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, PR China, tachyzoites of the highly pathogenic T. gondii RH strain (type I) were propagated via the BALB/c mice according to previous research (37).

Purchased from the Institute of Cell Biology, Chinese Academy Sciences, Shanghai, PR China, Human Embryonic Kidney 293-T (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 (penicillin–streptomycin solution, Gibco, Carlsbad, CA, USA) at 37°C in a 5% CO₂ atmosphere.

Obtained from the Model Animal Research Center of Nanjing University, Nanjing University, PR China, specific pathogen-free (SPF) BALB/c mice (18–22 g) and Sprague–Dawley rats (200–220 g) were strictly kept in an SPF environment. Supervised by the Animal Ethics Committee, Nanjing Agriculture University, China, all the animal-related operations were carried out in strict compliance with the requirements of the Ethics Procedures and Guidelines of the People’s Republic of China.

Total RNA Extraction kit (OMEGA Bio-Tek, Norcross, GA, USA) was used to isolate total RNA from 10⁶ tachyzoites of T. gondii according to the manufacturer’s protocol. By using a reverse transcription kit (Takara Biotechnology, Dalian, China), reverse transcription PCR (RT-PCR) was immediately employed to synthesize the cDNA. To amplify the complete open reading frame (ORF) of TgP2, primers were designed based on the conserved domain sequences (CDS) of TgP2 (GenBank: XM_002364187.2). Along with the Kozak translation initiation sequence and the restriction endonuclease sites (EcoRI and XhoI), primers, 5ʹ-CCG GAATTC GCCACC ATGGCAATGAAATACTTCGCTG-3ʹ (forward) and 5ʹ-CCG CTCGAG TTAGTCGAAGAGCGAGAAGCCC-3ʹ (reverse), were synthesized by Tsingke Biological Technology (Nanjing, China). Based on the instruction (Vazyme Biotech, Nanjing, China), PCR was performed in a volume of 50 μl, and the PCR amplification was conducted as follows: 1 cycle of 95°C for 5 min and then 35 cycles of 95°C for 30 s, 62.7°C for 30 s, and 72°C for 30 s. The final primer extension time was extended to 5 min at 72°C. The amplicons were detected using electrophoresis on a 1.0% agarose gel containing 0.01% ethidium bromide. The expected DNA bands were purified by Gel Extraction Kit (OMEGA Bio-Tek, Norcross, GA, USA), digested by EcoRI and XhoI restriction endonuclease (Takara Biotechnology, Dalian, China), and inserted into a linear pVAX1 vector (Invitrogen Biotechnology, Shanghai, China) by T4 DNA ligase (Takara Biotechnology, Dalian, China). The constructed plasmids were first determined by double restriction enzyme digestion and then sequenced by ABI PRISM™ 3730 XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA). The correct plasmids were transferred into E. coli DH5α (Invitrogen Biotechnology, Shanghai, China). To obtain the plasmids in large quantity, E. coli DH5α carrying the correct plasmids was much duplicated in Luria Bertani (LB) medium containing 100 μg/ml of kanamycin monosulfate until the OD600 reached 0.6 (37°C and shaking at 180 rpm). The Endo-free DNA plasmid extraction kit (Vazyme Biotech, Nanjing, China) and ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GeneScript, Piscataway, NJ, USA) was employed to obtain the endo-free DNA plasmids. Before being stored at −20°C, a nanodrop microvolume spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to determine the concentration of obtained plasmids.

To obtain the soluble tachyzoite antigens (STAg) of T. gondii, the previously described procedure was conducted with minor modifications (38). Briefly, the peritoneal lavage fluids were collected and passed through a 5-μm filtering membrane (Millipore, Billerica, MA, USA). After being centrifuged at 2,500 rpm for 5 min, 5 × 10⁶ tachyzoites were redissolved in 1,000 μl of phosphate-buffered saline (PBS). The protease and phosphatase inhibitor (Beyotime, Shanghai, China) were then added according to the instructions, and then tip sonication (Scientz Biotechnology, Ningbo, China) was carried out in a continuous mode for 2 s at 2 s (10 min in total) under an output power of 30 W. Before being at −20°C, the total amount of protein was conducted by bicinchoninic acid (BCA) assay (Thermo Scientific, Waltham, MA, USA).

To obtain the polyclonal antibody against T. gondii STAg, two Sprague–Dawley rats were first immunized with 500 μg of STAg mixed with complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Two weeks later, quartic immunizations were conducted using 500 μg of STAg mixed with incomplete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA) at 1-week intervals. All immunizations were conducted subcutaneously in the back skin with multiple points. One week after the last injection, blood was harvested at the eye socket, and sera containing polyclonal antibodies were collected. Blank sera were also harvested from rats immunized with PBS using the same immunization strategy. All sera were kept at −20°C until use.

To determine the expression of constructed plasmids in vitro, 1 × 10⁶ HEK 293-T cells were cultured in a six-well plate (Costar, Cambridge, MA, USA) and were transfected with the endo-free plasmids using the Lipofectmine™ 3000 reagent (Invitrogen Biotechnology, Shanghai, China) according to the manufacturer’s guidelines. At 48-h stationary cultivation after transfection, the supernatants were discarded, and the cells were fixed with 1 ml of 4% paraformaldehyde for 12 h at 4°C after being rinsed three times with PBS. Then the cells were permeabilized with tris-buffered saline (TBS) containing 0.1% Triton X-100 (TBSx), blocked with TBSx containing 5% bovine serum albumin (BSA), and incubated with TBSx containing 5% BSA and serum against STAg (1:100 dilution). Rinsed 5 min in TBSx, the samples were incubated with CY3-conjugated anti-rat IgG (1:500 dilution, Sigma, Saint Louis, MO, USA) and subsequently with 4′,6-diamidino-2-phenylindole (DAPI) staining solution (500 μl/well, Beyotime, Shanghai, China). The images were immediately recorded by a Nikon A1 plus laser scanning confocal microscopy (Nikon Corporation, Tokyo, Japan).

To further confirm the expression of constructed plasmids in vitro, the transfected HEK 293-T cells were incubated at 37°C for 48 h. The cells were scraped into 500 μl of radioimmunoprecipitation assay (RIPA) lysis (Beyotime, Shanghai, China) containing protease and phosphatase inhibitor. Added with 10× loading buffer, HEK 293-T cell lysates were incubated at 95°C for 5 min and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Cell lysates were transferred to methanol-activated polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) using a Semi-Dry Transfer system (Bio-Rad, Hercules, CA, USA). Membranes were blocked in TBS containing 0.05% (v/v) Tween 20 and 5% (w/v) non-fat skimmed milk powder on a rotary shaker with 70 rpm at 37°C for 2 h. Rinsed 5 min in TBST (TBS containing 0.05% (v/v) Tween 20), the membranes were incubated with polyclonal antibody against T. gondii STAg in TBST (1:100 dilution) at 4°C overnight with constant shaking. The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated anti-rat IgG (1:8,000 dilution, eBioscience, San Diego, CA, USA) on a rotary shaker with 70 rpm at 37°C for 2 h. Rinsed 5 min in TBST, the blots were visualized by newly prepared 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO, USA).

To synthesize the PLGA nanospheres, the double emulsion solvent evaporation technique (w/o/w) was conducted based on the previous research with minor modifications (39, 40). To obtain the organic solution, 50 mg of PLGA (molecular weight (MW) 40,000–75,000 Da, LA/GA: 65/35, Sigma, Saint Louis, MO, USA) was weighed in a 50-ml centrifuge tube containing a magnetic stirring bar. Then 1 ml of dichloromethane (DCM; Sigma, Saint Louis, MO, USA) was added with the tube plugged and stirred until the complete dissolution of the polymer. Subsequently, polyvinyl alcohol (PVA; MW 31,000–75,000 Da, Sigma, Saint Louis, MO, USA) was dissolved in double-distilled water to prepare the 6% (w/v) PVA solution. Before use, the 6% PVA solution was passed through a 0.22-μm filtering membrane (Millipore, Billerica, MA, USA). At room temperature, 2 ml of 6% PVA solution was stirred using a magnetic stirrer (400 rpm), and 4 mg of endo-free DNA plasmids (concentration was 1 mg/ml) was dropwise dissolved by means of the Pipetman^® P1000L (Gilson, Shanghai, China). The aqueous solution was obtained after a subsequent mix on a vortex mixer for 5 min. The organic solution was dropped into the aqueous solution using a magnetic stirrer (400 rpm). Tip sonication (Scientz Biotechnology, Ningbo, China) was then carried out in a continuous mode for 2 s at 2 s (5 min in total) under an output power of 40 W under 4°C to synthesize the w/o emulsions. The w/o emulsions were dropped into 3 ml of 6% PVA solution using a magnetic stirrer (400 rpm). Tip sonication was again conducted with the same parameters to synthesize the w/o/w emulsions. The resulting w/o/w emulsions were then transferred into a 50-ml centrifuge tube using a magnetic stirrer (400 rpm) overnight to evaporate DCM. The w/o/w emulsions were centrifuged at 40,000 rpm for 15 min at 4°C, and PLGA nanospheres (precipitation) were collected in double-distilled water. In addition, the supernatant was also collected. After being passed through a 0.22-μm filtering membrane, the obtained PLGA nanospheres were then frozen. Once completely congealed, the nanospheres were transferred to a vacuum freeze-drier (Labconco, Kansas City, MO, USA) until completely freeze-dried. The freeze-dried PLGA nanospheres were stored at 4°C under sealed conditions until use.

To synthesize the chitosan nanospheres, the ionic gelation technique was conducted based on previous research with minor modifications (41). To make the chitosan solution, 100 mg of chitosan (MW 50–190 kDa, Sigma, Saint Louis, MO, USA) was dissolved in 50 ml of 1.0% (v/v) aqueous acetic acid solution and left in agitation until complete dissolution. Then the pH value was adjusted to 5.0 by NaOH solution. To prepare a 2 mg/ml sodium tripolyphosphate (TPP; Aladdin, Shanghai, China) solution, 20 mg of TPP was dissolved in 10 ml of double-distilled water and passed through a 0.22-μm filtering membrane (Millipore, Billerica, MA, USA). Chitosan solution measuring 10 ml was transferred into a 50-ml centrifuge tube using a magnetic stirrer (400 rpm) in an incubator at 30°C, and then 2 mg of endo-free DNA plasmids (concentration was 1 mg/ml) was added dropwise. Subsequently, 2 ml of TPP solution was dropped, and a 20-min constant stirring was conducted. Then tip sonication was then performed in a continuous mode for 2 s at 2 s (3 min in total) under an output power of 50 W. Centrifuged at 40,000 rpm for 15 min at 4°C, the chitosan nanospheres (precipitation) were collected in double-distilled water. The supernatant was also collected for further analysis. Passed through a 0.22-μm filtering membrane, the obtained CS nanospheres were then frozen. Once completely congealed, the nanospheres were transferred to the vacuum freeze-drier until completely freeze-dried. The freeze-dried CS nanospheres were stored at 4°C under sealed conditions until use.

To determine the loading capacity (LC) and encapsulation efficiency (EE) of synthesized nanospheres, the concentration of non-bound plasmids in the supernatant obtained in Section 2.4 was determined by the nanodrop microvolume spectrophotometer. LC and EE in PLGA and chitosan nanospheres were evaluated by applying formula (1) and formula (2), respectively.

To investigate the morphological shape, the formulated nanospheres were sent to the College of Life Science, Nanjing Agriculture University, PR China, for scanning electron microscopy (SEM) observation (Hitachi SU8010, Hitachi Ltd., Tokyo, Japan). ImageJ software (version 1.8, NIH Image, Bethesda, MD, USA) was employed to characterize the average diameter of PLGA and chitosan nanospheres.

The release characteristics from PLGA and chitosan nanospheres in vitro were investigated in PBS (pH 7.4) as previously described (42). In brief, synthesized PLGA and chitosan nanospheres were dispersed in PBS (pH 7.4) at 37°C under mild shaking (180 rpm). At a 12-h interval, samples of the supernatant were collected by centrifugation and stored at −20°C. The settled nanospheres were resuspended in the original solution after each collection. After the last collection, the plasmid concentration in the samples was measured by a nanodrop microvolume spectrophotometer, and the cumulative release (CR) was calculated by applying formula (3).

To evaluate the toxicity of synthesized nanospheres, 35 BALB/c mice were randomly divided into seven groups (n = 5 mice): Blank (immunized with an equal volume of PBS), Control (immunized with pVAX1 plasmids), pVAX1/CS (immunized with pVAX1/CS nanospheres), pVAX1/PLGA (immunized with pVAX1/PLGA nanospheres), TgP2-pVAX1 (immunized with TgP2-pVAX1 plasmids), TgP2-pVAX1/CS (immunized with TgP2-pVAX1/CS nanospheres), and TgP2-pVAX1/PLGA (immunized with TgP2-pVAX1/PLGA nanospheres) groups. Each animal was injected intramuscularly with a dose containing 300 μg of plasmids, which was tripled in the immune dosage compared with the regular dosage. Three days later, a booster vaccination was conducted with the same dosage and strategy. Sera were harvested from the eye socket 1 day after treatment, and the levels of creatinine (Cr) in serum and the blood urea nitrogen (BUN) were evaluated by the sarcosine oxidase method (Solarbio, Beijing, China) and urease-indophenol method (Solarbio, Beijing, China). During the test period, their physical health and mental status were observed and recorded.

To investigate the protective immunity of two synthesized nanospheres, BALB/c mice weighing 18–22 g (7 weeks old) were randomized into seven groups (n = 28 mice), all animals were intramuscularly immunized twice in the leg muscles with multipoint at 2-week intervals ( Table 1 ). Blood samples were harvested through the eye socket before immunization, and the sera were separated and stored at −20°C until use. Blank or vaccinated animals were challenged with a lethal dose (10³ tachyzoites) of T. gondii RH strain intraperitoneally as indicated previously (43). Seven days after the challenge, infected animals were anesthetized and then sacrificed under the supervision of the Animal Ethics Committee, Nanjing Agriculture University, China, and the cardiac tissue was isolated and stored at −20°C until use.

To investigate the titers of T. gondii-specific serum antibody in the sera, ELISAs were conducted as described previously (44). Briefly, each well (96-well plates, Costar, Cambridge, MA, USA) was coated with 1 μg of STAg (dissolved in 100 μl of carbonate buffer pH 9.6) overnight at 4°C. After 5-min rinsing in TBST, each well was blocked with TBST containing 5% BSA (Sangon Biotech, Shanghai, China) at 37°C for 1 h. Sera from mice were diluted (1:100) in TBST containing 5% BSA and added into each well after being rinsed in TBST for 5 min. Incubated at 37°C for 1 h, each well of the 96-well plates was rinsed 5 min in TBST and incubated with HRP-conjugated anti-mouse IgG, IgG1, or IgG2a (1:8,000, eBioscience, San Diego, USA) at 37°C for 1 h. Finally, tetramethylbenzidine (TMB; Tiangen, Beijing, China) as substrate was used to evaluate the immunoreaction, and the reaction in each well was stopped by 100 μl of 2 M newly prepared H₂SO₄. The absorbance was determined at 450 nm by a microplate reader (Thermo Scientific, Waltham, MA, USA).

To determine cytokine secretions in animals’ sera, commercially available ELISA kits (Yifeixue Biotech, Nanjing, China) based on double antibody sandwich method were used to evaluate the concentrations of interferon-gamma (IFN-γ), interleukin (IL) 4 (IL-4), IL-10, and IL-17 referenced to known amounts of mouse recombinant cytokines.

At weeks 2 and 4 (before immunization or challenge), five mice from each group were euthanized, and the splenic lymphocytes were harvested by the lymphocyte separation kit (Solarbio, Beijing, China). To investigate the surface markers of dendritic cells (DCs), the separated lymphocytes were cultured in DMEM containing 10% FBS and 1% double antibiotics overnight. The non-adhering cells were discarded, and the attached cells were collected gently after being washed three times in PBS. Cells were then adjusted to 10⁶ cells in 100 μl of PBS and stained with anti-mouse CD11c-APC, CD86-FITC, and CD83-PE (eBioscience, San Diego, CA, USA) for 40 min at 4°C in the dark. After washing, centrifugation, and collection, cells were sorted by flow cytometry (Beckman Coulter Inc., Brea, CA, USA). Before cell sorting, the fluorescence compensation trial was performed to determine adequate compensation according to the instructions.

For the MHC molecules changes in DCs, the separated lymphocytes were directly adjusted to 10⁶ cells in 100 μl of PBS and stained with anti-mouse CD11c-PE, MHC-I-FITC, and MHC-II-APC (eBioscience, San Diego, CA, USA) under the same conditions mentioned above. Then the flow cytometry analysis was conducted using the same strategies described previously.

To analyze the proportion of CD4⁺ and CD8⁺ T lymphocytes subsets, 2 × 10⁶ splenic lymphocytes were equally dissolved in two tubes containing 100 μl of PBS. One tube was stained with anti-mouse CD3e-PE and CD4-FITC (eBioscience, San Diego, CA, USA) for CD4⁺ T lymphocyte subset detection, while another tube was stained with anti-mouse CD3e-PE and CD8-FITC (eBioscience, San Diego, CA, USA) for CD8⁺ T lymphocyte subset detection. The staining strategy and flow cytometry analysis were conducted using the same strategies described previously.

Three days after the booster immunization, three mice from each group were euthanized to separate the splenic lymphocytes using the same method described in Section 2.8. The splenic lymphocytes were adjusted to 10⁵ cells/well in a 96-well plate. The transfected cells described in Section 2.3 were scraped into 1 ml of sterile PBS, and then tip sonication was conducted to obtain the cell lysates in a continuous mode for 2 s at 2 s (10 min in total) under an output power of 30 W. After protein determination by BCA assay, 50 μg/well cell lysates was added into each well in a 96-well plate. The splenic lymphocytes with cell lysates were incubated at 37°C in a 5% CO₂ atmosphere for 48 h. Cell Counting Kit 8 (CCK-8; Beyotime, Shanghai, China) measuring 10 μl was added according to the instructions. Incubated for 2 h, the plate was subsequently measured at 450 nm by a microplate reader to illustrate the lymphocyte proliferation.

To demonstrate the parasite burden in mice, 30.0 mg of cardiac tissue obtained in Section 2.6 was lysed for genomic DNA extraction following the guidelines (OMEGA Bio-Tek, Norcross, Georgia, USA), and the DNA extracts were stored at −20°C until use. Absolute quantitative real-time PCR (qPCR) was carried out to investigate the 529-bp fragments in the extracts according to the published research (45). The plasmids containing the amplified 529-bp fragments were also constructed, and an online tool (http://cels.uri.edu/gsc/cndna.html) was employed to calculate the copy numbers of the plasmids. For qPCR amplification, 6.0 μl of 2× ChamQ Universal SYBR qPCR MasterMix (Vazyme, Nanjing, China), 0.24 μl of each primer, 1 μl of DNA extracts, and double-distilled water to make a final volume of 12.0 µl were involved in each reaction. Amplification for each reaction was performed on an Applied Biosystems 7500 (Life Technologies, Carlsbad, CA, USA) by 30-s incubation at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. At the end of the reaction between 60°C and 95°C with an increment of 0.05°C/s, a melting curve analysis was performed. Before further analysis, the melting curve of each amplification was identified with one uniform peak as expected.

Conducted by GraphPad 6.0 software (GraphPad Prism, San Diego, CA, USA), a one-way ANOVA was employed to illustrate differences between groups. Pairwise comparisons among TgP2-pVAX1, TgP2-pVAX1/PLGA, and TgP2-pVAX1/CS group were estimated by ANOVA following Bonferroni’s correction. The results were considered as significant at p < 0.05 and represented as mean ± SD for each group. CytExpert software (version 2.3, Beckman Coulter Inc., Brea, CA, USA) was recruited to conduct the flow cytometric analysis.

The TgP2-pVAX1 plasmid was successfully constructed as described previously. To verify the constructed plasmid, the double enzyme digestion was performed with EcoRI and XhoI, yielding two fragments of 354 and 2,966 bp, respectively ( Figure 1A ). The sequence results also indicated that the insert in the constructed plasmid was the ORF of TgP2. In summary, all these results proved that the TgP2-pVAX1 plasmid was correctly constructed.

The HEK 293-T eukaryotic cells were transfected with an endo-free TgP2-pVAX1 plasmid. As illustrated in Figure 1B , cells transfected with TgP2-pVAX1 plasmid showed specific red fluorescence, whereas cells transfected with pVAX1 plasmid did not display red fluorescence, indicating the expression of the TgP2 protein. The Western blotting results revealed a single band (approximately 24 kDa, Figure 1C ), indicating an MW that is twice as expected (11.77 kDa). The pH value of resolving gel used in the current research was 8.8, and TgP2 has been proved to oligomerize at higher pH values (46). According to the protein sequence of TgP2, it lacks cysteines altogether. Thus, it is expected that the recombinant TgP2 protein should not exist in the disulfide bond, indicating the dimerization of TgP2 in resolving gel. Similar results have been reported in the previous study (18). In addition, no band was detected in pVAX1 transfected cells, and all these findings indicated that the recombinant TgP2 protein could be expressed by TgP2-pVAX1 in HEK 293-T cells.

After encapsulation of TgP2-pVAX1 plasmid in PLGA and chitosan nanospheres, the morphology of nanospheres was examined under SEM. As observed by SEM ( Figure 2 ), the morphology of PLGA and chitosan nanospheres was both spherical in shape, with small round convex particles detected on its surface. According to five arbitrary nanospheres in the SEM images, the average diameter of TgP2-pVAX1/PLGA nanospheres synthesized by using 6% PVA was determined to be 112.31 ± 15.60 nm, while the mean diameter of TgP2-pVAX1/CS nanospheres formulated by using 2 mg/ml of TPP was 100.93 ± 12.36 nm. Based on five independent trials, the LC of PLGA and chitosan nanospheres was 1.91% and 4.16%, respectively, while the EE was 59.18% and 85.74%, respectively.

Referenced to the blank PLGA or chitosan nanospheres under the same circumstance, the release characteristics of TgP2-pVAX1/PLGA and TgP2-pVAX1/CS nanospheres were investigated. As demonstrated in Figure 3 , the burst release of two types of nanospheres was both observed in the first 2 days, and the TgP2-pVAX1/CS nanospheres showed a steadier release profile as compared to the TgP2-pVAX1/PLGA nanospheres; 10.40% ± 5.67% of TgP2-pVAX1 were released from TgP2-pVAX1/PLGA nanospheres on the first day, while only 5.71% ± 5.17% plasmids were detected in the supernatant of TgP2-pVAX1/CS nanospheres on the first day. After the fourth day, the release profile of TgP2-pVAX1/PLGA nanospheres became flat, while the release curve became steep 6 days later (after the tenth day). However, the release curve of TgP2-pVAX1/CS nanospheres was slow-released at the first 7 days and became less steep after about 8 days. In addition, the CR of TgP2-pVAX1/PLGA nanospheres has crept up to over 100% on the twelfth day; such phenomenon may be caused by the existence of calculation error.

Based on the sarcosine oxidase and urease-indophenol method, the levels of Cr ( Figure 4A ) and BUN ( Figure 4B ) were analyzed to assess the toxicity of nanospheres. The levels of Cr and BUN remained at an acceptable range, and all groups were statistically similar (p > 0.05) to the blank or control group. In addition, no adverse reaction was observed in all animals, and no abnormal changes in the physical health and mental status. All the results indicated that the synthesized nanospheres were nontoxic.

To determine whether the nanomaterial can promote the efficacy of TgP2-pVAX1 plasmids after multiple immunizations, mice were vaccinated with either TgP2-pVAX1 plasmids alone or nanospheres loaded with TgP2-pVAX1 plasmids one or two times. As expected, vaccinations of PLGA and chitosan nanospheres could induce higher levels of T. gondii-specific IgG ( Figure 5A , p < 0.001) as compared with naked TgP2-pVAX1 plasmids at one and two immunizations. After the second immunization, animals immunized with TgP2-pVAX1/CS nanospheres could generate higher levels of IgG (p < 0.01) when compared to the animals immunized with TgP2-pVAX1/PLGA nanospheres. Furthermore, immunizations with the DNA vaccines entrapped in nanospheres as well as DNA vaccines alone could elicit higher levels of T. gondii-specific IgG1 ( Figure 5B ) and IgG2a ( Figure 5C ) antibody responses as compared with the blank or control group. When compared with naked TgP2-pVAX1 plasmids, immunizations with the TgP2-pVAX1/CS nanospheres could bolster statistically higher levels of IgG1 and IgG2a, regardless of immunization times. However, the TgP2-pVAX1/PLGA nanospheres could elicit significantly higher levels of IgG1 (p < 0.05) after the second immunizations and remarkable higher levels of IgG2a (p < 0.05) after the first immunization, when compared to the naked TgP2-pVAX1 plasmids.

Following TgP2-pVAX1 plasmid immunization with or without nanospheres, sera of animals were harvested after the first and second immunizations to determine cytokine production. Immunizations with TgP2-pVAX1 plasmids or its nanomaterial encapsulations could enhance the production of cytokines IFN-γ ( Figure 6A ), IL-4 ( Figure 6B ), IL-10 ( Figure 6C ), and IL-17 ( Figure 6D ) when compared with blank or control group, regardless of the number of immunizations. In TgP2-pVAX1/CS group, animals receiving two immunizations could generate higher levels of IFN-γ, IL-4, IL-10, and IL-17 than those in the TgP2-pVAX1 group, while animals in TgP2-pVAX1/PLGA group receiving two immunizations could generate equal levels of four tested cytokines when compared with animals in TgP2-pVAX1 group (p > 0.05). Notably, animals in TgP2-pVAX1/CS group showed significantly higher levels of IFN-γ and IL-4 than those in the TgP2-pVAX1/PLGA group after the booster immunizations. All the obtained results suggested that TgP2-pVAX1 plasmids, TgP2-pVAX1/CS nanospheres, and TgP2-pVAX1/PLGA nanospheres could mediate the production of cytokines, and the concentrations in the sera were dependent on different nanospheres and the number of immunizations. In addition, the effects of PLGA or chitosan nanospheres in promoting the secretions of cytokines were also evaluated.

Splenic lymphocytes were collected from animals immunized once or twice with either naked TgP2-pVAX1 plasmids or nanospheres loaded with plasmids to determine the effect of nanosphere on DC maturation. As illustrated in Figures 7A, C , animals immunized with TgP2-pVAX1 plasmids, TgP2-pVAX1/CS nanospheres, or TgP2-pVAX1/PLGA nanospheres were detected with high expressions of CD83 on the surfaces of DCs (p < 0.001). Elicited by DNA vaccines and their nanospheres, high CD86 frequencies (p < 0.001) were also observed after the first or second immunization when compared with the blank or control group ( Figures 7B, C ). When compared with TgP2-pVAX1 plasmids, TgP2-pVAX1/PLGA nanospheres exhibited better effects in inducing CD83 and CD86 molecules according to the data analysis. Furthermore, statistical differences were observed between TgP2-pVAX1/CS and TgP2-pVAX1/PLGA group in promoting CD83 and CD86 molecules, with greater capacity of TgP2-pVAX1/PLGA nanospheres in inducing two tested molecules. All the obtained results indicated that the synthesized nanospheres as well as the TgP2-pVAX1 plasmids played a crucial role in inducing the CD83 and CD86 molecules of DCs, and the ability in enhancing surface molecule expression was dependent on the type of nanospheres and number of immunizations.

PLGA and chitosan nanospheres are known to be the delivery systems for antigens. To further evaluate the effects of TgP2-pVAX1/CS and TgP2-pVAX1/PLGA nanospheres on antigen presentation, murine lymphocytes were collected and analyzed by flow cytometry. Compared with the blank group ( Figures 8A, C ), TgP2-pVAX1/PLGA nanosphere vaccination induced higher levels of MHC-I molecules (p < 0.05) after booster immunization. However, the expressions of MHC-II molecules were significantly (p < 0.001) promoted by naked TgP2-pVAX1 plasmids or two types of nanospheres after the primary and booster immunizations ( Figures 8B, C ). In addition, TgP2-pVAX1/PLGA nanospheres exhibited higher capacity (p < 0.001) in eliciting MHC-II molecules after the booster immunizations, when compared with TgP2-pVAX1 plasmids. These obtained results indicated that the antigen presentation effects of DCs could be activated by TgP2-pVAX1 plasmids and their nanospheres, and an obvious promotion could be presented by the PLGA nanomaterial.

The activated DCs are known to be highly effective stimulators that activate T lymphocytes. To determine whether the splenic T lymphocytes were effectively activated, splenic lymphocytes were separated 3 days after the booster immunization, and the lymphocyte proliferation was determined. As evaluated in Figure 9 , animals immunized with TgP2-pVAX1 plasmids, TgP2-pVAX1/CS nanospheres, or TgP2-pVAX1/PLGA nanospheres could generate a significant proliferation when compared with the blank or control group. Compared with the naked TgP2-pVAX1 plasmids, TgP2-pVAX1/CS nanospheres could remarkably promote the proliferation of splenic lymphocytes (p < 0.001). Moreover, the significance could be also detected between animals immunized with TgP2-pVAX1/CS and TgP2-pVAX1/PLGA nanospheres (p < 0.01), indicating that the chitosan nanospheres were superior in inducing the proliferation of T lymphocytes.

To demonstrate the PLGA and chitosan nanosphere effects on response inductions of T lymphocytes, splenic lymphocytes were harvested from animals immunized once or twice. Vaccination with TgP2-pVAX1 plasmids, TgP2-pVAX1/CS nanospheres, and TgP2-pVAX1/PLGA nanospheres induced higher levels of CD4+ ( Figure 10A ) and CD8+ T lymphocytes ( Figure 10B ) in animals’ spleen. Induced CD4⁺ T and CD8⁺ T lymphocytes were proportional to the number of immunizations, with animals receiving the booster immunizations showing better responses than animals immunized once. In addition, TgP2-pVAX1/PLGA nanospheres were better in CD4⁺ T lymphocyte enhancement when compared with the TgP2-pVAX1/CS nanospheres after the first and second immunization, and TgP2-pVAX1 plasmids, TgP2-pVAX1/CS nanospheres, and TgP2-pVAX1/PLGA nanospheres were equal in activating CD8⁺ T lymphocyte (p > 0.05).

To determine the effects of DNA vaccines and their nanospheres on improving protective efficacy, immunized animals were challenged with a lethal dose of T. gondii RH strain 2 weeks after the booster immunization. Parasite burden in heart tissue of challenged animals was investigated 7 days after challenge infection. As presented in Figure 11 , animals immunized with the naked TgP2-pVAX1 plasmids and two types of nanospheres gained an advantage in resisting T. gondii over the unimmunized control (p < 0.001). In addition, compared with the naked TgP2-pVAX1 plasmids, both TgP2-pVAX1/CS (p < 0.001) and TgP2-pVAX1/PLGA nanospheres (p < 0.01) exhibited strong anti-T. gondii effect, and nearly two-fold differences of parasite burden were observed. All these obtained results suggested the importance of nanomaterials in inducing immune protection.