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c mice induced by a suicidal DNA vaccine of the MIC3 gene of Toxoplasma gondii
Veterinary Parasitology 164 (2009) 134–140
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Protective immune response in BALB/c mice induced by a suicidal DNA vaccine of the MIC3 gene of Toxoplasma gondii Rui Fang a,b, Hao Nie a,b, Zhengsong Wang a,b, Pan Tu a,b, Danna Zhou a,b, Lixia Wang a,b, Lan He a,b, Yanqin Zhou b,c,*, Junlong Zhao a,b,** a b c
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China Key Laboratory Preventive Veterinary of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 January 2009 Received in revised form 3 June 2009 Accepted 11 June 2009
To evaluate the protective efficiency of a suicidal DNA vaccine against protozoal parasite Toxoplasma gondii, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+ respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay and real time RT-PCR. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferation response and IFN-g expression level induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P < 0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P < 0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P > 0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Toxoplasma gondii MIC3 Suicidal DNA vaccine Protective immune response Conventional DNA vaccine
1. Introduction
* Corresponding author at: Key Laboratory Preventive Veterinary of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China. Tel.: +86 27 8728 0787; fax: +86 27 8728 0408. ** Corresponding author at: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China. Tel.: +86 27 8728 1810; fax: +86 27 8728 0408. E-mail addresses: [email protected] (Y. Zhou), [email protected] (J. Zhao). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.06.012
Toxoplasma gondii is an apicomplaxan protozoal parasite infecting most warm-blooded animals and humans and causes toxoplasmosis. Toxoplasmosis is endemic in most areas of the world and may cause abortion or congenital disease in humans, sheep and goats (Tentera et al., 2000; Montoya and Liesenfeld, 2004). Almost 80–90% primary infections are asymptomatic and the chronic form exists in up to one-third of the human population worldwide. Only very small number of cases of
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T. gondii infection are symptomatic (Ferreira et al., 2008). Primary infection usually confers long-term protective resistance to reinfection. However in the immunocompromised individual, especially one with acquired immunodeficiency syndrome (AIDS), T. gondii is responsible for the development of a variety of clinical syndromes, the most frequent of which is toxoplasmic encephalitis (TE). In this condition, rapid multiplication of tachyzoites destroys the neural tissue resulting in the pathologic changes associated with TE (Kasper and Buzoni-Gatel, 1998). Therefore, T. gondii is of veterinary and medical importance. A vaccine against human infection with T. gondii is not available, but an effective vaccine preventing infection in animals used for human consumption would block the main transmission route to humans (Girish, 2003; Liu et al., 2008). Thus, development of an effective and safe vaccine for controlling T. gondii infection in those animals is an important goal for scientists worldwide. So far, only a live attenuated vaccine has been available for veterinary use for several years in some countries, but it is expensive, causes side effects, and has the possibility to revert to a pathogenic strain (Mateus-Pinilla et al., 1999; Liu et al., 2008). In order to overcome these problems, subunit, recombinant and DNA vaccines were proposed recently (Bout et al., 2002). Among these new kinds of vaccines, DNA vaccines become a focus as such vaccines have been shown to elicit potent, long-lasting humoral and cell-mediated immunity (Alarcon et al., 1999). In spite of the advantages of DNA vaccines, conventional DNA vaccines raise certain concerns such as potential integration into the host genome and cell transformation as well as their potency since they do not have the intrinsic ability to amplify in vivo as viral vaccines do (Hsu et al., 2001). In order to overcome the possible problems caused by conventional DNA vaccines, more recently, a new generation of animal cell expression vector systems based on the replicon of alphaviruses, particularly Semliki Forest virus (SFV), has been developed. It has some prominent advantages. For example, it has a wide range of animal cell hosts, high efficiency and is ease of use (Liljestrom and Garoff, 1991). It could induce higher level humoral and cell-mediated immunity against a variety of antigens, and the immunized animals developed more pronounced immune response than those received a conventional DNA vaccine encoding the same antigen (Kirman et al., 2003). It could break immunological tolerance by activating innate antiviral pathways (Leitner et al., 2003) and had higher biosafety than the conventional DNA vaccines (Berglund et al., 1998). All these advantages indicate that suicidal DNA vaccine is a good delivery vehicle and could be used to replace conventional DNA vaccines. Membrane-associated surface antigen SAG1, SAG2, excreted-secreted dense-granule proteins GRA1, GRA2, GRA4, and GRA7, rhoptry proteins ROP1, ROP2, micronemal protein MIC1, MIC2, MIC3 are the T. gondii putative vaccine candidates (Buxton et al., 1991; Ismael et al., 2003; Zhou et al., 2007; Jongert et al., 2007; Cui et al., 2008). Among all those antigens, the micronemal protein MIC3 is particularly promising because it is a potent adhesin of T. gondii, and expressed in all three infectious stages of T. gondii
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(tachyzoites, bradyzoites, and sporozoites) and elicits early and powerful immune responses in mice (Ismael et al., 2003). pcDNA3.1+ plasmid DNA encoding the MIC3 protein of T. gondii induced strong specific humoral and cellular immune responses, as well as effective, highly significant protection in immunized CBA/J mice (Ismael et al., 2003). In our previous study, MIC3 also display excellent immunogenicity (Jiang et al., 2008). Therefore, the aim of this study was to assess the immunogenic properties and protection value of a suicidal DNA vaccine of MIC3 gene against T. gondii challenge. 2. Materials and methods 2.1. Parasites and cell Tachyzoites of the highly virulent T. gondii (RH) strain were harvested from the peritoneal fluid of BALB/c mice after infected 5–6 days earlier by injecting 1 ml of 0.1 M phosphate buffered saline (PBS) pH 7.2 as described by Diab and El-Bahy (2008). The exudate was separated by low speed centrifugation (100 g for 5 min) at 4 8C to remove the cellular debris. The parasites in the supernatant were precipitated by centrifugation at 600 g for 10 min and then washed in 0.1 M PBS, pH 7.2, counted in a hemocytometer and adjusted to 5 104 parasite/ml. BHK-21 cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; HyClone), 100 U/ml penicillin, and 100 mg/ ml streptomycin at 37 8C in a 5% CO2 (Sun et al., 2007; Xiao et al., 2004). (Tachyzoites of the RH strain and BHK21 cells were obtained from National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China). 2.2. Construction of recombinant vectors To construct the suicidal DNA vaccine encoding T. gondii MIC3, pSCA1 based on the replicon of Semliki Forest virus (SFV) was used (a generous gift from Dr. Rod Bremner at the University of Toronto). This pSCA1 vector contains human cytomegalovirus (hCMV) promoter immediately upstream of SFV replicon. The subgenomic promoter is located after the SFV replicon, upstream of a multiple cloning site for insertion of genes of interest (DiCiommo and Bremner, 1998). MIC3 gene (GeneBank accession no. AJ132530) was amplified from nucleotide positions 696–1775 by PCR and inserted into SmaI sites of pSCA1 and resulted in the suicidal DNA vaccine pSCA/ MIC3. MIC3 gene was inserted into HindIII and XbalI sites of pcDNA3.1+ and resulted in the conventional DNA vaccine pcDNA/MIC3 (Fig. 1). The right open-reading frames of these constructs were confirmed by restriction digestion and sequence analysis. The expression plasmid was transformed into Escherichia coli DH5a and largescale plasmid production was performed using EndoFree Plasmid Mega kit columns (Qiagen). (pSCA1 and pcDNA3.1+ were obtained from National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China).
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2.6. Lymphocyte proliferation assay
Fig. 1. Schematic diagrams of plasmids used for expression analysis in vitro. The individual transcriptional control element comprising the conventional DNA vaccine plasmid (pcDNA3.1+) and the suicidal DNA vaccine plasmid (pSCA1) based on SFV replicon are indicated. PCMV, the major immediate-early promoter/enhancer of hCMV (human cytomegalovirus); polyA, polyadenylation signal; MIC3, full-length of microneme protein 3 gene of Toxoplasma gondii RH; nsps, the nonstructural protein genes of SFV (Semliki Forest Virus); 26S, the subgenomic promoter of SFV.
2.3. In vitro plasmid expression Recombinant plasmids pSCA/MIC3 and pcDNA/MIC3 (25–50 mg/well) were introduced into BHK-21 cells in 24well tissue culture plates when the cells reached approximately 50–70% confluence using Lipofect AMINE2000 Plus reagent (Invitrogen). Forty-eight hours after transfection, cells were collected and fixed with absolute methanol and processed for indirect immunofluorescence assay (IFA) for detection of MIC3 proteins. The cells were incubated with rabbit anti-T. gondii serum (37 8C for 30 min) in a humid box and then with fluorescein-conjugated goat anti-rabbit serum (Sigma) for 30 min at 37 8C (Xiao et al., 2004; Guo et al., 2005; Wang et al., 2007). Fluorescence was observed under a Nikon microscope. At the same time point (48 h posttransfection), the expression of MIC3 was examined by RT-PCR to check whether MIC3 mRNA was successfully transcribed in the transfected cells. At the same time, total RNA of the transfected BHK-21 cells was extracted and one microgram of RNA was reverse transcribed using the Rever Tra Ace1 kit (ToYoBo, Shanghai, China) in a 20 ml reaction mixture. The cDNA product (5 ml) was amplified in a 20 ml reaction mixture. 2.4. Immunization of mice 5–6 weeks old female BALB/c mice (purchased from the Animal Center, Institute of Medicine, Hubei Province) were randomly divided into four groups (sixteen mice each group) including three experimental groups and one control group. These four groups of mice were injected intramuscularly with 100 ml of PBS containing 100 mg pSCA/MIC3, 100 ml of PBS containing 100 mg pcDNA/MIC3, 100 ml of PBS containing 100 mg pSCA1 vector only and 100 ml of PBS only, respectively. All groups of BALB/c mice were inoculated three times at 2-week intervals. The diluted DNA was injected into the quadriceps muscle of both rear legs using a syringe and needle. Serum was collected at weeks 0, 2, 4 and 6 post-immunization (p.i.).
Splenocyte suspensions were collected from immunized mice on 10th day after last immunization, and as described by Desolme et al. (2000), 3 106 cells per well in 150 ml culture medium alone were cultured in 96-well plates in triplicate in RPMI-1640 (GIBCO) supplemented with 5% fetal calf serum (FCS), 2 mM L-glutamine, 10 mM HEPES, 50 mM b-mercaptoethanol, 1 mM sodium pyruvate, 100 U of penicillin/ml, and 100 mg of streptomycin per ml. The cell culture was stimulated with 5 mg/ml MIC3, or unstimulated, respectively, for 72 h at 37 8C in 5% CO2, then 10 ml of 5 mg/ml methyl thiazolyl tetrazolium (MTT, Sigma) was added to each well, and incubated for 4 h. Thereafter, the contents of all wells were discarded, and 100 ml dimethy sulfoxide (DMSO) was added to every well until the deposit was diluted, and the plates were read at 570 nm by a BioRad ELISA reader. Lymphocyte proliferative responses were quantitated by stimulation index (SI), calculated as the ratio of OD570 nm of stimulated cells to OD570 nm of unstimulated cells (Liu et al., 2008). 2.7. Analyses of IFN-g and IL-4 mRNA expression by real-time PCR Ten days after the last immunization, freshly isolated spleen cells (3 106 cells/ml) were cultured in 24-well plates as described in 2.6 for 24 h, with or without 25 mg/ml recombinant MIC3 fusion protein. Total RNA was extracted and one microgram of RNA was reverse transcribed using the Rever Tra Ace1 kit (ToYoBo, Shanghai, China) in a 20 ml reaction mixture as described (Wang et al., 2007). The cDNA product (0.6 ml) was amplified in a 25 ml reaction mixture containing SYBR1 Green Realtime PCR Master Mix (from 10 stock, ToYoBo), 0.3 mM each of the forward and reverse gene-specific primers (for IFN-g: forward primer, 50 TCAAGTGGCATAGATGTGGAAGAA-30 and reverse primer, 50 -TGGCTCTGCAGGATTTTCATG-30 ; for IL-4: forward primer, 50 -TCCTCACAGCAACGAAGAACACCACA-30 and reverse primer, 50 -GAAGCCCTACAGACGAGCTCA-30 ; for b-actin: forward primer, 50 -CACTGCCGCATCCTCTTCCTCCC-30 and 50 -CAATAGTGATGACCTGGCCGT-30 ) were aliquoted into 96well plates (Applied Biosystems, Foster City, CA), and sealed with optical sealing tape (ABI). Each cDNA sample was performed in triplicate. PCR amplifications were performed using an Applied Biosystems 7500 Real-Time PCR System (ABI). Thermal cycling conditions were 2 min at 50 8C, 10 min at 94 8C, and 40 cycles of 15 s at 94 8C and 1 min at 60 8C. Gene expression was measured by relative quantity, which compares the threshold cycle (Ct) of the sample of interest to the Ct generated by a reference sample referred to as the calibrator (non-stimulated splenocytes incubated for the same period). 2.8. Mouse protection test (MPT)
2.5. ELISA for detection of MIC3-specific antibodies Serum samples collected from BALB/c mice were evaluated by an indirect ELISA test using the recombinant GST-MIC3 protein of T. gondii produced in a previous study (Jiang, 2006; Jiang et al., 2008).
Ten days after the last immunization, eight mice were selected randomly from each group and injected intraperitoneally with 1 104 tachyzoites of T. gondii (RH strain). Tachyzoites were prepared as described in 2.1, counted in a hemocytometer and adjusted to 5 104 parasite/ml with
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Fig. 2. Expression analyses of MIC3 proteins in transfected BHK-21 cells by IFA (A–E) and RT-PCR (F) on 48 h of transfection: (A) BHK-21 cells transfected with pcDNA/MIC3; (B) BHK-21 cells transfected with pSCA/MIC3; (C) BHK-21 cells transfected with pSCA1; (D) BHK-21 cells transfected with pcDNA3.1+; (E) untransfected BHK-21 cells. (F) Lane 1: BHK-21 cells transfected with pcDNA/MIC3; lane 2: BHK-21 cells transfected with pSCA/MIC3; lane 3: BHK-21 cells transfected with pcDNA3.1+; lane 4: BHK-21 cells transfected with pSCA1; lane 5: BHK-21 cells untransfected.
0.1 M phosphate buffered saline (PBS) pH 7.2 Then the symptom was observed and the survival time was recorded. 2.9. Statistical analysis For evaluation the protective immune responses among the different groups, all groups’ mean values were compared by analysis of variance (ANOVA) and Student’s t-test using SPSS v.12.0 software. P-Values of <0.05 were considered statistically significant. 3. Results
3.2. MIC3-specific antibodies in immunized mice To evaluate the immunogenicity of the suicidal DNA vaccines, serum collected at 0, 2, 4 and 6 weeks after immunization were tested for the antibodies against MIC3 by an indirect ELISA test using the recombinant MIC3 protein. Specific antibodies against T. gondii MIC3 appeared following injection of pSCA/MIC3 and pcDNA/MIC3. The mean antibody titers of the pSCA/MIC3-vaccinated group and pcDNA/MIC3-vaccinated group were significantly higher (P < 0.05) than those of the pSCA1, pcDNA3.1+ and PBS control group (Fig. 3). There was no significant difference in IgG responses between the groups immunized with pSCA/MIC3 and pcDNA/MIC3 (P > 0.05).
3.1. Expression of pSCA/MIC3 plasmid in BHK-21 cells In order to demonstrate expression of the T. gondii MIC3 proteins, transfected BHK-21 cells were examined by IFA and RT-PCR. Cells transfected with pSCA/MIC3 and pcDNA/MIC3 showed specific green fluorescence but the negative controls, which were transfected with the same amount of pSCA1 or pcDNA3.1+, without the insert, and non-transfected cells, respectively, did not show any fluorescent emission (Fig. 2(A)–(E)). At the same time, specific PCR band were detected in samples from cells transfected with pSCA/MIC3 or pcDNA/MIC3 on agarose gel but there were not any PCR bands detected in the negative controls which were transfected with the same amount of pSCA1 or pcDNA3.1+ and the blank BHK-21 cells (Fig. 2(F)).
Fig. 3. Anti-MIC3 antibody levels in BALB/c mice after immunization. Sera were tested for antibodies at a 1:160 dilution. The result was obtained from mean ELISA absorbance values of eight sera from each group.
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Fig. 4. On the 10th day after the last immunization, BALB/c mice (eight per group) splenocytes were isolated and used for lymphocyte proliferation assay as described in Section 2.6 (Fig. 4).
3.3. Lymphocyte proliferation assay To investigate whether pSCA/MIC3 could also enhance the cell-mediated immune responses, splenocyte suspensions were collected for lymphocyte proliferative responses assay on 10th day after last immunization. The lymphocyte proliferative responses assay were performed as described in Section 2.6 using recombinant MIC3 protein as stimulator and ConA as positive control. As shown in Fig. 4, the specific proliferative response was significantly higher in mice immunized with pSCA/MIC3 and pcDNA/MIC3 (P < 0.05) than in mice immunized with empty vectors and PBS, respectively. However the mean lymphocyte stimulation index (SI) of the pSCA/MIC3vaccinated group was higher than pcDNA/MIC3-vaccinated group, but this was not statistically significant (P > 0.05). 3.4. IFN-g and IL-4 expression assay The IFN-g and IL-4 expression in splenocytes stimulated by MIC3 were analyzed by real-time RT-PCR at the same time. As shown in Fig. 5(A), the production of IFN-g in pSCA/MIC3 or pcDNA/MIC3 groups were significantly
Fig. 6. Survival time of the immunized mice after T. gondii challenge. Each group of mice (n = 8) was immunized (i.m.) with 100 mg of DNA at 0, 2, 4 and 6 weeks. On the 10th day after the last immunization, mice were challenged with 104 tachyzoites of RH strain. Survival was monitored daily for 15 days after challenge.
higher (P < 0.05) than that of control groups, but there was no significant difference in IFN-g responses between pSCA/MIC3 group and pcDNA/MIC3 group (P > 0.05). In contrast, as shown in Fig. 5(B), the production of IL-4 in pSCA/MIC3 or pcDNA/MIC3 groups were significantly lower (P < 0.05) than that of control groups, there was no significant difference in IL-4 responses between the pSCA/MIC3 group and pcDNA/MIC3 group (P > 0.05). 3.5. Lethal challenge test in BALB/c mice To test whether this vaccine could induce protective effect against a lethal T. gondii infection, the mice were intraperitoneally infected with 104 tachyzoites on tenth day after the last immunization. The survival time was shown in Fig. 6, the survival time of mice immunized with pSCA/MIC3 and pcDNA/MIC3 were significant longer (P < 0.05) in comparison to control groups, and pSCA/ MIC3-vaccinated group’s survival time was longer than pcDNA/MIC3-vaccinated group’s, but this was not statistically significant (P > 0.05).
Fig. 5. IFN-g mRNA expression assay (A) and IL-4 mRNA expression assay (B). On the 10th day after the last immunization, BALB/c mice (eight per group) splenocytes were isolated. They were used in IFN-g and IL-4 mRNA expression assay as described in Section 2.7.
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4. Discussion In the present study, the in vitro expression study indicated that the recombinant plasmids pSCA/MIC3 and pcDNA/MIC3 could express the MIC3 protein faithfully. Then in vivo study in BALB/c mice showed that MIC3specific immune responses were induced when they were injected intramuscularly with suicidal DNA vaccine pSCA/ MIC3 or conventional DNA vaccine pcDNA/MIC3, and higher antibody titers were detected in mice immunized with pSCA/MIC3 and pcDNA/MIC3. The differences in stimulation index (SI) of lymphocyte proliferation assay between suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were statistically significant in comparison to control groups and suicidal DNA vaccine pSCA/MIC3 could stimulate highest stimulation index than others; IFN-g detection showed that pSCA/ MIC3 and pcDNA/MIC3 could significantly increased the production of IFN-g, but there was no significant difference in IFN-g responses between pSCA/MIC3 group and pcDNA/ MIC3 group (P > 0.05). In contrast, as shown in Fig. 5(B) pSCA/MIC3 or pcDNA/MIC3 (P < 0.05) significantly decreased the production of IL-4, there was no significant difference in IL-4 responses between the pSCA/MIC3 group and pcDNA/MIC3 group (P > 0.05). These findings indicate that a Th1 immune response was induced by pSCA/MIC3 or pcDNA/MIC3 immunization. Lethal challenge experiments showed that pSCA/MIC3 and pcDNA/MIC3 provided significantly longer survival time (P < 0.05) against T. gondii challenge than control groups, but the difference between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P > 0.05), although pSCA/MIC3 group’s survival rate was higher than that of pcDNA/MIC3 group’s. All these results showed that suicidal DNA vaccine pSCA/MIC3 could induce high humoral and cell-mediated immune responses. Compared with conventional DNA vaccine pcDNA/MIC3, pSCA/MIC3 also provided favourable efficacy. However, it could improve safety of the conventional DNA vector. This is the first report on a suicidal DNA construct strategy as a potential DNA vaccine against T. gondii challenge. A good vaccine should always have several essential elements including safety and low cost in manufacture, storage, and administration (Doel, 1999). Under this standardization, suicidal DNA vaccine came into our eyes. Based on the alphavirus replicon, suicidal DNA vaccines have emerged as an important strategy to enhance immunogenicity and to improve the biosafety of conventional vaccines (Doel, 1999). Among suicidal DNA vaccines, pSCA1 is a layered DNA/RNA construct. The first layer of this vector system utilizes a RNA polymerase II-dependent promoter, it stimulates the transcription of the alphaviral replicon RNA transcript. Translation of this RNA molecule produces the alphaviral replicase complex, which catalyzes cytoplasmic self-amplification of recombinant RNA, and high-level expression of the heterologous antigens (Berglund et al., 1998). Furthermore, when a suicidal DNA vaccine is transfected into cells, it leads eventually to apoptosis of the transfected cells (DiCiommo and Bremner, 1998; Leitner et al., 2000), which is particularly important in alleviating the concerns of potential integration and cell
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transformation generated by the use of conventional DNA vaccines (DiCiommo and Bremner, 1998; Gurunathan et al., 2000). Several groups have demonstrated the ability of suicidal DNA vaccines to induce higher level of humoral and cell-mediated immunity against several pathogens causing infections including foot-and-mouth disease (Guo et al., 2005; Wang et al., 2007), swine vesicular disease virus (Sun et al., 2007), louping ill virus (Fleeton et al., 2000), etc. The results from the present study showed that the suicidal DNA vaccine has the ability of induce higher level humoral and cell-mediated immunity against protozoal parasite too. In the present study, the level of IFN-g mRNA expression was higher in mice immunized with pSCA/ MIC3 or pcDNA/MIC3, while IL-4 mRNA expression were lower in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 than control group. These results are consistent to other reports in T. gondii infection. For example, Liu et al. (2008) demonstrated that BALB/c mice vaccinated with rPRV/SAG1 developed significant levels of IFN-g, while IL-4 was not detected. These findings indicate that both pSCA/MIC3 and pcDNA/MIC3 induce an enhanced Th1-type response. Further, this suicidal DNA vaccine pSCA/MIC3 will be used to immunize animals of food source, its protection against T. gondii infection in those animals will be investigated. It was expected that it could help to reduce worldwide jeopardizing for public health and economics caused by toxoplasmosis. In conclusion, a T. gondii suicidal DNA vaccine pSCA/ MIC3 was successfully constructed for the first time. Compared to control groups, suicidal DNA vaccine pSCA/ MIC3 and conventional DNA vaccine pcDNA/MIC3 were able to elicit a significant humoral and cellular immune response and increased survival time in mice challenged with the lethal RH tachyzoites, but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P > 0.05). The difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/ MIC3 was not statistically significant (P > 0.05). The findings demonstrated that as conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provides favourable efficacy. However, based on the alphavirus replicon, suicidal DNA vaccines leads eventually to apoptosis of the transfected cells, which is particularly important in alleviating the concerns of potential integration and cell transformation generated by the use of conventional DNA vaccines, so it could improve safety of the conventional DNA vector. In this case, the design of vaccines against T. gondii based on suicidal DNA approach is feasible and effective.
Acknowledgments This study was in part supported by the national special research programs for non-profit-trades (No. 200803017) and National Natural Science Foundation of China (NSFC, the Grant No. 30800818). Dr. S.B. Xiao was thanked for skillful technical assistance. Dr. M. Hu (Department of
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Agricultural Sciences, La Trobe University, Australia) and two anonymous reviewers were thanked for their constructive suggestions and correction of this manuscript.
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Ismael, A.B., Sekkai, D., Collin, C., Bout, D., Me´ve´lec, M.N., 2003. The MIC3 gene of Toxoplasma gondii is a novel potent vaccine candidate against toxoplasmosis. Infect. Immun. 71, 6222–6228. Jiang, T., 2006. Study on molecular diagnosis methods and gene immunity for swine toxoplasmosis [D]. Huazhong Agric. Univ. Libr. 63–65. Jiang, T., Gong, D.H., Ma, L.A., Nie, H., Zhou, Y.Q., Yao, B.A., Zhao, J.L., 2008. Evaluation of a recombinant MIC3 based latex agglutination test for the rapid serodiagnosis of Toxoplasma gondii infection in swines. Vet. Parasitol. 158, 51–56. Jongert, E., Craeye, S.D., Dewit, J., Huygen, K., 2007. GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. Parasite Immunol. 29, 445–453. Kasper, L.H., Buzoni-Gatel, D., 1998. Some opportunistic parasitic infections in AIDS: candidiasis, pneumocystosis, cryptosporidiosis, toxoplasmosis. Parasitol. Today 14, 150–156. Kirman, J.R., Turon, T., Su, H., Li, A., Kraus, C., Polo, J.M., Belisle, J., Morris, S., Seder, R.A., 2003. Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an Alphavirus Plasmid Replicon Expressing Antigen 85A. Infect. Immun. 71, 575–579. Leitner, W.W., Hwang, L.N., Deveer, M.J., Zhou, A., Silverman, R.H., Williams, B.R., Dubensky, T.W., Ying, H., Restifo, N.P., 2003. Alphavirusbased DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat. Med. 9, 33–39. Leitner, W.W., Ying, H., Driver, D.A., Dubensky, T.W., Restifo, N.P., 2000. Enhancement of tumor-specific immune response with plasmid DNA replicon vectors. Cancer Res. 60, 51–55. Liljestrom, P., Garoff, H., 1991. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (NY) 9, 56–61. Liu, Q., Gao, S.Y., Jiang, L., Shang, L.M., Men, J.T., Wang, Z., Zhai, Y.J., Xia, Z.P., Hu, R.L., Zhang, X.C., Zhu, X.Q., 2008. A recombinant Pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice. Microb. Infect. 8, 1–8. Mateus-Pinilla, N.E., Dubey, J.P., Choromanski, L., Weigel, R.M., 1999. A field trial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T. gondii exposure for swine. J. Parasitol. 85, 855–860. Montoya, J.G., Liesenfeld, O., 2004. Toxoplasmosis. Lancet 363, 1965– 1976. Sun, S.Q., Liu, X.T., Guo, H.C., Yin, S.H., Shang, Y.J., Feng, X., Liu, Z.X., Xie, Q.G., 2007. Protective immune responses in guinea pigs and swine induced by a suicidal DNA vaccine of the capsid gene of swine vesicular disease virus. J. Gen. Virol. 88, 842–848. Tentera, A.M., Heckeroth, A.R., Weiss, L.M., 2000. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30, 1217–1258. Wang, S.P., Fang, L.R., Chen, H.C., Xiao, S.B., 2007. Construction and immunogenicity of pseudotype baculovirus expressing GP5 and M protein of porcine reproductive and respiratory syndrome virus. Vaccine 25, 8220–8227. Xiao, S.B., Chen, H.C., Fang, L.R., Liu, C.S., Zhang, H., Jiang, Y.B., 2004. Comparison of immune responses and protective efficacy of suicidal DNA vaccine and conventional DNA vaccine encoding glycoprotein C of pseudorabies virus in mice. Vaccine 22, 345–351. Zhou, H.Y., Gu, Q.M., Zhao, Q.L., Zhang, J.Q., Cong, H., Li, Y., He, S.Y., 2007. Toxoplasma gondii expression and characterization of a recombinant protein containing SAG1 and GRA2 in Pichia pastoris. Parasitol. Res. 100, 829–835.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Protective immune response in BALB/c mice induced by a suicidal DNA vaccine of the MIC3 gene of Toxoplasma gondii Rui Fang a,b, Hao Nie a,b, Zhengsong Wang a,b, Pan Tu a,b, Danna Zhou a,b, Lixia Wang a,b, Lan He a,b, Yanqin Zhou b,c,*, Junlong Zhao a,b,** a b c
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China Key Laboratory Preventive Veterinary of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 January 2009 Received in revised form 3 June 2009 Accepted 11 June 2009
To evaluate the protective efficiency of a suicidal DNA vaccine against protozoal parasite Toxoplasma gondii, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+ respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay and real time RT-PCR. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferation response and IFN-g expression level induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P < 0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P < 0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P > 0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Toxoplasma gondii MIC3 Suicidal DNA vaccine Protective immune response Conventional DNA vaccine
1. Introduction
* Corresponding author at: Key Laboratory Preventive Veterinary of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China. Tel.: +86 27 8728 0787; fax: +86 27 8728 0408. ** Corresponding author at: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China. Tel.: +86 27 8728 1810; fax: +86 27 8728 0408. E-mail addresses: [email protected] (Y. Zhou), [email protected] (J. Zhao). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.06.012
Toxoplasma gondii is an apicomplaxan protozoal parasite infecting most warm-blooded animals and humans and causes toxoplasmosis. Toxoplasmosis is endemic in most areas of the world and may cause abortion or congenital disease in humans, sheep and goats (Tentera et al., 2000; Montoya and Liesenfeld, 2004). Almost 80–90% primary infections are asymptomatic and the chronic form exists in up to one-third of the human population worldwide. Only very small number of cases of
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T. gondii infection are symptomatic (Ferreira et al., 2008). Primary infection usually confers long-term protective resistance to reinfection. However in the immunocompromised individual, especially one with acquired immunodeficiency syndrome (AIDS), T. gondii is responsible for the development of a variety of clinical syndromes, the most frequent of which is toxoplasmic encephalitis (TE). In this condition, rapid multiplication of tachyzoites destroys the neural tissue resulting in the pathologic changes associated with TE (Kasper and Buzoni-Gatel, 1998). Therefore, T. gondii is of veterinary and medical importance. A vaccine against human infection with T. gondii is not available, but an effective vaccine preventing infection in animals used for human consumption would block the main transmission route to humans (Girish, 2003; Liu et al., 2008). Thus, development of an effective and safe vaccine for controlling T. gondii infection in those animals is an important goal for scientists worldwide. So far, only a live attenuated vaccine has been available for veterinary use for several years in some countries, but it is expensive, causes side effects, and has the possibility to revert to a pathogenic strain (Mateus-Pinilla et al., 1999; Liu et al., 2008). In order to overcome these problems, subunit, recombinant and DNA vaccines were proposed recently (Bout et al., 2002). Among these new kinds of vaccines, DNA vaccines become a focus as such vaccines have been shown to elicit potent, long-lasting humoral and cell-mediated immunity (Alarcon et al., 1999). In spite of the advantages of DNA vaccines, conventional DNA vaccines raise certain concerns such as potential integration into the host genome and cell transformation as well as their potency since they do not have the intrinsic ability to amplify in vivo as viral vaccines do (Hsu et al., 2001). In order to overcome the possible problems caused by conventional DNA vaccines, more recently, a new generation of animal cell expression vector systems based on the replicon of alphaviruses, particularly Semliki Forest virus (SFV), has been developed. It has some prominent advantages. For example, it has a wide range of animal cell hosts, high efficiency and is ease of use (Liljestrom and Garoff, 1991). It could induce higher level humoral and cell-mediated immunity against a variety of antigens, and the immunized animals developed more pronounced immune response than those received a conventional DNA vaccine encoding the same antigen (Kirman et al., 2003). It could break immunological tolerance by activating innate antiviral pathways (Leitner et al., 2003) and had higher biosafety than the conventional DNA vaccines (Berglund et al., 1998). All these advantages indicate that suicidal DNA vaccine is a good delivery vehicle and could be used to replace conventional DNA vaccines. Membrane-associated surface antigen SAG1, SAG2, excreted-secreted dense-granule proteins GRA1, GRA2, GRA4, and GRA7, rhoptry proteins ROP1, ROP2, micronemal protein MIC1, MIC2, MIC3 are the T. gondii putative vaccine candidates (Buxton et al., 1991; Ismael et al., 2003; Zhou et al., 2007; Jongert et al., 2007; Cui et al., 2008). Among all those antigens, the micronemal protein MIC3 is particularly promising because it is a potent adhesin of T. gondii, and expressed in all three infectious stages of T. gondii
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(tachyzoites, bradyzoites, and sporozoites) and elicits early and powerful immune responses in mice (Ismael et al., 2003). pcDNA3.1+ plasmid DNA encoding the MIC3 protein of T. gondii induced strong specific humoral and cellular immune responses, as well as effective, highly significant protection in immunized CBA/J mice (Ismael et al., 2003). In our previous study, MIC3 also display excellent immunogenicity (Jiang et al., 2008). Therefore, the aim of this study was to assess the immunogenic properties and protection value of a suicidal DNA vaccine of MIC3 gene against T. gondii challenge. 2. Materials and methods 2.1. Parasites and cell Tachyzoites of the highly virulent T. gondii (RH) strain were harvested from the peritoneal fluid of BALB/c mice after infected 5–6 days earlier by injecting 1 ml of 0.1 M phosphate buffered saline (PBS) pH 7.2 as described by Diab and El-Bahy (2008). The exudate was separated by low speed centrifugation (100 g for 5 min) at 4 8C to remove the cellular debris. The parasites in the supernatant were precipitated by centrifugation at 600 g for 10 min and then washed in 0.1 M PBS, pH 7.2, counted in a hemocytometer and adjusted to 5 104 parasite/ml. BHK-21 cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; HyClone), 100 U/ml penicillin, and 100 mg/ ml streptomycin at 37 8C in a 5% CO2 (Sun et al., 2007; Xiao et al., 2004). (Tachyzoites of the RH strain and BHK21 cells were obtained from National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China). 2.2. Construction of recombinant vectors To construct the suicidal DNA vaccine encoding T. gondii MIC3, pSCA1 based on the replicon of Semliki Forest virus (SFV) was used (a generous gift from Dr. Rod Bremner at the University of Toronto). This pSCA1 vector contains human cytomegalovirus (hCMV) promoter immediately upstream of SFV replicon. The subgenomic promoter is located after the SFV replicon, upstream of a multiple cloning site for insertion of genes of interest (DiCiommo and Bremner, 1998). MIC3 gene (GeneBank accession no. AJ132530) was amplified from nucleotide positions 696–1775 by PCR and inserted into SmaI sites of pSCA1 and resulted in the suicidal DNA vaccine pSCA/ MIC3. MIC3 gene was inserted into HindIII and XbalI sites of pcDNA3.1+ and resulted in the conventional DNA vaccine pcDNA/MIC3 (Fig. 1). The right open-reading frames of these constructs were confirmed by restriction digestion and sequence analysis. The expression plasmid was transformed into Escherichia coli DH5a and largescale plasmid production was performed using EndoFree Plasmid Mega kit columns (Qiagen). (pSCA1 and pcDNA3.1+ were obtained from National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China).
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2.6. Lymphocyte proliferation assay
Fig. 1. Schematic diagrams of plasmids used for expression analysis in vitro. The individual transcriptional control element comprising the conventional DNA vaccine plasmid (pcDNA3.1+) and the suicidal DNA vaccine plasmid (pSCA1) based on SFV replicon are indicated. PCMV, the major immediate-early promoter/enhancer of hCMV (human cytomegalovirus); polyA, polyadenylation signal; MIC3, full-length of microneme protein 3 gene of Toxoplasma gondii RH; nsps, the nonstructural protein genes of SFV (Semliki Forest Virus); 26S, the subgenomic promoter of SFV.
2.3. In vitro plasmid expression Recombinant plasmids pSCA/MIC3 and pcDNA/MIC3 (25–50 mg/well) were introduced into BHK-21 cells in 24well tissue culture plates when the cells reached approximately 50–70% confluence using Lipofect AMINE2000 Plus reagent (Invitrogen). Forty-eight hours after transfection, cells were collected and fixed with absolute methanol and processed for indirect immunofluorescence assay (IFA) for detection of MIC3 proteins. The cells were incubated with rabbit anti-T. gondii serum (37 8C for 30 min) in a humid box and then with fluorescein-conjugated goat anti-rabbit serum (Sigma) for 30 min at 37 8C (Xiao et al., 2004; Guo et al., 2005; Wang et al., 2007). Fluorescence was observed under a Nikon microscope. At the same time point (48 h posttransfection), the expression of MIC3 was examined by RT-PCR to check whether MIC3 mRNA was successfully transcribed in the transfected cells. At the same time, total RNA of the transfected BHK-21 cells was extracted and one microgram of RNA was reverse transcribed using the Rever Tra Ace1 kit (ToYoBo, Shanghai, China) in a 20 ml reaction mixture. The cDNA product (5 ml) was amplified in a 20 ml reaction mixture. 2.4. Immunization of mice 5–6 weeks old female BALB/c mice (purchased from the Animal Center, Institute of Medicine, Hubei Province) were randomly divided into four groups (sixteen mice each group) including three experimental groups and one control group. These four groups of mice were injected intramuscularly with 100 ml of PBS containing 100 mg pSCA/MIC3, 100 ml of PBS containing 100 mg pcDNA/MIC3, 100 ml of PBS containing 100 mg pSCA1 vector only and 100 ml of PBS only, respectively. All groups of BALB/c mice were inoculated three times at 2-week intervals. The diluted DNA was injected into the quadriceps muscle of both rear legs using a syringe and needle. Serum was collected at weeks 0, 2, 4 and 6 post-immunization (p.i.).
Splenocyte suspensions were collected from immunized mice on 10th day after last immunization, and as described by Desolme et al. (2000), 3 106 cells per well in 150 ml culture medium alone were cultured in 96-well plates in triplicate in RPMI-1640 (GIBCO) supplemented with 5% fetal calf serum (FCS), 2 mM L-glutamine, 10 mM HEPES, 50 mM b-mercaptoethanol, 1 mM sodium pyruvate, 100 U of penicillin/ml, and 100 mg of streptomycin per ml. The cell culture was stimulated with 5 mg/ml MIC3, or unstimulated, respectively, for 72 h at 37 8C in 5% CO2, then 10 ml of 5 mg/ml methyl thiazolyl tetrazolium (MTT, Sigma) was added to each well, and incubated for 4 h. Thereafter, the contents of all wells were discarded, and 100 ml dimethy sulfoxide (DMSO) was added to every well until the deposit was diluted, and the plates were read at 570 nm by a BioRad ELISA reader. Lymphocyte proliferative responses were quantitated by stimulation index (SI), calculated as the ratio of OD570 nm of stimulated cells to OD570 nm of unstimulated cells (Liu et al., 2008). 2.7. Analyses of IFN-g and IL-4 mRNA expression by real-time PCR Ten days after the last immunization, freshly isolated spleen cells (3 106 cells/ml) were cultured in 24-well plates as described in 2.6 for 24 h, with or without 25 mg/ml recombinant MIC3 fusion protein. Total RNA was extracted and one microgram of RNA was reverse transcribed using the Rever Tra Ace1 kit (ToYoBo, Shanghai, China) in a 20 ml reaction mixture as described (Wang et al., 2007). The cDNA product (0.6 ml) was amplified in a 25 ml reaction mixture containing SYBR1 Green Realtime PCR Master Mix (from 10 stock, ToYoBo), 0.3 mM each of the forward and reverse gene-specific primers (for IFN-g: forward primer, 50 TCAAGTGGCATAGATGTGGAAGAA-30 and reverse primer, 50 -TGGCTCTGCAGGATTTTCATG-30 ; for IL-4: forward primer, 50 -TCCTCACAGCAACGAAGAACACCACA-30 and reverse primer, 50 -GAAGCCCTACAGACGAGCTCA-30 ; for b-actin: forward primer, 50 -CACTGCCGCATCCTCTTCCTCCC-30 and 50 -CAATAGTGATGACCTGGCCGT-30 ) were aliquoted into 96well plates (Applied Biosystems, Foster City, CA), and sealed with optical sealing tape (ABI). Each cDNA sample was performed in triplicate. PCR amplifications were performed using an Applied Biosystems 7500 Real-Time PCR System (ABI). Thermal cycling conditions were 2 min at 50 8C, 10 min at 94 8C, and 40 cycles of 15 s at 94 8C and 1 min at 60 8C. Gene expression was measured by relative quantity, which compares the threshold cycle (Ct) of the sample of interest to the Ct generated by a reference sample referred to as the calibrator (non-stimulated splenocytes incubated for the same period). 2.8. Mouse protection test (MPT)
2.5. ELISA for detection of MIC3-specific antibodies Serum samples collected from BALB/c mice were evaluated by an indirect ELISA test using the recombinant GST-MIC3 protein of T. gondii produced in a previous study (Jiang, 2006; Jiang et al., 2008).
Ten days after the last immunization, eight mice were selected randomly from each group and injected intraperitoneally with 1 104 tachyzoites of T. gondii (RH strain). Tachyzoites were prepared as described in 2.1, counted in a hemocytometer and adjusted to 5 104 parasite/ml with
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Fig. 2. Expression analyses of MIC3 proteins in transfected BHK-21 cells by IFA (A–E) and RT-PCR (F) on 48 h of transfection: (A) BHK-21 cells transfected with pcDNA/MIC3; (B) BHK-21 cells transfected with pSCA/MIC3; (C) BHK-21 cells transfected with pSCA1; (D) BHK-21 cells transfected with pcDNA3.1+; (E) untransfected BHK-21 cells. (F) Lane 1: BHK-21 cells transfected with pcDNA/MIC3; lane 2: BHK-21 cells transfected with pSCA/MIC3; lane 3: BHK-21 cells transfected with pcDNA3.1+; lane 4: BHK-21 cells transfected with pSCA1; lane 5: BHK-21 cells untransfected.
0.1 M phosphate buffered saline (PBS) pH 7.2 Then the symptom was observed and the survival time was recorded. 2.9. Statistical analysis For evaluation the protective immune responses among the different groups, all groups’ mean values were compared by analysis of variance (ANOVA) and Student’s t-test using SPSS v.12.0 software. P-Values of <0.05 were considered statistically significant. 3. Results
3.2. MIC3-specific antibodies in immunized mice To evaluate the immunogenicity of the suicidal DNA vaccines, serum collected at 0, 2, 4 and 6 weeks after immunization were tested for the antibodies against MIC3 by an indirect ELISA test using the recombinant MIC3 protein. Specific antibodies against T. gondii MIC3 appeared following injection of pSCA/MIC3 and pcDNA/MIC3. The mean antibody titers of the pSCA/MIC3-vaccinated group and pcDNA/MIC3-vaccinated group were significantly higher (P < 0.05) than those of the pSCA1, pcDNA3.1+ and PBS control group (Fig. 3). There was no significant difference in IgG responses between the groups immunized with pSCA/MIC3 and pcDNA/MIC3 (P > 0.05).
3.1. Expression of pSCA/MIC3 plasmid in BHK-21 cells In order to demonstrate expression of the T. gondii MIC3 proteins, transfected BHK-21 cells were examined by IFA and RT-PCR. Cells transfected with pSCA/MIC3 and pcDNA/MIC3 showed specific green fluorescence but the negative controls, which were transfected with the same amount of pSCA1 or pcDNA3.1+, without the insert, and non-transfected cells, respectively, did not show any fluorescent emission (Fig. 2(A)–(E)). At the same time, specific PCR band were detected in samples from cells transfected with pSCA/MIC3 or pcDNA/MIC3 on agarose gel but there were not any PCR bands detected in the negative controls which were transfected with the same amount of pSCA1 or pcDNA3.1+ and the blank BHK-21 cells (Fig. 2(F)).
Fig. 3. Anti-MIC3 antibody levels in BALB/c mice after immunization. Sera were tested for antibodies at a 1:160 dilution. The result was obtained from mean ELISA absorbance values of eight sera from each group.
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Fig. 4. On the 10th day after the last immunization, BALB/c mice (eight per group) splenocytes were isolated and used for lymphocyte proliferation assay as described in Section 2.6 (Fig. 4).
3.3. Lymphocyte proliferation assay To investigate whether pSCA/MIC3 could also enhance the cell-mediated immune responses, splenocyte suspensions were collected for lymphocyte proliferative responses assay on 10th day after last immunization. The lymphocyte proliferative responses assay were performed as described in Section 2.6 using recombinant MIC3 protein as stimulator and ConA as positive control. As shown in Fig. 4, the specific proliferative response was significantly higher in mice immunized with pSCA/MIC3 and pcDNA/MIC3 (P < 0.05) than in mice immunized with empty vectors and PBS, respectively. However the mean lymphocyte stimulation index (SI) of the pSCA/MIC3vaccinated group was higher than pcDNA/MIC3-vaccinated group, but this was not statistically significant (P > 0.05). 3.4. IFN-g and IL-4 expression assay The IFN-g and IL-4 expression in splenocytes stimulated by MIC3 were analyzed by real-time RT-PCR at the same time. As shown in Fig. 5(A), the production of IFN-g in pSCA/MIC3 or pcDNA/MIC3 groups were significantly
Fig. 6. Survival time of the immunized mice after T. gondii challenge. Each group of mice (n = 8) was immunized (i.m.) with 100 mg of DNA at 0, 2, 4 and 6 weeks. On the 10th day after the last immunization, mice were challenged with 104 tachyzoites of RH strain. Survival was monitored daily for 15 days after challenge.
higher (P < 0.05) than that of control groups, but there was no significant difference in IFN-g responses between pSCA/MIC3 group and pcDNA/MIC3 group (P > 0.05). In contrast, as shown in Fig. 5(B), the production of IL-4 in pSCA/MIC3 or pcDNA/MIC3 groups were significantly lower (P < 0.05) than that of control groups, there was no significant difference in IL-4 responses between the pSCA/MIC3 group and pcDNA/MIC3 group (P > 0.05). 3.5. Lethal challenge test in BALB/c mice To test whether this vaccine could induce protective effect against a lethal T. gondii infection, the mice were intraperitoneally infected with 104 tachyzoites on tenth day after the last immunization. The survival time was shown in Fig. 6, the survival time of mice immunized with pSCA/MIC3 and pcDNA/MIC3 were significant longer (P < 0.05) in comparison to control groups, and pSCA/ MIC3-vaccinated group’s survival time was longer than pcDNA/MIC3-vaccinated group’s, but this was not statistically significant (P > 0.05).
Fig. 5. IFN-g mRNA expression assay (A) and IL-4 mRNA expression assay (B). On the 10th day after the last immunization, BALB/c mice (eight per group) splenocytes were isolated. They were used in IFN-g and IL-4 mRNA expression assay as described in Section 2.7.
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4. Discussion In the present study, the in vitro expression study indicated that the recombinant plasmids pSCA/MIC3 and pcDNA/MIC3 could express the MIC3 protein faithfully. Then in vivo study in BALB/c mice showed that MIC3specific immune responses were induced when they were injected intramuscularly with suicidal DNA vaccine pSCA/ MIC3 or conventional DNA vaccine pcDNA/MIC3, and higher antibody titers were detected in mice immunized with pSCA/MIC3 and pcDNA/MIC3. The differences in stimulation index (SI) of lymphocyte proliferation assay between suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were statistically significant in comparison to control groups and suicidal DNA vaccine pSCA/MIC3 could stimulate highest stimulation index than others; IFN-g detection showed that pSCA/ MIC3 and pcDNA/MIC3 could significantly increased the production of IFN-g, but there was no significant difference in IFN-g responses between pSCA/MIC3 group and pcDNA/ MIC3 group (P > 0.05). In contrast, as shown in Fig. 5(B) pSCA/MIC3 or pcDNA/MIC3 (P < 0.05) significantly decreased the production of IL-4, there was no significant difference in IL-4 responses between the pSCA/MIC3 group and pcDNA/MIC3 group (P > 0.05). These findings indicate that a Th1 immune response was induced by pSCA/MIC3 or pcDNA/MIC3 immunization. Lethal challenge experiments showed that pSCA/MIC3 and pcDNA/MIC3 provided significantly longer survival time (P < 0.05) against T. gondii challenge than control groups, but the difference between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P > 0.05), although pSCA/MIC3 group’s survival rate was higher than that of pcDNA/MIC3 group’s. All these results showed that suicidal DNA vaccine pSCA/MIC3 could induce high humoral and cell-mediated immune responses. Compared with conventional DNA vaccine pcDNA/MIC3, pSCA/MIC3 also provided favourable efficacy. However, it could improve safety of the conventional DNA vector. This is the first report on a suicidal DNA construct strategy as a potential DNA vaccine against T. gondii challenge. A good vaccine should always have several essential elements including safety and low cost in manufacture, storage, and administration (Doel, 1999). Under this standardization, suicidal DNA vaccine came into our eyes. Based on the alphavirus replicon, suicidal DNA vaccines have emerged as an important strategy to enhance immunogenicity and to improve the biosafety of conventional vaccines (Doel, 1999). Among suicidal DNA vaccines, pSCA1 is a layered DNA/RNA construct. The first layer of this vector system utilizes a RNA polymerase II-dependent promoter, it stimulates the transcription of the alphaviral replicon RNA transcript. Translation of this RNA molecule produces the alphaviral replicase complex, which catalyzes cytoplasmic self-amplification of recombinant RNA, and high-level expression of the heterologous antigens (Berglund et al., 1998). Furthermore, when a suicidal DNA vaccine is transfected into cells, it leads eventually to apoptosis of the transfected cells (DiCiommo and Bremner, 1998; Leitner et al., 2000), which is particularly important in alleviating the concerns of potential integration and cell
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transformation generated by the use of conventional DNA vaccines (DiCiommo and Bremner, 1998; Gurunathan et al., 2000). Several groups have demonstrated the ability of suicidal DNA vaccines to induce higher level of humoral and cell-mediated immunity against several pathogens causing infections including foot-and-mouth disease (Guo et al., 2005; Wang et al., 2007), swine vesicular disease virus (Sun et al., 2007), louping ill virus (Fleeton et al., 2000), etc. The results from the present study showed that the suicidal DNA vaccine has the ability of induce higher level humoral and cell-mediated immunity against protozoal parasite too. In the present study, the level of IFN-g mRNA expression was higher in mice immunized with pSCA/ MIC3 or pcDNA/MIC3, while IL-4 mRNA expression were lower in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 than control group. These results are consistent to other reports in T. gondii infection. For example, Liu et al. (2008) demonstrated that BALB/c mice vaccinated with rPRV/SAG1 developed significant levels of IFN-g, while IL-4 was not detected. These findings indicate that both pSCA/MIC3 and pcDNA/MIC3 induce an enhanced Th1-type response. Further, this suicidal DNA vaccine pSCA/MIC3 will be used to immunize animals of food source, its protection against T. gondii infection in those animals will be investigated. It was expected that it could help to reduce worldwide jeopardizing for public health and economics caused by toxoplasmosis. In conclusion, a T. gondii suicidal DNA vaccine pSCA/ MIC3 was successfully constructed for the first time. Compared to control groups, suicidal DNA vaccine pSCA/ MIC3 and conventional DNA vaccine pcDNA/MIC3 were able to elicit a significant humoral and cellular immune response and increased survival time in mice challenged with the lethal RH tachyzoites, but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P > 0.05). The difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/ MIC3 was not statistically significant (P > 0.05). The findings demonstrated that as conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provides favourable efficacy. However, based on the alphavirus replicon, suicidal DNA vaccines leads eventually to apoptosis of the transfected cells, which is particularly important in alleviating the concerns of potential integration and cell transformation generated by the use of conventional DNA vaccines, so it could improve safety of the conventional DNA vector. In this case, the design of vaccines against T. gondii based on suicidal DNA approach is feasible and effective.
Acknowledgments This study was in part supported by the national special research programs for non-profit-trades (No. 200803017) and National Natural Science Foundation of China (NSFC, the Grant No. 30800818). Dr. S.B. Xiao was thanked for skillful technical assistance. Dr. M. Hu (Department of
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Agricultural Sciences, La Trobe University, Australia) and two anonymous reviewers were thanked for their constructive suggestions and correction of this manuscript.
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