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Infection and Immunity, April 1999, p. 1677-1682, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transformed Toxoplasma gondii
Tachyzoites Expressing the Circumsporozoite Protein of
Plasmodium knowlesi Elicit a Specific Immune Response in
Rhesus Monkeys
Manlio
di Cristina,1
Firman
Ghouze,1
Clemens H. M.
Kocken,2
Silvia
Naitza,1
Pamela
Cellini,3
Dominique
Soldati,4
Alan W.
Thomas,2 and
Andrea
Crisanti1,*
Department of Biology, Imperial College, SW7
2BB London, United Kingdom1; Department
of Parasitology, Biomedical Primate Research Centre, 2288 GJ Rijwijk,
The Netherlands2; Istituto di
Parassitologia, Università di Roma "La Sapienza," 00185 Rome, Italy3; and Zentrum für
Molekulare Biologie, Universität Heidelberg, Heidelberg 6900, Germany4
Received 13 July 1998/Returned for modification 23 September
1998/Accepted 8 December 1998
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ABSTRACT |
Toxoplasma gondii tachyzoites were transformed with the
coding sequence of the circumsporozoite (CS) protein of the
primate malaria parasite Plasmodium knowlesi. A single
inoculation of live transformed tachyzoites elicited an antibody
response directed against the immunodominant repeat epitope
(EQPAAGAGG)2 of the P. knowlesi CS protein
in rhesus monkeys. Notably, these animals failed to show a positive
serum conversion against T. gondii. Antibodies against
Toxoplasma antigens were detected only after a
second inoculation with a higher number of transformed tachyzoites. This boost induced an increased antibody response against the P. knowlesi CS protein associated with immunoglobulin class
switching, thus demonstrating the establishment of immunological
memory. These results indicate that the
Toxoplasma-derived CS protein is efficiently
recognized by the monkey immune system and represents an
immunodominant antigen in transformed parasites.
| |
INTRODUCTION |
The circumsporozoite (CS)
protein uniformly covers the entire surface of the malaria
sporozoite in a dense layer (27). Immunity to the CS
protein blocks sporozoite invasion of hepatocytes in vitro
(21) and exerts a potent antiparasitic effect in rodent malaria models (25, 27, 29). Recently, a recombinant form of
Plasmodium falciparum CS protein fused to the hepatitis B
surface antigen was reported to induce protective immunity against a
sporozoite challenge in the great majority of immunized human
volunteers (33). This experiment has shown the feasibility
of developing a malaria sporozoite vaccine in humans by using the
CS protein formulated together with potent adjuvants. Previous efforts
aimed at inducing protection in humans by using different CS
protein-based vaccines have failed or resulted in incomplete protection
(4, 6, 26, 30). Plasmid DNA (3, 15, 18, 20) and
attenuated recombinant poxvirus vectors (2, 23, 35) are also
being employed in the effort to generate effective malaria vaccines. The availability of gene transfer technology for the protozoan parasite
Toxoplasma gondii may offer an additional
opportunity to express and deliver the CS protein in a highly
immunogenic form.
T. gondii shares with Plasmodium species the
host cell invasion machinery and subcellular organelles. The
phylogenetic relationship among these parasites (24)
suggests that malaria antigens expressed in
Toxoplasma would most likely assume their
natural conformation. Transgenic T. gondii
tachyzoites also have the potential to induce a long-lasting
immunity against malaria antigens. In normal individuals with an
intact immune system, T. gondii causes a mild and
self-limited flu-like disease that stimulates a potent humoral and
cell-mediated immune response (13, 14). Such an immune
response efficiently protects exposed individuals from
subsequent T. gondii infections for the rest of their
lives, although the parasite may persist in the form of tissue cysts.
The pathogenicity of Toxoplasma for immunocompromised individuals and nonimmune fetuses (16)
currently limits the use of the wild-type parasite as an antigen
delivery system. The development of nonvirulent T. gondii strains that have lost the ability to form tissue cysts
under normal conditions may ultimately circumvent this problem (8,
9, 11).
To investigate the ability of T. gondii to function as
an antigen delivery system for Plasmodium antigens, we
have generated tachyzoites expressing the CS protein of the primate
malaria parasite Plasmodium knowlesi. We report here on the
ability of these transgenic parasites to elicit a specific immune
response against the P. knowlesi CS protein in
rhesus monkeys, an experimental host for P. knowlesi and a well-defined model for human immune responses.
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MATERIALS AND METHODS |
Parasite cultures.
T. gondii was
propagated by serial passages over a monolayer of human foreskin
fibroblasts in Dulbecco's modified Eagle medium (Gibco) containing
10% NuSerum (Collaborative Biomedical Products). The clonal parasite
isolate RH (34), which lacks the ability to form tissue
cysts, was used for the genetic manipulation and the immunization procedures.
Transformation vector.
A DNA fragment encompassing the
coding sequence of the P. knowlesi CS gene
(28) from nucleotide 61 to 1053 was amplified by PCR with
P. knowlesi genomic DNA as the template and the
primer combination of pk1 (5'-CCG GCC ATG GCT CAC TTC GAA CAT AAT GTA G-3') and pk2 (5'-CCG GTT AAT TAA TTG AAT AAT GCT AGG AC-3'), containing at their 5' ends an NcoI and a PacI
restriction site, respectively. The amplified P. knowlesi CS sequence was designed to encode the full-length
parasite protein with the exception of its signal sequence. The
P. knowlesi CS-coding sequence was cloned together
with the signal sequence of the T. gondii SAG 1 gene in
the plasmid Bluescript between the SAG 1 promoter (32) and a
300-nucleotide untranslated sequence flanking the 3' end of the SAG 1 gene. The sequence coding for the epitope c-Myc (10) was
cloned between the SAG 1 signal sequence and the CS sequence, generating the transformation construct pSPc-myc/PkCS.
Transformation of T. gondii
tachyzoites.
Transformation experiments were carried out with
the construct pSPc-myc/PkCS together with the vector pT/230, which
contains the selectable chloramphenicol acetyltransferase marker
(22, 31). In brief, 2 × 107 freshly
harvested tachyzoites were resuspended in 700 µl of solution (cytomix) containing 100 µg of pSPmyc/CS, 10 µg of pT/230, and 100 U of BamHI (5). The two plasmids were
linearized with the restriction enzyme BamHI prior to
electroporation (5) and mixed in a 10:1 mass ratio.
Electroporated parasites were subjected to chloramphenicol selection as
described previously (22). After 7 to 10 days, stable
populations of parasites resistant to chloramphenicol emerged, and
single clones were isolated by limiting dilution.
Immunoblot analysis.
Protein lysates of extracellular
tachyzoites were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to nitrocellulose filters. Nonspecific absorption of antibodies was
prevented by incubating the filters with 1% bovine serum albumin (BSA)
in phosphate-buffered saline (PBS) for 2 h at 37°C. The presence
of the recombinant CS protein was revealed with monoclonal antibody
(MAb) 9E10, directed against the c-Myc epitope (Sigma). Antibody
bound to the filter was detected with a goat anti-mouse immunoglobulin
conjugated to alkaline phosphatase (Sigma).
To assess the specifity of human immunoglobulin reagents against rhesus
monkey antibodies, serum proteins from either macaques, humans, or rats
were separated under nonreducing conditions by SDS-8% PAGE or under
reducing conditions by SDS-12.5% PAGE and electroblotted onto
nitrocellulose filters. Blotted proteins were incubated with either
goat anti-human immunoglobulin G heavy plus light chains [IgG(H+L)],
goat anti-human IgG (
-chain specific), or goat anti-human IgM
(µ-chain specific) alkaline phosphotase-conjugated immunoglobulins
(Sigma). Antibody bound to the filter was detected with a goat
anti-mouse immunoglobulin conjugated to alkaline phosphatase (Sigma).
Immunofluorescence.
Extracellular tachyzoites were
washed in PBS, air dried on slides, fixed for 10 min in 1%
formaldehyde-PBS, and permeabilized in acetone at 
20°C for 5 min.
The slides were blocked for 30 min in 1% BSA-PBS and then incubated
at room temperature with MAb 9E10 for 1 h. Antibodies bound to
Toxoplasma cells were revealed with goat anti-mouse
fluorescein isothiocyanate-conjugated immunoglobulins (Becton
Dickinson). Slides were washed in PBS, mounted with Vectashield (Vector), and analyzed by using a Bio-Rad 600 confocal microscope.
Recombinant bacterial proteins and synthetic peptides.
The
recombinant constructs PkCS 1.0 and HRPIIr were amplified by PCR with,
as templates, P. knowlesi and P. falciparum genomic DNAs, respectively. PkCS 1.0 encompasses the
entire sequence of the P. knowlesi CS with the
exception of the signal peptide and the hydrophobic
glycosylphosphatidylinositol (GPI) anchor. HRPIIr encompasses the
sequence of P. falciparum histidine-rich protein II
(HRPII) lacking only its signal peptide. The sequences coding for PkCS
1.0 and HRPIIr were cloned in the vector pDS56/RBSII,6XHis. The
expression unit of this vector is under the control of an IPTG
(isopropyl-
-D-galactopyranoside)-inducible promoter and yields fusions between a stretch of six histidines and the amino terminus of the inserted sequence (19). The recombinant
proteins PkCS 1.0 and HRPIIr containing the histidine tail were
purified by nickel chelate chromatography. The synthetic peptide
(EQPAAGAGG)2 was purchased from the Imperial College
peptide synthesis service. As control, we employed the synthetic
peptide Der pI (FGISNYCQIYPPNANKIREALAQPQRYCR), which encompasses an
epitope from the house dust mite allergen Der P1.
Selection of animals and immunization.
Sera from 20 rhesus
monkeys (Macaca mulatta) were screened by immunoblotting
against a T. gondii protein lysate at a 1:50 dilution.
Seven of the 20 tested rhesus monkeys showed very strong reactivity
with multiple protein bands, suggesting previous exposure to
Toxoplasma. Seven animals were completely negative,
and the remaining ones reacted only very weakly with one or more
protein bands. Ten animals in the same age and weight range were
allocated to two groups, with each group consisting of three
nonreactive and two weakly reactive (9305 and L167 in group A and L175
and 2BWI in group B) animals. Rhesus monkeys were immunized by
intravenous infection with 5 × 106 freshly prepared
tachyzoites at day 0. Group A received Tx-PkCS7, and group B
received RH strain tachyzoites. Both groups received a booster
infection with 30 × 106 Tx-PkCS7 tachyzoites at
day 130.
Serum reactivity.
Blood samples (5 ml) were withdrawn from
each monkey 2 weeks before the first inoculation and at monthly
intervals thereafter. The detection of specific antibodies to
recombinant P. knowlesi CS protein was done on
96-well microtiter plates (Falcon 3912) coated with either purified
PkCS 1.0 or HRPIIr (1 µg/ml) at 4°C overnight in 2× PBS, pH
7.3. After blocking for 2 h at 37°C with 1% BSA in 2×
PBS, the wells were incubated for 1 h at room temperature with different dilutions of the monkey sera in 2× PBS. Antibodies bound to antigens were revealed by using an anti-human goat
immunoglobulin antibody conjugated to alkaline phosphatase (Promega).
The IgG and IgM antibody fractions were detected by using goat
anti-human IgG (Fc specific) and goat anti-human IgM (µ-chain
specific) alkaline phosphotase-conjugated immunoglobulins (Sigma),
respectively. The competition experiments were performed by
preincubating the monkey sera, diluted 1:50 in 2× PBS, for 30 min
either with increasing concentrations (0.16, 0.8, 4, and 20 µg/ml) of
the P. knowlesi CS synthetic peptide
(EQPAAGAGG)2 or with 20 µg of the peptide Der pI per ml.
The reactivity of the monkey sera against Toxoplasma was analyzed by using a diagnostic kit (Radim Diagnostics, Rome, Italy)
that has been developed to detect human IgG directed against an extract
of parasite proteins.
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RESULTS AND DISCUSSION |
Expression of P. knowlesi CS protein in
T. gondii.
Toxoplasma tachyzoites
were transformed with the vector pSPc-myc/PkCS (Fig.
1A), containing (i) the tachyzoite
stage-specific promoter of the Toxoplasma
major surface antigen SAG 1 (32), (ii) a chimeric
P. knowlesi CS protein gene (myc/PkCS), and (iii) a 300-nucleotide untranslated sequence flanking the 3' end
of the SAG 1 gene (7). The myc/PkCS gene encoded a
polypeptide encompassing the signal peptide of SAG 1 (7), the c-Myc epitope (10), and the
P. knowlesi Nuri strain CS sequence from residue 21 to 351 (28). The malaria protein contained the GPI anchor and lacked its own signal peptide. The promoter and the signal sequence
of the SAG 1 gene were inserted in the myc/PkCS expression unit to
facilitate correct transcription, translation, and processing of the
recombinant malaria protein in Toxoplasma
tachyzoites. The c-Myc epitope was inserted between the
signal peptide of SAG 1 and the P. knowlesi
CS-coding sequence in order to facilitate analysis of expression of
the malaria protein.
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FIG. 1.
(A) Schematic representation of the construct
pSPc-myc/PkCS. (B) Immunoblot analysis of total protein lysates of RH
(left lane) and transformed Tx-PkCS7 (right lane) tachyzoites
developed by using MAb 9 E10, directed against the c-Myc epitope.
The migrations (in kilodaltons) of low-molecular-mass standards from
Sigma are indicated on the right.
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Eight transformed parasite clones were isolated and analyzed in
immunoblots with MAb 9E10, directed against the c-Myc epitope
(
10). One
T. gondii clone (Tx-PkCS7)
expressed significant amounts
of the
P. knowlesi CS
protein that migrated as two bands with
the apparent molecular masses
of 44 and 54 kDa (Fig.
1B). This
observation is in agreement with a
previous report showing that
the CS protein from
P. knowlesi sporozoites migrated as multiple
bands with
apparent molecular masses of 42, 50, and 52 kDa (
28),
with
the largest two molecules representing intracellular precursors
of the
final 42-kDa surface protein. These findings suggest that
T. gondii tachyzoites efficiently expressed CS of
P. knowlesi.
Immunofluorescence of extracellular transformed
tachyzoites indicated
that the
P. knowlesi CS
protein was localized in large granules
within the cytoplasm of the
parasites (Fig.
2). This analysis
also
revealed the presence of large quantities of
P. knowlesi CS protein-reactive material in the parasitophorous
vacuoles of
intracellular tachyzoites (not shown). The localization
of the
P. knowlesi CS protein in intra- and
extracellular tachyzoites
is remarkably similar to the
immunofluorescence pattern observed
for the
Toxoplasma-encoded GRA molecules (
1,
12).
Accordingly,
in transformed tachyzoites the
P. knowlesi CS protein may be stored
in the dense granules and
released after parasite invasion of
host cells. On the basis of the
structural features of the construct
myc/PkCS, we expected a
localization of the malaria protein on
the surface of the
tachyzoites; however, such localization was
not observed. Recent
reports indicate that a specific signal is
not required to target
T. gondii protein to the plasma membrane
and that the
presence of a GPI anchor is sufficient to bring any
reporter protein to
the plasma membrane. We therefore concluded
that the
P. knowlesi
CS protein GPI anchor sequence may not be
efficiently recognized
by the tachyzoite-modifying enzymes, resulting
in its mistargeting.
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FIG. 2.
Immunolocalization of the P. knowlesi CS protein in Tx-PkCS7 tachyzoites. Confocal
immunofluorescence (A and C) and transmission (B and D)
photomicrographs of Tx-PkCS7 (A and B) and nontransformed (C and D)
tachyzoites stained by MAb 9E10 are shown. Magnification, ×630;
zoom factor, 2.6 for image acquisition.
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Immunogenicity of transformed T. gondii.
The
transformed Toxoplasma clone Tx-PkCS7 was assessed
for its ability to induce a specific immune response against the
P. knowlesi CS protein in rhesus monkeys (M. mulatta). Two groups of five animals were injected intravenously
with 5 × 106 freshly cultured extracellular
tachyzoites of either the Tx-PkCS7 clone (group A) or its
nontransformed parental strain (group B). Four months after the first
inoculation, both groups were injected with 30 × 106
Tx-PkCS7 parasites. Serum samples were collected from each monkey 2 weeks before the first inoculation and thereafter at monthly intervals
for 5 months. The antibody responses elicited by the immunization
regimens were evaluated by using an enzyme-linked immunosorbent assay
(ELISA) developed with the recombinant bacterial construct PkCS 1.0. This construct spans the entire coding sequence of the P. knowlesi CS protein with the exception of the signal peptide
and the GPI anchor sequence. As a control, the rhesus monkey sera were
also analyzed by ELISA against an unrelated malaria recombinant
construct which encompasses the coding sequence of HRPII of
P. falciparum. The specific reactivity of each serum against the construct PkCS 1.0 was expressed as net optical
density (OD) (ODPkCS 1.0 ODHRPIIr). One
month after the first inoculation, we detected in sera from the monkeys
inoculated with live Tx-PkCS7 tachyzoites significant amounts of
antibodies directed against the P. knowlesi CS
protein (Fig. 3A). At this time point,
three of five monkeys (L167, 49b, and 94003) showed a predominant IgG response, whereas high IgM levels were detected in the two
remaining animals (94002 and 9305) (Fig. 3A and B). The specific
antibody levels in group A monkeys steadily decreased in the
following 3 months while still remaining significantly higher
(P < 0.02) than those in group B. After boosting
with Tx-PkCS7 tachyzoites, all monkeys of group A showed
elevated levels of antibodies (exclusively IgG) directed against the
P. knowlesi CS protein. Monkeys from group B, which
were initially inoculated with the parental nontransformed RH
tachyzoites, failed to show any specific reactivity against the
malaria antigen during the 4 months following primary immunization (Fig. 3). In this group of animals a subsequent inoculation with Tx-PkCS7 tachyzoites was able to induce specific antibodies
directed against the P. knowlesi CS protein,
thus indicating that the previous exposure to T. gondii
parasites did not impair the ability of the monkeys to respond to the
transgenic malaria antigen. The dynamics of the IgM and IgG isotype
responses after the first and second inoculations strongly indicated
that the Tx-PkCS7 tachyzoites induced immunological memory against
the malaria antigen associated with immunoglobulin class switch.
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FIG. 3.
Development of humoral immunity against the
P. knowlesi CS protein. Monkeys in groups A (solid
lines) and B (dashed lines) were inoculated (day 0, arrowhead 1) either
with Tx-PkCS7 tachyzoites or with the nontransformed parental RH
line, respectively. Both groups of monkeys were inoculated 4 months
later (day 130, arrowhead 2) with Tx-PkCS7 tachyzoites. Specific
antibodies against the P. knowlesi CS protein were
revealed by comparing the reactivities of each monkey serum against the
recombinant constructs PkCS 1.0 and HRPIIr. The net OD at 405 nm was
calculated by subtracting the OD values in the HRPIIr ELISA from those
in the P. knowlesi CS ELISA. The assays were
carried out by using labelled secondary antibodies recognizing IgG(H+L)
(A), IgG (B), or IgM (C) human isotypes. Each serum was tested in four
replicates at a 1/200 dilution. Letter-and-number codes refer to
individual monkeys. The standard error for each determination was less
than 10%.
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The kinetics of the antibody response in rhesus monkeys was analyzed by
using secondary antibodies directed against human
immunoglobulins. The
specificities of these reagents were investigated
by immunoblotting
against human, rhesus monkey, and rat serum
proteins (Fig.
4). This analysis revealed that the
antibodies
directed against IgG(H+L) recognize heavy and light chains
of
both human and monkey immunoglobulins. The IgG and IgM secondary
antibodies were shown to selectively recognize, in both human
and
monkey sera, bands which migrate with the molecular sizes
of IgG and
IgM, respectively (Fig.
4). These findings rule out
the possibility
that the use of these reagents has introduced
a bias in the analysis of
the monkey immune response.
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FIG. 4.
Specificity analysis of anti-human immunoglobulin
antibodies against human, rhesus monkey, and rat serum proteins under
nonreducing (A) and reducing (B) conditions by immunoblotting with
anti-IgG(H+L), anti-IgG, or anti-IgM secondary antibodies. The
migrations (in kilodaltons) of high-molecular-mass standards from Sigma
are indicated on the left.
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Previous experiments have shown that the immunodominant region of the
P. knowlesi CS protein is contained within the
tandem
repeat peptide (
17), although additional
conformational epitopes
have also been described
(
36). To investigate the specificity
of the antibody
response induced by Tx-PkCS7 tachyzoites, we have
assessed the
reactivities of the monkey sera against the
P. knowlesi CS protein in the presence of an excess of the
synthetic peptide
(EQPAAGAGG)
2, encompassing the repeat
epitope (Fig.
5). Our results
indicated that a relevant fraction of the
P. knowlesi CS antibodies
elicited by Tx-PkCS7 were directed
against this repeat epitope.
In all sera analyzed, the
peptide (EQPAAGAGG)
2 significantly inhibited,
in a
dose-dependent manner, antibody binding to the PkCS 1.0 recombinant
protein (Fig.
5). However, the serum reactivity
was never completely
abolished, even in the presence of the
highest concentration (20
µg/ml) of peptide, indicating that
additional epitopes are also
recognized by the immunized monkeys.
The unrelated peptide Der
pI did not show a significant inhibitory
effect (
P < 0.001) when
compared to the highest
concentration of the peptide (EQPAAGAGG)
2.
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FIG. 5.
Fine specificity of serum reactivity against the
P. knowlesi CS protein. Monkey sera collected at 5 months were assessed by ELISA (1/50 dilution) against the recombinant
construct PkCS 1.0 in the presence of increasing concentrations of the
synthetic peptide (EQPAAGAGG)2 ( ). The
unrelated peptide Der pI ( ) was employed as a
specifity control and used at a concentration of 20 µg/ml. The
results are the averages of four determinations normalized to control
binding in the absence of synthetic peptide.
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We have also analyzed the monkey sera by using a commercially available
ELISA kit that detects human IgG against
Toxoplasma antigens. Notably, after the first
inoculation, all five monkeys
injected with Tx-PkCS7 and four of five
animals that received
nontransformed parasites failed to react against
Toxoplasma antigens
in the ELISA kit (Fig.
6). Antibodies directed against
Toxoplasma antigens were detected only after
injection of a much higher number
of tachyzoites, thus ruling out
the possibility that the lack
of reactivity was due to limitations in
the ELISA kit employed.
This pattern of reactivity was also confirmed
by immunoblot analysis
of the rhesus monkey sera against
Toxoplasma protein lysates (not
shown). It is
most likely that the first inoculation of tachyzoites
did
not result in a sustained and autonomous infection of
T. gondii in the monkeys and stimulated a weak immune response
against
Toxoplasma antigens. This conclusion was
supported by the observation that
none of the blood samples collected
from the monkeys at days 3,
6, and 10 induced toxoplasmosis in
susceptible BALB/c mice.
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FIG. 6.
Development of humoral immunity against
Toxoplasma antigens in immunized rhesus monkeys.
Solid and dashed lines indicate group A and B monkeys, respectively.
Serum reactivity was analyzed by using a Toxoplasma
ELISA kit developed for the diagnosis of human toxoplasmosis.
Toxoplasma antibody levels represent the means of
duplicate determinations at a 1/200 dilution.
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For group A monkeys, the comparison of the antibody responses
against the
P. knowlesi CS protein and
T. gondii antigens is
striking. The inoculation
of Tx-PkCS7 tachyzoites elicited a significant
humoral response to
the
P. knowlesi CS protein; however, all monkey
sera had
T. gondii antibody levels lower than the World
Health
Organization standard positive level for humans of 15 IU per ml
(
37). This finding strongly indicated that the malaria
protein
is efficiently recognized by the monkey immune system and
represents
the major immunodominant antigen in transformed
T. gondii parasites.
Toxoplasma-derived
Plasmodium antigen
preparations and/or attenuated
live
T. gondii-transformed parasite strains may represent invaluable
sources of highly immunogenic malaria
antigens.
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ACKNOWLEDGMENTS |
Manlio Di Cristina and Firman Ghouze contributed equally to this work.
This work was supported by a grant from the INCO Programme of the
European Commission.
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FOOTNOTES |
*
Corresponding author. Mailing address: Imperial
College, Department of Biology, Prince Consort Rd., SW7 2BB London,
United Kingdom. Phone: 44 171 5945426. Fax: 44 171 5945439. E-mail:
acrs{at}bio.ic.ac.uk.
Editor:
J. R. McGhee
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REFERENCES |
| 1.
|
Achbarou, A.,
O. Mercereau Puijalon,
A. Sadak,
B. Fortier,
M. A. Leriche,
D. Camus, and J. F. Dubremetz.
1991.
Differential targeting of dense granule proteins in the parasitophorous vacuole of Toxoplasma gondii.
Parasitology
3:321-329.
|
| 2.
|
Aidoo, M.,
A. Lalvani,
H. C. Whittle,
A. V. Hill, and K. J. Robson.
1997.
Recombinant vaccinia viruses for the characterization of Plasmodium falciparum-specific cytotoxic T lymphocytes: recognition of processed antigen despite limited re-stimulation efficacy.
Int. Immunol.
9:731-737[Abstract/Free Full Text].
|
| 3.
|
Allsopp, C. E.,
M. Plebanski,
S. Gilbert,
R. E. Sinden,
S. Harris,
G. Frankel,
G. Dougan,
C. Hioe,
D. Nixon,
E. Paoletti,
G. Layton, and A. V. Hill.
1996.
Comparison of numerous delivery systems for the induction of cytotoxic T lymphocytes by immunization.
Eur. J. Immunol.
26:1951-1959[Medline].
|
| 4.
|
Ballou, W. R.,
S. L. Hoffman,
J. A. Sherwood,
M. R. Hollingdale,
F. A. Neva,
W. T. Hockmeyer,
D. M. Gordon,
I. Schneider,
R. A. Wirtz,
J. F. Young, et al.
1987.
Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine.
Lancet
1:1277-1281[Medline].
|
| 5.
|
Black, M.,
F. Seeber,
D. Soldati,
K. Kim, and J. C. Boothroyd.
1995.
Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii.
Mol. Biochem. Parasitol.
74:55-63[Medline].
|
| 6.
|
Brown, A. E.,
P. Singharaj,
H. K. Webster,
J. Pipithkul,
D. M. Gordon,
J. W. Boslego,
K. Krinchai,
P. Suarchawaratana,
C. Wongsrichanalai,
W. R. Ballou, et al.
1994.
Safety, immunogenicity and limited efficacy study of a recombinant Plasmodium falciparum circumsporozoite vaccine in Thai soldiers.
Vaccine
12:102-108[Medline].
|
| 7.
|
Burg, J. L.,
D. Perelman,
L. H. Kasper,
P. L. Ware, and J. C. Boothroyd.
1988.
Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii.
J. Immunol.
141:3584-3591[Abstract].
|
| 8.
|
Buxton, D., and E. A. Innes.
1995.
A commercial vaccine for ovine toxoplasmosis.
Parasitology
110:S11-S16.
|
| 9.
|
Buxton, D.,
K. M. Thomson,
S. Maley,
S. Wright, and H. J. Bos.
1993.
Experimental challenge of sheep 18 months after vaccination with a live (S48) Toxoplasma gondii vaccine.
Vet. Rec.
133:310-312[Abstract].
|
| 10.
|
Chan, S.,
H. Gabra,
F. Hill,
G. Evan, and K. Sikora.
1987.
A novel tumour marker related to the c-myc oncogene product.
Mol. Cell Probes
1:73-82[Medline].
|
| 11.
|
Dubey, J. P.
1996.
Strategies to reduce transmission of Toxoplasma gondii to animals and humans.
Vet. Parasitol.
64:65-70[Medline].
|
| 12.
|
Dubremetz, J. F.,
A. Achbarou,
D. Bermudes, and K. A. Joiner.
1993.
Kinetics and pattern of organelle exocytosis during Toxoplasma gondii/host-cell interaction.
Parasitol. Res.
79:402-408[Medline].
|
| 13.
|
Frenkler, J. K.
1988.
Pathophysiology of toxoplasmosis.
Parasitol. Today
4:273.
[Medline] |
| 14.
|
Gazzinelli, R. T.,
E. Y. Denkers, and A. Sher.
1993.
Host resistance to Toxoplasma gondii: model for studying the selective induction of cell-mediated immunity by intracellular parasites.
Infect. Agents Dis.
2:139-149[Medline].
|
| 15.
|
Gerloni, M.,
W. R. Baliou,
R. Billetta, and M. Zanetti.
1997.
Immunity to Plasmodium falciparum malaria sporozoites by somatic transgene immunization.
Nat. Biotechnol.
15:876-881[Medline].
|
| 16.
|
Girdwood, R. W.
1989.
`Protozoan' infections in the immunocompromised patient the parasites and their diagnosis.
J. Med. Microbiol.
30:3-16[Free Full Text].
|
| 17.
|
Godson, G. N.,
J. Ellis,
P. Svec,
D. H. Schlesinger, and V. Nussenzweig.
1983.
Identification and chemical synthesis of a tandemly repeated immunogenic region of Plasmodium knowlesi circumsporozoite protein.
Nature
305:29-33[Medline].
|
| 18.
|
Gramzinski, R. A.,
D. C. Maris,
D. Doolan,
Y. Charoenvit,
N. Obaldia,
R. Rossan,
M. Sedegah,
R. Wang,
P. Hobart,
M. Margalith, and S. Hoffman.
1997.
Malaria DNA vaccines in Aotus monkeys.
Vaccine
15:913-915[Medline].
|
| 19.
|
Hochuli, E.,
W. Bannwarth,
H. Döbeli,
R. Gentz, and D. Stüber.
1988.
Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent.
Bio/Technology
6:1321-1325.
|
| 20.
|
Hoffman, S. L.,
D. L. Doolan,
M. Sedegah,
J. C. Aguiar,
R. Wang,
A. Malik,
R. A. Gramzinski,
W. R. Weiss,
P. Hobart,
J. A. Norman,
M. Margalith, and R. C. Hedstrom.
1997.
Strategy for development of a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use.
Vaccine
15:842-845[Medline].
|
| 21.
|
Hollingdale, M. R.,
E. H. Nardin,
S. Tharavanij,
A. L. Schwartz, and R. S. Nussenzweig.
1984.
Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells; an in vitro assay of protective antibodies.
J. Immunol.
132:909-913[Abstract].
|
| 22.
|
Kim, K.,
D. Soldati, and J. C. Boothroyd.
1993.
Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker.
Science
262:911-914[Abstract/Free Full Text].
|
| 23.
|
Lanar, D. E.,
J. A. Tine,
C. de Taisne,
M. C. Seguin,
W. I. Cox,
J. P. Winslow,
L. A. Ware,
E. B. Kauffman,
D. Gordon,
W. R. Ballou,
E. Paoletti, and J. C. Sadoff.
1996.
Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria.
Infect. Immun.
64:1666-1671[Abstract].
|
| 24.
|
Levine, N. D.
1988.
Progress in taxonomy of the apicomplexan protozoa.
J. Protozool.
35:518-520[Medline].
|
| 25.
|
McGregor, I. A.
1993.
Towards a vaccine against malaria.
Br. J. Biomed. Sci.
50:35-42[Medline].
|
| 26.
|
Nussenzweig, R. S., and V. Nussenzweig.
1989.
Antisporozoite vaccine for malaria: experimental basis and current status.
Rev. Infect. Dis.
11:S579-S585.
|
| 27.
|
Nussenzweig, V., and R. S. Nussenzweig.
1989.
Rationale for the development of an engineered sporozoite malaria vaccine.
Adv. Immunol.
45:283-334[Medline].
|
| 28.
|
Ozaki, L. S.,
P. Svec,
R. S. Nussenzweig,
V. Nussenzweig, and G. N. Godson.
1983.
Structure of the Plasmodium knowlesi gene coding for the circumsporozoite protein.
Cell
34:815-822[Medline].
|
| 29.
|
Reed, R. C.,
V. Louis Wileman,
R. L. Wells,
A. F. Verheul,
R. L. Hunter, and A. A. Lal.
1996.
Re-investigation of the circumsporozoite protein-based induction of sterile immunity against Plasmodium berghei infection.
Vaccine
14:828-836[Medline].
|
| 30.
|
Sherwood, J. A.,
R. S. Copeland,
K. A. Taylor,
K. Abok,
A. J. Oloo,
J. B. Were,
G. T. Strickland,
D. M. Gordon,
W. R. Ballou,
J. D. Bales, Jr.,
R. A. Wirtz,
J. Wittes,
M. Gross,
J. U. Que,
S. J. Cryz,
C. N. Oster,
C. R. Roberts, and J. C. Sadoff.
1996.
Plasmodium falciparum circumsporozoite vaccine immunogenicity and efficacy trial with natural challenge quantitation in an area of endemic human malaria of Kenya.
Vaccine
14:817-827[Medline].
|
| 31.
|
Soldati, D., and J. C. Boothroyd.
1993.
Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii.
Science
260:349-352[Abstract/Free Full Text].
|
| 32.
|
Soldati, D., and J. C. Boothroyd.
1995.
A selector of transcription initiation in the protozoan parasite Toxoplasma gondii.
Mol. Cell. Biol.
15:87-93[Abstract].
|
| 33.
|
Stoute, J. A.,
W. R. Ballou,
N. Kolodny,
C. D. Deal,
R. A. Wirtz, and L. E. Lindler.
1995.
Induction of humoral immune response against Plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library.
Infect. Immun.
63:934-939[Abstract].
|
| 34.
|
Suresh, K.,
J. W. Mak, and H. S. Yong.
1991.
Long term maintenance of Toxoplasma gondii (Rh strain) in Vero cell line and use of harvested antigens for immunodiagnosis.
S. E. Asian J. Trop. Med. Pub. Health
22:124-128.
|
| 35.
|
Tine, J. A.,
D. E. Lanar,
D. M. Smith,
B. T. Wellde,
P. Schultheiss,
L. A. Ware,
E. B. Kauffman,
R. A. Wirtz,
C. De Taisne,
G. S. Hui,
S. P. Chang,
P. Church,
M. R. Hollingdale,
D. C. Kaslow,
S. Hoffman,
K. P. Guito,
W. R. Ballou,
J. C. Sadoff, and E. Paoletti.
1996.
NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.
Infect. Immun.
64:3833-3844[Abstract].
|
| 36.
|
Vergara, U.,
R. Gwadz,
D. Schlesinger,
V. Nussenzweig, and A. Ferreira.
1985.
Multiple non-repeated epitopes on the circumsporozoite protein of Plasmodium knowlesi.
Mol. Biochem. Parasitol.
14:283-292[Medline].
|
| 37.
|
World Health Organization.
1977.
Biological substances. International standards, reference preparations, and reference reagents, p. 20-21.
World Health Organization, Geneva, Switzerland.
|
Infection and Immunity, April 1999, p. 1677-1682, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
