Immunogenicity of Duffy Binding-Like Domains That Bind Chondroitin Sulfate A and Protection against Pregnancy-Associated Malaria

Abstract

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is implicated in pathological outcomes of pregnancy-associated malaria (PAM). P. falciparum isolates that sequester in the placenta primarily bind chondroitin sulfate A (CSA). Following exposure to malaria during
pregnancy, women in areas of endemicity develop immunity, and so multigravid women are less susceptible to PAM than primigravidae.
Protective immunity to PAM is associated with the development of antibodies that recognize diverse CSA-binding, placental
P. falciparum isolates. The epitopes recognized by such protective antibodies have not been identified but are likely to lie in conserved
Duffy binding-like (DBL) domains, encoded by var genes, that bind CSA. Immunization of mice with the CSA-binding DBL3γ domain encoded by var1CSA elicits cross-reactive antibodies that recognize diverse CSA-binding P. falciparum isolates and block their binding to placental cryosections under flow. However, CSA-binding isolates primarily express var2CSA, which does not encode any DBLγ domains. Here, we demonstrate that antibodies raised against DBL3γ encoded by var1CSA cross-react with one of the CSA-binding domains, DBL3X, encoded by var2CSA. This explains the paradoxical observation made here and earlier that anti-rDBL3γ sera recognize CSA-binding isolates and
provides evidence for the presence of conserved, cross-reactive epitopes in diverse CSA-binding DBL domains. Such cross-reactive
epitopes within CSA-binding DBL domains can form the basis for a vaccine that provides protection against PAM.

Full text

INFECTION AND IMMUNITY, Oct. 2006, p. 5955–5963 Vol. 74, No. 10
0019-9567/06/$08.00⫹0 doi:10.1128/IAI.00481-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Immunogenicity of Duffy Binding-Like Domains That Bind Chondroitin
Sulfate A and Protection against Pregnancy-Associated Malaria
Nivedita Bir,
1
Syed Shams Yazdani,
1
Marion Avril,
2
Corinne Layez,
2
Ju¨rg Gysin,
2
and Chetan E. Chitnis
1
*
Malaria Group, International Centre for Genetic Engineering, and Biotechnology (ICGEB), New Delhi 110067, India,
1
and
Unite´ de Parasitologie Expe´rimentale, Faculte´deMe´decine, Universite´delaMe´diterrane´e, Marseille, France
2
Received 24 March 2006/Returned for modification 4 June 2006/Accepted 3 July 2006
Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is implicated in pathological
outcomes of pregnancy-associated malaria (PAM). P. falciparum isolates that sequester in the placenta pri-
marily bind chondroitin sulfate A (CSA). Following exposure to malaria during pregnancy, women in areas of
endemicity develop immunity, and so multigravid women are less susceptible to PAM than primigravidae.
Protective immunity to PAM is associated with the development of antibodies that recognize diverse CSA-
binding, placental P. falciparum isolates. The epitopes recognized by such protective antibodies have not been
identified but are likely to lie in conserved Duffy binding-like (DBL) domains, encoded by var genes, that bind
CSA. Immunization of mice with the CSA-binding DBL3␥domain encoded by var1CSA elicits cross-reactive
antibodies that recognize diverse CSA-binding P. falciparum isolates and block their binding to placental
cryosections under flow. However, CSA-binding isolates primarily express var2CSA, which does not encode any
DBL␥domains. Here, we demonstrate that antibodies raised against DBL3␥encoded by var1CSA cross-react
with one of the CSA-binding domains, DBL3X, encoded by var2CSA. This explains the paradoxical observation
made here and earlier that anti-rDBL3␥sera recognize CSA-binding isolates and provides evidence for the
presence of conserved, cross-reactive epitopes in diverse CSA-binding DBL domains. Such cross-reactive
epitopes within CSA-binding DBL domains can form the basis for a vaccine that provides protection against
PAM.
Following repeated exposure to Plasmodium falciparum in-
fections, adults in areas of endemicity develop immunity to
clinical malaria (42, 46). However, women in areas of ende-
micity are uniquely susceptible to P. falciparum malaria during
pregnancy (7, 36). Infection with P. falciparum is an important
cause of maternal anemia and increases the risk of abortion,
premature delivery, low birth weight, neonatal mortality, and
infant anemia, especially in primigravidae (8, 28, 31, 35, 52). P.
falciparum infections during pregnancy are frequently charac-
terized by the sequestration of infected erythrocytes (IEs) in
placental blood spaces (35), which can lead to inflammatory
responses (54), deposition of fibrinoid material (57), and re-
duced blood flow to the fetus (18).
There has been considerable interest in understanding the
molecular mechanisms that mediate placental sequestration of
IEs and the reasons for the apparent lack of immunity to P.
falciparum malaria in primigravidae residing in areas of ende-
micity. Multigravid women appear to be protected against the
deleterious effects of P. falciparum infection during pregnancy
(20, 36), suggesting that strain-transcending immunity devel-
ops rapidly following exposure to placental P. falciparum iso-
lates. The mechanisms that mediate protective immunity
against pregnancy-associated malaria (PAM) are not com-
pletely understood.
Adhesion studies have revealed that IEs derived from pla-
centas predominantly bind chondroitin sulfate A (CSA) (1, 16,
24, 43). Binding to hyaluronic acid and normal immunoglobu-
lins (Igs) may also play a minor role in placental sequestration
(5, 6, 16, 23). In contrast, IEs derived from peripheral blood of
P. falciparum-infected pregnant women or from that of non-
pregnant donors, including children and adult men, commonly
bind other endothelial receptors, such as CD36 (24). These
findings suggest the possibility that the placenta may select for
rare CSA-binding P. falciparum variants that are not commonly
found in infected children or nonpregnant adults.
The cytoadherence of IEs to the host endothelium is medi-
ated by variant surface proteins that belong to the P. falcipa-
rum erythrocyte membrane protein-1 (PfEMP-1) family (13).
The P. falciparum genome contains ⬃60 var genes that encode
diverse PfEMP-1 variants (3, 48, 49, 53). Expression of PfEMP-1
undergoes antigenic variation due to the switching of var gene
expression during blood-stage growth (48). Immune adults re-
siding in areas of endemicity acquire antibodies that recognize
diverse PfEMP-1 variants and agglutinate diverse P. falciparum
isolates (33). Antibodies directed against PfEMP-1 are thought
to be important components of naturally acquired immunity to
P. falciparum malaria (10). While sera from immune adult men
and primigravid women residing in areas of endemicity recog-
nize a wide range of peripheral P. falciparum isolates, they
exhibit poor recognition of placental P. falciparum isolates (4,
25) and CSA-binding laboratory strains (41, 51). Following P.
falciparum infection during pregnancy, women develop anti-
bodies that show improved recognition of a wide range of
placental isolates and CSA-binding laboratory strains (4, 25,
41, 51). The levels of antibodies recognizing placental isolates
or CSA-binding laboratory strains are significantly correlated
with parity (4, 25, 41, 51). This indicates the development of
* Corresponding author. Mailing address: Malaria Group, Interna-
tional Centre for Genetic Engineering and Biotechnology (ICGEB),
Aruna Asaf Ali Marg, New Delhi 110067, India. Phone and fax: 91 11
2618 7695. E-mail: cchitnis@icgeb.res.in.
5955

antibodies that recognize conserved epitopes on the IE sur-
faces of diverse placental and CSA-binding isolates. The iden-
tity of such conserved epitopes has not yet been defined, but
they are likely to lie within PfEMP-1 variants that mediate
adhesion to CSA. The PfEMP-1 variants that were initially
implicated in CSA binding include var1CSA from P. falcipa-
rum FCR3CSA (9) and CS2var from P. falciparum CS2 (39,
40). Adhesion to CSA is mediated by the DBL3␥domain of
var1CSA (9) and CS2var (39, 40). Monoclonal antibodies
raised against CHO cells expressing DBL3␥of var1CSA and
antisera raised against recombinant DBL3␥expressed in insect
cells recognize a wide range of placental P. falciparum isolates,
suggesting that DBL3␥contains conserved, cross-reactive
epitopes shared by diverse CSA-binding placental isolates (15,
32). However, although var1CSA was initially implicated as the
var gene responsible for CSA binding in P. falciparum
FCR3CSA, subsequent studies demonstrated that the expres-
sion of another var gene, var2CSA, and not that of var1CSA,is
upregulated in diverse CSA-binding parasite lines and placen-
tal isolates (19, 21, 30, 44, 56). The var2CSA gene implicated in
CSA binding does not encode any DBL␥domains. The re-
ported reactivity of anti-rDBL3␥sera with placental CSA-
binding P. falciparum isolates is thus paradoxical.
Here, we have produced recombinant DBL3␥(rDBL3␥)of
var1CSA in its functional form and examined its immunoge-
nicity. We demonstrate that immunization with rDBL3␥does
elicit sera that cross-react with a wide range of placental iso-
lates and block their binding to placental cryosections under
static as well as physiologically relevant flow conditions. Im-
portantly, we show that anti-rDBL3␥sera cross-react with one
of two CSA-binding DBL domains, namely, the DBL3X do-
main of var2CSA. This observation suggests that the CSA-
binding DBL domains DBL3␥and DBL3X share conserved
B-cell epitopes and explains the paradoxical observations
that anti-rDBL3␥sera recognize CSA-binding P. falciparum
isolates even though these parasites express var2CSA that
does not encode any DBL␥domains. Such conserved
epitopes within diverse CSA-binding DBL domains may
provide the basis for the development of vaccines that elicit
cross-reactive antibodies against CSA-binding isolates and
protect against PAM.
MATERIALS AND METHODS
Production and characterization of functional rDBL3␥.DNA encoding the
DBL3␥domain of var1CSA from P. falciparum FCR3 fused to a C-terminal
six-histidine tag was amplified by PCR using primers 5⬘ACT TGC CCA TGG
GAA AAC GAT GGA AAG AAA C 3⬘and 5⬘ACG AGT GCG GCC GCT
CAG TGA TGG TGA TGG TGA TGC CTG TTC AAG TAA TCT GTT G 3⬘
and a cloned fragment of the FCR3 var1CSA gene (kindly provided by Artur
Scherf, Institut Pasteur, Paris, France) (9) as the template. The PCR product was
cloned as a NcoI-NotI fragment downstream of the T7 promoter in expression
vector pET28a⫹(Novagen) and transformed into Escherichia coli BL21(DE3)
(Novagen). Expression of rDBL3␥with a C-terminal six-histidine tag was in-
duced by the addition of 1 mM isopropyl-1-thio-␤-D-galactopyranoside (IPTG)
to cultures of E. coli BL21(DE3)(pET28a⫹DBL3␥) grown to mid-logarithmic
phase. Induced cultures were harvested after4hofgrowth at 37°C and lysed by
sonication. Inclusion bodies were collected by centrifugation and solubilized in 6
M guanidine-hydrochloride. His-tagged rDBL3␥was purified under denaturing
conditions by metal affinity chromatography using a Ni-nitrilotriacetic acid col-
umn (QIAGEN), refolded by the method of rapid dilution, and purified further
to homogeneity by ion-exchange chromatography using SP-Sepharose (Pharma-
cia) and gel filtration chromatography using Superdex 75 as described earlier for
other DBL domains (37, 47).
Refolded and purified DBL3␥was separated by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) before and after reduction with 5
mM dithiothreitol and detected by silver staining. The homogeneity of rDBL3␥
was analyzed by reverse-phase chromatography on a C
8
column as previously
described (37, 47). The presence of free thiols was detected by Ellman’s method
with 5⬘,5⬘-dithiobis(2-nitrobenzoic acid) (DTNB) (22, 37, 47).
Assessment of binding of rDBL3␥to biotinylated CSA immobilized on
streptavidin-coated wells by ELISA. CSA, CSB, and CSC (Calbiochem) were
biotinylated using a biotinylation kit (Pierce) as described by the manufacturer.
Biotinylated CSA (Bio-CSA), Bio-CSB, and Bio-CSC (100 ␮lof5␮gml
⫺1
) were
immobilized in streptavidin-coated wells (Streptawell-96; Roche). Residual free
sites were saturated with 3% bovine serum albumin (BSA) in phosphate-buffered
saline (PBS) with 0.05% Tween 20 (PBST) for1hat37°C. rDBL3␥(5 ␮gml
⫺1
)
was added to wells and allowed to bind overnight at 4°C. Unbound rDBL3␥was
removed by washing with PBST four times. A mouse monoclonal antibody
against penta-histidine (penta-His) (QIAGEN) was added to each well at a
1:5,000 dilution and incubated at 37°C for1htodetect bound rDBL3␥. Bound
mouse anti-penta-His monoclonal antibodies were detected with goat anti-
mouse IgG peroxidase conjugate (Sigma) (1: 5,000 dilution) and O-phenylene-
diamine (OPD) (Sigma). The optical density at 490 nm was measured using an
enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Devices)
to estimate relative binding efficiencies. Biotinylated hyaluronic acid (Bio-HA)
and the recombinant receptor for complement component C1q, also known as
hyaluronic acid-binding protein 1 (gC1qR/HABP1) (17) (kindly provided by
Anup Biswas and Kasturi Datta, Jawaharlal Nehru University, New Delhi, India)
were used as controls.
Immunization of BALB/c mice. BALB/c mice were immunized intraperitone-
ally with 25 ␮g of rDBL3␥formulated in 250 ␮l Freund’s complete adjuvant and
subsequently given three booster immunizations with 25 ␮g of rDBL3␥formu-
lated with Freund’s incomplete adjuvant on days 21, 42, and 73. Sera were
collected on days 14, 35, 56, 70, and 87 and stored at ⫺80°C until use.
Inhibition of binding of CSA to rDBL3␥by anti-rDBL3␥sera raised in mice.
rDBL3␥(100 ␮lof5␮gml
⫺1
per well) was coated in 96-well polystyrene
microtiter plates (Nunc) by incubation overnight at 4°C followed by blocking with
3% BSA. Microtiter plate wells were incubated for1hat37°C with different
dilutions of mouse sera raised against rDBL3␥or human sera from areas of
endemicity. Pooled preimmune mice sera were used as controls. One hundred
microliters of Bio-CSA (5 ␮gml
⫺1
) was added to each well and allowed to bind
for1hat37°C. Following four washes with PBST, bound Bio-CSA was detected
with Extravidin peroxidase and OPD (Sigma). The optical densities at 490 nm
were used to estimate the relative binding efficiencies to CSA in the presence of
test sera compared to those of controls.
Recognition of P. falciparum IEs by anti-rDBL3␥mouse sera using FACS.
Recognition of P. falciparum IEs by mouse anti-rDBL3␥sera was evaluated by
fluorescence-assisted cell sorting (FACS) as described earlier (50). Briefly, 5 ⫻
10
6
erythrocytes from asynchronous P. falciparum cultures were washed twice
with RPMI medium, pH 6.8, and incubated for 30 min at 37°C with ethidium
bromide (5 ␮gml
⫺1
). After two washes, cells were incubated with anti-rDBL3␥
mouse sera diluted 1:20 for 30 min at 4°C. Preimmune mouse serum was used as a
negative control. Cells were washed two times with RPMI medium, pH 6.8, and
incubated with anti-mouse IgG (heavy plus light chains) chicken at a dilution of
1:200 for 30 min on ice. Erythrocytes were washed twice with RPMI medium and
incubated with a goat anti-chicken IgY conjugated to Alexa-488 (Molecular Probes)
at a dilution of 1:200 for 30 min on ice. The recognition of intact IEs by antibodies
was quantified with a Coulter EPICS flow cytometer and expressed as the mean
Alexafluor intensity of ethidium bromide-gated IEs. Mouse sera raised against
rDBL3␥were tested at a 1:20 dilution by both FACS and liquid immunofluores-
cence assay (L-IFA). Preimmune mouse sera were used as controls.
Inhibition of cytoadhesion of IE under static conditions by anti-rDBL3␥sera.
Static cytoadherence assays were performed as described previously (26). Briefly,
Saimiri monkey brain endothelial C2 (SBEC C2) cells were grown to confluence
on 12-well slides (Bio-Rad). Trophozoite- and schizont-stage IEs (20 ␮lof10
7
IEs ml
⫺1
) were added to each well to allow binding. Heat-inactivated (56°C)
anti-rDBL3␥mouse sera (dilution of 1:20) and soluble CSA (100 ␮gml
⫺1
)
(Calbiochem) were tested for inhibition of binding. Cytoadherence medium and
mouse preimmune serum served as controls. In another experiment, SBEC C2
cells were preincubated with rDBL3␥(200 ␮gml
⫺1
) prior to the addition of IEs.
Adherent IEs were detected by Giemsa staining and scored by light microscopy.
Results were expressed as percent inhibition of binding with respect to the
appropriate controls.
Inhibition of cytoadherence of IEs to placental cryosections with anti-rDBL3␥
mouse sera under flow conditions. The cytoadherence of IEs on placental cryo-
sections was tested under flow conditions as described earlier (2, 26). Briefly,
5956 BIR ET AL. INFECT.IMMUN.

slides of four cryosections from each of two different placentas were mounted in
a cell adhesion flow chamber (CAF-10; Immunetics) held in place by vacuum.
The system was connected to an infusion/withdrawal pump (model 210P; KD
Scientific) to control the flow of IE suspension or cell-free medium through the
perfusion chamber. Reservoirs containing the IE suspension or cytoadhesion
medium (RPMI medium, pH 6.8) were connected to the outlet of the perfusion
chamber through a three-way valve. The cryosections were perfused with IEs at
5% parasitemia (mature stages) and 25% hematocrit in RPMI medium for 10
min at a shear stress of 0.05 Pa. The chamber was then flushed with RPMI
medium to remove nonadherent IEs. Adherent IEs were observed with an
inverted microscope (Eclipse TE 200; Nikon) with a PlanFluor 40/0.60 objective
(Nikon). Bound IEs were counted on 10 randomly distributed fields with an area
of 0.081 mm
2
. Assays were performed in the presence of anti-rDBL3␥mouse
serum (1:20 dilution) to test the ability of the IEs to block adhesion. Results were
expressed as percent inhibition of binding with respect to binding in presence of
preimmune sera.
Parasites and cells. P. falciparum laboratory strains as well as field isolates
were cultured in O
⫹
erythrocytes in RPMI 1640 (Sigma, France) in candle jars
as described previously (55). P. falciparum laboratory strains FCR3CSA (9) and
BC-1-CSA (kindly provided by Artur Scherf, Institut Pasteur, Paris, France) and
P. falciparum placental isolates 24-CSA, 42-CSA, 42DJ-CSA, 193-CSA, 938-
CSA, and 939-CSA (29), all of which bind CSA, were used for the study. In
addition, P. falciparum strains FCR3CD36 (9), D10, and T996 (kindly provided
by David Walliker, University of Edinburgh) and peripheral isolates RAJ-68,
RAJ-104, and JDP8 (collected from regions of India where malaria is endemic
and kindly provided by C. R. Pillai, Malaria Research Centre, Delhi, India),
which do not bind CSA, were used as controls. The CSA-binding ability of
placental isolates and CSA-binding laboratory strains was maintained by periodic
panning on Sc17 cells (29).
Placental cryosections. Four cryosections from each of two different normal
placentas collected in France were mounted sideways in the centers of 76- by
25-mm superfrost glass slides (Menzel-Glaser, Germany) for cytoadherence
analysis under flow conditions. Placental cryosections (7 ␮m) were cut with a
cryotome (CM 3050; Leica, Germany), air dried, fixed in 2.5% paraformaldehyde
in PBS, pH 7.2, for 30 min, washed with PBS, dried, and preserved in an airtight
box at ⫺80°C until use. Before being used in adhesion assays, the slides were air
dried quickly to prevent the condensation of water. Informed consent was ob-
tained from all the participants. Biological material was sampled in strict accor-
dance with the Mattei Law 666-8.
Identification of CSA-binding DBL domains of var2CSA by expression on the
surface of mammalian cells. Plasmids were constructed to express the DBL
domains (DBL1X to DBL6ε) of var2CSA of P. falciparum 3D7 (PFL0030c) on
the surface of mammalian cells as described previously for other DBL domains
(12, 34, 38). Amino acid boundaries of each DBL domain construct were as
follows: for DBL1X, amino acids (aa) 46 to 343; for DBL2X, aa 535 to 934; for
DBL3X, aa 1214 to 1562; for DBL4ε, aa 1581 to 1931; for DBL5ε, aa 1983 to
2291; and for DBL6ε, aa 2333 to 2617. Briefly, the DBL domains were fused to
the signal sequence and transmembrane domain of herpes simplex virus glyco-
protein D (HSV gD) to allow targeting to the mammalian cell surface as de-
scribed previously (12, 34, 38). The plasmid pRE4 (kindly provided by Roselyn
Eisenberg and Gary Cohen), which contains the gene for HSV gD (14), was
digested with PvuII and ApaI to excise the central region encoding amino acids
33 to 248 of HSV gD. DNA fragments encoding DBL domains of 3D7 var2CSA
were amplified by PCR using Pyrococcus furiosus DNA polymerase (Stratagene)
by use of var2CSA-specific primers and cloned into PvuII and ApaI sites in the
vector pRE4 as previously described (12) to yield plasmids pRE4-DBL1X,
pRE4-DBL2X, pRE4-DBL3X, pRE4-DBL4ε, pRE4-DBL5ε, and pRE4-DB6ε.
The DNA sequence of the insert in each expression plasmid was confirmed by
sequencing with an ABI310 automated DNA sequencer. The sequence of each
insert was identical to that reported for 3D7 var2CSA (PFL0030c; GenBank
accession no. NP_701371). Mammalian 293T cells were transfected as described
below to express the DBL domains (DBL1X to DBL6ε) on the surface. Trans-
fected cells were tested for binding to CSA as described previously (34). CSA-
FIG. 1. Characterization of refolded and purified DBL3␥domain (rDBL3␥) of FCR3 var1CSA. (A) Mobility of rDBL␥as determined by
SDS-PAGE. Refolded rDBL␥has lower mobility in SDS-PAGE after reduction with dithiothreitol (⫹DTT), indicating the presence of disulfide
linkages. Molecular mass markers (M) in kDa are shown. (B) Reverse-phase chromatography profile of rDBL3␥. Refolded DBL3␥elutes as a
single, symmetric peak upon reverse-phase chromatography on a C
8
column, indicating that it is conformationally homogenous. AU, absorbance
units (280 nm).
FIG. 2. Binding of rDBL3␥to CSA. Binding of rDBL3␥to Bio-
CSA, Bio-CSB, Bio-CSC, and Bio-HA immobilized on streptavidin-
coated microwells was detected using a mouse monoclonal antibody
against penta-His. Recombinant PvRII, the binding domain of PvDBP,
and recombinant gC1qR/HABP1, which binds HA, were used as con-
trol ligands. rDBL3␥binds CSA but not CSB, CSC, or HA. gC1qR/
HABP1 binds HA but not CSA, CSB, or CSC. PvRII does not bind any
of the receptors tested. OD, optical density.
VOL. 74, 2006 CSA-BINDING DBL DOMAINS AND PAM 5957

binding assays were performed in the presence of anti-rDBL3␥mouse sera and
preimmune sera to test the abilities of sera to block binding.
Mammalian cell culture, transfection, and immunofluorescence assays. 293T
cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) with 10%
heat-inactivated fetal calf serum in a humidified CO
2
(5%) incubator at 37°C. Fresh
monolayers of 40 to 60% confluent 293T cells growing in 35-mm-diameter wells
were transfected with 2 ␮g of plasmid DNA using Lipofectamine Plus reagent
(Invitrogen) as indicated by the manufacturer. Transfected cells were used for
immunofluorescence and binding assays 36 to 40 h after transfection (34). Immu-
nofluorescence assays using mouse monoclonal antibody DL6 (kindly provided by
Roselyn Eisenberg and Gary Cohen), which reacts with amino acids 272 to 279 (14)
of HSV gD, or anti-rDBL3␥mouse sera were performed as described earlier (12) to
detect the expression of the fusion proteins. The binding of Bio-CSA to transfected
293T cells was tested as previously described (34).
Binding of refolded and purified rDBL3␥to biotinylated CSA immobilized on
streptavidin-coated microwells by ELISA. One hundred microliters of Bio-CSA,
Bio-CSB, or Bio-CSC was immobilized on each well of streptavidin-coated mi-
crowell plate (Stretawell-96; Roche Applied Science) as indicated by the man-
ufacturer. Five hundred nanograms of refolded and purified rDBL3␥was incu-
bated overnight at 4°C, and unbound rDBL3␥was removed by washing four
times with PBST. Residual free sites in the wells were saturated with 3% BSA in
PBST for1hat37°C, and the wells were washed four times with PBST. One
hundred microliters of anti-penta-His antibody (Sigma) diluted to 1:5,000 was
added to each well, and the wells were incubated at 37°C for 1 h, followed by four
washes with PBST. One hundred microliters of Extravidin peroxidase (Sigma)
(1:5,000 dilution) was incubated in each well for1hat37°C. Bound immuno-
complexes were detected with OPD (Sigma). Absorbance was measured at 490
nm using an ELISA reader (Molecular Devices).
Inhibition of binding of rDBL3␥-CSA binding by anti-rDBL3␥sera raised in
mice. Anti-rDBL3␥mouse sera were tested for inhibition of the rDBL3␥-CSA
interaction. Microtiter plate wells were coated with 100 ␮l of rDBL3␥(5 ␮g
ml
⫺1
). Plates were incubated at 4°C overnight and blocked with 3% BSA as
described above. Microtiter plate wells were incubated with different dilutions of
mouse antibodies raised against rDBL3␥. Preimmune sera were used as con-
trols. Five hundred nanograms of Bio-CSA was incubated in each well at 37°C
for 1 h. After being washed with PBST four times, the well was incubated with
Extravidin peroxidase (Sigma) for1hat37°C. Results were expressed as the
percentages of binding observed with respect to the values for controls.
RESULTS
Purity, homogeneity, and functional activity of rDBL3␥.The
DBL3␥domain of var1CSA derived from P. falciparum FCR3
was expressed in E. coli, purified from inclusion bodies under
denaturing conditions by metal affinity chromatography, re-
folded by rapid dilution, and purified to homogeneity by ion-
exchange and gel filtration chromatography. Refolded and pu-
rified rDBL3␥was analyzed for purity, homogeneity, and
functional activity. A single band of the expected size (⬃38
kDa) was detected on silver-stained SDS-PAGE gels (Fig. 1).
rDBL3␥migrated slower on SDS-PAGE gels after reduction
with dithiothreitol, indicating that the refolded protein con-
tained disulfide linkages (Fig. 1). The free thiol content in
rDBL3␥, which contains 10 cysteines, was measured using
Ellman’s method (22). The detection limit for free thiols by
this assay was 30 ␮M. No free thiols were detected in rDBL3␥
at a protein concentration of 50 ␮M, indicating that ⬎94% of
cysteines were disulfide linked. The homogeneity of refolded
rDBL3␥was analyzed by reverse-phase chromatography.
rDBL3␥eluted from a C
8
column as a single symmetric peak,
suggesting that refolded rDBL3␥was conformationally homoge-
neous (Fig. 1).
The functional activity of rDBL3␥was examined by testing
the binding of rDBL3␥with its receptor, CSA. Biotinylated
FIG. 3. Recognition of diverse P. falciparum isolates by mouse sera
raised against rDBL3␥of FCR3 var1CSA by use of flow cytometry. Mouse
sera raised against rDBL3␥were tested for recognition of P. falciparum
FCR3CD36 (A) and T996 (B), which do not bind CSA, and of FCR3CSA
(C) and 193 (D), which bind CSA. Gray areas represent signals from preim-
mune sera.
TABLE 1. Recognition of P. falciparum IEs by anti-DBL3␥mouse
sera by L-IFA and FACS and inhibition of adhesion of P. falciparum
IEs to SBEC C2 cells by CSA and recombinant DBL3␥
Parasite
strain
a
Source
% Inhibition of
binding to SBEC
C2 cells under
static conditions
(avg. ⫾SD) by
b
:
% Recognition
by
c
:
rDBL3␥CSA L-IFA FACS
FCR3CSA Lab strain 94 ⫾296⫾1 65 46.1
BC-1-CSA Lab strain 89 ⫾294⫾1 32 24.3
D10 Lab strain NT NT 0 2.83
T996 Lab strain NT NT 0 0.86
FCR3CD36 Lab strain NT NT 0 3.57
24-CSA Placental isolate 84 ⫾392⫾2 59 44.2
42-CSA Placental isolate 87 ⫾590⫾3 87 72.9
42DJ-CSA Placental isolate 83 ⫾392⫾2 39 44.1
193-CSA Placental isolate 90 ⫾295⫾1 94 87.1
938-CSA Placental isolate 88 ⫾393⫾3 42 21.9
939-CSA Placental isolate 86 ⫾294⫾2 51 32.23
RAJ-68 Peripheral isolate NT NT 0 2.67
RAJ-104 Peripheral isolate NT NT 0 3.54
JDP8 Peripheral isolate NT NT 0 2.3
a
P. falciparum peripheral and placental field isolates as well as P. falciparum
laboratory strains were tested for binding to SBEC C2 cells. P. falciparum lab-
oratory (Lab) strains FCR3CD36, D10, and T996 and field isolates RAJ-68,
RAJ-104, and JDP8 collected from peripheral blood of nonpregnant donors do
not bind SBEC C2 cells and were not tested (NT) for inhibition of binding with
CSA and rDBL3␥.
b
Number of IEs bound to SBEC C2 cells in control wells was in the range of
90 to 125 bound IEs per field. Results represent inhibition efficiencies (average ⫾
standard deviation) determined from three independent experiments. Each assay
was performed in duplicate wells in each experiment.
c
Mouse sera raised against rDBL3␥of FCR3 var1CSA were tested for rec-
ognition of P. falciparum laboratory strains and field isolates by L-IFA and
FACS. The percentages of IEs that reacted with anti-rDBL3␥sera by L-IFA and
FACS are reported.
5958 BIR ET AL. INFECT.IMMUN.

receptors, namely, Bio-CSA, Bio-CSB, Bio-CSC, and Bio-HA,
were immobilized on streptavidin-coated ELISA plate wells
and incubated with refolded rDBL3␥to allow binding. Bound
rDBL3␥was detected using a mouse monoclonal antibody
against the C-terminal penta-histidine tag. Binding of rDBL3␥
was observed only for wells coated with Bio-CSA (Fig. 2). The
lack of binding to CSB, CSC, and HA indicates that rDBL3␥
bound CSA with specificity. The human endothelial receptor
gC1qR/HABP1, which binds the globular head of complement
component C1q as well as HA (17), and the receptor-binding
domain, region II (PvRII), of P. vivax Duffy binding protein
(12), were used as control ligands. PvRII did not bind CSA,
CSB, CSC, or HA (Fig. 2). Recombinant gC1qR/HABP1
bound HA as expected but not CSA, CSB, or CSC (Fig. 2).
Mouse sera raised against rDBL3␥recognize diverse CSA-
binding P. falciparum laboratory strains and placental field
isolates. Pooled sera from five mice immunized with rDBL3␥
detected rDBL3␥by ELISA up to a dilution of 1:100,000 and
blocked the binding of rDBL3␥to CSA in an ELISA-based
binding assay with 50% inhibition at a dilution of 1:500. Anti-
rDBL3␥mouse sera were tested for recognition of diverse P.
falciparum laboratory strains and field isolates by FACS and
L-IFA. The P. falciparum laboratory strains and field isolates
were first tested for binding to SBEC C2 cells, which display
CSA on their surface (26). P. falciparum placental isolates
bound SBEC C2 cells (Table 1). The addition of CSA (100 ␮g
ml
⫺1
) inhibited the binding of placental isolates and CSA-
binding laboratory strains to SBEC C2 cells, confirming that
these parasites used CSA as a receptor for adhesion to SBEC
C2 cells (Table 1). Preincubation of SBEC C2 cells with
rDBL3␥also inhibited the adhesion of IEs (Table 1). Anti-
rDBL3␥mouse sera recognized CSA-binding P. falciparum
laboratory strains and placental isolates by FACS as well as
L-IFA (Fig. 3 and 4; Table 1). In contrast, anti-rDBL3␥mouse
sera did not recognize P. falciparum laboratory strains or field
isolates that did not bind CSA (Fig. 3; Table 1). In the FACS
studies, specific shifts characteristic of Alexafluor-stained IEs
were observed in the cases of CSA-binding laboratory strains,
such as FCR3CSA, and placental isolates, such as 193-CSA
(Fig. 3). The fraction of erythrocytes with specific shifts was
higher for CSA-binding isolates than for laboratory isolates
such as FCR3CD36 and T996 and peripheral isolates RAJ68,
RAJ104, and JDP8, which did not bind CSA (Fig. 3; Table 1).
The pattern of recognition of CSA-binding IEs by anti-
rDBL3␥sera by L-IFA clearly indicated reactivity with the
surface of IEs (Fig. 4). Anti-rBL3␥sera thus recognized com-
FIG. 4. Recognition of surfaces of P. falciparum IEs by anti-rDBL3␥mouse sera using L-IFA. Mouse sera raised against rDBL3␥of FCR3 var1CSA react
with the IE surface of P. falciparum laboratory strain FCR3CSA and that of placental isolate 193CSA, which bind CSA, but not with that of P. falciparum
laboratory strain FCR3CD36 or that of peripheral field isolate JDP8, which do not bind CSA. Parasites were stained with DNA-intercalating dye DAPI (blue)
to identify IEs and with anti-rDBL3␥mouse sera followed by Alexafluor 488-conjugated chicken anti-mouse IgG (green).
VOL. 74, 2006 CSA-BINDING DBL DOMAINS AND PAM 5959

mon conserved epitopes that are shared by diverse CSA-bind-
ing P. falciparum isolates. Preimmune sera collected from mice
prior to immunization with rDBL3␥showed no reactivity with
any of the parasites when tested by FACS or L-IFA. While
reactivity with FCR3CSA was observed by FACS when anti-
mouse IgG goat serum coupled to fluorescein isothiocyanate
was used for secondary staining with anti-rBL3␥mouse sera
for primary staining, no reactivity was observed when fluo-
rescein isothiocyanate-coupled anti-mouse IgM goat serum
was used for secondary staining (data not shown). This rules
out the possibility that the reactivity of anti-rBL3␥mouse
sera with CSA-binding isolates was due to nonspecific bind-
ing of mouse IgM.
Mouse sera raised against rDBL3␥block adhesion of di-
verse CSA-binding P. falciparum laboratory strains and field
isolates to Saimiri monkey brain endothelial cells and placen-
tal cryosections. Anti-rDBL3␥mouse sera and preimmune
mouse sera were tested for inhibition of binding of IEs to
SBEC C2 cells and placental cryosections. Anti-rDBL3␥sera
inhibited the binding of diverse CSA-binding P. falciparum
laboratory strains and placental isolates to SBEC C2 cells
under static conditions (Table 2). Anti-rDBL3␥sera also
blocked the adhesion of diverse CSA-binding parasite isolates
to placental cryosections under flow conditions (Table 2).
rDBL3␥was thus immunogenic and elicited cross-reactive anti-
bodies that blocked the cytoadherence of diverse CSA-binding P.
falciparum laboratory strains and placental isolates under both
static and flow conditions.
Anti-rDBL3␥mouse sera recognize DBL3X of var2CSA and
block its binding to CSA. Initial studies indicated that var1CSA
is responsible for the CSA-binding phenotype of FCR3CSA
(9). However, subsequent studies demonstrated that transcrip-
tion of another var gene, var2CSA, is upregulated in CSA-
binding parasites (42, 56). Here, we have expressed the DBL
domains of var2CSA from P. falciparum 3D7 (DBL1X,
DBL2X, DBL3X, DBL4ε, DBL5ε, and DBL6ε) on the surface
of mammalian 293T cells and tested them for binding to Bio-
CSA. In control experiments, the DBL domains were tested for
binding to Bio-CSB and Bio-CSC. The DBL3␥domain of
var1CSA was used as a positive control in the binding studies.
The DBL2X and DBL3X domains of var2CSA specifically
bound Bio-CSA in this study (Table 3). Soluble CSA, but not
CSB or CSC, inhibited the binding of DBL2X, DBL3X, and
DBL3␥to Bio-CSA, confirming that these domains bound
CSA with specificity (Table 4). Mouse sera raised against
DBL3␥of var1CSA recognized DBL3␥as well as DBL3X
expressed on the surface of 293T cells (Table 3). Importantly,
anti-rDBL3␥mouse sera completely blocked the binding of
both DBL3␥and DBL3X domains with CSA at a dilution of
1:20 (Table 4). The DBL3␥domain of var1CSA thus shares
cross-reactive epitopes with the DBL3X domain of var2CSA.
Antisera elicited against DBL3␥did not recognize DBL2X of
var2CSA and did not block its binding to CSA (Tables 3 and
4). Anti-rBL3␥sera also did not recognize any of the other
DBL domains of var2CSA (Table 3).
TABLE 2. Inhibition of adhesion of P. falciparum isolates to SBEC
C2 cells under static conditions and to placental cryosections under
flow conditions by anti-rDBL3␥mouse sera
Parasite
strain
% Inhibition by anti-rDBL3 ␥mouse sera
(avg ⫾SD) of IE binding to:
SBEC C2 (static)
a
Placenta (flow)
b
FCR3CSA 86 ⫾176⫾5
BC1-1CSA 90 ⫾382⫾5
24-CSA 86 ⫾388⫾2
42DJ-CSA 86 ⫾479⫾9
42-CSA 90 ⫾593⫾3
193-CSA 91 ⫾193⫾9
938-CSA 88 ⫾390⫾4
939-CSA 89 ⫾383⫾4
a
Anti-rDBL3␥mouse sera (1:20 dilution) were tested for inhibition of binding
of IEs to SBEC C2 cells in static binding assays. Binding in the presence of
preimmune mouse sera (1:20 dilution) was used as a control to determine
inhibition efficiencies of anti-rDBL3␥sera. The number of IEs bound to SBEC
C2 cells was scored in five random fields in each well by Giemsa staining. Results
represent inhibition efficiencies (average ⫾standard deviation) determined from
three independent experiments. Each assay was performed in duplicate wells in
each experiment. The number of IEs bound to SBEC C2 cells in control wells
was in the range of 90 to 125 bound IEs per field.
b
Anti-rDBL3␥pooled mouse sera were tested at a dilution of 1:20 for the
inhibition of adhesion of IEs to placental cryosections under flow conditions.
Binding in the presence of preimmune mouse sera (1:20 dilution) was used as a
reference to determine the inhibition efficiencies of anti-rDBL3␥sera. Results
represent inhibition efficiencies (average ⫾standard deviation) determined from
two independent experiments. Assays were performed with two different placen-
tal cryosections, each of which was mounted in duplicate flow cells for each
experiment. The number of bound IEs was scored in 10 fields per flow cell in
both experiments. The number of bound IEs in the presence of preimmune
serum was used as a reference. The number of bound IEs in control wells was in
the range of 213 to 328 per field.
TABLE 3. Binding of DBL domains of 3D7 var2CSA to CSA and
recognition by mouse sera raised against DBL3␥of FCR3 var1CSA
Construct
Frequency of reactivity
(%)
a
Binding efficiency (%)
b
DL6 Anti-rDBL3␥
antibodies CSA CSB CSC
pRE4-DBL3␥74 ⫾463⫾472⫾500
pRE4-DBL1X 70 ⫾80 000
pRE4-DBL2X 60 ⫾6058⫾600
pRE4-DBL3X 66 ⫾857⫾360⫾10 0 0
pRE4-DBL4ε58 ⫾40 000
pRE4-DBL5ε63 ⫾70 000
pRE4-DBL6ε54 ⫾60 000
pRE4 83 ⫾40 000
a
The DBL3␥domain of FCR3 var1CSA and all the DBL domains (DBL1X to
DBL6ε) of 3D7 var2CSA were expressed on the surfaces of mammalian 293T
cells as fusions to HSV gD by use of the transfection vector pRE4. Mouse
monoclonal antibody DL6 directed against HSV gD sequences in the fusion
proteins and mouse polyclonal sera raised against DBL3␥of FCR3 var1CSA
were used to detect expression of DBL domains on 293T cell surfaces. Preim-
mune mouse serum does not react with any cells transfected with any of the DBL
constructs tested. Around 1,000 to 1,500 293T cells were scored for recognition
by DL6 and anti-rDBL3␥mouse sera in immunofluorescence assays, and the
frequencies of reactivity were determined for both antibodies. Results represent
the average and standard deviation of three independent experiments.
b
Transfected 293T cells were incubated with Bio-CSA, Bio-CSB, and Bio-
CSC to allow binding followed by incubation with anti-biotin sera raised in mice.
Anti-mouse IgG chicken IgY conjugated with Alexafluor 488 (Molecular Probes)
was used to detect binding of biotinylated CSA, CSB, and CSC. Around 1,000 to
1500 293T cells stained with DAPI were scored for staining with Alexafluor 488.
The binding efficiency was calculated as follows: binding efficiency (%) ⫽[(num-
ber of Alexafluor 488-stained 293T cells)/(number of DAPI-stained 293T cells] ⫻
100. Where the binding efficiency is reported as zero, no Alexafluor 488-stained
cells were seen in the entire well. Results reported are average ⫾standard
deviation from three independent experiments.
5960 BIR ET AL. INFECT.IMMUN.

DISCUSSION
The epidemiology of naturally acquired immunity against
placental malaria suggests that multigravid women who have
experienced placental malaria rapidly develop antibodies that
cross-react with a wide range of placental isolates. This sug-
gests that placental isolates may use a limited number of CSA-
binding domains that share common B-cell epitopes. The first
parasite-derived domain shown to bind CSA was the DBL3␥
domain of var1CSA from the CSA-binding laboratory strain
FCR3CSA (9). Later studies indicated that the expression of
var1CSA is not restricted to placental isolates and that the
expression of another gene, var2CSA, is upregulated in
FCR3CSA and other CSA-binding placental and laboratory
isolates (44, 45, 56). However, antibodies raised against
rDBL3␥of var1CSA were shown to cross-react with a wide
range of CSA-binding placental and laboratory isolates (15,
32). This result was paradoxical, given that CSA-binding par-
asites primarily express var2CSA, which does not contain any
DBL␥domains. Here, we have produced recombinant DBL3␥
from FCR3 var1CSA in its functional form and reexamined its
immunogenicity. Characterization of rDBL3␥confirmed that it
was pure, homogenous, and functional in that it bound CSA
with specificity. We demonstrate here that the immunization of
mice with rDBL3␥of FCR3 var1CSA elicited antibodies that
recognize the homologous parasite FCR3CSA as well as a wide
range of heterologous P. falciparum laboratory strains and
placental field isolates that bind CSA (Fig. 3 and 4; Table 1). In
contrast, anti-rBL3␥sera did not recognize the IE surfaces of
P. falciparum peripheral isolates and strains that do not bind
CSA (Fig. 3 and Table 1). Given the questions raised about the
role of var1CSA in CSA binding and placental malaria, our
confirmation of previous results demonstrating the cross-reac-
tivity of anti-rBL3␥sera with placental isolates is important
and reassuring.
However, how does one explain the observed cross-reactivity
of anti-rBL3␥sera with CSA-binding placental isolates and
laboratory strains, given that they primarily express var2CSA,
which does not contain any DBL␥domains? In order to inves-
tigate this paradox, we expressed the DBL domains of var2CSA
derived from P. falciparum 3D7 on the surface of 293T cells and
tested them for binding to CSA and recognition by anti-rBL3␥
sera. The DBL2X and DBL3X domains of var2CSA bound CSA
in these assays. DNA sequencing confirmed that the sequences of
the inserts in the different var2CSA expression constructs used
here were identical to the published 3D7 var2CSA sequence. A
previous study has reported that the DBL2X and DBL6εdo-
mains of 3D7 var2CSA have CSA-binding activity (27). The
differences observed between the CSA-binding activity of DBL
domains of var2CSA here and that found in the previous study
may be due to differences in the domain boundaries of the
DBL constructs or differences in the expression systems and
cells used for transfection in the two studies. Importantly, we
found that anti-rBL3␥sera cross-reacted with the DBL3X do-
main of var2CSA and blocked the binding of DBL3X with
CSA. Anti-rDBL3␥sera did not recognize any of the other
DBL domains of var2CSA. The recognition of DBL3X by
anti-rDBL3␥sera indicates the presence of common cross-
reactive B-cell epitopes in these two diverse CSA-binding DBL
domains and explains the paradoxical observations made here
and earlier (11, 15, 32) that anti-rDBL3␥sera recognize CSA-
binding parasites. We have demonstrated that in addition to
recognizing a wide range of placental parasites, anti-rDBL3␥
sera block adhesion of IEs to placental cryosections under
physiologically relevant flow conditions. This observation indi-
cates that it may be possible to develop prophylactic strategies
that block placental sequestration of malaria parasites.
The B-cell epitopes that are recognized by protective anti-
bodies from sera of multigravid women have not yet been
identified. Following the identification of var2CSA as the ex-
pressed var gene in CSA-binding laboratory strains and pla-
cental isolates, recent efforts to identify targets of antibodies
that protect against PAM have focused on CSA-binding DBL
domains of var2CSA. Here, we have demonstrated that the
CSA-binding DBL domains of var1CSA and var2CSA, namely,
DBL3␥and DBL3X, respectively, share common B-cell
epitopes. Such conserved epitopes that are shared by diverse
CSA-binding DBL domains may serve as targets for protective
antibodies and form the basis for development of a vaccine
against PAM.
ACKNOWLEDGMENTS
We thank Artur Scherf, Institut Pasteur, Paris, France, for providing
a plasmid with FCR3 var1CSA gene; Gary Cohen and Roselyn Eisen-
berg, University of Pennsylvania, Philadelphia, Pennsylvania, for pro-
viding plasmid pRE4 and monoclonal antibody DL6; Anup Biswas and
Kasturi Datta for providing recombinant gC1qR/HABP1; C. R. Pillai
for providing P. falciparum field isolates RAJ68, RAJ104, and JDP8;
Ire´ne Juhan-Vague, Laboratoire d’He´matologie, Marseille, France,
for access to FACS; and Catherine Lepolard and Bruno Pouvelle,
Universite´delaMe´diterrane´e, for parasite cultures.
This work was supported by an International Senior Research Fel-
lowship to C.E.C. from the Wellcome Trust, United Kingdom, and by
grants to J.G. from BIOMALPAR program, an FP6 funded network of
excellence; PAL ⫹2002 of the MENRT; ACI program, INSERM MIC
no. 0318; and the Malaria Antigen Discovery Program (MADP) Ma-
laria in Pregnancy Initiative, Bill and Melinda Gates Foundation, no.
29202.
REFERENCES
1. Achur, R. N., M. Vialyavetti, A. Alkhalil, C. F. Ockenhouse, and D. C.
Gowda. 2000. Characterization of proteoglycans of human placenta and
TABLE 4. Inhibition of binding of CSA to DBL3␥domain of
FCR3 var1CSA and DBLX domains of 3D7 var2CSA
with mouse sera raised against rDBL3␥
Construct
Transfection
efficiency
(%)
Efficiency of Bio-CSA binding (%) after
preincubation with
a
:
Preimmune
sera
Anti-
rDBL3␥
sera
CSA CSB CSC
pRE4-DBL3␥79 ⫾381⫾30076⫾12 72 ⫾6
pRE4-DBL1X 68 ⫾50 0000
pRE4-DBL2X 61 ⫾657⫾455⫾6054⫾664⫾5
pRE4-DBL3X 66 ⫾662⫾30068⫾963⫾8
pRE4-DBL4ε58 ⫾40 0000
pRE4-DBL5ε60 ⫾60 0000
pRE4-DBL6ε51 ⫾50 0000
a
Transfected 293T cells were preincubated with preimmune mouse sera (1:20
dilution), anti-rDBL3␥mouse sera (1:20 dilution), CSA (200 ␮gml
⫺1
), CSB (200
␮gml
⫺1
), and CSC (200 ␮gml
⫺1
) prior to incubation with Bio-CSA. The
efficiency of Bio-CSA binding was determined as described for Table 3. Results
reported are average ⫾standard deviation of three independent experiments.
Where the binding efficiency is reported as zero, no Alexafluor 488-stained cells
were seen in the entire well.
VOL. 74, 2006 CSA-BINDING DBL DOMAINS AND PAM 5961

identification of unique chondroitin sulfate proteoglycans of the intervillous
spaces that mediate the adherence of Plasmodium falciparum-infected eryth-
rocytes to the placenta. J. Biol. Chem. 275:40344–40356.
2. Avril, M., B. Traore, F. T. M. Costa, C. Lepolard, and J. Gysin. 2004.
Placenta cryosection for study of the adhesion of Plasmodium falciparum-
infected erythrocytes to chondroitin sulfate A in flow conditions. Microbes
Infect. 6:249–255.
3. Baruch, D. I., B. L. Pasloske, H. B. Singh, X. Bi, X. C. Ma, M. Feldman, T. F.
Taraschi, and R. J. Taraschi. 1997. Cloning the Plasmodium falciparum gene
encoding PfEMP1, a malarial variant antigen and adherence receptor on the
surface of parasitized human erythrocytes. Cell 82:77–87.
4. Beeson, J. G., G. V. Brown, M. E. Molyneix, C. Mhango, F. Dzinjalamala,
and S. Rogerson. 1999. Plasmodium falciparum isolates from infected preg-
nant women and children are associated with distinct adhesive and antigenic
properties. J. Infect. Dis. 180:464–472.
5. Beeson, J. G., S. J. Rogerson, B. M. Cooke, J. C. Reeder, W. Chai, A. M.
Lawson, M. E. Molyneux, and G. V. Brown. 2000. Adhesion of Plasmodium
falciparum-infected erythrocytes to hyaluronic acid in placental malaria. Nat.
Med. 6:86–90.
6. Beeson, J. G., and G. V. Brown. 2004. Plasmodium falciparum-infected
erythrocytes demonstrate dual specificity for adhesion to hyaluronic acid and
chondroitin sulfate A and have distinct adhesive properties. J. Infect. Dis.
189:169–179.
7. Brabin, B. J. 1983. An analysis of malaria in pregnancy in Africa. Bull.
W. H. O. 61:1005–1016.
8. Brabin, B. J., M. Hakimi, and D. Pelletier. 2001. An analysis of anemia and
pregnancy-related maternal mortality. J. Nutr. 131:604S–615S.
9. Buffet, P. A., B. Gamain, C. Scheidig, D. Baruch, J. D. Smith, and R.
Hernandez-Rivas. 1999. Plasmodium falciparum domain mediating adhesion
to chondroitin sulfate A: a receptor for human placental infection. Proc.
Natl. Acad. Sci. USA 96:12743–12748.
10. Bull, P. C., B. S. Lowe, M. Kortok, C. S. Molyneux, C. I. Newbold, and K.
Marsh. 1998. Parasite antigens on the infected red cell surface are targets for
naturally acquired immunity to malaria. Nat. Med. 4:358–360.
11. Chia, Y. S., C. Badaut, N. G. Tuikue Ndam, A. Khattab, S. Igonet, N. Fievet,
G. A. Bentley, P. Deloron, and M. Q. Klinkert. 2005. Functional and immu-
nological characterization of a Duffy-binding-like gamma domain from Plas-
modium falciparum erythrocyte membrane protein-1 expressed by a placen-
tal isolate. J Infect. Dis. 192:1284–1293.
12. Chitnis, C. E., and L. H. Miller. 1994. Identification of the erythrocyte
binding domains of Plasmodium vivax and Plasmodium knowlesi proteins
involved in erythrocyte invasion. J. Exp. Med. 180:497–506.
13. Chitnis, C. E., P. Sinnis, and L. H. Miller. 1999. The sporozoite, the mer-
ozoite and the infected red cell: parasite ligands and host receptors, p.
249–285. In P. Perlmann and M. Wahlgren (ed.), Malaria: molecular and
clinical aspects. Harwood Academic Publishers, Amsterdam, The Nether-
lands.
14. Cohen, G. H., W. C. Wilcox, D. L. Sodora, D. Long, J. Z. Levin, and R. J.
Eisenberg. 1988. Expression of herpes simplex virus type 1 glycoprotein D
deletion mutants in mammalian cells. J. Virol. 62:1932–1940.
15. Costa, F. T. M., T. Fusai, D. Parzy, Y. Sterkers, M. Torrentino, J. B. Douki,
B. Traore, S. Petres, A. Scherf, and J. Gysin. 2003. Immunization with
recombinant duffy binding-like-␥3 induces pan-reactive and adhesion block-
ing antibodies against placental chondroitin sulfate A-binding Plasmodium
falciparum parasites. J. Infect. Dis. 188:153–164.
16. Creasey, A. M., T. Staalsoe, A. Raza, D. E. Arnot, and J. A. Rowe. 2003.
Nonspecific immunoglobulin M binding and chondroitin sulfate A binding
are linked phenotypes of Plasmodium falciparum isolates implicated in ma-
laria during pregnancy. Infect. Immun. 71:4767–4771.
17. Deb, T. B., and K. Datta. 1996. Molecular cloning of human fibroblast
hyaluronic acid binding protein confirms its identity with P-32, a protein
co-purified with splicing factor SF2. Hyaluronic acid binding protein as P-32
protein, co-purified with splicing factor SF2. J. Biol. Chem. 271:2206–2212.
18. Dorman, E. K., C. E. Shulman, J. Kingdom, J. N. Bulmer, J. Mwendwa, N.
Peshu, and K. Marsh. 2002. Impaired uteroplacental blood flow in pregnan-
cies complicated by falciparum malaria. Ultrasound Obstet. Gynecol. 19:
165–170.
19. Duffy, M. F., T. J. Byrne, S. R Elliot, D. W. Wilson, S. J. Rogerson, J. G.
Beeson, R. Noviyanti, and G. V. Brown. 2005. Broad analysis reveals a
consistent pattern of var gene transcription in Plasmodium falciparum re-
peatedly selected for a defined adhesion phenotype. Mol. Microbiol. 56:774–
788.
20. Duffy, P. E., and M. Fried. 2003. Antibodies that inhibit Plasmodium falcip-
arum adhesion to chondroitin sulfate A are associated with increased birth
weight and the gestational age of newborns. Infect. Immun. 71:6620–6623.
21. Elliot, S. R., M. F. Duffy, T. J. Byrne, J. G. Beeson, E. J. Mann, D. W. Wilson,
S. J. Rogerson, and G. V. Brown. 2005. Cross-reactive surface epitopes on
chondroitin sulfate A-adherent Plasmodium falciparum-infected erythro-
cytes are associated with transcription of var2csa. Infect. Immun. 73:2848–
2856.
22. Ellman, G. L. 1959. Tissue sulfhydril groups. Arch. Biochem. Biophys. 82:
70–77.
23. Flick, K., C. Scholander, Q. Chen, V. Fernandez, B. Pouvelle, J. Gysin, and
M. Wahlgren. 2001. Role of nonimmune IgG bound to PfEMP1 in placental
malaria. Science 293:2098–2100.
24. Fried, M., and P. E. Duffy. 1996. Adherence of Plasmodium falciparum to
chondroitin sulfate A in the human placenta. Science 272:1502–1504.
25. Fried, M., F. Nosten, A. Brockman, B. J. Brabin, and P. E. Duffy. 1998.
Maternal antibodies block malaria. Nature 395:851–852.
26. Fusai, T., D. Parzy, D. Spillmann, F. Eustacchio, B. Pouvelle, C. Lepolard,
A. Scherf, and J. Gysin. 2000. Characterisation of the chondroitin sulphate
of Saimiri brain microvascular endothelial cells involved in Plasmodium
falciparum cytoadhesion. Mol. Biochem. Parasitol. 108:25–37.
27. Gamain, B., A. R. Trimnell, C. Scheidig, A. Scherf, L. H. Miller, and J. D.
Smith. 2005. Identification of multiple chondroitin sulfate A (CSA)-binding
domains in the var2CSA gene transcribed in CSA-binding parasites. J. Infect.
Dis. 191:1010–1013.
28. Garner, P., and A. M. Gulmezoglu. 2003. Drugs for preventing malaria-
related illness in pregnant women and death in the newborn. Cochrane
Database Syst. Rev. 1:CD000169.
29. Gysin, J., B Pouvelle, N. Fievet, C. Lepolard, and A. Scherf. 1999. Ex vivo
desequestration of Plasmodium falciparum-infected erythrocytes from hu-
man placenta by chondroitin sulfate A. Infect. Immun. 67:6596–6602.
30. Kyes, S. A., Z. Christodoulou, A. Raza, P. Horrocks, R. Pinches, J. A. Rowe,
and C. I. Newbold. 2003. A well-conserved Plasmodium falciparum var gene
shows an unusual stage-specific transcript pattern. Mol. Microbiol. 48:1339–
1348.
31. le Cessie, S. F., H. Verhoeff, G. Mengistie, P. Kazembe, R. Broadhead, and
B. J. Brabin. 2002. Changes in haemoglobin levels in infants in Malawi: effect
of low birth weight and fetal anaemia. Arch. Dis. Child. Fet. Neonat. Ed.
86:F182–F187.
32. Lekana Douki, J. B., B. Traore, F. T. M. Costa, T. Fusaı¨, B. Pouvelle, Y.
Sterkers, A. Scherf, and J. Gysin. 2002. Sequestration of Plasmodium falcip-
arum-infected erythrocytes to chondroitin sulfate A, a receptor for maternal
malaria: monoclonal antibodies against the native parasite ligand reveal
pan-reactive epitopes in placental isolates. Blood 100:1478–1484.
33. Marsh, K., and R. J. Howard. 1986. Antigens induced on erythrocytes by P.
falciparum: expression of diverse and conserved determinants. Science 231:
150–153.
34. Mayor, A., N. Bir, R. Sawhney, S. Singh, P. Pattnaik, S. Singh, A. Sharma,
and C. E. Chitnis. 2004. Receptor-binding residues lie in central regions of
Duffy-binding-like domains involved in red cell invasion and cytoadherence
by malaria parasites. Blood 105:2557–2563.
35. McGregor, I. A., M. E. Wilson, and W. Z. Billewicz. 1983. Malaria infection
of the placenta in The Gambia, West Africa; its incidence and relationship
to stillbirth, birthweight and placental weight. Trans. R. Soc. Trop. Med.
Hyg. 77:232–244.
36. McGregor, I. A. 1984. Epidemiology, malaria and pregnancy. Am. J. Trop.
Med. Hyg. 33:517–525.
37. Pandey, K. C., S. Singh, P. Pattnaik, C. R. Pillai, U. Pillai, A. Lynn, S. K.
Jain, and C. E. Chitnis. 2002. Bacterially expressed and refolded receptor
binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibi-
tory antibodies. Mol. Biochem. Parasitol. 123:23–33.
38. Ranjan, A., and C. E. Chitnis. 1999. Mapping regions containing binding
residues within functional domains of Plasmodium vivax and Plasmodium
knowlesi erythrocyte-binding proteins. Proc. Natl. Acad. Sci. USA 96:14067–
14072.
39. Reeder, J. C., A. F. Cowman, K. M. Dvern, J. G. Beeson, J. K. Thompson,
S. J. Rogerson, and G. V. Brown. 1999. The adhesion of Plasmodium falcip-
arum-infected erythrocytes to chondroitin sulfate A is mediated by P. fal-
ciparum erythrocyte membrane protein 1. Proc. Natl. Acad. Sci. USA 96:
5198–5202.
40. Reeder, J. C., A. N. Holder, J. G. Beeson, and G. V. Brown. 2000. Identifi-
cation of glycosaminoglycan binding domains in Plasmodium falciparum
erythrocyte membrane protein 1 of a chondroitin sulfate A-adherent para-
site. Infect. Immun. 68:3923–3926.
41. Ricke, C. H., T. Staalsoe, K. Koram, B. D. Akanmori, E. M. Riley, T. G.
Theander, and L. Hviid. 2000. Plasma antibodies from malaria-exposed
pregnant women recognize various surface antigens of Plasmodium falcipa-
rum-infected erythrocytes in a parity-dependent manner and block parasite
adhesion to chondroitin sulfate A. J. Immunol. 165:3309–3316.
42. Riley, E. M., L. Hviid, and T. G. Theander. 1994. Malaria, p. 119–143. In F.
Kierszenbaum (ed.), Parasitic infections and the immune system. Academic
Press, New York, N.Y.
43. Rogerson, S. J., S. C. Chaiyaroj, K. Ng, J. C. Reeder, and G. V. Brown. 1995.
Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-
infected erythrocytes. J. Exp. Med. 1821:15–20.
44. Salanti, A., T. Staalsoe, T. Lavstsen, A. T. R. Jensen, M. P. K. Sowa, D. E.
Arnot, L. Hviid, and T. G. Theander. 2003. Selective upregulation of a single
distinctly structured var gene in chondroitin sulfate A-adhering Plasmodium
falciparum involved in pregnancy associated malaria. Mol. Microbiol. 49:
179–191.
45. Salanti, A., M. Dahlback, L. Turner, M. A. Nielsen, L. Barford, P. Magis-
trado, A. T. Jensen, T. Lavstsen, M. F. Ofori, K. Marsh, L. Hviid, and T. G.
5962 BIR ET AL. INFECT.IMMUN.

Theander. 2004. Evidence for the involvement of VAR2CSA in pregnancy-
associated malaria. J. Exp. Med. 200:1197–1203.
46. Sharma, S. K., R. Chattopadhyay, K. Chakrabarti, S. Pati, V. K. Srivastava,
P. Tyagi, S. Mahanty, S. K. Misra, T. Adak, B. S. Das, and C. E. Chitnis.
2004. Epidemiology of malaria transmission and development of natural
immunity in a malaria endemic village, San Dulakudar, in Orissa state, India.
Am. J. Trop. Med. Hyg. 71:457–465.
47. Singh, S., K. Pandey, R. Chattopadhayay, S. S. Yazdani, A. Lynn, A. Bharadwaj,
A. Ranjan, and C. E. Chitnis. 2001. Biochemical, biophysical, and functional
characterization of bacterially expressed and refolded receptor binding do-
main of Plasmodium vivax Duffy-binding protein. J. Biol. Chem. 276:17111–
17116.
48. Smith, J. D., C. E. Chitnis, A. G. Craig, D. J. Roberts, D. E. Hudson-Taylor,
D. S. Peterson, R. Pinches, C. I. Newbold, and L. H. Miller. 1995. Switches
in expression of Plasmodium falciparum var genes correlate with changes in
antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82:101–
110.
49. Smith, J. D., G. Subramanian, B. Gamain, D. I. Baruch, and L. H. Miller.
2000. Classification of adhesive domains in the Plasmodium falciparum
erythrocyte membrane protein 1 family. Mol. Biochem. Parasitol. 110:
293–310.
50. Staalsoe, T., H. A. Giha, D. Dodoo, T. G. Theander, and L. Hviid. 1999.
Detection of antibodies to various antigens of Plasmodium falciparum-in-
fected erythrocytes by flow cytometry. Cytometry 35:329–336.
51. Staalsoe, T., R. Megnekou, N. Fievet, C. H. Ricke, H. D. Zoring, R. Leke,
D. W. Taylor, P. Deloron, and L. Hviid. 2001. Acquisition and decay of
antibodies to pregnancy-associated variant antigens on the surface of Plas-
modium falciparum-infected erythrocytes that protect against placental para-
sitemia. J. Infect. Dis. 184:618–626.
52. Steketee, R. W., B. L. Nahlen, M. E. Parise, and C. Menendez. 2001. The
burden of malaria in pregnancy in malaria-endemic areas. Am. J. Trop. Med.
Hyg. 64:28–35.
53. Su, X. Z., V. M. Heatwole, S. P. Wertheimer, F. Guinet, J. A. Herfeldt, D. S.
Peterson, J. A. Ravetch, and T. E. Wellems. 1995. The large diverse gene
family var encodes proteins involved in cytoadherence and antigenic varia-
tion of Plasmodium falciparum infected erythrocytes. Cell 82:89–100.
54. Suguitan, A. L. J., R. J. Leke, G. Fouda, A. Zhou, L. Thuita, S. Metenou, J.
Fogako, R. Megnekou, and D. W. Taylor. 2003. Changes in the levels of
chemokines and cytokines in the placentas of women with Plasmodium
falciparum malaria. J. Infect. Dis. 188:1074–1082.
55. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continuous
culture. Science 193:673–675.
56. Tuikue Ndam, N. G., A. Salanti, G. Bertin, M. Dahlbeck, N. Fievet, L.
Turner, et al. 2005. High level of var2csa transcription by Plasmodium fal-
ciparum isolated from the placenta. J. Infect. Dis. 192:331–335.
57. Walter, P. R., Y. Garin, and P. Blot. 1982. Placental pathologic changes in
malaria. A histologic and ultrastructural study. Am. J. Pathol. 109:330–342.
Editor: J. L. Flynn
VOL. 74, 2006 CSA-BINDING DBL DOMAINS AND PAM 5963