Animals and Toxoplasma gondii strains.
Female BALB/c mice were purchased from DaeHan BioLink Co. (Chungcheongbuk-do, South Korea). All mice were maintained under specific-pathogen-free conditions and were 6 to 8 weeks of age when first immunized. Animal studies were carried out under the authority of the Chungnam National University Animal Ethnics Committee (application number 2010-2-33). Two T. gondii strains were used. The tachyzoites of the virulent RH strain were used for plasmid construction and the preparation of soluble tachyzoite antigen (STAg), and the cyst-forming Me49 strain was used to orally infect mice to evaluate the length of survival (in days) and brain cyst numbers.
Human retinal pigment epithelial cells (ARPE-19; ATCC, Rockville, MD) were maintained in a 1:1 mixture of Dulbecco's modified Eagle medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA) and nutrient mixture F12 (DMEM-F12) containing 10% heat-inactivated fetal bovine serum (FBS), 0.348% sodium bicarbonate, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cell cultures were maintained at 37°C and 5% CO2, and the medium was changed every 3 to 4 days.
Isolation of T. gondii tachyzoites and cysts.
Tachyzoites of T. gondii strain RH were infected into ARPE-19 cells (parasite/cell ratio, 5:1) and incubated at 37°C with 5% CO2 for 2 to 3 days. Following spontaneous host cell rupture, lysed tachyzoites and host cellular debris were centrifuged at 900 × g for 10 min using Percoll (Sigma Chemical Co., St. Louis, MO) to pellet parasites. The final pellet was suspended in cold phosphate-buffered saline (PBS), and the suspension was passed through a 5.0-μm-pore-size filter (Millipore, Bedford, MA). Purified tachyzoites were used in all experiments.
The cysts of T. gondii strain Me49 were obtained from the brains of infected mice. Each brain was suspended in PBS to a final volume of 5 ml and homogenized by serial passages through 18-gauge syringe needles. Mean cyst numbers in the brain were determined in triplicate by counting 10-μl samples of the homogenate under a microscope at ×200 magnification.
Preparation of STAg.
Purified tachyzoites were centrifuged at 5,000 × g for 3 min and disrupted by three cycles of freezing at −20°C and thawing at 4°C. Finally, the lysate was sonicated on ice at 60 W/s and centrifuged for 40 min at 100,000 × g. The supernatants were pooled and sterile filtered (Gelman Sciences, Ann Arbor, MI), and the protein concentration was determined via the Bradford method using bovine serum albumin (BSA) as the standard. STAg was stored in aliquots at −70°C until use.
Construction of DNA vaccine plasmid.
The mammalian expression vector pCMV-Tag2B was used as a DNA vaccine vector. To construct the GRA7-ROP1 fusion expression plasmid, the GRA7 gene (786 bp, no stop codon; amino acid [aa] residues 17 to 225) and ROP1 gene (1,023 bp; aa residues 22 to 381) were amplified by PCR from cDNA of T. gondii (RH strain) using the following primers: GRA7 forward, 5′-CGCGGATCCGCGGCGGCTTTGCCCCAGTT-3′, and reverse, 5′-AATCTGCAGAGGCACCTCTTGCTCGAGTG-3′ (recognition sites for BamHI and PstI, respectively, are underlined); ROP1 forward, 5′-CCCAAGCTTGCCGCCCTTTCGAGCCACAA-3′, and reverse, 5′-CCGCTCGAG GCCCTCCTCGCCATTAGTTC-3′ (HindIII and XhoI recognition sites, respectively, are underlined). PCR products were cloned into the pGEM-T Easy vector (Promega Corporation, Madison, WI), digested with the appropriate restriction enzyme, and purified from agarose gels. GRA7 and ROP1 gene fragments were inserted into the mammalian expression vector pCMV-Tag2B, generating pCMV-Tag2B-GRA7 (pGRA7) and pCMV-Tag2B-ROP1 (pROP1). The BamHI/PstI fragment encoding GRA7 was excised and cloned into the BamHI/PstI sites of the pROP1 vector to produce pCMV-Tag2B-GRA7-ROP1 (designated pGRA7-ROP1). All recombinant plasmids were propagated in Escherichia coli DH5α and confirmed by restriction analysis and PCR sequencing (Solgent, Daejeon, South Korea). The murine IL-12 expression plasmid containing the p35 and p40 sequences, designated pUMVC3-mIL-12 (pIL12), was provided by Alexander Rakhmilevich (University of Wisconsin–Madison).
Plasmid extraction and purification.
Large-scale plasmid DNA was prepared using the Endotoxin-Free Mega kit according to the manufacturer's instructions (Qiagen, Hilden, Germany), and concentrations were determined by A260/A280 measurement. The ratios of the optical densities at 260 and 280 nm (OD260 and OD280, respectively) were 1.8 to 2.0, indicating no major protein contamination. Plasmid DNA was dissolved (1 mg/ml) in sterile endotoxin-free PBS and stored at −20°C until use.
Expression of the compound gene in vitro and in vivo.
ARPE-19 cells were transfected with pGRA7, pROP1, pGRA7-ROP1, or pCMV-Tag2B (control) using Lipofectamine LTX and Plus reagents (Invitrogen Life Technologies, Carlsbad, CA). Six-well culture plates were seeded with ARPE-19 cells (2 × 105 cells/well) and then cultured to 50 to 80% confluence. DNA (2.5 μg) in 500 μl DMEM and 2 μl Plus reagent was mixed gently and incubated at room temperature for 5 min. Lipofectamine LTX was mixed with diluted DNA and incubated at room temperature for 30 min. DNA-lipid complexes were added to cells that had been rinsed with serum-free medium. After incubation for 6 h at 37°C in a 5% CO2 incubator, medium was exchanged with medium containing 10% FBS. Following a further 48 h of incubation, cells were washed with PBS and harvested.
To examine the level of in vivo expression, total RNA was isolated from spleens of DNA-vaccinated mice 4 weeks postinoculation and were analyzed by multiplex reverse transcription-PCR (M-RT-PCR) with GRA7- and ROP1-specific primers. PCRs were carried out in a 50-μl total volume. Each PCR mixture contained 0.25 μl of TaKaRa Ex Taq (5 U/μl), 5 μl of 10× Ex Taq buffer, 4 μl of deoxynucleoside triphosphate (dNTP) mixture (2.5 mM each), 2 μl of each primer (10 pmol/μl), and 3 μl of cDNA. All PCRs were performed in a MyCycler (Bio-Rad Laboratories, CA). The denaturation of DNA (94°C for 30 s) was followed by 30 cycles of amplification (98°C for 10 s, 60°C for 30 s, and 72°C for 2 min) and ended by a 10-min extension at 72°C. Amplified products were electrophoresed in 1.2% agarose gel and visualized by ethidium bromide staining. These PCR products also were confirmed by sequencing.
SDS-PAGE and Western blot analysis.
After 48 h, pellets of transfected cells were suspended in 150 μl SDS-PAGE sample buffer and then sonicated and heated at 100°C for 5 min. Lysates were separated on a 12% polyacrylamide gel, transferred to polyvinylidene difluoride membranes, and then blocked with Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) containing 0.1% Tween 20 (TBST) and 5% skim milk. Membranes were washed three times with TBST and then incubated with diluted mouse anti-FLAG primary antibody (Sigma-Aldrich, St. Louis, MO) for 2 h at room temperature. Unreacted antibody was washed out with TBST, followed by incubation with goat anti-mouse IgG-peroxidase conjugate (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:10,000 in 5% skim milk–TBST. Peroxidase activity was detected using the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham-Pharmacia, Freiburg, Germany).
Construction of bacterial expression plasmids and purification of recombinant protein.
To construct pGEX-GRA7 and pGEX-ROP1 expression plasmids, the coding sequence of the T. gondii GRA7 and ROP1 genes (GenBank accession no. DQ459443.2 and M71274.1, respectively) were amplified by PCR (using Pfu-X DNA Polymerase; Solgent, Daejeon, South Korea) from cDNA of T. gondii strain RH with a pair of oligonucleotide primers (GRA7 forward primer, 5′-CGCGGATCCATGGCCCGACACGCAATTTT-3′, and reverse primer, 5′-TCCCCCGGGCTACTGGCGGGCATCCTCCC-3′; ROP1 forward primer, 5′-CGCGGATCCATGGAGCAAAGGCTGCCAA-3′; ROP1 reverse primer, 5′-GGCCTCGAGTTATTGCGATCCATCATCC-3′; recognition sites [GRA7, BamHI and SmaI; ROP1, BamHI and XhoI] are underlined). The PCR products were digested with the appropriate restriction enzyme and cloned into the BamHI/SmaI or BamHI/XhoI sites of the pGEX-4T-1 expression vector containing an N-terminal glutathione S-transferase (GST). The resulting plasmids were named pGEX-GRA7 and pGEX-ROP1.
Bacterially expressed recombinant T. gondii soluble proteins were purified with a Pierce GST spin purification kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Purified GST fusion proteins were dialyzed against a dialysis buffer (PBS containing 2 mM EDTA and 1 mM dithiothreitol [DTT]), the protein concentration was determined by the Bradford assay method using BSA as the standard, and the solution was stored at −70°C until use.
DNA immunization and experimental design.
Nine groups (23 mice per group) were vaccinated twice at 2-week intervals with empty vector, pIL12, empty vector with pIL12 (vector+pIL12), pGRA7, pROP1, pGRA7-ROP1, pGRA7 with pIL12 (pGRA7+pIL12), pROP1 with pIL12 (pROP1+pIL12), or pGRA7-ROP1 with pIL12 (pGRA7-ROP1+pIL12). Mice were inoculated with an injection of 50 μg plasmid DNA (in 50 μl sterile endotoxin-free PBS) into the tibialis anterior muscles of both hind legs (100 μg/per mouse) using a 26-gauge needle. Blood and spleens were collected to assess serum IgG, in vitro T cell proliferation, and cytokine levels at 0, 2, 4, and 8 weeks after immunization. Four weeks after the final immunization, mice were challenged with Me49 strains to determine the number of days of survival of mice and parasite burdens in the brain.
Evaluation of survival length and parasite burdens in the brain.
Four weeks after the final immunization, six mice were challenged with a lethal dose (1,500 cysts per mouse) of T. gondii
strain Me49 according to previous reports (10
), and mortality was monitored daily for 4 weeks. In addition, five mice per group were orally challenged with a nonlethal dose of strain Me49 (20 cysts per mouse) 4 weeks after final immunization. Four weeks after the challenge, mice were euthanized and the brain cysts were counted as indicated above. All samples were counted in triplicate. Cyst reduction rate to vector control was calculated with the formula [1 − (brain cyst numbers of vaccinated mice/brain cyst number of vector control)] × 100.
Determination of T. gondii-specific IgG and IgG subclass titers.
T. gondii-specific serum IgG, IgG1, and IgG2a antibody levels were determined by enzyme-linked immunosorbent assay (ELISA). The 96-well plates were coated with 10 μg/ml of STAg, purified GST, and recombinant GRA7 and/or ROP1 (5 μg/ml) in 50 mM carbonate buffer (pH 9.6) (100 μl per well) at 4°C overnight. Plates were washed twice with PBS (pH 7.4) containing 0.05% Tween 20 (PBS-T20) and blocked with PBS containing 1% BSA (PBS–1% BSA) for 2 h at room temperature. Sera were diluted in PBS–0.1% BSA and incubated for 2 h. After being washed with PBS-T20, plates were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (diluted 1:8,000 in PBS–1% BSA), IgG1, and IgG2a (1:2,000) for 2 h. After washing with PBS-T20, 200 μl of substrate solution (10 mg of O-phenylenediamine and 10 μl of 30% H2O2 in 25 ml of 0.1 M citrate-phosphate buffer, pH 5.0) was added. Plates then were incubated in the dark for 30 min, and the reaction was stopped by the addition of 3N HCl (50 μl). The OD then was measured by an ELISA reader at 490 nm.
We also determined the IgG2a titers against recombinant T. gondii GRA7 and ROP1 by endpoint dilution. Each serum sample was subjected to conventional 2-fold serial dilutions starting at 1:100, and we determined the OD levels using the same ELISA methods as those described above. The mean OD plus 5 standard deviations (SD) from 10 uninfected healthy mouse sera was used as the cutoff between anti-T. gondii IgG2a antibody-positive and -negative results, which made significant differences of IgG2a titers between uninfected mouse sera and T. gondii-infected mouse sera. The endpoint titer was the highest serum dilution that yielded an OD greater than the value that defined the cutoff. For convenience, titers are expressed as reciprocal values.
In vitro T cell proliferation assay.
In vitro T cell proliferation was measured using a chemiluminescent bromodeoxyuridine (BrdU) ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN). Spleens from immunized mice were collected under aseptic conditions in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA). Erythrocytes were removed by lysis, and the remaining cells were washed and suspended in RPMI 1640 medium supplemented with 10% FBS, HEPES (10 mM), l-glutamine (2 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 mM), gentamicin (50 mg/ml), penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells then were seeded in triplicate in flat-bottomed 96-well microtiter plates (Corning Inc., Corning, NY) at 5 × 106 cell/ml in 100 μl complete medium; thereafter, 10 μg/ml of STAg was added. The plates were incubated at 37°C in 5% CO2. After 72 h, BrdU labeling solution (10 μl) was added to each well and incubated for 2 h. The plates were centrifuged, and the labeling medium was removed. The cells were dried, FixDenat (200 μl) was added, and the plates were incubated for 30 min at room temperature. After removing the FixDenat solution, 100 μl of anti-BrdU-POD (peroxidase conjugate) working solution was added, followed by incubation for 90 min. After removing antibody conjugate, the wells were rinsed three times with PBS (pH 7.4). After 30 min of incubation with 100 μl substrate solution, the absorbance of the samples was evaluated by an ELISA reader at 370 nm with a 492-nm reference.
Spleen cell cultures and cytokine quantification.
Four weeks after the last vaccination, 3 mice per group were euthanized, and their spleens were isolated. Single-cell suspensions of splenocytes were stimulated by incubation with purified GST antigen alone or in combination with recombinant GRA7 and ROP1 antigens (5 μg/ml). Cell-free supernatants were harvested and assayed for IL-4 activity at 24 h, for IL-10 activity at 72 h, for gamma interferon (IFN-γ) activity at 96 h, and for tumor necrosis factor alpha (TNF-α) activity at 48 h. The IL-4, IL-10, IFN-γ, and TNF-α concentrations were evaluated using a commercial ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Cytokine concentrations were determined by reference to standard curves constructed with known amounts of mouse recombinant IL-4, IL-10, IFN-γ, or TNF-α. The sensitivity limits for the assays were 2 pg/ml for IL-4, 4.0 pg/ml for IL-10, 2 pg/ml for IFN-γ, and 1.88 pg/ml for TNF-α.
Results are expressed as the means ± SD for each group. The statistical evaluation of the differences in survival rates was checked by Kaplan-Meier test, and then a log-rank test was done. Statistical differences in parasite burdens, antibody titers, in vitro T cell proliferation, and cytokine levels were determined using the Kruskal-Wallis test. Differences among the various groups were considered significant when P < 0.05.