PfSPATR, a Plasmodium falciparum Protein Containing an Altered Thrombospondin Type I Repeat Domain Is Expressed at Several Stages of the Parasite Life Cycle and Is the Target of Inhibitory Antibodies*
Rana Chattopadhyay,
Dharmendar Rathore
,
Hishasi Fujioka
,
Sanjai Kumar,
Patricia de la Vega,
David Haynes,
Kathleen Moch,
David Fryauff,
Ruobing Wang,
Daniel J. Carucci and
Stephen L. Hoffman ¶
From the
Malaria Program, Naval Medical Research Center, Silver Spring, Maryland
20910-7500,
Laboratory of Malaria and Vector
Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425,
and the
Institute of Pathology, Case Western
Reserve University, Cleveland, Ohio 44106
Received for publication, January 27, 2003
, and in revised form, April 11, 2003.
 |
ABSTRACT
|
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The annotated sequence of chromosome 2 of Plasmodium falciparum
was examined for genes encoding proteins that may be of interest for vaccine
development. We describe here the characterization of a protein with an
altered thrombospondin Type I repeat domain (PfSPATR) that is expressed in the
sporozoite, asexual, and sexual erythrocytic stages of the parasite life
cycle. Immunoelectron microscopy indicated that this protein was expressed on
the surface of the sporozoites and around the rhoptries in the asexual
erythrocytic stage. An Escherichia coli-produced recombinant form of
the protein bound to HepG2 cells in a dose-dependent manner and antibodies
raised against this protein blocked the invasion of sporozoites into a
transformed hepatoma cell line. Sera from Ghanaian adults and from a volunteer
who had been immunized with radiation-attenuated P. falciparum
sporozoites specifically recognized the expression of this protein on
transfected COS-7 cells. These data support the evaluation of this protein as
a vaccine candidate.
 |
INTRODUCTION
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Plasmodium parasites cause malaria and are transmitted by the bite
of infected mosquitoes. Numerous candidate genes and strategies have been
evaluated in an attempt to develop malaria vaccines
(15),
yet there is currently no licensed malaria vaccine, and it is unlikely that
there will be one for at least a decade
(6). The complex life cycle of
the parasite with its distinct morphological and antigenic stages has been a
major hurdle in developing such vaccines. It is anticipated that use of data
from the recently completed genomic sequence of Plasmodium falciparum
(7) will lead to an increased
understanding of parasite biology that will eventually be translated into new
drugs and vaccines for malaria
(8).
Chromosome 2 was the first chromosome of P. falciparum to be
sequenced, and initial analysis indicated that there were 209 genes on this
chromosome (9). In an effort to
discover additional protein candidates for vaccine development, we sought to
characterize one of the genes from chromosome 2 of P. falciparum,
which had been annotated as a putative secreted protein containing a
thrombospondin Type I repeat
(TSR)1 domain
(9). The TSR is an ancient
eukaryotic domain (10) now
known to be present in more than 300 different proteins
(11), including surface
antigens of pathogenic microorganisms
(12). Numerous
Plasmodium surface antigens have been shown to possess the TSR domain
(1315),
and these proteins have been shown to be involved in ookinete and sporozoite
motility, host cell attachment, and invasion
(1619),
thus making them potentially good vaccine targets. In addition to a TSR
domain, we found that the predicted protein also possessed a cysteine-rich
signature that could represent a Type II EGF-like domain. The orthologue of
this protein is present in the murine malaria parasite Plasmodium
yoelii and named "secreted protein with altered thrombospondin
repeat" or SPATR
(20).
We have characterized its expression, localization, and function at
different stages of the Plasmodium life cycle. We report that this
protein is expressed at several stages of the life cycle, that it binds to
hepatoma cells, and that antibodies to this protein inhibit P.
falciparum sporozoite invasion of liver cells.
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EXPERIMENTAL PROCEDURES
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Reverse Transcription-PCR and CloningTotal RNA was isolated
from P. falciparum sporozoites, synchronized erythrocytic stages
(ring, trophozoite, and schizont), and gametocytes using a High Pure RNA
Isolation Kit (Roche Diagnostics). 2 µg of RNA from each of these stages
was reverse transcribed using random hexamers and Superscript II RNase
H reverse transcriptase (Invitrogen). 5 µl of the reverse
transcribed product of each of the above stages was subjected to PCR using
PfSPATR-specific primers. Primer design was based on the published sequence
with GenBankTM accession number AE001404
[GenBank]
(9). The amplified products
were cloned into TA Cloning vector (Invitrogen) and sequenced.
Recombinant Protein Expression and PurificationFor protein
expression, a 690-bp cDNA encoding the mature form of PfSPATR (without signal
sequence) was cloned as a BamHI and EcoRI fragment into
pGEX-3X (Amersham Biosciences), a GST-based Escherichia coli
expression vector. The recombinant protein was expressed in BL21
Escherichia coli cells, and the expression was induced with 1
mM isopropyl-1-thio-
-D-galactopyranoside. The
protein was purified on a glutathione-agarose column as per the manufacturer's
instructions (Amersham Biosciences).
Generation of Anti-PfSPATR Serum in MiceOutbred CD-1 mice
were immunized intraperitoneally with 10 µg of the purified protein in
Freund's complete adjuvant and boosted 3 and 6 weeks after the first
immunization with 10 µg of protein in Freund's incomplete adjuvant. Sera
were collected 12 days after the third dose. Anti-GST antibodies were depleted
by passing the sera through an immobilized GST column (Pierce), which was
confirmed by Western blot.
Immunofluorescence AssaySpots of P. falciparum
sporozoites and smears of erythrocytic stages and gametocytes were made on
glass slides. Anti-PfSPATR serum at 1:20 to 1:6400 dilutions were added and
incubated in a moist chamber at 37 °C. After 1 h, unbound material was
removed by washing, and anti-mouse IgG-fluorescein isothiocyanate conjugate
was added. Unbound conjugate was removed, and the slides were observed under
UV light in a fluorescence microscope. Pre-immune mice sera were used as
controls.
Immunoelectron MicroscopyImmunoelectron microscopy was
carried out on sporozoites isolated from infected mosquito salivary glands and
in vitro cultured blood stages of P. falciparum (Clone 3D7)
using 1:40 anti-PfSPATR antiserum as described
(21). Pre-immune sera were
used as control.
Expression of PfSPATR on COS-7 CellsDNA encoding the
full-length open reading frame of PfSPATR was cloned in plasmid pRE4
(22), a mammalian expression
vector, and the endotoxin-free plasmid was transfected into COS-7 cells using
Lipofectin. Expression of PfSPATR was evaluated by immunofluorescence using
murine anti-PfSPATR antibodies and human serum samples obtained from 1)
naturally immune adult, lifelong residents of P. falciparum
hyperendemic area in Ghana, 2) malaria-naive volunteers immunized with
irradiated P. falciparum sporozoites, and 3) their controls exposed
to the bite of uninfected mosquitoes
(23). All sera were used at
dilutions ranging from 1:50 to 1:400. The use of human serum samples for this
experiment was approved by the institutional review board at the Naval Medical
Research Center.
Cell Binding AssayThe hepatoma cell line HepG2 was used to
evaluate the binding activity of PfSPATR. Cells were seeded in a 96-well plate
a day before the start of the experiment. The next day the recombinant
proteins, 0 to 1.00 µM, were added to the paraformaldehyde-fixed
cells and incubated at 37 °C for 1 h. Unbound material was removed
followed by the addition of murine anti-protein antibodies. After a 1-h
incubation at 37 °C, alkaline phosphatase-conjugated goat anti-mouse
antibody was added, and the bound protein was measured by a fluorescence-based
assay using 4-methyllumbelliferyl phosphate as substrate
(24).
In Vitro Inhibition of Sporozoite Invasion (ISI) in HepG2
CellsThe ISI assay was performed as described
(19). Briefly, 50,000 HepG2
cells were placed in each of the eight wells of tissue culture slides 2 days
before the experiment. P. falciparum (NF54) sporozoites were isolated
from mosquito salivary glands using a discontinuous gradient as described
(25). 20,000 sporozoites were
added to the cells along with anti-PfSPATR serum at a final dilution of 1:50
in the presence (20 or 10 µg/ml) or absence of PfSPATR protein. Anti-P.
falciparum circumsporozoite protein (PfCSP) monoclonal antibody, NFS1, at
a concentration of 10 µg/ml was added as a positive control (NFS1
monoclonal antibody was diluted 1:600 to achieve this concentration). After a
3-h incubation at 37 °C, the numbers of sporozoites that had invaded the
hepatoma cells were counted. Percent inhibition was calculated by the
following formula: [(mean number of invaded sporozoites in negative controls)
(mean number of invaded sporozoites in test) / (mean number of invaded
sporozoites in negative controls)] x 100.
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RESULTS
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Selection, Cloning, and Expression of PfSPATRAnalysis of
the published DNA sequence of chromosome 2 of P. falciparum
identified a 1069-nucleotide sequence containing a two-exon gene encoding a
250-amino acid long putative secreted protein with an altered TSR domain
(Fig. 1). Application of
reverse transcriptase-PCR to assess its expression in sporozoites, infected
erythrocytes (rings, trophozoites, schizonts, and gametocytes) revealed that
the gene is transcribed in all the evaluated stages of the parasite life cycle
(Fig. 2). The amplified
fragment was 753 bp in length, and sequencing of the cDNA confirmed that the
correct mRNA had been amplified (data not shown). To obtain recombinant
PfSPATR protein, a 690-bp cDNA fragment encoding the mature protein was cloned
in plasmid pGEX-3X, a GST-based E. coli expression vector. The
construct was expressed in BL21 cells and induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h. Although the
coding sequence had a high AT content, a characteristic feature of P.
falciparum genes, we could detect the expression of the fusion protein on
a Coomassie Blue-stained polyacrylamide gel (data not shown). The fusion
protein was purified to homogeneity on a glutathione-agarose column (data not
shown). Purified protein was used to immunize outbred CD-1 mice, and
anti-PfSPATR serum was obtained.

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FIG. 1. Schematic representation of PfSPATR gene and protein. The mature
mRNA formed after removal of the intron region is 753 nucleotides in length
and translates into a 250-amino acid polypeptide. The probable Type II
EGF-like domain is boxed, whereas the altered thrombospondin domain
is underlined. The first 21 amino acids represent the putative signal
sequence and are shown in lowercase letters.
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FIG. 2. Transcription of PfSPATR gene at different stages of the P.
falciparum life cycle. The reaction was performed in the absence
(RT) and presence (+RT) of reverse transcriptase.
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Localization of PfSPATR Protein in Different Parasite
StagesTo determine whether the transcribed mRNA was associated
with protein expression, we evaluated the cellular expression and localization
of PfSPATR at different stages of the parasite life cycle. Immunofluorescence
assays using anti-PfSPATR antibodies produced in mice demonstrated binding in
all of the evaluated stages, viz. sporozoites, asexual erythrocytic
stages, and gametocytes, suggesting that the protein is expressed in several
stages of parasite life cycle (Fig.
3). The strongest reactivity was observed against sporozoites
where the protein was detectable even at dilutions of 1:6400 of the antiserum.
Sporozoites and asexual erythrocytic stages were further evaluated by
immunoelectron microscopy to determine the specific location of PfSPATR. In
longitudinal and cross-sections, PfSPATR was localized on the surface of
sporozoites and was not detected in the intracellular organelles such as
micronemes (Fig. 4, A and
B). In asexual erythrocytic stages, PfSPATR was
detectable around the rhoptries and to a lesser extent on the infected
erythrocyte membrane (Fig.
4C). Western blot using anti-PfSPATR antiserum on
sporozoite and blood-stage parasite lysates detected PfSPATR protein at its
expected size of
30 kDa (data not shown).

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FIG. 3. Immunofluorescence assay using anti-PfSPATR sera from mice immunized
with the recombinant protein to detect PfSPATR protein expression in different
stages of P. falciparum. A, sporozoite (1:6400).
B, trophozoite (1:640). C, asexual erythrocytic stage
schizont (1:640). D, merozoites escaping from a late stage schizont
(1:640). E, gametocyte (1:640). Dilutions of antisera used are in
parentheses.
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FIG. 4. Localization of PfSPATR in P. falciparum by immunoelectron
microscopy. A, longitudinal section of sporozoite. B,
cross-section of sporozoite. C, cross-section of an infected
erythrocyte containing a schizont. R, rhoptry; Mz,
merozoites; FV, food vacuole; Hz, hemozoin pigments;
E, erythrocyte membrane; Mn, micronemes.
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Reactivity of Sera from Malaria Endemic Regions with PfSPATR on COS-7
CellsHaving established expression, we investigated whether sera
from individuals exposed to P. falciparum parasites recognized
PfSPATR. Employing a plasmid expressing PfSPATR to transiently transfect COS-7
cells, it was found that the protein was expressed on the surface of the cells
and was readily recognized by the anti-PfSPATR serum
(Fig. 5A) but not by
the pre-immune serum (Fig.
5B). Sera from a malaria-naive volunteer who had been
immunized with radiation-attenuated P. falciparum sporozoites
(Fig. 5C) and five
clinically immune adults (Fig. 5,
EI) from a region of Ghana with intense P.
falciparum-malaria transmission recognized the PfSPATR expressed on COS-7
cells. However, serum from a volunteer who was also immunized with irradiated
sporozoites but had low anti-sporozoite antibodies
(Fig. 5D) as
characterized by immunofluorescence assay and sera from two nonimmune adults
(Fig. 5, J and
K) did not recognize PfSPATR expression.

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FIG. 5. Recognition of PfSPATR expression on COS-7 cells. A, serum
from mice immunized with PfSPATR-recombinant protein. B, serum taken
prior to immunization with PfSPATR-recombinant protein. C, serum from
a volunteer immunized with radiation-attenuated P. falciparum
sporozoites who had high levels of anti-sporozoite antibodies. D,
serum from a volunteer who had been immunized with irradiated P.
falciparum sporozoites whose serum was negative for anti-sporozoite
antibodies. EI, sera from semi-immune adults from Ghana and
Africa. JK, sera from nonimmune adults from the United
States.
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Biological Function of PfSPATRAs PfSPATR showed strong
surface localization on sporozoites, we investigated its possible involvement
in cell-cell interaction by evaluating its potential for binding to the human
hepatocyte cell line, HepG2. The protein demonstrated potent binding to HepG2
cells that was dose-dependent (Fig.
6) and comparable with that of Pf-CSP. In contrast, serum albumin
used as a negative control showed no binding (data not shown). This result
suggested that PfSPATR could function as another parasite ligand involved in
the interaction of sporozoites with liver cells and that a receptor for
PfSPATR was present on HepG2 cells.

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FIG. 6. Binding of PfSPATR protein to hepatoma cells. Binding activity of
PfSPATR (filled circles) and recombinant PfCSP (open
circles) was evaluated on HepG2 cells in a fluorescence-based assay.
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In Vitro Inhibition of Sporozoite InvasionAs PfSPATR
efficiently bound HepG2 cells, we investigated the ability of antibodies
against PfSPATR to prevent sporozoite invasion of human liver cells.
Anti-PfSPATR serum at a final dilution of 1:50 inhibited sporozoite invasion
by more than 80%. Nonimmune control serum showed no inhibition suggesting that
the inhibitory property of anti-PfSPATR serum was specific. The inhibitory
activity was comparable with that of an anti-PfCSP monoclonal antibody that
prevented invasion by more than 90% at 10 µg/ml. This invasion inhibition
was antigen-specific as shown by the addition of free recombinant protein in
the assay, which completely reversed the inhibition
(Fig. 7).

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FIG. 7. In vitro inhibition of sporozoite invasion into HepG2 cells by
anti-PfSPATR serum. The final dilution of anti-PfSPATR serum and control
antisera used was 1:50, and the NFS1 monoclonal antibody against PfCSP was
diluted 1:600 to give a final concentration of 10 µg/ml. The numbers of
P. falciparum sporozoites invading liver cells were counted in the
presence of anti-PfSPATR serum alone or with the recombinant-PfSPATR protein
(20 or 10 µg/ml) used as a competitor to neutralize the inhibitory affect
of anti-PfSPATR antibodies.
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DISCUSSION
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We investigated a protein of P. falciparum that is encoded by a
gene located on chromosome 2 of the parasite
(9). Based on protein sequence
homology, the protein had been suggested to possess an altered TSR domain
toward the carboxyl terminus
(20). The central motif
present in most TSR-family proteins is WSXW (where
"X" can be any amino acid) followed by CSXTCG
(26). As the CSXTCG
motif is absent in the SPATR sequence, it has been referred to as an
"altered thrombospondin domain"
(20). In Plasmodium,
TSR-containing genes show synteny linkage conservation between different
species (27). Along with the
altered TSR domain, the protein has a 15-amino acid region
(72NSRNCWCPRGYILCS86) that has characteristics
suggestive of a Type II EGF-like signature sequence. The BLOCKS protein domain
data base and search tool
(www.blocks.fhcrc.org)
that predicted this characteristic indicated that the PfSPATR sequence was not
typical of the Type II EGF signature sequence found in a diverse range of
proteins (28). Interestingly,
no other protein domain data base and search tool predicted this Type II
EGF-like signature. Although the precise roles of the TSR and potential EGF
domains are as yet unclear, these domains are located in the extracellular
region of membrane-bound proteins or in proteins known to be secreted
(29). Although TSR
(1315)
or EGF domains
(3032)
are individually present in a number of Plasmodium proteins, no
Plasmodium antigen has been reported in which these two domains are
predicted to be present together.
We found that the PfSPATR gene is transcribed during the sporozoite and the
major erythrocytic stages of the parasite life cycle
(Fig. 2). Sporozoites for our
studies were produced in mosquitoes, and the erythrocytic stage parasites were
produced in cultures of erythrocytes, so there could not have been any
cross-contamination. The expressed sequence matched perfectly with the
predicted gene structure of the protein, which also verified the predicted
exon-intron boundaries of the gene (Fig.
1). Only a few Plasmodium proteins have been reported to
be expressed during multiple stages of the parasite life cycle
(33).
We evaluated the cellular expression of PfSPATR by immunofluorescence assay
and by immunoelectron microscopy and found that the protein is present in the
sporozoite, asexual erythrocytic, and gametocyte stages of the life cycle
(Figs. 3 and
4). In sporozoites it was
exclusively located on the surface, whereas in asexual blood stages it was
present around the rhoptries of merozoites and on the membrane of infected
erythrocytes (Fig. 4). The
presence of this protein on the surface of the parasite in different stages
led us to investigate whether this protein was recognized by the host immune
system in P. falciparum-infected individuals. The PfSPATR protein
that was expressed on the cell surface of transfected COS-7 cells was
recognized by sera from naturally infected clinically immune adult Africans,
indicating that this protein was recognized by the host immune system
(Fig. 5). The fact that the
serum from an individual immunized with irradiated sporozoites recognized the
protein corroborates the expression and immunogenicity of the protein during
the early pre-erythrocytic stage of the parasite infection in humans. Serum
from a volunteer with low levels of anti-sporozoite antibodies and control
sera from two nonimmune donors did not recognize PfS-PATR expression on COS-7
cells indicating that this reaction was specific.
The expression of PfSPATR protein on the sporozoite surface and its
recognition by the host immune system in infected individuals led us to
investigate its biological role in the parasite life cycle. Other known
Plasmodium antigens with similar properties have been shown to be
involved in cell-cell interactions
(1618).
We hypothesized a role for this protein in the binding of sporozoites to liver
cells, a property known to be associated with other sporozoite proteins
possessing a TSR domain. PfSPATR bound to human liver cells, and its binding
was comparable with that of PfCSP, the predominant sporozoite surface protein
(Fig. 6). The binding of
PfSPATR appeared to be specific and is presumed to be involved in the
sporozoite invasion of liver cells as evidenced by inhibition of invasion by
anti-PfSPATR antibodies and reversal of inhibition by the addition of
recombinant PfSPATR protein (Fig.
7). It will be interesting to determine whether there are
anti-PfSPATR antibodies that block the invasion-inhibiting activity of
anti-PfSPATR antibodies as has been demonstrated for MSP-19
(34).
The presence of antibodies that partially inhibit the sporozoite invasion
of hepatocytes does not indicate that an individual will be protected against
P. falciparum infection. If a mosquito injects 20 sporozoites and 19
of them are inhibited from invading hepatocytes by antibodies to PfSPATR or
against any other sporozoite protein, the subject will not be protected
against developing P. falciparum infection, because within 1 week a
single successfully invaded sporozoite can give rise to 10,000 merozoites each
of which can invade erythrocytes. Immunization of volunteers with a number of
PfCSP-recombinant protein vaccines has elicited antibodies to sporozoites that
successfully inhibit sporozoite invasion of hepatoma cells in vitro
but fail to protect the volunteers against challenge
(35). Nonetheless, we know
from passive transfer studies in mice and monkeys
(36) that antibodies against
sporozoites can completely protect against sporozoite challenge. In those
cases, the invasion-inhibitory activity was generally >95%. We are
currently investigating the potential of anti-SPATR antibodies to protect
against infection in the P. yoelii rodent malaria model.
The SPATR protein is present in multiple Plasmodium species. It is
present in the transcriptome of P. yoelii sporozoites
(20), and we have also
identified its orthologue in Plasmodium knowlesi and Plasmodium
vivax species.2
The presence of this protein in human, simian, and rodent malaria parasite
species suggests that the protein plays an important role in the biology of
the parasite. Numerous efforts are currently under way to develop an effective
vaccine against malaria (37).
The complex life cycle of the parasite with distinct sets of antigens
expressed during various stages of development has made vaccine design and
development a major challenge to malaria researchers. We have described herein
a molecule that holds potential for investigation as a malaria vaccine
candidate. Its multistage expression by sporozoites, asexual erythrocytic
forms, and gametocytes, along with its possible role in liver cell invasion,
suggests that PfSPATR could be a valuable new vaccine component.
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FOOTNOTES
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* This work was supported by Naval Medical Research and Development Command
Work Unit 6000.RAD1.F.A0309. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
¶
To whom correspondence should be addressed: Sanaria, 308 Argosy Drive,
Gaithersburg, MD 20878. Tel.: 240-299-3178; Fax: 301-990-6370; E-mail:
SLHoffman{at}sanaria.com.
1 The abbreviations used are: TSR, thrombospondin Type I repeat; PfCSP,
Plasmodium falciparum circumsporozoite protein; PfSPATR,
Plasmodium falciparum-secreted protein with altered thrombospondin
repeat; EGF, epidermal growth factor; GST, glutathione
S-transferase. 
2 R. Chattopadhyay, unpublished results. 
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ACKNOWLEDGMENTS
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We thank Dr. Gary H. Cohen and Dr. Roselyn J. Eisenberg, University of
Pennsylvania, Philadelphia for providing the pRE4 plasmid, DL6 and ID3
monoclonal antibodies used in expression of PfSPATR in COS-7 cells work and
Dr. Yupin Charoenvit of the Malaria Program, United States Naval Medical
Research Center, Silver Spring, MD for providing sera from irradiated
sporozoite trial volunteers. We also acknowledge Dr. Stefan H. I. Kappe,
Department of Pathology, New York University School of Medicine, New York and
Dr. Mani Subramanian, Human Genome Sciences, Rockville, MD for their critical
comments and suggestions.
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