Vaccine 31 (2013) 6225–6231
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DNA immunization with eukaryotic initiation factor-2␣ of Toxoplasma gondii induces protective immunity against acute and chronic toxoplasmosis in mice Jia Chen a , Si-Yang Huang a , Dong-Hui Zhou a,∗ , Zhong-Yuan Li a , Eskild Petersen b , 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
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Article history: Received 6 July 2013 Received in revised form 20 September 2013 Accepted 8 October 2013 Available online 31 October 2013 Keywords: Toxoplasma gondii Toxoplasmosis Eukaryotic initiation factor-2␣ (TgIF2␣) DNA vaccine Protective immunity Mice
a b s t r a c t Toxoplasma gondii infection is a serious health problem of humans and animals worldwide. T. gondii eukaryotic initiation factor-2␣ (TgIF2␣) plays a crucial role in parasite viability and is an important virulence factor of T. gondii. To evaluate the vaccine potential of TgIF2␣, we constructed a novel eukaryotic plasmid pVAX-IF2␣ expressing TgIF2␣ from the RH strain and validated expression and immunogenicity in vitro in the Marc145 cell expression system by indirect immunofluorescence (IFA). Administration of pVAX-IF2␣ intramuscularly induced specific humoral immune responses including high levels of specific TgIF2␣ IgG antibody and a mixed IgG1/IgG2a response with a predominance of IgG2a production. The cellular immune response was elicited, showing significant production of IFN-␥ and IL-2 associated with Th1 type response, and thus strong cell-mediated cytotoxic activity with increased frequencies of IFN-␥ parameters analyzed in both CD4+ and CD8+ T cell compartments (CD4+ IFN-␥+ T cells and CD8+ IFN-␥+ T cells). Immunization resulted in partial protection against acute and chronic toxoplamosis in outbred Kunming mice, demonstrated by a significantly prolonged survival time (15.9 ± 4.6 days) after challenge with the virulent RH strain and significant reduction in brain cysts (44.1%) against chronic infection with PRU cyst in contrast to control mice. Our data suggested that pVAX-IF2␣ could be used as a DNA vaccine candidate against both acute and chronic T. gondii infection by the activation of effective humoral and cellular immune responses. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Toxoplasma gondii infects all warm-blooded animals and humans, with a worldwide distribution [1–4]. Most T. gondii infections in humans are asymptomatic, but may cause congenital infection and posterior uveritis [5,6]. Infection in immunocompromised individuals are often severe due to encephalitis and disseminated infection [6–9]. The infection is a major cause of abortion in livestock, especially in sheep and goats, and consumption of infected meat is the main route of transmission to humans [10–12].
∗ Corresponding authors 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. Tel.: +86 18793138037; fax: +86 931 8340977. E-mail addresses: [email protected] (D.-H. Zhou), [email protected], [email protected] (X.-Q. Zhu). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.10.034
Tremendous advances have been made in the studies of antiToxoplasma DNA-delivered vaccines inducing Th1 type and CD8+ cytotoxic T-lymphocyte (CTL) responses. Many T. gondii antigens have been identified as vaccine candidates, including dense granule antigen GRA7 [13], microneme proteins MIC13 [14], rhoptry proteins ROP16, ROP18 and ROP13 [15–17], PLP1 [18], IMP1 [19], NTPase II [20], RON4 [21] and eIF4A [22]. However, these single antigen vaccines only induced partial protection and new vaccine candidate antigens need to be identified. T. gondii eukaryotic initiation factor-2␣ (TgIF2␣) is identified to posses a regulatory serine residue (Ser-71) [23] and the phosphorylation of TgF2␣ is critical for parasite viability [24]. The TgIF2␣ probably is relevant to RH virulence due to the reduced virulence of TgIF2␣-S71A mutant parasites in vivo and a significant delayed acute toxoplasmosis in mice model [24]. This antigen may have the ability to induce considerable protective efficacy against T. gondii infection, in spite of its characteristics of a non-secreted translation factor.
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In the present study, we constructed a eukaryotic plasmid pVAX-IF2␣ expressing TgIF2␣, analyzed immune responses and protective efficacy in Kunming mice induced by pVAX-IF2␣ against lethal challenge with T. gondii RH strain (Type I) or chronic infection with T. gondii PRU strain (Type II).
immunofluorescence assay (IFA) followed by incubation with goat anti-T. gondii tachyzoites polyclonal antiserum and a FITC-labeled donkey-anti-goat IgG antibody (Proteintech Group Inc., Chicago, USA). The specific fluorescence was examined through a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany). Marc-145 cells transfected with empty pVAX I served as the negative control.
2. Materials and methods 2.1. Mice Specific-pathogen-free (SPF) grade female outbred Kunming mice of 6–8 week old were purchased from Lanzhou University Laboratory Animal Center (Lanzhou, China). All mice used for the experiments were raised and treated in strict accordance with good animal practices under the Animal Ethics Procedures and Guidelines of the People’s Republic of China, according to the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. 2.2. Parasites, cell and antigens Two T. gondii strains were used in this study, the virulent RH strain (Type I) and the cyst-forming PRU strain (Type II). Tachyzoites of the RH strain and cysts of the PRU strain were prepared according to the methods described previously [15,22,25]. The obtained tachyzoites of the RH strain were used for the production of the TgIF2␣ clones, the challenge of mice used tachyzoites of the RH strain and cysts of the PRU strain. Preparation of Toxoplasma lysate antigen (TLA) was performed as previously described by Chen et al. [22]. Monkey kidney cells (Marc-145; preserved in our laboratory) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS), 100 mg/ml streptomycin, and 100 IU/ml penicillin at 37 ◦ C with 5% CO2 . 2.3. Construction of DNA vaccine plasmid The eukaryotic expression vector pVAX I was used as a DNA vaccine vector. To clone the TgIF2␣ gene, the total RNA of tachyzoites of RH strain was prepared using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and the coding sequence of TgIF2␣ (1044 bp, GenBank accession no. AY518935.1) was obtained by RT-PCR amplification, with designed specific primers (forward primer: 5 -CCGGAATTCATGGAGGCGAGAGACGGCAC-3 , reverse primer: 5 -GCTCTAGATCACGCATTTCCGTCATCGTTA-3 ), in which EcoR I and Xba I restriction sites were introduced and underlined. The amplified RT-PCR products were inserted into pMD18-T vector (TaKaRa, China) and sequenced in both directions to ensure fidelity, formed recombinant plasmid pMD18-IF2␣. The TgIF2␣ fragment cleaved from pMD-IF2␣ by EcoR I/Xba I was sub-cloned into the corresponding sites of pVAX I (Invitrogen) using T4 DNA ligase, thus generated plasmid pVAX-IF2␣. The recombinant plasmid was propagated in Escherichia coli DH5␣ and confirmed by specific PCR, restriction analysis and DNA sequencing. The positive recombinant plasmid was processed as described previously by Chen et al. [22]. The concentration of pVAX-IF2␣ was determined by spectrophotometer at OD260 and OD280 . 2.4. Expression of pVAX-IF2˛ plasmid in vitro The recombinant plasmid pVAX-IF2␣ was transfected into Marc-145 cells, and the expression was examined as described previously [22]. In brief, 48 h post-transfection, cells were fixed with 100% acetone for 30 min and washed with PBS-0.1% TritonX-100 (PBST) for three times, and then were processed for indirect
2.5. DNA immunization and challenge infection Mice were divided into four groups (35 mice in each group), three groups of mice were injected intramuscularly with 100 g pVAX-IF2␣ DNA in 100 l sterile PBS, 100 g empty vector pVAX, PBS (100 l/each), respectively, and one group of mice were not inoculated to constitute blank control. All experimental groups were vaccinated three times at weeks 0, 2 and 4. Blood was collected from the tail vein prior to each immunization and challenge infection, and sera were separated and stored at −20 ◦ C until analyzed for specific antibodies. Two weeks after the last immunization, 15 mice of each group were challenged intraperitoneally (IP) with 1 × 103 tachyzoites of the RH strain. The survival time for each mice and the percentages of mice survived were recorded daily until a fatal outcome for all animals. Eight mice of each group were inoculated orally with 20 cysts of the PRU strain at day 14th after the third immunization. Mice were observed daily for mortality. Four weeks after infection, surviving mice were sacrificed and the mean number of cysts per brain was calculated as described previously [20,25]. In brief, the whole brain from each mouse was isolated, homogenized and diluted in 3 ml PBS, and 1 l of the homogenized brain was examined to calculate the number of T. gondii tissue cysts under an optical microscope. This procedure was carried out in triplicate, and the mean of three counts was obtained and was then used to calculate the total number of T. gondii tissue cysts in each brain sample. Thereafter, the remaining mice in all groups were sacrificed and splenocytes were aseptically harvested for lymphocyte proliferation assay, cytokine measurements, and flow cytometric analysis. This analysis was performed in three independent experiments. Pre-immune serum samples were used as negative controls. 2.6. Antibody analysis The levels of IgG, IgG1 and IgG2a antibodies in serum were measured by ELISA 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 g/ml; provided by the commercial Kit) in 100 l of phosphate buffered saline (pH7.4) overnight at 4 ◦ C. The plates were washed with PBS plus 0.05% Tween-20 (PBS-T) and blocked with PBS containing 1% BSA for 1 h. 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 l of horseradishperoxidase (HRP) conjugated anti-mouse IgG diluted in 1:250 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 l substrate solution (pH4.0) (1.05% citrate substrate buffer, 1.5% ABTS, 0.03% H2 O2 ) for 20 min. The absorbance was measured at 405 nm using an ELISA reader (Bio-TekEL 800×, USA). All samples were run in triplicate. 2.7. Lymphocyte proliferation assays The in vitro spleen cell proliferative response was measured as described previously [14,22], and the proliferative
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activity was measured using a 3-(4,5-dimethylthylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml, Sigma) dye assay, as described previously [26]. The stimulation index (SI) was calculated as the ratio of the average OD570 value of wells containing TLA-stimulated cells (OD570TLA ) to the average OD570 value of wells containing only cells with medium (OD570M ). All experimental and control samples were run in triplicate. 2.8. Cytokine assays Splenocytes from each group were cultured with different stimuli (TLA, ConA for positive control; medium alone for negative control) in flat-bottom 96-well microtiter plates as described for the lymphocyte proliferation assay. Culture supernatants were harvested and centrifuged for 5 min at 110 × g, and cytokine levels in cell-free supernatants were examined after 24 h for IL-2 and IL-4, 72 h for IL-10, and 96 h for IFN-␥ 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-␥, IL-2, IL-4 and IL-10. The sensitivity limits for the assays were 8.0 pg/ml for IFN-␥, 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. 2.9. Cell surface staining and intracellular staining of splenic lymphocytes Spleen cells were removed and collected as described above. The viability of the cells was evaluated using 0.04% trypan blue (viability > 90%). The cell concentration was adjusted to 1 × 106 cells/ml in PBS containing 2% FBS. After incubation with surface markers including phycoerythrin (PE)-labeled anti-mouse CD3, Allophycocyanin (APC)-labeled anti-mouse CD4 and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 (eBioscience) at 4 ◦ C for 30 min. In the dark, the cultures were washed using 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). For intracellular cytokine staining (ICS) assays, lymphocytes (2 × 106 ml−1 ) were restimulated with 30 mg/ml TLA in RPMI-1640 containing 10% fetal calf serum (FCS) at 37 ◦ C in 5% CO2 for 12 h, and brefeldin-A was added as recommended by the manufacturer (BD Biosciences) during the final 4 h of culture, which was processed by surface staining and then intracellular staining according to the methods as described previously [41,42]. In brief, the cultures were then washed with PBS, stained using LIVE/DEAD fixable Aqua Stain cell reagent (Invitrogen), and Fc receptors were blocked by incubation with anti-CD16/32 (15 min at 4 ◦ C). Then, the cells were surface-stained at 4 ◦ C for 30 min with MAbs (CD8-PE, CD4-PE; eBioscience) specific for cell surface molecules, fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) for 20 min in dark. The cells were intracellularly stained with IFN-␥APC (eBioscience) for 30 min at 4 ◦ C, and finally, labeled cells were washed and fixed in 1% formaldehyde-PBS, and then data were acquired on a BD FACScan flow cytometer. 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 Students’ t-test. Statistical analysis of the standard error was calculated using the
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function of “stdevp” in Microsoft Excell Software. The results in comparisons between groups were considered different if P < 0.05. 3. Results 3.1. Identification of the expressed product by IFA As shown in Fig. 1, specific green fluorescence was observed in Marc-145 cells transfected with pVAX-IF2␣, whereas no fluorescence was observed in the negative controls transfected with the same amount of empty pVAX I. 3.2. Humoral response induced by DNA immunization Antibody responses detected in sera from the immunized mice and control mice are shown in Fig. 2. A high total IgG antibody titers were induced in mice immunized with pVAX-IF2␣, which were 0.751 ± 0.007 at two weeks after last immunization in contrast to the group injected with PBS, pVAX I, or blank control (P < 0.05), and thus the increase of antibody titers occurred with successive DNA immunization (Fig. 2A). Both IgG1 and IgG2a were induced in the sera of mice immunized with pVAX-IF2␣, but a predominance of IgG2a over IgG1 (Fig. 2B). 3.3. Cellular immune responses Splenocyte proliferation was assessed two weeks after the last immunization. Proliferation stimulation index (SI) of mice immunized with pVAX-IF2␣ (4.16 ± 0.07) was significantly higher in comparison with pVAX I (1.05 ± 0.00), PBS (1.04 ± 0.07) and blank control (1.03 ± 0.03) groups, but there was no significant difference between three control groups (P > 0.05). The analysis of cellular immune responses was shown in Fig. 3, the percentage of CD3+ CD8+ CD4-T cells and CD3+ CD4+ CD8-T cells were significantly increased in mice immunized with pVAXIF2␣ compared with that in mice of the PBS, pVAX I or blank control groups. Similarly, DNA immunization with pVAX-IF2␣ significantly altered CD4+ or CD8+ T cell profiles in terms of IFN-␥ expression in comparison with all controls (Fig. 4). There was no significant difference between the three control groups (P > 0.05). 3.4. Production of cytokines by spleen cells The measurement of the concentration of cytokines in supernatants of splenocytes re-stimulated with TLA was shown in Fig. 5, the mice immunized with pVAX-IF2␣ produced a significant increase in secreted IFN-␥ and IL-2 compared with that in the pVAX I, PBS and the blank controls. Furthermore, small amounts of IL-4 and IL-10 were also secreted by restimulated splenocytes of mice immunized with pVAX-IF2␣ compared to the control groups (P < 0.05). 3.5. Assessment of protective efficacy of DNA immunized mice against T. gondii As shown in Fig. 6, immunization of mice with pVAX-IF2␣ prolonged survival time (15.9 ± 4.6 days) significantly after challenge with the virulent RH strain compared to mice in groups of pVAX I, PBS and blank control. All control mice died within 6 days after challenge, and no significant difference was observed among these control groups (P > 0.05). Protection against chronic infection with tissue cysts of T. gondii PRU strain was shown in Table 1. Mice immunized with pVAXIF2␣ showed a significant reduction (44.1%) in the number of cysts
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Fig. 1. Indirect immunofluorescence (IFA) detection of TgIF2␣ expression on Marc-145 cells at 48 h post-transfection. (A) Marc-145 cells were transfected with pVAX-IF2␣; (B) empty vector pVAX I.
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-IF2␣, 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 (n = 3) and statistically significant difference (P < 0.05) are indicated by (*).
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.
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Fig. 4. The expression of IFN-␥ on CD4+ and CD8+ T cells using flow cytometry analysis. (A) The percentages of IFN-␥+ CD4+ T lymphocytes in mice spleen cells. (B) The percentages of IFN-␥+ CD8+ T lymphocytes in mice spleen cells.
Fig. 5. Cytokine production by splenocytes of immunized Kunming mice after stimulation by Toxoplasma lysate antigen (TLA). Levels of cytokines in cell-free supernatants are represented in scatter dot plots with a line at the mean from 3 individual experiments with 3 mice per group. (A) IFN-␥. (B) IL-2. (C) IL-4. (D) IL-10. *P < 0.05.
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Fig. 6. 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. The mice immunized with pVAX-IF2␣ were dead from day 9 to day 22. Mice immunized with the pVAX-IF2␣ showed an increased survival time (15.9 ± 4.6 days) compared with mice in the control groups (pVAX I, PBS, blanking controls) died within 6 days after challenge (P < 0.05).
Table 1 Mean cyst burden per mouse brain 4 weeks after challenge with 20 cysts of T. gondii strain PRU per mouse. Group (n = 3) pVAX-IF2␣ pVAX1 PBS Blank control
No of brain cysts (means ± SD)* 1742 3067 3067 3117
± ± ± ±
A
119 125B 125B 140B
Reduction (%)a 44.1 1.6 1.6 –
a
The reduction of brain cysts were from the values for the blank control group. The same superscript letter means no difference (P > 0.05), whereas different superscript letters mean significant difference (P < 0.05). *
in the brain compared to control mice. There was no statistically significant protection in all those control groups (P > 0.05). 4. Discussion In the past years, despite attenuated T. gondii strains or subunit vaccines were demonstrated to defend against T. gondii infection effectively, the current attempts have been focused on the exploration of DNA vaccines, which is due to its advantages of costeffectiveness, safety, and the potentiality of inducing long-lasting immunity [27–29]. We have described previously that DNA-based vaccine using pVAX I as the eukaryotic expression vector, which has the capacity to express a variety of genes in vivo, can elicit both an effective humoral and cellular immune response [15,18,22]. This eukaryotic system used in the present study expressed the TgIF2␣ protein successfully, possessing good immunogenicity by IFA analysis. During natural T. gondii invasion, the generation of strong cellular immune responses determines the course of the infection [30,31]. The present study measured various mechanisms of specific cellular immune responses to pVAX-IF2␣ vaccination. In agreement with previous studies of eIF4A and ROP18 [17,22], we found that a T cell response was elicited by immunizing mice with the plasmid pVAX-IF2␣ in contrast with control groups, showing a vigorous lymphocyte proliferation. Furthermore, a primary T cell subsets of CD8+ T cells, in particular in synergy with CD4+ T cells, were involved in the control of development and spreading of T. gondii infection [32–34]. In agreement with this efficacy, we observed the increase of the percentage of CD8+ T cells and CD4+ lymphocytes in mice immunized with pVAX-IF2␣, which also suggested the activation of CD4+ and CD8+ T cells, and thus may be in synergy to contribute to cytotoxic activity against T. gondii. Additionally, the present study showed that CD4+ IFN-␥+ T and CD8+
IFN-␥+ T cells were up-regulated after the administration of pVAXIF2␣, suggesting the activated CTLs effect, which is ascribed to the theory that T. gondii-specific CTLs response is associated with IFN␥ producing CD4+ , and cytotoxic CD8+ T cells mainly are mediated via IFN-␥-producting CD8+ T cells [32,35,36]. The development of potent Th1-type immune responses is essential for the control of T. gondii infection [29,36]. As the indicator for activated Th1 lymphocytes, the pro-inflammatory cytokines including IFN-␥ and IL-2 are also involved in the protection against the infection [37,38]. Our results showed that in contrast with the controls, immunization with pVAX-IF2␣ induced the production of high levels of IL-2 and IFN-␥, which is associated with Th1-type mediated immunity. Nevertheless, Th2-type cytokines, both IL-4 and IL-10, may partially inhibit the secretion of pro-inflammatory cytokines, and prevent CD4+ T cell-mediated severe immunopathology during the acute and chronic stage of T. gondii invasion [30]. Thus, a slight increase in the release of IL-4 and IL-10 in combination with the high levels of IL-2 and IFN-␥ observed in the present study suggested the activation of an appropriate T helper response, but mainly specific Th1-biased cellular immune response after immunization with pVAX-IF2␣. The critical role of antibody in immunity to T. gondii has been recognized for a long time, referred to the ability of killing the parasite by the attachment of the parasite to the host cell receptors or resulting from the bindings to the complement protein [39,40]. Similar to previous studies [15,16,18,20,22], the present study showed that immunization of Kunming mice with pVAX-IF2␣ elicited high level of specific antibodies in sera by ELISA assay. In this study, vaccination with pVAX-IF2␣ induced a high ratio of IgG2a to IgG1 antibody titers, and thus indicated the induction of a mixed Th1/Th2 response. In this study, we used out bred Kunming mice, which are highly susceptible to T. gondii as the infection model, to test the protective effects of pVAX-IF2␣ vaccine in mice against challenge with T. gondii virulent RH strain (Type I) and avirulent PRU strain (Type II). Since our unpublished data showed a very low sequence variation (0.6%) in IF2␣ sequences between T. gondii Type I and Type II strains, the results from this study showed cross-protection induced by pVAX-IF2␣ DNA immunization against Type I and II strains successfully. Therefore, the results of this study showed that CD8+ T cell mediated response effects, Th1-type based immune response with high levels of IL-2 and IFN-␥ production, integrated with antibodies response drive the protection against T. gondii infection. pVAX-IF2␣elicited a protective efficacy which is similar to that of some secreted parasite proteins such as GRA7 [13], MIC13 [14], and ROP13 [15], suggesting again that TgIF2␣ may be responsible for the key biological role during the life cycle of T. gondii. In summary, the present study demonstrates that a plasmid expressing T. gondii IF2␣ has significant potentiality to elicit humoral and cellular immunity against acute and chronic toxoplasmosis, and suggests that the immune efficacy of IF2␣-based vaccines should be further evaluated in other apicomplexan parasites.
Acknowledgements Project support was provided by the National Natural Science Foundation of China (Grant Nos. 31230073, 31172316 and 31101812), the International Science and Technology Cooperation Project of Gansu Province (Grant No. 1204WCGA023), and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006).
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DNA immunization with eukaryotic initiation factor-2␣ of Toxoplasma gondii induces protective immunity against acute and chronic toxoplasmosis in mice Jia Chen a , Si-Yang Huang a , Dong-Hui Zhou a,∗ , Zhong-Yuan Li a , Eskild Petersen b , 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
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Article history: Received 6 July 2013 Received in revised form 20 September 2013 Accepted 8 October 2013 Available online 31 October 2013 Keywords: Toxoplasma gondii Toxoplasmosis Eukaryotic initiation factor-2␣ (TgIF2␣) DNA vaccine Protective immunity Mice
a b s t r a c t Toxoplasma gondii infection is a serious health problem of humans and animals worldwide. T. gondii eukaryotic initiation factor-2␣ (TgIF2␣) plays a crucial role in parasite viability and is an important virulence factor of T. gondii. To evaluate the vaccine potential of TgIF2␣, we constructed a novel eukaryotic plasmid pVAX-IF2␣ expressing TgIF2␣ from the RH strain and validated expression and immunogenicity in vitro in the Marc145 cell expression system by indirect immunofluorescence (IFA). Administration of pVAX-IF2␣ intramuscularly induced specific humoral immune responses including high levels of specific TgIF2␣ IgG antibody and a mixed IgG1/IgG2a response with a predominance of IgG2a production. The cellular immune response was elicited, showing significant production of IFN-␥ and IL-2 associated with Th1 type response, and thus strong cell-mediated cytotoxic activity with increased frequencies of IFN-␥ parameters analyzed in both CD4+ and CD8+ T cell compartments (CD4+ IFN-␥+ T cells and CD8+ IFN-␥+ T cells). Immunization resulted in partial protection against acute and chronic toxoplamosis in outbred Kunming mice, demonstrated by a significantly prolonged survival time (15.9 ± 4.6 days) after challenge with the virulent RH strain and significant reduction in brain cysts (44.1%) against chronic infection with PRU cyst in contrast to control mice. Our data suggested that pVAX-IF2␣ could be used as a DNA vaccine candidate against both acute and chronic T. gondii infection by the activation of effective humoral and cellular immune responses. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Toxoplasma gondii infects all warm-blooded animals and humans, with a worldwide distribution [1–4]. Most T. gondii infections in humans are asymptomatic, but may cause congenital infection and posterior uveritis [5,6]. Infection in immunocompromised individuals are often severe due to encephalitis and disseminated infection [6–9]. The infection is a major cause of abortion in livestock, especially in sheep and goats, and consumption of infected meat is the main route of transmission to humans [10–12].
∗ Corresponding authors 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. Tel.: +86 18793138037; fax: +86 931 8340977. E-mail addresses: [email protected] (D.-H. Zhou), [email protected], [email protected] (X.-Q. Zhu). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.10.034
Tremendous advances have been made in the studies of antiToxoplasma DNA-delivered vaccines inducing Th1 type and CD8+ cytotoxic T-lymphocyte (CTL) responses. Many T. gondii antigens have been identified as vaccine candidates, including dense granule antigen GRA7 [13], microneme proteins MIC13 [14], rhoptry proteins ROP16, ROP18 and ROP13 [15–17], PLP1 [18], IMP1 [19], NTPase II [20], RON4 [21] and eIF4A [22]. However, these single antigen vaccines only induced partial protection and new vaccine candidate antigens need to be identified. T. gondii eukaryotic initiation factor-2␣ (TgIF2␣) is identified to posses a regulatory serine residue (Ser-71) [23] and the phosphorylation of TgF2␣ is critical for parasite viability [24]. The TgIF2␣ probably is relevant to RH virulence due to the reduced virulence of TgIF2␣-S71A mutant parasites in vivo and a significant delayed acute toxoplasmosis in mice model [24]. This antigen may have the ability to induce considerable protective efficacy against T. gondii infection, in spite of its characteristics of a non-secreted translation factor.
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In the present study, we constructed a eukaryotic plasmid pVAX-IF2␣ expressing TgIF2␣, analyzed immune responses and protective efficacy in Kunming mice induced by pVAX-IF2␣ against lethal challenge with T. gondii RH strain (Type I) or chronic infection with T. gondii PRU strain (Type II).
immunofluorescence assay (IFA) followed by incubation with goat anti-T. gondii tachyzoites polyclonal antiserum and a FITC-labeled donkey-anti-goat IgG antibody (Proteintech Group Inc., Chicago, USA). The specific fluorescence was examined through a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany). Marc-145 cells transfected with empty pVAX I served as the negative control.
2. Materials and methods 2.1. Mice Specific-pathogen-free (SPF) grade female outbred Kunming mice of 6–8 week old were purchased from Lanzhou University Laboratory Animal Center (Lanzhou, China). All mice used for the experiments were raised and treated in strict accordance with good animal practices under the Animal Ethics Procedures and Guidelines of the People’s Republic of China, according to the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. 2.2. Parasites, cell and antigens Two T. gondii strains were used in this study, the virulent RH strain (Type I) and the cyst-forming PRU strain (Type II). Tachyzoites of the RH strain and cysts of the PRU strain were prepared according to the methods described previously [15,22,25]. The obtained tachyzoites of the RH strain were used for the production of the TgIF2␣ clones, the challenge of mice used tachyzoites of the RH strain and cysts of the PRU strain. Preparation of Toxoplasma lysate antigen (TLA) was performed as previously described by Chen et al. [22]. Monkey kidney cells (Marc-145; preserved in our laboratory) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS), 100 mg/ml streptomycin, and 100 IU/ml penicillin at 37 ◦ C with 5% CO2 . 2.3. Construction of DNA vaccine plasmid The eukaryotic expression vector pVAX I was used as a DNA vaccine vector. To clone the TgIF2␣ gene, the total RNA of tachyzoites of RH strain was prepared using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and the coding sequence of TgIF2␣ (1044 bp, GenBank accession no. AY518935.1) was obtained by RT-PCR amplification, with designed specific primers (forward primer: 5 -CCGGAATTCATGGAGGCGAGAGACGGCAC-3 , reverse primer: 5 -GCTCTAGATCACGCATTTCCGTCATCGTTA-3 ), in which EcoR I and Xba I restriction sites were introduced and underlined. The amplified RT-PCR products were inserted into pMD18-T vector (TaKaRa, China) and sequenced in both directions to ensure fidelity, formed recombinant plasmid pMD18-IF2␣. The TgIF2␣ fragment cleaved from pMD-IF2␣ by EcoR I/Xba I was sub-cloned into the corresponding sites of pVAX I (Invitrogen) using T4 DNA ligase, thus generated plasmid pVAX-IF2␣. The recombinant plasmid was propagated in Escherichia coli DH5␣ and confirmed by specific PCR, restriction analysis and DNA sequencing. The positive recombinant plasmid was processed as described previously by Chen et al. [22]. The concentration of pVAX-IF2␣ was determined by spectrophotometer at OD260 and OD280 . 2.4. Expression of pVAX-IF2˛ plasmid in vitro The recombinant plasmid pVAX-IF2␣ was transfected into Marc-145 cells, and the expression was examined as described previously [22]. In brief, 48 h post-transfection, cells were fixed with 100% acetone for 30 min and washed with PBS-0.1% TritonX-100 (PBST) for three times, and then were processed for indirect
2.5. DNA immunization and challenge infection Mice were divided into four groups (35 mice in each group), three groups of mice were injected intramuscularly with 100 g pVAX-IF2␣ DNA in 100 l sterile PBS, 100 g empty vector pVAX, PBS (100 l/each), respectively, and one group of mice were not inoculated to constitute blank control. All experimental groups were vaccinated three times at weeks 0, 2 and 4. Blood was collected from the tail vein prior to each immunization and challenge infection, and sera were separated and stored at −20 ◦ C until analyzed for specific antibodies. Two weeks after the last immunization, 15 mice of each group were challenged intraperitoneally (IP) with 1 × 103 tachyzoites of the RH strain. The survival time for each mice and the percentages of mice survived were recorded daily until a fatal outcome for all animals. Eight mice of each group were inoculated orally with 20 cysts of the PRU strain at day 14th after the third immunization. Mice were observed daily for mortality. Four weeks after infection, surviving mice were sacrificed and the mean number of cysts per brain was calculated as described previously [20,25]. In brief, the whole brain from each mouse was isolated, homogenized and diluted in 3 ml PBS, and 1 l of the homogenized brain was examined to calculate the number of T. gondii tissue cysts under an optical microscope. This procedure was carried out in triplicate, and the mean of three counts was obtained and was then used to calculate the total number of T. gondii tissue cysts in each brain sample. Thereafter, the remaining mice in all groups were sacrificed and splenocytes were aseptically harvested for lymphocyte proliferation assay, cytokine measurements, and flow cytometric analysis. This analysis was performed in three independent experiments. Pre-immune serum samples were used as negative controls. 2.6. Antibody analysis The levels of IgG, IgG1 and IgG2a antibodies in serum were measured by ELISA 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 g/ml; provided by the commercial Kit) in 100 l of phosphate buffered saline (pH7.4) overnight at 4 ◦ C. The plates were washed with PBS plus 0.05% Tween-20 (PBS-T) and blocked with PBS containing 1% BSA for 1 h. 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 l of horseradishperoxidase (HRP) conjugated anti-mouse IgG diluted in 1:250 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 l substrate solution (pH4.0) (1.05% citrate substrate buffer, 1.5% ABTS, 0.03% H2 O2 ) for 20 min. The absorbance was measured at 405 nm using an ELISA reader (Bio-TekEL 800×, USA). All samples were run in triplicate. 2.7. Lymphocyte proliferation assays The in vitro spleen cell proliferative response was measured as described previously [14,22], and the proliferative
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activity was measured using a 3-(4,5-dimethylthylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml, Sigma) dye assay, as described previously [26]. The stimulation index (SI) was calculated as the ratio of the average OD570 value of wells containing TLA-stimulated cells (OD570TLA ) to the average OD570 value of wells containing only cells with medium (OD570M ). All experimental and control samples were run in triplicate. 2.8. Cytokine assays Splenocytes from each group were cultured with different stimuli (TLA, ConA for positive control; medium alone for negative control) in flat-bottom 96-well microtiter plates as described for the lymphocyte proliferation assay. Culture supernatants were harvested and centrifuged for 5 min at 110 × g, and cytokine levels in cell-free supernatants were examined after 24 h for IL-2 and IL-4, 72 h for IL-10, and 96 h for IFN-␥ 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-␥, IL-2, IL-4 and IL-10. The sensitivity limits for the assays were 8.0 pg/ml for IFN-␥, 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. 2.9. Cell surface staining and intracellular staining of splenic lymphocytes Spleen cells were removed and collected as described above. The viability of the cells was evaluated using 0.04% trypan blue (viability > 90%). The cell concentration was adjusted to 1 × 106 cells/ml in PBS containing 2% FBS. After incubation with surface markers including phycoerythrin (PE)-labeled anti-mouse CD3, Allophycocyanin (APC)-labeled anti-mouse CD4 and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 (eBioscience) at 4 ◦ C for 30 min. In the dark, the cultures were washed using 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). For intracellular cytokine staining (ICS) assays, lymphocytes (2 × 106 ml−1 ) were restimulated with 30 mg/ml TLA in RPMI-1640 containing 10% fetal calf serum (FCS) at 37 ◦ C in 5% CO2 for 12 h, and brefeldin-A was added as recommended by the manufacturer (BD Biosciences) during the final 4 h of culture, which was processed by surface staining and then intracellular staining according to the methods as described previously [41,42]. In brief, the cultures were then washed with PBS, stained using LIVE/DEAD fixable Aqua Stain cell reagent (Invitrogen), and Fc receptors were blocked by incubation with anti-CD16/32 (15 min at 4 ◦ C). Then, the cells were surface-stained at 4 ◦ C for 30 min with MAbs (CD8-PE, CD4-PE; eBioscience) specific for cell surface molecules, fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) for 20 min in dark. The cells were intracellularly stained with IFN-␥APC (eBioscience) for 30 min at 4 ◦ C, and finally, labeled cells were washed and fixed in 1% formaldehyde-PBS, and then data were acquired on a BD FACScan flow cytometer. 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 Students’ t-test. Statistical analysis of the standard error was calculated using the
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function of “stdevp” in Microsoft Excell Software. The results in comparisons between groups were considered different if P < 0.05. 3. Results 3.1. Identification of the expressed product by IFA As shown in Fig. 1, specific green fluorescence was observed in Marc-145 cells transfected with pVAX-IF2␣, whereas no fluorescence was observed in the negative controls transfected with the same amount of empty pVAX I. 3.2. Humoral response induced by DNA immunization Antibody responses detected in sera from the immunized mice and control mice are shown in Fig. 2. A high total IgG antibody titers were induced in mice immunized with pVAX-IF2␣, which were 0.751 ± 0.007 at two weeks after last immunization in contrast to the group injected with PBS, pVAX I, or blank control (P < 0.05), and thus the increase of antibody titers occurred with successive DNA immunization (Fig. 2A). Both IgG1 and IgG2a were induced in the sera of mice immunized with pVAX-IF2␣, but a predominance of IgG2a over IgG1 (Fig. 2B). 3.3. Cellular immune responses Splenocyte proliferation was assessed two weeks after the last immunization. Proliferation stimulation index (SI) of mice immunized with pVAX-IF2␣ (4.16 ± 0.07) was significantly higher in comparison with pVAX I (1.05 ± 0.00), PBS (1.04 ± 0.07) and blank control (1.03 ± 0.03) groups, but there was no significant difference between three control groups (P > 0.05). The analysis of cellular immune responses was shown in Fig. 3, the percentage of CD3+ CD8+ CD4-T cells and CD3+ CD4+ CD8-T cells were significantly increased in mice immunized with pVAXIF2␣ compared with that in mice of the PBS, pVAX I or blank control groups. Similarly, DNA immunization with pVAX-IF2␣ significantly altered CD4+ or CD8+ T cell profiles in terms of IFN-␥ expression in comparison with all controls (Fig. 4). There was no significant difference between the three control groups (P > 0.05). 3.4. Production of cytokines by spleen cells The measurement of the concentration of cytokines in supernatants of splenocytes re-stimulated with TLA was shown in Fig. 5, the mice immunized with pVAX-IF2␣ produced a significant increase in secreted IFN-␥ and IL-2 compared with that in the pVAX I, PBS and the blank controls. Furthermore, small amounts of IL-4 and IL-10 were also secreted by restimulated splenocytes of mice immunized with pVAX-IF2␣ compared to the control groups (P < 0.05). 3.5. Assessment of protective efficacy of DNA immunized mice against T. gondii As shown in Fig. 6, immunization of mice with pVAX-IF2␣ prolonged survival time (15.9 ± 4.6 days) significantly after challenge with the virulent RH strain compared to mice in groups of pVAX I, PBS and blank control. All control mice died within 6 days after challenge, and no significant difference was observed among these control groups (P > 0.05). Protection against chronic infection with tissue cysts of T. gondii PRU strain was shown in Table 1. Mice immunized with pVAXIF2␣ showed a significant reduction (44.1%) in the number of cysts
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Fig. 1. Indirect immunofluorescence (IFA) detection of TgIF2␣ expression on Marc-145 cells at 48 h post-transfection. (A) Marc-145 cells were transfected with pVAX-IF2␣; (B) empty vector pVAX I.
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-IF2␣, 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 (n = 3) and statistically significant difference (P < 0.05) are indicated by (*).
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.
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Fig. 4. The expression of IFN-␥ on CD4+ and CD8+ T cells using flow cytometry analysis. (A) The percentages of IFN-␥+ CD4+ T lymphocytes in mice spleen cells. (B) The percentages of IFN-␥+ CD8+ T lymphocytes in mice spleen cells.
Fig. 5. Cytokine production by splenocytes of immunized Kunming mice after stimulation by Toxoplasma lysate antigen (TLA). Levels of cytokines in cell-free supernatants are represented in scatter dot plots with a line at the mean from 3 individual experiments with 3 mice per group. (A) IFN-␥. (B) IL-2. (C) IL-4. (D) IL-10. *P < 0.05.
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Fig. 6. 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. The mice immunized with pVAX-IF2␣ were dead from day 9 to day 22. Mice immunized with the pVAX-IF2␣ showed an increased survival time (15.9 ± 4.6 days) compared with mice in the control groups (pVAX I, PBS, blanking controls) died within 6 days after challenge (P < 0.05).
Table 1 Mean cyst burden per mouse brain 4 weeks after challenge with 20 cysts of T. gondii strain PRU per mouse. Group (n = 3) pVAX-IF2␣ pVAX1 PBS Blank control
No of brain cysts (means ± SD)* 1742 3067 3067 3117
± ± ± ±
A
119 125B 125B 140B
Reduction (%)a 44.1 1.6 1.6 –
a
The reduction of brain cysts were from the values for the blank control group. The same superscript letter means no difference (P > 0.05), whereas different superscript letters mean significant difference (P < 0.05). *
in the brain compared to control mice. There was no statistically significant protection in all those control groups (P > 0.05). 4. Discussion In the past years, despite attenuated T. gondii strains or subunit vaccines were demonstrated to defend against T. gondii infection effectively, the current attempts have been focused on the exploration of DNA vaccines, which is due to its advantages of costeffectiveness, safety, and the potentiality of inducing long-lasting immunity [27–29]. We have described previously that DNA-based vaccine using pVAX I as the eukaryotic expression vector, which has the capacity to express a variety of genes in vivo, can elicit both an effective humoral and cellular immune response [15,18,22]. This eukaryotic system used in the present study expressed the TgIF2␣ protein successfully, possessing good immunogenicity by IFA analysis. During natural T. gondii invasion, the generation of strong cellular immune responses determines the course of the infection [30,31]. The present study measured various mechanisms of specific cellular immune responses to pVAX-IF2␣ vaccination. In agreement with previous studies of eIF4A and ROP18 [17,22], we found that a T cell response was elicited by immunizing mice with the plasmid pVAX-IF2␣ in contrast with control groups, showing a vigorous lymphocyte proliferation. Furthermore, a primary T cell subsets of CD8+ T cells, in particular in synergy with CD4+ T cells, were involved in the control of development and spreading of T. gondii infection [32–34]. In agreement with this efficacy, we observed the increase of the percentage of CD8+ T cells and CD4+ lymphocytes in mice immunized with pVAX-IF2␣, which also suggested the activation of CD4+ and CD8+ T cells, and thus may be in synergy to contribute to cytotoxic activity against T. gondii. Additionally, the present study showed that CD4+ IFN-␥+ T and CD8+
IFN-␥+ T cells were up-regulated after the administration of pVAXIF2␣, suggesting the activated CTLs effect, which is ascribed to the theory that T. gondii-specific CTLs response is associated with IFN␥ producing CD4+ , and cytotoxic CD8+ T cells mainly are mediated via IFN-␥-producting CD8+ T cells [32,35,36]. The development of potent Th1-type immune responses is essential for the control of T. gondii infection [29,36]. As the indicator for activated Th1 lymphocytes, the pro-inflammatory cytokines including IFN-␥ and IL-2 are also involved in the protection against the infection [37,38]. Our results showed that in contrast with the controls, immunization with pVAX-IF2␣ induced the production of high levels of IL-2 and IFN-␥, which is associated with Th1-type mediated immunity. Nevertheless, Th2-type cytokines, both IL-4 and IL-10, may partially inhibit the secretion of pro-inflammatory cytokines, and prevent CD4+ T cell-mediated severe immunopathology during the acute and chronic stage of T. gondii invasion [30]. Thus, a slight increase in the release of IL-4 and IL-10 in combination with the high levels of IL-2 and IFN-␥ observed in the present study suggested the activation of an appropriate T helper response, but mainly specific Th1-biased cellular immune response after immunization with pVAX-IF2␣. The critical role of antibody in immunity to T. gondii has been recognized for a long time, referred to the ability of killing the parasite by the attachment of the parasite to the host cell receptors or resulting from the bindings to the complement protein [39,40]. Similar to previous studies [15,16,18,20,22], the present study showed that immunization of Kunming mice with pVAX-IF2␣ elicited high level of specific antibodies in sera by ELISA assay. In this study, vaccination with pVAX-IF2␣ induced a high ratio of IgG2a to IgG1 antibody titers, and thus indicated the induction of a mixed Th1/Th2 response. In this study, we used out bred Kunming mice, which are highly susceptible to T. gondii as the infection model, to test the protective effects of pVAX-IF2␣ vaccine in mice against challenge with T. gondii virulent RH strain (Type I) and avirulent PRU strain (Type II). Since our unpublished data showed a very low sequence variation (0.6%) in IF2␣ sequences between T. gondii Type I and Type II strains, the results from this study showed cross-protection induced by pVAX-IF2␣ DNA immunization against Type I and II strains successfully. Therefore, the results of this study showed that CD8+ T cell mediated response effects, Th1-type based immune response with high levels of IL-2 and IFN-␥ production, integrated with antibodies response drive the protection against T. gondii infection. pVAX-IF2␣elicited a protective efficacy which is similar to that of some secreted parasite proteins such as GRA7 [13], MIC13 [14], and ROP13 [15], suggesting again that TgIF2␣ may be responsible for the key biological role during the life cycle of T. gondii. In summary, the present study demonstrates that a plasmid expressing T. gondii IF2␣ has significant potentiality to elicit humoral and cellular immunity against acute and chronic toxoplasmosis, and suggests that the immune efficacy of IF2␣-based vaccines should be further evaluated in other apicomplexan parasites.
Acknowledgements Project support was provided by the National Natural Science Foundation of China (Grant Nos. 31230073, 31172316 and 31101812), the International Science and Technology Cooperation Project of Gansu Province (Grant No. 1204WCGA023), and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006).
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