BALB/c mice aged between 6 and 8 weeks were purchased from the Laboratory Animal Centre of Zhejiang Academy of Agricultural Sciences. All the mice were maintained under specific pathogen-free standard conditions with stable temperature (24°C ± 1°C), 50% ± 10% humidity, and a 12/12-h light–dark cycle; food and water were supplied ad libitum. All experiments were approved by the Animal Ethics Committee of Zhejiang Academy of Agricultural Sciences. BALB/c mice were used for the vaccination study, and Vero cells were used for maintenance and proliferation of T. gondii RH strain tachyzoites.

Toxoplasma lysate antigen (TLA) was obtained as previously described (Holec-Gasior et al., 2010). Briefly, 10⁷ tachyzoites were collected from Vero cells, washed three times with sterile phosphate buffered saline (PBS), and then centrifuged at 1,000 rpm for 10 min. The tachyzoites were disrupted using 10 freezing cycles at -80°C and thawing at 37°C. Then, the supernatant with TLA was collected, its concentration was measured using a bicinchoninic acid (BCA) Protein Assay Kit (Sangon Biotech, Shanghai, China), and it was stored at -80°C until use.

A total of 10⁷ T. gondii tachyzoites were collected, and total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions and then reverse transcribed into cDNA using the First Strand cDNA Synthesis Kit. The whole Coding sequence (CDS) of GRA7 was amplified from cDNA using PCR with primers containing EcoRI and BamHI restriction sites (underlined), 5′-gaattcATGGCCCGACACGCAATT-3′ (forward) and 5′-ggatccCTGGCGGGCATCCTCCCCATCTT-3′ (reverse). PCR amplification was performed as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a final extension time of 72°C for 10 min. The PCR product was detected by 1.5% agarose gel electrophoresis, the target band was purified and cloned into the pMD-19T vector, and the clone was sequenced by Sangon Biotech Company (Shanghai). The correct GRA7-pMD-19T sequence was cloned into the eukaryotic expression plasmid pEGFP-C2 using EcoRI and BamHI restriction enzymes. The recombinant plasmid GRA7-pEGFP-C2 was extracted using a Plasmid Purification Kit (Solarbio, China, Beijing), and its concentration was measured using a NanoDrop2000 Ultra Micro Spectrophotometer. Then, the preparation plasmid was stored at -20°C until use.

Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 100 μg/ml streptomycin/penicillin and 10% fetal bovine serum (FBS) at 37°C and 5% CO₂. Vero cells were cultivated in six-well plates with cell slides before transfection, and then the recombinant plasmid GRA7-pEGFP-C2 (4 μg) or the empty plasmid (pEGFP-C2) was transfected into Vero cells using 10 μl LipoFiterTM Liposomal Transfection Reagent (Hanbio Biotechnology, Shanghai, China). After inoculation for 48 h, the cell climbing tablets were removed from the six-well plates and washed with 0.1 M PBS three times and then fixed in 4% paraformaldehyde for 15–20 min. Fifty microliters of 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime, Shanghai, China) was added to the climbing tablets and incubated for 5 min. Finally, the expression of GRA7 in Vero cells was observed using a laser confocal microscope.

GRA7 protein expression from the Vero cells was analyzed by Western blot as follows. Vero cells transfected with GRA7-pEGFP-C2 and pEGFP-C2 were collected, and protein was isolated with radioimmunoprecipitation assay (RIPA) lysis buffer containing 1 mM phenyl-methanesulfonyl fluoride (PMSF; Beyotime Biotechnology, China), then the lysis solution was centrifuged at 12,000 rpm for 10 min at 4°C. Next, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane by electric transfer instrument (Bio-Rad, America). The membrane was sealed overnight at 4°C with 5% skimmed milk after washing three times with PBS with 0.05% Tween-20 (PBST), then coated with anti-T. gondii tachyzoite antigen mouse sera (diluted 1:1,000), followed by incubation for 2 h with a horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (Solarbio, China). Finally, the bands were detected using enhanced chemiluminescence (ECL; Thermo, America).

CaPNs were prepared as previously described (He et al., 2000). Briefly, 12.5 mM dibasic sodium phosphate, 12.5 mM calcium chloride, and 15.6 mM sodium citrate were mixed together slowly and stirred for 48 h. After sonication for 30 min, a dynamic light-scattering instrument (Anton Paar Litesizer 500) and transmission electron microscope were used to determine the average size distribution, and the particle morphology was observed by scanning electron microscopy (SEM). Subsequently, a GRA7 DNA vaccine coated with CaPNs was prepared by vortexing mixtures of GRA7-pEGFP-C2 and CaPNs for 60 min, with 100 μg plasmid plus 100 μg NPs. The particles were isolated by ultracentrifugation at 66,000 g for 30 min, the supernatant was collected, the particles were redispersed in 1 ml sterile ultrapure water, and the uncoated plasmid was removed by this purification method. Afterward, the concentration of the GRA7-pEGFP-C2 plasmid in the supernatant or in the particles was analyzed using a NanoDrop2000 Ultra Micro Spectrophotometer. The loading efficiency (LE) was determined using the following equation:

A total of five groups of female BALB/c mice (13 mice/group) were used for the immunization experiment, and these mice were injected three times with 100 μl of the purified GRA7-pEGFP-C2 plasmid DNA dissolved in 100 μl sterile 0.1 M PBS, empty vector (pEGFP-C2), or GRA7-pEGFP-C2+nano-adjuvant (CaPNs). At the same time, two control groups (PBS and CaPNs) were designed. For the second and third inoculations, mice were boosted using the same protocol on days 14 and 28. Tail blood was collected from each mouse on days 0, 14, 28, 42, and 63, and sera were obtained and stored at -20°C until use.

Two weeks after the last immunization, 10 mice from each group were intraperitoneally injected with T. gondii RH strain tachyzoites (1 × 10⁴/each) as previously described (Han et al., 2017; Song et al., 2020). The status of infected mice was monitored every day, and the survival rate was recorded.

To investigate the humoral immune response induced in all immunized mice, total immunoglobulin G (IgG), IgG1, and IgG2 were measured using enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer’s instructions (MultiSciences, Hangzhou, China). Briefly, 96-well microplates were coated with 100 μl/well of TLA (20 μg/ml) (Ahmadpour et al., 2017; Roozbehani et al., 2018). The microplates were blocked with 100 μl 5% skimmed milk in PBST for 2 h after overnight coating. Then, 100 μl of mouse serum (diluted 1:100 in 1% skimmed milk) was added to each well and incubated for 1 h at 37°C. After washing three times with PBST, the wells were incubated with HRP-conjugated anti-mouse IgG (diluted 1:2,000 in 1% skimmed milk), IgG1 (1:2,000), and IgG2a (1:2,000) for 40 min at 37°C. After five times of washing, a Tetramethylbenzidine (TMB) substrate solution was added and incubated for 15 min at 37°C and then was stopped by the addition of 2 M H₂SO₄. Finally, optical density (OD) values were measured at 450 nm. All samples were run in triplicate.

Two weeks after the last immunization, three mice from each group were euthanized, and splenocytes were collected and treated with red blood cell lysate and then cultured in a 96-well plate (1 × 10⁵ cells/well) in DMEM (100 μg/ml streptomycin/penicillin and 10% FBS). Thereafter, the cells were stimulated with 10 μg/ml TLA or 7.5 μg/ml concanavalin A (ConA) (positive control). As a negative control, media alone were added. The plates were incubated at 37°C in 5% CO₂ for 72 h, after which Cell Counting Kit (CCK)-8 solution was added (50 μl/well) and cultured for 4 h. Proliferative activity was evaluated by measuring the OD values at 450 nm using an ELISA reader. The splenocyte stimulation index (SI) was calculated as the ratio of the average absorbance of the TLA-treated samples to the average absorbance of the negative groups. All samples were run in triplicate.

Splenocytes were collected as mentioned above and cultured in 96-well microtiter plates. The supernatants were harvested and assayed for IL-4 at 24 h, IL-10 at 72 h, and IL-12 and interferon gamma (IFN-γ) at 96 h using ELISA kits according to the manufacturer’s instructions.

In order to evaluate tissue parasite burden, the heart, liver, spleen, and lung from three mice (each group) were removed. We collected the tissues using sterile scissors, and the tissues were divided into masses of equal quality (1 mg). Genomic DNA was extracted using the genomic DNA extraction kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Afterward, the parasite burdens were determined by quantitative real-time PCR using the repeated element (RE) primers (forward, 5-AGGGACAGAAGTCGAAGGGG-3; reverse, 5- GCAGCCAAGCCGGAAACATC-3) (Roozbehani et al., 2018). The final volume of the Q-PCR reaction was 20 μl containing 10 μl SYBR green master mix (TAKARA, Japan), 0.5 μl forward primer (10 pmol), 0.5 μl reverse primer (10 pmol), 1 μl DNA template, 8 μl RNase-free water. The amplification steps were an initial denaturation at 95°C for 10 min, and amplification consisted of 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and amplification at 72°C for 30 s. Melting curve analysis was performed to verify the specific amplification of the correct sequence. The standard curve was determined by the known concentration of the T. gondii RH tachyzoites DNA. The number of parasites in the samples was calculated from the threshold cycle (Ct) value according to the standard curve (Y = -3.48X + 32.326; R² = 0.987). The results were based on three independent experiments.

All statistical analyses were performed using GraphPad Prism Version 5. Antibody production and cytokine levels were analyzed using one-way ANOVA. Tukey’s Student range test was used when a significant difference appeared. P value of <0.05 was considered a significant difference.

The expression and localization of GRA7 in Vero cells and cells transfected with pEGFP-C2 were analyzed using laser confocal microscopy ( Figures 1A, B ). Green fluorescence was observed in the GRA7-pEGFP-C2 ( Figures 1A1, A2 )- and pEGFP-C2-transfected groups ( Figures 1B1, B2 ). A2 and B2 showed the single cell transfected with GRA7-pEGFP-C2 and pEGFP-C2 and A3 and B3 exhibited the cells transfected with GRA7-pEGFP-C2 pEGFP-C2 ( Figures 1A3, B3 ), which were detected under white light, whereas no fluorescence was observed in the untransfected cells. GRA7 protein expression in the transfected Vero cells was determined by Western blot analysis; as shown in Figure 1C , a specific band was detected in lysates of the GRA7-pEGFP-C2-transfected cells, whereas the negative control cells showed no bands. These results indicated that the GRA7-pEGFP-C2 recombinant plasmid was successfully transfected and expressed in Vero cells.

The average NP diameter was 47.28 nm, and the diffusion coefficient was approximately 4, indicating acceptable monodispersity ( Figures 2A, B ); a consistent size was observed by transmission electron microscopy (TEM) ( Figure 2C ). The morphology of adjuvant (CaPNs) NPs was analyzed by SEM, showing that most of them were circular in shape with a smooth surface ( Figure 2D ).

To determine the T. gondii-specific antibody response, sera from all vaccinated mice were collected, and the total IgG and IgG subclasses (IgG1 and IgG2a) were analyzed by ELISA. High levels of IgG were observed in the serum of the vaccine-immunized groups (GRA7-pEGFP-C2+nano-adjuvant, GRA7-pEGFP-C2) (P < 0.01); however, no significant difference was observed between these two vaccine groups (P > 0.05) ( Figure 3A ). IgG1 in the immunization groups was also significantly elevated compared to that in the control groups ( Figure 3B ) (P < 0.05 for both vaccine groups). The GRA7-pEGFP-C2+nanoadjuvant (CaPN) group exhibited a higher level of IgG2a ( Figure 3C ) (P < 0.01), and higher levels of IgG2a were also observed in GRA7-pEGFP-C2-immunized mice (P < 0.05) than that in the control groups. Meanwhile, the levels of IgG2a were significantly higher than IgG1 in the vaccine groups (P < 0.05, compared to control groups), and the ratios of IgG2a/IgG1 were higher in mice immunized with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) compared to those immunized with GRA7-pEGFP-C2 alone (P < 0.05) ( Figure 3D ). Taken together, these results showed that a Th1-type immune response was elicited in response to nanoadjuvant vaccine immunization.

Splenocytes were collected from immunized and control groups (5 weeks after the last immunization) to analyze their proliferation, and the cells were treated with TLA and ConA. As shown in Figure 4 , a significantly higher lymphocyte proliferation SI was obtained in the GRA7-pEGFP-C2+nano-adjuvant (CaPNs) and GRA7-pEGFP-C2 groups compared to the control groups (P < 0.05). In addition, the GRA7-pEGFP-C2+nano-adjuvant (CaPNs) group induced an almost 2-fold higher level of lymphocyte proliferation than the GRA7-pEGFP-C2 immunization group (P < 0.05).

To further explore T-cell responses to vaccination, splenocytes were collected 63 days after immunization, and supernatants were harvested to evaluate the expression of cytokines, including IL-12, IFN-γ, IL-4, and IL-10 ( Figure 5 ). Compared with the control groups, the IL-12 level of mice vaccinated with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) was statistically higher (P < 0.01) ( Figure 5A ). Moreover, the production of IFN-γ was significantly higher in the GRA7-pEGFP-C2+nano-adjuvant (CaPNs) group than that in the control groups (P < 0.01), and GRA7-pEGFP-C2-treated mice displayed higher levels of IFN-γ (P < 0.05) ( Figure 5B ). In contrast, IL-4 and IL-10 levels showed no statistically significant differences in any of the groups compared to the control groups (P > 0.05) ( Figures 5C, D ). These results confirmed that immunization with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) or GRA7-pEGFP-C2 promoted a Th1-type immune response.

To evaluate protective efficacy, mice from all groups were intraperitoneally challenged with 10⁴ tachyzoites of the T. gondii RH strains 5 weeks after the final vaccination, and the survival time was recorded. As shown in Figure 6 , mice in the control groups all died within 4 days (P > 0.05), while the survival times of mice immunized with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) (extending survival time to the 14th day) were significantly longer by comparison (P < 0.05); however, no significant difference was observed between the GRA7-pEGFP-C2+nano-adjuvant (CaPNs) and GRA7-pEGFP-C2 groups.

Three mice from each group were randomly selected after death to determine the parasite burden in tissues of T. gondii-infected mice in the heart, liver, spleen, and lung. SYBR-green real-time PCR was used to quantify parasite loads. As shown in Figure 7 , all tissues examined were T. gondii infection positive, but the vaccine immunization groups exhibited lower parasite loads than the control groups. Mice immunized with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) or GRA7-pEGFP-C2 exhibited significantly reduced parasite loads in the liver, spleen, and lung (P < 0.001) ( Figures 7B–D ), and the average parasite loads in the heart (GRA7-pEGFP-C2+nano-adjuvant group) were reduced by 3.15-fold (P < 0.05) compared with the control groups ( Figure 7A ). GRA7-pEGFP-C2+nano-adjuvant (CaPNs)-immunized mice showed decreased parasite loads compared to the GRA7 pEGFP-C2-immunized group in the spleen and lung (P < 0.05), and no significant differences were found in the control groups (P > 0.05).