As a relevant model used in this study, female ICR mice weighing 18–22 g (7 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and were cultured at a specific pathogen-free (SPF) environment. Purchased from the same company, female Wistar rats weighing 180–220 g (7–8 weeks old) were also cultured in the same SPF environment. The experimental design, animal management, injection, and operations strictly followed the Ethics Procedures and Guidelines of the People’s Republic of China and were supervised by the Animal Ethics Committee, Ningxia University, Yinchuan, China.

The purified T. gondii RH strains were obtained from Dr. Xiangrui Li at the Nanjing Agricultural University, and retained in liquid nitrogen at the Preventive Veterinary Laboratory, College of Veterinary Science, Ningxia University, Yinchuan, China. The parasites were propagated in mice as previously described (42).

The total RNA of 10⁷ T. gondii tachyzoites was purified by Trizol reagent (TAKARA Biotech, Dalian, China), and the cDNA was synthesized by using the reverse transcription kit (TAKARA Biotech, Dalian, China). PCR amplification (Applied Biosystems, Waltham, MA, USA) was then conducted to obtain the conserved domain sequences (CDS) of TgGAP45 (GenBank: TGME49_223940), using the following primers: forward, 5'-CCATGGCTGATATCGGATCCATGGGAAACGCGTGCAAGA-3', and reverse, 5'-TGTCGACGGAGCTCGAATTCTCAGTTCAACAAGGGTGCATCC-3'. Reverse PCR was also conducted with the blank pET-32a plasmid as the template by using the following primers: forward, 5'-GAATTCGAGCTCCGTCGACAAGC-3', and reverse, 5'-GGATCCGATATCAGCCATGG-3'. Phanta Max Master Mix (BioGlod Science, Zhejiang, China) was used for the PCR amplification mentioned above with the recommended protocol. Then, the recombinant plasmid, named pET-32a-TgGAP45, was constructed by using the homologous recombination system (Vazyme Biotech, Nanjing, China), and was transferred into Escherichia coli BL21 (DE3) cells (Tsingke Biotech, Xian, China). The E. coli BL21 (DE3) cells carrying pET-32a-TgGAP45 plasmids were propagated in Luria Bertani (LB) medium containing 100 μg/mL ampicillin. The plasmids were extracted by a commercial reagent kit (Omega Bio-Tek, Norcross, GA, USA), verified by double restriction enzyme digestion (TAKARA, Dalian, China), and sequenced by the ABI PRISM™ 3730 XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA). To collect purified recombinant TgGAP45 proteins, BL21 (DE3) cells were cultured in LB medium containing 100 μg/mL ampicillin, and 1.0 mM isopropyl β-D-thiogalactoside (IPTG, Aladdin, Shanghai, China) was added until the OD₆₀₀ (optical density at 600 nm) reached approximately 0.5. The chemical competent cells were collected, crushed by tip sonication (Scientz Biotechnology, Ningbo, China), and purified by a chelating column (Cytiva, Marlborough, MA, USA). Before subsequent analysis, the endotoxin in purified rTgGAP45 was eradicated by the Endotoxin Removal Kit (GeneScript, Piscataway, NJ, USA), and the endotoxin level of rTgGAP45 after eradication was detected by a commercial kit (GeneScript, Piscataway, NJ, USA). To analyze the purification and concentration of purified rTgGAP45, 12% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and the BCA assay were performed.

To collect the sera against rTgGAP45, purified rTgGAP45 was emulsified with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, USA), and each Wistar rat received a subcutaneous injection of emulsions containing 200 µg of purified rTgGAP45 in the back skin with multiple points in the first week. The emulsion mixture of purified rTgGAP45 and Freund’s incomplete adjuvant (Sigma-Aldrich, Saint Louis, USA) was vaccinated four times into rats through the same method mentioned above at 14-day intervals. Whole blood was harvested 1 week later after the last vaccination from the orbit, and the sera were separated and stored at −20°C. Blank sera were also collected from animals immunized with PBS using the same vaccination strategy. Furthermore, the soluble tachyzoite antigen (STAg) of the parasites was prepared as described previously (43). Briefly, 10⁸ T. gondii tachyzoites were collected and resuspended in RIPA lysis (Biosharp, Life Science, Hefei, China), then tip sonication was conducted. To obtain the sera against T. gondii, rats were injected with emulsions containing STAg and Freund’s adjuvant using the same immunization strategy. All sera were stored at −20°C until use.

Purified rTgGAP45 and STAg were first separated in 12% SDS-PAGE gel and were subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, IRL) via the Mini Trans-Blot Cell (Bio-Rad, Hercules, CA, USA). Membranes were then blocked in TBST [tris-buffered saline containing 0.5% (v/v) Tween 20] containing 5% (w/v) skimmed milk powder (Sangon Biotech, Shanghai, China) at 37°C for 2 h, and were separately incubated in TBST containing rats’ sera against STAg and in the TBST containing the sera against rTgGAP45 at 1:100 dilutions overnight at 4°C on a rotary shaker. Rinsed three times in TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rat IgG (1:5,000 dilutions, ABclonal Technology, Wuhan, China) for 2 h at room temperature. Finally, the membranes were visualized by the Electro-Chemi Luminescence (ECL) system (Tanon, Shanghai, China). The sera harvested from animals immunized with PBS were also conducted as a control.

Based on the double emulsion solvent evaporation technique (44), the TgGAP45PLGA nanoparticles (w/o/w nanoparticles) were synthesized with minor modifications. In short, to form PLGA solution, 0.50 g of PLGA (MW: 40,000–75,000 Da, LA/GA: 65/35, Macklin Biochemical Technology, Shanghai, China) was dissolved in 10.0 mL of dichloromethane (DCM, Macklin Biochemical Technology, Shanghai, China) at room temperature. Then, 20 mL of 5% (w/v) polyvinyl alcohol (PVA, MW: 31,000–75,000 Da, Macklin Biochemical Technology, Shanghai, China) was added dropwise into the PLGA solution under frequent stirring. The mixture was then kept in an ice bath, and tip sonication was carried out in continuous mode (duration, 2 s; interval time, 3 s) under an output power of 40 W, until the mixture turned milky white. After that, 40 mg of rTgGAP45 without endotoxin was dropwise added under frequent stirring at room temperature, and tip sonication was again conducted under the same criteria. To form w/o/w emulsions, 20 mL of 5% PVA was subsequently dropwise added, and tip sonication was performed again. Before centrifugation at 35,000 rpm for 20 min at 4°C, the obtained emulsions were passed through the 0.22-μm filter membrane (Millipore, Billerica, MA, USA) to remove unexpected impurities. The precipitates were collected and resuspended in deionized water, while the supernatants were also harvested. The resuspended solution was stored at −20°C overnight and was completely freeze-dried (Labconco, Kansas City, MO, USA) to remove DCM. The freeze-dried powder, also known as TgGAP45-PLGA nanoparticles, was stored at −20°C and diluted by 1 × PBS before use. To characterize the sustained-release properties of the nanoparticles, an in vitro release study was conducted under sink conditions. The experiment utilized PBS (pH 7.4) as a release medium with a volume exceeding 10-fold the saturation solubility of rTgGAP45, ensuring the protein concentration remained below 10% of its saturation limit throughout the assay. To accurately mimic physiological conditions, the release medium was maintained without replacement during the entire study period.

To obtain the TgGAP45-206VG emulsions (w/o/w emulsions), Montanide™ ISA-206VG (Seppic, Paris, France) was pre-incubated in a 50°C water bath before use, and the rTgGAP45 was dissolved in PBS (pH 7.4) to a final concentration of 1.0 mg/mL. According to the guidelines, the protein solution was gently added into Montanide™ ISA-206VG in equal volumes at a bath temperature of 31°C.

Afterward, the emulsions were stirred constantly until thickened at a temperature of 31°C. The formulated TgGAP45-206VG emulsions were shortly stored at 4°C until use (within 2 h).

To obtain the TgGAP45-660VG emulsions (w/o emulsions), the rTgGAP45 was dissolved in PBS (pH 7.4) to a final concentration of 1.0 mg/mL. Based on the instructions, the protein solution was directly added into Montanide™ ISA-660VG in a 2:3 (w/w) ratio at room temperature. Then, the emulsions were stirred constantly until thickened, and the synthesized emulsions, also known as TgGAP45-660VG emulsions, were also shortly stored at 4°C until use (within 2 h).

To analyze the surface morphology of TgGAP45-PLGA nanoparticles, the synthesized nanoparticles were imaged by a scanning electron microscope (SEM, SU8010, Hitachi, Tokyo, Japan) at Nanjing Agriculture University (Nanjing, China). TgGAP45-PLGA nanoparticles in the SEM images were randomly measured, and the average diameter of synthesized nanoparticles was calculated by ImageJ software (version 1.8.0, NIH, Bethesda, MD, USA). Using Zetasizer nanoseries nano-ZS (Malvern Panalytical Ltd., Malvern, UK), the polydispersity index (PDI) of synthesized nanoparticles was examined using the dynamic light scattering technique in Saisijuxin Biotechnology (Yinchuan, China).

To investigate the encapsulation efficiency (EE) of prepared nanoparticles, the synthesized TgGAP45-PLGA nanoparticles were separated from the supernatant via high-speed centrifugation at 40,000 rpm for 20 min at 4°C (as described in Section 2.4). The total volume of the supernatant was evaluated by a measuring cylinder, and EE was calculated based on Equations 1 and 2.

To investigate the sustained release behavior of TgGAP45-PLGA nanoparticles, the cumulative release assay was also performed in vitro according to the previously reported method with minor modification (45). Synthesized nanoparticles were dissolved in 1 × PBS (pH 7.4) and kept on a shaker under 37°C in the centrifuge tubes, then 20 μL of supernatant as the sample was collected at an interval of 12 h after centrifugation at 15,000 rpm for 20 min. Nanoparticle solution was resuspended and the total volume was recorded; samples were kept at −20°C until use. After the last record, the total concentration of free proteins in the samples was demonstrated by BCA assay, and the in vitro cumulative release (CR) profile was constructed by using Equation 3. A least-square 233 fitting method was conducted to construct the fit line.

To demonstrate the toxicity of TgGAP45-PLGA nanoparticles, 25 ICR mice were randomly divided into four groups: Blank (administered with equal volume of 1 × PBS), Control (administered with pET-32a vector protein), TgGAP45 (administered with rTgGAP45), and TgGAP45-PLGA (administered with TgGAP45-PLGA nanoparticles). Each animal received a subcutaneous injection with a dose containing 300 μg of antigen, which was three times higher than usual. A booster vaccination was also conducted 7 days later using the same strategy, and the sera were harvested from the eye sockets of animals. Based on the indophenol blue method and the creatine oxidase 234 method, the concentrations of blood urea nitrogen (BUN) and creatinine (Cr) were analyzed by the 250 commercial kits (Solarbio, Beijing, China). During the trials, the animals’ physical status and behaviors were constantly monitored.

ICR mice were randomly allocated into nine groups with 21 replicates in each group. Animals were immunized subcutaneously in the back with multipoint, and the maximum dosage for single injection was controlled within 500 μL. The booster vaccination using identical criteria was performed 14 days after the primary immunization ( Table 1 ). Animals in the experimental group received an injection containing 100 μg of purified rTgGAP45, while animals in the control group received the same dosage without rTgGAP45 in the same manner. To estimate the protective efficacy generated by immunizations as described previously (4), eight animals from each group received an intraperitoneal injection with a lethal dose of RH strain (100 tachyzoites per mouse) 10 days after booster vaccination ( Figure 1 ). All the animal trials were conducted under the supervision of the Animal Ethics Committee, Ningxia University, Yinchuan, China.

On days 10 (10 days after the first immunization) and 20 (10 days after the booster immunization), bloods were harvested from the eye sockets, and the sera were prepared. To determine the titers of T. gondii-specific serum antibody in the sera, enzyme-linked immunosorbent assays (ELISAs) were carried out as previously mentioned (46). In brief, each well of the 96-well plates (Biosharp, Life Science, Hefei, China) was incubated with 1 μg of T. gondii STAg (dissolved in 100 μL carbonate buffer pH 9.6) overnight at 4°C. After being rinsed in TBST for 5 min, each well was blocked with 100 μL of TBST containing 5% (w/v) skimmed milk power at 37°C for 1 h. After being washed in TBST for three times, plates were incubated with 100 μL of the animals’ sera (each well) for 1 h at 37°C. Plates were incubated with HRP-conjugated anti-mouse IgG (ABclonal Technology, Wuhan, China) at 1:10,000 dilutions (100 μL each well) at 37°C for 1 h. After being washed, each well was incubated with 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB, Beyotime Biotech, Shanghai, China) to develop colors at room temperature, and the reaction in each well was stopped by adding 100 μL of 2 M H₂SO₄ solutions. Then, the absorbance at OD₄₅₀ was investigated by a microplate photometer (Bio Tek Instruments, VT, USA) within 30 min. Each group contained five replicates, and each replication was measured once.

To further investigate the humoral immunity of immunized animals, animals’ sera collected on day 20 (10 days after the booster immunization) were conducted by commercially available ELISA kits (Enzyme-linked Biotechnology, Shanghai, China). Based on double antibody sandwich ELISA, the levels of interferon-gamma (IFN-γ), interleukin-4 (IL-4), transforming growth factor-β (TGF-β), IL-6, IL-10, and IL-17 in the animals’ sera were investigated in strict accordance with the requirements. Each group contained five replicates, and each replication was measured once.

On day 19 (1 day before the challenge), three animals from each group were euthanized to isolate splenic lymphocytes using the separation solution (Solarbio, Beijing, China) according to the instructions. The obtained lymphocytes were adjusted to 10⁵ cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco Life Technologies, Carlsbad, CA, USA) containing 20% (v/v) fetal bovine serum (FBS, Gibco Life Technologies, Carlsbad, CA, USA) and 20 μg/mL purified TgGAP45 proteins, and cultured in a 96-well plate. After a 72-h incubation at 37°C in a 5% (v/v) CO₂ atmosphere, each well was added with 10 μL of Cell Counting Kit 8 reagent (CCK-8, Beyotime Biotech, Shanghai, China), and was sequentially cultured for 4 h. Then, the absorbance at OD₄₅₀ was determined by a microplate photometer. Each group contained three animals, the lymphocytes from each animal were divided into three replicates, and each replication was measured once.

On days 10 (10 days after the first immunization) and 20 (10 days after booster immunization), five animals from each group were sacrificed to harvest splenic lymphocytes by using the same separation solution described in Section 2.8, and the obtained lymphocytes were resuspended in 1 × PBS. To evaluate the surface marker of dendritic cells (DCs), lymphocytes were first incubated in DMEM media containing 10% (v/v) fetal bovine serum and 1% (w/v) double antibiotics at 37°C in a 5% (v/v) CO₂ atmosphere overnight. Subsequently, 10⁶ attached lymphocytes were gently harvested and re-dissolved in 100 μL of 1 × PBS, and cells were stained with APC-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD86, and PE-conjugated anti-mouse CD83 (eBioscience, San Diego, CA, USA) for 1 h at 4°C in the dark. After washing in 1 × PBS, cells were re-collected and sorted by flow cytometry (Beckman Coulter Inc, Brea, CA, USA). Before cell sorting, adequate fluorescence compensation was adjusted according to the fluorescence minus one (FMO) controls, and the gating strategies are shown in Supplementary Figure S1 .

To analyze the MHC molecule changes in DCs, 10⁶ splenic lymphocytes were resuspended in 100 μL of 1 × PBS, stained with PE-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse MHC-I, and APC-conjugated anti-mouse MHC-II (eBioscience, San Diego, CA, USA) under the same criteria, and sorted by the flow cytometry using the same strategies described previously.

As for the proportion of CD4⁺ and CD8⁺ T lymphocytes, 10⁶ splenic lymphocytes were also resuspended in 100 μL of 1 × PBS and stained with FITC-conjugated anti-mouse CD3e, PE-conjugated anti-mouse CD8, and FITC-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA). The staining strategy and flow cytometry analysis were performed using the same strategies mentioned previously.

Indirect immunofluorescence assay (IFA) was recruited to determine T. gondii burdens in the spleen. In brief, on day 24 (4 days after the challenge), three animals from each group were euthanized to isolate the spleen. The obtained spleen was fixed in 10% neutral-buffered formalin, embedded in paraffin, and sliced by a tissue slicer (Leica Biosystems, Deer Park, TX, USA). After dehydration in graded ethanol and vitrification by dimethylbenzene (Macklin Biochemical Technology, Shanghai, China), sections were immersed in repair buffer (Servicebio, Wuhan, China) and incubated at 95°C for 20 min. After antigen retrieval, sections were blocked in TBST containing 5% (w/v) skimmed milk powder for 1 h at 37°C, and stained with rabbit anti-T. gondii (Abcam, Waltham, MA, USA) at 1:1,000 dilutions in TBST overnight at 4°C on a rotary shaker. Rinsed in TBST for three times, sections were then incubated with CY3-conjugated anti-rabbit IgG (1: 5,000 dilutions, ABclonal Technology, Wuhan, China) for 1 h at 37°C. Before sealing, sections were stained with DAPI dihydrochloride (Beyotime Biotech, Shanghai, China) for 5 min at room temperature. Finally, three visual fields were randomly chosen from one section, and visualized by Pannoramic MIDI (3DHistech Ltd, Budapest, Hungary). Each group contained three animals, and one section was made from each tissue.

To analyze the immune protective efficacy elicited by vaccinations, absolute quantitative real-time PCR (qPCR) was performed to investigate T. gondii burdens in the cardiac tissue of challenged animals. Five animals from each group were sacrificed to isolate the cardiac tissue on day 26 (6 days after the challenge), and 30.0 mg of collected tissue was lysed to obtain genomic DNA per the guidelines of a commercial kit (OMEGA Bio-tek, Norcross, Georgia, USA). As previously described (47), the 529-bp fragments in the extracts were amplified by ChamQ Universal SYBR qPCR MasterMix (Vazyme, Nanjing, China) using a CFX96 amplifier (Bio-Rad, Hercules, CA, USA). The reference amplification was also conducted by using the constructed plasmids as DNA template, and the melting curve analysis was also carried out at the end of amplifications to remove unexpected amplification. Each group contained 10 replications, and each replication was detected once.

Data analysis was conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Statistical differences between two groups were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Comparisons among TgGAP45, TgGAP45-PLGA, TgGAP45-206VG, and TgGAP45-660VG groups were illustrated by ANOVA following Bonferroni’s correction. Significance was considered at p< 0.05, and values in all graphs were presented as mean ± standard deviation (SD).

The recombinant pET-32a-TgGAP45 plasmid was well constructed as previously described ( Supplementary Figure S2a ). Double enzyme digestion was conducted by using BamHI and EcoRI to verify the constructed vector, yielding two fragments of 744 and 5,894 bp ( Figure 2a ). Furthermore, plasmid sequencing also indicated that the inserts of the recombinant vector were correct ( Supplementary Figure S2b ). According to the instructions of the pET-32a vector, the recombinant TgGAP45 protein generated by the constructed vector consisted of a tag protein (17.7 kDa) and TgGAP45 (27.3 kDa). Thus, the theoretical molecular weight of recombinant TgGAP45 was 45.0 kDa, which matched the obtained result shown in Figure 2b . By endotoxin eradication assay, the endotoxin level of purified rTgGAP45 decreased to 0.01 EU/mL. Moreover, evaluated by immunoblot analysis shown in Figure 2c , recombinant TgGAP45 protein could be identified by the rat’s serum against STAg, and the natural TgGAP45 could be detected by the serum against rTgGAP45, indicating a satisfactory antigenicity of rTgGAP45, which could induce the immune response in host.

To validate whether the synthesized nanoparticles were in nanoscale, the TgGAP45-PLGA nanoparticles were imaged and the results showed that TgGAP45-PLGA nanoparticles were spherical and well-proportioned with a rough surface ( Figure 3 ). Based on the SEM images, the average diameter of TgGAP45-PLGA nanoparticles was determined to be 65.93 ± 3.13 nm (n = 10), with a PDI of 0.296 ± 0.03. Furthermore, the EE was 74.97%, according to three independent experiments.

Analyzed by a sustained slow release over a 1-week period, the release profile of TgGAP45PLGA was constructed. As demonstrated in Figure 4 , a burst release with approximately 9.23% of rTgGAP45 was observed at the initial stage, and such phenomenon may be caused by the recombinant proteins coated to the surface of PLGA nanoparticles. Obtained by a fit curve constructed with a coefficient of determination (R ²) of 0.8741, similar results were also indicated. In addition, the release curve was relatively steep in the first 4 days, while the trend became smoother after the fourth day.

As a premise of in vivo research, the safety of nanoparticles is of major concern before application, even if it involves nontoxic nanomaterials with biodegradable characteristics. Thus, we tested the toxicity of synthesized nanoparticles in mice ( Figure 5 ). The levels of BUN and Cr in animals’ sera were maintained in an acceptable level, and no significant difference occurred (p > 0.05) in all immunized animals, indicating that the recombinant TgGAP45 proteins and their PLGA nanoparticles could not affect the function of animals’ liver and kidney. Furthermore, the mental health of each animal was also recorded, and no adverse reaction was observed during the trials. The induction of a balanced Th1/Th2 immune response is critical for effective defense against intracellular pathogens like T. gondii. Our findings demonstrate that “TgGAP45-PLGA nanoparticles elicited a mixed Th1/Th2 profile,” characterized by elevated IFN-γ (Th1) and IL-4 (Th2) levels ( Figure 6c ), which aligns with the dual functionality required for parasite containment (cellular immunity) and humoral protection (antibody-mediated neutralization). All these obtained results suggested that the recombinant TgGAP45 proteins as well as their nanoparticles were nontoxic to mice.

To determine the levels of IgG, IgG1, and IgG2a induced by nanoparticles and two types of emulsions, animals were immunized according to the group assignment and vaccination program mentioned in Table 1 , and sera were collected from all immunized mice. As shown in Figure 7 , animals immunized with TgGAP45 as well as its preparations could remarkably induce higher levels of total IgG and IgG1 (p< 0.001), while naked proteins and its preparations could equally enhance the release of IgG2a after the first immunization (p< 0.01). As for the isotypes illustrated in Figures 7b, c , TgGAP45-PLGA nanoparticles could significantly promote the generation of IgG1 and Ig2a compared with the naked proteins (p< 0.01). Compared with the TgGAP45 group, animals in the TgGAP45-206VG group could elicit statistically higher levels of IgG1 after the booster immunization (p< 0.05), while both tested emulsions could increase the generation of IgG2a after the first immunization (p< 0.01).

To further analyze humoral immunity in vaccinated animals, sera were collected from each group and analyzed by the commercially available ELISA kits. As for the Th1 immune response exhibited in Figures 6a, c , TgGAP45-PLGA nanoparticles could enhance the secretion of IFN-γ and IL-4 when compared with the control or naked TgGAP45 group (p< 0.01). Only TgGAP45-206VG emulsions could both stimulate the generations of IFN-γ and IL-4 when compared with the control group (p< 0.01). As for the Th2 immune response demonstrated in Figures 6b, e , TgGAP45-PLGA nanoparticles and TgGAP45-206VG emulsions could inhibit the secretion of IL-10 when compared with control or recombinant proteins (p< 0.001). In addition, no significant difference was detected in all prepared agents in eliciting TGF-β (p > 0.05). Regarding the Th17 immune response demonstrated in Figure 6f , all nanoparticles and emulsions could promote higher levels of IL-17 than the controls in animals’ sera (p< 0.05), while only TgGAP45-PLGA nanoparticles could generate higher IL-17 when compared with the naked proteins (p< 0.001). Regarding the inflammatory cytokine displayed in Figure 6d , all prepared agents containing recombinant TgGAP45 proteins generated similar effects in eliciting IL-17 in mice (p > 0.05).

On days 10 (10 days after the first immunization) and 20 (10 days after booster immunization), five mice from each group were sacrificed to harvest splenic lymphocytes to evaluate the maturation of DCs by flow cytometry. As illustrated in Figures 8a–c , the synthesized nanoparticles as well as the prepared emulsions could significantly induce the expressions of CD83 and CD86 molecules in splenic DCs when compared with the controls (p< 0.01). However, compared with the naked proteins, only TgGAP45-PLGA nanoparticles and TgGAP45-206VG emulsions could enhance the proportions of CD11c⁺CD86⁺ cells in the spleens (p< 0.05).

Mature DCs would differentiate and present antigens. Flow cytometry was conducted to analyze the MHC molecule changes in DCs isolated from immunized mice. As shown in Figures 9a, c , only animals in the TgGAP45-PLGA group were detected with higher expressions of MHC-I molecules when compared with those in the control group (p< 0.05). As for the MHC-II molecules displayed in Figures 9b, c , all agents containing rTgGAP45 could significantly promote its expressions in CD11c⁺ cells (p< 0.05). Moreover, mice in TgGAP45-PLGA nanoparticles could elicit higher proportions of CD11c⁺MHC-II⁺ cells than those in the TgGAP45 group (p< 0.05), while two types of emulsions, after the first immunization, could only enhance the percentage of CD11c⁺MHC-II⁺ cells when compared with the naked proteins (p< 0.05).

On day 19 (1 day before the challenge), three mice from each group were euthanized to isolate splenic lymphocytes. Then, the effects of TgGAP45 proteins as well as its nanoparticles and emulsions in promoting T-cell proliferation were investigated by using a commercial CCK-8 reagent. As shown in Figure 10 , vaccinations with TgGAP45 and its nanoparticles and emulsions could significantly enhance lymphocyte proliferation when compared with the controls (p< 0.01).

Furthermore, TgGAP45-PLGA nanoparticles generated the ability to enhance cell proliferation when compared with the naked proteins (p< 0.001).

Ten days after the first (day 10) and the booster immunization (day 20), lymphocytes were separated by the commercial separation solution and sorted by flow cytometry. As evaluated in Figures 11a, c , PLGA nanoparticles as well as two types of emulsions could remarkably enhance the proportions of CD4⁺ T lymphocytes in animals’ spleens (p< 0.05). When compared with rTgGAP45, both TgGAP45-PLGA nanoparticles and TgGAP45-206VG emulsions exhibited a capability in enhancing the proportions of CD3e⁺CD4⁺ cells. As for the CD8⁺ T lymphocytes demonstrated in Figures 11b, c , three types of vaccines could notably upregulate the proportions of CD8⁺ T lymphocytes (p< 0.05), while TgGAP45-PLGA nanoparticles and TgGAP45-206VG emulsions could enhance the proportions of CD8⁺ T lymphocytes higher than rTgGAP45 itself after the second immunization (p< 0.05).

All groups of mice were challenged intraperitoneally with 100 tachyzoites on day 20 (10 days after the booster immunizations). Four days later (day 24), three randomly selected animals from each group were euthanized to isolate the spleen tissues, and the indirect IFA was carried out to analyze T. gondii burdens. As demonstrated in Figure 12 , animals immunized with TgGAP45 and its preparations were detected with less parasite burdens in the spleen tissues, which suggested that three types of preparations could provide partial resistance against T. gondii to some extent.

To further investigate the parasite burdens in immunized animals, five mice were euthanized 6 days after the challenge (day 26), and the cardiac tissues were collected. Then, qPCR was performed to analyze T. gondii burdens in the cardiac tissues of challenged animals. Compared with controls, the parasite burdens in TgGAP45, TgGAP45-PLGA, TgGAP45-206VG, and TgGAP45-660VG groups were respectively reduced by 44.45%, 86.76%, 57.80%, and 72.47% ( Figure 13 ), and naked TgGAP45 proteins as well as their preparations could notably reduce the parasite burdens in cardiac tissues when compared with controls (p< 0.01). In addition, in comparison with recombinant TgGAP45 proteins alone, TgGAP45-PLGA nanoparticles could remarkably decrease the replications of T. gondii in immunized animals (p< 0.05).