Cross-Reactive Surface Epitopes on

Chondroitin Sulfate A-Adherent

Plasmodium falciparum-Infected
Erythrocytes Are Associated with
Transcription of var2csa
Salenna R. Elliott, Michael F. Duffy, Timothy J. Byrne,
James G. Beeson, Emily J. Mann, Danny W. Wilson,

Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV

Stephen J. Rogerson and Graham V. Brown
Infect. Immun. 2005, 73(5):2848. DOI:
10.1128/IAI.73.5.2848-2856.2005.

Updated information and services can be found at:

http://iai.asm.org/content/73/5/2848

These include:
REFERENCES This article cites 58 articles, 34 of which can be accessed free
at: http://iai.asm.org/content/73/5/2848#ref-list-1

CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new
articles cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml

To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
INFECTION AND IMMUNITY, May 2005, p. 2848–2856 Vol. 73, No. 5
0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.5.2848–2856.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cross-Reactive Surface Epitopes on Chondroitin Sulfate A-Adherent

Plasmodium falciparum-Infected Erythrocytes Are Associated with
Transcription of var2csa
Salenna R. Elliott,* Michael F. Duffy, Timothy J. Byrne, James G. Beeson,† Emily J. Mann,
Danny W. Wilson,† Stephen J. Rogerson, and Graham V. Brown
Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Victoria, Australia

Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV

Received 18 November 2004/Returned for modification 30 December 2004/Accepted 7 January 2005

Malaria in pregnancy is associated with placental accumulation of Plasmodium falciparum-infected eryth-

rocytes (IE) that adhere to chondroitin sulfate A (CSA). Adhesion is mediated by P. falciparum erythrocyte
membrane protein 1 (PfEMP1), a variant parasite protein expressed on the surface of IE and encoded by var
genes. Rabbit antiserum was generated against the CSA-adherent P. falciparum line CS2, in which the
dominant var transcribed is var2csa, a relatively conserved var gene that has been associated with CSA
adhesion. Anti-CS2 recognized genetically distinct CSA-adherent P. falciparum lines but not CD36-adherent
parent lines. Reactivity with anti-CS2 correlated with the level of adhesion to CSA. Fluorescence-activated cell
sorting according to binding of anti-CS2 showed reactivity was associated with CSA adhesion and transcription
of var2csa. These data are consistent with the hypothesis that var2csa encodes a PfEMP1 expressed on the
surface of IE, which mediates adhesion to CSA and is relatively conserved between genetically distinct strains
of P. falciparum.

During pregnancy, women are more susceptible to the con-
sequences of Plasmodium falciparum malaria infection. Ma-
laria in pregnancy is associated with increased risk of maternal
anemia and fetal intrauterine growth retardation (11, 54) and
is characterized by accumulation of P. falciparum-infected
erythrocytes (IE) in the placenta (59).
Adhesion of IE to multiple host receptors is mediated by P.
falciparum erythrocyte membrane protein 1 (PfEMP1) (2, 12,
15, 16, 43; for a review see reference 40), a variant parasite
protein expressed on the IE surface and encoded by the var
multigene family (3, 51, 55). Transcriptional switching between
var genes is associated with changes in parasite antigenic and
adhesion phenotypes (51, 55). IE from nonpregnant individu-
als commonly bind to the endothelial receptors CD36 and
intercellular adhesion molecule 1 (ICAM-1) (5, 26, 42). Pla-
cental IE have a unique phenotype, adhering to chondroitin
sulfate A (CSA) and hyaluronic acid (HA), which are ex-
pressed on syncytiotrophoblasts (1, 5, 8, 26).
Partial immunity to pregnancy-associated malaria develops
after several pregnancies. This is associated with acquisition of
antibodies to variant parasite antigens expressed on the IE
surface, which inhibit adhesion of placental IE to CSA (7, 28,
39, 45, 53). PfEMP1 is a potential vaccine candidate for preg-
nancy-associated malaria. There are approximately 60 var
genes per parasite genome (30), and since var genes are highly
polymorphic (55), the diversity of PfEMP1 molecules is likely
to be large. However, the range of PfEMP1 molecules ex-
* Corresponding author. Mailing address: Department of Medicine,
University of Melbourne, Royal Melbourne Hospital, Victoria 3050 Aus-
tralia. Phone: 613 8344 3267. Fax: 613 9347 1863. E-mail: salenna@
unimelb.edu.au.
† Present address: The Walter and Eliza Hall Institute of Medical
Research, Melbourne, Victoria, Australia.
pressed by placental IE is probably restricted. Sera from ma-
laria-exposed multigravid women can inhibit adhesion of pla-
cental IE to CSA even when IE and sera are obtained from
geographically distinct locations, suggesting conservation of
CSA binding domains (28).
Several var genes purported to encode CSA-binding do-
mains have been described, including varCS2 (43) and
FCR3varCSA (12, 50). Binding to CSA has been localized to
DBL␥ domains of these genes (12, 18, 32, 38, 43, 44). However,
recent studies of FCR3varCSA failed to show a correlation
between transcription and the CSA adhesion phenotype or
specific transcription in placental-type parasites (21, 27, 35,
47). FCR3varCSA (12, 50) and varCS2 (43) were originally
identified in CSA-adherent parasite lines using reverse tran-
scription PCR (RT-PCR). Since single trophozoites can tran-
scribe multiple var genes simultaneously (21), this approach
might detect genes that do not encode the expressed PfEMP1
protein.
A recent study using quantitative RT-PCR showed that se-
lection of laboratory lines for CSA adhesion was associated
with increased transcription of the var gene var2csa (49). The
var2csa gene was transcribed at higher levels in placental iso-
lates than in isolates from children. Unlike many var genes,
var2csa shows a high level of homology in genetically distinct
isolates (33, 49). Recent mass spectrometric analysis failed to
identify the var2csa protein in IE membrane extracts of CSA-
selected or placental isolates (29). However, antisera gener-
ated against domains of 3D7 var2csa recognize CSA-selected
IE, suggesting that the PfEMP1 encoded by var2csa is ex-
pressed on the infected erythrocyte surface (48).
In this study, we examined genetically distinct CSA-adherent
P. falciparum lines, looking for an association between surface
antigenic phenotype and var2csa transcription. Using rabbit
antiserum generated against trophozoites of the CSA-adherent

2848
VOL. 73, 2005
FIG. 1. Summary of P. falciparum lines used in this study. Three
genetically distinct laboratory lines, FAF-EA8 (derived by selection of
ItG on human umbilical vein endothelial cells [HUVEC]), 3D7, and
HM (from a traveler infected in Southeast Asia), were used. (A) FAF-
EA8 was selected for adhesion to CSA to generate CS2, selected on
HA to generate E8B-HA, and selected on ICAM-1 to generate E8B-
ICAM. Rabbit antiserum was generated against the CSA-adherent line
CS2 (boldface box). (B) 3D7 was selected on CSA to generate 3C,
which in turn was selected on ICAM-1 to produce 3CI and then
reselected on CSA to derive 3CIC. Direct selection of 3D7 on ICAM-1
produced 3D7-ICAM. (C) The patient isolate HM was selected on
CSA to generate HCS3. The adhesion phenotype of each line is indi-
cated in italics.
line CS2, in which the dominant var transcript is var2csa (22),
we linked the presence of conserved or cross-reactive epitopes
on the surface of CSA-adherent IE with transcription of
var2csa.
MATERIALS AND METHODS
Parasite culture. Parasites were cultured as previously described (4, 57). Par-
asite lines were regularly tested for Mycoplasma contamination and genotyped
(MSP1 and MSP2 alleles). Gelatin enrichment of knob-expressing parasites (31)
and sorbitol synchronization (37) were performed every 1 to 2 weeks.
Parasite lines. Three genetically different P. falciparum lines were used (Fig.
1): ItG (shown to be identical to FCR3 [21, 46, 58]), 3D7, and HM (isolated from
a traveler infected in Southeast Asia). FAF-EA8 (E8B) (Fig. 1A) was from a
clone of ItG2F6 (FAF6) (9, 14) and bound predominantly to CD36. CS2 was
obtained by repeated selection of FAF-EA8 on Chinese hamster ovary cells and
purified CSA (17) (Fig. 1A). CS2 also binds HA (8). E8B-HA was derived from
FAF-EA8 by selecting for adhesion to HA and also binds CSA (8) (Fig. 1A).
E8B-ICAM was selected from FAF-EA8 by panning on ICAM-1 (Fig. 1A). 3D7
SURFACE ANTIGENS AND var2csa IN MALARIA 2849
lines were selected for adhesion to CSA and ICAM-1 as described previously
(41) (Fig. 1B). 3D7 lines selected for adhesion to CSA do not bind HA (4).
3D7-ICAM was obtained by repeated selection of 3D7 on purified ICAM-1 (Fig.
1B). HM was selected on purified CSA to generate HCS3 (6) (Fig. 1C), which
binds HA as well (4).
Characterization of adhesion phenotype. Adhesion assays were performed as
described previously (5). Receptors were diluted in phosphate-buffered saline
(PBS) and coated in triplicate spots on the bases of 25-ml plastic petri dishes
(Falcon 1058; Becton Dickinson) by overnight incubation at 4°C (CD36 purified
from platelets [a gift from M. Berndt, Baker Institute, Australia]), soluble
ICAM-1 [from transfected Chinese hamster ovary cells], CSA [from bovine
trachea {Sigma}; 10 ␮g/ml), and HA [from bovine vitreous humor {Sigma}; 100
␮g/ml). After blocking for 30 min with PBS/1% bovine serum albumin, plates
Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV
were washed twice in RPMI-HEPES, and trophozoites (3 to 12% parasitemia)
were resuspended at 7% hematocrit in adhesion medium (RPMI-HEPES sup-
plemented with 50 ␮g/ml hypoxanthine, 2.5 ␮g/ml gentamicin, and 10% pooled
human serum). Thirty-five microliters of this suspension was added per receptor
spot and incubated at 37°C for 45 min. Four to six washes with RPMI-HEPES
were used to remove unbound cells. Bound cells were fixed by incubation for at
least 2 h in 2% glutaraldehyde in PBS and then stained with Giemsa stain. The
IE bound in eight fields on each receptor spot were counted, and the mean and
standard error of the mean for these 24 counts are given below.
Parasite selection for adhesion. Parasite selection for adhesion was performed
as described previously (41). Receptors were diluted in PBS (CD36, ICAM-1,
CSA, and HA as described above), filter sterilized, and added to a sterile 25-ml
petri dish (Falcon 1058; Becton Dickinson) either as 10-␮l spots covering the
base of the dish (CD36, ICAM-1) or as 50 ml of diluted receptor (CSA, HA).
After overnight incubation, the plate was blocked for 30 min using culture
medium containing 10% human serum and then washed twice with RPMI-
HEPES. Seventy to 100 ml of cultured parasites (5 to 10% mid-trophozoites) was
Percoll purified, resuspended in 50 ml of sterile adhesion medium (as described
above), and added to the receptor-coated dish. After 45 to 60 min of incubation
at 37°C in 1% O2, 4% CO2, 95% N2, unbound cells were removed by four to six
washes in RPMI-HEPES until no unbound IE were observed. Directly pipetting
10 ml of culture medium onto bound IE allowed the IE to be removed and
transferred to a petri dish for culture with fresh red blood cells. After several
selections to obtain the initial phenotype, repeated reselection was required for
most parasite lines after 3 to 4 weeks in continuous culture to maintain optimum
adhesion.
Generation of anti-CS2 rabbit antiserum (R1945). A rabbit was immunized
with synchronized whole CS2 trophozoites as described previously (43) (ap-
proved by the Animal Ethics Committee, The Walter and Eliza Hall Institute of
Medical Research). Rabbit antiserum was heat inactivated (45 min at 56°C) and
preabsorbed against normal human erythrocytes for 1 h at room temperature.
Malaria-exposed human serum. Serum from malaria-exposed donors (from
Malawi or Papua New Guinea) or serum from unexposed Australian controls
was heat inactivated (45 min at 56°C). Ethical clearance was obtained from the
Human Research Ethics Committee, Royal Melbourne Hospital, the College of
Medicine Research Ethics Committee, Malawi, and the Medical Research Ad-
visory Committee, Papua New Guinea.
Characterization of antigenic phenotype by flow cytometry. Trophozoites were
incubated with anti-CS2 rabbit antiserum or malaria-exposed human serum as
described previously (7). Anti-CS2 (1/20) was detected with fluorescein isothio-
cyanate (FITC)-conjugated goat anti-rabbit immunoglobulin (Ig) (1/20) (Sile-
nus) or with goat anti-rabbit IgG (1/50) (Jackson ImmunoResearch) and FITC-
conjugated anti-goat IgG (1/20) (Caltag). Human serum was detected by
sequential incubation with goat anti-human IgG (Caltag) (1/50) and FITC-
conjugated anti-goat IgG (1/20) (Caltag or Chemicon). Quadrants were posi-
tioned based on staining with serum from unimmunized rabbits or malaria-naive
human serum performed in parallel for each experiment. The percentage of IE
specifically recognized by anti-CS2 or human serum was calculated by subtracting
the percentage of uninfected erythrocytes that stained positive.
Cell sorting. E8B-HA IE were enriched by gelatin sedimentation. After two
washes in RPMI-HEPES and three washes in sterile PBS/1% fetal calf serum,
staining with anti-CS2 was performed as described previously, with adjustment
for the greater volume of cells (6 ml of cells at 0.1% hematocrit). All reagents
were filter sterilized before use, and the FITC-conjugated anti-rabbit Ig (Silenus)
was dialyzed against PBS overnight to remove sodium azide. Staining with
ethidium bromide was omitted. Parasitized erythrocytes were sorted at 4°C,
collected into culture medium with 10% human serum, and returned to culture.
The phenotypes of sorted populations were characterized after 2 weeks of con-
tinuous culture (to generate sufficient numbers of IE).
2850 ELLIOTT ET AL. INFECT. IMMUN.

Agglutination assay. Agglutination assays were performed as described previ-

ously (13). A parasite culture (5 to 10% trophozoites and 3% hematocrit) was
spun down and resuspended in an equivalent volume of PBS with ethidium
bromide (10 ␮g/ml). IE were incubated with serum at a 1/10 dilution on a
rotating wheel at room temperature for 45 min. After gentle resuspension,
aliquots were transferred to slides and examined by fluorescence microscopy.
Samples were considered positive if more than three agglutinates consisting of
more than five IE were observed in the absence of agglutinates of uninfected
erythrocytes.
Adhesion inhibition. The ability of anti-CS2 to inhibit adhesion of CS2 tro-
phozoites to CSA was determined as described previously (7). CS2 trophozoites
were washed in RPMI-HEPES, resuspended at 0.5% hematocrit in RPMI-
HEPES with anti-CS2 or normal rabbit serum (1/10 dilution), and incubated for
30 min at room temperature. Thirty microliters of the parasite suspension was

Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV

then added to CSA spots for 20 min at 37°C. Otherwise the assay was performed
as described above for the adhesion assays.
Nucleic acid isolation and manipulation. Standard phenol-chloroform extrac-
tion techniques were used to prepare CS2 genomic DNA (gDNA). RNA was
extracted using Trizol reagent (Sigma) as previously described (52). Precipitated
RNA was dissolved in either formamide (for Northern blots) or water (for
reverse transcription reactions).
Northern blots. Northern blotting was performed as previously described (34,
56) using 0.9% agarose gels and random prime [␣-32P]dATP-labeled probes.
Northern blots probed with the var exon 2 sequence were washed at low strin-
gency with 2⫻ SSC and 0.1% sodium dodecyl sulfate at 55°C (1⫻ SSC is 0.15 M
NaCl plus 0.015 M sodium citrate). Northern blots probed with the specific
var2csa probe were washed at a higher stringency with 0.5⫻ SSC and 0.1%
sodium dodecyl sulfate at 60°C.
Quantitative RT-PCR (Q-RT-PCR). Contaminating gDNA in RNA from each
parasite line was removed by repeated digestion with DNase I (DNA-free;
Ambion). Ten to 20 ␮g of DNase I-digested RNA from each parasite line was
made to 0.3 mg/ml in a solution containing 0.8 mM each of dATP, dCTP, dGTP,
and dTTP and 20 ␮g/ml random hexamers. The solution was incubated at 65°C
for 5 min and then chilled on ice, made to 20 ␮l containing 50 mM Tris-Cl (pH
8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 40 U of Rnasin
(Promega), and incubated first at 25°C for 10 min and then at 42°C for 2 min.
Two hundred units of SuperScript II RNase H reverse transcriptase was then
added, and the solution was incubated first at 42°C for 50 min and then at 70°C
for 15 min. The cDNA was diluted and stored as single-use aliquots at ⫺80°C.
Forward and reverse primers to amplify var2csa (c10) (49) were used at a
concentration of 2 ␮M. 18S rRNA primers (10) were used at concentrations of
0.7 ␮M (forward) and 6 ␮M (reverse). Each 10-␮l PCR mixture contained 5 ␮l
of SYBR Green PCR master mixture (PE Biosystems) and was amplified using
the ABI Prism 7900HT sequence detection system. PCR mixtures were incu-
bated at 95°C for 10 min and then subjected to 40 cycles of 95°C for 15 s and 60°C
for 1 min. A final incubation of 95°C for 2 min was followed by a dissociation step
at 60°C for 2 min with a 2% ramp rate to 95°C for 2 min. The specificity of each
primer pair was determined by dissociation curve analysis.
DNA from var2csa was cloned into the pGEM-T EASY plasmid (Promega),
and the concentration of the recombinant plasmid DNA was accurately deter- FIG. 2. Rabbit antiserum generated against CS2 trophozoites (an-
mined using a fluorescent DNA quantitation kit (Bio-Rad). The plasmid was ti-CS2) was screened against the CSA-adherent ItG lines CS2 (A) and
thawed and diluted 10-fold five times to generate a six-point standard curve, and E8B-HA (B) and the CD36-adherent parent line FAF-EA8 (C) by flow
2 ␮l of each dilution was used per Q-RT-PCR mixture, enabling quantitation of cytometry. Background staining of CS2 with normal rabbit serum
1 ⫻ 106 to 10 cDNA copies. CS2 gDNA at 50 ␮g ml⫺1 was also stored as (NRS) from an unimmunized rabbit is also shown (A). Binding of
single-use aliquots at ⫺80°C and diluted 10-fold five times immediately prior to malaria-exposed human serum (HS) to FAF-EA8 IE (C) demon-
assaying to generate a six-point standard curve. Two microliters of each dilution strated the presence of variant surface antigens. The percentage of
was used per Q-RT-PCR mixture to generate a gDNA standard curve that trophozoites bound by anti-CS2 is indicated in the upper right quad-
spanned 50 ␮g ml⫺1 to 0.5 ng ml⫺1. This standard curve was used to quantitate rant of each dot plot. The bar graph in each panel shows the adhesion
the 18S rRNA levels that were used to normalize the var2csa data such that phenotype for each line (mean and standard error of the mean). The
cDNA from equivalent numbers of cells were compared for all parasite lines. percentage of parasitemia (trophozoites) used in each adhesion assay
Absolute quantitation was performed by using the PE Biosystems instructions by is shown on each graph. Representative results from individual exper-
interpolation of threshold cycle values for unknown samples onto standard iments are shown.
curves of quantity plotted against threshold cycle.

of another ItG CSA-adherent line, E8B-HA (Fig. 1), also

RESULTS
Specificity of anti-CS2 rabbit antiserum. When flow cytom-
etry was used, the majority of CS2 IE were recognized by
anti-CS2 (mean ⫾ standard deviation, 90.8% ⫾ 5.7% [n ⫽ 10
experiments]) (Fig. 2A). There was minimal binding of serum
from an unimmunized rabbit (2.5% ⫾ 0.9% [n ⫽ 10]). Most IE
bound to anti-CS2 (83.3% ⫾ 5.1% [n ⫽ 5]) (Fig. 2B), whereas
few IE from the CD36-adherent parent line, FAF-EA8, were
recognized (6.5% ⫾ 3.4% [n ⫽ 8]) despite expressing variant
surface antigens (demonstrated by the binding of malaria-ex-
posed human serum) (Fig. 2C).
To exclude nonspecific binding of rabbit IgG to PfEMP1,
VOL. 73, 2005
FIG. 3. Anti-CS2 was screened against the 3D7-derived CSA-ad-
herent lines 3C (A) and 3CIC (B) and the CD36-adherent 3D7 line
(C) by flow cytometry. Binding of malaria-exposed human serum (HS)
to 3D7 IE (C) indicated the presence of variant surface antigens. The
percentage of trophozoites bound by anti-CS2 is indicated in the upper
right quadrant of each dot plot. The bar graph in each panel shows the
adhesion phenotype for each line (mean and standard error of the
mean). The percentage of parasitemia (trophozoites) used in each
adhesion assay is shown on each graph. Representative results from
individual experiments are shown.
CS2 trophozoites were incubated with purified rabbit IgG (10
mg/ml) in place of serum. The level of binding of purified
rabbit IgG to CS2 trophozoites was similar to the level of
binding of serum from unimmunized rabbits (results not
shown). Antiserum against the conserved cytoplasmic acidic
terminal sequence domain of PfEMP1 showed levels of bind-
ing to CS2 trophozoites similar to those of normal rabbit se-
rum, confirming that only surface antigens were being detected
by anti-CS2 (results not shown). A three-step staining protocol
using anti-rabbit IgG secondary antibody gave results similar to
the results of the two-step protocol with anti-rabbit Ig de-
scribed previously (results not shown).
Anti-CS2 agglutinated CS2 trophozoites but not the CD36-
binding parent line, FAF-EA8, and inhibited adhesion of CS2
to CSA (results not shown).
Binding of anti-CS2 to genetically different CSA-adherent
parasite lines. Anti-CS2 bound to two CSA-adherent 3D7-
derived parasite lines, 3C and 3CIC (Fig. 1), with a high pro-
portion of IE recognized (for 3C, 83.7% ⫾ 10.6% [n ⫽ 5]; for
3CIC, 64.5% ⫾ 10.2% [n ⫽ 7]) (Fig. 3A and B). Few 3D7 IE,
SURFACE ANTIGENS AND var2csa IN MALARIA 2851
Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV
FIG. 4. Anti-CS2 was screened against the CSA-selected line
HCS3 (A) and the Southeast Asian isolate from which it was derived,
HM (B), by flow cytometry. Binding of malaria-exposed human serum
(HS) to HM IE (B) confirmed the presence of variant surface antigens.
The percentage of trophozoites bound by anti-CS2 is indicated in the
upper right quadrant of each dot plot. The bar graphs in panels A and
B show the adhesion phenotype for each line (mean and standard error
of the mean). The percentage of trophozoites used in each adhesion
assay is shown on each graph. Representative results from individual
experiments are shown. (C) Northern blots of HM and HCS3, ob-
tained using probes against the conserved var exon 2 (which should
detect all var genes) and var2csa.
which adhered to CD36, were recognized by anti-CS2 (5.6% ⫾
3.1% [n ⫽ 6]) (Fig. 3C).
Expression of variant surface antigens by 3D7 was confirmed
by showing binding of malaria-exposed human serum (Fig.
3C).
A third line, HCS3, derived by selection of the Southeast
Asian isolate HM on CSA (Fig. 1), also had a high proportion
of IE that bound to anti-CS2 (91.2% ⫾ 5.6% [n ⫽ 4]) (Fig.
4A). Anti-CS2 did not recognize the majority of trophozoites
from the parent line, HM, although there was low-level binding
to a subpopulation (27.1% ⫾ 4.6% [n ⫽ 3]) (Fig. 4B). In
2852 ELLIOTT ET AL. INFECT. IMMUN.

Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV

FIG. 5. E8B-HA IE with mixed adhesion phenotypes after several weeks in culture (A) were reselected on CD36 (B), CSA (C), or HA (D),
and their recognition by anti-CS2 was determined. The percentage of trophozoites bound by anti-CS2 is indicated in the upper right quadrant of
each dot plot. The bar graph in each panel shows the adhesion phenotype for each line (mean and standard error of the mean). The percentage
of trophozoites used in adhesion assays is shown on each graph. Representative results from individual experiments are shown.

parallel assays, the mean fluorescence intensity for anti-CS2

binding to this subpopulation of HM IE was significantly lower
than that for binding to HCS3 IE (75.3 ⫾ 21.4 for HM, com-
pared with 383.1 ⫾ 100.3 for HCS3 [n ⫽ 3; P ⬍ 0.05]), sug-
gesting that fewer epitopes on the surface of HM IE were
recognized by anti-CS2. A subpopulation of HM (52.9% ⫾
5.4% [n ⫽ 2]) showed high-level binding of malaria-exposed
human serum (mean fluorescence intensity, 590 ⫾ 12.8) (Fig.
4B).
In a recent study, the dominant var transcripts in CSA-
adherent lines derived from ItG and 3D7 hybridized with a
var2csa-specific probe on Northern blots (22). In the present
study, Northern blotting of HCS3 probed with a conserved var
exon 2 sequence revealed two dominant var transcripts, which
also hybridized to the var2csa probe (Fig. 4C). In contrast, the
dominant var transcripts that hybridized to the conserved var
exon 2 probe in the parent line, HM, did not hybridize to the
var2csa probe.
Binding of anti-CS2 was related to CSA adhesion. Most
parasite lines selected for adhesion lose their phenotype in
continuous culture and require reselection. Reduced adhesion
to CSA in selected lines derived from ItG, 3D7, and HM was
associated with a decrease in the percentage of IE recognized
by anti-CS2, which increased if parasite lines were reselected
on the appropriate receptor (results not shown). After a period
of continuous culture, E8B-HA showed reduced adhesion to
CSA and HA, increased adhesion to CD36, and decreased
recognition by anti-CS2 compared with the phenotype imme-
diately after reselection on HA. This E8B-HA was reselected
on CD36, CSA, or HA (Fig. 5). Reselection on either CSA or
HA was associated with restoration of adhesion to both CSA FIG. 6. E8B-HA IE with mixed phenotypes after several weeks in
and HA and enrichment of IE recognized by anti-CS2. There culture (A) were cell sorted according to recognition by anti-CS2.
was no significant change in anti-CS2 binding associated with Anti-CS2-positive IE (B) and anti-CS2-negative IE (C) were grown for
approximately 2 weeks, and the adhesion phenotypes were deter-
reselection on CD36. mined. The percentage of trophozoites bound by anti-CS2 is indicated
To further establish that recognition by anti-CS2 was linked in the upper right quadrant of each dot plot. Binding of malaria-
to CSA adhesion, fluorescence-activated cell sorting according exposed human serum (HS) to anti-CS2 negative IE (C) demonstrated
to recognition by anti-CS2 was performed on the same the presence of variant surface antigens. The bar graph in each panel
shows the adhesion phenotype for each line (mean and standard error
E8B-HA IE with mixed adhesion phenotypes (Fig. 6A), and of the mean). The percentage of trophozoites used in each adhesion
the sorted populations were characterized. The anti-CS2-pos- assay is shown on each graph. Representative results from individual
itive population showed a dominant CSA and HA adherence experiments are shown.
VOL. 73, 2005
FIG. 7. (A) Northern blot of ring-stage RNA from E8B-HA sorted
lines (anti-CS2 pos, anti-CS2 neg) and selected lines (sel CSA, sel
CD36, sel HA [see Fig. 5 and 6]) using a var exon 2 probe (which
should detect all var genes) and a var2csa-specific probe. The dominant
var2csa transcript in anti-CS2-positive, CSA-selected (sel CSA), and
HA-selected (sel HA) lines is indicated (arrow). (B) Quantitative RT-
PCR using var2csa-specific primers with E8B-HA sorted and selected
lines (means and standard deviations).
phenotype (Fig. 6B). Adhesion to CD36 was also detected. In
contrast, IE that were not recognized by anti-CS2 adhered
predominantly to CD36, with minimal CSA or HA adhesion
(Fig. 6C). Binding of malaria-exposed human serum to anti-
CS2-negative IE confirmed that their lack of recognition by
anti-CS2 was not related to an absence of variant surface
antigens.
Binding of anti-CS2 was associated with transcription of
var2csa. var2csa is the dominant var transcript detected on
Northern blots in the ItG lines CS2 and E8B-HA (22). After
continuous culture and before reselection of E8B-HA (as de-
SURFACE ANTIGENS AND var2csa IN MALARIA 2853
scribed above), low levels of the var2csa transcript were de-
tected by Northern blotting (Fig. 7A) and Q-RT-PCR (Fig.
7B). Following reselection on CSA or HA, which increased the
proportions of IE recognized by anti-CS2, var2csa was the
dominant var transcript that hybridized with the conserved var
exon 2 probe on Northern blots (Fig. 7A), and increased tran-
scription of var2csa was detected by Q-RT-PCR (Fig. 7B). In
IE reselected on CD36, which did not enrich for IE recognized
by anti-CS2, transcription of var2csa was minimal (Fig. 7B).
Cell-sorted E8B-HA IE recognized by anti-CS2 showed in-
creased transcription of var2csa by Q-RT-PCR. However, on
Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV
Northern blots, in addition to var2csa, two other var transcripts
were detected. Unlike var2csa, these var genes were not de-
tected on Northern blots of the E8B-HA lines reselected on
CSA or HA (Fig. 7A). var2csa transcription was decreased in
anti-CS2-negative IE compared with the parent E8B-HA.
Taken together, these observations suggested that the major
antigenic determinant on the trophozoite surface recognized
by anti-CS2 rabbit antiserum was associated with adhesion to
CSA and might be the protein product of var2csa.
Antiserum reactive with CSA-binding isolates may cross-
react with surface antigens on IE having a different adhesion
phenotype. Unexpectedly, two 3D7 lines selected for ICAM-1
adhesion, 3CI and 3D7-ICAM (Fig. 1), also showed high-level
recognition by anti-CS2 (for 3CI, 64.8% ⫾ 14.1% [n ⫽ 5]; for
3D7-ICAM, 74.8% ⫾ 10.1% [n ⫽ 4]) without detectable ad-
hesion to CSA (Fig. 8A and B). The proportion of IE recog-
nized by anti-CS2 correlated with the level of adhesion to
ICAM-1 (results not shown). The dominant var that hybridized
with the var exon 2 probe in 3D7-ICAM and 3CI did not
hybridize with the var2csa probe (Fig. 8C). A genetically dis-
tinct ICAM-1-adherent line, E8B-ICAM, was not recognized
by anti-CS2 (13.4% ⫾ 5.1% [n ⫽ 2]) (Fig. 8D).
DISCUSSION
Cross-reactive epitopes were detected on the surface of
erythrocytes infected with genetically distinct CSA-adherent P.
falciparum by rabbit antiserum generated against the CSA-
adherent P. falciparum line CS2. Similar to placental isolates,
CS2 is recognized by sera from pregnant women in a parity-
dependent manner and is rarely recognized by sera from adult
men (7). Recent work has shown that var2csa is the dominant
var gene transcribed in CS2 (22); therefore, this antiserum
should detect the PfEMP1 product of var2csa if it is expressed
on the IE surface.
We and others have shown that infected erythrocytes of
CSA-adherent P. falciparum lines derived from ItG, 3D7, and
the patient isolate HM are recognized by serum antibodies
from pregnant women in a parity-dependent manner (7, 48;
Beeson et al., unpublished data). These observations suggest
that infected erythrocytes of these CSA-selected lines express
antigens similar to those expressed during pregnancy-associ-
ated malaria. In the current study, anti-CS2 recognized CSA-
adherent IE from all three P. falciparum lines, ItG, 3D7, and
HM. In contrast, anti-CS2 did not bind at high levels to CD36-
adherent parent lines. We established that the parent lines
expressed variant surface antigens that were not recognized by
anti-CS2 by demonstrating binding of malaria-exposed human
serum and adhesion to CD36. Binding of anti-CS2 was asso-
2854 ELLIOTT ET AL.
FIG. 8. Anti-CS2 was screened against the 3D7 lines selected for
adhesion to ICAM-1, 3CI (A) and 3D7-ICAM (B). The percentage of
trophozoites bound by anti-CS2 is indicated in the upper right quad-
rant of each dot plot. The bar graphs in panels A, B, and D show the
adhesion phenotype for each line (mean and standard error of the
mean). The percentage of trophozoites used in each adhesion assay is
shown on each graph. Representative results from individual experi-
ments are shown. (C) Northern blot of ring-stage RNA from 3D7-
ICAM, 3CI, and HCS3 (included as a positive control) using a var exon
2 probe (which should detect all var genes) and a var2csa-specific
probe. (D) Anti-CS2 screened against the genetically distinct ICAM-
1-adherent line E8B-ICAM.
ciated with adhesion to CSA. Reselection of the ItG line
E8B-HA on either CSA or HA confirmed that this process
selected for IE recognized by anti-CS2.
CSA-adherent IE have been shown to bind nonimmune hu-
man IgM (20) and IgG (25) by a mechanism that is not antigen
INFECT. IMMUN.
specific. Immunoglobulins in nonimmune rabbit serum may
also bind P. falciparum IE (19). We excluded nonspecific bind-
ing of rabbit IgM or IgG as an explanation for cross-reactivity
of anti-CS2 with different CSA-adherent P. falciparum lines by
showing that serum from unimmunized rabbits and purified
rabbit IgG gave staining only marginally greater than the stain-
ing in the absence of serum. Comparable levels of staining with
anti-Ig and anti-IgG secondary antibodies established that
binding of nonspecific IgM did not contribute significantly to
reactivity. Similar levels of staining with antiserum against the
cytoplasmic acidic terminal sequence domain of PfEMP1 con-
Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV
firmed that anti-CS2 was binding to surface antigens and not to
cytoplasmic proteins.
Binding of anti-CS2 was associated with transcription of
var2csa in CSA-adherent lines. Recently, var2csa has been
shown to be the dominant var transcript in CS2, E8B-HA, 3C,
and 3CIC (22), and in the current study it was also found to be
the dominant var gene transcribed in HCS3. All these lines
were recognized by anti-CS2. This association was confirmed
by cell sorting IE of the ItG line E8B-HA according to recog-
nition by anti-CS2 and showing that var2csa was transcribed at
high levels in anti-CS2-positive IE but not in anti-CS2-negative
IE. Interestingly, anti-CS2-positive IE transcribed two var
genes in addition to var2csa. Because sorted IE had to be
cultured for several weeks before phenotyping, a subpopula-
tion of anti-CS2-positive IE may have switched var gene ex-
pression. This could account for the anti-CS2-negative and
CD36-adherent IE detected in the anti-CS2-positive popula-
tion. Alternatively, PfEMP1 proteins encoded by these var
genes might be antigenically cross-reactive with the var2csa
PfEMP1, resulting in binding of anti-CS2.
A level of cross-reactivity between surface antigens ex-
pressed by CSA-adherent and non-CSA-adherent IE was evi-
dent. The CD36- and ICAM-1-adherent line HM contained a
subpopulation that was recognized by anti-CS2, but the level of
antibody binding (indicated by mean fluorescence intensity)
was lower than the level of binding to CSA-selected HCS3
parasites. Anti-CS2 also bound to IE of the 3D7 ICAM-1-
adherent lines 3CI and 3D7-ICAM but not to the genetically
distinct line E8B-ICAM. Binding by anti-CS2 correlated with
the level of adhesion to ICAM-1 in the 3D7 lines, suggesting
that it was domains of PfEMP1 that were recognized. Finally,
CD36-selected E8B-HA showed low-level reactivity with anti-
CS2 in the absence of adhesion to CSA or transcription of
var2csa. We suggest that our findings are explained by cross-
reactivity between domains of PfEMP1 separate from the func-
tional domains mediating parasite adhesion, although the pos-
sibility of binding to other variant proteins on the infected
erythrocyte surface, such as rifins (24, 36), cannot be excluded.
In summary, using an antiserum against a P. falciparum line
in which the dominant var gene transcribed is var2csa, we
detected cross-reactive epitopes on the surface of genetically
distinct CSA-adherent malaria parasite lines, the presence of
which was associated with increased transcription of var2csa.
These findings are consistent with the recently published ob-
servations of Salanti et al. (48). Both studies suggest that the
3D7 and ItG lines selected for adhesion to CSA express the
protein product of var2csa, which is antigenically cross-reactive
between these lines. Additionally, we showed that a third CSA-
adherent line, HCS3, transcribes var2csa and expresses variant
VOL. 73, 2005
surface antigens that are cross-reactive with those of CSA-
adherent 3D7 and ItG lines. Our observation that cell sorting
infected erythrocytes with anti-CS2 reactivity selected for
CSA-adherent IE that transcribed var2csa confirmed the close
link between transcription of var2csa and the antigenic surface
phenotype associated with CSA adhesion.
Given that women in malaria-endemic areas are susceptible
to pregnancy-associated malaria, despite preexisting antima-
larial immunity, a dedicated vaccine is required for this condi-
tion. Antibodies specific for variant surface antigens expressed
by placental isolates are associated with protection against the
consequences of pregnancy-associated malaria, namely, low
birth weight and maternal anemia (53), and probably function
by inhibiting parasite adhesion to CSA (23, 28). Ideally, a
vaccine for pregnancy-associated malaria would induce anti-
bodies specific for domains of surface proteins that mediate
CSA adhesion. Together with recent observations (48), our
data suggest that the PfEMP1 encoded by var2csa is expressed
on the surface of CSA-adherent malaria-infected erythrocytes
and is relatively conserved between genetically distinct P. fal-
ciparum lines, which is consistent with the high level of homol-
ogy reported in the var2csa gene between isolates (33, 49).
Recent data (48) indicate that antibodies specific for
VAR2CSA are associated with a reduced risk of low birth
weight. If VAR2CSA is the dominant protein responsible for
CSA adhesion by placental parasites and is highly conserved, it
might be a suitable vaccine candidate for malaria in pregnancy.
Further studies using fresh placental isolates should be per-
formed to establish the prevalence of VAR2CSA expression
and its level of conservation in a natural setting.
ACKNOWLEDGMENTS
This work was supported by funding from the National Health and
Medical Research Council of Australia (grant 215201).
We thank Aphrodite Caragounis for technical assistance.
REFERENCES
1. Achur, R. N., M. Valiyaveettil, A. Alkhalil, C. F. Ockenhouse, and D. C.
Gowda. 2000. Characterization of proteoglycans of human placenta and
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. Baruch, D. I., J. A. Gormely, C. Ma, R. J. Howard, and B. L. Pasloske. 1996.
Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized
erythrocyte receptor for adherence to CD36, thrombospondin, and intercel-
lular adhesion molecule 1. Proc. Natl. Acad. Sci. USA 93:3497–3502.
3. Baruch, D. I., B. L. Pasloske, H. B. Singh, X. Bi, X. C. Ma, M. Feldman, T. F.
Taraschi, and R. J. Howard. 1995. Cloning the P. 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., and G. V. Brown. 2004. Plasmodium falciparum-infected eryth-
rocytes demonstrate dual specificity for adhesion to hyaluronic acid and
chondroitin sulfate A and have distinct adhesive properties. J. Infect. Dis.
189:169–179.
5. Beeson, J. G., G. V. Brown, M. E. Molyneux, C. Mhango, F. Dzinjalamala,
and S. J. Rogerson. 1999. Plasmodium falciparum isolates from infected
pregnant women and children are associated with distinct adhesive and
antigenic properties. J. Infect. Dis. 180:464–472.
6. Beeson, J. G., W. Chai, S. J. Rogerson, A. M. Lawson, and G. V. Brown. 1998.
Inhibition of binding of malaria-infected erythrocytes by a tetradecasaccha-
ride fraction from chondroitin sulfate A. Infect. Immun. 66:3397–3402.
7. Beeson, J. G., E. J. Mann, S. R. Elliott, V. M. Lema, E. Tadesse, M. E.
Molyneux, G. V. Brown, and S. J. Rogerson. 2004. Antibodies to variant
surface antigens of Plasmodium falciparum-infected erythrocytes and adhe-
sion inhibitory antibodies are associated with placental malaria and have
overlapping and distinct targets. J. Infect. Dis. 189:540–551.
8. 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.
SURFACE ANTIGENS AND var2csa IN MALARIA 2855
9. Biggs, B. A., R. F. Anders, H. E. Dillon, K. M. Davern, M. Martin, C.
Petersen, and G. V. Brown. 1992. Adherence of infected erythrocytes to
venular endothelium selects for antigenic variants of Plasmodium falciparum.
J. Immunol. 149:2047–2054.
10. Blair, P. L., A. Witney, J. D. Haynes, J. K. Moch, D. J. Carucci, and J. H.
Adams. 2002. Transcripts of developmentally regulated Plasmodium falcipa-
rum genes quantified by real-time RT-PCR. Nucleic Acids Res. 30:2224–
2231.
11. Brabin, B. J. 1983. An analysis of malaria in pregnancy in Africa. Bull.
W. H. O. 61:1005–1016.
12. Buffet, P. A., B. Gamain, C. Scheidig, D. Baruch, J. D. Smith, R. Hernandez-
Rivas, B. Pouvelle, S. Oishi, N. Fujii, T. Fusai, D. Parzy, L. H. Miller, J.
Gysin, and A. Scherf. 1999. Plasmodium falciparum domain mediating ad-
hesion to chondroitin sulfate A: a receptor for human placental infection.
Proc. Natl. Acad. Sci. USA 96:12743–12748.
Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV
13. 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.
14. Chaiyaroj, S. C., R. L. Coppel, S. Novakovic, and G. V. Brown. 1994. Mul-
tiple ligands for cytoadherence can be present simultaneously on the surface
of Plasmodium falciparum-infected erythrocytes. Proc. Natl. Acad. Sci. USA
91:10805–10808.
15. Chen, Q., A. Barragan, V. Fernandez, A. Sundstrom, M. Schlichtherle, A.
Sahlen, J. Carlson, S. Datta, and M. Wahlgren. 1998. Identification of
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the
rosetting ligand of the malaria parasite P. falciparum. J. Exp. Med. 187:15–
23.
16. Chen, Q., A. Heddini, A. Barragan, V. Fernandez, S. F. Pearce, and M.
Wahlgren. 2000. The semiconserved head structure of Plasmodium falcipa-
rum erythrocyte membrane protein 1 mediates binding to multiple indepen-
dent host receptors. J. Exp. Med. 192:1–10.
17. Cooke, B. M., S. J. Rogerson, G. V. Brown, and R. L. Coppel. 1996. Adhesion
of malaria-infected red blood cells to chondroitin sulfate A under flow
conditions. Blood 88:4040–4044.
18. Costa, F. T., T. Fusai, D. Parzy, Y. Sterkers, M. Torrentino, J. B. Douki, B.
Traore, S. Petres, A. Scherf, and J. Gysin. 2003. Immunization with recom-
binant duffy binding-like-gamma3 induces pan-reactive and adhesion-block-
ing antibodies against placental chondroitin sulfate A-binding Plasmodium
falciparum parasites. J. Infect. Dis. 188:153–164.
19. Crandall, I., N. Guthrie, and I. W. Sherman. 1996. Plasmodium falciparum:
naturally occurring rabbit immunoglobulins recognize human band 3 peptide
motifs and malaria-infected red cells. Exp. Parasitol. 82:45–53.
20. 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.
21. Duffy, M. F., G. V. Brown, W. Basuki, E. O. Krejany, R. Noviyanti, A. F.
Cowman, and J. C. Reeder. 2002. Transcription of multiple var genes by
individual, trophozoite-stage Plasmodium falciparum cells expressing a chon-
droitin sulphate A binding phenotype. Mol. Microbiol. 43:1285–1293.
22. Duffy, M. F., T. J. Byrne, S. R. Elliott, D. W. Wilson, S. J. Rogerson, J. G.
Beeson, R. Noviyanti, and G. V. Brown. Broad analysis reveals a consistent
pattern of var gene transcription in Plasmodium falciparum repeatedly se-
lected for a defined adhesion phenotype. Mol. Microbiol., in press.
23. Duffy, P. E., and M. Fried. 2003. Antibodies that inhibit Plasmodium falci-
parum adhesion to chondroitin sulfate A are associated with increased birth
weight and the gestational age of newborns. Infect. Immun. 71:6620–6623.
24. Fernandez, V., M. Hommel, Q. Chen, P. Hagblom, and M. Wahlgren. 1999.
Small, clonally variant antigens expressed on the surface of the Plasmodium
falciparum-infected erythrocyte are encoded by the rif gene family and are
the target of human immune responses. J. Exp. Med. 190:1393–1404.
25. 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.
26. Fried, M., and P. E. Duffy. 1996. Adherence of Plasmodium falciparum to
chondroitin sulfate A in the human placenta. Science 272:1502–1504.
27. Fried, M., and P. E. Duffy. 2002. Two DBLgamma subtypes are commonly
expressed by placental isolates of Plasmodium falciparum. Mol. Biochem.
Parasitol. 122:201–210.
28. Fried, M., F. Nosten, A. Brockman, B. J. Brabin, and P. E. Duffy. 1998.
Maternal antibodies block malaria. Nature 395:851–852.
29. Fried, M., J. P. Wendler, T. K. Mutabingwa, and P. E. Duffy. 2004. Mass
spectrometric analysis of Plasmodium falciparum erythrocyte membrane pro-
tein-1 variants expressed by placental malaria parasites. Proteomics 4:1086–
1093.
30. Gardner, M. J., N. Hall, E. Fung, O. White, M. Berriman, R. W. Hyman,
J. M. Carlton, A. Pain, K. E. Nelson, S. Bowman, I. T. Paulsen, K. James,
J. A. Eisen, K. Rutherford, S. L. Salzberg, A. Craig, S. Kyes, M. S. Chan, V.
Nene, S. J. Shallom, B. Suh, J. Peterson, S. Angiuoli, M. Pertea, J. Allen, J.
Selengut, D. Haft, M. W. Mather, A. B. Vaidya, D. M. Martin, A. H. Fair-
lamb, M. J. Fraunholz, D. S. Roos, S. A. Ralph, G. I. McFadden, L. M.
Cummings, G. M. Subramanian, C. Mungall, J. C. Venter, D. J. Carucci,
2856 ELLIOTT ET AL.
S. L. Hoffman, C. Newbold, R. W. Davis, C. M. Fraser, and B. Barrell. 2002.
Genome sequence of the human malaria parasite Plasmodium falciparum.
Nature 419:498–511.
31. Goodyer, I. D., J. Johnson, R. Eisenthal, and D. J. Hayes. 1994. Purification
of mature-stage Plasmodium falciparum by gelatine flotation. Ann. Trop.
Med. Parasitol. 88:209–211.
32. Khattab, A., J. Kun, P. Deloron, P. G. Kremsner, and M. Q. Klinkert. 2001.
Variants of Plasmodium falciparum erythrocyte membrane protein 1 ex-
pressed by different placental parasites are closely related and adhere to
chondroitin sulfate A. J. Infect. Dis. 183:1165–1169.
33. Kraemer, S. M., and J. D. Smith. 2003. Evidence for the importance of
genetic structuring to the structural and functional specialization of the
Plasmodium falciparum var gene family. Mol. Microbiol. 50:1527–1538.
34. Kyes, S., R. Pinches, and C. I. Newbold. 2000. A simple RNA analysis
method shows var and rif multigene family expression patterns in Plasmo-
dium falciparum. Mol. Biochem. Parasitol. 105:311–315.
35. 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.
36. Kyes, S. A., J. A. Rowe, N. Kriek, and C. I. Newbold. 1999. Rifins: a second
family of clonally variant proteins expressed on the surface of red cells
infected with Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 96:9333–
9338.
37. Lambros, C., and J. P. Vanderberg. 1979. Synchronization of Plasmodium
falciparum erythrocytic stages in culture. J. Parasitol. 65:418–420.
38. Lekana Douki, J. B., B. Traore, F. T. Costa, T. Fusai, B. Pouvelle, Y.
Sterkers, A. Scherf, and J. Gysin. 2002. Sequestration of Plasmodium falci-
parum-infected erythrocytes to chondroitin sulfate A, a receptor for mater-
nal malaria: monoclonal antibodies against the native parasite ligand reveal
pan-reactive epitopes in placental isolates. Blood 100:1478–1483.
39. Maubert, B., N. Fievet, G. Tami, M. Cot, C. Boudin, and P. Deloron. 1999.
Development of antibodies against chondroitin sulfate A-adherent Plasmo-
dium falciparum in pregnant women. Infect. Immun. 67:5367–5371.
40. Newbold, C. I., A. G. Craig, S. Kyes, A. R. Berendt, R. W. Snow, N. Peshu,
and K. Marsh. 1997. PfEMP1, polymorphism and pathogenesis. Ann. Trop.
Med. Parasitol. 91:551–557.
41. Noviyanti, R., G. V. Brown, M. E. Wickham, M. F. Duffy, A. F. Cowman, and
J. C. Reeder. 2001. Multiple var gene transcripts are expressed in Plasmo-
dium falciparum infected erythrocytes selected for adhesion. Mol. Biochem.
Parasitol. 114:227–237.
42. Ockenhouse, C. F., M. Ho, N. N. Tandon, G. A. Van Seventer, S. Shaw, N. J.
White, G. A. Jamieson, J. D. Chulay, and H. K. Webster. 1991. Molecular
basis of sequestration in severe and uncomplicated Plasmodium falciparum
malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1.
J. Infect. Dis. 164:163–169.
43. Reeder, J. C., A. F. Cowman, K. M. Davern, J. G. Beeson, J. K. Thompson,
S. J. Rogerson, and G. V. Brown. 1999. The adhesion of Plasmodium falci-
parum-infected erythrocytes to chondroitin sulfate A is mediated by P. fal-
ciparum erythrocyte membrane protein 1. Proc. Natl. Acad. Sci. USA 96:
5198–5202.
44. Reeder, J. C., A. N. Hodder, 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.
Editor: W. A. Petri, Jr.
INFECT. IMMUN.
45. 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 variant surface antigens on Plasmodium falcipa-
rum-infected erythrocytes in a parity-dependent manner and block parasite
adhesion to chondroitin sulfate A. J. Immunol. 165:3309–3316.
46. Robson, K. J., J. R. Hall, L. C. Davies, A. Crisanti, A. V. Hill, and T. E.
Wellems. 1990. Polymorphism of the TRAP gene of Plasmodium falciparum.
Proc. R. Soc. Lond. B Biol. Sci. 242:205–216.
47. Rowe, J. A., S. A. Kyes, S. J. Rogerson, H. A. Babiker, and A. Raza. 2002.
Identification of a conserved Plasmodium falciparum var gene implicated in
malaria in pregnancy. J. Infect. Dis. 185:1207–1211.
48. Salanti, A., M. Dahlback, L. Turner, M. A. Nielsen, L. Barfod, P. Magis-
trado, A. T. Jensen, T. Lavstsen, M. F. Ofori, K. Marsh, L. Hviid, and T. G.
Theander. 2004. Evidence for the involvement of VAR2CSA in pregnancy-
associated malaria. J. Exp. Med. 200:1197–1203.
Downloaded from http://iai.asm.org/ on June 15, 2014 by CHULALONGKORN UNIV
49. Salanti, A., T. Staalsoe, T. Lavstsen, A. T. Jensen, M. P. Sowa, D. E. Arnot,
L. Hviid, and T. G. Theander. 2003. Selective upregulation of a single
distinctly structured var gene in chondroitin sulphate A-adhering Plasmo-
dium falciparum involved in pregnancy-associated malaria. Mol. Microbiol.
49:179–191.
50. Scherf, A., R. Hernandez-Rivas, P. Buffet, E. Bottius, C. Benatar, B. Pou-
velle, J. Gysin, and M. Lanzer. 1998. Antigenic variation in malaria: in situ
switching, relaxed and mutually exclusive transcription of var genes during
intra-erythrocytic development in Plasmodium falciparum. EMBO J. 17:
5418–5426.
51. 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.
52. Smith, J. D., S. Kyes, A. G. Craig, T. Fagan, D. Hudson-Taylor, L. H. Miller,
D. I. Baruch, and C. I. Newbold. 1998. Analysis of adhesive domains from the
A4VAR Plasmodium falciparum erythrocyte membrane protein-1 identifies
a CD36 binding domain. Mol. Biochem. Parasitol. 97:133–148.
53. Staalsoe, T., C. E. Shulman, J. N. Bulmer, K. Kawuondo, K. Marsh, and L.
Hviid. 2004. Variant surface antigen-specific IgG and protection against
clinical consequences of pregnancy-associated Plasmodium falciparum ma-
laria. Lancet 363:283–289.
54. 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.
55. Su, X. Z., V. M. Heatwole, S. P. Wertheimer, F. Guinet, J. A. Herrfeldt, 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.
56. Taylor, H. M., S. A. Kyes, D. Harris, N. Kriek, and C. I. Newbold. 2000. A
study of var gene transcription in vitro using universal var gene primers. Mol.
Biochem. Parasitol. 105:13–23.
57. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continuous
culture. Science 193:673–675.
58. Triglia, T., S. J. Foote, D. J. Kemp, and A. F. Cowman. 1991. Amplification
of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen
as multiple independent events. Mol. Cell. Biol. 11:5244–5250.
59. 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.