Induction of CD8+ T cell-mediated protective immunity against Trypanosoma cruzi
Yasushi Miyahira,
Seiki Kobayashi1,
Tsutomu Takeuchi1,
Tsuneo Kamiyama2,
Takeshi Nara,
Junko Nakajima-Shimada and
Takashi Aoki
Department of Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,Tokyo 113-8421, Japan
1 Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
2 Department of Veterinary Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
Correspondence to:
Y. Miyahira
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Abstract
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Trypanosoma cruzi was transformed with the Plasmodium yoelii gene encoding the circum-sporozoite (CS) protein, which contains the well-characterized CD8+ T cell epitope, SYVPSAEQI. In vivo and in vitro assays indicated that cells infected with the transformed T. cruzi could process and present this malaria parasite-derived class I MHC-restricted epitope. Immunization of mice with recombinant influenza and vaccinia viruses expressing the SYVPSAEQI epitope induced a large number of specific CD8+ T cells that strongly suppressed parasitemia and conferred complete protection against the acute T. cruzi lethal infection. CD8+ T cells mediated this immunity as indicated by the unrelenting parasitemia and high mortality observed in immunized mice treated with anti-CD8 antibody. This study demonstrated, for the first time, that vaccination of mice with vectors designed to induce CD8+ T cells is effective against T. cruzi infection.
Keywords: CD8+ T cells, Chagas' disease, protective immunity, recombinant influenza virus, recombinant vaccinia virus, Trypanosoma cruzi
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Introduction
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Chagas' disease, which is caused by a protozoan parasite Trypanosoma cruzi, is endemic in Central and South America, infecting up to 1618 million people and making 90 million people at risk of infection (13). Approximately 10% of the infected individuals would develop a severe symptomatic acute illness with encephalitis and myocarditis (4). Between 20 and 40% of infected individuals never clear the parasites and develop the characteristic features of chronic Chagas' disease some years later (24).
After T. cruzi invade the cells, they escape from phago-lysosome into cytosol, where they proliferate. At this stage, infected cells could process and present MHC class I epitopes, and eventually stimulate antigen-specific CD8+ T cells. In fact, results from several studies including immuno-histochemical analyses (57), in vivo T cell depletion using specific mAb (8,9) and the use of gene knockout mice (1013) indicated that CD8+ T cells are induced after T. cruzi infection. Furthermore, these studies also strongly suggested that these T cells could play a protective role against this parasitic infection (14).
To further characterize the role of CD8+ T cells in T. cruzi infection, it would be necessary to identify parasite-derived epitopes recognized by these T cells. In view of the difficulties encountered to achieve this task, we transfected T. cruzi with a well-characterized model antigen, the murine malarial circumsporozoite (CS) protein (15), which contains the MHC class I-restricted CD8+ T cell epitope, SYVPSAEQI (16).
By using this model system, we demonstrated that epitope-specific CD8+ T cells induced by immunization with the recombinant viruses expressing the malaria epitope protect mice against the acute phase of lethal T. cruzi infection. This report is the first study to demonstrate that immunization with vectors designed to induce CD8+ T cells is effective against the causative agent of Chagas' disease.
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Methods
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Parasites and mice
The Tulahuen strain of T. cruzi (17) was used throughout the study. Epimastigotes were maintained in liver infusion tryptose medium (18) supplemented with 10% FCS (LIT) at 27°C. Metacyclogenesis of the parasite was induced by maintaining them at the stationary state in culture for 1 week. These metacyclic trypomastigotes were used for the production of tissue-culture trypomastigotes as described below. Inbred BALB/c mice (H-2d), 58 weeks old, were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan), and used for immunizations and protection assays.
Cells and media
P815 (H-2d) cells were used for immunological assays. Swiss 3T3 cells were used for the production of tissue-culture trypomastigotes. These cells are maintained in DMEM with high glucose (Life Technologies/BRL, Rockville, MD) supplemented with 10% FCS (Life Technologies/BRL), 2 g/l sodium bicarbonate (Sigma, St Louis, MO), 200 mg/l L-arginine hydrochloride (Life Technologies/BRL), 36 mg/l L-asparagine (Life Technologies/BRL), 2.6 g/l HEPES (Sigma), 105 M 2-mercaptoethanol (Sigma) and antibiotics (complete DMEM). NIH 3T3 cells transformed with pcDNA3 (Invitrogen, San Diego, CA) were generously supplied by Dr K. Ishidoh (Department of Biochemistry, Juntendo University School of Medicine). The cell line was maintained in DMEM supplemented with 10% bovine calf serum (CC Laboratories, Cleveland, OH) and 400 µg/ml of G418 (Sigma), and other supplementation was the same as in complete DMEM. Y26 CD8+ T cell clone, which is specific for SYVPSAEQI derived from the P. yoelii CS (PYCS) protein (19), was supplied by Dr F. Zavala (New York University) and are maintained in the complete DMEM supplemented with 0.5% phorbol myristate acetate-stimulated EL4 cell supernatant as a source of 30 U/ml IL-2 (complete DMEM-IL2).
Plasmid vectors and transformation protocol
The transformation vectors for T. cruzi were either pTEX (20) or pBS:CnFc (21) as described previously. The P. yoelii CS (PYCS) gene (15) was cloned into the BamHI site of the pTEX vector, and into the XbaI at the 5'-end and the BamHI at the 3'-end sites of the pBS:CnFc vector. After the confirmation of the DNA sequence of the PYCS gene by performing the standard dye deoxy terminator method, XL-1 Blue competent cells (Stratagene, La Jolla, CA) were transformed with each plasmid DNA. DNA was prepared and purified by using the Wizard Plus Maxipreps DNA purification system (Promega, Madison, WI). PTEX-PYCS plasmid DNA was used as circular DNA for transfection, while pBS:CnFc-PYCS was linearized with BsiWI and AflII before the electroporation. The T. cruzi Tulahuen strain grown at the log phase in LIT was harvested and washed with ice-cold PBS 3 times, then resuspended in ice-cold PBS with 0.5 mM magnesium acetate and 0.1 mM CaCl2 at a cell concentration of 1x108/ml. Then 25 µg of plasmid DNA was dissolved in 50 µl of 10 mM Tris and 1 mM EDTA (pH 8.0), mixed with 400 µl of cell suspension (containing 4x107 cells), and placed in a disposable cuvette with a 2 mm electrode gap (BioRad, Hercules, CA) on ice for 10 min. The cells were pulsed 3 times in close succession with a BioRad Gene Pulser set at 300 V and 500 µF. Then 3 ml of ice-cold LIT was added into the cuvette immediately after the electroporation. The parasites were placed in an incubator at 27°C for 2 days in 5 ml LIT. G418 (Sigma) was then added into the culture at the final concentration of 250 µg/ml. G418-resistant parasites were obtained 34 weeks later. Tissue-culture trypomastigotes of transformed T. cruzi were first produced by using the Swiss 3T3 cells. The trypomastigotes were then transferred into the G418-resistant NIH 3T3 tissue culture, which enabled the maintenance of parasites in the presence of 400 µg/ml G418.
ELISPOT and standard 51Cr-release assays
Standard ELISPOT assay was performed as described previously with slight modification (22). Multiscreen Immobilon-P 96-well filtration plates (Millipore, Bedford, MA) were used for the detection of antigen-specific IFN-
-secreting cells. The concentration of secondary antibody, biotinylated anti-mouse IFN-
mAb, XMG1.2 (PharMingen, San Diego, CA), was 2.5 µg/ml. Streptavidin peroxidase was purchased from Sigma and used at 1:800 dilution. The spots were counted with the aid of a stereomicroscope and the results were described as the mean number of IFN-
-secreting cells per 106 spleen cells. A standard 51Cr-release assay was performed as described elsewhere (19). P815 cells (106) were labeled with 250 µCi of 51Cr in FCS for 1 h at 37°C. Effector cells (immune spleen cells) were incubated, at different ratios, with 2.5x103 51Cr-labeled P815 cells, in the presence or absence of 1 µM of peptide SYVPSAEQI. After 5 h incubation at 37°C, the supernatants were collected with the aid of a semi-automatic harvester (Skatron, Sterling, VA) and the number of counts determined. The percentage of specific lysis was calculated as described previously (19).
Recombinant influenza and vaccinia viruses bearing a PYCS antigen
The recombinant influenza virus contains the class I MHC-restricted NEDSYVPSAEQI epitope of the PYCS protein in the hemagglutinin molecule, while the recombinant vaccinia virus expresses the whole CS protein. Both recombinant viruses have been described in detail in previous publications (2325) and were generously supplied by Dr F. Zavala (New York University). The respective wild-type viruses were used as controls in several studies.
Amplification of the PYCS gene by PCR
To confirm the presence of the PYCS gene in T. cruzi, PCR analyses were performed as follows. Transformed T. cruzi (3x106) were washed with PBS 3 times, resuspended in 250 µl of distilled water and then immediately immersed in boiled water for 15 min. The suspension was centrifuged at 15,000 r.p.m. for 10 min at 4°C and 1 µl of the supernatant was used as template DNA for PCR reaction (Perkin-Elmer Cetus, Norwalk, CT). Specific primers for the amplification of the PYCS gene from pTEX-PYCS were P15 and P17. P15: 5'-ATG AAG AAG TGT ACC ATT TTA GTT GT-3'. P17: 5'-CCT CTA AGG TCA AAT TTT CTG GTT GC-3'. The PCR cycle consisted of 30 cycles of 95°C for 40 s, 56°C for 1 min and 72°C for 2 min. Primers P4 and P5 were used for the PYCS gene amplification from pBS:CnFc-PYCS. P4: 5'-GCT CTA GAA TGA AGA AGT GTA CC-3'. P5: 5'-CGG GAT CCT TAA TTA AAG AAT AC-3'. The PCR cycle consisted of 30 cycles of 95°C for 40 s, 60°C for 1 min and 72°C for 2 min.
Infection of cells with T. cruzi in vitro
P815 cells (4x106) were placed in 60 mm tissue culture Petri dishes (Becton Dickinson Labware, Lincoln Park, NJ) and 2x107 of either transformed or wild-type T. cruzi was inoculated into the culture (host cell:parasite ratio was 1:5). After 30 h incubation in the CO2 incubator at 37°C, the ELISPOT assay using Y26 T cell clone as effector cells and T. cruzi-infected P815 cells as target cells was performed.
Infection of mice with T. cruzi
BALB/c mice were administered i.m. with different doses of transformed or wild-type T. cruzi, given 100 mg/kg of benznidazole orally into each mouse 2 weeks later for three consecutive days and sacrificed 28 days later to examine the presence of epitope-specific CD8+ T cells in spleens. Isolated spleen cells (5x107) were co-cultured with 3x106 irradiated and peptide-pulsed P815 cells for 1 week in complete DMEM-IL2, and then tested for the presence of CD8+ T cells specific for SYVPSAEQI.
Immunizations and challenge infections with T. cruzi
BALB/c mice were immunized with 5x103 p.f.u. of recombinant influenza or wild-type virus s.c. at the base of tail. The immunizing dose for recombinant vaccinia or wild-type virus was 2x107 p.f.u. per mouse, administered i.p. In mice receiving two immunizing doses of different viruses, the second dose was administered 2 weeks after the first. All the immunized mice were challenged with the transformed or wild-type T. cruzi, i.m., 14 or 18 days after the last immunizing dose. The detailed experimental protocol including the challenge doses and the time-schedule are described in each figure legend.
In vivo depletion of T cells
The rat IgG mAb used for the in vivo T cell depletion was anti-CD8 (53-6.72, IgG2a), which was kindly supplied by Dr S. Waki (Gunma Prefectural College of Health Sciences) (26). Each mouse received, i.p., daily doses of 1 mg of anti-CD8 or purified rat IgG (Sigma) for three consecutive days and was challenged on the final day with the pBS:CnFc-PYCS-transformed T. cruzi.
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Results
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Transformation of T. cruzi
Two transformed T. cruzi strains were produced by electroporation (27) with two different plasmid DNAs containing the CS gene. For this purpose, we used vectors pTEX and pBS:CnFc, which previous studies have shown to be effective for the transfection of T. cruzi (20,21). Transfected parasites were selected by resistance to G418 and the presence of the PYCS gene in the selected parasites was confirmed by PCR analysis from tissue culture trypomastigote infecting the G418-resistant NIH 3T3 cell line. As shown in Fig. 1
, using CS gene-specific primers, a single band was obtained after PCR amplification from the pBS:CnFc-transformed parasites, but not from the wild-type T. cruzi. Similar results were obtained by using DNA from the pTEX-transformed parasites (data not shown).

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Fig. 1. Amplification of the PYCS gene by PCR from the pBS:CnFc-PYCS-transformed T. cruzi tissue culture trypomastigotes. Protocols for DNA extraction and PCR are described in Methods. After the amplification, DNAs were run on 1% agarose gel and stained with ethidium bromide. Lane 1, amplified DNA in the PCR reaction using plasmid DNA pBS:CnFc-PYCS as DNA template. Lane 2, amplified DNA from T. cruzi Tulahuen strain transformed with pBS:CnFc-PYCS gene. Lane 3, amplified DNA from T. cruzi Tulahuen strain wild-type.
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Expression of the CS protein in cells infected with transformed T. cruzi
To determine the expression of SYVPSAEQI, the class I MHC-restricted epitope in T. cruzi-infected cells, P815 cells were infected with either transformed or wild-type T. cruzi for 30 h and then used as target cells in an ELISPOT assay using an epitope-specific CD8+ T cell clone (Y26) (19). The Y26 cells recognized the P815 cells infected with the pBS:CnFc-PYCS-transformed parasites but not those infected with wild-type T. cruzi (Fig. 2
). Cells infected with pTEX-PYCS-transformed T. cruzi were also recognized by the Y26 CD8+ T cells (data not shown).
Induction of epitope-specific CD8+ T cells in mice infected with transformed T. cruzi
Next, we infected BALB/c mice with the transformed T. cruzi strains using sub-lethal doses ranging from 10 to 103 parasites per mouse. Twenty-eight days post-infection, the mice were sacrificed and their spleens were removed. After in vitro incubation for 1 week in the presence of antigen, spleen cells were used to perform ELISPOT assays and also standard 51Cr-release assays to detect the presence of epitope-specific CD8+ T cells. Figure 3
shows the results obtained in both assays performed with spleen cells from mice after the infection with pBS:CnFc-PYCS-transformed parasites. The number of epitope-specific IFN-
-secreting cells detected in ELISPOT assays ranged from 15,000 to 30,000 cells per 106 spleen cells at each infecting dose (Fig. 3A
). 51Cr-release assay confirmed the presence of SYVPSAEQI-specific cytolytic T cells in the spleen cells derived from mice infected with the transformed T. cruzi (Fig. 3B
). Similar results were obtained when using spleen cells from mice infected with the pTEX-PYCS-transformed parasites. No epitope-specific CD8+ T cells were detected in spleen cells from the wild-type T. cruzi-infected mice in both assays (Fig. 3C and D
).
Protection of mice from lethal T. cruzi infection by immunization with recombinant viruses
Both the in vitro and in vivo studies described above demonstrated the expression of malarial CS protein in T. cruzi, the processing and presentation of the MHC class I-restricted epitope by infected cells, and the induction of antigen-specific CD8+ T cells. We therefore, as a next step, initiated immunization experiments to determine whether the epitope-specific CD8+ T cells induced by recombinant viruses expressing the SYVPSAEQI epitope could be protective against T. cruzi infection. Previous studies demonstrated that optimal induction of antigen-specific CD8+ T cells could be obtained by priming with recombinant influenza virus, followed by a booster injection of recombinant vaccinia virus (2325). Using this protocol, we determined that ~3000 antigen-specific CD8+ T cells per 106 spleen cells were induced after the combined immunization with these recombinant viruses. In contrast, recombinant influenza or recombinant vaccinia alone induced 130 and 270 CD8+ T cells respectively (Fig. 4A
). These results are comparable to those described in previous reports. Eighteen days after the booster injection of recombinant vaccinia, each mouse was challenged with 4x105 of pBS:CnFc-PYCS-transformed T. cruzi, i.m. Parasitemia in all four non-immunized mice reached >104 parasites per 5 µl tail vein blood (TVB) (Fig. 4B
) and all of them died by 23 days post-infection (Fig. 4C
). In contrast, the parasitemia in four immunized mice reached <102 per 5 µl TVB and decreased to microscopically undetectable levels 1 month later. All immunized mice survived at least 100 days after infection (Fig. 4B and C
). Similar results regarding the time course of parasitemia and the survival rate were observed in infection with the pTEX-PYCS-transformed T. cruzi. In contrast, no differences in the time course of parasitemia and the survival rate were observed between immunized and non-immunized mice after challenge with the wild-type T. cruzi (data not shown).
This combined recombinant virus immunization was clearly more effective at inducing protective immunity than immunization with a single virus. In mice receiving combined immunization and challenged with 8x105 pBS:CnFc-PYCS-transformed T. cruzi, the parasitemia reached 768 in 5 µl TVB, peaked at day 14, disappeared to undetectable levels by 39 days post-infection and all of them survived for >100 days. In contrast, immunization with only one recombinant virus, either with recombinant influenza or recombinant vaccinia, showed only partial protective capacity compared to the combined immunization. In mice immunized with recombinant vaccinia, the level of parasitemia reached ~2x104 per 5 µl TVB and 7x103 per 5 µl TVB in mice immunized with recombinant influenza (Fig. 5A
). The rising parasitemia was suppressed afterwards in both groups; however, some of them eventually died despite this suppression (Fig. 5B
). All naïve control mice exhibited parasitemia reaching >104 parasites per 5 µl TVB (Fig. 5A
) after 10 days and all of them died by 13 days post-infection (Fig. 5B
). The combined immunization with wild-type influenza and vaccinia viruses, which do not express the CS protein, did not confer protective immunity against the T. cruzi challenge infection, as the control group of mice exhibited a time course of parasitemia and mortality identical to that observed in non-immunized mice (Fig. 5A and B
).




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Fig. 5. CD8+ T cells as a main mediator for the protection against the lethal infection of pBS:CnFc-PYCS-transformed T. cruzi. After immunizations and/or treatments with antibodies, all mice were challenged with 8x105 pBS:CnFc-PYCS-transformed T. cruzi, i.m., 16 days after the last immunizing dose. Then 5 µl TVB was collected at days indicated in the figure, and the time course of parasitemia (A and C) and the survival rate against the infection (B and D) of each group were monitored. (A and B) Twelve mice in three groups, four mice in each group, were treated as follows. One group received 5x103 p.f.u. of recombinant influenza virus (circles), while another group received 2x107 p.f.u. of recombinant vaccinia virus for immunization (triangles). The remaining four mice were first immunized with 5x103 p.f.u. wild-type influenza virus, s.c. at the base of tail, and then given a booster injection of 2x107 p.f.u. wild-type vaccinia virus, i.p., 2 weeks later (squares). (C and D) Twelve mice in three groups were first immunized with 5x103 p.f.u. recombinant influenza virus, s.c. at the base of tail, and then given a booster injection of 2x107 p.f.u. recombinant vaccinia virus, i.p., 2 weeks later. Following this combined immunization, two groups received either the anti-CD8 mAb (triangles) or normal rat IgG (circles) treatment respectively for three consecutive days until the day of challenge infection. The time course of parasitemia and the survival rate against the challenge infection of two recombinant virus-immunized (diamonds) and non-immunized BALB/c mice (squares) are described in all figures. Parasitemia was described as mean numbers of parasites per 5 µl TVB ± SE.
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CD8+ T cells mediated the protective immunity
The depletion of CD8+ T cells in vivo, after administration of the specific mAb, revealed that these T cells mediated the protective immunity induced after immunization. As shown in Fig. 5
, the depletion of CD8+ T cells in immunized mice completely abrogated the protective effect, resulting in unrelenting parasitemia (Fig. 5C
) and 100% mortality by 12 days post-infection (Fig. 5D
). In contrast, treatment of immunized mice with normal rat IgG did not alter the time course of parasitemia or the survival rate (Fig. 5C and D
).
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Discussion
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In the last few years, the results of various experimental studies suggested that CD8+ T cells could play an important protective role against T. cruzi infection (513); however, the lack of information regarding the cytotoxic T lymphocyte epitopes present in T. cruzi antigens severely limited studies aimed at further characterizing the role of these T cells. The current study circumvented this problem by generating transformed T. cruzi strains expressing the malarial CS protein, which contains a well-defined CD8+ T cell epitope (19). The expression of the CD8+ T cell epitope in T. cruzi-infected cells was confirmed by using a well-characterized anti-CS CD8+ T cell clone, Y26, which recognized cells infected with transformed parasites (Fig. 2
). In addition, we also determined that mice infected with T. cruzi transformants were capable of generating a CD8+ T cell response against the SYVPSAEQI epitope (Fig. 3
).
These results are compatible with a previous report, in which T. cruzi transformed with the ovalbumin (OVA) gene was used to infect cells. It demonstrated the MHC class I antigen processing and presentation of the OVA epitope in T. cruzi infected cells and also the induction of MHC class I-restricted OVA-specific CD8+ T cells in mice infected with the transformed T. cruzi strain (28).
Still, it remained to be determined whether immunization with vectors designed to induce preferentially CD8+ T cells could generate a response that may confer protective immunity against T. cruzi infection. To investigate this matter, we used recombinant viruses, which have become a powerful tool for the induction of protective host immune responses against various pathogens including viral and parasitic infections (2325,2933). The results of our study demonstrated that immunization with recombinant viruses could protect mice from lethal T. cruzi infection and that conventional vaccination strategies may be applicable to combat Chagas' disease. We showed that, while all non-immunized mice died after T. cruzi infection, all mice which were immunized with recombinant influenza followed by recombinant vaccinia cleared the moderate level of parasitemia and survived. The protective immunity was mediated by CD8+ T cells, as the depletion of this T cell subset completely reversed the course of parasitemia and survival rate to the level of non-immunization. The immunization with only one recombinant virus, either recombinant influenza or recombinant vaccinia, conferred less protective immunity, reflecting the importance of the number of antigen-specific CD8+ T cells that needs to be induced to reduce parasitemia and mortality. Still, CD8+ T cells induced by immunization with recombinant influenza virus alone which expresses only the minimal 9mer (SYVPSAEQI) CD8+ T cell epitope induced an immune response in three out of four mice, which survived the T. cruzi infection despite initial high levels of parasitemia.
In both humans and mice, immune control of T. cruzi is reported to be mediated by several cell populations besides CD8+ T cells (34). Very recently, some class I MHC-restricted epitopes located in sialidase/trans-sialidase (35) and in amastigote surface proteins (36) of T. cruzi have been reported. It has also been shown that adoptive transfer of the CD8+ T cell line against the epitope in sialidase/trans-sialidase (35) into mice significantly reduced parasitemia and mortality. In addition, there are several reports of potential T. cruzi target molecules recognized by antibodies (37) and CD4+ T cells (38). It is likely that the protective efficacy would be improved by combining the induction of protective immune responses such as humoral immunity and CD4+ T cells. Finally, it would be most important to determine whether any immunological intervention shown to be effective against the acute phase of T. cruzi infection would be also effective against chronic Chagas' disease.
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Acknowledgments
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We thank Dr F. Zavala (New York University) for his generous support. The authors express sincere appreciation to Dr T. Nozaki (Keio University) for supplying the pTEX vector, Dr W. C. Van Voorhis (University of Washington, Seattle) for supplying the pBS:CnFc vector, and K. Saiga, A. Kawasaki and H. Matsuda (Juntendo University) for their help in immunological studies.
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Abbreviations
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CS | circumsporozoite |
LIT | liver infusion tryptose medium supplemented with 10% FCS |
OVA | ovalbumin |
PY | Plasmodium yoelii |
TVB | tail vein blood |
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Notes
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Transmitting editor: R. Nussenzweig
Received 3 August 1998,
accepted 13 October 1998.
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