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Original Research

DNA vaccination with genes encoding Toxoplasma gondii antigens ROP5 and GRA15 induces protective immunity against toxoplasmosis in Kunming mice Expert Rev. Vaccines 14(4), 617–624 (2015)

Jia Chen1‡, Zhong-Yuan Li1,2‡, Eskild Petersen3, Si-Yang Huang1, Dong-Hui Zhou1 and Xing-Quan Zhu*1,2,4 1 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 2 College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China 3 Department of Infectious Diseases and Clinical Microbiology, Faculty of Health Sciences, Institute for Clinical Medicine, Aarhus University, Aarhus, Denmark 4 Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu Province 225009, PR China *Author for correspondence: Fax: +86 931 834 0977 [email protected]

Authors contributed equally

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Objectives: To evaluate the protective efficacy of a DNA vaccine encoding Toxoplasma gondii rhoptry protein 5 (ROP5) and GRA15 antigens. Methods: We constructed eukaryotic plasmids expressing pVAX-ROP5 and pVAX-GRA15, and measured the immune responses to these DNA vaccines. Results: Kunming mice immunized with pVAX-ROP5 or pVAX-GRA15 showed significantly increased serum IgG2a titers; Th1 responses association with the production of IFN-g, IL-2, IL12 p40 and IL-12 p70; cell-mediated cytotoxic activity with increased frequencies of IFN-g secreting CD8+ T cells (CD8+ IFN-g+ T cells), as well as prolonged survival time (19.4 ± 4.9 days for ROP5; 17.8 ± 3.8 days for GRA15) and brain cyst reduction (57.4% for ROP5; 65.9% for GRA15) compared to control mice. Co-administration with pVAX-ROP5 and pVAX-GRA15 boosted the cellular and humoral immune responses, and significantly increased cyst reduction (79%) and prolonged the survival of immunized mice (22.7 ± 7.2 days). Conclusion: Co-immunization of pVAX-ROP5 and pVAX-GRA15 increase the protective efficacy. KEYWORDS: DNA vaccine . GRA15 . Kunming mice . protective immunity . ROP5 . Toxoplasma gondii . toxoplasmosis

Toxoplasma gondii is a major global public health problem and is an opportunistic protozoan parasite infecting mammals and birds, including humans [1–3]. Infections in immune-competent individuals are usually subclinical or asymptomatic but may occasionally cause retinochoroiditis; however, in pregnant women it may cause miscarriage, hydrocephalus, blindness, mental retardation and posterior uveitis [4–6]. In the chronic phase, recrudescence of a latent infection may lead to severe or even lethal damage in immune-compromised patients [7,8]. T. gondii infections in animals pose an economic risk to the livestock industry from abortion mainly in sheep and goats [9]. Meat products from infected livestock with

10.1586/14760584.2015.1011133

tissue cysts of T. gondii can transmit T. gondii to humans [10,11]. No treatments can control all lifecycle stages of T. gondii, in particular the cyst stages is not susceptible to antibiotics. Thus, as safe and efficient vaccine inducing protective immunity in humans and animals, preventing cyst formation in animals and infection in humans including protection against eye disease and congenital infection is needed. T. gondii can activate the hosts’ immune system in response to both surface and secreted antigens, and it is a priority to define if these antigen targets could be vaccine candidates [12]. Dense-granule antigens (GRAs), rhoptry antigens (ROPs) and microneme proteins (MICs),

 2015 Informa UK Ltd

ISSN 1476-0584

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Original Research

Chen, Li, Petersen, Huang, Zhou & Zhu

such as ROP9, ROP18, ROP13, GRA1, GRA2, MIC6 and MIC13, have recently been studied [13–19]. Similar to the critical role of ROP16 and ROP18, both ROP5 and GRA15 are other important virulence determinants in mice [20,21], as well as master regulators of the host inflammatory response [21,22]. Moreover, ROP5 is confirmed to be responsible for the virulence of all Type I strains, and GRA15 mediates host’s pro-inflammatory responses in Type II infections [21,22]. It is therefore of particular importance to study ROP5 and GRA15 as vaccine candidates. Accumulating evidence show that DNA vaccines with several antigens can be developed and will induce a better protective immunity in contrast to vaccines based on a single antigens [23–25]. Therefore, in this study, we used eukaryotic plasmid vectors expressing T. gondii ROP5 of RH strain (Type I) and T. gondii GRA15 of PRU strain (Type II) alone and in combination for the induction of protective immunity in a mice model. Materials & methods Animals

Six- to eight-week-old specific pathogen-free female Kunming mice were purchased from Lanzhou University Laboratory Animal Center (Lanzhou, China). All animals were handled in strict accordance with good animal practice requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. The study was approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC2012-009). Parasites

Tachyzoites of T. gondii highly virulent RH strain (Type I, originated from human in France) and tissue cysts of the PRU strain (Type II, originated from human in France) were propagated, harvested and used, respectively, for preparation of Toxoplasma lysate antigen (TLA), mice challenge and total RNA extraction as described in our previous studies [26]. Table 1. The proliferative response of lymphocytes of immunized Kunming mice after stimulation by Toxoplasma lysate antigen.

Construction of the eukaryotic expression plasmids

The complete open-reading frame of TgROP5 and TgGRA15 was obtained by RT-PCR amplification from total RNA using specific primers F1 (forward primer: 5¢-CCGGAATTCATGGCGACGA AGCTCGCTAGACT-3¢, reverse primer: 5¢-GCTCTAGATCAA GCGACTGAGGGCGCAG-3¢) and F2 (forward primer: 5¢-CC GGAATTCATGGTGACAACAACCACGCCAAC-3¢, reverse primer: 5¢-GCTCTAGATCATGGAGTTACCGCTGATTGT G-3¢), in which BamH I and Xba I restriction sites, and EcoR I and Xba I were introduced and underlined, respectively. The amplification reaction was carried out in a volume of 25 l containing 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 250 M each of dNTP, 0.2 M of each primer, 100–200 ng of template cDNA and 0.25 U La Taq polymerase (TaKaRa). Amplification of cDNA templates was carried out in a thermocycler (Bio-Rad, Hercules, CA, USA) under the following conditions: denaturation at 94 C for 10 min (initial denaturation), followed by 35 cycles consisting of 94 C for 30 s (denaturation), 59.5 C for 35 s (for GRA15) or 60.0 C for 35 s (for ROP5) (annealing), 72 C for 2 min (extension) and a final extension step was at 72 C for 10 min. After purification (TIANquick Midi Purification Kit, TIANGEN, China), the PCR products were inserted into corresponding restriction sites of pVAX I (Invitrogen) using T4 DNA ligase and generated recombinant plasmids pVAXROP5 and pVAX-GRA15, respectively. The recombinant plasmids were then purified from transformed Escherichia coli DH5a cells by anion exchange chromatography (EndoFree plasmid giga kit, Qiagen Sciences, Maryland, USA) following the manufacturer’s instructions, dissolved in sterile endotoxinfree TE buffer and stored at -20 C until use. The expression of recombinant plasmid in vitro

Marc-145 cells grown in six-well plates were transfected with recombinant plasmids pVAX-ROP5 and GRA15 using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions, and the expression in vitro was assayed by indirect immunofluorescence assay at 48 h after transfection as described previously [26,27]. In brief, the transfected cells were fixed with 100% acetone and washed with phosphate buffered saline (PBS) 0.1% Triton-X-100 (PBST), and then incubated with goat antiT. gondii polyclonal antibody (diluted 1:50 in PBST) and fluorescein isothiocyanate-labeled rabbit anti-goat mouse IgG antibody (1:2000, Sigma, St. Louis, Missouri, USA). The specific fluorescence was examined using a Zeiss Axio-plan fluorescence microscope (Carl Zeiss, Germany). Marc-145 cells transfected with empty pVAX I served as the negative control.

Group (n = 3)

Proliferation (SI)

ROP5 + GRA15

4.45 ± 0.05A

ROP5

3.32 ± 0.05B

GRA15

3.01 ± 0.07B

pVAX I

1.02 ± 0.03C

PBS

1.03 ± 0.03C

Immunization & challenge

Control

1.02 ± 0.03C

Six groups of 30 mice each were used for the present study, including three control groups (n = 30  3: empty pVAX I vector, PBS and blank control) and three vaccination groups (n = 30  3: pVAX-ROP5, pVAX-GRA15 and pVAX-ROP5+ pVAX-GRA15) (TABLE 1). The female Kunming mice were immunized intramuscularly with three doses of 100 g plasmid

Splenocytes from three mice were harvested 2 weeks after the last immunization. The same capital letter on the shoulders of experimental data means no statistically significant difference (p > 0.05) between different experimental groups from the same measurement, while different letters mean statistically significant difference (p < 0.05). ROP5: Rhoptry protein 5; SI: Stimulation index.

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Original Research

Protective efficacy of a DNA vaccine encoding T. gondii ROP5 and GRA15 antigens

B

C

Figure 1. Indirect immunofluorescence detection of TgROP5 and TgGRA15 expression on Marc-145 cells at 48 h posttransfection. (A) Marc-145 cells were transfected with pVAX-GRA15; (B) empty vector pVAX I; (C) Marc-145 cells were transfected with pVAX-ROP5. ROP5: Rhoptry protein 5.

dissolved in 100 l sterile PBS, such as pVAX-ROP5, pVAXGRA15 or pVAX-ROP5+ pVAX-GRA15 and boosted with a 2-week interval. Mice injected with empty pVAX I vector (100 g/100 l) or PBS (100 l) served as negative control groups, and the blank control group received nothing. Blood was collected from the tail vein prior to each immunization and challenge (at weeks 0, 2, 4, and 6), and sera were separated and stored at –20 C until analyzed. Pre-immune (at weeks 0) serum samples were used as negative controls. Two weeks after the last immunization, a total of 10 mice per group were sacrificed and splenocytes were aseptically harvested for cytokine measurements (five mice) and flow cytometric analysis (another five mice). Also, 2 weeks after the third inoculation, 10 mice in the vaccinated and control groups were challenged intraperitoneally with 1  103 tachyzoites of the virulent T. gondii RH strain, and the survival periods were recorded daily until all mice were dead. Meanwhile, six mice of A 1.4

pVAX/ROP5+pVAX/GRA15

pVAX/ROP5

pVAX/GRA15

pVAXI

PBS

Control

each group were inoculated orally with 10 PRU tissue cysts, and the mean number of cysts per brain was determined by counting three samples of 10 l aliquots of each homogenized brain under an optical microscope at 4 weeks after the challenge as described in our previous studies [27,28]. This analysis was performed in three independent experiments. T. gondii-specific antibody response

T. gondii-specific IgG, IgG1 and IgG2a antibodies in serum sample storage at -20 C mentioned above were analyzed by ELISA using SBA Clonotyping System-HRP Kit (Southern Biotech Co., Ltd, Birmingham, USA) according to the manufacture’s instruction as described previously [27,29]. In brief, ELISA plates were coated with capture antibody (10 g/ml) overnight at 4 C and then blocked with PBS containing 1% BSA for 1 h and washed with PBS containing 0.05% Tween 20 (PBS-T). Mouse serum diluted in PBS (1:5) were added to the wells and B *

1.2 Mean+/-S.D. OD405

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Figure 2. Humoral response in Kunming mice induced by DNA vaccination. (A) Determination of IgG antibodies in the sera of Kunming mice immunized with pVAX-ROP5, pVAX-GRA15, pVAX-ROP5 + pVAX-GRA15, 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 2 weeks after the last immunization. Results are expressed as means of the OD405 ± SD (n = 3), and the different letters mean statistically significant difference (p < 0.05). *Statistically significant difference (p > 0.05) between different experimental groups. ROP5: Rhoptry protein 5.

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5 A1

incubated at room temperature for 1 h with gentle shaking. Then, the wells were incubated with 100 l of horseradish-peroxidase-conjugated anti-mouse Ig diluted in 1:250, or anti-mouse IgG1 or IgG2a in 1:500 overnight at 4 C. The absorbance was measured at 405 nm after adding substrate solution (pH 4.0) (1.05% citrate substrate buffer; 1.5% ABTS; 0.03% H2O2) for 20 min. All samples were run in triplicate.

P5 RO

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Figure 3. Cytokine production by splenocytes of immunized Kunming mice after stimulation by Toxoplasma lysate antigen. *p < 0.05.

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IL-12p40 400.00 500.00

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* * IFN-γ 1000.00

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Original Research

The level of in vitro proliferative response of spleen cells were estimated using the 3-(4,5-dimethylthylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) dye assay as described previously [27,29]. In brief, 2 weeks after the last immunization, spleens were aseptically removed from three mice from each group, in Hank’s balanced salt solution (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 splenocytes were resuspended in Dulbecco’s modified eagle’s medium supplemented with 10% fetal calf serum. Cells were plated at a density of 5  105 cells per well in 96-well costar plates after counting the number with a hemocytometer by 0.04% Trypan Blue dye exclusion technique and stimulated with TLA (10 g/ml), concanavalin A (ConA; 5 g/ml; Sigma) or medium alone, at 37 C in a 5% CO2 incubator. The proliferative activity was measured using a 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 5 mg/ml, Sigma) dye assay, as previously described [30]. The stimulation index was calculated as the ratio of the average OD570 value of wells containing TLA-stimulated cells (OD570TLA) to ConAstimulated cells (OD570ConA) compared with medium (OD570M) respectively. That is, stimulation index = (OD570TLA/OD570M): (OD570ConA/OD570M). All experimental and control samples were run in triplicate. Characterization of IFN-g+ spleen lymphocytes by flow cytometry

Lymphocytes were isolated from spleen as described previously [26,27,29]. Analysis of percentages of CD4+ and CD8+ T lymphocytes and intracellular cytokine staining assays were performed as described before [26]. Surface staining was performed by incubation with surface markers, including phycoerythrin-labeled anti-mouse CD3 (2 g/l), allophycocyanin-labeled anti-mouse CD4 (2 g/l) and fluorescein isothiocyanate-labeled antimouse CD8 (2 g/l; all from eBioscience). For IFN-g detection, surface staining was performed by staining with CD8-phycoerythrin (eBioscience) as above, and then fixed and permeabilized with Cytofix/Cytoperm solution (all from BD Biosciences) before incubated with IFN-g-allophycocyanin (2 g/l; eBioscience). All data were Expert Rev. Vaccines 14(4), (2015)

Protective efficacy of a DNA vaccine encoding T. gondii ROP5 and GRA15 antigens

acquired on a BD FACScan flow cytometer. The analysis was performed with the data from three independent experiments.

Table 2. Flow cytometry analysis of the percentages of lymphocyte subpopulations in the spleen cells between the experimental and control groups. Group (n = 3)

CD3+CD4+CD8-(%)

CD3+CD8+CD4-(%)

CD8+IFNg+ (MFI)

The prepared splenocytes without red pVAX-ROP5 + 35.2 ± 0.2A 18.9 ± 0.0A 75.1 ± 26.9A blood cell from each group were copVAX/GRA15 cultured with TLA (10 g/ml) for positive pVAX/GRA15 28.3 ± 1.0B 14.3 ± 0.3B 63.4 ± 21.5B control and medium alone for negative control in microtiter plates as described pVAX-ROP5 28.2 ± 0.4B 14.5 ± 0.7B 52.9 ± 14.7C previously [29]. Culture supernatants were 13.1 ± 1.0C 7.5 ± 0.2C 16.9 ± 5.1D harvested at 24 h for determination of pVAX I IL-2 and IL-4, 72 h for IL-10 and 96 h for PBS 13.4 ± 0.6C 7.4 ± 0.1C 17.2 ± 6.2D IL-12(p40), IFN-g and IL-12(p70) using Control 13.2 ± 0.3C 7.2 ± 0.2C 16.6 ± 4.5D commercial ELISA kits according to the Note: The same capital letter on the shoulders of experimental data means no statistically significant difference manufacturer’s instructions (Biolegend, (p > 0.05) between different experimental groups from the same measurement, while different letters mean statistically significant difference (p < 0.05). USA). Cytokine concentrations were deterMFI: Median fluorescence intensity; ROP5: Rhoptry protein 5. mined by reference to standard curves constructed with known amounts of mouse recombinant IFN-g, IL-2, IL-4, IL-10 or IL-12p70. The sensitiv- pVAX-ROP5 + pVAX-GRA15 in contrast to PBS, pVAX I or ity limits for the assays were 8.0 pg/ml for IFN-g, 0.9 pg/ml for blank control (p < 0.05), and the antibody levels increased with IL-2, 0.5 pg/ml for IL-4 and IL-12p70, 23.8 pg/ml for IL-10, successive DNA immunizations (FIGURE 2A). In order to characterize whether a Th1 and/or Th2 response respectively. The analysis was performed with the data from three was elicited, the distribution of the IgG1 and IgG2a isotypes was independent experiments, including three replicates per spleen. analyzed 2 weeks after the last immunization. As shown in FIGURE 2B, both IgG1 and IgG2a were detected in all immunized Statistical analysis All statistical analyses were performed by SPSS13.0 Data Editor mice with a high IgG2a to IgG1 ratio, suggesting that Th1-type (SPSS, Inc, Chicago, IL, USA). Differences (e.g., antibody immune response were elicited by the DNA immunization. responses and lymphoproliferation assays) were compared by Injection of pVAX-ROP5 (1.51 ± 0.03) induced a higher IgG2a/ Students’ t-test, and thus the differences of cytokine production IgG1 ratio compared to the pVAX-GRA15 (1.29 ± 0.02) immuwere compared by non-parametric statistical tests. Statistical anal- nized animals, and co-injection of pVAX-ROP5 with pVAXysis of the standard error was calculated using the function of GRA15 (1.72 ± 0.03) induced the highest IgG2a/IgG1 ratio. ‘stdevp’ in Microsoft Excel Software. The results were considered significantly differ120.0 ent if p < 0.05. pVAX/ROP5+pVAX/GRA15 100.0

Results Expression of the plasmids in Marc-145 cells

GRA15- and ROP5-specific green fluorescence in Marc-145 cells transfected with pVAX-ROP5 and pVAX-GRA15 by immunofluorescence assay, but not in the cells transfected with the same amount of empty pVAX I (FIGURE 1), showing that TgROP5 and TgGRA15 protein were successfully expressed in Marc-145 cells. Humoral immune responses induced by DNA immunization

All immunized mice induced a significant IgG response, with higher antibody levels in pVAX-ROP5 immunized compared to pVAX-GRA15 immunized, and with the highest antibody levels in the group of informahealthcare.com

Survival (%)

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Cytokine assays

Original Research

80.0

pVAX/ROP5 pVAX/GRA15 pVAXI PBS Control

60.0 40.0 20.0 0.0 7 8 9 10 11 1213 1415 16 17 18 19 20 2122 23 2425 26 27 28 29 303132 33 34 Days after challenge

Figure 4. Protection of Kunming mice against Toxoplasma gondii infection. Survival rate of mice immunized with PBS, pVAX I, pVAX-GRA15, pVAX-ROP5, pVAXROP5 + pVAX-GRA15 or blank control after challenge with 1  103 tachyzoites of T. gondii RH strain. Each group had 10 mice. Three control groups (PBS, pVAX I and blank control) had 0% survival at day 9. ROP5: Rhoptry protein 5.

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Table 3. Mean cyst burden per mouse brain 4 weeks after challenge with 10 cysts of Toxoplasma gondii strain PRU per mouse.

As shown in TABLE 3, immunization with pVAX-ROP5, pVAX-GRA15 and pVAX-ROP5 + pVAX-GRA15 induced high levels of immune responses mentioned above, but only partial protection against T. gondii tissue cyst formation, and the average parasite burden was reduced significantly by 57.4, 65.9 and 79.0% (p < 0.05), respectively, but relatively low change of 2.3 and 1.1% in the group of pVAX I and PBS, respectively (p > 0.05), in comparison with the control groups (TABLE 3; p < 0.05).

Group (n = 3)

Number of brain cysts (Mean ± SD)*

% reduction†

Control

1956 ± 44A



PBS

1933 ± 67

1.1

pVAX I

1911 ± 42

2.3

pVAX-ROP5

B

833 ± 24

57.4

Discussion

pVAX/GRA15

667 ± 65C

65.9

pVAX-ROP5+ pVAX/GRA15

411 ± 20D

79.0

DNA vaccines have been demonstrated to stimulate both CD4+ and CD8+ T cells and induce a Th1-biased response, which is required for protective immunity to T. gondii infection [31,32]. We previously described that two virulence proteins, ROP18 and ROP16, were effective DNA vaccine candidates inducing a Th1-reponse in mice, although they did not induce complete protection [13,14]. In the present study, immunization with pVAX-ROP5 + pVAX-GRA15 increased the predominance of IgG2a over IgG1 induced by injection with pVAX-ROP5 or pVAXGRA15 alone. These observations were in consistence with previous reports in which DNA immunization could readily induce a mixed Th1/Th2 immune response, and even more heterogeneity in IgG2a production, but also dominance of Toxoplasma-specific IgG2a over IgG1 was greater in multiplegene compared to single-gene vaccinations [33]. A vigorous Th1 cell-mediated response associated with high levels of IFN-g and IL-2 are necessary for control of acute and chronic T. gondii infections [34]. IL-12 is required for resistance to acute or chronic toxoplasmosis dependent upon parasite genotype, especially both IL-12p40 and IL-12p70 [35]. In this study, high levels of Th1-type cytokines, including IFN-g, IL-2, IL-12p40 and IL-12p70 that were produced by immunization with pVAX-ROP5 or pVAX-GRA15, confirmed an activated Th1-biased immune responses. Also, the strongest Th1 responses also induced by the pVAX-ROP5 + pVAXGRA15 vaccine, which is in accordance with previous studies, emphasized again that Th1 immune responses can be enhanced by DNA immunization with cocktails of more than one antigen [28,36]. Once the T lymphocyte response are primed, the major effector cells, the CD8+ T cell subpopulation, are considered to be responsible for the control of development and spread of T. gondii infection, in synergy with CD4+ T cells [37,38]. Consistent with studies on DNA vaccination with ROP18 [13] and IF2a [26], we found that the proliferative response of lymphocytes were induced and the percentages of CD8+ T cells and CD4+ T cells significantly were increased in the groups of pVAX-ROP5 and pVAX-GRA15 or cocktailed pVAX-ROP5 with pVAX-GRA15 in contrast to control groups, demonstrating that DNA vaccination induced the activation of CD4+ and CD8+ T cells. It was well known that CTLs targeted to T. gondii secrete IFN-g, which is associated with IFN-g-producing CD8+ T cells [35,39]. So, in order to analyze whether the CD8+

A A

*The same letter on the shoulders of experimental data means no statistically significant difference (p > 0.05) between different experimental groups, while the different letters mean statistically significant difference (p < 0.05). † The reduction of brain cysts is relative to that of the blank control group. ROP5: Rhoptry protein 5.

Cellular immune responses induced by DNA immunization

High levels of the Th1 cytokines IFN-g, IL-2, IL-12p70 and IL-12p40 (FIGURE 3) and thus the proliferative response of lymphocytes were observed in spleen cells from all immunized mice compared with the pVAX I, PBS and blank control groups (TABLE 1; p < 0.05). Spleen cells from mice immunized with pVAX-ROP5 produced significantly more IFN-g and IL-2 than in the group immunized with pVAX-GRA15, but immunization with pVAXGRA15 induced significantly more IL-12p70 and IL-12p40 than in the group of pVAX-ROP5. The levels of IL-4 and IL-10 were increased in supernatants from spleen cells of pVAX-ROP5, pVAX-GRA15 and pVAX-ROP5 + pVAX-GRA15 compared to these control groups (p < 0.05). Co-injection of pVAX-ROP5 and pVAX-GRA15 induced the highest Th1 cytokine levels compared with the all controls (p < 0.05). + + TABLE 2 shows that the percentages of CD3 CD8 CD4 T cells + + – and CD3 CD4 CD8 T cells in pVAX-ROP5, pVAX-GRA15 and pVAX-ROP5 + pVAX-GRA15 immunized animals were significantly increased, most in the pVAX-ROP5+ pVAXGRA15 immunized animals compared, but there was no significant difference between three control groups (p < 0.05). There were no difference between changes in CD8+ and CD4+ T cell profiles in terms of IFN-g secretion. Similarly, DNA immunization with pVAX-ROP5, pVAX-GRA15 and pVAX-ROP5 + pVAX-GRA15 significantly altered CD8+T cell profiles in terms of IFN-g expression in comparison with all controls (p < 0.05). Assessment of protective activity FIGURE 4 shows that immunized mice had a prolonged survival time (mean survival days ± standard deviation (S.D.); n = 10) compared to mice receiving pVAXI, PBS or blank controls (all these mice dead in 9 days, p > 0.05), followed by challenge with the virulent RH strain, with 19.4 ± 4.9 days in the group of pVAX-ROP5, 16.2 ± 1.3 days in the group of pVAX-GRA15 and 22.7 ± 7.2 days in the group of pVAX-ROP5 + pVAX-GRA15 (p < 0.05).

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Protective efficacy of a DNA vaccine encoding T. gondii ROP5 and GRA15 antigens

T cell-mediated cytotoxic T cell response has been activated by DNA immunization, we have detected the secretion of IFN-g from CD8+ T cells. Thus, our results confirm that immunization with pVAX-ROP5 and/or pVAX-GRA15 up-regulate IFN-g production by CD8+ T cell subsets, as a strong effective function of the CD8+ T cell-mediated immune response, which are similar to that in DNA vaccination with pVAX-IF2a [26]. Previous studies have found sequence variation in ROP5 and GRA15 between Type I and Type II of T. gondii [20–22], and our results show that immunization with pVAX-ROP5 of the RH strain or pVAX-GRA15 of the PRU strain did induce effective cross-protection between genotype I and II. In addition, DNA vaccination with pVAX-ROP5 induced a significantly longer survival time than that in the other study with recombinant ROP5 protein immunization [40], suggesting that DNA vaccination could confer significantly stronger protective efficacy compared to a protein-based vaccine. In conclusion, our results demonstrate that a potential DNA vaccine expressing the T. gondii ROP5 or GRA15 elicit Th1-biased responses, as well as CD8+ T cell-mediated cytotoxic T cell response, against acute or chronic T. gondii

Original Research

infection in mice. A combination of TgROP5 and TgGRA15 boosted the immune responses and increased protective efficacy against T. gondii infection compared to single antigen vaccines. Nevertheless, further studies are needed focusing on inducing a stronger immune protection by combination with the other antigens, for instance ROP18 and ROP16. Financial & competing interests disclosure

The study was approved by the animal ethics committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No, LVRIAEC2012-009). Project support was provided by the National Natural Science Foundation of China (Grant Nos. 31230073 and 31172316), the international science and technology cooperation project of Gansu province (Gran No. 1204WCGA023), and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006) The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Key issues .

Vaccination with Toxoplasma gondii rhoptry protein 5 (ROP5) and or GRA15 DNA vaccine induced specific humoral and cellular responses associated with increased frequencies of IFN-g parameters analyzed in CD8+ T-cell compartments (CD8+ IFN-g+ T cells).

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Significant production of IFN-g, IL-2, IL12 p40 and IL-12 p70 associated with a Th1 type response was observed after DNA vaccination.

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DNA vaccination resulted in a significantly prolonged survival time of immunized mice.

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Significant reduction in brain cyst number was observed after vaccination.

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Co-administration with pVAX-ROP5 and pVAX-GRA15 boosted the cellular and humoral immune responses and thus the protective efficacy.

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Both T. gondii ROP5 and pVAX-GRA15 are potential vaccine candidates against both acute and chronic T. gondii infection and coinjection of pVAX-ROP5 and pVAX-GRA15 could increase the protective efficacy.

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Expert Rev. Vaccines 14(4), (2015)

DNA vaccination with genes encoding Toxoplasma gondii antigens ROP5 and GRA15 induces protective immunity against toxoplasmosis in Kunming mice.

To evaluate the protective efficacy of a DNA vaccine encoding Toxoplasma gondii rhoptry protein 5 (ROP5) and GRA15 antigens...
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