DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice

Background Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. Currently, the main control strategy depends on the usage of drugs that target the acute stage of the infection, however, drawbacks were encountered while applying this method; therefore, development of an alternative effective method would be important progress. Deoxyribose Phosphate Aldolase (TgDPA) plays an important role supporting cell invasion and providing energy for the parasite. Methods TgDPA was expressed in Escherichia coli and the purified recombinant protein was used to immunize rats. The antibodies obtained were used to verify in vitro expression of TgDPA. The vector pVAX1 was utilized to formulate a DNA vaccine designated as pTgDPA, which was used to evaluate the immunological changes and the level of protection against challenge with the virulent RH strain of T. gondii. Results DNA vaccine, TgDPA revealed that it can induce a strong humoral as well as cellular mediated response in mice. These responses were a contribution of TH1, TH2 and TH17 type of responses. Following challenge, mice immunized with TgDPA showed longer survival rates than did those in control groups. Conclusions Further investigation regarding TgDPA is required to shed more light on its immunogenicity and its possible selection as a vaccine candidate.


Background
Toxoplasma gondii is an important zoonotic parasite infecting a wide range of warm blooded hosts, causing a pathological condition known as toxoplasmosis [1]. The infection is mainly acquired either by using water contaminated with oocysts released by the final host or handling intermediate host tissues infested with the asexual cysts [2,3]. In humans, there are two types of the infection according to symptoms; the first type is the asymptomatic form, resulting in a latent infection with tissue cysts. This form is less frequently seen in immunologically intact individuals. However, the infection could be severe in specific groups of patients, such as immunologically impaired individuals (AIDS or organ transplants) or congenitally infected fetuses and newborns [4,5].
Currently, the strategies of toxoplasmosis control mainly rely on the application of chemotherapeutics targeting the acute phase of the infection, however, some drawbacks were found to be associated with drug application, e.g; rapid re-infection besides toxic effects of the drugs [6,7]. Such issues 'blew the whistle', shifting the research directions into the area of vaccine development as an alternative control strategy for toxoplasmosis, with DNA vaccines receiving considerable attention [6].
Recent important progress has been made identifying anti-toxoplasma vaccine candidates that can stimulate an immunological response, with most of the work focusing on tachyzoite surface antigens, namely SAG1, SAG2 and SAG3, and SAG1 was recognized to be the most promising candidate in this group [8][9][10][11]. In the same context, T. gondii excretory secretory antigens like GRA molecules, have also been reported to demonstrate significant immunogenic capabilities [12][13][14]. Vaccination with DNA vaccines has been found to induce effective humoral and cellular immune responses, with both CD4 + T helper cells and CD8 + cytotoxic T cells included in these responses [15]. Such elements are important for understanding the mechanisms through which the parasite modulates the host immune response during both acute and chronic phases of the disease [16].
Deoxyribose phosphate aldolase, a glycolytic enzyme, functionally mediates in host cell invasion, acting as a bridge linking actin filaments to the parasite's surface adhesion microneme protein 2. Furthermore, aldolase plays an essential role providing carbon and energy sources for the organism, as part of the glycolysis cycle, on which the parasite gliding motility depends during the invasion process [17][18][19][20].
Blocking the parasite from invading the cell and consequently preventing the parasite form multiplying may help in reducing the parasitic burden and leave the parasite exposed to other immunological elements, thus in this study we demonstrated the immunological changes after vaccination of mice with a DNA vaccine encoding TgDPA followed by challenge with virulent T. gondii RH strain.

Animals and parasite
Six to eight week-old female Swiss Webster (SW) mice were purchased from The Center of Comparative Medicine, Yangzhou University (Yangzhou, China) and maintained under specific-pathogen-free standard conditions. All animal experiments were approved by the Animal Ethics Committee of Nanjing Agricultural University (Approval number 200709005). Toxoplasma gondii strain RH (Type I), was provided by The Laboratory of Veterinary Molecular and Immunological Parasitology, Nanjing Agricultural University, China. To maintain the parasite, as described by [21], intraperitoneally injected SW mice were infected with the parasite tachyzoites. Every 3 days, the tachyzoites were harvested and recovered from peritoneal washings of infected mice to be used for re-infection.

Construction of the prokaryotic plasmid
According to the manufacturer's protocol Trizol reagent (Takara, Life Technologies), total RNA of T. gondii was extracted from T. gondii tachyzoites, followed by construction of the cDNA. The open reading frame (ORF) of Deoxyribose Phosphate Aldolase (TgDPA) gene (XM_002365690.1) was obtained from T. gondii cDNA by PCR amplification using the following synthetic primers in which recognition sites were inserted as underlined below.
The PCR product of TgDPA was inserted into the pMD-18 T Vector (TaKaRa) to generate prokaryotic plasmid pMD-TgDPA. The recombinant plasmid was used to transform the bacteria E. coli DH5α (JM109). Insertion was confirmed by sequencing in both directions. After purification, pMD-TgDPA recombinant plasmid was double digested with appropriate restriction enzymes (BamH I/Hind III) and sub-cloned into the matching sites of pET28a (+) vector (Novagen). Following the screening by enzymatic cleavage, the positive clones were sequenced in both directions to ensure the plasmid designated as pET28a/TgDPA was successfully constructed.

Expression and purification of TgDPA recombinant protein
The recombinant plasmid designated as pET28a/TgDPA was used to transform E. coli bacteria strain BL21 (DE3), and protein expression was induced by addition of 0.8 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) after the OD 600 of the bacterial culture reached 0.6 at 37°C. The cells were incubated at 37°C for 5 hr and harvested by centrifugation. The cell pellet was lysed using lysozyme (10 μg/ml) followed by disruption of the cells using sonication. Expression of the protein was analyzed by 12% (w/v) Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE).
The recombinant protein was purified by Ni 2+ -nitrilotriacetic acid (Ni 2+ -NTA) column (GE Healthcare) according to the manufacturer's instructions. Purity of the protein was detected by 12%SDS-PAGE. The concentration of the protein was determined according to the Bradford procedure using bovine serum albumin (BSA) as a standard. The purified protein was used to develop anti-rTgDPA sera and the rest of the protein was stored at −20°C for later applications.

Construction of the eukaryotic plasmid
The restriction enzymes BamH I and Xho I were used to digest the recombinant plasmid pET28a/TgDPA and the target gene DNA fragment was directionally sub-cloned into the pVAX1 vector (Invitrogen, Life Technologies), which was previously linearized with similar enzymes. The resultant recombinant plasmid designated pTgDPA was verified and confirmed by sequencing in both directions and also with double enzyme digestion. Plasmids were then purified from transformed E. coli DH5 (JM109) cells by anion exchange chromatography (EndoFree Plasmid mega Kit Qiagen) following the manufacturer's instructions, dissolved in sterile endotoxin-free H 2 O and the concentration was determined by spectrophotometer at OD 260 and OD 280 . The recombinant plasmid was stored at −20°C until use.
Plasmid in vitro translation of pTgDPA BHK cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) with 10% Fetal Bovine Serum (FBS), 100 mg/ml streptomycin and 100 IU/ml penicillin at 37°C in the presence of 5% CO 2 . Before transfection, BHK cells were transferred in a 6-well plate (Costar, USA). When the confluency of the cells reached 80%-90%, 5 μg of the recombinant eukaryotic plasmid (pTgDPA) was used to transfect the cells using Lipofectamine 2000 regent (Invitrogen) according to the manufacturer's guidance. The empty vector pVAX1 (5 μg) was also transfected into BHK cells as a negative control. Lipofectamine 2000 reagent was respectively mixed with pTgDPA and pVAX1 at a concentration of 10 μg/ml in DMEM without Fetal Bovine Serum (FBS) and antibiotics, and was incubated at room temperature for 30 min. The mixture of lipofectamine and plasmid was then added into BHK cells. The cells were incubated with the transfection mix for 6 hr at 37°C in the presence of 5% CO 2 . At the end of incubation, fresh growing medium was supplemented and plates were returned to the cell incubator for further incubation. After 48 hr of incubation, BHK cells were collected and expression of the gene was evaluated by Western blotting analysis.

Mice immunization and challenge
To observe the immunogenicity of DPA, mice were randomly divided into four groups (25/group). Before vaccination, plasmids were diluted and suspended in sterile phosphate buffered saline (PBS pH 7.4) to a final concentration of 100 μg/ml. All experimental groups were injected intramuscularly (quadriceps muscle), tw{Capron, 1988 #496}o times at weeks 0 and 2. Control groups received PBS or empty plasmid or no treatment. Blood samples of mice were collected before vaccination (negative control), as well as at the time of the second injection and 2 weeks after the second injection. The sera were stored at −20°C for antibody evaluation and cytokine measurement. Two weeks after the last injection, the mice in the four groups were challenged intraperitoneally (i.p) with 2 × 10 4 tachyzoites of T. gondii RH strain. The survival times of the mice were observed and recorded on a daily basis.

Determination of antibodies and isotype distribution by ELISA
Enzyme-linked immunosorbent assay, ELISA technique, was used to determine the levels of total IgG in sera samples collected at week 0, 2 and 4, while levels of IgA, IgM, IgE and subclasses IgG 1 and IgG 2a , were determined in sera samples collected at week 4. The microtiter plates (Costar, USA) were coated with 5 μg rTgDPA recombinant protein in 50 mM carbonate buffer (pH 9.6) and incubated at 4°C overnight. After three washes, the plates were blocked with 2% skimmed milk for 1 h at 37°C and subsequently incubated with the mouse sera diluted (1:100) in the same blocking buffer for 1 h at 37°C. HRP-conjugated goat anti-mouse of IgA, IgM, IgE, IgG, IgG 1 and IgG 2a (Santa Cruz Biotechnology) were used as secondary antibody (1:1000). Finally, the immune complexes were developed by incubating with 3,3,5,5-Tetramethylbenzidine (TMB) for 30 min. The reaction was stopped by adding 2MH 2 SO 4 , and the absorbance was measured at 450 nm with an automated ELISA reader (MULTISKAN FC, Thermo scientific), all samples were run in duplicate.

Assay of cytokines
To assay cytokine production levels, sera from each experimental group were obtained as described previously. Interferon Gamma (IFN-γ), Interleukin-4 (IL-4), Interleukin-17 (IL-17) and Transformation Growth Factor-β1 (TGF-β1) were measured using ready ELISA kits according to the manufacturer's instructions (Boster Systems, Wuhan, China). Cytokine concentrations were determined by reference to standard curves constructed with known amounts of mouse recombinant IL-4, IL-17, IFN-γ and TGF-β1. The analysis was performed with the data from three independent experiments.

Flow cytometry analysis of T cell subsets and MHC molecules
The percentages of T cells subsets CD4 + and CD8 + , beside MHC-I and MHC-II molecules in the spleenocytes of mice in the test groups, pTgDPA and pVAX1, PBS and blank, were analyzed using the flow cytometry technique as described by [22].
Splenocytes suspensions (1 × 10 6 cells/ml) were dually stained with anti-mouse CD3e-FITC + anti-mouse CD8-PE, anti-mouse CD3e-FITC + anti-mouse CD4-PE, anti-mouse CD3e-FITC + anti-mouse MHC-I-PE or anti-mouse CD3e-FITC + anti-mouse MHC-II-PE (eBioscience) for 30 min at room temperature in the dark. Cell population analysis was conducted by FACScan flow cytometry with CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA). A lymphocyte specific gating was set according to forward and side scatters profiles. The percentages of CD4 + and CD8 + T lymphocytes, MHC-I and MHC-II molecules in mice spleenocytes were determined as described by [23].

Statistical analysis
All statistical analyses were performed by Graphpad Prism 5.Ink software. The differences of the data between all the groups were compared by one-way ANOVA. Survival rate of the mice was compared using the Kaplan-Meier method. The results in comparisons between groups were considered different if P < 0.05.

TgDPA recombinant plasmids expression
Following sequence analysis confirming that the TgDPA DNA fragment was directionally inserted; pET28a/TgDPA was successfully constructed ( Figure 1A). Expression of the recombinant protein took place by IPTG induction, followed by purification, the product was analyzed using SDS-PAGE (Figure 2A). The purified protein was used to develop polyclonal antibodies (anti-rTgDPA) and western blotting technique was used to verify the results ( Figure 2B).
As for the eukaryotic plasmid pTgDPA, a separate trial was conducted to screen in vitro expression. Lysates of the BHK cells transfected with pTgDPA were probed with anti-rTgDPA polyclonal antibodies revealing successful expression of the protein ( Figure 2C), while cells transfected with empty pVAX1 exposed no specific bands and remained negative ( Figure 2D).

Antibody response in immunized mice and subclass determination
The titers of total IgG, beside subclasses IgG 1 and IgG 2a were measured prior to and after immunization, using standard ELISA. As shown in Figure 3A, specific total IgG antibodies were detected in the experimental group vaccinated with pTgDPA. There was a significant difference at (p < 0.05) between pTgDPA group after first immunization (0.651 ± 0.04) and 2 nd immunization (0.752 ± 0.03), compared to the control groups of pVAX1 (0.073 ± 0.011), PBS (0.050 ± 0.07) and Blank (0.07 ± 0.03).
IgG isotype determination revealed that, both IgG 1 (1.33 ± 0.485) and IgG 2a (0.506 ± 0.029) were significantly (P < 0.05) stimulated after delivering the antigen ( Figure 3B and C). Moreover, the difference between the levels of these isotypes was found to be significant at (P < 0.05), for the advantage of IgG 1 ( Figure 3G). Regarding IgA, IgM and IgE, and when compared to the control groups, dynamics of the first two antibody types demonstrated high OD values (P < 0.05) in the immunized group (0.974 ± 0.33) and (1.55 ± 0.26) respectively ( Figure 3D and E). However, IgE activity showed no significant changes at the time of evaluation (Figure 3 F).

Cytokine production
Sera samples collected at weeks 0, 2 and 4 were used to measure the amounts of IFN-γ, IL-4, IL-17 and TGF-β1 produced in the different experimental groups. As shown in Figure 4A, mice vaccinated with pTgDPA generated significant levels of IFN-γ at (P < 0.05) compared to mice in the control groups, peak production was reached 2 weeks after the last immunization (697.0 ± 8.39).
IL-4 and IL-17 of the pTgDPA group showed a significant difference (P < 0.05) against the control groups ( Figure 4B and C). The peak of production was also at 2 weeks after the booster dose (91.6 ± 1.34) for IL-4 and  (62.2 ± 2.83) for IL-17. Additionally, TGF-β1 ( Figure 4D) displayed a different activity. Immunized groups showed a significant peak after the first immunization (70.4 ± 6.66), which was dramatically decreased (34.2 ± 2.26) two weeks after the last immunization. Compared to the control groups both time points were significant at (P < 0.05).
After both prime and booster immunizations, MHC-I molecules of the immunized group displayed sustained high significant readings (33. Figures 5C & 6C). Concerning MHC-II molecules, a gradually increasing pattern was noticed in the vaccinated group ( Figures 5D & 6D) starting at week 2 of the experiment reaching a peak point (5.81 ± 0.87) at week 4. Compared to control groups (1.85 ± 0.74), (1.74 ± 0.65) and (1.99 ± 0.82) the difference between these values was found significant at (P < 0.05).

Protection of vaccinated mice against challenge with T. gondii RH strain
In order to evaluate the protective effect of pTgDPA DNA vaccine against acute toxoplasmosis, vaccinated and control mice groups were challenged with lethal T. gondii tachyzoites within the second week after booster immunization. Mortality was observed daily until all the mice died and survival curves of different groups were generated and are shown in (Figure 7). Significantly longer survival time (20 days) was observed in mice immunized with pTgDPA against the control group, who died within 8-9 days after challenge (p < 0.05).

Discussion
In this study, we have demonstrated that a DNA vaccine encoding DPA of T. gondii could elicit a considerable specific immune response, as well as providing significant levels of protection against T. gondii challenge.
In this report, humoral response was analyzed after vaccination with TgDPA. Immunized mice generated specific high titers of IgG, in contrast to control groups. Analysis of IgG isotypes revealed that the levels of IgG 1 were significantly higher than that of IgG 2a . Similar results were detected after T. gondii antigens like cathepsin proteases, protein kinase 3 and GRA4 were evaluated [24][25][26]. IgG 2a and IgG 1 are characteristic of T H 1 and T H 2 immunity, TGF-β1, in sera samples (n = 5) collected at weeks 0, 2 and 4, and the comparison results were expressed as means ± SD of pg/ml. The asterisk designates statistically significant differences (p < 0.05) between groups. Results presented here were from three independent experiments.  respectively [27]. Higher IgG 1 levels in this research indicated that TgDPA induced mainly T H 2 responses. Immunoglobulins IgA, IgM and IgE were reported to participate in the immunological responses against T. gondii infection. However, less attention has been placed on these immunoglobulins during vaccination trials against T. gondii [28,29]. IgA is an important immunoglobulin to act on neutralization of toxins and pathogenic microbes, beside regulating interaction between specific receptors and immune mediators [30,31]. With specific relation to T. gondii infection, IgM was reported to enhance the phagocytic capacity of neutrophils and activate the complement cascade which might result in killing of T. gondii as well as reducing the spread of T. gondii by blocking cell invasion [32][33][34]. In our research, high titers of IgA and IgM were detected in the immunized group. This suggested that both IgA and IgM played roles in the protective responses induced by the TgDPA.
IgE was recognized during the infection of toxoplasmosis [35,36]. However, our data revealed no significant traces of this immunoglobulin after vaccination with TgDPA. Our results were consistent with previous studies [37].
IFN-γ is a key cytokine of T H 1 type immune response and is known to play an important role in resistance against T. gondii. This cytokine supports many immunological mechanisms, interferes with survival and multiplication of intracellular pathogenic organisms and leads to the eradication of pathogenic organisms [38][39][40][41][42]. Remarkable levels of IFN-γ were detected in this study. This result and the release of IgG 2a isotype substantiated the involvement of T H 1 response against TgDPA. This finding agrees with many studies in which significant production of IFN-γ was detected after immunization with T. gondii antigens [25,26,[43][44][45][46][47].
IL-4 is known as an immune regulatory cytokine of T H 2 type of immune response. In this research, the immunized group showed a significant release of IL-4 compared to the control groups. The positive increase of this cytokine during this investigation agrees with previous reports highlighting the role of this cytokine during vaccination trials using T. gondii antigens [48,49]. The significant release of IL-4, together with the release of IgG 1 showed that the T H 2 type response was involved in the protection provided by TgDPA against the T. gondii challenge [27,50,51].
The T H 1/T H 2 immune response pattern has dominated the studies of cell-mediated immune resistance to infections like toxoplasmosis [52]. Recently, a new lineage of T helper cells recognized for producing proinflammatory cytokines, such as IL-17, IL-21, and IL-22 [53], had been identified and designated as T H 17. Cytokines related to T H 17 are associated with recruitment and activation of neutrophils during inflammatory diseases [54,55]. However, evaluation of T H 17 responses during immunization trials against T. gondii received less attention compared to T H 1 and T H 2. In this investigation, after the booster immunization, a significant increase of IL-17 concentration was detected. This finding indicated that TgDPA was capable of inducing T H 17 differentiation and resulted in an inflammatory reaction. This result also showed that T H 17 response obviously played an important role during immunization with TgDPA.
TGF-β is a typical cytokine of T reg cells and usually plays inhibitory roles in the immune responses [56][57][58][59]. The inhibitory function of T reg cells was also demonstrated during toxoplasmosis infection [60]. In this study, Figure 7 Survival curves of mice in pTgDPA, pVAX1, PBS and Blank groups, following challenge with T. gondii RH strain. Survival time was monitored daily after lethal challenge with 2 × 10 4 tachyzoites of virulent T. gondii RH strain, 2 weeks after the last immunization. The mice immunized with pTgDPA were dead from day 11 to day 20, showing an increased survival time compared with mice in the control groups (pVAX1, PBS, blanking controls), which died within 8-9 days after challenge (P < 0.05).
the immune group displayed significantly low concentrations of TGF-β1 after the booster immunization compared to the control groups. It indicated that immunization with TgDPA down-regulated T reg cells response. This character of TgDPA will be beneficial to its potential as a vaccine candidate.
Resistance against T. gondii parasite is characterized by the induction of specific CD4 + and CD8 + T cells, which eventually lead to the killing of the parasite [61,62]. In murine models, CD4 + T cells were crucial regulators of the immune response during resistance against toxoplasmosis, while in humans CD4 + displayed cytotoxic activity against T. gondii infected cells [15,63,64]. On the other hand, CD8 + subtype were considered to be the major effector cytotoxic T lymphocyte (CTL) cells mediating lysis of T. gondii infected host cells [65][66][67]. In this investigation, our data demonstrated that both cell subtypes were significantly accumulated in response to immunization with TgDPA. This result corresponds with reports regarding immunological responses to T. gondii antigens [16,[68][69][70][71][72].
In this study, results that further support the increments of both CD4 + and CD8 + T cell subsets were the simultaneous significant increases of the ratios of MHC-I and MHC-II. Activation of CD4 + T cells requires the endocytosis or phagocytosis of secreted or exogenous proteins entering into the MHC class II pathway, while the activation of CD8 + depends on the recognition of antigens restricted by MHC class I molecules [27,73]. The findings in this study demonstrated that TgDPA antigen was presented through MHC-I and MHC-II.
As a result of these significant immunological changes, pTgDPA vaccinated mice survived for a longer time compared to the control groups in this research. However, due to uncontrolled parasite replication, pTgDPA mice ultimately succumb during late acute infection. It indicated that the DNA vaccine of pTgDPA did not provide complete protection. However, investigations concerning this protein should further be conducted.
One of the prominent advantages of DNA vaccine application is their induction of CTL cells. CTL cells kill the pathogen infected cells mainly by inducing apoptosis [74]. T. gondii maintains its survival and replication by interfering with infected cells apoptosis, blocking an important pathway known as caspase cascade [75][76][77][78]. Measurement of damaged infected cells was an important tool to measure CTL function [74,79,80]. Application of such methods is required in further investigations regarding TgDPA antigen to highlight its role in CTL response stimulation and resistance development against T. gondii infection.

Conclusion
Our study demonstrated that the pTgDPA delivered as a single protein is an antigen with the potential of inducing and regulating significant levels of humoral as well as cellular (T H 1, T H 2 and T H 17) immune responses against acute T. gondii infection. This finding may encourage more investigations in evaluating the immunogenicity of DPA based vaccines against Toxoplasmosis.