Intranasal immunization with recombinant Toxoplasma gondii actin depolymerizing factor confers protective efficacy against toxoplasmosis in mice

Specific-pathogen-free female BALB/c mice (4–6 weeks of age) were purchased from the Chinese Academy of Medical Science Animal Center (Beijing, China). All mice were bred with food and water provided ad libitum. Tachyzoites of the T. gondii RH strain were stored in liquid nitrogen in the laboratory and maintained by intraperitoneal passaging in BALB/c mice, as described previously [19]. This study was conducted according to the Guidelines for the Laboratory Animal Use and Care Committee of the Ministry of Health, China and the Ethics Committee on Animal Research of Xuzhou Medical University (No. SCXK < SU > 2014–0003).

Total RNA of T. gondii tachyzoites was extracted using the TRIzol reagent (life,CA, USA). The complete open reading frame (ORF) of TgADF (GenBank: U62146.1) was amplified using polymerase chain reaction (PCR) from the cDNA template with specific primers (forward primer: 5′-ACGCGGATCCATGGCGTCCGGAATGGGTG-3′ and reverse primer: 5′-ACCGCTCGAGCGCGAGGGGTGCGAGGTC-3′), in which BamHI and XhoI were introduced (underlined). The following PCR conditions were used: 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s and 72 °C for 7 min. The PCR products were cloned into the prokaryotic expression vector pET30a(+) to obtain pET-30a(+)-TgADF used to transform Escherichia coli DH5α competent cells. The positive plasmids were confirmed using restriction enzyme digestion, PCR, and DNA sequencing.

E. coli BL21/DE3 transformed with pET-30a(+)-TgADF were grown in Luria-Bertani (LB) medium supplemented kanamycin (50 μg/mL) at 37 °C until an optical density (OD₆₀₀) of 0.4–0.6 was achieved. The expression was induced by adding 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and maintaining the medium at 25 °C for 12 h. The bacterial pellets were harvested and lysed by sonication in an iced water bath for 15 min (power, 200 W; pulse on, 5 s; pulse off, 15 s). To prevent protein degradation, all subsequent steps were performed at 4 °C. After centrifugation at 8,000 × g for 15 min, the supernatant was mixed with Ni-NTA agarose (Qiagen, Germany) for 1 h. The column was washed with a buffer containing 20 mM imidazole, 50 mM NaH₂PO₄, and 300 mM NaCl. The target protein was eluted by increasing the imidazole concentration to 150 mM. The endotoxin was removed and detected using the ToxinEraser™ Endotoxin Removal Kit and Chromogenic End-point Endotoxin Assay Kit (Chinese Horseshoe Crab Reagent Manufactory, Xiamen, China). Purified rTgADF was stored at –80 °C until further use.

The mice were randomly divided into 5 groups (8 mice per group). Four groups were intranasally administered with 10, 20, 30, or 40 μg of rTgADF that was separately dissolved in 20 μL of phosphate-buffered saline (PBS). The control group was immunized with PBS. On days 0, 14, and 21, the nostrils of the mice were slowly instilled with rTgADF protein solution (10 μL per nostril). Two weeks after the last immunization, blood was drawn from the retro-orbital plexus of the mice, centrifuged at 3,000 × g for 10 min, and the serum was isolated. The nasopharyngeal region, small intestine, and urinary bladder of all the mice were exposed and washed according to a previously described method [20]. Nasal, intestinal, and vesical washes were centrifuged at 3,000 × g for 10 min, and collected in Eppendorf tubes. All samples were stored at –20 °C for evaluation of antibody content.

Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of rTgADF-specific antibody, IgG in the serum, and secretory IgA (sIgA) in the mucosal washes [21]. A 96-well plate was coated with rTgADF at a concentration of 5 μg/mL. The serum was diluted 1:300 for IgG, and the mucosal washes were not diluted for sIgA. HRP-conjugated goat anti-mouse IgG (diluted 1:2500) or IgA (diluted 1:1000) was used as the secondary antibody. Absorbance was read at 492 nm by using a microplate reader (Bio-Tek, VT, USA). The average value of three independent experiments was recorded for each sample.

The immunized mice were dissected under aseptic conditions. The spleens were harvested, gently ground, and then passed through stainless-steel meshes in PBS. Splenocyte suspensions were prepared after the removal of erythrocytes [22]. The cells were seeded in 24-well microtiter plates at a density of 1.5 × 10⁶ cells per well and stimulated with rTgADF (10 μg/mL). The plates were incubated in 5 % CO₂ at 37 °C. Concentrations of IL-2 and IL-4 at 24 h, IL-10 at 72 h, and IFN-γ at 96 h in the harvested cell-free supernatants were assayed using ELISA kits (PeproTech, USA), according to the manufacturer’s instructions.

The isolated splenocytes were plated at 5 × 10⁵ cells per well in 96-well plates and incubated with a cell-culture medium, concanavalin A (Con A; 5 μg/mL), and rTgADF (10 μg/mL). Cell proliferative activity was measured using the cell counting kit CCK-8 (Dojindo, Japan), according to the manufacturer’s instructions. Absorbance was measured at 450 nm. The stimulation index (SI) was calculated as follows: SI = average OD₄₅₀ values from stimulated cultures/average OD₄₅₀ values from non-stimulated cultures.

Two groups of BALB/c mice (30 mice per group) were immunized with PBS or 30 μg of rTgADF, as mentioned above. Twenty-two mice from each group were orally infected (intragastric administration) with a dose of 4 × 10⁴ tachyzoites for the acute assay. Thereafter, survival times of the infected mice were recorded daily. The other 8 mice per group were challenged with 1 × 10⁴ tachyzoites for the chronic model. Four weeks later, real-time PCR was used to quantify tachyzoite loads in the liver and brain for detecting the SAG1 gene, as previously described [23].

The full-length ORF sequence of TgADF was amplified using RT-PCR, and the expected size was 357 bp (Fig. 1a). TgADF was inserted into pET-30a(+) and verified using PCR, restriction endonuclease digestion, and sequencing. After induction with 0.1 mM IPTG at 25 °C, rTgADF was expressed successfully in E. coli and then purified using Ni-NTA agarose. SDS-PAGE showed that the isolated protein was soluble and approximately 20 kDa, which is the molecular weight of the expressed rTgADF protein and a polyhistidine tag (Fig. 1b). Western blotting indicated that the purified rTgADF protein was able to combine with the anti-His antibody and rabbit anti-T. gondii serum (Fig. 1c).

We estimated nasal, intestinal, and vesical sIgA as indexes for mucosal immune response. Significantly higher levels of sIgA titers were observed in the nasal, intestinal, and vesical washes in mice immunized with 20, 30, or 40 μg of rTgADF than in the control groups (P < 0.05; Fig. 2). The sIgA levels in three mucosal washes in the 30- and 40-μg groups were prominently higher than that in the 20-μg group. Moreover, an apparent predominance of the 30-μg group over the 40-μg group was observed on the basis of the vesical washes. Results of the sIgA antibody levels in the mucosal washes demonstrated that 30 μg of rTgADF elicited the strongest response.

Humoral and cellular immune responses were evaluated by analyzing IgG antibody levels in the serum, splenocyte proliferation, and cytokine production. High IgG titers were detected in the serum samples of all immunized mice (P < 0.05; Fig. 3). The OD values for IgG were continuously increased following an increase in the immunization dosage, and the 40-μg group showed the highest titer among all the immunized groups (P < 0.01). Splenocytes from the 30-μg and 40-μg groups exhibited a significantly greater proliferative response to rTgADF than splenocytes from the control group. No statistically significant differences were detected in the 10-μg, 20-μg, and control groups. Meanwhile, splenocytes from all groups proliferated to comparable levels in response to ConA (Table 1). In addition, we observed that splenocytes from all the immunized groups secreted significantly high levels of IFN-γ and IL-2 when compared with the control group, and the highest levels were elicited by 30 μg of rTgADF (Fig. 4a and b). In contrast, IL-4 and IL-10 levels displayed no significant changes between the immunized and control groups (P > 0.05; Fig. 4c and d).

Survival curves of the mice are shown in Fig. 5a. After the oral challenge with T. gondii, the mice showed symptoms such as vertical hair, lassitude, and lethargic and decreased appetite. Most of the mice in the control group were dead within 9 days post-infection, with the last mouse dying on the 23rd day. However, 36.36 % of the mice immunized with 30 μg of rTgADF remained alive 30 days post-infection. Similarly, rTgADF immunization protected against chronic infection. The tachyzoite load in the livers and brains was significantly lower in the immunized mice {13.89 ± 1.27 (10⁵/g) and 6.33 ± 0.43 (10⁵/g), respectively)} than in the control mice {43.09 ± 3.03 (10⁵/g) and 12.92 ± 3.30 (10⁵/g), respectively}, which showed a reduction in the tachyzoite load by 67.77 % in the liver and 51.01 % in the brain when compared with the control group (Fig. 5b).