Experimental Parasitology 139 (2014) 42–48

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Toxoplasma gondii: Protective immunity induced by rhoptry protein 9 (TgROP9) against acute toxoplasmosis Jia Chen a,1, Dong-Hui Zhou a,1, Zhong-Yuan Li a,c, Eskild Petersen b, Si-Yang Huang a, Hui-Qun Song a, Xing-Quan Zhu a,c,⇑ a State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China b Department of Infectious Diseases, Clinical Institute, and Institute of Medical Microbiology and Immunology, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark c College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 pVAX-ROP9 induced high level of

humoral immune response.  pVAX-ROP9 induced lymphocyte

proliferative response and Th1-type immune response significantly.  pVAX-ROP9 increased the percentages of CD4+ T and CD8+ cells significantly.  Immunization with pVAX-ROP9 prolonged survival time in mice (12.9 ± 2.9 days).

a r t i c l e

i n f o

Article history: Received 28 September 2013 Received in revised form 17 January 2014 Accepted 9 February 2014 Available online 3 March 2014 Keywords: Toxoplasma gondii Toxoplasmosis DNA vaccine Rhoptry protein 9 (ROP9) Protective immunity Mice

a b s t r a c t Toxoplasma gondii rhoptry protein 9 (ROP9) is involved in the early stages of host invasion, and contains B cell epitopes. The aim of this study was to evaluate the immune protective efficacy of a DNA vaccine encoding TgROP9 gene against acute T. gondii infection in mice. A DNA vaccine (pVAX-ROP9) encoding TgROP9 inserted into eukaryotic expression vector pVAX I was constructed, and the efficacy of intramuscular vaccination of Kunming mice with pVAX-ROP9 was analyzed. Mice immunized with pVAX-ROP9 induced a high level of specific anti-T. gondii antibodies, as well as a mixed IgG1/IgG2a response with predominance of IgG2a production. Also, injection of pVAX-ROP9 induced a specific lymphocyte proliferative responses and Th1-type cellular immune response with production of IFN-c and interleukin-2. The percentages of CD4+ and CD8+ T cells were significantly increased in mice immunized with pVAX-ROP9, compared to empty vector, PBS or blank controls. Immunization with pVAX-ROP9 significantly (P < 0.05) prolonged survival time (12.9 ± 2.9 days) after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 6 days. DNA vaccination with pVAX-ROP9 triggered strong humoral and cellular responses, and induced effective protection in mice against acute T. gondii infection, indicating that TgROP9 is a promising vaccine candidate against acute toxoplasmosis. Ó 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China. Fax: +86 931 8340977. E-mail address: [email protected] (X.-Q. Zhu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.exppara.2014.02.016 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.

J. Chen et al. / Experimental Parasitology 139 (2014) 42–48

1. Introduction Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution, which can invade diverse endothermic vertebrate species including humans (Montoya and Liesenfeld, 2004; Dubey, 2010; Chen et al., 2012; Robert-Gangneux and Dardé, 2012). In humans, T. gondii infection can cause severe disease in pregnant women and immunocompromised individuals (Weiss and Dubey, 2009), although the vast majority of infections in adults are asymptomatic (Montoya and Liesenfeld, 2004). Infection in livestock is of great economic importance, as well as a source of transmission to humans due to food-borne outbreaks (Fayer et al., 2004; Dubey et al., 2005; Innes, 2010). So far, there are no available drugs that can effectively eliminate T. gondii infections, especially persistent tissue cysts in hosts. (Coombs and Muller, 2002; Bhopale, 2003; Innes and Vermeulen, 2006). Thus, the development of an effective vaccine against T. gondii would be an attractive alternative to prevent and control T. gondii infections (Zhang et al., 2013). In the past years, several types of experimental vaccines have been studied including subunit vaccines, genetically engineered vaccines and a live, attenuated vaccine (T. gondii S48), but so far with inadequate protective efficacy (Innes and Vermeulen, 2006; Wang et al., 2007). Currently, considerable efforts have been devoted to exploit DNA vaccines owing to their advantages of costeffective to produce, low cost and safe, and the abilities of longlasting immunity (Gurunathan et al., 2000; Kur et al., 2009). So, it would be considerable value to identify novel T. gondii antigens for DNA immunization. Rhoptry protein 9 (ROP9) is a soluble rhoptry protein which is expressed only in tachyzoite stage and might be involved in the early stages of invasion (Reichmann et al., 2002). The protein contains putative B cell epitopes and triggers an exclusive CD4+ T cell response (Reichmann et al., 2002). The objective of this study was to evaluate the immunoprotective immunity induced by T. gondii ROP9 (TgROP9) by constructing a novel eukaryotic plasmid expressing T. gondii ROP9 (pVAX-ROP9), and investigating murine immune responses and protection against lethal challenge with the T. gondii RH strain (Type I). 2. Materials and methods 2.1. Animals and parasites Specific-pathogen-free (SPF) grade female 6- to 8-week-old Kunming mice were purchased from Lanzhou University Laboratory Animal Center (Lanzhou, China). Tachyzoites of the highly virulent RH strain of T. gondii were used to prepare Toxoplasma lysate antigen (TLA) and challenge mice. The RH strain was preserved in our laboratory (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), maintained by serial intraperitoneal passage in Kunming mice and purified according to the method previously described (Yuan et al., 2011a,b). All mice were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China, and the study was approved by the Animal Ethics Committee of Chinese Academy of Agricultural Sciences (permit CAASAEM-2012-8). 2.2. Preparation of Toxoplasma lysate antigen (TLA) Toxoplasma lysate antigen was prepared as described previously (Chen et al., 2013a). In brief, purified tachyzoites of T. gondii RH strain were disrupted by three cycles of frozen and thawed, and then sonicated on ice at 60 W/s. The extract was centrifuged for

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30 min at 10,000g at 4 °C, and then the supernatants were sterile filtered with 0.2 lm sterile nitrocellulose filters (Sartorius). The TLA samples concentration was determined via the Bradford method using bovine serum albumin (BSA) as the standard, aliquoted and stored at 70 °C until use. 2.3. Construction of the eukaryotic expression plasmid The eukaryotic expression vector pVAX I was used as the DNA vaccine vector. To construct eukaryotic expression plasmid, the coding sequence of TgROP9 gene was obtained by PCR amplification from the cDNA of T. gondii RH strain tachyzoites, with a pair of specific primers: forward primer, 50 -CCGGAATTCATGACGCACCCAAATCCCCTT-30 , and reverse primer, 50 -GCTCTAGATCACTGCA TGATCAACGAGG-30 (recognition sites for EcoR I and Xba I, respectively, are underlined). The amplified PCR product was inserted into pMD18-T vector (TaKaRa, China) and sequence analysis was performed to confirm that no PCR mutations were introduced. The TgROP9 gene was recovered from the pMD18-T vector by digestions using EcoR I and Xba I and subcloned into the EcoR I/ Xba I sites of pVAX I (Invitrogen) and generated plasmid pVAXROP9. Plasmids were then purified from transformed Escherichia coli DH5a cells by anion exchange chromatography (EndoFree plasmid giga kit, Qiagen Sciences, MD, USA) following the manufacturer’s instructions, dissolved in sterile endotoxin-free TE buffer and stored at 20 °C until use. The concentration of pVAX-ROP9 was determined by spectrophotometer at OD260 and OD280. 2.4. Expression of pVAX-ROP9 plasmid in vitro Marc-145 cells grown in 6-well plates were transfected with recombinant plasmid pVAX-ROP9 with lipofectamine™ 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Fortyeight hours after transfection, the cells were fixed with cool acetone for 15 min, and washed with PBS-0.1% Triton-X-100 (PBST) for three times. The plasmid pVAX-ROP9 expression in the cells was detected using the indirect immunofluorescence assay (IFA). In brief, the cells were incubated with goat anti-T. gondii tachyzoite polyclonal antiserum (kindly provided by Professor Delin Zhang, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, China) diluted 1:50 in PBST at 37 °C for 60 min and washed by PBST, followed by a FITC-labeled donkey-anti-goat IgG antibody diluted 1:100 in PBST at 37 °C for 45 min (Proteintech Group Inc., Chicago, USA). After extensive washings with PBST, the specific fluorescence was imaged through a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany). Marc-145 cells transfected with empty pVAX I was prepared as the negative control. 2.5. Immunization and challenge Female Kunming mice (25 per experimental group) were immunized by bilateral intramuscular injection into the quadriceps three times at two week intervals with 100 lg plasmid encoding pVAX-ROP9 dissolved in 100 ll sterile phosphate buffered saline (PBS). For controls, groups of mice were injected with empty pVAX I vector, PBS respectively, and one group of mice were not inoculated to constitute blank control. Blood was collected from the mouse tail vein prior to immunization and challenge, and sera were separated and stored at 20 °C until analyzed for specific antibodies. Two weeks after the last immunization, three mice per group were sacrificed and splenocytes were aseptically harvested for lymphocyte proliferation assay, cytokine measurements, and flow cytometric analysis, and thereafter the mice in all groups were

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intraperitoneally (IP) challenged with 1  103 tachyzoites of the virulent T. gondii RH strain. Pre-immune serum samples were used as negative controls. 2.6. Determination of antibody titers and isotype TgROP9 antigen-specific IgG, IgG1 and IgG2a antibodies in serum samples were determined using SBA Clonotyping System-HRP Kit according to the manufacture’s instruction (Southern Biotech Co., Ltd., Birmingham, USA). In brief, microtiter plates were coated with capture antibody (10 lg/ml; provided by the commercial Kit) in 100 ll of phosphate buffered saline (pH 7.4) overnight at 4 °C. The plates were washed with PBS containing 0.05% Tween 20 (PBS-T) and then blocked with PBS containing 1% BSA for 1 h. Mouse serum samples diluted in PBS were added to the wells and incubated for 1 h at 37 °C. After washing with PBS-T, the wells were incubated with 100 ll of horseradish-peroxidase (HRP) conjugated anti-mouse IgG diluted in 1:500 for 60 min at 37 °C, or anti-mouse IgG1 or IgG2a in 1:500, which were used for determination of antibody levels and isotype analysis, respectively. Binding was visualized by incubating with 100 ll substrate solution (pH 4.0) (1.05% citrate substrate buffer; 1.5% ABTS; 0.03% H2O2) for 20 min. The absorbance was measured at 405 nm using ELISA reader (Bio-TekEL  800, USA). All samples were run in triplicate. 2.7. Lymphocyte proliferation assay The level of in vitro proliferative response of spleen cells were estimated using the MTT assay. In brief, two weeks after the last immunization, spleens were aseptically removed from 3 mice of each group in Hank’s balanced salt solution (HBSS, Sigma), and splenocyte suspensions were prepared by pushing the spleens through a wire mesh. After the erythrocytes were lysed using erythrocyte lysis buffer (0.15 M NH4Cl, 1.0 M KHCO3, 0.1 mM EDTA, pH 7.2), the homogeneous splenocytes were resuspended in DMEM medium supplemented with 10% fetal calf serum (FCS). Cells were plated at a density of 2  105 cells per well in 96-well costar plates after counting the number with a haemocytometer by Trypan blue dye exclusion technique, and stimulated with TLA (10 lg/ml) and concanavalin A (ConA; 5 lg/ml; Sigma) or cultured in medium alone served as positive and negative controls, respectively, at 37 °C in a 5% CO2 incubator. After 72 h, the proliferative activity was measured using a 3-(4,5-dimethylthylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml, Sigma) dye assay, according to the method previously described (Bounous et al., 1992). The stimulation index (SI) was calculated as the ratio of the average OD570 value of wells containing antigen-stimulated cells to the average OD570 value of wells containing cells only with medium. All experimental and control samples were run in triplicate.

2.8. Analysis of percentages of CD4+ and CD8+ T lymphocytes by flow cytometry In order to analyze the percentages of CD4+ and CD8+ T lymphocytes, suspensions of splenocytes were obtained as described above, and the viability of the cells was evaluated using 0.04% Trypan blue. The cell concentration was adjusted to 1  106 cells/ml in PBS containing 2% FBS, and cell surface staining was employed using fluorochrome-labeled mAbs. Following incubation with optimal concentrations of phycoerythrin (PE)-labeled anti-CD3 antibody (5 lg/ml), allophycocyanin (APC)-conjugated anti-CD4 antibody (5 lg/ml), and fluorescein isothiocyanate (FITC)-labeled anti-CD8 antibody (5 lg/ml) (eBioscience) at 4 °C for 30 min in the dark, the cultures were washed by 2 ml PBS, then fixed with FACScan buffer (PBS containing 1% FCS and 0.1% Sodium azide) and 2% paraformaldehyde. The samples were analyzed of fluorescence profiles on a FACScan flow cytometer (BD Bio-sciences) by SYSTEM II software (Coulter). 2.9. Cytokine assays Two weeks after the third immunization, splenocytes from each experimental group were obtained and cultured with different stimuli as described for lymphocyte proliferation assay. Culture supernatants were harvested at 24 h for interleukin-2 (IL-2) and interleukin-4 (IL-4), 72 h for interleukin-10 (IL-10), 96 h for gamma-interferon (IFN-c) and cytokine levels were assayed using commercial ELISA kits according to the manufacturer’s instructions (Biolegend, USA). Cytokine concentrations were determined by reference to standard curves constructed with known amounts of mouse recombinant IFN-c, IL-2, IL-4 or IL-10. The sensitivity limits for the assays were 8.0 pg/ml for IFN-c, 0.9 pg/ml for IL-2, 0.5 pg/ ml for IL-4 and 23.8 pg/ml for IL-10, respectively. The analysis was performed with the data from three independent experiments. 2.10. Statistical analysis All statistical analyses were performed by SPSS13.0 Data Editor (SPSS Inc., Chicago, IL, USA). The differences of the data (e.g. antibody responses, lymphoproliferation assays and cytokine production) between all the groups were compared by one-way ANOVA. The results in comparisons between groups were considered different if P < 0.05. 3. Results 3.1. Expression of pVAX-ROP9 plasmid in Marc-145 cells In vitro expression of pVAX-ROP9 was evaluated by IFA at 48 h post-transfection. As shown in Fig. 1, specific green fluorescence

Fig. 1. Detection of TgROP9 expression on Marc-145 cells by indirect immunofluorescence (IFA) at 48 h post-transfection. (A) Marc-145 cells were transfected with pVAXROP9. (B) Negative control.

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Fig. 2. Humoral response in Kunming mice induced by DNA vaccination. (A) Determination of IgG antibodies in the sera of Kunming mice immunized with pVAX-ROP9, pVAX I, PBS and blank controls on weeks 0, 2, 4, 6. (B) Determination of IgG subclass profile (IgG1 and IgG2a) in the sera of the immunized Kunming mice two weeks after the last immunization. Results are expressed as mean of the OD450 ± SE, which represent the experiment run in three technical triplicates using samples from three separate mice (3  3) and statistically significant difference (P < 0.05) are indicated by (⁄).

Table 1 Cytokine production by splenocytes of immunized Kunming mice after stimulation by Toxoplasma lysate antigen (TLA). Group (n = 3)

pVAX-ROP9 pVAX1 PBS Blank control

Cytokine production (pg/ml)

Proliferation (SI)

IFN-c

IL-2

IL-4

IL-10

466.62 ± 12.72a 56.10 ± 5.18b 52.58 ± 2.46b 52.40 ± 2.08b

305.88 ± 15.02a 52.50 ± 1.37b 51.07 ± 2.55b 50.80 ± 1.96b

231.72 ± 9.89a 51.50 ± 1.65b 50.33 ± 1.24b 50.56 ± 2.04b

212.00 ± 16.23a 51.64 ± 1.28b 50.34 ± 1.40b 50.12 ± 2.29b

3.21 ± 0.05a 1.05 ± 0.00b 1.04 ± 0.07b 1.03 ± 0.03b

SI stands for stimulation index. Splenocytes from 3 mice were harvested 2 weeks after the last immunization. Values for IFN-c are for 96 h, values for IL-2 and IL-4 are for 24 h, and values for IL-10 are for 72 h. The same superscript letter means no difference (P > 0.05), whereas different superscript letters mean significant difference (P < 0.05).

was observed in Marc-145 cells transfected with pVAX-ROP9, but not in the negative controls transfected with the same amount of empty pVAX I. 3.2. Evaluation of humoral immune responses To investigate whether mice immunized with pVAX-ROP9 induced humoral immune response, all serum samples were tested by ELISA. As shown in Fig. 2A, significantly high levels of antibody IgG were detected in the sera of mice immunized with pVAX-ROP9, and the antibody levels increased with successive DNA immunization. In contrast, mice injected with pVAX I or PBS failed to elicit humoral immune responses and did not induce IgG response. There were no statistically significant differences in the levels of antibodies between the three control groups (P > 0.05). To study whether a Th1 and/or Th2 response was elicited after immunization with pVAX-ROP9, the levels of IgG1 and IgG2a isotype responses were analyzed. As depicted in Fig. 2B, both IgG1 and IgG2a were detected, but with a high ratio (1.69) of IgG2a to IgG1 demonstrating that immunization of pVAX-ROP9 primarily induced a Th1 type response.

CD4+ and CD8+ T cells in the spleen of mice immunized with pVAX-ROP9, pVAX I, or PBS were evaluated by flow cytometry analysis. As shown in Fig. 3, the percentages of CD3+CD8+ T cells were significantly increased in mice immunized with pVAX-ROP9 compared with that in PBS group, pVAX I or the blank control. Meanwhile, there were significant differences in the percentages of CD4+ lymphocytes between the pVAX-ROP9group and the controls (P < 0.05). There were no statistically significant differences between the three control groups (P > 0.05). 3.4. Evaluation of cytokine productions As shown in Table 1, spleen cells from mice immunized with pVAX-ROP9 produced large amounts of IFN-c (466.62 ± 12.72 pg/ ml), IL-2 (305.88 ± 15.02 pg/ml), but no significant production of these two cytokines were observed in the groups of pVAX I, PBS and the blank control. In the meanwhile, low levels of IL-4 (231.72 ± 9.89 pg/ml) and IL-10 (212.00 ± 16.23 pg/ml) showed a slight proliferative response from the splenocytes from mice immunized with pVAX-ROP9 compared to control mice (P < 0.05). These results further confirmed the results of the IgG subclasses as shown above.

3.3. Cellular immune response induced by DNA immunization 3.5. Protection of mice against challenge with T. gondii RH strain As shown in Table 1, the mice immunized with pVAX-ROP9 produced a stronger splenocyte proliferative response than the other mice (P < 0.05). However, there were no statistically significant differences in stimulation index (SI) between the three control groups (P > 0.05). In order to determine CD8+ T and/or CD4+ T cell mediated cellular immune response after DNA vaccination, the percentages of

To evaluate the protective effect of DNA immunization with pVAX-ROP9 against acute T. gondii infection, the immunized and control Kunming mice were challenged with 103 tachyzoites of lethal T. gondii tachyzoites two weeks after last immunization and their survival was monitored daily until all immunized and challenged mice died. Survival curves of different groups of mice

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Fig. 3. Detection of lymphocyte subpopulations using flow cytometry analysis. (A) The percentages of CD3+CD4+CD8- T lymphocytes (CD3 gated) in mice spleen cells. (B) The percentages of CD3+CD8+CD4- T lymphocytes (CD3 gated) in mice spleen cells.

Fig. 4. Protection of Kunming mice against T. gondii infection. Survival curves of immunized Kunming mice after lethal challenge with 1  103 tachyzoites of virulent T. gondii RH strain 2 weeks after the last immunization. Mice immunized with pVAX-ROP9 were dead from day 9 to 18. Mice immunized with the pVAXROP9 showed an increased survival time (12.9 ± 2.9 days) compared with mice in the control groups (pVAX I, PBS and blank controls) died within 6 days after challenge (P < 0.05).

are shown in Fig. 4. The mice immunized with pVAX-ROP9 had prolonged survival time (12.9 ± 2.9 days) compared to mice in groups of pVAX I, PBS and blank control, which all died within 6 days. No significant difference was observed among these control groups (P > 0.05). 4. Discussion Immunization with DNA vaccines coding for pathogen-specific antigens can elicit effective humoral and cellular immune responses that produce specific antibodies and both CD4+ T helper

cells and CD8+ cytotoxic T cells against pathogens in animal models (Robinson, 1999; Gurunathan et al., 2000; Kur et al., 2009). Therefore, in recent years, the immunogenic efficacy of DNA vaccine candidates against T. gondii infection has been extensively evaluated (Peng et al., 2009; Liu et al., 2010; Rashid et al., 2011; Dziadek et al., 2012; Huang et al., 2012; Chuang et al., 2013), in particular, rhoptry proteins of T. gondii have recently been demonstrated to be the potential vaccine candidates due to their key biological roles in the parasite, such as ROP13 (Wang et al., 2012), ROP16 (Yuan et al., 2011a), and ROP18 (Yuan et al., 2011b; Qu et al., 2013). In order to determine the immunological mechanisms of protective immunity evoked by DNA vaccination with pVAX-ROP9, we analyzed both the levels and characteristics of immune responses. Generally, it is well established that T cell-mediated immunity to the intracellular parasite T. gondii is critical to resistance to T. gondii (Jongert et al., 2010). In our studies, mice immunized with pVAX-ROP9 showed a significant T. gondii specific splenocyte proliferation, demonstrating that an adaptive immune response against T. gondii was elicited. Furthermore, we observed differences in the relative proportion of CD4+ and CD8+ T cells between the vaccinated and control groups, in particular an increase of both percentages of CD4+ and CD8+ T cells in immunized mice, suggesting that immunization with pVAX-ROP9 may induce the activation of CD4+ and CD8+ T cells, which may be synergistic to contribute to cytotoxic activity against T. gondii infection. Accumulating evidence showed that IFN-c produced by T cells plays a central role in the control of T. gondii infection (Suzuki et al., 1998; Suzuki et al., 1989; LaRosa et al., 2008). Also, some other cytokines such as IL-2 and IL-12 are involved in the protection against T. gondii (Matowicka-Karna et al., 2009). By re-stimulation in vitro with TLA, we showed that a significant increase of both IFN-c and IL-2 was produced followed by the pVAX-ROP9 DNA vaccination. Since the CD4+ lymphocytes can act as helper

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cells in induction of immunity against T. gondii infection, especially, the potent Th1-type immune responses are essential for the limiting of T. gondii infection (Garcia, 2009; Gigley et al., 2009). As markers for Th1 type cells, the high levels of both IFNc and IL-2 produced by splenocyte suggested that Th1-type cellular immune response was mainly activated. Moreover, Th2 type cytokines including IL-4 and IL-10 are crucial to prevent severe immunopathology during the acute and chronic stage of infection (Dupont et al., 2012). In the present study, a slight increase of both IL-4 and IL-10 was also observed, suggesting that low levels of antiinflammatory response were activated. Furthermore, consistent with previous reports (Chen et al., 2013a,b), immunization with pVAX-ROP9 induced high titers of total specific IgG antibodies, which may play an important role in killing intracellular parasites (Kang et al., 2000; Correa et al., 2007). Subsequently, using ELISA assay, mice immunized with pVAX-ROP9 exhibited a mixed Th1/Th2 response, with a high ratio of IgG2a (Th1) to IgG1 (Th2) antibody titers, again emphasizing that Th1 mediated cells were mainly activated. In order to investigate whether pVAX-ROP9 vaccination could positively influence the outcome of T. gondii infection, we challenged Kunming mice intraperitoneally with 1  103 tachyzoites of the highly virulent T. gondii RH strain. In this acute infection model, mice immunized with pVAX-ROP9 significantly prolonged survival time, demonstrating effective protective efficacy, which was similar to results of our previous studies employing several other single gene DNA vaccines (Liu et al., 2010; Yan et al., 2011), but it is not as good as that of other pVAX vaccination candidates such as pVAX-ROP8 (Parthasarathy et al., 2013), pVAXROP16 (Yuan et al., 2011a), pVAX-ROP13 (Wang et al., 2012), pVAX-eIF4A (Chen et al., 2013a) and pVAX-MIC13 (Yuan et al., 2013). In conclusion, the results of the present study showed that immunization with a TgROP9 plasmid induced strong humoral and cellular Th1-type immune responses, and prolonged survival time against lethal challenge. Althoguh TgROP9 elicited only partial protection against acute toxoplasmosis, it could be used as a potential vaccine candidate in further studies of multi-component T. gondii vaccines against toxoplasmosis in the mice model. Acknowledgments Project support was provided, in part, by the International Science and Technology Cooperation Project of Gansu Province (Grant No. 1204WCGA023), the National Natural Science Foundation of China (Grant Nos. 31230073 and 31172316) and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006). References Bhopale, G.M., 2003. Development of a vaccine for toxoplasmosis: current status. Microbes Infect. 5, 457–462. Bounous, D.I., Campagnoli, R.P., Brown, J., 1992. Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes. Avian Dis. 36, 1022–1027. Chen, J., Xu, M.J., Zhou, D.H., Song, H.Q., Wang, C.R., Zhu, X.Q., 2012. Canine and feline parasitic zoonoses in China. Parasites Vectors 5, 152. Chen, J., Huang, S.Y., Li, Z.Y., Yuan, Z.G., Zhou, D.H., Petersen, E., Zhang, N.Z., Zhu, X.Q., 2013a. Protective immunity induced by a DNA vaccine expressing eIF4A of Toxoplasma gondii against acute toxoplasmosis in mice. Vaccine 31, 1734–1739. Chen, J., Huang, S.Y., Zhou, D.H., Li, Z.Y., Petersen, E., Song, H.Q., Zhu, X.Q., 2013b. DNA immunization with eukaryotic initiation factor-2a of Toxoplasma gondii induces protective immunity against acute and chronic toxoplasmosis in mice. Vaccine 31, 6225–6231. Chuang, S.C., Ko, J.C., Chen, C.P., Du, J.T., Yang, C.D., 2013. Encapsulation of chimeric protein rSAG1/2 into poly(lactide-co-glycolide) microparticles induces longterm protective immunity against Toxoplasma gondii in mice. Exp. Parasitol. 134, 430–437.

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