All animal experiments were approved by the Animal Ethics committee of responsible authority from the College of Veterinary Medicine, Nanjing Agricultural University, China (permission: NJAU. No20210315014).

Specific pathogen-free (SPF) BALB/c mice (18–22 g, female) and Sprague–Dawley (SD) rats (200–220 g, female) were purchased from Model Animal Research Center of Nanjing University, Nanjing University, China. All animals were strictly housed in a specific pathogen-free environment following the requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China.

Human embryonic kidney 293-T (HEK 293-T) cells were obtained from the Institute of Cell Biology, Chinese Academy Sciences, Shanghai, China, and preserved in our laboratory. HEK 293-T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% double antibiotics (Carlsbad, Gibco, CA, USA) at 37°C in a 5% CO₂ atmosphere.

Tachyzoites of the violent T. gondii RH strain (type I) were maintained in the Ministry of Education (MOE) Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. Tachyzoites used in this study were replicated in BALB/c mice according to a previous study (41).

Strictly following the instructions, the total RNA of 10⁶ tachyzoites of T. gondii was isolated by using the Total RNA Extraction kit (OMEGA Bio-tek, Norcross, GA, USA). Reverse transcription PCR (RT-PCR) was immediately conducted to synthesize the cDNA by a reverse transcription kit (Takara Biotechnology, Dalian, China). Primers amplifying the complete open reading frame (ORF) of TgSDRO were designed based on the conserved domain sequences (CDS) of the TgSDRO gene (GenBank: AAYL02000041). Along with the restriction endonuclease sites (EcoRI and XhoI) and a Kozak translation initiation sequence, the forward and reverse primers, 5’-CCG GAATTC GCCACC ATGGATATTCAAGCGGTTGGGA-3’ and 5’-CCGCTCGAGTCAGGTGGAATGCGGGGG-3’, were synthesized by the company (Tsingke Biological Technology, Nanjing, China). For PCR amplification, 1.25 U Ex Taq DNA polymerase (Takara Biotechnology, Dalian, China), 5 µl 10 × Ex Taq buffer, 4 µl dNTP mixtures (Mg²⁺ plus), 20 pmol of each primer, 2 ng cDNA template, and ddH₂O to make a final volume of 50 µl were involved in each reaction. The amplification was determined by an amplifier (Thermo Scientific, Waltham, MA, USA) using the following program: preheating (5 min at 95°C), amplification for 35 cycles of 30 s at 95°C, 30 s at 64°C and 70 s at 72°C, and the final extension (5 min at 72°C). Then, 10 μl PCR amplicons were analyzed in a 1.0% agarose gel containing 0.01% ethidium bromide. The single band with expected base pairs was then purified by using a Gel Eradication Kit (OMEGA Bio-tek, Norcross, GA, USA). After being digested with EcoRI and XhoI restriction endonuclease (Takara Biotechnology, Dalian, China), the obtained PCR amplicons were inserted into the linear pVAX1 vector (Invitrogen Biotechnology, Shanghai, China) by using T4 DNA ligase (Takara Biotechnology, Dalian, China). The recombinant plasmids were identified by double restriction enzyme digestion and then sequenced by the ABI PRISM™ 3730 XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA). The positive plasmid was transferred to Escherichia coli (E. coli) DH5α (Invitrogen Biotechnology, Shanghai, China).

To obtain a large quantity of recombinant plasmid, E. coli DH5α carrying the recombinant plasmid was amplified in a Luria Bertani (LB) medium containing 100 μg/ml kanamycin monosulfate until the OD600 reached 0.6 (37°C and shaking at 180 rpm). Endo-free DNA plasmid was extracted by a commercially available kit (Vazyme, Nanjing, China) in a large quantity, and then the level of endotoxin was determined by a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GeneScript, Piscataway, NJ, USA). After concentration determination by a nanodrop microvolume spectrophotometer (Thermo Scientific, Waltham, MA, USA), the recombinant plasmids were stored at -20°C until use.

The liposomal transfection method was used for the delivery of plasmids in HEK 293-T cells (1 × 10⁶ cells) cultured in a 6-well plate (Costar, Cambridge, MA, USA) with 70–90% confluence, then a Lipofectmine™ 3000 reagent (Invitrogen Biotechnology, Shanghai, China) was used as the transfection reagent for making the plasmid–lipid complex according to the instructions. The transfected cells were incubated for 15 min before replacing the medium with 2 ml of fresh medium. Three days later, the cultured medium was removed, and the cells were washed with phosphate buffered saline (PBS) for three times. Then cells were scraped into 250 μl RIPA lysis buffer (Beyotime, Shanghai, China) containing 10 μl protease and phosphatase inhibitor (Beyotime, Shanghai, China). Tip sonication (Scientz Biotechnology, Ningbo, China) was conducted in a continuous mode for 2 s at 2 s (5 min in total) under the output power of 20 W, and then the cell lysates were stored at -20°C until use.

To obtain the cell culture medium containing recombinant protein, the culture medium was first renewed after a 3-day cultivation, and then the transfected cells were scraped into the medium; then the tip sonication was conducted in the same continuous mode mentioned above under sterile condition. The cell culture medium was collected and stored at -20°C after centrifugation at 12,000 rpm for 5 min at 4°C.

To obtain the serum against T. gondii, two SD rats were challenged with 10³ tachyzoites of T. gondii RH strain intraperitoneally. One month later, the serum containing the antibodies against T. gondii was harvested. All sera were kept at -20°C until use.

The transfected HEK 293-T cells were prepared following the method described in the section Recombinant Plasmids Transfection in Vitro. With 3-day stationary cultivation after transfection in the medium, cells were washed with PBS for three times and fixed with 1 ml 4% paraformaldehyde for 12 h at 4°C. Permeabilized with tris buffered saline (TBS) containing 0.1% Triton X-100 (TBSx) for 3 min on a rotary shaker at 50 rpm, cells were subsequently incubated with TBSx containing 5% BSA and 1% serum obtained from T. gondii-infected rats for 1 h on a rotary shaker at 37°C. Diluted 1:500 in TBSx containing 5% BSA, CY3-conjugated anti-rat IgG (Sigma, Saint Louis, MO, USA) was added to the cells before washing three times in TBSx. After being incubated for 1 h at 37°C on a rotary shaker, 500 μl of 4’,6-diamidino-2-phenylindole (DAPI) staining solution (Beyotime, Shanghai, China) was added in each well. After being stained for 5 min at 37°C on a rotary shaker, DAPI staining solution was removed, and the cells were washed three times in TBSx. Cover lips were mounted with 300 μl anti-fade mounting medium (Beyotime, Shanghai, China) and the image was taken by a Nikon A1 plus laser scanning confocal microscopy (Nikon Corporation, Tokyo Metropolis, Japan).

HEK 293-T cell lysates were added with 10 × loading buffer and heated at 95–100°C for 5 min. Separated by 12% SDS-PAGE, HEK 293-T cell lysates were electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then incubated with 5% (w/v) BSA in TBS containing 0.5% (v/v) tween 20 (TBST) for 2 h at 37°C on a rotary shaker with 50 rpm. After three times washing in TBST, the membranes were subsequently incubated with TBST containing 1% (v/v) rat serum at 4°C overnight with constant shaking. Washed with TBST in triplicate, the membranes were incubated with HRP-conjugated anti-rat IgG (1:5,000, eBioscience, San Diego, CA, USA) dissolved in TBST for 1 h at 37°C on a rotary shaker. After three times washing in TBST, the membranes were incubated with newly prepared 3,3’-diaminobenzidine (DAB, Sigma-Aldrich, Saint Louis, MO, USA) for 10 min.

According to a previous study (42), the double emulsion solvent evaporation technique (w/o/w) was used for the synthesis of PLGA nanospheres loaded with TgSDRO-pVAX1 plasmid (TgSDRO-pVAX1/PLGA nanospheres) with minor modifications. Briefly, to construct the organic phase, 5 mg of PLGA (MW: 40,000–75,000 Da, LA/GA: 65/35, Sigma, Saint Louis, MO, USA) was dissolved in 1 ml dichloromethane (DCM, Sigma, Saint Louis, MO, USA). Subsequently, 2 mg (the concentration was 1 mg/ml) purified plasmids were dropwise dissolved in 2 ml of 6% (w/v) polyvinyl alcohol (PVA, MW: 31,000–75,000 Da, Sigma, Saint Louis, MO, USA) with a plastic pipette tip at room temperature to construct the aqueous phase. For the 6% PVA solution, PVA was dissolved in double-distilled water and passed through a 0.22-μm filtering membrane (Millipore, Billerica, MA, USA). After the aqueous phase was thoroughly mixed on a vortex mixer for 2 min, the organic phase was dropped into the aqueous phase on a magnetic stirrer (400 rpm) using a plastic pipette tip. To form the w/o/w emulsions, tip sonication (Scientz Biotechnology, Ningbo, China) was then conducted in a continuous mode for 5 s at 5 s (4 min in total) under the output power of 50 W in an ice bath. To evaporate DCM, the w/o/w emulsions were stirred at 400 rpm on a magnetic stirrer at 4°C overnight. After being centrifuged at 35,000 rpm for 25 min at 4°C, the nanospheres were collected and dissolved in double-distilled water, and the supernatant was also obtained to calculate the total amount of plasmids. The obtained nanospheres were passed through a 0.22-μm filtering membrane, then frozen at -80°C for at least 2 h. The frozen PLGA nanospheres were quickly transferred to a vacuum freeze dryer (Labconco, Kansas City, MO, USA) until completely freeze-dried. The empty PLGA nanospheres were also prepared by replacing the TgSDRO-pVAX1 plasmid with PBS, and the synthesized PLGA nanospheres were stored at 4°C until use.

The ionic gelation technique was conducted for the synthesis of chitosan nanospheres loaded with TgSDRO-pVAX1 plasmid (TgSDRO-pVAX1/CS nanospheres) according to a previous study (43). Briefly, 0.1 g of chitosan (MW 50–190 kDa, Sigma, Saint Louis, MO, USA) was dissolved in 50 ml of 1% (v/v) aqueous solution of acetic acid. The chitosan solution was then thoroughly mixed on a magnetic stirrer to make chitosan fully dissolved, then the pH value was adjusted by 2 M NaOH solution. Sodium tripolyphosphate (0.02 g; TPP, Aladdin, Shanghai, China) was dissolved in 10 ml double-distilled water and passed through a 0.22-μm filtering membrane (Millipore, Billerica, MA, USA). TPP solution (4 ml) was dropwise added to 20 ml of chitosan solution on a magnetic stirrer at a bath temperature of 30°C, and then 4 mg (the concentration was 1 mg/ml) purified plasmids were dropwise added in. Constant stirring continued at least for 20 min to make chitosan fully dissolved. Then tip sonication was performed in a continuous mode for 5 s at 5 s (2 min in total) under an output power of 50 W in an ice bath. After being centrifuged at 40,000 rpm for 25 min at 4°C, the nanospheres were collected and dissolved in double-distilled water, and the supernatant was also collected to calculate the total amount of plasmids. Before freezing at -80°C for at least 2 h, the nanospheres were passed through a 0.22-μm filtering membrane. The frozen chitosan nanospheres were then transferred to a vacuum freeze dryer until completely freeze-dried. To synthesize empty chitosan nanospheres, PBS, instead of TgSDRO-pVAX1 plasmid, was conducted. The synthesized chitosan nanospheres were stored at 4°C until use.

To investigate the encapsulation efficiency (EE) and the loading capacity (LC), the concentration of free plasmids in the supernatant was quantified by a nanodrop microvolume spectrophotometer, and the total amount of free plasmids in the supernatant could be obtained. The EE and LC can be calculated by formula (1) and formula (2), respectively.

To characterize the features of TgSDRO-pVAX1/PLGA and TgSDRO-pVAX1/CS nanospheres, the nanospheres were sent to Nanjing Agriculture University for scanning electron microscope (SEM) observation by using Hitachi SU8010 (Tokyo, Japan). By using the ImageJ software version 1.8 (NIH Image, Bethesda, MD, USA), the average diameter of nanospheres was determined by measuring five arbitrary nanospheres.

To investigate the integrity of prepared nanospheres, synthesized PLGA and chitosan nanospheres were preserved at 4°C for more than 3 months, and the image was taken by SEM as described above. The integrity of two types of nanospheres was then evaluated by analyzing SEM pictures captured at different stages.

As referenced to a previous study (44), the release characteristics of TgSDRO-pVAX1/PLGA and TgSDRO-pVAX1/PLGA nanospheres were conducted with minor modifications. Briefly, after being dissolved in 1 ml of PBS (pH 7.4), 2 mg nanospheres were dispersed on a rotary shaker (180 rpm) at 37°C. The time interval between two sample points was 12 h. To record the total volume of the supernatant and sample the supernatant, the nanospheres solution was centrifuged at 12,000 rpm for 5 min. The nanospheres that settled at the bottom were resuspended, and the tubes were put back. Referenced to the same PBS used for dissolving empty PLGA or chitosan nanospheres (loaded with PBS, instead of TgSDRO-pVAX1 plasmid), the concentrations of plasmid in the supernatant were immediately measured. Then, the cumulative release (CR) was calculated by formula (3). Each group had three replications, and each replication was measured once.

Three BALB/c mice were challenged with 200 tachyzoites of T. gondii RH strain intraperitoneally. Seven days later, infected mice were sacrificed under the supervision of the Animal Ethics Committee, Nanjing Agriculture University, China. Then tissue samples from the intestine, heart, leg muscle, brain, liver, spleen, lung, and kidney were collected and store at -20°C until use.

To detect T. gondii in different tissues, PCR was conducted to amplify the internal transcribed spacer 1 (ITS-1) sequence based on a published paper (45). Briefly, 30 mg of tissue samples was used for genomic DNA extraction according to the instruction (OMEGA Bio-tek, Norcross, Georgia, USA), and the extracts were stored at -20°C until use. The PCR system was the same as that described in the section Construction of the Eukaryotic Expression Plasmid, and the amplification was determined by using the following program: preheating (5 min at 95°C), amplification for 40 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C, and the final extension (5 min at 72°C). The positive genomic DNA was obtained from the tachyzoites described in the section Animals, Cell Lines, and Parasites using the same method described above. In each amplification, both positive and negative controls (no-DNA control) were included. Then, 4 μl PCR amplicons were visualized in a 1.0% agarose gel containing 0.01% ethidium bromide.

To evaluate the parasitic load in tissues, absolute quantitative real-time PCR (qPCR) was conducted to illustrate the 529 bp repeat element in the extracts referenced to a published study (46)‘ absolute quantitative real-time PCR (qPCR) was conducted to illustrate the 529 bp repeat element in the extracts. The recombinant vector containing the amplified region was also constructed, and the copy numbers of cloned vectors were calculated by using an online tool (http://cels.uri.edu/gsc/cndna.html). For qPCR amplification, 10 μl of 2 × ChamQ SYBR qPCR MasterMix (Vazyme, Nanjing, China), 0.4 pmol of each primer, 0.4 μl 50×ROX Reference Dye 2, 1 μl of DNA extracts, and ddH₂O to make a final volume of 20 µl were involved in each reaction. Each reaction was amplified by Applied Biosystems 7500 (Life Technologies, Carlsbad, USA) using the following program: pre-denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. A melting curve analysis was performed at the end of the reaction between 60°C and 95°C with an increment of 0.05°C/s. The melting curve of each amplification was ensured with one uniform peak at the expected temperature before further analysis. Before amplification, the OD260/OD280 value of each sample was analyzed by a nanodrop microvolume spectrophotometer. Each tissue had three replications, and each replication was performed once. Moreover, the recombinant vectors with a known copy number were also conducted in triplicate.

BALB/c mice were randomly divided into five groups (28 mice/group), and all mice were immunized intramuscularly in the leg muscles with multipoint at 2-week intervals. For single immunization, the immunized dosage of each mouse did not exceed 0.2 ml. Mice were immunized with an equal volume of PBS that was used as the blank control (Table 1). To investigate the adverse reactions in animals, the living conditions of animals were observed weekly from the first immunization to challenge.

Two weeks after the last immunization, the immune protections were evaluated by a method described previously (47). In short, five mice in every group were challenged with a lethal dose (10³ tachyzoites) of T. gondii RH strain intraperitoneally. The infected mice in each group were sacrificed under supervision 7 days after the challenge, and the specific tissue was collected and stored at -20°C until use.

Serum samples were collected from the eye socket at weeks 0, 2, and 4 before immunization and challenge. All serum samples were kept in -20°C before use. To obtain the soluble tachyzoite antigens (STAg), a previously described procedure was conducted (48).

According to a previous study (49), enzyme linked immunosorbent (ELISA) assays were performed to determine the T. gondii-specific serum antibody levels. Briefly, the 96-well plates (Costar, Cambridge, MA, USA) were coated with 1 μg STAg (diluted to 10 μg/ml with carbonate buffer pH 9.6) for each well overnight at 4°C. After being washed three times with TBST (TBS containing 0.05% Tween 20), each well was blocked with TBS containing 5% skimmed milk at 37°C for 1 h. Then mouse sera were diluted (1:100 in TBS) and added to each well for 1 h at 37°C after three times washing in TBST. After being subsequently rinsed 5 min in TBST, each well was incubated with HRP-conjugated anti-mouse IgG, IgG1, or IgG2a (1:5,000, eBioscience, San Diego, USA) at 37°C for 1 h to detect bound antibodies. Finally, 3,3’,5,5’-tetramethylbenzidine (TMB, Tiangen, Beijing, China) was used to evaluate the enzymatic activity; the reaction was stopped by adding 100 μl, 2 M newly prepared H₂SO₄. The absorbance was measured at 450 nm with a microplate photometer (Thermo Scientific, Waltham, USA). Each group had five replications, and each replication was measured once.

To assay the cytokine production levels in sera, commercially available ELISA kits (Senbeijia, Nanjing, China) were used strictly following the manufacturer’s instructions. Referenced to standard curves based on known amounts of mouse recombinant cytokines, the concentrations of interferon-gamma (IFN-γ) and interleukin (IL) 4 (IL-4), IL-10, and IL-17 were measured. The analysis was performed based on five replications in each group, and each replication was measured once.

One week after the booster immunization, three mice of each group (without challenge) were sacrificed under supervision to obtain the splenic lymphocytes by using a lymphocyte separation kit (Solarbio, Beijing, China). The splenic lymphocytes were adjusted to 10⁵ cells/well in the medium containing recombinant protein obtained in the section Recombinant Plasmids Transfection in Vitro and cultured in a 96-well plate. The plate was incubated for 72 h at 37°C. Then 10 μl of Cell Counting Kit 8 (CCK-8, Beyotime, Shanghai, China) was added according to the instructions. After 2-h incubation, the lymphocyte proliferation absorbance was measured at 450 nm with a microplate photometer. Each group had three replications, and each replication was measured once.

At weeks 0, 2, and 4, five mice from each group (before immunization and challenge) were euthanized to obtain the splenic as described in the section Determination of Lymphocyte Proliferation. The splenic lymphocytes were divided into several parts. For the maturation detection of dendritic cells (DCs), the splenic lymphocytes were cultured in DMEM containing 10% FBS and 1% double antibiotics for 12 h, and the non-adhering cells were discarded. After the plate was washed three times using PBS, the attached cells were collected by pipetting repeatedly and gently. Cells were then stained with anti-mouse CD11c-APC, CD86-FITC, and CD83-PE (eBioscience, San Diego, CA, USA). To investigate the MHC molecule changes in DCs, the splenic lymphocytes were stained with anti-mouse CD11c-PE, MHC-I-FITC, and MHC-II-APC (eBioscience, San Diego, CA, USA). To analyze the percentages of CD4⁺ T lymphocyte subsets, the splenic lymphocytes were stained with anti-mouse CD3e-PE and CD4-FITC (eBioscience, San Diego, CA, USA). To detect the percentages of CD8⁺ T lymphocyte subsets, the splenic lymphocytes were stained with anti-mouse CD3e-PE and CD8-FITC (eBioscience, San Diego, CA, USA). All stained procedures were conducted at 4°C for 40 min. After centrifugation, collection, and washing, cells were sorted by a flow cytometry (Beckman Coulter Inc, Brea, CA, USA). Each group had five replications, and each replication was measured once.

To determine the parasitic loads in mice, 30 mg of specific tissue obtained in the section Immunization and Challenging Schedules in Mice was used for genomic DNA extraction and qPCR as described in the section Quantification of T. gondii in Tissue. Each group had five replications, and each replication was performed in triplicate; the recombinant vectors with a known copy number were also conducted in triplicate.

Significance analysis was conducted by the SPSS 25.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad 6.0 software (GraphPad Prism, San Diego, CA, USA). Based on one-way analysis of variance (ANOVA), differences between groups were considered as significant at p < 0.05. Based on the homogeneity of variance, values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared with the independent t-test. The flow cytometric analysis was illustrated by the CytExpert software (version 2.3, Beckman Coulter Inc, Brea, CA, USA).

The recombinant plasmid TgSDRO-pVAX1 was successfully constructed as mentioned above. To verify the recombinant plasmid, an enzyme digestion was performed with EcoRI and XhoI, yielding two fragments of 1,119 and 2,966 bp (Figure 1A). The sequence analysis also proved that the insert in the recombinant plasmid was the ORF of TgSDRO. Together, all these results indicate that the recombinant plasmid was constructed correctly.

The endotoxin free DNA plasmids were purified by a commercial kit, and the endotoxin level of purified TgSDRO-pVAX1 plasmids fell to 0.1 EU/ml. Then, immunofluorescence assay was conducted to evaluate the in vitro expression of TgSDRO-pVAX1 at 72 h after transfection of HEK 293-T cells. Cells transfected with TgSDRO-pVAX1 showed specific red fluorescence, whereas cells transfected with pVAX1 did not display red fluorescence (Figure 1B). The western blotting results revealed a single band (approximately 41 kDa) of the expected molecular size (40.37 kDa), and no protein band was observed in cells transfected with the empty pVAX1 vector (Figure 1C). All these findings indicated that recombinant TgSDRO protein was expressed in HEK 293-T cells.

The SEM images showed that both TgSDRO-pVAX1/PLGA and TgSDRO-pVAX1/CS nanospheres were spherical in shape with round convex particles in the surface (Figure 2). The mean diameter of TgSDRO-pVAX1/PLGA nanospheres formed using 6% PVA was about 100.96 ± 21.58 nm (n = 5), while the mean diameter of TgSDRO-pVAX1/CS nanospheres formed using 2 mg/ml TPP was about 115.77 ± 24.87 nm (n = 5). Based on five independent trails, the EE and LC of TgSDRO-pVAX1/PLGA nanosphere formulation reached 67.39% and 4.38% with the concentration of plasmids at 1 mg/ml, while those were 82.42% and 1.23% in TgSDRO-pVAX1/CS nanosphere formulations. Preserved at least 3 months (Figures 2C, D), TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres were similar with those prepared freshly in morphologic characteristics. This finding suggested that both freeze-dried TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres could keep the integrality over a long period of time at 4°C.

By using a nanodrop microvolume spectrophotometer, the release characteristics of TgSDRO-pVAX1/PLGA and TgSDRO-pVAX1/CS nanospheres were further evaluated. TgSDRO-pVAX1/CS nanospheres showed a more favorable release profile compared to TgSDRO-pVAX1/PLGA nanospheres (Figure 3). Only 15.75 ± 4.19% of the antigens were released from TgSDRO-pVAX1/CS nanospheres immediately and slowly released over the following days, finally reaching 82.58 ± 6.79%. A quickly releasing phase of TgSDRO-pVAX1/PLGA nanospheres could be observed approximately during the first week with the initial CR at 7.09 ± 2.98%; then the release profile became flat and finally reached 90.50 ± 6.28% in the following days.

To determine T. gondii infection in different tissues, PCR amplification was conducted based on the ITS-1 sequence. As described in Figure 4A, T. gondii was observed in the intestine, heart, liver, spleen, and lung, while the leg muscle, brain, and kidney tissue were not detected. To determine the amount of T. gondii in different tissues, absolute qPCR was conducted based on the 529 bp repeat element. Before qPCR amplification, the OD260/OD280 value of each sample was quantified, and all samples were in the range of 1.6–1.8. As illustrated in Figure 4B, the concentration of T. gondii reached a maximum in the intestine tissue (35.055 ×10⁶ copies/μl), followed by heart (25.179 ×10⁶ copies/μl), lung (14.085 ×10⁶ copies/μl), liver (4.529 ×10⁶ copies/μl), and spleen tissue (3.706 ×10⁶ copies/μl).

The titers of total IgG, IgG1, and IgG2a induced in mice were measured by standard ELISA procedure. Demonstrated in Figure 5A, enhanced secretions of total IgG antibodies were obtained significantly in TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA after the first (week 2) and booster (week 4) immunization. Mice immunized with TgSDRO-pVAX1/CS could generate the highest titer of total IgG after the second immunization (week 4). Compared with the blank and control group, mice immunized with TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA could generate higher titers of isotype IgG1 (Figure 5B) and IgG2a (Figure 5C) after the first and second immunizations. Furthermore, no significant difference (p > 0.05) was evaluated in the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups.

Strictly following the instruction, the concentrations of IFN-γ, IL-4, IL-10, and IL-17 in the sera were determined by commercial ELISA kits based on the double antibody sandwich method. As demonstrated in Figures 6A, B, remarkable increases could be observed in the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups after the first immunization (week 2), while the TgSDRO-pVAX1 group remained unchanged (p > 0.05). After the second immunization (week 4), all mice from the treatment group could generate a significant (p < 0.001) level of IFN-γ, and mice immunized with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA could generate a significant (p < 0.01) level of IL-4. As for IL-17 showed in Figure 6D, significant secretions can be obtained in mice immunized with TgSDRO-pVAX1 and TgSDRO-pVAX1/CS after the first immunization, and after the second immunization, secretions of IL-17 were significantly enhanced in all treatment groups. Furthermore, equivalent IL-10 (p > 0.05, Figure 6C) secretions were observed in all immunized animals.

The expression of CD83 and CD86 on DCs was examined by flow cytometry. After the first and second immunizations (Figures 7A, C), treatment with TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA showed significant improvements in the percentage of CD11c⁺CD83⁺ cells when compared with the blank or control group. Furthermore, statistical improvements in the percentage of CD11c⁺CD86⁺ cells could be detected in the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups (Figures 7B, C) after the first and second immunizations. Noticeably, no statistical difference (p > 0.05) was revealed between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups in DC maturation analysis.

In order to investigate the percentage of MHC molecules on splenic DCs, five mice from each group were sacrificed, and the spleen lymphocytes were analyzed. Described in Figures 8A, C, the ratio of MHC-I molecules in the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups was enhanced after the first and second immunizations, while the TgSDRO-pVAX1 group remained unchanged (p > 0.05). For the ratio of MHC-II molecules in Figures 8B, C, significant enhancement was detected after the first and second immunizations. Furthermore, the statistics also revealed that mice immunized with TgSDRO-pVAX1/PLGA could generate significantly higher percentage (p < 0.001) of MHC-II molecules after a booster immunization compared to those immunized with TgSDRO-pVAX1/CS.

Spleen lymphocytes were prepared from different groups 1 week after the second immunization, and the proliferative responses were analyzed. Evaluated in Figure 9, mice immunized with TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA produced a significant (p < 0.001) proliferation when compared with the blank and control group. Moreover, the TgSDRO-pVAX1/PLGA group could generate a higher (p < 0.05) proliferation than the TgSDRO-pVAX1/CS group.

Five mice from each group were sacrificed and the splenocytes were stained, then the percentages of CD4⁺ T and CD8⁺ T lymphocytes of each group were conducted. Illustrated in Figure 10A, the percentage of CD4⁺ T cells in the TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA groups was significantly increased at weeks 2 and 4 when compared with the blank and control group. In the case of the percentage of CD8⁺ T cells (Figure 10B), the TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA groups generated a significant increase when compared with the blank and control group at weeks 2 and 4. For the percentages of CD4⁺ T and CD8⁺ T lymphocytes, mice in the TgSDRO-pVAX1/CS group could generate statistically similar (p > 0.05) to those in the TgSDRO-pVAX1/PLGA group after the first and second immunizations.

The animals’ health condition was investigated through the clinical observation, and the significant toxicity of nanospheres was not evaluated. All immunized animals were similar in physical health and stable in mental status over the duration of the trials compared with the animals immunized with PBS.

To obtain a more accurate analysis of parasite burden in mice, the method of absolute qPCR was conducted to detect the 529 bp repeat element of T. gondii in cardiac muscles from the tip of the heart. Before the qPCR amplification, the OD260/OD280 value of each sample was quantified, and all samples were in the range of 1.6–1.8. Presented in Figure 11, the parasite burden was significantly (p < 0.001) inhibited in the TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA groups compared to the blank and control group. Furthermore, significance (p < 0.05) also could be observed in the 529 bp repeat element between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups.