A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection.

Abstract

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.

Full text

Page 1

INFECTION AND IMMUNITY, Jan. 2003, p. 309–316
0019-9567/03/$08.00?0
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 1
DOI: 10.1128/IAI.71.1.309–316.2003
A GRA1 DNA Vaccine Primes Cytolytic CD8?T Cells To Control
Acute Toxoplasma gondii Infection
T. Scorza,† S. D’Souza,‡ M. Laloup,§ J. Dewit,§ J. De Braekeleer,§ H. Verschueren,?
M. Vercammen,# K. Huygen,‡ and E. Jongert*
Department of Toxoplasmosis, Pasteur Institute of Brussels, 1180 Brussels, Belgium
Received 2 June 2002/Returned for modification 27 July 2002/Accepted 4 October 2002
Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma
interferon (IFN-?). The contribution of CD4?and CD8?T-lymphocyte subsets to protective immune responses
against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell
depletion experiments indicated that both CD4?and CD8?T-cell subsets produced IFN-? upon restimulation
with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8?T cells that were shown to have
cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized
with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the
empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that
CD8?T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii
infection, while depletion of CD4?T cells led to an increase in brain cyst burden during the chronic phase of
infection.
In immunocompetent individuals, Toxoplasma gondii gener-
ally induces a mild asymptomatic infection that is associated
with the rapidly dividing tachyzoite form of the parasite. Res-
olution of the infection in the host occurs through induction of
strong and persistent cell-mediated immunity that results in
the control of T. gondii tachyzoites (11, 46). In humans, this
relatively benign infection may reactivate under conditions of
immunosuppression, resulting in toxoplasma encephalitis and
other complications (11). A primary T. gondii infection and
subsequent transplacental transmission during pregnancy can
result in miscarriage or in severe disease in the infant (26).
These pathological consequences associated with congenital
toxoplasmosis not only represent a threat to humans but also
are a cause of economic losses due to abortions in farm ani-
mals (17). Therefore, a vaccine capable of controlling the
tachyzoite multiplication associated with the acute primary
infection is important and has been the subject of study in our
laboratory.
Reports on DNA vaccination against experimental T. gondii
infection in mice have been accumulating, and the antigens
that have been tested now include T. gondii membrane-asso-
ciated surface antigen SAG1 (1, 33), excreted-secreted dense
granule proteins GRA1 (43), GRA4 (16), and GRA7 (43), and
rhoptry proteins ROP1 (23) and ROP2 (30, 43). For all these
antigens, immunity was associated with Th1-type responses,
which are characterized by production of gamma interferon
(IFN-?). It was reported previously that DNA vaccination with
three distinct immunogenic Toxoplasma antigens (GRA1,
GRA7, and ROP2) induced partial immunity in C3H/HeN
mice and that the responses were associated with a Th1-type
profile (43). The GRA1 antigen (p24), a product of T. gondii
tachyzoites and bradyzoites, is a promising vaccine candidate
(3, 7, 18, 43). This antigen induces humoral and cellular im-
mune responses in mice and humans in the chronic phase of
the infection (7, 19). Vaccination with GRA1 has been shown
to be protective in two animal models of infection (18, 38).
Adoptive transfer of T cells from rats vaccinated with GRA1-
expressing vaccinia virus partially protected nude rats against
lethal challenge with the virulent T. gondii RH strain (18). In
addition, immunization of sheep with recombinant Mycobacte-
rium bovis BCG producing and secreting GRA1 resulted in
specific, partially protective cellular immune responses char-
acterized by the production of IFN-? (38).
It is well established that IFN-? and T cells play a central
role in host resistance to T. gondii during both the acute and
chronic phases of infection (11, 46). However, the concerted
interplay of several other cytokines may be necessary to main-
tain the delicate balance between protection and immunopa-
thology caused by excessive inflammation (22, 32, 35). Another
crucial interaction for protection against a T. gondii infection is
the one between CD8?and CD4?T cells (21). Although some
reports have suggested that CD8?T cells mediate their effect
through IFN-? production (5, 14, 27), CD8?T cells with cy-
totoxic activity against infected cells have been described in
humans (9, 31, 34, 45) and mice (8, 12, 25, 27, 37).
In the present study we compared the contributions of
* Corresponding author. Mailing address: Laboratory of Toxoplas-
mosis, Pasteur Institute of Brussels, Engelandstraat 642, 1180 Brussels,
Belgium. Phone:32-2-373-33-77.
ejongert@pasteur.be.
† Present address: Institute of Parasitology, McGill University, Ste-
Anne-de-Belle-Vue, H9X 3V9 Montreal, Quebec, Canada.
‡ Present address: Laboratory of Mycobacterial Immunology, Pas-
teur Institute of Brussels, Brussels, Belgium.
§ Present address: Laboratory of Toxoplasmosis, Pasteur Institute of
Brussels, Brussels, Belgium.
? Present address: Department of Molecular Biology, Flanders In-
teruniversity Institute of Biotechnology, University of Ghent, Ghent,
Belgium.
# Present address: Department of Clinical Chemistry, Academic
Hospital of the Free University of Brussels, Brussels, Belgium.
Fax:32-2-373-32-82.E-mail:
309

Page 2

CD4?and CD8?T cells in the protection conferred by the
pVR1020-GRA1 DNA vaccine. Both CD4?and CD8?T cells
produced IFN-?, and the CD8?T-cell subset had T. gondii-
specific cytolytic activity. Our results suggest that although
CD4?T cells are the strongest producers of IFN-?, CD8?T
cells are the major effectors of the vaccine-induced protection
against acute toxoplasmosis.
MATERIALS AND METHODS
Plasmid construction. The DNA construct used for vaccination was based on
the plasmid vector VR1020, obtained from Vical, Inc. (San Diego, Calif.). The
gene encoding GRA1 was amplified by PCR from cloned DNA fragments and
inserted into pVR1020 as described previously (43). Briefly, sense and antisense
primers were designed to contain a BamHI restriction site. The gene was cloned
into the BamHI site of the expression vector pVR1020 to generate an in-frame
fusion with the vector-encoded signal sequence of human tissue plasminogen
activator. All plasmids were propagated in Escherichia coli DH1. DNA for
vaccination was purified by anion-exchange chromatography (EndoFree plasmid
giga kits; Qiagen GmBH, Hilden, Germany) and was dissolved in sterile endo-
toxin-free phosphate-buffered saline (PBS) (BioWhittaker Europe, Verviers,
Belgium). Plasmid integrity was checked by agarose gel electrophoresis after
digestion with appropriate restriction enzymes. The DNA concentration was
determined by absorbance at 260 nm.
Vaccination of experimental animals. Female inbred C3H/HeN mice (H-2k)
that were 6 to 8 weeks old were purchased from Harlan (Horst, The Nether-
lands). The C3H/HeN mouse model is relevant for investigation of the induction
of protective anti-T. gondii immune responses because the mice are moderately
resistant to acute infection and develop T. gondii brain cysts and progressive
toxoplasma encephalitis (39). These features allow workers to monitor the acute
phase as well as the chronic phase of infection in response to vaccination. The
mice received three injections of 100 ?g of pVR1020-GRA1 DNA (separated by
2-week intervals) in both tibialis anterior muscles administered with a 0.3-ml
syringe (BD Biosciences, San Diego, Calif.). Mice injected with the empty vector
pVR1020 were used as negative controls. Three weeks after the third DNA
injection, the vaccinated mice were bled from the tail vein, and sera were
analyzed with a GRA1-specific enzyme-linked immunosorbent assay (ELISA) as
described previously (43). All mice vaccinated with pVR1020-GRA1 were shown
to have GRA1-specific antibodies after the third injection. This study was con-
ducted in compliance with the regulations concerning the use of laboratory
animals at the Pasteur Institute, Brussels, Belgium.
Parasite and antigen preparation. The T. gondii type II IPB-G and IPB-M
strains (43) were isolated from the placentas of women who gave birth to infants
with congenital toxoplasmosis. Tachyzoites of the IPB-M and IPB-G strains were
obtained from the ascites of C3H mice infected by intraperitoneal injection and
subcutaneously treated with hydrocortisone acetate (25 ?g; Laboratoires Rous-
sel, Paris, France) at 2-day intervals for 1 week. Expression of GRA1 in both
strains was confirmed by Western blotting.
To prepare toxoplasma lysate (TLA), tachyzoites from the virulent T. gondii
RH strain were obtained from the peritoneal fluid of infected Swiss mice as
described previously (43). The material was passed twice through a 26-gauge
needle. The parasites were washed, resuspended in PBS, and sonicated (1-min
burst, 1 min of cooling, 150 W) with an Ultrasonic disintegrator (MSE, Leicester,
United Kingdom). The protein concentration of TLA was determined by the
Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, Calif.).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot
analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried
out as described by Laemmli (28) by using a 12% polyacrylamide gel and the
Bio-Rad minigel system (Bio-Rad Laboratories). The BenchMark prestained
protein ladder (Life Technologies, Grand Island, N.Y.) was used for molecular
weight standards. Electrophoretic transfer onto nitrocellulose membranes (Hy-
bond-C; Amersham Biosciences, Uppsala, Sweden) was done with a mini Trans-
Blot electrophoretic cell system (Bio-Rad Laboratories) as instructed by the
manufacturer. The membrane was blocked by incubation with 3% bovine serum
albumin (Merck, Darmstadt, Germany) in Tris-buffered saline containing 0.1%
Tween 20 for 1 h at room temperature. For detection of recombinant GRA1 in
BW-Sp3 transfectants, a polyclonal pool of sera from five T. gondii-infected C3H
mice, diluted 1:200, was used as the primary antibody; it was incubated overnight
at 4°C and subsequently with a peroxidase-labeled rat anti-mouse immunoglob-
ulin G (IgG) (Amersham Biosciences) as the secondary antibody for 1 h at room
temperature. The chemiluminescent compound Supersignal (Pierce, Rockford,
Ill.) was used as the substrate according to the manufacturer’s instructions. For
detection of native GRA1 in T. gondii IPB-M- and IPB-G-derived TLA, a
monoclonal antibody (MAb) against GRA1, MAb BATO 35 (36), was used at a
dilution of 1:1,000, and pools of sera from three seropositive pVR1020-GRA1-
vaccinated C3H mice or pVR1020-vaccinated C3H mice, diluted 1:300, were also
used. These primary antibodies were incubated overnight at 4°C with the mem-
brane strips. A peroxidase-labeled rat anti-mouse IgG (Sigma, St. Louis, Mo.)
was used as the secondary antibody for 1 h at room temperature. The chromo-
genic reaction was performed with 4-chloro-naphthol substrate tablets (Sigma)
according to the manufacturer’s instructions. The reaction was stopped by wash-
ing the preparations in water.
In vitro spleen cell cultures. Animals were sacrificed 2 months after the third
DNA injection. Single-cell suspensions of splenocytes were prepared, and red
blood cells were lysed with RBC Lysing buffer (Sigma). The cell suspension
recovered was washed in RPMI 1640 (GIBCO, Life Technologies, Paisley,
United Kingdom) and plated on RPMI 1640 supplemented with 10% fetal calf
serum (FCS), 2 mM glutamine 1640 (GIBCO, Life Technologies), 0.05 mM
2-mercaptoethanol (Sigma), 1? nonessential amino acids (GIBCO, Life Tech-
nologies), 1 mM sodium pyruvate (GIBCO, Life Technologies), and 100 U of
penicillin-streptomycin (Life Technologies) per ml. Splenocytes (3 ? 106cells/
ml) were stimulated with TLA (25 ?g/ml) and cultured for 4 days in 24-well
plates (Nunc, Roskilde, Denmark). The optimal TLA concentration (25 ?g/ml)
and the optimal time of culture were determined previously on the basis of a
kinetics experiment (data not shown).
In vitro depletion of CD4?and CD8?T cells and IFN-? measurement. For in
vitro and in vivo depletion experiments, culture supernatants of the anti-mouse
CD4-producing GK1.5 hybridoma (American Type Culture Collection) and the
anti-mouse CD-8?-producing H35-17.2 clone (kindly provided by Anja Geldhof,
Free University of Brussels) were obtained and concentrated by passage through
protein G-Sepharose columns (Amersham Biosciences) by using an Econopump
and a UV monitor (Bio-Rad Laboratories). The concentrates were exhaustively
dialyzed against PBS, and the protein content was measured by the Bio-Rad DC
protein assay (Bio-Rad Laboratories). The MAbs were divided into aliquots and
stored at ?80°C until they were used. Selective in vitro depletion of CD4?or
CD8?T cells was achieved by incubating splenocytes (2 ? 107cells) either with
20 ?g of the anti-mouse CD4 MAb per ml or with 20 ?g of the anti-mouse CD8?
MAb per ml at 4°C for 30 min. The cells were washed, the cell concentration was
adjusted to the original concentration in medium containing 5% FCS, and each
preparation was incubated by using a ratio of sheep anti-rat IgG M-450 dyna-
beads (Dynal Biotech, Oslo, Norway) to cells of 4:1 for 1 h at 37°C with constant
rotation. CD4?and/or CD8?T cells were removed by magnetic sorting. Finally,
the cells were washed twice and resuspended at a concentration of 3 ? 1054
cells/ml in complete medium (RPMI 1640 containing 2 mM glutamine, 1?
nonessential amino acids [GIBCO, Life Technologies], 1 mM sodium pyruvate
[GIBCO, Life Technologies], 100 U of penicillin/ml, 100 ?g of streptomycin/ml,
and 50 ?M 2-mercaptoethanol). The efficiency of depletion of each T-cell subset
was monitored by cytofluorometric analysis before and after depletion, as well as
after the 4 days of in vitro stimulation with TLA, and was found to be at least
90% for each T-cell subset. Both T-cell subsets remained at these levels on day
4 of the experiment (data not shown), when the supernatants were harvested.
The IFN-? content was measured by ELISA, as previously described (43).
Generation of BW-Sp3(GRA1) for CTL assays. In order to obtain target cells
expressing GRA1 in an H-2kcontext, expression vector pcDNA3.1 (Invitrogen,
Life Technologies) containing the GRA1 gene was transfected into BW-Sp3, a
subline of the BW5147T lymphoma cell line of AKR mice (42). BW-Sp3 was
obtained by in vivo selection and has higher levels of expression of both Kkand
Dkantigens than the parental line. The full-length GRA1 fragment was recov-
ered from the pVR1020-GRA1 construct by restriction with SalI and BglII and
was ligated into plasmid pcDNA3.1 digested with BamHI and XhoI. The result-
ing construct was electroporated into BW-Sp3 cells, and positive clones were
selected by using neomycin. Expression of the GRA1 gene was checked by
reverse transcription (RT)-PCR by using a sense primer (AGATGATGGGGA
ACACGTATCG) starting at position 185 from the ATG codon and an antisense
primer (AGGAACCCAATGTCATCC) ending at position 555 from the ATG
codon. Thus, the PCR product contained a 370-bp fragment from the carboxy-
terminal end of the mature GRA1 gene. GRA1 expression of a PCR-positive
clone was confirmed at the protein level by Western blotting. Clone BW-GRA1
was used as the target for cytotoxic T-lymphocyte (CTL) assays.
Preparation of bone marrow macrophages as CTL targets. Bone marrow was
obtained from the femurs and tibias of 8- to 12 week-old female C3H mice in
Hanks balanced salt solution (GIBCO, Life Technologies) supplemented with 10
mM HEPES and 100 U of penicillin-streptomycin (GIBCO, Life Technologies)
per ml. Cells were recovered, washed, and plated on 100-mm tissue culture petri
310 SCORZA ET AL.INFECT. IMMUN.

Page 3

dishes at a concentration of 10 ? 106cells to 15 ? 106cells/10 ml in each plate
(Falcon, Becton Dickinson, San Diego, Calif.). The culture medium consisted of
Dulbecco modified Eagle medium (GIBCO, Life Technologies) supplemented
with 10% FCS, sodium pyruvate, and penicillin-streptomycin, as well as 30%
supernatant from confluent cultures of L929 fibroblasts (grown in 45% RPMI
1640–45% Dulbecco modified Eagle medium–10% FCS) as a source of macro-
phage colony-stimulating factor. After 6 days of incubation at 37°C in the pres-
ence of 5% CO2, the plates were gently washed to remove nonadherent cells. The
plates each contained 3 ? 106to 5 ? 106macrophages that were ?95% pure.
Adherent macrophages were infected overnight with RH tachyzoites attenuated
by exposure to UV light. The dose and efficiency of attenuation were determined
previously (data not shown). Two hundred microcuries of Na251CrO4was simul-
taneously added to each plate of macrophages. The following day, extracellular
tachyzoites, as well as unincorporated radioactivity, were removed by four gentle
washes with RPMI 1640. The adherent macrophages were detached with a
rubber policeman, washed, counted, and used as targets in a Cr release assay.
Noninfected macrophages were included as a control.
Generation of effector cells for CTL assays. To generate effector cells for CTL
assays, two different experiments were performed. In the first experiment, sus-
pensions containing 6 ? 106splenocytes from pVR1020-GRA1- and pVR1020-
vaccinated mice per 2 ml were stimulated in vitro with irradiated BW-
Sp3(GRA1) cells (7,000 rads; ratio of effectors to stimulators, 50:1) for 6 days.
The cells were harvested, layered over a Ficoll-Paque gradient (Amersham
Biosciences), and centrifuged at 300 ? g for 25 min. The cells in the interface
were collected, washed twice, and used as effectors in a standard51Cr release
assay with BW-Sp3(GRA1) cells (1 ? 105cells/ml) as the target cells. In the
second experiment, splenocytes from pVR1020-GRA1- and pVR1020-vacci-
nated mice were stimulated with live tachyzoites from the T. gondii IPB-M strain
at a multiplicity of infection of 1:10 (ratio of parasites to splenic cells). The T.
gondii IPB-M strain is characterized by its mild virulence in mice and slow growth
in monolayers of Vero cells (unpublished data). Following 6 days of in vitro
culture, the viable splenic T cells were used as effectors and RH-infected syn-
geneic bone marrow macrophages were used as targets in a standard51Cr release
assay. In order to confirm the identity of the CTL population, immediately
before use in cytotoxicity assays CD8?T cells were depleted in vitro by magnetic
sorting as described above, and the remaining cells were used as effectors.
In vivo depletion of CD4?and CD8?cells. Mice received 0.5 mg of anti-mouse
CD4 depleting MAb GK1.5 or anti-mouse CD8? depleting MAb H35.17.2 in-
traperitoneally (i.p.) 1 day prior to i.p. challenge with 40 cysts of the IPB-G strain
and then 0.25 mg every 6 days for 3 weeks. Depletion had a long-lasting effect (at
least 7 days) for a dose of 0.25 mg (data not shown). There was a period of at
least 3 weeks between the last DNA vaccination and the onset of the in vivo
depletion. Survival of undepleted mice was evaluated in parallel. In a previous
study (43), peroral challenge studies were performed with vaccinated C3H mice.
In further studies, we did not observe any differences in the susceptibilities of
vaccinated C3H mice challenged by the oral and i.p. routes. As the i.p. route
allows better control over the dose of cysts administered, this challenge route was
used in the experiments. The experiments were repeated twice with experimental
groups consisting of four mice. The extent of in vivo depletion was monitored
prior to challenge by cytofluorometric analysis of blood samples. In earlier
depletion experiments fluorescence-activated cell sorting (FACS) analysis
showed that depletion of blood lymphocytes was highly correlated with the
depletion of splenic lymphocytes (unpublished data).
Cytofluorometric analysis. For intracellular IFN-? staining of effector CD8?T
cells amplified for the CTL assays, Golgi Plug (BD Biosciences Pharmingen, San
Diego, Calif.) was added to cultures of stimulated splenocytes. Five hours later,
the cells were harvested and washed twice in FACS wash buffer (5% FCS in
PBS). A total of 106cells for each condition were incubated with anti-CD8 MAb
(clone 53.6.7; BD Biosciences) for 30 min. on ice. The cells were washed and
fixed with 4% paraformaldehyde for 20 min at 4°C, washed twice with FACS
wash buffer containing 0.1% saponin (Sigma) to permeabilize the cell membrane,
and incubated with R-phycoerythrin (PE)-labeled anti-IFN-? MAb (clone
XMG1.2; BD Biosciences) for 30 min at 4°C. Prior to cytofluorometric analysis,
the lymphocytes were washed and resuspended in 300 ?l of PBS.
For ex vivo determination of CD4?and CD8?T-cell populations 4 days after
the last in vivo CD4?T-cell depletion, whole blood was collected in 2% EDTA
to prevent coagulation. Red blood cells were lysed by incubation with ammonium
chloride lysis buffer (150 mM NH4Cl, 10 mM NaHCO3, 0.4% EDTA). Blood
lymphocytes were washed twice in FACS wash buffer and directly labeled with
anti-CD4 fluorescein isothiocyanate (FITC) (clone RM4-4) or anti-CD8 FITC
(clone 53.6.7) and anti-CD3 PE (clone 145-2C11) MAbs (BD Biosciences).
Background fluorescence was measured by using FITC- or PE-labeled isotype
control antibodies (BD Biosciences). Cytofluorometric analysis was performed
with a FACScalibur cytofluorometer (Becton Dickinson). Calculations were per-
formed on the basis of an appropriate lymphocyte gate for 5 ? 104counts.
Enumeration of T. gondii cysts in mouse brains. Mice challenged with T. gondii
parasites were sacrificed 6 weeks after infection, and their brains were homog-
enized in 2 ml of PBS. Four samples of the suspension were counted by using a
phase-contrast microscope at a magnification of ?40.
Statistical analysis. For statistical evaluation of data obtained from IFN-?
production and brain cyst counting analysis, the data from pVR1020-GRA1-
vaccinated mice were compared to control data by using a two-sided Student t
test. The survival curves for vaccinated mice were compared to those for controls
by the Mantel-Haenszel test. Statistical analysis and graphics were carried out by
using the Prism 3 software (GraphPad, San Diego, Calif.).
RESULTS
GRA1 expression in the T. gondii IPB-G and IPB-M strains.
To confirm that GRA1 is expressed in the IPB-G and IPB-M
strains, TLA was prepared from tachyzoites isolated from as-
cites fluid obtained after cortisone treatment of IPB-G- and
IPB-M-infected mice and analyzed by Western blotting. In
both strain IPB-G and strain IPB-M, the 23-kDa GRA1 anti-
gen could be detected with the anti-GRA1 MAb BATO 35 and
serum from pVR1020-GRA1-vaccinated mice (Fig. 1).
Production of IFN-? by CD4?and CD8?T cells from mice
vaccinated with GRA1 DNA. In order to assess the relative
contribution to the production of IFN-? by the CD4?or CD8?
T-cell subset, in vitro depletion experiments were performed.
Splenocytes frompVR1020-GRA1-seropositive
pVR1020-vaccinated mice were cultured in the presence of
TLA. As reported previously (43), production of IFN-? by
splenocytes from pVR1020-GRA1-vaccinated mice was maxi-
mal after 4 days, while IFN-? production by splenocytes from
control pVR1020-vaccinated mice remained at the background
level (data not shown). Compared with the results obtained
with undepleted splenocyte cultures from pVR1020-GRA1-
vaccinated mice, CD4?T-cell depletion resulted in a more-
than-fourfold decrease in IFN-? production, whereas after de-
pletion of CD8?T cells IFN-? production decreased less than
twofold (Fig. 2).
Induction of GRA1-specific cytotoxic T lymphocytes after
and
FIG. 1. Presence of GRA1 in total lysates of T. gondii strains
IPB-G and IPB-M confirmed by detection with a MAb against GRA1
(MAb BATO 35). In addition, serum from pVR1020-GRA1-vacci-
nated C3H mice was shown to recognize GRA1 in these lysates,
whereas serum from C3H mice vaccinated with the empty vector did
not. Lanes 1, 3, and 5, IPB-G TLA; lanes 2, 4, and 6, IPB-M TLA.
GRA1 was detected with MAb BATO 35 (lanes 1 and 2), serum from
pVR1020-GRA1-vaccinated mice (lanes 3 and 4), and serum from
control pVR1020-vaccinated mice (lanes 5 and 6).
VOL. 71, 2003T. GONDII GRA1 DNA VACCINE EVOKES CD8?T CELLS 311

Page 4

vaccination with pVR1020-GRA1. As our in vitro depletion
experiments indicated that CD8?T cells could be primed by
GRA1 DNA vaccination, it was of interest to determine
whether these cells had T. gondii-specific cytolytic activity.
In a first approach, a GRA1-transfected lymphoma cell line
was generated. The presence of GRA1 mRNA and the pres-
ence of protein in the BW-Sp3 cell line were verified by RT-
PCR (Fig. 3A) and Western blotting with pooled sera from T.
gondii-infected C3H mice (Fig. 3B), respectively. Splenocytes
from pVR1020-GRA1-vaccinated and control pVR1020-vacci-
nated mice were amplified with irradiated BW-Sp3(GRA1)
cells. Radioisotope-labeled BW-Sp3(GRA1) cells were then
used as targets in cytotoxic assays. When a ratio of effectors to
targets of 60:1 was used, the percentage of specific lysis was
more than 70%, and significant lytic activity was observed even
at a ratio of 10:1 (Fig. 4A). The magnitude of the lysis exerted
by vaccine-induced CTLs was the same as the magnitude of the
lysis observed with splenocytes from chronically infected mice
(data not shown). The levels of lysis of untransfected control
BW-Sp3 cells remained at background levels (data not shown).
Significant CTL activity was also measured by using a second
approach. Splenocyte cultures from vaccinated mice were in-
fected with live IPB-M tachyzoites to amplify effector cells,
while
fected with UV radiation-attenuated RH parasites served as
target cells. When a ratio of effectors to targets of 40:1 was
used, the percentage of specific lysis was more than 45% (Fig.
4B). The levels of lysis of uninfected bone marrow macro-
phages remained at background levels (data not shown).
Using both approaches, we demonstrated that vaccination
51Cr-labeled syngeneic bone marrow macrophages in-
FIG. 2. IFN-? production in CD4?and CD8?T-cell-depleted
splenocyte cultures from GRA1 DNA-vaccinated mice. Spleen cells
were depleted in vitro with anti-CD4 or anti-CD8 MAb and cultured
for 96 h in the presence of TLA. IFN-? levels in the culture superna-
tants were measured by ELISA. The data are the averages of three
independent depletion experiments. For undepleted mice, the average
IFN-? level reached 16,600 ? 1,852 pg/ml, while the average IFN-?
level dropped after CD4?and CD8?T-cell depletion to 3,467 ? 1,815
and 11,300 ? 755 pg/ml, respectively (P ? 0.001 and P ? 0.02, respec-
tively).
FIG. 3. BW-Sp3(GRA1) transfectants were generated by electro-
poration of pcDNA3.1 containing the GRA1 gene into BW-Sp3, and
the positive clones were selected with neomycin. Expression of the
GRA1 gene was further confirmed by RT-PCR (A) and Western
blotting (B). The size of the cDNA generated corresponds to the
calculated size (370 bp) of the amplified fragment from pCDNA3.1-
GRA1, while sera from T. gondii-infected mice detected GRA1 cor-
responding to the known molecular weight. (A) RT-PCR. Lane 1,
BW-Sp3; lane 2, BW-Sp3(GRA1). (B) Western blotting. Lane 1, TLA;
lane 2, lysate of BW-Sp3(GRA1); lane 3, lysate of BW-Sp3.
FIG. 4. Vaccination with pVR1020-GRA1 induced CD8?T cells
with specific GRA1 cytolytic activity. (A) Cytotoxic activities of effec-
tors from pVR1020-vaccinated (I), pVR1020-GRA1-vaccinated (?),
and pVR1020-GRA1-vaccinated CD8?T-cell-depleted (F) C3H mice
against BW-Sp3(GRA1) targets. Effector cells were stimulated for 6
days with irradiated BW-Sp3(GRA1) cells. The data are the means
from three independent experiments. (B) Cytotoxic activities of effec-
tors from pVR1020-vaccinated (I), pVR1020-GRA1-vaccinated (?),
and pVR1020-GRA1-vaccinated CD8?T-cell-depleted (F) C3H mice
against T. gondii-infected syngeneic bone marrow macrophages. Effec-
tor cells were stimulated for 6 days with tachyzoites from the T. gondii
IPB-M strain. The data are the means from three independent exper-
iments. E, effector cells; T, target cells.
312 SCORZA ET AL.INFECT. IMMUN.

Page 5

with pVR1020-GRA1 induced genuine CTLs, which could lyse
GRA1-transfected lymphoma cells and T. gondii-infected syn-
geneic bone marrow macrophages. In order to identify the
T-cell subset with CTL activity, we also included in vitro CD8?
T-cell-depleted splenocytes from pVR1020-GRA1-vaccinated
mice. The T-cell-mediated cytotoxicity was completely elimi-
nated after in vitro depletion of CD8?T cells (Fig. 4). No
significant IFN-? production by CD8?T effector cells from
pVR1020-GRA1-vaccinated mice was detected by cytofluoro-
metric analysis in either of the two approaches used (data not
shown). Interestingly, CD8?T cells from splenocyte cultures
of T. gondii-infected mice produced IFN-? only when they
were stimulated with live IPB-M tachyzoites, not when they
were stimulated with the irradiated BW-Sp3(GRA1) target
cells (data not shown).
The protective effect of the GRA1 DNA vaccine was elimi-
nated by in vivo CD8?T-cell depletion. In order to evaluate
the relative contribution of CD8?T cells to the protection
conferred by the pVR1020-GRA1 vaccine, in vivo depletion
experiments were performed. The induced protection was
monitored by comparison of the survival curves of pVR1020-
and pVR1020-GRA1-vaccinated mice after i.p. challenge with
40 cysts of the T. gondii IPB-G strain. Confirming a previous
report (43), we found that GRA1 DNA vaccination resulted in
significant protection. Data from two independent experiments
are shown in Fig. 5. In the first experiment (Fig. 5A), all
control pVR1020-vaccinated mice succumbed to infection,
while 75% of the mice vaccinated with pVR1020-GRA1 were
protected against infection with IPB-G (P ? 0.02). In vivo
CD8?T-cell depletion at the onset of challenge resulted in
75% mortality of pVR1020-GRA1-vaccinated mice and 100%
mortality of pVR1020-vaccinated mice. There was no signifi-
cant difference between the survival curves of these two
groups. (P ? 0.05). In the second experiment (Fig. 5B), all
mice vaccinated with GRA1 DNA survived, while 75% of the
mice vaccinated with control DNA succumbed to infection (P
? 0.05), and again, CD8?T-cell depletion completely elimi-
nated the protective effect of the GRA1 DNA vaccine. No
significant differences were observed between the survival
curves of GRA1-vaccinated and control mice (50 and 25%
survival, respectively; P ? 0.05) after CD8?T-cell depletion.
Contribution of CD4?T cells from vaccinated mice to brain
cyst development after infection with T. gondii. In contrast to
CD8?T-cell depletion, in vivo depletion of CD4?T cells did
not influence the survival of pVR1020-GRA1-vaccinated mice
during the acute phase of T. gondii infection (Fig. 5). All CD4?
T-cell-depleted GRA1-vaccinated mice survived infection, and
the data were similar to the 75 and 100% survival rates of
undepleted GRA1-vaccinated mice (Fig. 5) (P ? 0.05 for both
experiments). As CD4?T-cell depletion was reported to in-
fluence the development of brain cysts (6), we assessed the
contribution of CD4?T cells to T. gondii brain cyst develop-
ment during the acute and early chronic phases of infection. As
described previously (43), after infection with T. gondii
pVR1020-GRA1-vaccinatedmice
lower numbers of brain cysts than pVR1020-vaccinated mice
developed. In additional in vivo CD4?T-cell depletion exper-
iments, mice were challenged with a sublethal dose of IPB-G
(20 cysts i.p.). Depletion of CD4?T cells resulted in a signif-
icant increase in the numbers of brain cysts in pVR1020-
developedsignificantly
GRA1-vaccinated mice (4,540 ? 932 cysts/brain; n ? 4) com-
pared to the numbers of brain cysts in undepleted pVR1020-
GRA1-vaccinated mice (1,247 ? 195 cysts/brain; n ? 4) (P ?
0.01). Furthermore, no significant differences between the
numbers of brain cysts in CD4?T-cell-depleted pVR1020-
GRA1-vaccinated mice and the numbers of brain cysts in
CD4?T-cell-depleted pVR1020-vaccinated mice (5,793 ? 347
cysts per brain; n ? 4) were detected. Interestingly, in these in
vivo CD4?T-cell depletion experiments, decreased numbers
of CD8?T cells were observed by cytofluorometric analysis in
both GRA1- and control-vaccinated mice. Compared to unde-
pleted splenocytes from pVR1020-GRA1-vaccinated mice, the
CD4?T-cell population decreased by 93%, while the CD8?
FIG. 5. Survival curves for in vivo T-cell-depleted vaccinated C3H
mice from two independent experiments (A and B). Mice were vacci-
nated with pVR1020 or pVR1020-GRA1 and challenged i.p. with 40
cysts of the T. gondii IPB-G strain (solid symbols). Mice that were
subjected to CD8?or CD4?T-cell depletion (starting 1 day prior to
challenge) are represented by open symbols. The depletion efficiency
was monitored by cytofluorometric analysis of blood samples and was
found to be 98 to 100%.
VOL. 71, 2003 T. GONDII GRA1 DNA VACCINE EVOKES CD8?T CELLS 313