Ndiop, a group
of 60 villagers 4 to 71 years of age was enrolled in this study. The villagers were
selected so as to be representative of all age groups and were clinically asymp-
tomatic at the time of the study. The main epidemiological features of these two
villages have been reported previously (26, 27, 32). Entomological and parasi-
tological surveys showed that Dielmo, with approximately 200 to 300 infected
mosquito bites per person per year, is an area where malaria is holoendemic and
characterized by high and perennial parasite transmission (13), whereas Ndiop is
a mesoendemic area where malarial transmission is seasonal, approximately 10
times lower, with ca. 20 to 30 infected bites per person per year (14). Clinical data
were recorded on a daily basis year-round, and malaria attacks were defined as
a fever of ?38.5°C associated with a parasite density over an age-dependent
* Corresponding author. Mailing address: Bio-Medical Parasitol-
ogy Unit, Institut Pasteur, 25 rue du Dr Roux, Paris 75015, France.
Phone: 331 45 68 85 78. Fax: 331 45 68 86 40. E-mail: druilhe
?Published ahead of print on 12 January 2009.
threshold defined for each age group (32). In the present study, malaria attacks
recorded for months following blood sampling were used for the statistical
After informed consent from each individual or their legal representative was
obtained, venous blood samples were collected during winter, i.e., during the
lowest transmission season in both villages, using 10-ml Vacutainers in which 250
IU of preservative-free heparin (Liquemine; Roche) had been added. Blood
samples were transferred to our laboratory in Dakar, Senegal, within 4 to 5 h at
a temperature of 20°C to 25°C.
This study was examined and approved by the National Senegalese Ethical
Peptides. Seventeen overlapping long synthetic peptides (LSP1 to LSP17) 45
to 186 amino acids in length, spanning most of the P. falciparum LSA-3 protein
(Fig. 1) were synthesized by solid-phase 9-fluorenylmethoxy carbonyl (Fmoc)
chemistry as described previously (18, 24, 25). The purity of the LSP was deter-
mined by high-performance liquid chromatography and mass spectrometry and
ranged from 70% to 80%. Impurities consisted mostly of either smaller frag-
ments or full-length peptides differing by one or two t-butyl groups [-C-(CH3)3].
No indication of bacterial endotoxin contamination was found in the peptide
preparations following the use of Limulus amebocyte lysate (Pyrogent Plus;
Cambrex Bio Science). Two peptides, LSP with amino acids 44 to 119 (LSP
44-119) (LSP2) and LSP 100-222 (LSP1), represent most of the nonrepeat region
I (NR-I) from the N-terminal part of the protein. Three peptides, namely, LSP
501-596 (LSP14), LSP 646-706 (LSP13), and LSP 692-854 (LSP12), contain
sequences representative of the repeat region R2. Eight out of the 12 remaining
peptides spanning amino acids 840 to 1517 correspond to sequences from non-
repeat region II (NR-II). One LSP, LSP3 (LSP 1503-1575) covers the repeat
block R3, and the last three peptides spanning residues1601 to 1786 represent
the nonrepeat C-terminal region (NR-III). Each peptide overlapped the follow-
ing or previous one by 15 amino acids, except the sequences 1503-1575 and
1601-1712 for which 26 amino acids were missing in the repeat region R3 (Fig.
1). In addition, three smaller peptides derived from the recombinant protein
DG729 (positions 176 to 325) used previously in immunization experiments and
inducing protection (1, 4, 21), covering part of the NR-I region of the protein
(LSA-3-NRI 176-201 and NR-II 198-222) and part of the R2 region (LSA-3-Re
300-327) (4), were tested in parallel.
ELISAs. Preliminary assays showed that 5 ?g/ml and 1 ?g/ml concentrations of
the LSA-3 short and long synthetic peptides, respectively, gave optimal enzyme-
linked immunosorbent assay (ELISA) results. The peptides, diluted in PBS were
added to each well of 96-well flat-bottomed polystyrene microtiter plates (Im-
mulon) and incubated at 4°C overnight. After the wells were washed five times
with PBS containing 1% Tween, 200 ?l of PBS with 1% bovine serum albumin
blocking buffer was added to each well, and the plates were incubated for 1 h at
Each plasma sample was tested in duplicate in peptide-coated wells for total
immunoglobulin G (IgG) and each IgG subclass (IgG1 to IgG4). The plasma
samples, diluted 1/100, were added to the wells (100 ?l/well), and the plates were
incubated at 37°C for 90 min. After the wells were washed, the plates were
incubated at 37°C for 1 h in the presence of peroxidase-conjugated goat F(ab?)2
fragment to human IgG Fc (gamma chain specific; at 1/6,000) (Cappel, France).
For the immunoglobulin isotype determination, the monoclonal antibodies were
purchased from Unipath (Bedford, United Kingdom) and corresponded to
mouse anti-IgG1 (clone NL16) at 1/2,000, anti-IgG3 (clone ZG4) at 1/10,000,
and anti-IgG4 (clone GB7B) at 1/30,000. Anti-IgG2 antibody (clone HP6002)
was used at 1/10,000 and purchased from Sigma. Plates were treated as previ-
ously described (1), and optical density (OD) values at 450 nm were read by using
Titertek multiscan apparatus (Flow Laboratories). The ELISA reader was set so
as to subtract the reading of a blank control from the test samples. Each plasma
sample was analyzed in the presence of 10 negative-control and 10 positive-
control plasma samples. In addition, a negative-control standard (corresponding
to a pool of plasma samples from naïve French donors) and a positive-control
standard (corresponding to a pool of positive plasma samples from Senegalese
donors) were included in each assay. A sample was considered positive (i.e.,
above the threshold of positivity) if the mean OD reading of the duplicates was
higher than the mean OD value plus 3 standard deviations (SD) of the negative
samples. Therefore, each absorbance value above the threshold was considered
a positive OD.
Lymphoproliferation assays. Peripheral blood mononuclear cells were iso-
lated from heparinized whole blood by Histopaque-1077 density gradient (Sigma
Diagnosis, St. Louis, MO) and washed three times in PBS. Cultures of peripheral
blood mononuclear cells were grown in 96-well round-bottomed plates (Nun-
clon) at a final concentration of 106cells/ml and in a total volume of 200 ?l of
RPMI 1640 culture medium supplemented with 10% (vol/vol) heat-inactivated
pooled human serum AB (from the French National Center for Blood Transfu-
sion, France), 1 mM glutamine, 35 mM HEPES, 1% sodium pyruvate, 2 g/liter
sodium bicarbonate, and 10 mg/liter gentamicin. The peptides tested were added
at a final concentration of 10 ?g/ml. The plates were thereafter incubated at 37°C
in a water-saturated atmosphere containing 5% CO2. Unstimulated (control)
and stimulated cells were cultured in parallel. For a positive control, we used
tuberculin (purified protein-derived tuberculin from the Statens Serum Institute,
Copenhagen, Denmark) at a final concentration of 2.5 ?g/ml. Cultures were
pulsed on day 6 with [3H]thymidine (1 ?Ci per well) 16 h before harvesting, and
the radioactivity incorporated was evaluated using a liquid scintillation counter.
Cells from five unexposed donors tested under the same conditions with the
various peptides were included as negative controls (no significant proliferative
response was observed using lymphocytes of the unexposed donors). Results are
expressed as stimulation indices (SI), defined as the mean counts per minute
(cpm) of cells recovered from triplicate wells containing antigen divided by the
mean cpm of control cells recovered from triplicate wells without antigen. The
cutoff value for positivity was a SI value higher than 2 and a change in cpm of
Statistical analysis. Mean antibody responses were determined on antibody
“responders,” i.e., on individuals with positive OD values. The Spearman rank
correlation test was used for comparison of antibody responses in the different
age groups (Stat View software; SAS Institute Inc., Cary, NC). The relationship
between all the antibody responses (using continuous data) and the risk of
FIG. 1. Schematic representation of the Plasmodium falciparum LSA-3 protein showing the localization of the peptides employed, the 17 long
synthetic peptides (LSP1 to LSP17) and the 3 smaller peptides (LSA-3-NRI, LSA-3-NRII, and LSA-3-RE) encompassing most of the LSA-3
protein. The localization of DG729 is indicated. The numbers are amino acid positions.
1190 TOURE-BALDE ET AL.INFECT. IMMUN.
malaria attack occurrence during the 12 months following the blood sampling
was tested using a Poisson regression model where the number of malaria attacks
was tested as the dependent variable and the number of days of follow-up (i.e.,
exposure to P. falciparum infection) as well as antibody responses as the inde-
pendent variables (Stata software; Stata Corporation, College Station, TX). The
relative risk associated with the different variables within each age group was
estimated by the generalized estimating equation model. This model is based on
the inclusion or exclusion of the antibody responses tested, and in each case, the
fraction of the clinical immunity acquired in each age group and attributable to
each LSP peptide response was calculated. The effect of antibody response was
tested separately for each of the 17 LSP and was considered significant only when
its P value was below 0.003 (? 0.05/17; i.e., after Bonferroni’s correction) to take
into account the multiplicity of tests.
Antigenicity of the nonrepeat (LSA-3-NRI and LSA-3-NRII)
and repeat (LSA-3-Re) peptides of LSA-3. We first determined
in the inhabitants of Dielmo, Senegal, the cellular and humoral
responses directed against three short peptides, NRI, NRII,
and RE, which cover DG297, the recombinant protein with
which most protection data has been obtained in primates as
described previously (1, 4, 21). Results from lymphoprolifera-
tive assays carried out in various age groups are shown in Table
1. Out of 143 Dielmo inhabitants tested, 39% responded to
peptide LSA-3-NRI, 37% responded to LSA-3-NRII, and 44%
responded to LSA-3-Re. The prevalence of responders was
high in children less than 10 years old, slightly lower in ado-
lescents (11 to 15 years old), and highest in young adults (16 to
20 years old). However, the prevalence decreased in subjects
older than 20 years, in contrast with responses to many other
malarial antigens (26–28). The association between a decrease
in proliferative response and an increase in age for the three
synthetic peptides tested was more prominent in the 143 vil-
lagers tested (as indicated by Spearman rank correlation tests)
for LSA-3-Re (? ? ?0.287; P ? 0.0005) than for LSA-3-NRI
(? ? ?0.218; P ? 0.008) and for LSA-3-NRII (? ? ?0.152;
P ? 0.067).
Antibody responses determined by ELISAs showed a
marked difference between repeat and nonrepeat regions. Al-
though the prevalence of antibody responses against LSA-3-
NRI and LSA-3-NRII was high (i.e., 40% and 60%, respec-
tively, of the individuals tested [data not shown]), the mean
antibody responses were low and did not change much with
age. Of note, both the prevalence and level of antibodies di-
rected to the repeat region LSA-3-Re were high, with a prev-
alence of ca. 70% in children ?5 years old, and the mean OD
values increased with age as illustrated in Fig. 2.
Immune responses to 17 long synthetic peptides spanning
the LSA-3 protein. Further studies relied on 17 LSP covering
most of the 210-kDa LSA-3 protein, and the antigenicity of
LSA-3 was tested in inhabitants of the Dielmo and Ndiop
villages in Senegal.
T-cell assays of the inhabitants of Dielmo, Senegal, showed
that with the notable exception of LSP14 and LSP16, which
were recognized by only 3.1% and 9.4% of the individuals,
TABLE 1. Results of the lymphoproliferative assays carried out in
Dielmo, Senegal, using three peptides encompassing the repeat
and nonrepeat subregions of LSA-3
% of responders
Mean SI ? SDb
3.77 ? 0.90
3.74 ? 1.20
3.96 ? 1.38
3.78 ? 0.92
3.55 ? 1.23
3.48 ? 1.21
3.32 ? 0.86
3.61 ? 0.91
4.86 ? 1.24
3.45 ? 1.05
3.42 ? 0.89
2.46 ? 0.43
3.84 ? 1.33
3.88 ? 0.96
4.35 ? 1.38
3.78 ? 1.42
3.15 ? 0.77
3.72 ? 1.24
Total 39.20 (56/143)3.73 ? 1.093.62 ? 1.103.77 ? 1.17
aFor each age group, the percentage of responders to one peptide or all three
peptides is shown. The number of responding individuals (n) and the total
number of subjects tested in each age group (N) is shown in parentheses.
bThe mean stimulation index ? standard deviation is shown for each peptide.
The mean SI of mononuclear cell proliferation is given with reference to the
control wells (i.e., autologous cells maintained in complete culture medium but
without antigen). LSA-3-NRI and LSA-3-NRII correspond to the nonrepeat
regions of LSA-3, and LSA-3-Re corresponds to the repeat region of the antigen.
FIG. 2. Prevalence of individuals having antibodies to the LSA-3-Re repeat and trend for increase with age in the mean levels of antibodies
in Dielmo, Senegal. The percentages of responders to LSA-3-Re (A) and the mean levels of antibody responses to LSA-3-Re antigen (B) are shown
for various age groups (age in years). The total number of villagers tested was 143, and the number of individuals included in each age group is
the same as that indicated in Table 1. The bars in panel A correspond to 95% confidence intervals of the percentages, and the bars in panel B
correspond to standard deviations.
VOL. 77, 2009ANTIGENICITY OF PLASMODIUM FALCIPARUM LSA-3 IN HUMANS1191
respectively, the other 15 LSP induced a lymphoproliferative
response in 17% to 50% of the inhabitants of Dielmo studied
(Table 2). The highest stimulation indices were found in
Dielmo for LSP13, a peptide derived from the repeat region of
LSA-3 (mean SI ? SD, 14.2 ? 21.3). A somewhat different
pattern was found in 43 inhabitants from Ndiop, Senegal (Ta-
ble 2) in whom the highest prevalences of cellular responses
were found for LSP10, LSP11, and LSP12, followed by LSP17,
LSP16, LSP4, and LSP9. Prevalences of responses to LSP14
and -16 were markedly higher in Ndiop (reaching 25.6 and
48.8%) than in Dielmo (P ? 0.072 and P ? 0.0071 for LSP14
and LSP16, respectively) despite the lower transmission pre-
vailing in Ndiop. T-cell responses were thus detected in the two
villages to every single peptide tested, sometimes at preva-
lences above 55% in Ndiop. The combined pattern of indi-
vidual responses suggests that each peptide likely defines an
individual, non-cross-reactive epitope. Finally, the T-cell an-
tigenicity of the protein is supported by the fact that the
prevalences of some proliferative responses in the presence
of certain LSA-3 peptides were as high in inhabitants from
Ndiop receiving 10-fold-less infected mosquito bites than in
IgG antibodies directed against the 17 LSP were evaluated
in the plasma samples from 91 individuals living in Dielmo,
Senegal, and 60 individuals from Ndiop, Senegal. As shown in
Table 3, each of the LSP defined at least one B-cell epitope but
with a wide range of prevalences and of antibody levels. In
agreement with the results initially obtained with the smaller
LSA-3-Re peptide, the highest antibody levels were obtained
in response to LSP12 in Dielmo and in response to a peptide
situated in the R2 repeat region of LSA-3 in Ndiop. Antibody
prevalence was also maximal to this peptide in Dielmo. How-
ever, both repeat and nonrepeat regions of LSA-3 were tar-
TABLE 2. Proliferative responses of mononuclear cells obtained
from inhabitants of the villages of Dielmo and Ndiop in
Senegal by using 17 long synthetic peptidesa
Mean SI ? 1 SD
Mean SI ? 1 SD
4.6 ? 3.3
4.6 ? 3.3
2.2 ? 0.3
14.2 ? 21.3
6.1 ? 5.7
5.8 ? 5.4
4.2 ? 2.7
4.4 ? 3.2
4.4 ? 3.9
12.9 ? 19.6
6.9 ? 8.9
7.9 ? 9.8
7.5 ? 12.6
4.0 ? 2.4
3.4 ? 1.2
2.3 ? 0.3
4.1 ? 1.5
11.3 ? 15.8
4.9 ? 3.4
3.2 ? 1.3
4.1 ? 1.9
9.9 ? 13.3
14.5 ? 15.6
10.0 ? 8.8
12.1 ? 13.2
8.4 ? 4.9
5.6 ? 4.0
4.3 ? 2.9
9.4 ? 12.3
9.1 ? 10.1
11.2 ? 13.2
2.1 ? 0.1
8.2 ? 7.5
8.1 ? 8.9
PPD74.3 (52/70)19.2 ? 21.890.7 (39/43)89.9 ? 110.4
aMononuclear cells were cultured in the presence of the various LSA-3-
bThe 17 LSP tested (LSP1 to LSP17, which encompass most of the LSA-3
protein and are shown in order from the N terminus to the the C terminus of
LSA-3) are shown. Purified protein-derived tuberculin (PPD) was used as a
cThe percentage of responders to each peptide is shown. The number of
responding individuals (n) and the total number of subjects tested in each age
group (N) is shown in parentheses.
TABLE 3. Prevalence of IgG antibodies to the different LSP corresponding to subregions of LSA-3 antigen in inhabitants of the villages of
Dielmo and Ndiop in Senegala
Mean OD ? 1 SD for
% of responders (n/N)c
Mean OD ? 1 SDd
% of responders (n/N)Mean OD ? 1 SD
0.523 ? 0.459
0.699 ? 0.328
0.690 ? 0.406
0.581 ? 0.557
1.620 ? 0.726
0.418 ? 0.477
0.848 ? 0.748
0.195 ? 0.237
0.399 ? 0.420
0.639 ? 0.517
0.777 ? 0.625
0.668 ? 0.558
0.885 ? 0.655
0.903 ? 0.697
0.354 ? 0.250
0.632 ? 0.368
0.0550.180 ? 0.05
0.421 ? 0.25
0.200 ? 0.09
0.170 ? 0.05
0.230 ? 0.11
0.160 ? 0.05
0.160 ? 0.03
0.190 ? 0.04
0.150 ? 0.04
0.160 ? 0.05
0.220 ? 0.09
0.210 ? 0.09
0.250 ? 0.02
0.190 ? 0.07
0.240 ? 0.11
0.370 ? 0.14
0.400 ? 0.15
0.686 ? 0.266
0.408 ? 0.274
0.147 ? 0.130
0.907 ? 0.534
0.336 ? 0.286
0.759 ? 0.588
0.307 ? 0.322
0.513 ? 0.495
0.690 ? 0.560
0.775 ? 0.601
0.761 ? 0.632
0.722 ? 0.498
0.420 ? 0.507
0.515 ? 0.419
0.394 ? 0.218
aThe optical density values of the IgG responses (corresponding to the mean OD tests minus the mean OD of the negative controls plus 3 standard deviations) are
bLSP1 to LSP17, which encompass most of the LSA-3 protein, are shown in order from the N terminus to the C terminus of LSA-3.
cFor each of the different antigens tested (LSP1 to LSP17), the percentage of responders is shown. The number of responding individuals (n) and the total number
of individuals (N) are shown in parentheses.
dFor each of the different antigens tested (LSP1 to LSP17), the mean net IgG OD ? 1 standard deviation is indicated.
eTen negative controls.
1192TOURE-BALDE ET AL.INFECT. IMMUN.
geted by specific antibodies, with comparable, high preva-
lences. This was seen for instance for the nonrepeat peptides
LSP4, -7, and -10 encompassing a nonrepeat region, compared
to the three peptides LSP12, -13, and -14 encompassing the R2
repeat region, which is an unusual finding. Here as well, the
combined pattern of individual responses suggests that each
peptide likely defines at least one individual, B-cell epitope,
not cross-reactive with the remaining epitopes as described
previously (20), except for the three peptides (LSP12, -13, and
-14) derived from the R2 repeat region. IgG isotype-specific
antibodies were analyzed in a subset of 33 individuals from
Dielmo. Antibodies to the 17 peptides were found to include
each of the four IgG subclasses, with a high prevalence of
IgG3. The highest IgG3 levels were found in response to
LSP12, with 30 out of 33 individuals responding specifically to
this long synthetic peptide, but no particular indication regard-
ing the pattern of anti-LSA-3 IgG subclass responses was iden-
tified (data not shown).
Age-specific patterns in subjects exposed to medium or high
malaria transmission. Since the inhabitants of the Dielmo and
Ndiop villages in Senegal differ markedly in their level of
exposure to infectious mosquito bites (13, 14) and the pattern
of immune responses (27), we then compared anti-LSA-3 an-
tibody responses as a function of age in both villages. A trend
for an increase in antibody responses to several LSP was ob-
served associated with age in Dielmo and Ndiop villages. An-
tibody responses to the repeat region, particularly to LSP12,
-13, and -14 increased with age in Dielmo. There was also an
increase of antibodies to several peptides from the nonrepeat
region (i.e., LSP3 to LSP-7 and LSP10) with increasing age
that indicated that higher doses of these immunogens were
required to raise antibodies compared to LSP from the repeat
region. The general patterns of age-dependent increase in an-
tibody to the 17 peptides was largely superimposed for each
LSP in the two villages (Fig. 3A and B), but the maximal levels
reached were higher in Dielmo and a trend for a higher per-
centage of villagers with detectable antibody responses to each
LSP was found in this village. Therefore, these results indicate
that most parts of LSA-3 (with a few exceptions, such as LSP2,
LSP15, and LSP16) are antigenic under natural conditions of
exposure and elicit antibody responses both under medium or
high malaria transmission conditions.
Relationship between antibody responses and acquired clin-
ical resistance to malaria. The availability of very detailed
FIG. 3. Levels of antibody responses to the 17 different LSP encompassing the LSA-3 protein in different age groups of inhabitants of the
Dielmo and Ndiop villages in Senegal. The numbering of the different LSP tested correspond to that shown in Fig. 1, i.e., they are indicated from
the N terminus to the C terminus of LSA-3. The absorbance values (AU) indicated in the figure correspond to the difference between test OD
values minus the mean of the control OD values plus 3 SD.
VOL. 77, 2009ANTIGENICITY OF PLASMODIUM FALCIPARUM LSA-3 IN HUMANS1193
clinical data led us to analyze antibody responses to each
LSA-3 peptide in relation to the clinical attacks recorded over
a period of 1 year following collection of the plasma samples.
To this end, the 90 subjects from Dielmo, Senegal, were strat-
ified into four age groups of 3 to 6 years of age (n ? 12), 7 to
11 years of age (n ? 10), 12 to 15 years of age (n ? 11), and
?16 years of age (n ? 57).
The incidence of clinical malaria attacks occurring during
the year following the blood sampling was recorded actively on
a daily basis (Table 4). All the individuals were enrolled in this
study at the same time in the same place, were equally exposed
to malaria transmission, and had the same access to health
services. No self-treatment, chemoprophylaxis, or effective an-
tivectorial intervention was used by this population. The inci-
dence of clinical malaria has been analyzed in a multivariate
Poisson regression model including age in four groups (chil-
dren aged 3 to 6 years old as reference group) and the LSA-
3-specific antibody responses. In comparison to the children
aged 3 to 6 years old, the acquired protection, i.e., immunity
acquired with age was 41% (relative risk, 0.59), 77% (relative
risk, 0.23), and 96% (relative risk, 0.04) by individuals aged 7 to
11, 12 to 15, and ?16 years old, respectively.
Independent of age, antibody response to LSP10 was found
to be significantly associated with a reduction by 2.8 (95%
confidence interval, 1.5 to 5.3; P ? 0.001 ? 0.003 ? 0.05/17,
Bonferroni’s correction for multiple tests) of the risk of ma-
laria attack. The level of resistance to malaria due to age only,
i.e., age-associated immunity independent of the antibody re-
sponse to LSA-3 or LSP10, was estimated to be 32% (1 to
0.68), 59% (1 to 0.41), and 94% (1 to 0.06) in individuals aged
7 to 11, 12 to 15, and ?15 years old, respectively. Therefore,
the fraction of the immunity acquired by the children aged 7 to
15 years old in comparison to children aged 3 to 6 years old
that could be associated with anti-LSP10 antibody response
was estimated to be 22.7% (i.e., 41.4% ? 32%)/41.4% in chil-
dren 7 to 11 years of age and 22.9% [i.e., (76.7% ? 59.1%)/
76.7%] in children 12 to 15 years of age, whereas it was only
marginal, 2% [i.e., (96.1% ? 94%)/96.1%], for the individuals
aged 16 years or more.
A detailed immunoepidemiological study of LSA-3, a Plas-
modium falciparum preerythrocytic stage antigen with demon-
strated potential as a malaria vaccine (4), was conducted in two
villages of Senegal (West Africa). We used 3 short and 17 long
synthetic peptides spanning almost all of the LSA-3 protein.
The latter peptides rely on a technology which accommodates
the successful synthesis of long sequences and led us to gather
an estimate of the antigenic content of all regions from this
large protein and to further document our preliminary anti-
genic characterization of LSA-3 (1, 4).
Overall, lymphocyte and antibody assays indicate that each
of the 17 overlapping long synthetic peptides spanning almost
all of LSA-3 contain both B- and T-cell epitopes within the
same sequence, which is not surprising given the relatively
large size of each peptide. The prevalence of responses is high,
and some individuals exposed to malaria recognized most re-
gions of LSA-3.
At the B-cell level, each individual studied had detectable
antibodies against a minimum of 6 polypeptides and a maxi-
mum of 15 of the 17 polypeptides studied. Both the preva-
lences and levels of antibodies to the repeat and nonrepeat
regions were high, and on some occasions, they were of com-
parable magnitude. This observation challenges the current
concept that dominant B-cell epitopes segregate in repetitive
regions (3, 7). These results differ from those reported for the
circumsporozoite protein (CS), in which the repeat region is
the main target of antibodies in subjects living in areas where
malaria is endemic (5–7, 17), and conversely, in which immu-
nodominant T-cell epitopes are located in the most polymor-
phic nonrepetitive C terminus of the protein (8, 15, 30, 33, 34).
In this respect, LSA-3 stands closer to Plasmodium falciparum
thrombospondin-related adhesive protein (PfTRAP) than to
CS. Using 50 overlapping PfTRAP peptides, 26 Th cell
epitopes along the entire protein were described using lym-
phocytes from Gambian adults (12). However, in the case of
PfTRAP, only 10 out of 26 epitopes were found to be con-
served, whereas the available data for LSA-3 showed conser-
vation of the epitopes identified so far (4).
At the T-cell level, each of the individuals studied showed
lymphocyte responses against at least 2 and up to 12 out of the
17 LSP analyzed. Although the prevalence of LSA-3-specific
proliferative responses was expectedly lower than that of B-cell
responses, the pattern of proliferative responses roughly par-
alleled that of antibody responses. The T-cell responses to the
N-terminal LSP1 peptide confirm previous data gathered using
TABLE 4. Fraction of clinical immunity attributable to LSA-3- or LSP10-specific antibody responses calculated for the different age groups
of inhabitants living in Dielmo, Senegal
Age group (yr)
No. of malaria
No. of days of
rate of malaria
Age variation of the relative risk of malaria
attack/year (95% confidence interval)b
% of clinical immunity
attributable to LSP10-
aThe incidence rates of malaria attacks in inhabitants living in Dielmo, Senegal, were determined for different age groups.
bThe age-dependent changes in the relative risk of malaria attacks when excluding the effect of anti-LSP10 specific antibody responses (LSP10?) or when taking
into account the effect of anti-LSP10 specific antibody responses (LSP10?) are shown. The total age variation of the relative risk of malaria attack/year (95% confidence
interval) for LSP10 was 2.83 (1.52–5.29).
1194 TOURE-BALDE ET AL.INFECT. IMMUN.
short peptides within the same sequence (i.e., LSA-3-NRI and
LSA-3-NRII) (1). However, the peak prevalence occurred
early in age and exposure (11 to 15 years) and remained similar
in older individuals. Finally, and again in contrast with the CS
protein, the repeat region of LSA-3 was found to define a
strong T-cell epitope. Comparison of the SI and prevalence of
proliferating cells tested in the two villages in Senegal led to a
consistent difference between Dielmo and Ndiop. Two pep-
tides (LSP7 and LSP13) were inducing the highest SI in
Dielmo, whereas LSP2, LSP3, LSP9, LSP10, and LSP11 which
gave comparatively lower SI values were more strongly recog-
nized in Ndiop. Obviously, different peptides were found as-
sociated with the most sustained proliferative responses ob-
served in each of the two villages, which differ not only by
malaria transmission rate but also by the genetic background
of the inhabitants, with Serere dominating in Dielmo and
Wolof and Fulani dominating in Ndiop. Nevertheless, the pre-
cise explanation of the differences observed in proliferative
responses would need more investigations.
The prevalences of both B- and T-cell responses were not
directly dependent on transmission levels, an observation
which is also in agreement with the high antigenicity of each
region of the protein. In Ndiop, Senegal, an area where ma-
laria transmission is strictly seasonal, the pattern of responses
to LSA-3 was largely similar to that found in Dielmo, Senegal
(where malaria transmission is perennial and approximately 10
to 20 times higher than in Ndiop). Yet immune responses,
particularly antibody levels, increased as a function of age and
hence exposure to infected mosquito bites, at least during
childhood and adolescence, to most peptides and thereafter
reached a plateau. This phenomenon, and the lack of differ-
ence between Ndiop and Dielmo, suggest that the protein is
highly antigenic and that exposure to low numbers of sporo-
zoites in Ndiop, i.e., small amounts of antigen, is sufficient to
induce consistent immune responses.
The epidemiological relationship observed between anti-
LSP10 antibodies and acquired clinical protection is an addi-
tional point of interest, although its biological basis remains to
be investigated further. The data showing that a significant
percentage of acquired immunity between two age groups
could be associated with LSA-3 antibody responses led us to
formulate the hypothesis that LSA-3 is not only antigenic but
that immune responses to this antigen may also correlate with
the level of individual protection developed during the pro-
gressive process of its acquisition through cumulative exposure
to malaria transmission. Of note, the potential association be-
tween anti-LSP10 antibodies and protection was consistently
detected in the age groups during which the transition between
nonprotection toward protection status was observed in these
areas where malaria is endemic. In addition, and in line with
other observations carried out in the same settings, the poten-
tial association between protection and antibodies to other
antigens was also found to be stronger in the young age groups
(26, 28). Anti-LSP10 antibodies could be either a surrogate
marker of protection against the preerythrocytic stage or an
effective component of a defense mechanism. Since LSA-3 was
found to be able to induce protection (2, 4, 21, 29) and since
anti-LSA-3 antibodies strongly inhibit P. falciparum sporozoite
invasion into human hepatocytes, as well as P. yoelii sporozoite
invasion into mouse hepatocytes (2) and can passively transfer
protection in vivo (2), an antibody-mediated mechanism tar-
geting the LSP10 region is a plausible hypothesis that would
now deserve to be investigated. If confirmed by further studies,
LSP10 should be incorporated into a vaccine so as to induce or
boost existing responses in young children. Overall, the present
results add further critical information to our initial immuno-
logical characterization of LSA-3 (1, 2, 4). This study led to the
identification of numerous domains within LSA-3 that are
highly antigenic in individuals exposed to malaria which might
therefore correspond to immune effector targets and further
document the potential of LSA-3 as a valuable malaria
preerythrocytic stage vaccine candidate.
We are grateful to the villagers of Dielmo and Ndiop in Senegal for
their active and continuing participation in the project, to the medical
staff involved in both villages, and to E. M. Fall for his technical
assistance. We particularly thank the national authorities in Senegal
for their continuous support to the project.
This study was supported by a VIHPAL grant from the French
Ministry of Research.
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Editor: J. F. Urban, Jr.
1196TOURE-BALDE ET AL.INFECT. IMMUN.