T. gondii RH strain (type I) was gifted by Prof Li Xiangrui from Nanjing Agricultural University and stored at the laboratory. According to previous studies, tachyzoites of the highly pathogenic T. gondii RH strain (type I) were intraperitoneally injected into mice for propagation to obtain a purified strain of T. gondii. HFF cells were preserved in the research group and maintained 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, New Cell & Molecular Biotech, Suzhou, China) at 37°C in a 5% atmosphere, were used for T. gondii generation.

Specific pathogen-free (SPF) ICR mice (18-22 g, female, approximately 7-8 weeks) and Wistar rats (200-220g, female, approximately 6-8 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd and kept in a strict SPF environment. ICR mice are internationally recognized as closed-group mice, and widely used in safety evaluation, pharmacology, toxicology, and immunology research. This study was approved by the Ningxia University Technology Ethics Committee (No: NXU-2023-085, No: NXU-2023-086). Under the supervision of the Ningxia University Technology Ethics Committee, all relevant animal operations were performed in strict compliance with the Chinese legislation regarding the use and care of research animals (GB/T35823-2018).

The FreeZol Reagent (Vazyme Biotech Co., Ltd, Nanjing, China) was used to extract total RNA from 10⁷ T. gondii tachyzoites based on the instructions provided. cDNA preparation was completed according to the reverse transcription kit protocol (ABclonal, Wuhan, China). To amplify the complete open reading frame (ORF) of TgRPS2, a pair of specific primers were designed based on the conserved domain sequences (CDS) of TgRPS2 (GenBank: TGRH88_017180): forward primer, 5’-CCGGAATTCATGGCAGAACGCGGCAGC-3’; reverse primer, 5’-CCCAAGCTTCTAGGCGAGCGGTCGCGAC-3’ (The underlined sequences respectively represent the restriction endonuclease of EcoRI and HindIII), were synthesized by Sangon Biotech (Xian, China). Following the instructions of 2 × Phanta Max Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), CDS fragments of TgRPS2 were obtained by PCR amplification. Subsequently, the appropriate bands were identified by 1.5% agarose gel electrophoresis containing 0.01% ethidium bromide, and the correct bands were purified by Gel Extraction Kit (Vazyme Biotech Co., Ltd, Nanjing, China). Then, purified PCR products and the pET32a vector (Takara Biotechnology, Dalian, China) were digested by restriction endonucleases EcoRI and HindIII (Takara Biotechnology, Dalian, China) and ligated using T4 DNA ligase (Takara Biotechnology, Dalian, China) to obtain the pET32a-RPS2 recombinant plasmid. The constructed plasmids were validated by double restriction enzyme digestion and then sequenced on an ABI PRISM™ 3730 XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA) by Sangon Biotech (Xian, China).

After obtaining the correct recombinant plasmid pET32a-RPS2, it was transformed into Escherichia coli BL21 (DE3) (Tsingke Biological Technology, Nanjing, China) and then amplified and cultured in Luria Bertani medium (LB) (37°C with shaking at 180 rpm). Subsequently, it was expressed under the induction of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Analysis of recombinant protein expression by 12.5% sodium salt polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. Then, based on the distribution of proteins, they were filtered with 0.22 μm membrane (BioSharp, Hefei, China) and purified by a chelating nickel column (Cytiva, Logan, UT, USA). Purified proteins were concentrated by the addition of polyethylene glycol (PEG) powder and the purification effect was confirmed by SDS-PAGE. The endotoxin was removed from the purified protein following the instructions provided with the ToxinEraser™ Endotoxin Removal Kit (GeneScript, Piscataway, NJ, USA). Subsequently, the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GeneScript, Piscataway, NJ, USA) was employed to assess the endotoxin levels in the recombinant proteins before further analysis. The recombinant protein concentration was determined by the Bicin-choninic Acid (BCA) protein quantification kit (New Cell & Molecular Biotech, Suzhou, China) and stored at -80°C.

The polyclonal anti-rTgRPS2 antibody was produced in Wistar rats. Before immunization, blank sera were collected from rat eye sockets as a control. Then, rats were first immunized subcutaneously at four different sites with 300 μg of purified rTgRPS2 protein emulsified with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Two weeks later, rats were immunized with the same dose of purified rTgRPS2 protein emulsified with Freund’s incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) at one-week intervals. A total of eight immunizations were administered, and blood containing polyclonal antibodies was collected from the rat eye sockets every 7 days after the sixth immunization. All sera were stored at -20°C until use.

In order to obtain soluble tachyzoite antigens (STAg) of T. gondii, 5×10⁶ tachyzoites were resuspended in 1 ml of phosphate-buffered saline (PBS). Subsequently, protease and phosphatase inhibitors (Beyotime, Shanghai, China) were added, and the tip sonication (Scientz Biotechnology, Ningbo, China) was carried out in continuous mode for 2 s (15 min in total) at 30 W output power. The obtained proteins were quantified using the BCA protein quantification kit (Beyotime, Shanghai, China), and were stored at -20°C until use. Similarly, we obtained polyclonal antibodies against T. gondii STAg using the same immunization strategy as in 2.3.

To analyze the reactogenicity between the natural TgRPS2 proteins and the polyclonal antibodies against the recombinant TgRPS2 proteins, the purified rTgRPS2 protein or T. gondii STAg were first separated by 12.5% SDS-PAGE gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Subsequently, TBST (TBS containing 0.5% Tween 20) containing 5% (w/v) skim milk powder was used to block non-specific binding sites at 37°C for 1.5 hours. After that, being washed in TBST for three times, the membranes were incubated with sera against STAg (1:100 dilution) or sera against rTgRPS2 (1:100 dilution) overnight at 4 °C. After being rinsed in TBST for five minutes, the membranes were stained with HRP-conjugated goat anti-Rat IgG (1:8000 dilution, ABclonal, Wuhan, China) at 37°C for 1 hour. Rinsed again in TBST for five minutes, the membranes were finally imaged by Tanon™ Femto-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) under a chemiluminescent image analysis system (Tanon, Shanghai, China).

PLGA nanospheres were synthesized using the double emulsion solvent evaporation technique (w/o/w) (27, 28). Briefly, 0.6 g of polyvinyl alcohol (PVA, MW 31,000–75,000 Da, Macklin, Shanghai, China) was weighed and dissolved in an appropriate amount of deionized water. Then a container was placed on a magnetic stirrer stirring at 500 rpm until the PVA was completely dissolved. Adjust the volume to 20 ml to prepare a 3% PVA solution. Subsequently, 100 mg of PLGA was mixed with 1 ml of dichloromethane (DCM, Sigma, Saint Louis, MO, USA) into a 50 ml centrifuge tube to form the organic phase. At room temperature, 2 ml of 3% PVA solution was stirred using a magnetic stirrer, and 2 ml of rTgRPS2 protein (at a concentration of 1 mg/ml) was dripped with stirring. After mixing, tip sonication was carried out at 4°C for 6 min in continuous mode at an output power of 35 W for 2 s at a time to synthesize w/o emulsion by Tip sonication (Scientz Biotechnology, Ningbo, China). Then 3 ml of 3% PVA solution was dropped into the w/o emulsion, which was mixed by a magnetic stirrer and then subjected to tip sonication to synthesize the w/o/w emulsion. Subsequently, the obtained w/o/w emulsion was transferred to a 50 ml centrifuge tube for overnight evaporation of DCM. Then, centrifuge at 40,000 r/min at 4°C for 20 minutes to separate the supernatant from the precipitate. Resuspend the precipitate in deionized water and adjust the volume to 4 ml. Transfer the resuspension to a lyophilization vial, freeze it at -80°C for at least 2 hours, and then transfer it to a vacuum freeze-drier (Yiheng, Shanghai, China) for 24 hours until completely freeze-dried. The freeze-dried PLGA nanospheres were stored at 4°C under sealed conditions until use.

The desired CS nanospheres were synthesized using the ionic gelation technique with slight modifications (29). Briefly, 100 mg of CS (MW 50-190 kDa, Macklin, Shanghai, China) was dissolved in 50 ml of 1.0% (v/v) aqueous acetic acid solution on a magnetic stirrer (400 rpm) until complete dissolution. Subsequently, the solution pH was adjusted to 4.6-5 by adding appropriate 20 wt% aqueous sodium hydroxide solution. 40 mg of TPP was dissolved in 20 ml of double-distilled water, and the completed 2 mg/ml sodium tripolyphosphate (TPP, Aladdin, Shanghai, China) solution was prepared by passing it through a 0.22 μm filter membrane (BioSharp, Hefei, China). 10 ml of CS solution was transferred to a 50 ml centrifuge tube, and then 2 mg of rTgRPS2 protein (at a concentration of 1 mg/ml) was cautiously dripped to 2 ml of TPP solution for 20 min on a magnetic stirrer stirring at 500 rpm. Then, the tip sonication was carried out for 5s at 2 s intervals (5 min total) under output power of 45 W. After sonication, the samples were centrifuged at 40,000 rpm for 20 min at 4°C, and the obtained nanospheres were rinsed and resuspended in double-distilled water and passed through a 0.22 μm filter membrane. After freezing at -80°C, the CS nanospheres were transferred to a vacuum freeze dryer until completely freeze-dried, and then the freeze-dried CS nanospheres were stored at 4°C under sealed conditions until use.

The morphological characteristics of synthesized nanospheres were analyzed using scanning electron microscopy (SEM) from the College of Life Science, Nanjing Agriculture University, PR China, and the average diameter of the PLGA and CS nanospheres was quantified using ImageJ software (version 1.8, NIH Image, Bethesda, MD, USA).

To determine the loading capacity (LC) and encapsulation efficiency (EE) of the synthesized nanospheres, the concentration of non-bound proteins in the supernatants of the collected PLGA and CS nanospheres was determined using the BCA protein quantification kit (New Cell & Molecular Biotech, Suzhou, China). The LC and EE of PLGA and CS nanospheres were calculated according to (Equations 1, 2), respectively.

To investigate the release characteristics of TgRPS2-PLGA and TgRPS2-CS nanospheres in vitro, two nanospheres were dispersed in PBS (pH 7.4) under magnetic stirring at 180 rpm at 37°C. Every 12 h, the supernatant samples were collected by centrifugation and stored at -20°C. After each collection, the settled nano drops were resuspended with the original solution. After the last collection, the protein concentration in the sample was measured using the BCA protein quantification kit (New Cell & Molecular Biotech, Suzhou, China), and the cumulative release (CR) was calculated using the (Equation 3).

A total of 35 ICR mice were randomly allocated to seven experimental groups (n = 5 mice): Blank (immunized with an equal volume of PBS), Control (immunized with an equal volume of the pET32a-label protein), PLGA (immunized with an equal volume of PLGA nanospheres), CS (immunized with an equal volume of CS nanospheres), TgRPS2 (immunized with an equal volume of TgRPS2 protein), TgRPS2-PLGA (immunized with an equal volume of TgRPS2-PLGA nanospheres), and TgRPS2-CS (immunized with an equal volume of TgRPS2-CS nanospheres). Mice were immunized with a triple dose of the regular dosage, and 300 μg of protein was injected intramuscularly in each mouse. Three days later, a booster immunization was administered using the same strategy. One day after the completion of the immunization procedure, sera were collected from the eye sockets of the mice. The serum levels of creatinine (Cr) and blood urea nitrogen (BUN) were assessed using the sarcosine oxidase assay (Solarbio, Beijing, China) and the urease-indole phenol assay (Solarbio, Beijing, China). During the trials, the physical health and mental state of the mice were observed and recorded daily.

ICR mice were randomly divided into 7 groups (n=26). Before immunization, all TgRPS2 proteins and synthesized nanospheres were diluted and suspended in PBS (pH 7.4). Then mice were immunized by multipoint intramuscular injections according to the immunization schedule ( Table 1 ). Briefly, on the 1 and 15 days, mice were given their first and booster immunizations according to their groups. Simultaneously, blood and splenic lymphocytes were collected from the mice after 10 days of each immunization, and serum samples were preserved at -20°C. After 14 days of the booster immunization (on the 29th day), all mice were challenged with 100 tachyzoites from highly virulent T. gondii RH strains intraperitoneally as indicated previously (30). Four days after the challenge experiment (on the 33th day), three mice were anesthetized and sacrificed under the supervision of the Ningxia University Technology Ethics Committee, and spleen tissues were isolated in 10% neutral buffered formalin. Six days after the challenge (on the 35th day), ten mice were anesthetized and sacrificed under supervision, and cardiac muscles were collected and stored at -20°C.

The titers of T. gondii-specific antibodies in mice serum were determined using an indirect enzyme-linked immunosorbent assay (ELISA). Briefly, each well in the ELISA plate was coated with 1 μg of STAg (diluted to 10 μg/ml with carbonate buffer pH 9.6) overnight at 4°C. After rinsing in TBST 5 times, each well was blocked with TBST containing 5% bovine serum albumin (BSA) at 37°C for 1 h (Sangon Biotech, Shanghai, China).

The plates were washed three times with TBST. The wells were then incubated with sera (diluted 1:100 in TBST containing 5% BSA) from immunized mice at 30°C for 1 h. After being washed with TBST three times, the plates were incubated with HRP-conjugated anti-mouse IgG, IgG1, or IgG2a (1:5000, ABclonal, Wuhan, China) at 37°C for 1 h. The plates were washed again, and the enzymatic activity was assessed using 3,3′,5,5′-tetramethylbenzidine (TMB, Tiangen, Beijing, China) as the substrate. The reaction was conducted in the dark at room temperature for 10 minutes. Finally, the reaction was stopped with 100 μl of freshly prepared H₂SO₄. A microplate photometer (BioTek, Burlington, VT, USA) was used to quantify the titers of antibodies at an absorbance of 450 nm.

The secretion of cytokines interferon-γ (IFN-γ), transforming growth factor β (TGF-β), IL-4, IL-6, IL-10, and IL-17 in mice serum was determined using a commercial ELISA kit (Enzyme Linked Biotechnology Co., Ltd, Shanghai, China) according to the instructions.

After 10 days of the first and the booster immunization, respectively, five mice from each group were sacrificed before immunization or challenge, and splenic lymphocytes were collected using a lymphocyte isolation kit (Solarbio, Beijing, China). The percentages of CD4⁺ and CD8⁺ T cells of mice were detected using flow cytometry. Isolated spleen lymphocytes were cultured overnight in DMEM containing 10% FBS and 1% double antibiotics. Non-adherent cells were discarded, and adherent cells were collected after washed with PBS three times. Then, cells were adjusted to 10⁶ cells in 100 μl PBS. The spleen lymphocytes were, respectively, stained with anti-mouse CD3e-PE and CD4-FITC (eBioscience, San Diego, CA, USA), and anti-mouse CD3e-PE and CD8-FITC (eBioscience, San Diego, CA, USA) in the dark for 40 minutes at 4°C. In order to investigate the changes of CD83, CD86, and MHC molecules in splenic DCs, isolated lymphocytes were double-stained with anti-mouse CD11c-APC, CD86-FITC, CD83-PE, and anti-mouse CD11c-PE, MHC-I-FITC, and MHC-II-APC antibodies (eBioscience, San Diego, CA, USA) in the dark for 40 minutes at 4°C. After being washed three times in PBS, centrifugation, and collection, cells were sorted using flow cytometry (Beckman Coulter Inc., Brea, CA, USA).

The day before the challenge experiment (On the 28th day), three mice from each group were sacrificed, and spleen lymphocytes were isolated using the same method as described in 2.11. Splenic lymphocytes were seeded into a 96-well plate at a density of 5 × 10⁵ cells per well. The cells in each well were stimulated with rTgRPS2 (10 μg/ml) and incubated for 48 h at 37°C in a 5% CO₂ incubator. Afterward, Cell Counting Kit 8 (CCK-8, Beyotime, Shanghai, China) was added to each well according to the manufacturer’s instructions and incubated for 4 h. Subsequently, the absorbance at 450 nm was measured using a microplate reader (BioTek, Burlington, VT, USA) to assess lymphocyte proliferation.

After 14 days of the booster immunization (on the 29th day), all mice were challenged with 100 tachyzoites from highly virulent T. gondii RH strains intraperitoneally. Four days after the challenge, three mice were sacrificed under the supervision, and spleen tissues were isolated from mice, and fixed in 10% neutral buffered formalin for 24-48 hours. Subsequently, dehydrate the tissue gradually in increasing ethanol concentrations for 1-2 hours each, followed by clearing in xylene for 30 minutes. Embed the spleen tissue in paraffin and sectioned at a 6 μm thickness using Minux^® Rotary Microtomes S710 (RWD Life Science, Shenzhen, China). The sections were dehydrated and then dried for 24 h at 37°C, and inactivated with 3% H₂O₂ for 25 min under dark conditions to inhibit endogenous peroxidase activity. Subsequently, the sections were blocked with blocking solution (5% bovine serum albumin) at 37°C for 30 min and incubated with anti-T. gondii antibody (1:500 dilution, Abcam, Cambridge, UK) at 4°C overnight. After being washed with PBS three times, the sections were incubated with HRP-conjugated goat anti-rabbit IgG (1:8000 dilution, ABclonal, Wuhan, China) at room temperature for 1 h. Finally, the sections were detected by prepared 3,3′-diaminobenzidine (DAB; Sangon Biotech, Shanghai, China) for 5 min and were counterstained with hematoxylin, then dehydrated and sealed with neutral balsam. The sections were examined under a microscope to assess the distribution of T. gondii in the spleen tissues.

To analyze the immune protective efficacy elicited by nano vaccines, absolute quantitative real-time PCR (qPCR) was performed to investigate T. gondii burdens in the cardiac tissue of challenged animals. Six days after the challenge, ten mice were sacrificed under the supervision, and the cardiac tissue was collected. Then, genomic DNA was extracted from 30 mg of cardiac tissue collected from the challenged animals following the manufacturer’s instructions for the genomic DNA extraction kit (OMEGA Bio-tek, Norcross, GA, USA). The purity of DNA samples was evaluated by measuring the OD260/OD280 ratio using a nanodrop microvolume spectrophotometer (Thermo Scientific, Waltham, MA, USA). The recombinant vector was constructed according to previous studies (31), and the copy number was calculated. The 529 bp fragments in the extracts were amplified by ChamQ Universal SYBR qPCR MasterMix (Vazyme Biotech Co., Ltd, Nanjing, China) using a CFX96 amplifier (Bio-rad, Hercules, CA, USA). Before further analysis, the melting curve of each amplification was verified, and each displayed a single uniform peak as expected.

One-way ANOVA with multiple comparisons was conducted using GraphPad Prism 9.5 software (GraphPad Prism, San Diego, CA, USA) to account for differences between groups. Statistical significance was determined at p < 0.05, and the results are presented as mean ± SD for each group. Flow cytometry analysis was conducted using CytExpert software (version 2.3, Beckman Coulter Inc., Brea, CA, USA).

The obtained pET32a-RPS2 plasmid was first double-digested by EcoRI and HindIII, and then verified in 1% agarose gel electrophoresis, respectively generating two fragments of 816 bp and 5881 bp ( Figure 1A ). In accordance with the expected result, the obtained results revealed that the inserted fragment was the correct ORF of TgRPS2. In addition, DNA sequencing showed that the target fragments were correctly inserted in the pET32a vectors. In conclusion, all the results mentioned above indicated that the pET32a-RPS2 plasmid was constructed correctly.

The molecular weight of recombinant TgRPS2 was 47.3 kDa, which included pET32a vector protein (18.0 kDa) and native TgRPS2 protein (29.3 kDa). As shown in Figure 1B , the results of SDS-PAGE showed that the molecular weight of the purified rTgRPS2 protein was about 47 kDa, which conformed to the expected result. Furthermore, western blot analysis revealed that anti-T. gondii STAg antibodies could react with the rTgRPS2 protein ( Figure 1C ), emphasizing that the rTgRPS2 protein could be recognized by anti-T. gondii STAg antibodies. The interaction of STAg from T. gondii with serum containing anti-rTgRPS2 antibodies demonstrated that the anti-rTgRPS2 antibodies could effectively recognize the STAg from T. gondii ( Figure 1D ). All these results suggest that rTgRPS2 exhibits good immunogenicity, which can be used as the vaccine antigen.

After encapsulation of the rTgRPS2 protein in PLGA and CS nanospheres, the morphology of the synthesized nanospheres was observed using SEM. As illustrated in Figure 2 , the nanospheres appeared spherical with uneven surface textures, and slight irregularities were also detected. Employed by Image J software, the diameters were analyzed according to five randomly selected nanospheres. The average diameter of TgRPS2-PLGA and TgRPS2-CS nanospheres were 79.46 ± 6.14 nm and 93.98 ± 14.37 nm by analyzing the SEM pictures (n = 5). By conducting the concentration of nonbinding proteins and total proteins in the supernatant using BCA protein quantification kit, the LC values of PLGA and CS nanospheres were 0.94% and 5.15% (n = 3) respectively, while EE values were 70.33% and 41.45% (n = 3).

The release characteristics of the synthesized TgRPS2-PLGA and TgRPS2-CS nanospheres in vitro were evaluated by calculating different CR values at different points. As shown in Figure 3 , two types of nanospheres exhibited a rapid release at the first three days, compared with the fourth to seventh day. The CR value of TgRPS2-CS nanospheres at the first detection was higher compared with TgRPS2-PLGA nanospheres, while the average CR value of TgRPS2-PLGA nanospheres was investigated higher during the third to seventh day. After the fourth day, the release curves of both nanospheres showed a slow and steady trend, continuing until the seventh day. The experimental results in vitro suggested the evidence that both obtained nanospheres could provide one-week-term stable release, which could enhance the effectiveness of immunizations.

In order to investigate whether the nanospheres were poisonous, the levels of Cr and BUN in the collected sera were assessed based on the sarcosine oxidase assay and the urease-indole phenol assay. The results showed that the levels of Cr and BUN are within acceptable ranges, and no statistically significant difference was investigated among the immunization groups and the control group (p > 0.05, Figure 4 ). In addition, the behavior, diet, and physiological status of the animals were normal during the experiment, and no adverse reactions were observed. All these obtained results demonstrated that the synthesized nanospheres are non-toxic.

Based on the group assignment displayed in 2.9, sera were collected from all immunized mice to evaluate IgG, IgG2a, and IgG1 levels. As expected, vaccinations with two types of nanospheres and naked TgRPS2 protein could induce significantly higher levels of T. gondii-specific IgG when compared with the control group (p < 0.01, Figure 5A ). Furthermore, TgRPS2-PLGA and TgRPS2-CS nanospheres could induce significantly higher levels of T. gondii-specific IgG when compared with naked TgRPS2 after booster immunization (p < 0.001). As shown in Figures 5B, C , mice immunized with recombinant proteins and its nanospheres generated higher levels of T. gondii-specific IgG1 and IgG2a when compared with the animals in the control group. It is noteworthy to note that regardless of the number of immunizations, immunization with TgRPS2-CS and TgRPS2-PLGA nanospheres could generate statistically enhanced IgG1 levels compared to the naked TgRPS2 protein (p < 0.01, Figure 5B ).

After the booster immunization, the levels of IFN-γ, TGF-β, IL-4, IL-6, IL-10, and IL-17 in sera were measured by commercial ELISA kits to explore the effects of nanospheres on inflammation and immune response in the body. As demonstrated in Figures 6A, C, F , mice immunized with TgRPS2-PLGA and TgRPS2-CS nanospheres showed significantly increased secretions of IFN-γ, IL-4, and IL-17 compared with the controls (p < 0.01). The obtained results also showed that mice immunized with recombinant proteins generated significantly higher levels of IL-6 compared with the controls (p < 0.01, Figure 6D ). In addition, the levels of IL-10 in the nanospheres-immunized groups, especially the TgRPS2-PLGA group, were significantly down-regulated compared to the control group (p < 0.001, Figure 6E ). In addition, no statistically significant difference was detected in the levels of TGF-β between the control group and the immunized groups (p > 0.05, Figure 6B ).

Dendritic cells (DCs) subpopulations in the collected spleen lymphocytes were determined to evaluate the maturation of DCs by flow cytometry. Compared with the controls, TgRPS2 protein, TgRPS2-PLGA, and TgRPS2-CS nanospheres could significantly induce the expression levels of CD83 and CD86 in lymphocytes (p < 0.001, Figures 7A–C ). Moreover, the expressions of CD86 in the TgRPS2-PLGA and TgRPS2-CS groups were significantly higher than that in the TgRPS2 group after the booster vaccination (p < 0.001, Figure 7B ). All these results suggested that synthetic nanospheres and TgRPS2 proteins play an important role in stimulating lymphocytes to generate CD83 and CD86 molecules.

To investigate the effects of PLGA and CS nanospheres on antigen presentation, the expression of MHC-I and MHC-II in all immunized groups was analyzed by flow cytometry. As shown in Figures 8A, C the expression levels of MHC-I in DCs collected from the TgRPS2-CS group were detected significantly higher after the enhanced immunization when compared with the control group (p < 0.05). Compared with the controls, TgRPS2 protein, TgRPS2-PLGA, and TgRPS2-CS nanospheres could significantly promote the expression levels of MHC-II in DCs (p < 0.05, Figures 8B, C ). Furthermore, the expressions of MHC-II in TgRPS2-PLGA groups were significantly higher than that in the TgRPS2 group after the booster vaccination (p < 0.01, Figure 8B ). These results suggested that the TgRPS2 protein and its nanospheres, especially PLGA nanospheres, could activate the antigen-presenting effect of DCs.

CD4⁺ and CD8⁺ T lymphocytes played a pivotal role in the control of T. gondii infections. To investigate the inductions of PLGA and CS nanospheres on T lymphocyte response, the expression levels of CD4⁺ and CD8⁺ T lymphocytes in the collected spleen lymphocytes were evaluated by flow cytometry. As shown in Figures 9A–C , compared with the controls, TgRPS2 protein, TgRPS2-PLGA, and TgRPS2-CS nanospheres could significantly increase the expression levels of CD4⁺ and CD8⁺ T lymphocytes in lymphocytes (p < 0.05). After the initial immunizations, the expression levels of CD4⁺ and CD8⁺ T lymphocytes in the nanospheres-immunized groups, especially the TgRPS2-PLGA group, were significantly higher compared to the TgRPS2 protein-immunized group (p < 0.05, Figures 9A, B ). These obtained findings demonstrated that the TgRPS2 protein, as well as its two types of nanospheres, could stimulate lymphocytes to generate CD4 and CD8 molecules, and PLGA nanospheres exhibited a great advantage in activating T lymphocytes.

The day before the challenge experiment (on the 28th day), spleen lymphocytes were isolated from the immunized mice, and the status of lymphocyte proliferation was assessed using commercial CCK-8 kits. As shown in Figure 10 , lymphocyte proliferation was significantly increased in the nanospheres-immunized group compared with the control group (p < 0.001). In addition, compared with the TgRPS2 protein, TgRPS2-CS nanospheres could statistically induce the proliferation of lymphocytes (p < 0.001). These observations were indicative of a great effect of TgRPS2 protein, and its two types of nanospheres, in stimulating T-lymphocyte proliferation.

After 14 days of the booster immunization (on the 29th day), 100 highly virulent T. gondii RH strain tachyzoites were intraperitoneally injected into mice for challenge experiments. Four days later (On the 33 days), the mice were sacrificed under the supervision of the Ningxia University Technology Ethics Committee, and spleen tissues were collected and processed into paraffin sections. As demonstrated in Figure 11 , mice immunized with TgRPS2 protein, TgRPS2-PLGA, and TgRPS2-CS nanospheres exhibit reduced spleen infection by T. gondii. These findings suggested that nanospheres-immunization could provide immune protection in mice against toxoplasmosis, and such resistance could inhibit the growth of T. gondii tachyzoites to some extent.

After 14 days of the booster immunization (on the 29th day), all groups of mice were challenged with 100 highly virulent T. gondii RH strains. Then, six days after the challenge (On the 35th day), qPCR was conducted to evaluate the parasite burdens in heart tissues harvested from immunized mice. Compared to the control group, the parasite burdens in TgRPS2, TgRPS2-PLGA, and TgRPS2-CS nanospheres groups were significantly reduced by 60.7%, 91.7%, and 86.7%, respectively. In addition, the parasite burdens in the TgRPS2-PLGA and TgRPS2-CS groups were statistically reduced compared with the TgRPS2 group (p < 0.01, Figure 12 ). The obtained results demonstrated that the immune response generated by synthesized nanospheres played an essential role in controlling T. gondii infection.