Development of a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V and determination of its protective efficacy against acute toxoplasmosis

Background Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. The aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii. Methods 49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites. Results In mice vaccinated with hexavalent vaccine, strong total IgG (P < 0.0001) and IgG2a (P < 0.001) responses were induced compared to controls, the ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.001). The survival time of the vaccinated mice increased to 8.38 ± 2.13 days which was significantly higher than controls (P < 0.01). Conclusions Altogether, these results show that the hexavalent vaccine which is developed for the first time against T. gondii induced strong and balanced Th1 and Th2 immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model.


Background
Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in all warm blooded animals and humans. It is reported that one third of the world's population is estimated to be infected with T. gondii. T. gondii usually causes an asymptomatic infection in healthy people, but can be life-threating in immunecompromised patients (organ transplant recipients, Acquired Immunodeficiency Syndrome patients, and cancer patients). Congenital toxoplasmosis may cause abortion, neonatal death or foetal abnormalities in the foetus [1]. Farm animals such as sheep, goats, and pigs have also been shown to be susceptible to T. gondii infection. Toxoplasmosis infection in farm animals causes significant economic losses as a result of prenatal death, abortion, and neonatal death. Moreover, T. gondii is linked to mental disorders and may affect human behaviour, personality, and other phenotypic traits [2].
Cats and other felines are the definitive hosts of T. gondii [3]. In the life cycle of T. gondii the main infective forms are tissue cysts (containing bradyzoites) and oocysts (containing sporozoites). The infection sources for humans are consumption of vegetables, fruits or water contaminated with faeces of infected cats containing oocysts and raw or undercooked meat contaminated with tissue cysts [4]. For these reasons, development of a safe and protective vaccine against T. gondii infection that can be used in animals and humans has utmost importance.
Recombinant protein vaccines are safe and efficient, and have a great potential for prevention or eradication of diseases. One of the most important issues during a recombinant protein vaccine development is the selection of the vaccine candidate antigen(s) [5]. Antigens to be used in vaccine formulations against toxoplasmosis should actively induced strong immune response, produce long-lasting immunity, and be antigenic in each stage of the parasite [6]. In our study group's previous studies, we used protein microarray containing 2870 candidate exon products of T. gondii to screen well-characterized sera from acute and chronic toxoplasmosis human cases and murine model infected orally with oocysts or tissue cysts. During these studies, screening human sera prioritized 240 antigenic proteins and screening these 240 antigens with murine sera prioritized 49 proteins based on their immunogenicity [6][7][8].
In this study, we selected six proteins based on their antigenic epitopes using bioinformatics and protein expression level in E. coli. Thereafter, we developed a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V and administered to a murine model to determine its immunogenicity and protection efficiency against lethal toxoplasmosis.

Methods
Animals 6-8 week old female healthy Swiss outbred mice were obtained from the Bornova Veterinary Control Institute Animal Production Facility and used during the experiments. Animals were housed under standard and suitable conditions. Specifically rooms had ambient temperature and humidity, adequate light cycle, and diet was specific for each animal type. All animals were checked for humane endpoints every day such as rapid weight loss more than~20% of gross body weight, inability to assess water or food, or loss of skin elasticity indicative of dehydration. In any of these circumstances, we pre-euthanized the animals with ketamine hydrochloride (2 mg/kg) and 2% xylazine (3 mg/kg) and then euthanized with cervical dislocation.

Determination of vaccine candidate antigens
Based on the data obtained from previous studies [6][7][8], 49 proteins were selected based on their immunogenicity in humans and murine sera (Table 1). In order to determine the vaccine candidate antigens, expression levels of these proteins were analysed using western blot and moreover, a comprehensive bioinformatics analyses was performed.

Expression levels of 49 proteins in E. coli
The gene sequences of the 49 different genes were accessible from the Toxoplasma Genomic Resource (http:// www.toxodb.org/toxo/). The plasmids expressing these 49 recombinant proteins were constructed as previously described [6][7][8]. Thereafter, the plasmids were cloned into chemically competent Escherichia coli (E. coli) BL21 Star (DE3) pLysS cells according to the manufacturer's protocol (Invitrogen, USA).
E. coli BL21 Star (DE3) pLysS cells containing 49 different plasmids were incubated at 37°C with shaking at 225 rpm for 16-24 h until the optical density (OD600) reached an absorbance of 0,4 ng/μL. Then, recombinant protein expression was induced with 0.5 mM IPTG (isopropyl-D-thiogalactopyranoside) and the bacterial cultures were incubated for 4 h, 37°C with shaking at 225 rpm. Next, cell cultures were harvested by centrifugation at 5000×g for 10 min. The pellets were homogenized with lysis buffer [0.1% Triton X-100, 50 mM Tris-Cl and 0.3 M NaCl (pH: 7.4)] and were freeze-thawed 3 times. The homogenates were incubated on a rotator at room temperature for 15 min and then were centrifuged at 30.000×g for 20 min.
The supernatants were incubated with 1 ml Ni-NTA Superflow beads (Qiagen, USA) for 30 min with shaking at 100 rpm. At the end of incubation, the suspension was centrifuged at 4000 rpm for 1 min and the supernatant was discarded. Then, the Ni-NTA beads were washed with 50 mM Tris-Cl and 0.3 M NaCl and 25 mM imidazole (pH: 7.4) for 30 min with shaking at 100 rpm. Thereafter, the suspension was centrifuged at 4000 rpm for 1 min and the supernatant was discarded. Next, Ni-NTA beads were incubated with 50 mM Tris-Cl and 0.3 M NaCl and 0.5 M imidazole (pH: 7.4) for 30 min with shaking at 100 rpm. Finally, the suspension was centrifuged at 4000 rpm for 1 min and supernatants were analysed with Western blot as described below to determine the expression level of recombinant proteins. The most abundantly expressed recombinant proteins were selected for vaccine development.

Bioinformatics analyses of 49 recombinant proteins for antigen mapping and glycosylation
The protective immune response against toxoplasmosis is conferred by mainly by cellular immune response and through humoral immune response [6,9]. In addition, the expression of 49 recombinant proteins of T. gondii will be performed in E. coli. T. gondii is a eukaryotic obligate intracellular parasite. Protein post-translational modifications are common events in most eukaryotes, such as glycosylation. Glycosylation of peptide effects protein immunogenicity and major histocompatibility complex (MHC) binding [10]. Thus, apart from the determination of the expression levels, 49 proteins will analysed by bioinformatics tools to determine the presence of antigenic epitopes as well as glycosylation sites. At the end of the bioinformatics analyses, we aimed to select the proteins that have cellular and humoral immune response inducing epitopes with the least glycosylation sites.
The cells were centrifuged at 5000×g for 10 min and the pellet was resuspended with 300 ml pre-chilled loading buffer (50 mM Tris-Cl, 0.3 M NaCl, pH 7.5) and homogenized with a blender for 10 s (Waring, USA). Then, the homogenized cells were disrupted twice using a microfluidizer processor (Microfluidics M-110 L Pneumatic, USA) at a low temperature under internal pressure of 18,000 psi and centrifuged at 30,000 g for ½ h at 4°C. The homogenates were centrifuged at 30,000×g for 30 min at 4°C. The clarified supernatants were filtered using 0.45 μm pore filter (Corning, USA). The filtered samples were purified with AKTA Fast Protein Liquid Chromatography (FPLC) system, controlled by UNI-CORN™ software (GE Health, USA), using 5 ml HiTrap Ni 2+ Chelating HP column (GE Health, USA). Approximately 100 ml filtered supernatant was loaded to the HiTrap Ni 2+ Chelating HP column. After binding, the column was washed with buffers containing increasing concentrations of imidazole (50 mM, 100 mM, 250 mM). The recombinant proteins (rH2, rA4, rE4, rD6, rE6, and rH6) were eluted by raising the imidazole concentration to 0.5 M. Purity and identity of the purified proteins were analysed by 12% (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) SDS-PAGE and Western Blot analysis and concentrated with Vivaspin 20 (Sartorius, Germany).
The proteins were further purified by FPLC on a Superdex 200/10-300 GL [100000-600,000 Molecular weight cut-off (MWCO)] column (GE Health, USA) to remove excess endotoxin. The subsequent protein fractions were pooled, concentrated and quantitated by Bradford method (Pierce, USA).

SDS-PAGE and Western blot analysis
To observe the expression levels, purity and immunoreactivity, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The separated proteins were transferred to polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P, Millipore, MA), blocked by 6.25% non-fat dry milk for 1 h at room temperature. The membranes were probed with a 1/ 3333 dilution of monoclonal anti-polyhistidine antibody (Sigma-Aldrich, USA) for 1.5 h at room temperature. Then the membranes were probed with a 1/3333 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) antibody (Sigma-Aldrich, USA) for 1 h at room temperature. The blot was developed with diethanolamine buffer (10% Diethanolamine, 4 M HCl pH: 9.8,

Determination and removal of endotoxin from purified recombinant proteins
The amount of endotoxin in the purified vaccine candidate 6 recombinant proteins were determined with Limulus Ameobocyte Lysate (LAL) Gel-Clot test using Pyrotell single test vials according to the manufacturer's protocol (Cape Cod Inc., USA). The test sensitivity was 0.25 Endotoxin Units (EU)/mL. E. coli O113:H10 control standard endotoxin (Cape Cod Inc., USA) was used for positive control and LAL reagent water was used for negative control. The concentration of endotoxin-depleted, purified recombinant protein samples were calculated with Bradford method (Pierce, USA) and stored at − 80°C until use.
Vaccination and T. gondii challenge infection 6-8 weeks old female Swiss Webster outbred mice were randomly divided into four groups, each group consisting of eleven mice (n:11). Mice were vaccinated intraperitoneally (i.p.) twice at 3 weeks intervals. Vaccines and control groups as shown in Table 2. The first group was immunized with hexavalent (6 antigens) recombinant proteins adjuvanted with Montanide ISA 50 V (Seppic, France) prepared according to the manufacturer's protocols. Montanide ISA 50 V was used as adjuvant due to its efficiency in inducing both humoral and cellular immune response. Three groups were considered as control; one control group was administered only with hexavalent recombinant proteins without Montanide ISA 50 V adjuvant. The adjuvant control group was inoculated with only Montanide ISA 50 V [100 μl Montanide ISA 50 V adjuvant + 100 μl Phosphate buffered saline (PBS)] and the last control group was inoculated only PBS (200 μl PBS). Tail bleeds were performed 3 weeks after each vaccination.
Nine weeks after first vaccination, eight mice from all four groups were challenged intraperitoneally with 1 × 10 5 tachyzoites of T. gondii local strain called Ankara [22]. Thereafter, the infected mice were observed for the symptoms of toxoplasmosis such as loss of fur brightness and appetite and survival times were recorded on a daily basis.
Detection of total IgG and IgG subclass antibody response using rec-ELISA Determination of T. gondii specific total IgG, IgG1 and IGg2a antibodies in vaccinated mice were performed by recombinant Enzyme-Linked ImmunoSorbent Assay (Rec-ELISA) as described [23,24]. In brief, each well of microplates (Nunc, USA) were coated with 100 μl of recombinant proteins solution (containing 5 μg/ml of each rH2, rA4, rE4, rD6, rE6 and rH6 in 1 × PBS) and incubated overnight at 4°C. Next day, plates were washed 3 times with 300 μl PBS-T (0.05% Tween 20 in PBS) and then blocked with 5% nonfat dry milk containing 0.05% PBS-T for 2 h at room temperature. Mice sera were diluted to 1/100 with blocking buffer supplemented with E. coli lysate at a final concentration of 10 mg/ml protein to block anti-E. coli antibodies and incubated for 30 min at 37°C. Then, the mouse sera were added to the wells in duplicate and incubated for 2 h at 37°C with gentle shaking. After three washes with 300 μl PBS-T, the plates were incubated with 100 μl of anti-mouse IgG

Determination of extracellular cytokine by ELISA
To evaluated cytokine production, three mice from per group were euthanized 6 weeks after the prime vaccination and their spleens were removed. Single-cell suspensions of splenocytes were prepared as previously described [24,25]. Aliquots of 5 × 10 5 viable splenocytes in growth medium [1 × RPMI 1640 supplemented with 10% FCS (NBCS, HyClone, Thermo Fisher Scientific, USA), 2 mM l-glutamine (Gibco, Invitrogen,USA) penicillin (100 U/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich, USA), 0.1 mM non-essential amino acid and 1  Thereafter, the cells were permeabilized and labelled with PE conjugated rat anti-mouse IFN-γ (BD Biosciences, USA) or PE conjugated rat anti-mouse IL-4 antibodies (BD Biosciences, USA) according to the manufacturer's recommendations. Antibodies were diluted in Perm/Wash solution (BD Biosciences, USA) for intracellular staining. All antibodies were used at a final concentration of 0.5 μg/ 10 6 cells.

Statistical analysis
Data obtained during the experiments were processed using Microsoft Excel Software 2010 and Prism 3.03 program (GraphPad, San Diego, CA). A two-tailed unpaired t-test with 95% confidence interval was used to determine the significance between the vaccination groups. Kaplan-Meier survival curves were constructed to illustrate protection from lethal toxoplasmosis. Humoral and cellular immune responses and survival time were expressed as mean ± standard deviation (S.D.).

Expression and purification of vaccine candidate 6 antigenic proteins
Based on the protein expression levels and MHC-I, MHC-II, and B cell epitope prediction results, rH2, rA4, rE4, rD6, rE6, and rH6 were grown in big batches of LB using a bioreactor, purified in on a HiTrap Ni2+ chelating column and then polished on a gel filtration column to remove excess endotoxin. The purity of rH2, rA4, rE4, rD6, rE6 and rH6 were assessed by SDS-PAGE and western blotting as shown in Fig. 3a and b. The purified rH2, rA4, rE4, rD6, rE6 and rH6 had apparent molecular weight of approximately 26.7 kDa, 33.9 kDa, 38.8 kDa, 78.1 kDa, 81.2 kDa, and 110.2 kDa, respectively. Some of the proteins gave several bands possibly due to multimerization of recombinant protein, degradation or stalling of protein synthesis in E. coli.

Humoral immune response
To determine total IgG, IgG1 and IGg2a antibodies against rH2, rA4, rE4, rD6, rE6, and rH6 proteins in adjuvanted and control group mice serum samples, Rec-ELISA was performed. According to the results, total IgG response detected at day 42 was significantly higher in sera of mice administered with hexavalent recombinant protein mixture (+) Montanide ISA 50 V vaccine and the control group administered with only hexavalent recombinant protein mixture compared to the pre-vaccination sera (P < 0.0001, ***). In control groups administered with PBS or Montanide ISA 50 V didn't induce a significant total IgG response. Significantly high levels of total IgG immune response was detected in the Hexavalent recombinant protein mixture (+) Montanide ISA 50 V when compared with the mice administered with Hexavalent recombinant protein mixture (P = 0.002, **) (Fig. 4).
To assess the polarization of IgG1/IgG2a which is a preliminary marker of whether vaccine induced the Th1 or Th2 immune response, the IgG1 and IgG2a response was analysed by Rec-ELISA. The polarization of IgG1 and IgG2a response is shown in Fig. 5. Hexavalent recombinant protein mixture (+) Montanide ISA 50 V vaccine induced significantly high levels of IgG1 and IgG2a at day 42 compared to the controls groups (P < 0.001, **). Overall, in mice administered with Hexavalent recombinant protein mixture (+) Montanide ISA 50 V, IgG2a and IgG1 levels showed a strong and balanced immune response. In control groups, PBS or Montanide ISA 50 V didn't induce a significant IgG1 or IgG2a response. (Fig. 5).

Cellular immune response
Single-cell suspensions of splenocytes obtained from mice administered with hexavalent recombinant protein mixture (+) Montanide ISA 50 V and controls were stimulated with purified rH2, rA4, rE4, rD6, rE6, and rH6 proteins. Concentration of extracellular cytokine IL-4 and IFN-γ were determined using ELISA. According to the results, IFN-γ level was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) Montanide ISA 50 V compared to mice vaccinated with only Montanide ISA 50 V (P = 0.001,***) or PBS (P = 0.0007,***) (Fig. 6a). On the other side, IL-4 level was also significantly high in mice vaccinated with the hexavalent recombinant protein mixture (+) Montanide ISA 50 V compared to control groups administered with PBS (P = 0.0133, *) or Hexavalent recombinant protein mixture (P = 0.0183, *) (Fig. 6b). Flow cytometry analysis was used to determine the ratio of CD8 + T lymphocytes secreting IFN-γ and CD4 + secreting IFN-γ and IL-4 in vaccinated and control groups. Protection against intracellular parasite like T. gondii is primarily achieved by CD8 + T lymphocytes secreting IFN-γ. For this reason, CD8 + T cell response is important for an ideal vaccine against toxoplasmosis [11,26,27]. T cell population has been gated with CD3 + staining. In mice vaccinated with Hexavalent recombinant protein mixture (+) Montanide ISA 50 V vaccine, the ratio of CD4 + T lymphocytes secreting IFN-γ increased 2, 1.6 and 1.45 times compared to control groups vaccinated with PBS, Montanide ISA 50 V, and only Hexavalent recombinant protein mixture (Fig. 7a). Besides, the ratio of CD8 + T lymphocytes secreting IFN-γ increased 2.2 and 1.6 times in mice vaccinated with Hexavalent recombinant protein mixture (+) Montanide ISA 50 V compared to control groups vaccinated with PBS, Montanide ISA 50 V, and only Hexavalent recombinant protein mixture (Fig. 7b). On the other hand, the ratio of CD4 + T lymphocytes secreting IL-4 in mice vaccinated with Hexavalent recombinant protein mixture (+) Montanide ISA 50 V increased 3.07, 2.88, and 1.1 times compared to control groups vaccinated with PBS, Montanide ISA 50 V, and only Hexavalent recombinant protein mixture (Fig. 7c). CD3 + T cells from all experimental groups proliferated to comparable ratios in response to ConA (Data not shown).

Protective effect of mice immunized against challenge with lethal T. gondii
Protection against lethal toxoplasmosis in mice vaccinated with Hexavalent recombinant protein mixture (+) Montanide ISA 50 V was determined using T. gondii Ankara strain tachyzoites. Eight from each  The results are summarized in Fig. 8.

Discussion
Recently, our study group has discovered some T. gondii proteins that can be used as vaccine candidate against toxoplasmosis using in silico and immunoscreening approaches based on protein microarrays [6,8]. We selected the antigens from this screening approach because antigens selected without a screening approach ended with disappointing clinical results such as the malaria vaccine RTS,S which is a hybrid protein particle designed in 1980s. The Major Surface Glycoprotein (MSG) containing hybrid protein was formulated in a multi-component adjuvant (AS01) and showed 39% protection in East African children in 2011 [28]. For these reasons, development of a multivalant recombinant protein vaccine using some of these discovered proteins was the aim of this study.
For this purpose, we used the data from protein microarray screening and prioritized 49 proteins based on their immunogenicity. To further analyse these proteins for their availability for vaccine development against toxoplasmosis, we conducted small scale protein expression experiments in conjunction with bioinformatics analyses. According to protein expression levels 6 proteins were suitable to be used in the vaccine development. Among them, rH2 (TGME49_019310_8) is a DnaK family protein, rA4 (TGME49_058660_1) is ROP6, rE4 (TGME49_073130_2) is a SAG-related sequence SRS30A, rD6 (TGME49_055180_7) is an Fig. 6 Extracellular a IFN-γ and b IL-4 levels elicited by hexavalent recombinant protein mixture (+) Montanide ISA 50 V and control groups. The red bars represent IFN-γ response and the green bars, IL-4 response. Each bar represents the mean ± SD value of IFN-γ and IL-4 responses of 3 mice from each group. Each bar represents the mean ± SD value of IFN-γ and IL-4 responses of 3 mice from each group. Single cell suspensions were stimulated with rH2, rA4, rE4, rD6, rE6, and rH6 recombinant proteins with a final concentration of 100 μg/ml. In figure, *** represent P ≤ 0.001 and * represent P ≤ 0.05 ubiquitin carboxyl-terminal hydrolase, rE6 (TGME49_ 025340_13) is a hypothetical protein, and rH6 (TGME49_013390_5) is a plectin.
The protection against T. gondii infection is dependent on CD8 + T-cytotoxic lymphocytes which play a significant role in cell mediated protection as well as B cells which is important in humoral immune response [11][12][13][15][16][17][18][19]. For this reason, MHC-I, MHC-II, and B-cell epitopes of the vaccine candidate proteins were predicted by bioinformatics. SVMTriP online service was used to analyse the B-cell epitopes of rE6, rD6, rH6, rE4, rA4, and rH2 proteins. As shown in Fig. 1c, the presence of B-cell epitopes on the 6 vaccine candidate proteins suggests that they have a strong potential to act as a Bcell antigen. We also used the online service IEDB to analyse MHC-I and MHC-II epitopes of rE6, rD6, rH6, rE4, rA4, and rH2 proteins and found MHC-I and MHC-II epitopes on the 6 vaccine candidate proteins ( Fig. 1a and b).
Protein glycosylation has great importance in terms of protein stability, three-dimensional structure, surface expression, activity, and antigenicity [29]. Previously, it was shown that T. gondii contains 11 N-linked glycosylated proteins and 15 O-linked glycosylated proteins by lectin-probed western blot analysis. Moreover, it is reported that N and O-linked glycosylated proteins are found throughout the secretory pathway of the T. gondii and N-linked glycosylation of proteins is essential for the survival of parasite [20]. For this reason, Nand O-linked glycosylation sites of 49 antigenic proteins, which may be candidates for vaccination, was predicted by bioinformatics. The results suggest that the 6 vaccine candidate proteins contain N-linked glycosylation sites except for the rH2 and rE4 proteins (Fig. 2b). O-linked glycosylation sites are found on rE6, rD6, rH6, rE4, rA4, and rH2 proteins (Fig. 2a).
At this stage, we further examined the N-and Olinked glycosylation sites in B cell, MHC-I, and MHC-II epitopes of the 6 vaccine candidate antigens by bioinformatics. The results demonstrate that B-cell epitopes of 6 vaccine candidate proteins were not containing N-linked glycosylation sites. B-cell epitopes of rE6, rD6, rH6, rE4, rA4 were O-glycosylated ( Table 3). The N-linked glycosylation site in the MHC-I epitopes were only found in the rE6 and rH6 proteins and the O-linked glycosylation is detected in the rE4, rE6, rH6, and rD6 proteins ( Table 3). The N-linked glycosylation site in the MHC-II epitopes were similarly found in the rE6 and rH6 proteins and the O-linked glycosylation is detected in the rE6, rD6, rH6, rE4, and rH2 proteins (Table 3). These results show that the 6 vaccine candidate proteins and their epitopes are glycosylated at various ratios and their antigenicity is high.
Thereafter, we developed a hexavalent recombinant protein protein vaccine adjuvanted with Montanide ISA 50 V which has shown to induce strong cellular and humoral immune response. After vaccination of Swiss Webster outbred mice, strong total IgG, IgG1 and IgG2a responses were detected in mice administered with hexavalent recombinant protein mixture (+) Montanide ISA 50 V compared to control groups vaccinated with only Montanide ISA 50 V or PBS (P < 0.0001) indicating strong and balanced Th1 and Th2 responses (Figs. 4 and 5). The production of extracellular IFN-γ was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) Montanide ISA 50 V compared to mice vaccinated with only Montanide ISA 50 V or PBS (P < 0.001) (Fig. 6a). Flow cytometry results were also in compatible with extracellular ELISA in which hexavalent recombinant protein mixture (+) Montanide ISA 50 V showed increment in ratio of CD8 + and CD4 + T lymphocytes secreting IFN-γ ( Fig. 7a and b).
CD4+ T lymphocytes secreting IL-4 were also increased according to flow cytometry results as well as there was an increase in extracellular IL-4 secretion according to ELISA (Figs. 6b and 7c). Flow cytometry results, specifically CD4 + T lymphocytes secreting IL-4 cell ratio is bigger than CD4 + T lymphocytes secreting IFN-γ which contradict with ELISA results in which IFN-γ was higher than IL-4 secretion. This discrepancy can interpreted as IFN-γ levels detected by ELISA can be related to CD8 + T lymphocytes secreting and macrophages other than CD4 + T lymphocytes. On the other side, the main protective cells against toxoplasmosis are conferred by CD8 + T lymphocytes secreting IFN-γ which has increased with recombinant protein mixture (+) Montanide ISA 50 V compared to controls. The protective efficacy of hexavalent recombinant protein mixture (+) Montanide ISA 50 V against lethal toxoplasmosis was evaluated by infecting mice intraperitoneally with T. gondii Ankara strain tachyzoites. T. gondii Ankara strain is Africa 1 genotype and causes death in mice in approximately 4-5 days [22]. Challenging study showed that survival was prolonged from 4 to 5 days which was observed in control group mice administered with only Montanide ISA 50 Vand PBS to more than 11 days in two mice vaccinated with hexavalent recombinant protein mixture (+) Montanide ISA 50 V (Fig. 8).
In this study, Montanide ISA 50 V was selected as an adjuvant due to its efficiency in inducing both humoral and cellular immune responses. Montanide ISA 50 V was used as adjuvant in previous studies against Bovine herpesvirus 5, Boophilus microplus, Foot and Mouth Disease Virus (FMDV), and Leishmania major [30][31][32][33]. In the vaccine trial against FMDV, Boophilus microplus, and Leishmania major, Montanide ISA 50 V induced significant levels of protective cytokine production and/ or antibody response. During the vaccine trial with recombinant glycoprotein D of Bovine herpesvirus 5, a mixed Th1/Th2 response was elicited [30]. In this study, hexavalent recombinant protein mixture (+) Montanide ISA 50 V showed strong and balanced Th1 and Th2 responses. Overall, the Th1 part of the immune response elicited by hexavalent recombinant protein mixture (+) Montanide ISA 50 V induced significant levels of CD4 + and CD8 + T lymphocytes secreting IFN-γ and conferred significant protection in Swiss Outbred mice challenged with lethal dose of T. gondii Ankara strain tachyzoites.
In all of the recombinant protein or DNA vaccine studies against toxoplasmosis, murine models are being used to determine the immunity and protection conferred by vaccinations. Michima et al., 2001 vaccinated mice with a mixture of rSAG1, rSAG2, rSAG3, rSRS1, and rP54 proteins adjuvanted with Freund and achieved 17% survival up to 120 days after i.p. challenging with T. gondii Beverley strain bradyzoites [34]. Golkar et al., 2007 vaccinated mice with a mixture of rGRA2 and rGRA6 proteins adjuvanted with monophosphoryl lipid A and achieved 48.2% decrease in brain cyst formation after i.p. challenging with T. gondii Pru strain cysts [35]. In one study, rROP2, rGRA5, rGRA7 proteins and cholera toxin were administered through intranasal route to mice and oral challenge with VEG cysts resulted in 58.3% decrease in brain cysts compared to controls [36]. In a study that used antigenic epitopes of SAG1, GRA1, and MAG1 proteins adjuvanted with Freund decreased the brain cysts formation by 89% [37]. Dziadek et al., 2011, evaluated rROP2, rROP4, rGRA4, and rSAG1 in three vaccine formulations adjuvanted with incomplete Freund administered subcutaneously to mice and challenge with T. gondii DX cysts results with 71 to 90% decrease in parasite burden [38]. In another study, a synthetic peptide, generated using B-cell and two T-cell epitopes derived from SAG1, GRA4, and GRA1 antigens was adjuvanted with Freund and challenge with GJS tachyzoites increased survival time [39]. In other study, mice were vaccinated with the mixture of rROP5 and rSAG1 adjuvanted with Freund and challenged with the T. gondii RH strain. The results showed that T. gondiispecific IgG antibodies levels and lymphocyte proliferative responses are increased in vaccine group and conferred more efficient protection compared to the control groups [40]. Sun et al., 2014 vaccinated mice with a mixture of rBAG1, rSRS4, and rSRS9 proteins adjuvanted with Freund or recombinant mindin. The results showed that vaccine using mindin as an adjuvant efficiently stimulated humoral and cellular responses, including antigen-specific IgG1 and IgG2a, as well as lymphocyte proliferation. Also the improved protection against T. gondii infection was observed in the mindin adjuvanted vaccine group compared with the other controls [41]. In another study, T-and B-cells epitopes of AMA1, RON2, and RON4 proteins was used to develop multivalent peptide vaccine formulations. The IgA levels were increased in the mice immunized with single rRON2, while the IgG levels were higher in the mice immunized with rAMA1 (+) rRON. Significant level of IFN-γ was detected in mice immunized with rAMA1 (+) rRON2. Infection with T. gondii is naturally occurs through the oral route by water or food contaminated with tissue cysts (containing bradyzoites) or oocysts (containing sporozoites). Interestingly, in this study, the mice were challenged orally with 4 × 10 4 tachyzoites of T. gondii RH strain. It was reported that the control mice died within 13 days. The mice immunized with A1 + R2 achieved 70% survival rates. The mice immunized with AMA1, A1 + R2 + R4, and RON2 achieved 60, 50, and 40% survival rates, respectively [42]. Another vaccine trial using T-and B-cells epitopes of SAG1, GRA2, GRA7, and ROP16 proteins resulted with higher levels of IgG and IgG2a subclass titters, significant production of IFN-γ, percentage of T lymphocyte subsets and longer survival times against intraperitoneal challenge with 10 3 T. gondii RH strain tachyzoites [43]. rGRA1 and rBAG1 were used in a multivalent recombinant protein vaccine adjuvanted with Alum. Significant increase in IgG, IgG2a subclass titters as well as significant increment in ratio of IFN-γ secreting CD4 + and CD8 + T lymphocyte subsets were achieved. Mice were challenged orally with 10-12 T. gondii PRU strain tissue cysts and the amount of tissue cysts in vaccinated group decreased 10.5% compared to control groups. In sum, multistage and multivalent rBAG1 and rGRA1 vaccine increased immune response but induced partial protection against toxoplasmosis [24].
Picchio et al., 2018 vaccinated mice with rTgPI-1, rROP2, and rGRA4 proteins ajuvanted with Alum intradermally or with CpG-ODN 1826 intranasally and mice were orally challenged with the T. gondii ME49tissue cyst. P + R + G vaccine formulations induced significant decreases in the number of cysts per brain compared to the control group. According to the levels of IgG1 and IgG2a subclasses P + R + G vaccine groups showed a mixed Th1/Th2 immunity [44].

Conclusions
Overall, in the present study a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 was first time developed and administered to mice to protect against lethal toxoplamosis. Moreover, the immunogenic and protective efficiency of rROP6, DnaK family protein, SRS30A, ubiquitin carboxyl-terminal hydrolase, and plectin was first time tested in an animal model. In addition, Montanide ISA 50 V was first time used as an adjuvant in mice model against toxoplasmosis. Apart from these, multiplexing recombinant proteins induced strong and balanced Th1 and Th2 immune responses and improved protection against toxoplasmosis and thus showing the the importance of using multivalant recombinant protein vaccines in future vaccine development studies against ruminants, cats or humans.