Protective Efficacy of a Toxoplasma gondii Rhoptry Protein 13 Plasmid DNA Vaccine in Mice

Mice and parasites.Six- to 8-week-old specific-pathogen-free (SPF) female Kunming mice, the most widely used outbred strain in China, were purchased from Yat-Sen University Laboratory Animal Center, Guangzhou, China. All mice were bred under specific-pathogen-free conditions and had free access to a commercial basal diet and tap water ad libitum.

Two T. gondii strains were used: the virulent RH strain (type I) and the brain cyst-forming PRU strain (type II). Tachyzoites of the highly virulent RH strain of T. gondii were preserved in our laboratory and maintained by serial intraperitoneal passage in Kunming mice. To prepare soluble tachyzoite antigens (STAg) for the detection of antibodies, the tachyzoites of the RH strain were collected from the peritoneal fluids, washed by centrifugation, and then suspended in sterile phosphate-buffered saline (PBS) and sonicated. The supernatant was considered STAg and was kept at −80°C until further use.

For the preparation of T. gondii cysts, mice were infected with 20 cysts of the PRU strain. About 1 month after infection, their brains were isolated, homogenized, and diluted in PBS (pH 7.2) in order to obtain 10 tissue cysts in 100 μl.

Construction of the eukaryotic expression plasmid.To construct the pVAX-ROP13 expression plasmid, the coding sequence of the T. gondii ROP13 gene (GenBank accession no. DQ096560.1), 1,203 bp from sequence positions 1 to 1203, was amplified by PCR from genomic DNA of T. gondii strain RH with a pair of oligonucleotide primers (forward primer, 5′-CGCGGATCCATGAAGAGAACAGAGCTTTG-3′, and reverse primer, 5′-GCTCTAGATCACAATAGCCTCAAGGAATTC-3′), and BamH I and XbaI recognition sites were introduced. The PCR product was cloned in pGEM-T easy vector (Promega) and sequenced in both directions to ensure fidelity, generating pGEM-ROP13. The ROP13 fragment was cleaved by BamH I/XbaI from pGEM-ROP13 and cloned into the BamH I/XbaI sites of pVAX I (Invitrogen). The resulting plasmid was named pVAX-ROP13. The plasmid pVAX/IL-18 was extracted previously in our laboratory (23). The concentrations of pVAX-ROP13 and pVAX/IL-18 were determined using a spectrophotometer at optical densities at 260 and 280 nm (OD₂₆₀ and OD₂₈₀).

In vitro pVAX-ROP13 plasmid expression.Marc-145 cells were transfected with pVAX-ROP13 using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, the cells were fixed with cool acetone for 15 min, and ROP13 expression was detected using the indirect immunofluorescence assay (IFA), with anti-T. gondii polyclonal antiserum (goat) and a fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat IgG (Proteintech Group Inc., Chicago, IL). Evans blue (Fisher) was included in the secondary-antibody solution as a counterstain. Coverslips were rinsed three times with PBS. The monolayers binding the marker were covered with glycerol and examined for specific fluorescence under a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany).

Immunization and challenge infection.Mice were divided into five groups of 40 mice per group. The mice were injected intramuscularly with 100 μg of plasmid DNA suspended in 100 μl sterile PBS (100 μl in each thigh skeletal muscle), whereas control mice received PBS alone. The mice in groups I and II were injected with PBS and empty pVAX I vector, respectively, as controls; the mice in group III and group V were injected with pVAX-ROP13 alone (100 μg) or combined with pVAX/IL-18 (50 μg/each) and those in group IV with pVAX/IL-18. The mice in all groups were vaccinated three times at weeks 0, 2, and 4.

Two weeks after the final immunization, 17 mice in all groups were challenged intraperitoneally (i.p.) with 1 × 10³ tachyzoites of the virulent T. gondii RH strain, and the other 17 mice were inoculated orally with 10 cysts of the PRU strain. The mice were observed daily for mortality. One month after the challenge, the surviving mice were sacrificed and their brains were removed. Each brain was homogenized in 2 ml of PBS. The mean number of cysts per brain was determined by counting in three samples of 25-μl aliquots of each homogenized brain under an optical microscope. Blood was collected from the mouse tail vein before each immunization. Sera were separated and stored at −20°C for enzyme-linked immunosorbent assay (ELISA).

Antibody assays.Antigen-specific antibody levels were measured with an anti-T. gondii IgG ELISA kit according to the manufacture's instructions (Combined Biotech Co., Ltd., Shenzhen, China). In brief, plates were coated overnight at 4°C with 1 μg of STAg in 100 μl of carbonate buffer, pH 9.2. The blood of three mice was collected from the mouse tail vein, and the sera diluted with PBS were applied to the wells; then, the bound antibodies were detected with horseradish peroxidase-conjugated anti-mouse IgG diluted 1:2,000. Immune complexes were revealed by incubation with orthophenylene diamine and 0.15% H₂O₂ for 30 min. The reaction was stopped by the addition of 1 M H₂SO₄, and the absorbance was measured at 450 nm using an ELISA reader (Bio-TekEL × 800). All samples were run in triplicate.

Lymphoproliferation assay.Briefly, splenocyte suspensions were prepared from each of the three mice per group by pushing the spleens through a wire mesh 1 day before the challenge. After the red blood cells (RBC) were removed using RBC lysis solution (Sigma), splenocytes were resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells were then plated in 96-well Costar plates at a density of 5 × 10⁵ cells per well and cultured with STAg (10 μg/ml) or ConA (5 μg/ml; Sigma) or medium alone (negative control) at 37°C with 5% CO₂. The proliferative activity was measured using a 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml; Sigma) dye assay (16). The stimulation index (SI) was calculated as the ratio of the average OD₅₇₀ value of wells containing antigen-stimulated cells to the average OD₅₇₀ value of wells containing only cells with medium. All assays were performed in triplicate.

Cytokine assays.To evaluate cytokine production, splenocytes from three immunized mice were cultured with different stimuli as described for the lymphoproliferation assay 2 weeks after the last immunization. Cell-free supernatants were harvested and assayed for IL-2 and IL-4 activities at 24 h, for IL-10 activity at 72 h, and for IFN-γ activity at 96 h. The IL-2, IL-4, IL-10, and IFN-γ concentrations were evaluated using a commercial ELISA kit according to the manufacturer's instructions (R&D Systems). Cytokine concentrations were determined by reference to standard curves constructed with known amounts of mouse recombinant IFN-γ, IL-2, IL-4, or IL-10. The sensitivity limits for the assays were 20 pg/ml for IFN-γ, 50 pg/ml for IL-2, and 10 pg/ml for IL-4 and IL-10.

Statistical analysis.Data, including antibody responses, lymphoproliferation assays, and cytokine production, were compared between the different groups by one-way analysis of variance (ANOVA). All data were processed by analysis with SPSS13.0 Data Editor (SPSS Inc., Chicago, IL). P values of <0.05 were considered to be statistically significant.