Epitope analysis, expression and protection of SAG5A vaccine against Toxoplasma gondii

Acta Tropica 146 (2015) 66–72

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Epitope analysis, expression and protection of SAG5A vaccine against Toxoplasma gondii Gang Lu a,# , Lin Wang a,# , Aihua Zhou b,∗ , Yali Han a , Jingjing Guo a , Pengxia Song a , Huaiyu Zhou a , Hua Cong a , Qunli Zhao a , Shenyi He a,∗ a

Department of Parasitology, Shandong University School of Medicine, Jinan, Shandong Province 250012, People’s Republic of China Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University School of Medicine, Jinan, Shandong Province 250021, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 1 December 2014 Received in revised form 6 March 2015 Accepted 9 March 2015 Available online 16 March 2015 Keywords: DNA vaccine Immunity Peptide SAG5A Toxoplasma gondii

a b s t r a c t Bioinformatics approaches were used to identify B-cell epitopes and T-cell epitopes on SAG5A protein. Compared to SAG1, SAG5A with good B-cell epitopes and T-cell epitopes had a potentiality to become a more successful vaccine against Toxoplasma gondii. Thereafter, SAG5A DNA vaccine was constructed successfully and was injected into mice with peptide to evaluate the immunoprotection. Compared to the control groups, the vaccine (DNA/peptide) could induce more effective cellular and humoral immune responses in immunized mice. Furthermore, a significant reduction of brain cyst was detected in the mice vaccinated with peptide (732 ± 160), pSAG5A (815 ± 197), or pSAG5A/peptide (436 ± 174) compared by the mice injected by PBS (1260 ± 241) or pEGFP-C1 (1350 ± 268). The number of cysts in brains was 35% reduced in the mice immunized with DNA/peptide than in the control mice treated by PBS. The results indicated that the DNA vaccine encoding SAG5A significantly induced immune responses and enhanced protection against cysts of PRU strain, especially with the help of peptide. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The phylum Apicomplexa includes a large number of obligate intracellular parasites, many of which are important sources of morbidity and mortality in humans and animals. Toxoplasma is a leading source of congenital neurological birth defects and a prominent opportunistic infection in HIV-infected patients (Hill and Dubey, 2002). Toxoplasmosis causes abortions, stillbirths, and neonatal deaths in livestock, leading to serious economic losses, and infected livestock is a major source of infection to humans (Bhopale, 2003). Many studies indicate that DNA vaccines could be an excellent solution because the vaccines could elicit long-lasting humoral and cell-mediated immunity, as well as provide protection against parasitic infections (Alarcon et al., 1999). In addition, previous studies largely focused on tachyzoite of virulent strain (RH strain) and

∗ Corresponding author. Tel.: +86 0531 88382042. E-mail addresses: [email protected] (G. Lu), [email protected] (L. Wang), [email protected] (A. Zhou), [email protected] (Y. Han), guojingjing [email protected] (J. Guo), [email protected] (P. Song), [email protected] (H. Zhou), [email protected] (H. Cong), [email protected] (Q. Zhao), [email protected] (S. He). # Equal contribution http://dx.doi.org/10.1016/j.actatropica.2015.03.013 0001-706X/© 2015 Elsevier B.V. All rights reserved.

used the method of intraperitoneal injection to evaluate the level of immunoprotection (Meng et al., 2012; Wang et al., 2014; Zhao et al., 2013). Bradyzoite plays a decisive role in spreading infection among mammals, so it is necessary to construct a vaccine against the bradyzoite stage of T. gondii. The parasite has evolved novel mechanisms for invasion and intracellular survival, and secretion from “apical organelles” is associated with establishment of an intracellular “parasitophorous vacuole” (Carruthers and Sibley, 1997; Bradley and Sibley, 2007). Notably, the surface proteins of parasite play an important role in the initial recognition of the target cell. The molecules associated with host-cell attachment have proven to be glycosylphosphatidylinositol (GPI)-anchored polypeptides exposed on the plasma membrane of tachyzoites and bradyzoites in T. gondii. Most of these polypeptides were from the SAG1 family, a group of molecules sharing sequence and structural similarity (Lekutis et al., 2001). SAG5, SAG1-like sequence branch, contains five subtypes from SAG5A to SAG5E. SAG5D protein is not expressed in bradyzoites (Tinti et al., 2003), while SAG5A, -5B, and -5C are all transcribed in T. gondii tachyzoites and bradyzoites (Elsheikha and Zhao, 2008; Elsheikha et al., 2008). Furthermore, the genes were demonstrated to be expressed on the surface of bradyzoites and played an important role in persistence of cysts in the host. In order to verify the antigenicity and immune protection, bioinformatics

G. Lu et al. / Acta Tropica 146 (2015) 66–72

approaches were used to analyze the SAG5 genes and the proteins. Bioinformatics is playing a significant role in predicting protein structures and biological characteristics, and has been fully used in the analysis of protein epitopes. In the present study, the linear-B cell epitopes and Th-cell epitopes of SAG5A were analyzed and compared with SAG1 using bioinformatics. Furthermore, we constructed the recombinant eukaryotic plasmid DNA vaccine pEGFP-SAG5A (pSAG5A) and evaluated the ability to protect mice from the cyst-forming PRU strain of T. gondii. In order to enhance the immune response of the vaccine, the DNA prime-peptide boost vaccination regime (Meng et al., 2013) was used.

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The PCR product amplified from the SAG5A cDNA was inserted into a pEASY-T1 vector (TransGen Biotech, China) to build a recombinant cloning plasmid. After sequencing, SAG5A was subcloned into a eukaryotic expression plasmid pEGFP-C1 (Novagen, USA) to obtain pSAG5A. Lastly, the new recombinant plasmids were transfected into HEK 293-T cells using LipofectamineTM 2000 reagent (Invitrogen, USA). A good supply of plasmids was obtained by the Endotoxin-Free Mega kit in accordance with the manufacturer’s instructions (Qiagen, Hilden, Germany), and the concentrations of pEGFP-C1 and pSAG5A were detected by A260/A280 measurement. All the plasmids were diluted into 1 mg/ml by sterile endotoxin-free PBS and stored at −20 ◦ C before use.

2. Materials and methods 2.1. Prediction of linear-B cell epitopes and Th-cell epitopes Epitopes are the bases of protein antigenicity that determines antigen specificity (Gao et al., 2012; Van Regenmortel, 2009). The linear-B cell epitopes of SAG5A were analyzed by DNASTAR software. The PROTEAN subroutine was used to predict antigenic index and surface probability of SAG5A. The peptides that had good antigenic index and surface probability were chosen. Moreover, DNAMAN software was used to search for linear-B cell epitopes on SAG5A amino acid sequences. Given T. gondii is a kind of intracellular parasite, cellular immunity mediated by T cells plays an important role in T. gondii infection (El-Kady, 2011). To construct a positive vaccine against T. gondii, it is essential to explain which type of Th cellmediated immune response. The Immune Epitope Database (IEDB) (http://tools.immuneepitope.org/mhcii) online service was used to analyze the half maximal inhibitory concentration (IC50) values of peptides that bind to the major histocompatibility complex (MHC) class II molecules of SAG5A (Table 2). 2.2. The mice and parasites Eight-week-old female BALB/c mice were purchased from Shandong University Laboratory Animal Center. They all were bred in groups of 10 per cage under specific-pathogen-free conditions and had free access to diet and tap water. All of the animal experiments were approved by the Ethics Committee on Animal Experiments of the Medical School of Shandong University. The low virulent strain (PRU strain) of T. gondii was maintained in our laboratory by passage of cysts in Kunming mice. The T. gondii tachyzoites were used to create soluble tachyzoite antigens after washed by centrifugation and resuspended in sterile PBS. The parasite suspension was sonicated and centrifuged at 1000 rpm for 20 min. Supernatant containing soluble tachyzoites antigens (STAg) was collected and kept at −70 ◦ C for further use (Zhang et al., 2013). 2.3. Plasmid construction and preparation The entire SAG5A open reading frame (ORF) was amplified by PCR from the cDNA of T. gondii (PRU strain) tachyzoites with primer (5 - cggGGTACCATGAAAAATACTCTGTTGTC -3 ) (forward) and primer (5 - cgGGATCCTCAAAATTGATGCGTTCT -3 ) (reverse). The two primers contain Kpn I and BamH I restriction sites (underlined), respectively. Trans TagTM High Fidelity DNA Polymerase (TransGen Biotech, China) was used in PCR amplification. The amplification was performed with the selected conditions: 1 cycle of 95 ◦ C for 5 min then 30 cycles of 95 ◦ C for 30 s, 60 ◦ C for 45 s, and 72 ◦ C for 30 s. Final primer extension was extended to 10 min at 72 ◦ C. PCR product was detected by electrophoresis on 1.0% agarose gel.

2.4. Preparation of polypeptide In this study, B-cell epitopes and T-cell epitopes of SAG5A were analyzed with softwares and online service. Peptide sequence containing outstanding B-cell epitopes and T-cell epitopes were selected and synthesized. Peptide 325–339 (HAPTPSFLGLLAVVF) of SAG5A protein was synthesized by Sheng Gong Biotechnology Company (Shanghai, China), and the analytic HPLC (High Performance Liquid Chromatography) was used to purify the peptide. 2.5. Expression of SAG5A in HEK 293-T cells HEK 293-T cells were maintained in a humidified 5% CO2 atmosphere at 37 ◦ C in six-well plates (Costar, USA) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with streptomycin (100 mg/ml), penicillin (100 IU/ml) and 10% fetal Bovine serum (FBS). When the density of HEK 293-T cells reached 80–90%, the recombinant eukaryotic expression plasmids (pSAG5A) and the original vectors (pEGFP-C1) were transfected into cells with the Lipofectamine 2000 regent (Invitrogen, USA) according to the manufacturer’s guidance. Plasmids were mixed with lipofectamine 2000 reagent at a concentration of 10 ␮g/ml in DMEM without antibiotics and FBS. The mixed solutions were incubated at room temperature for 20 min before added into HEK 293-T cells drop by drop. The cells were incubated with the solutions for 6 h in a humidified 5% CO2 atmosphere at 37 ◦ C. Finally, fresh cell culture fluid was added and the six-well plates were returned to the cell incubator for 48 h incubation. The cells from different groups (control, pEGFPC1 and pSAG5A) were, respectively, observed using fluorescence microscope under blue laser after incubation. 2.6. DNA immunization and challenge All the female mice were divided randomly into five groups (16 mice per group). Mice were injected twice at 2-week intervals with PBS (100 ␮l/each), pEGFP-C1 (100 ␮g/each), peptide (100 ␮g/each), pSAG5A (100 ␮g/each), or pSAG5A (100 ␮g/each)/peptide (100 ␮g/each). And all groups were injected intramuscularly four times. Furthermore, the last group was immunized by pSAG5A for the first two times and injected with peptide for the after two times. Blood samples of all groups were collected and stored at −20 ◦ C before assessing serum IgG levels at weeks 2, 4, 6 and 8. Four mice per group were sacrificed and splenocytes were harvested in aseptic condition for cytokine detections after 8 weeks. Two weeks after the last injection, mice were infected intragastrically with 20 cysts of T. gondii PRU strain. The brains were taken out from cranial cavities of mice and homogenized in 1 ml PBS. The number of cysts in each brain was detected by counting three samples of 10 ␮l mixture, and the average value was used to evaluate the effect of the vaccine against T. gondii.

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G. Lu et al. / Acta Tropica 146 (2015) 66–72

2.7. Assays of antibodies The antibody levels of T. gondii were detected using enzymelinked immunosorbent assay (ELISA) in the light of previous introduction. Briefly, the 96-well plates (Costar, USA) were coated by STAg (10 ␮g/well) and incubated at 4 ◦ C overnight. Plates were washed three times with special ELISA lotion and blocked with PBS containing 1% Bovine Serum Albumin (BSA) for 2 h at room temperature. After washing further three times, the plates were incubated with the mice sera diluted with PBS for 1 h at 37 ◦ C. After washing, plates were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (diluted 1:4000 in PBS – 1% BSA), IgG1 (1:2000), and IgG2a (1:2000) for 1 h at 37 ◦ C. After washing by ELISA lotion, orthophenylene diamine (Sigma, USA) and 0.15% H2 O2 were added. Plates then were incubated in the dark for 30 min at 37 ◦ C, and the reaction was stopped by adding 2 M H2 SO4 . The OD was measured at 490 nm using an ELISA reader (ELX800, USA). All samples were run for four times. 2.8. Measurement of cytokine In order to detect the levels of cytokine productions, spleens were isolated from four mice per group after 8 weeks. The spleen cells were cultured in 96-well plates at 37 ◦ C in 5% CO2 . Cell-free supernatants were harvested and assayed for interleukin-4 (IL-4) activity at 24 h, for interleukin-10 (IL-10) activity at 72 h, for gamma interferon (IFN-␥) activity at 96 h. The IL-4, IL-10, and IFN-␥ concentrations were measured by a commercial ELISA kit according to the manufacturer’s instructions (R&D Systems, USA). All samples were run for four times. 2.9. Statistical analysis SPSS 17.0 was used in the statistical analysis. Antibody levels and cytokine productions among the diverse groups were determined with a one-way analysis of variance. When a significant difference (P = 0.05) was observed among treatments, Tukey’s studentized range test was used for post-test comparisons. The difference was considered statistically significant if P < 0.05.

laboratory animals. All mice were maintained in specific pathogenfree conditions, and all efforts were made to minimize suffering. Humane endpoints to reduce pain or distress in mice were used via euthanasia. Mice were sacrificed immediately using CO2 gas before the brains were removed. Generally, mice were placed in a chamber and CO2 was administered at a concentration of 60–70% over a 5-min exposure time, after which the cervical dislocation method was sometimes used to ensure that effective euthanasia had occurred. 3. Results 3.1. Prediction of epitopes Epitopes are chemical groups which determine the feature of an antigen. T-cell antigen receptor and B-cell antigen receptor recognize T-cell epitopes and B-cell epitopes, respectively, in immune response. In the present study, antigenic index and surface probability were predicted by DNASTAR software. As shown in Fig. 1, SAG1, a good vaccine candidate protein, had excellent antigenic index and surface probability. The prediction result indicated that antigenic index of SAG5A was better than SAG1. Moreover, SAG5A had more significant surface probability than SAG1 in the analysis result. In addition, we used DNAMAN software to search for linear-B cell epitopes in SAG5A amino acid sequence. The result of the prediction indicated the presence of 16 potential epitopes on SAG5A in Table 1. The epitopes with scores above 1 were selected to be potential epitopes. In order to analyze Th-cell epitopes, the Immune Epitope Database (http://tools.immuneepitope.org/mhcii) online service was used. The half maximal inhibitory concentration (IC50) values of peptides binding to the major histocompatibility complex (MHC) class II molecules of SAG5A were analyzed by the online service. The Th-cell epitopes on SAG5A that were identified by bioinformatic analyses were predicted to have the ability to bind strongly to MHC class II molecules (Table 2). As shown in Table 2, the minimum percentile ranks of each MHC II alleles on SAG5A were chosen and listed.

2.10. Ethics statement

3.2. Identification and expression of the constructed plasmid

This study was approved by the Institutional Animal Care and Use Committee of Shandong University under Contract 2011-0015, and the animals were kept and the experiments were performed in accordance with committee’s criteria for the care and use of

The SAG5A gene was amplified by polymerase chain reaction and ligated into the eukaryotic expression vector pEGFP-C1 with suitable restriction enzymes to generate a new recombinant plasmid. Restriction analysis was performed to identify the plasmid.

Fig. 1. The linear-B cell epitopes of SAG1 and SAG5A predicted by DNASTAR in antigenic index and surface probability rules. The red line on SAG5A was the position of peptide 325–339 (HAPTPSFLGLLAVVF) used in this study. The prediction was run for three times. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

G. Lu et al. / Acta Tropica 146 (2015) 66–72

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Fig. 2. Direct immunofluorescence detection of the fusion protein in transfected HEK 293-T cells. (A) Cells transfected with pEGFP-C1 were detected under blue light; (B) Cells transfected by pSAG5A were detected under blue light; (C) Untransfected cells were detected under blue light. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

HEK 293-T cells were successfully transfected with pSAG5A or empty plasmid pEGFP-C1 for 48 h. In pSAG5A- and vector pEGFPC1-transfected cells, proteins emitted green fluorescence upon exposure to blue laser as observed using fluorescence microscopy (Fig. 2A,B and C), whereas no fluorescence was observed in control cells.

The levels of T. gondii-specific IgG antibodies induced by peptide and plasmids in mice were assayed by ELISA at weeks 2, 4, 6 and 8. As shown in Fig. 3, significantly high levels of IgG antibodies were assayed in the sera of mice vaccinated by peptide, pSAG5A, or pSAG5A/peptide following immunization, as compared to the control groups (P < 0.05). Mice injected with PBS or pEGFP-C1 did not generate T. gondii-specific IgG antibodies. Furthermore, the mice

Table 1 The linear-B cell epitopes in SAG1 and SAG5A amino acid sequences predicted by DNAMANa .

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

MHC II alleleb

HLA-DRB1*01:01 H2-IAb

3.3. Assays of antibody responses

Order

Table 2 IC50 values for SAG1 and SAG5A peptide binding to MHC class II molecules obtained using the immune epitope databasea .

Amino acid position

Scoreb

SAG1

SAG5A

SAG1

SAG5A

20–63 136–162 167–193 314–333 4–14 292–302 107–123 276–287 66–72 197–206 79–85 211–223 227–233 88–96 98–105 244–250

178–192 321–359c 7–47 195–204 50–63 295–310 236–247 253–265 82–104 281–287 146–161 209–217 163–169 123–130 108–117 132–138

1.179 1.175 1.172 1.164 1.157 1.156 1.138 1.131 1.127 1.109 1.109 1.103 1.075 1.075 1.070 1.063

1.230 1.228 1.212 1.185 1.180 1.176 1.152 1.149 1.148 1.130 1.128 1.122 1.113 1.095 1.092 1.034

a The prediction was run for three times. Two or more amino acids condense into a peptide. b High score = high binding. c The used peptide 325–339 (HAPTPSFLGLLAVVF) embedded in the sequence 321–359.

H2-IAd H2-IEd

Start–stopc

Percentile rankd

SAG1

SAG5A

SAG1

SAG5A

12–26 35–49 26–40 297–313 21–35 168–182 14–28 34–48

339–353 325–339e 330–344 132–146 8–22 325–339 161–175 318–332

0.88 2.74 2.15 2.81 0.34 1.22 18.45 30.62

0.42 1.53 1.77 3.71 0.48 0.73 17.38 20.23

a The immune epitope database (http://tools.immuneepitope.org/mhcii). The prediction was run for three times. b H2-IAb, H2-IAd and H2-IEd alleles are mouse MHC class II molecules; the HLADRB1*01:01 allele is a human MHC class II molecule. c We chose 15 amino acids for analysis each time. d Low percentile = high level binding, high percentile = low level binding. e The sequence of the peptide (HAPTPSFLGLLAVVF) used in the study.

Fig. 3. Measurement of specific IgG antibodies in sera of immunized mice. Sera were collected 2 days prior to each immunization and determined by ELISA. All samples were run for four times. The results are mean of 10 independent experiments and expressed as the mean of OD 490 ± SD. The statistical differences (P < 0.05) are indicated by * compared with PBS or pEGFP-C1.

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G. Lu et al. / Acta Tropica 146 (2015) 66–72 Table 4 The cysts in injected mice after challenge by cysts of PRU strain. Challenged groupa

Brain cysts per mouse (mean ± SD)b

PBS pEGFP-C1 pSAG5A peptide pSAG5A/peptide

1260 1350 815 732 436

± ± ± ± ±

241 268 197* 160* 174* , #

*

Compared with PBS or pEGFP-C1 group, P < 0.05. Compared with pSAG5A or peptide, P < 0.05. Ten mice from each group were challenged intragastrically by 20 cysts 2 weeks after the last immunization. All samples were run for four times. b The mean number of cysts of each group was obtained from every mice brain cysts in the group. #

a

Fig. 4. Detection of IgGl and IgG2a levels in the vaccinated mouse sera by ELISA. Immune sera were collected at 2 weeks after the last immunization and determined by ELISA. All samples were run for four times. The results are mean of 10 independent experiments and expressed as the mean of OD 490 ± SD. The statistical differences (P < 0.05) are indicated by * as compared to control groups.

injected with pSAG5A/peptide generated higher T. gondii-specific IgG antibodies than the other four groups (P < 0.05). There was no statistical difference between PBS and pEGFP-C1, pSAG5A and peptide (P > 0.05). The results strongly illustrated that a new plasmid encoding T. gondii SAG5A protein induced a strong T. gondii-specific IgG antibody response in mice. The levels of IgG subclass (IgG1 and IgG2a) in all groups at the second week after the last immunization were detected to determine whether a Th1 and/or Th2 response was elicited as presented in Fig. 4. An apparent predominance of IgG2a over IgG1 was assayed in the sera of vaccine-immunized mice, which demonstrated a shift toward the Th1 type response. Moreover, the level of IgG2a in mice vaccinated with pSAG5A/peptide was significantly increased, compared by mice injected using PBS, peptide, pEGFP-C1, or pSAG5A. There was no statistical difference in IgG2a levels between the groups immunized with pSAG5A or peptide (P > 0.05). The result indicated that peptide and pSAG5A immunized mice mainly generated a Th1 immune response. 3.4. Assay of cytokine production In order to determine whether the DNA vaccine augmented the Th1 or Th2 cytokine response, culture supernatants of splenocytes from injected mice were obtained 2 weeks after the final immunization and assayed for the production of IFN-␥, IL-4 and IL-10 activities. As shown in Table 3, compared to PBS (55.45 ± 11.7) and pEGFP-C1 (52.36 ± 11.6) immunized groups, the levels of IFN-␥ in mice injected by peptide (489.7 ± 98.9), pSAG5A (505.3 ± 71.62), Table 3 Cytokine production by splenocytea cultures from immunized BALB/c mice. Group

Cytokine production (pg/mL)b IFN-␥

PBS pEGFP-C1 pSAG5A peptide pSAG5A/peptide *

55.5 52.4 505.3 489.7 721.0

± ± ± ± ±

IL-4 11.7 11.6 71.6* 98.9* 99.6* , #

38.2 38.2 40.0 39.5 42.8

IL-10 ± ± ± ± ±

7.4 9.4 7.1 7.5 7.4

39 38.5 41.1 38.0 40.1

± ± ± ± ±

7.1 8.5 6.3 7.3 7.5

Compared with PBS or pEGFP-C1 group, P < 0.05. Compared with pSAG5A or peptide, P < 0.05. a Splenocytes from four mice per group 2 weeks after the final immunization. All samples were run for four times. b Values for IFN-␥ at 96 h, IL-4 at 24 h, IL-10 at 72 h are expressed as mean ± SD. #

or pSAG5A/peptide (721.03 ± 99.59) were significantly higher (P < 0.05). The big error in peptide (489.7 ± 98.9) should not be ignored. In addition, the level of IFN-␥ in mice immunized with pSAG5A/peptide was significantly higher than that of mice injected by peptide or pSAG5A (P < 0.05). There was no statistical difference in IFN-␥ levels between the groups immunized by PBS and pEGFP-C1. The highest level of IL-4 was assayed in mice vaccinated by pSAG5A/peptide, and no statistically significant difference was found between pSAG5A/peptide and others (P > 0.05). On the other hand, the levels of IL-10 detected in the experimental and control groups indicated no statistically significant differences between the groups (P > 0.05). 3.5. Protection of DNA vaccine against T. gondii (PRU strain) Two weeks after the last immunization, each mouse was challenged intragastrically with 20 cysts of PRU strain of T. gondii to evaluate the effect of immunoprotection induced by the DNA vaccine. As shown in Table 4, a significant reduction of brain cyst was detected in the mice vaccinated with peptide (732 ± 160), pSAG5A (815 ± 197), or pSAG5A/peptide (436 ± 174) compared by the mice injected by PBS (1260 ± 241) or pEGFP-C1 (1350 ± 268). It was worth noting that a big error was found in pSAG5A/peptide (436 ± 174). The number of brain cyst in pSAG5A/peptide treated mice reduced to 35% of the control. Moreover, there was no statistically significant difference between peptide and pSAG5A (P > 0.05). No statistical difference was observed between groups immunized with PBS and pEGFP-C1 (P > 0.05). 4. Discussion Bioinformatics, an interdisciplinary science, has been widely used to predict protein structures, functions and epitopes (Romano et al., 2011). Prediction of the epitopes plays an important role in design of immunogenicity of peptides and new vaccines (Martin et al., 2004; Bai et al., 2012). In this study, the B-cell epitopes and T-cell epitopes of SAG5A were fully analyzed by related softwares and online services. On the one hand, DNAMAN software was used to analyze the sequence of SAG5A protein following DNAStar software. Sixteen predicted epitopes were found in SAG5A and SAG1 proteins, and the scores of the former were higher than the latter. The result suggested that SAG5A had great potential to become a good B-cell antigen. On the other hand, we used online service IEDB to analyze T-cell epitopes of SAG5A and found several potential Tcell epitopes on the protein. As presented in Table 2, a lower value of IC50 indicated a higher affinity, and a higher affinity indicated a better T-cell epitope. Obviously, compared to SAG1, SAG5A had much lower value of IC50 on the whole, which demonstrated that the latter could be an excellent T-cell antigen. In short, SAG5A with good B-cell epitopes and T-cell epitopes had great potentiality to become a successful vaccine against T. gondii.

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After the analysis of epitopes on SAG5A with bioinformatics, the SAG5A gene was cloned and inserted into a eukaryotic expression plasmid pEGFP-C1. The observation of fluorescence in transfected cells indicated that the eukaryotic expression vector was constructed successfully. The results showed that the humoral and cellular immune responses were successfully induced in mice vaccinated with peptide and pSAG5A. Immunotherapy may be one of the most promising approaches to prevent the infection of T. gondii. The DNA vaccine encoding SAG5A could induce a specific Th1 immune response, and DNA vaccines often cannot produce enough protection against T. gondii challenge (Desolme et al., 2000; Chen et al., 2009). As a powerful vaccine approach, prime-boost strategy was developed to strongly induce cellular immunity. The strategy contains the administration of two different vaccines expressing the same antigen (Kim et al., 2007). Several reports have demonstrated the effect of prime-boost vaccine strategies in producing protective immune response to several pathogens (Gilbert et al., 2002; Gonzalo et al., 2002; Pancholi et al., 2003; Alekseeva et al., 2009). Priming with polypeptide and boosting with DNA is an approach, while priming with DNA and boosting with polypeptide is another approach (Meng et al., 2013). Peptides was used to study the immunization of T. Gondii in some reports. Peptides derived from SAG1 were used to immunize mice and got positive results (Siachoque et al., 2006). In another report, P30 and ROP18 peptides were detected in human toxoplasmosis (Torres-Morales et al., 2014). Though the peptides in these reports were not used combining with DNA vaccine, they played an important role in immunization of toxoplasmosis. In the present study, priming with DNA (SAG5A) and boosting by peptide (HAPTPSFLGLLAVVF) was used and stimulated strong humoral and cellular responses in the body of mice. Cellular and humoral immunity play a decisive role in host resistance against T. gondii. The infection of T. gondii leads to a strong and persistent Th1 immune response with lots of IgG antibody and a large number of cytokines, which are essential for the control of infection (Gazzinelli et al., 1993; Machado et al., 2010). Some reports indicate that a good DNA vaccine should be able to arouse a Th1 rather than a Th2 response (Zheng et al., 2013). The specific IgG antibodies play an important role in protecting mice against being infected by T. gondii. Furthermore, the IgG antibodies can prevent the parasite from the attaching to the host cell (Kang et al., 2000). In the present study, the DNA and peptide vaccines produced humoral and cellular immune responses in the immunized mice. The high ratios of IgG2a to IgG1 antibody titers were detected in the vaccinated mice. Furthermore, a higher level of IgG antibodies was assayed in mice injected with DNA/peptide compared to other groups. The results suggested that the Th1 cells had been activated with the vaccines in the study. In addition, in order to further characterize the polarization of the immune response, the levels of cytokine productions (IFN-␥, IL-4 and IL-10) from spleen cells of mice were detected. The cytokines play their respective roles in host resistance against T. gondii. The immunity mediated by IFN-␥ plays an important role in controlling the replication of the protozoan (Sher et al., 1995). Of course, IFN-␥ is important in restricting the growth of T. gondii in the acute phase of the infection and preventing reactivation of parasites from dormant cysts (Suzuki et al., 1988). Furthermore, reports indicated that the Th2 cytokines (IL-4 and IL-10) produced in T. gondii infection play an important role in immune response. IL-4 was considered to prevent the host from succumbing to early Th1-polarized hyperactive immune response, and the production of IFN-␥ was subtracted by high level of IL-4 (Gazzinelli et al., 1996; Dawson et al., 2005). In our study, compared to the control groups, the level of IFN-␥ induced in the experimental groups were higher. However, the levels of IL-4 and IL-10 in experimental groups were same as control groups. Obviously, the levels of IFN-␥ of experimental mice were remarkably higher than those of

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IL-4 and IL-10, which suggested that the immune response stimulated with SAG5A was mainly specific Th1-type immune response. In order to evaluate the efficiency of protection against T. gondii, 20 cysts of PRU strain were used to challenge vaccinated BALB/c mice. The PRU strain of T. gondii is significantly less virulent in mice and no vaccines have been reported to provide full protection against intragastrical challenge by cysts of T. gondii. Compared to the mice of control group, the cyst burden reduction rate reached to 77% in mice immunized with three DNA plasmids (Zhang et al., 2013). The recombinant vaccine (rROP2 + rROP4 + rSAG1) reduced brain cysts by 90% and 71% relative to control group in C57BL/6 mice and C3H/HeJ mice (Dziadek et al., 2011). These indicated that the species of mice might be a factor that influenced vaccine’s protection. In this study, a significant reduction of brain cyst was detected in DNA/peptide BALB/c mice (35%), which indicated that the vaccine of DNA/peptide could provide positive effect in protection against T. Gondii in BALB/c mice. The decrease in the cysts loading in brains of rROP2 + rROP4-vaccinated mice reached 46% compared to control PBS injected mice (Dziadek et al., 2009). The vaccine (rROP2 + rROP4 + rGRA4) induced a significant reduction (84%) in brain cyst number compared to control mice (Dziadek et al., 2012). These reports indicated that multigene vaccine may be more effective than a single gene vaccine. Although the immunizations did not provide complete protection against cysts challenge with T. gondii, the number of cysts was drastically reduced. In Meng’s report, peptide and DNA were used to stimulate the immune response in mice and got good protection against virulent strain (Meng et al., 2013). In the present study, the immunization strategy was adopted and obtained efficacious immune protection against low virulent strain. In addition, some parameters (like animal species, feeding, and management) might hinder the effective evaluation of the protection. In further studies, considering more parameters could obtain more encouraging results to evaluate the immune response induced with vaccines. 5. Conclusion In the present study, bioinformatics approaches were used to identify B-cell epitopes and T-cell epitopes on SAG5A protein. Thereafter, DNA vaccine and peptide were injected into mice to evaluate the immunoprotection induced by such vaccines. The vaccine could induce effective cellular and humoral immune response in vaccinated mice. The number of cysts in brains of the mice immunized with DNA/peptide reduced to 35% of mice treated PBS. The vaccine was proven to be positive in protection against cysts of PRU strain. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was supported, in part, by grants from the National Natural Science Foundation of China (Grant Nos. 81071373 and 81271857), the State Key Laboratory of Veterinary Etiological Biology (Grant No. SKLVEB2011KFKT005) and the Shandong Provincial Natural Science Foundation (Grant No. ZR2009CM079). References Alarcon, J.B., Waine, G.W., McManus, D.P., 1999. DNA vaccines: technology and application as anti-parasite and anti-microbial agents. Adv. Parasitol. 42, 343–410. Alekseeva, E., Sominskaya, I., Skrastina, D., Egorova, I., Starodubova, E., Kushners, E., Mihailova, M., Petrakova, N., Bruvere, R., Kozlovskaya, T., Lsaguliants, M., Pumpens, P., 2009. Enhancement of the expression of HCV core gene does not enhance core-specific immune response in DNA immunization: advantages of

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