Vaccination with toxofilin DNA in combination with an alum-monophosphoryl lipid A mixed adjuvant induces significant protective immunity against Toxoplasma gondii

The gene and amino acid sequences of toxofilin were identified from GenBank (http://​www.​ncbi.​nlm.​nih.​gov/​genbank), the primary characteristics of the gene encoding protein were analyzed by ProtParam (http://​web.​expasy.​org/​protparam/​), the primary amino acid sequences of toxofilin homologs were downloaded from the National Center for Biotechnology Information, and the multiple sequence alignment was generated using the Clustal W method in DNASTAR MegAlign software.

B-cell epitopes are recognized in their native structure by B-cell receptors or antibodies. Continuous B cell epitope prediction is mainly according to the amino acid properties. However the discontinuous B cell epitope prediction requires 3D structure of the antigen [21‐23]. In this study, the complete amino acid sequence and the secondary structure of toxofilin were analyzed using DNASTAR [24]. A higher score of toxofilin corresponded to a higher probability of the residue being involved in the epitope, using DNAMA software. The SOPMA and I-TASSER online services were used to construct the three-dimensional (3D) structures of the toxofilin epitopes [25].

The Immune Epitope Database online service (http://​tools.​immuneepitope.​org/​analyze/​html/​mhc_​II_​binding.​html) was used to predict MHC II molecules of toxofilin. It is important to note, however, that such binding to MHC is necessary but not sufficient for recognition by T cells [26].

HEK239T cells stored at −80 °C were thawed for 1 min at 37 °C followed by culture in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 IU/mL penicillin at 37 °C in 5% CO₂.

Macrophages were cultured at 37 °C in DMEM with 4 mM l-glutamine (Dutscher) supplemented with 10% heat-inactivated fetal calf serum (Dutscher) and Hepes 10 mM (Dutscher), in a 5% CO₂ atmosphere.

Six- to eight-week-old female BALB/c mice were purchased from Shandong University Laboratory Animal Centre (Jinan, China). All animal experiments were approved with the Animal Ethics Committee of Shandong University under Contract 2011–0015.

We used two strains of T. gondii (RH and PRU) in this study. The low virulent strain (PRU strain) of T. gondii was kept in our laboratory by passage of cysts in Kunming mice. The tachyzoites of the RH strain of T. gondii, which was preserved at −80 °C, were obtained by passage on macrophages. About 1 × 10⁴ tachyzoites of intermediate virulence were collected from one 225-cm² culture flask, corresponding to 1 μg of TE. Tachyzoites of highly virulent T. gondii were collected to challenge mice.

The T. gondii toxofilin gene coding sequence [GenBank: AJ132777.2] was amplified by PCR from genomic parasite DNA using the following specific primer pair. Toxofilin for eukaryotic expression plasmid construction: forward primer: 5′-CGGGGTACCATGGCGCAATACAAGTCACG-3’(Kpn I), reverse primer: 5’-CG GGATCCTTACGACGAGGGCATAGCG-3’(BamH I). Toxofilin for prokaryotic expr -ession plasmid construction: forward primer: 5′-CGGGGTACCATGGCGCAA TACAAGTCACG-3’(Kpn I), reverse primer: 5′-CGGCGGCCGCTTACGACGA GGGCATAGCG-3’′ (Not I) (recognition sites for Kpn I, BamH I and Not I, respectively, are underlined). The amplified PCR products were cloned into the eukaryotic expression vector pEGFP-C1 (TransGen, China) and the prokaryotic expression plasmid pET-30a(+) to formed a recombinant plasmid, pEGFP-toxofilin and pET30-toxofilin.

pEGFP-toxofilin plasmids were transformed into Escherichia coli DH5 by the way of hot shot (42 °C.). Expression of two genes in transformed DH5α cells was screened on LK solid medium, followed by choosing a single colony to cultivate for harvest of recombinant plasmids. All the recombinant plasmids were extracted by an endotoxin-free plasmid purification kit (Tiangen, China) and stored at −20 °C until use. The plasmids concentrations were determined by A260/A280 measurement.

HEK239T cells grown in 6-well plates were respectively transfected with recombinant eukaryotic plasmid (pEGFP-toxofilin) or an empty plasmid pEGFP-C1 with the assistance of LipofectamineTM 2000 reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. Protein expression in vitro was observed by specific green fluorescence on HEK293T cells after incubation for 24 h at 37 °C in a 5% CO₂ incubator. At 36 h post-transfection, the cells were rinsed twice with PBS, handled with lysis buffer (Sigma, USA), and centrifuged at 16,000 × g for 30 min. The lysates of transfected cells were analyzed by western blotting.

Escherichia coli DH5α strain cells were transformed by recombinant pET30-toxofilin and grown in Luria Bertani (LB) at 37 °C until the cell density reached an absorbance of 0.6 (at λ = 600 nm). Synthesis of recombinant pET30-toxofilin protein was induced with 1 mM isopropyl-β-d-thiogalactoside (IPTG) for 6 h and 8 h at 25 °C and the growth for the next 3 h at 37 °C. The cells were lysed and the protein was purified. The endotoxin was removed with the ToxinEraser™ Endotoxin Removal Kit and measured by the Chromogenic End-point Endotoxin Assay Kit (Chinese Horseshoe Crab Reagent Manufactory, Xiamen, China). Expression of the pET30-toxofilin protein was analyzed by western blotting and kept at −80 °C for further use.

The immunization experiments that pEGFP-toxofilin DNA was used as the vaccine were performed in 6- to 8-week-old female BALB/c mice, which were randomly divided into eight groups (n = 25/group). Mice were injected intramuscularly three times at fortnightly intervals with the following four experimental immunizations: non-adjuvanted toxofilin (50 μg toxofilin DNA + 100 μL PBS/mouse); alum-toxofilin (50 μg toxofilin DNA + 50 μg alum + 50 μL PBS/mouse); MPLA-toxofilin (50 μg toxofilin DNA + 50 μg MPLA + 50 μL PBS/mouse); alum-MPLA-toxofilin (50 μg toxofilin DNA + 50 μg MPLA + 50 μL PBS/mouse); and 4 control groups, with mice receiving 150 μL PBS, 150 μg alum, 150 μg MPLA, 150 μg alum-MPLA. The blood of mice in each group was collected at 0, 2, 4, and 6 weeks by orbital blood and sera were stored at −20 °C until used for subsequent analysis. Two weeks after the final inoculation, 12 mice in each group were challenged i.p. with 1 × 10⁴  T. gondii RH strain tachyzoites, and the remaining mice in all groups were inoculated with 20 cysts of the attenuated virulent PRU strain orally. The individual survival time injected with RH strain along with the overall percentage survival were recorded. The dead mice infected RH strain were treated by high pressure sterilization and sent to the animal experiment center for further processing. The brain cysts were determined one month later after the challenge infection with 20 cysts of the attenuated virulent PRU strain orally for a one time. Each brain was homogenized in 2 ml PBS, and the mean number of cysts per brain was calculated. This analysis was performed in three independent experiments.

Anti-T. gondii IgG, IgG1, and IgG2a antibodies were detected using enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer’s instructions (Southern Biotech Co., Ltd, Birmingham, USA). Briefly, microtiter 96-well plates were coated overnight at 4 °C with pET30-toxofilin protein antigen (diluted in 0.1 M carbonate buffer (pH 9.5) for optimal binding. The plates were washed three times with PBS containing 0.05% Tween20 (PBST) and then blocked with 1% bovine serum albumin for 1 h at 37 °C with gentle shaking. After three washes with PBST, the plates were incubated with mouse sera diluted 1:100 in PBS for 1 h, followed by incubation with goat anti-mouse IgG, IgG1, or IgG2a secondary antibodies for 1 h to detect the target antibody and isotype control, respectively. Finally, the immune enzymatic reaction complexes were visualized by incubating with ortho-phenylenediamine (Sigma, USA) and 0.15% H₂O₂ for 30 min. Reactions were stopped by adding 2 M H₂SO_4, and the results were read at OD450 nm using an ELISA plate reader. An average of three independent ELISAs for each serum sample was recorded.

Cytokine levels were determined as previously described [16]. Three mice per group were euthanized and their splenocytes aseptically harvested after two weeks after the last immunization, Cells were dispensed into 96-well plates at a density of 5 × 10⁵ cells at 37 °C with 95% relative humidity in 5% CO₂. Cell-free supernatants were harvested and assayed for interleukin (IL)-4, IL-10, and IFN-γ at 24 h, 72 h, and 96 h, respectively, using a commercial ELISA kit. All measurements were run in triplicate.

By flow cytomety analysis, the percentage of CD4+ and CD8+ T cell in spleen were determined. The cell concentration was adjusted to 1 × 10⁶ cells/ml in PBS containing 2% FBS. After incubation with FITC-conjugated anti-mouse CD4+ monoclonal antibody (mAb), PE-conjugated anti-mouse CD3+ mAb and Cy5.5-conjugated anti-mouse CD8+ mAb (eBioscience) at 4 °C for 30 min in the dark. The cultures were washed by 2 mL PBS and the suspensions were analyzed using SYSTEM II software (Coulter) through FACScan flow cytometer (BD Biosciences, San Jose, CA, USA).

The toxofilin protein of T. gondii (Tg-toxofilin) is a 245 amino acid protein with a molecular weight of 27.1317 kDa. Its physical and chemical properties include a theoretical PI of 9.57, an instability index of 60.41, an aliphatic index of 84.98, and a grand average of hydropathicity of −0.63. For four different T. gondii strains, the protein sequence alignment is shown in Fig. 1. and share 96.53% similarity, with toxofilin of the T. gondii RH strain sharing 100%, 91.84%, and 94.29% sequence identity with GT1, ME49, and VANDPR strains, respectively.

B-cell epitopes are the portions of antigens that are recognized by the variable regions of antibodies. Accurately predicting epitopes is of great help for designing immunogenic peptides and new vaccines. In the present study, the DNASTAR software was used to predict antigenic index and surface probability, as shown in Fig. 2. Based on this analysis, the peptides with good hydrophilicity, high accessibility, high flexibility, and strong antigenicity were chosen as antigen epitopes. To further confirm these results, the DNAMAN software was used to analyze these sequences and nine potential epitopes were chosen with the highest antigen index scores, as shown in Table 1.

A potentially antigenic region of a protein increases the epitope prediction and promote a better understanding the 3D structure, contributing to the elucidation of the relationship between structure and function. The SOPMA and I-TASSER online services were used to predict the 3D structures of toxofilin alone and in complex with mammalian actin (Fig. 3 A1 and A2).

The Th-cell epitopes identified on toxofilin by bioinformatic analyses reportedly have the ability to bind strongly to MHC class II molecules (Table 2). It is known that the binding force increases can influence Th-cell differentiation and promote more cells to differentiate into Th-1 cells [15]. Therefore, we speculate that toxofilin has the potential to induce Th-1 cell-mediated immune responses.

Forty-eight hours after transfection of HEK cells, the presence of pEGFP-toxofilin or pEGFP-C1 was determined by fluorescence microscopy. As shown in Fig. 4(A1) and (A2), green fluorescence was observed in HEK293T cells transfected with pEGFP-toxofilin and pEGFP-C1, whereas there was no signal in the untransfected cells Fig. 4 (A3). Western blot analysis of these proteins was shown in Fig. 4B; a specific protein band (about 27 kDa) was recognized in cells transfected with pEGFP-toxofilin by incubation with a T. gondii antibody (lanes 2), whereas the negative controls transfected with empty pEGFP vector showed no specific bands upon incubation with a T. gondii antibodies (lane 1). The purified recombinant pET30-toxofilin vector was used to produce toxofilin protein antigen. Western blot technique by using a T. gondii antibody was performed to verify the toxofilin antigen (lane4).

Sera induced in T. gondii immunized mice were tested post-immunization by standard ELISA at weeks 0, 2, 4, and 6. As shown in Fig. 5, significantly higher levels of total IgG antibodies were detected in the sera of mice immunized in the experimental groups compared with the control immunized groups, especially after the third immunization. Moreover, the mean titers of IgG antibodies induced in the alum-MPLA- toxofilin group were significantly higher after both priming and booster immunization compared with the levels induced by toxofilin alone DNA vaccine or with single adjuvant. In contrast, the total IgG levels were similar between alum-toxofilin and MPLA-toxofilin groups.

Serum levels of IgG1 and IgG2a, which are characteristic of Th2 and Th1 responses respectively, were analyzed by ELISA after the last immunization. As shown in Fig. 6, the non-adjuvanted toxofilin DNA vaccine group demonstrated similar levels of IgG1 and higher levels of IgG2a compared with control groups. However, compared with the non-adjuvanted toxofilin DNA vaccine, the alum-toxofilin group showed a significant improvement in the IgG1 level, indicative of a Th2-skewed immune response. Conversely, the MPLA-toxofilin group induced higher levels of IgG2a, and is therefore more likely to induce a Th1-biased immune response. Importantly, significantly higher serum levels of IgG2a and a slight increase in IgG1 were detected in the alum-MPLA-toxofilin immunized group when compared with other groups, suggesting that this combination of DNA vaccine and adjuvant may be most efficacious in raising a Th1 response following immunization.

The sera samples collected were used to measure the levels of IFN-γ, IL-4, and IL-10 induced by immunization with toxofilin DNA vaccine in the presence or absence of adjuvants. Generally, IFN-γ favors Th1-type immune responses, whereas IL-4 and IL-10 drive Th2 skewing [16]. As demonstrated in Table 3, values of IFN-γ in toxofilin DNA vaccine, alum- toxofilin, and MPLA- toxofilin immunization groups were 607.29 ± 63.09 pg/mL, 694.39 ± 88.64 pg/mL, and 3043.54 ± 350.72 pg/mL, respectively, which were significantly higher than in the control group of PBS (48.54 ± 2.40 pg/mL), alum (49.58 ± 2.37 pg/mL), MPLA (218.34 ± 21.34 pg/mL), alum-MPLA (267.34 ± 24.35 pg/mL). Furthermore, the highest level of IFN-γ was detected at 3859.42 ± 380.67 pg/mL in the alum-MPLA-toxofilin group. Similarly, elevated levels of IL-4 and IL-10 were detected in the alum-toxofilin, MPLA- toxofilin, and alum-MPLA-toxofilin groups. However, significantly higher concentrations of both cytokines (IL-4 236.473 ± 24.05 pg/mL, IL-10 212.32 ± 30.27 pg/mL) were detected in the alum-toxofilin group, compared with the other groups.

The Splenocyte proliferation was evaluated in mice two weeks after the last immunization. As shown in Table 4, CD8+ T cells accounted for a significantly higher proportion than that in the control groups and a significant higher percentage of CD8+ T cells were dramatically increased in the alum-MPLA- toxofilin mixture group. The result also showed same statistically difference in the percentage of CD4+ T cells between experimental groups (toxofilin, alum-toxofilin, MPLA-toxofilin and alum -MPLA -toxofilin immunization groups) and the control groups.

The survival curves against acute toxoplasmosis are shown in Fig. 7, and notably prolonged survival times were recorded in the experimentally immunized mice in comparison with the control groups (P < 0.05). Among the different experimental groups, mice vaccinated with the alum-MPLA-toxofilin had obviously prolonged survival, with 50% of mice surviving to day 24 of challenge.

“One month after challenge with 20 cysts of PRU strain of T. gondii, surviving mice were killed and brain cysts were enumerated.” The cyst number (1034.49 ± 45.87) in the mice immunized with alum-MPLA-toxofilin was significantly reduced compared to other groups (Table 5).