Toxoplasma gondii vaccine candidates: a concise review.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis. It has been shown that the severity of symptoms depends on the functioning of the host immune system. Although T. gondii infection typically does not lead to severe disease in healthy people and after infection, it induces a stable immunity, but it can contribute to severe and even lethal Toxoplasmosis in immunocompromised individuals (AIDS, bone marrow transplant and neoplasia). The antigens that have been proposed to be used in vaccine candidate in various studies include surface antigens and secretory excretions that have been synthesized and evaluated in different studies. In some studies, secretory antigens play an important role in stimulating the host immune response. Various antigens such as SAG, GRA, ROP, ROM, and MAG have been from different strains of T. gondii have been synthesized and their protective effects have been evaluated in animal models in different vaccine platforms including recombinant antigens, nanoparticles, and DNA vaccine. Four bibliographic databases including Science Direct, PubMed Central (PMC), Scopus, and Google Scholar were searched for articles published up to 2020.The current review article focuses on recent studies on the use and usefulness of recombinant antigens, nanoparticles, and DNA vaccines.

Introduction

Toxoplasmosis is a parasitic disease caused by the intracellular protozoan Toxoplasma gondii. The infection is transmitted through drinking of water contaminated by definitive host’s feces containing oocysts or through consumption of the definitive or intermediate hosts’ tissues containing tissue cysts [1]. The parasite life cycle includes the following steps in summary: the oocysts sporulation step in the environment that makes them infective takes 1–5 days. The next step is the infection of intermediate hosts in nature (including sheep, pigs, cattle, birds, rodents, and humans) after ingesting soil, water, or plant material contaminated with oocysts. In this step, oocysts transform into tachyzoites shortly after ingestion [2].

Toxoplasmosis can present in two forms in human. The first form is asymptomatic and occurs by latent infections due to tissue cysts consumption. The second form is severe infections that occur in immunocompromised hosts (e.g., AIDS and organ transplant recipients) or in fetus or newborn with congenital toxoplasmosis [3].

The toxoplasmosis adverse consequences are due to the ability of the parasite to destroy host cells especially cells from critical organs such as brain and eye [4]. Furthermore, the parasite is capable of crossing the placenta; therefore, it can cause fatal or severe and debilitating morbidity in the fetus and newborn [5]. Infection in pregnant women especially during the first trimester can lead to spontaneous abortion, preterm labor, or severe congenital defects such as hydrocephalus, mental retardation, and chorioretinitis [6]. Toxoplasmosis and its prevention is also an important issue in the veterinary medicine and livestock industry as the infection not only decreases the meat production by causing abortion especially in sheep and goat but also can be both a source for human infections and a reservoir for the parasite [7]. Currently, toxoplasmosis control strategies are largely based on treatment by medications in the acute phase of the infection [8].

However, the current medications have drawbacks including toxic effects, limited accessibility, and high costs. Furthermore, reinfections can occur after treatment due to the complicated life cycle of the T. gondii and the presence of its infectious life stages in the environment [6, 8, 9]. In addition, the drug is not effective on the tissue cysts and is not applicable in the primary stages when the infected individuals are asymptomatic [10, 11]. The parasite exists in three forms depending on the stage in its life cycle: tachyzoites, bradyzoites, and sporozoites that are similar in ultrastructure but with differences in intracellular organelles [12]. The parasite life cycle can be divided into two phases: (1) the intestinal or isospora stage in definitive feline hosts. (2) Extra-intestinal stage in both the definitive and intermediate hosts [13]. The prevalence of congenital toxoplasmosis dramatically varies from 1 to 100 per 10,000 live births in different countries and even in different regions or communities in one country [14]. It is estimated that approximately 30–50% of the world population is infected by the T. gondii [15]. The severity of the disease by the parasite is determined by the host resistance, the parasite’s variants and the antigenic variations. The main antigens of the T. gondii are membrane, cytoplasmic, and soluble antigens that the latter results from a combination of cytoplasmic shedding, active secretion by the parasite, and lysis due to the immune system responses [16]. The parasite secretory antigens that are produced by three parasite’s organelles microneme, rhoptry, and dense granule comprise 90% of the soluble antigens [17, 18]. The T. gondii DNA vaccine studies are mainly focused on four families of molecules. These are surface antigens (SAGs), microneme antigens (MIC), rhoptry antigens (ROP), and dense granules antigens (GRA) [19, 20]. Identification of the molecules that are crucial in pathogenesis and immune protection is a bottleneck in efficient vaccine development. Excretory/secretory antigens (ESAs) produced by T. gondii in tachyzoites and bradyzoites forms have an important role in immune system stimulation [21]. These antigens are mainly GRA that is suggested as a candidate antigen for vaccine development [22]. The microneme secretions contain cell surface adhesion molecules that are involved in the first step of parasite adhesion and invasion to the host [23, 24]. The rhoptry antigens are secreted into the expanding parasitophorous vacuoles during the parasite invasion [24]. The immune response to the T. gondii depends on the clinical presentation of the infection. The CD4 + and CD8 + cells are crucial for protection against the infection [25]. These cells are involved in protection by secretion of inflammatory cytokines such as IFN-γ, TNF-α, IL-1, and IL-6. Toxoplasmosis can induce CD8 + cytotoxic lymphocytes in both human and mice that can destroy the infected cells [26]. It has been found that all the mice strains can develop a strong T helper cell type 1 (Th1) immune response against the T. gondii. Furthermore, macrophages, natural killer (NK) cells, dendritic cells (DCs), antibodies, and other immune effector factors are involved in the prevention of the infection [27]. So far, a variety of T. gondii antigens have been identified by different methods and the molecular characteristics of many of them have been evaluated to be used in diagnostic, therapeutic, immunization, and vaccine development applications [28].

In people with a healthy immune system, the symptoms of the infection usually are similar to mild flu symptoms, while in immunocompromised patients, it can cause severe and even life-threatening complication such as encephalitis and severe ocular complications. In addition, in pregnant women, toxoplasmosis may cause abortion or congenital toxoplasmosis with manifestations such as neurological or ocular in the fetus [6, 29]. It is estimated that approximately 50% of untreated maternal infections are transmitted to the fetus in which approximately 60% are subclinical, 30% have severe damage such as hydrocephalus, intracerebral calcification, retinochoroiditis (Classical triad) and mental retardation and 9% are fatal for the fetus [30]. Generally, in immunocompetent women with one experience of T. gondii infection related abortion or fetal infection [31], the subsequent pregnancies are safe regarding T. gondii reinfection and manifestations; however, there is occasional reports implying transmission of congenital toxoplasmosis by immunocompetent women infected before conception [32, 33]. The available therapeutics for the treatment of toxoplasmosis are not completely safe and effective [6, 20]. Recently considerable progress has been made in designing toxoplasma vaccine candidates that can efficiently stimulate the immune responses [34]. In the current study, we reviewed the T. gondii candidate vaccines that include a heterogeneous collection of studies with different methodologies including recombinant antigens, micro/nanoparticles displaying the antigens and DNA vaccine.

Methods

Database search

Four bibliographic databases including Science Direct, PubMed Central (PMC), Scopus, and Google Scholar were searched for articles published up to 2020. The following MeSH (Medical Subject Headings) keywords were considered in the initial search strategy: “Toxoplasmosis,” “nanoparticles -based vaccines,” “Recombinant vaccines,” and “DNA vaccines of T. gondii” with employing the Boolean operators ‘OR’ and/or ‘AND.’

Vaccine design based on recombinant antigens

Toxoplasmosis in a healthy host that is immunocompetent stimulates a lifelong protective immunity that prevents reinfection. The immunogenic proteins that can induce these protective responses have been identified that are candidates for vaccine development for T. gondii. These immunogenic proteins include surface antigens (SAG), dense granule antigens (GRA), rhoptry proteins (ROP), and microneme proteins (MIC) [35]. The use of recombinant antigens as a candidate vaccine against toxoplasmosis has been considered since the 1990s [36]. At the beginning, the SAG 1 antigen, which is on the surface of the parasite, as well as the (GRA)1 antigen were studied [37]. Subsequently, after 2000, other secretory antigens such as GRA7, ROP2, MICs, and other proteins such as heat shock proteins considered as candidates of vaccines. In recent years, more than 10 genes have been cloned into eukaryotic and bacterial expression systems, as follows: ROP 18 [38, 39], GRA4 [40-42], and ROP4 [40, 41, 43, 44], ROP2 [41-46], SAG1 [41, 45, 47, 48], actin depolymerizing factor (ADF),GRA6 [49, 50], ROP5 [48], GRA2 [50, 51], SAG2 [52], Toxoplasma gondii Hsp70 (TgHSP 70) [53], Toxoplasma tissue cyst matrix protein (MAG1) [44], T. gondii serin protease inhibitor-1 (TgPI-1) [42], GRA5 [51]. Many of these antigens have been used to detect specific antibodies in the serum of mice, pigs, and cats and to assess the immune response in the host. The results of some studies have shown that immunization using these recombinant antigens responds well and effectively induce the immune responses. Table 1 shows the immunogenicity of recombinant antigens produced in different hosts such as mice, pigs, and cats.

Table 1

Recombinant Toxoplasma antigens based on vaccine studies

Antigen	Toxoplasma strain	Expression system	Fusion type	Processing and purification method	Route of injection and the location and dose
ROP18	RH strain	pET30a system in E. coli BL21	HIS fusion	HIS-sepharose columns (and then dialyzed)	Subcutaneously injected with 100 µg rROP18 and Re
GRA 4 and ROP4 proteins	RH strain of virulent T. gondii	pcDNA3 Escherichia coli strain M15	Histidine-linked tag fusion	Nitrilotriacetic acid-Ni2_ columns	10 µg of either rROP2, rGRA4, or a mix of 10 µg of each recombinant antigen adsorbed to 0.5 mg of Al(OH)3 by intramuscular injection into the hindquarters
Recombinant ROP2 and SAG1 fusion protein (compared to their DNA vaccine)	RH strain of virulent T. gondii	For recombinant protein: pET28b vector E. coli strain of BL21-Codon Plus (DE3)-RIL For DNA vaccine: pcDNA3.1vector (for DNA vaccine) E. coli strain DH5α	HIS tag	Ni2 + chelated affinity column	Subcutaneously with 2.5 μg rROP2-SAG1 protein mixed with equal volume of Freund’s complete adjuvant (FCA) intramuscularly with 100 μg pcROP2-SAG1 DNA three times
Recombinant SAG1 Protein	T. gondii C56, a mildly virulent strain	P. pastoris (no more elaboration)	Not mentioned	Not mentioned	Subcutaneously with 10 mg (500 ml) of recombinant SAG1, formulated with the SBAS1 adjuvant
Recombinant ROP2 and ROP4	Low virulent DX strain (lysates from highly virulent BK strain)	Escherichia coli Top 10 (for gene cloning) TA vector (pGEM-Teasy) Escherichia coli BL21(DE3) (for protein expression) poly histone vector	pHis tag	Affinity Ni2 + -chromatography using His-Bind columns	Subcutaneous injection either rROP2, rROP4 or a mix of 10 µg of each recombinant antigen emulsified with complete Freund’s adjuvant
ROP2, ROP4, GRA4 and SAG1	Low-virulence DX T. gondii strain	Escherichia coli Top 10 (for gene cloning) TA vector (pGEM-Teasy) Escherichia coli BL21(DE3) (for protein expression) poly histone vector	pHis tag	Affinity Ni2 + -chromatography using His-Bind columns	Subcutaneous injection 10 µg.of recombinant and antigen ncomplete Freund’s adjuvant
Actin depolymerizing factor (ADF)	Challenge by T. gondii of highly virulent RH strain	E. coli Rosetta host bacteria cells, vector pET-28a (+)	Not mentioned	Centrifuged at 12,000 g Ni2 + column	Intramuscular injections rADF (100 µg/each)
GRA2 and GRA6	RH strain tachyzoites	E. coli BL21 (DE3) pLysS competent cells pUET1expression plasmid	2 HisX6 tags	Centrifuged at 12,000 × g, the supernatants were passed through 0.2 µm filters, Ni2 + -NTA agarose column	Injected subcutaneously (s.c.) in their hind footpad with either 20 µg of rGRA2, 20 µg of rGRA6, or with a mix of 10 µg of each antigen, all formulated in 50 µl of monophosphoryl lipid A (MPL) adjuvant
ROP5 alone or in combination with rSAG1	Lethal challenge with the T. gondii RH strain	Escherichia coli BL21 (DE3) cells Into pHis vector	His tag	Ni2 + -NTAagarose columns	Subcutaneous injection 100 µg of recombinant antigens
SAG2	Orally challenged with 3,000 sporulated oocysts of the Me49 strain of T. gondii, or orally with 10 tissue cysts of the C56 strain	Escherichia coli pGEX-2 T vector	Glutathione-S-transferase (GST) fusion protein	Affinity to glutathione agaros	Subcutaneously in a volume of 0.2 ml either 0.65 mg v22 iscom or 0.8 mg GST iscom combind with approximately 10 mg Quil-A
TgPI-1, ROP2 and GRA4 proteins	Orally challenged with 20 ME49 strain tissue cysts (sublethal dose)	Escherichia coli Strain E15 pQE	Six-histidine tag	Ni2 + -NTAagarose columns	Intradermally 10 μg each of rTgPI-1, rROP2, rGRA4 Ags with 0,125 mg of aluminum (Al(OH)3, or intranasally with 10 μg of CpG-ODN 1826
TgHSP70	TgHSP70 from T. gondii RH TgHSP70 gene oral challenge ME-49 T. gondii infection	E. coli pGEx-4 T-2 plasmid	Fusion with GST tag	Purified by glutathione and polimixin B affinity chromatography	Injected subcutaneously with 10 μg of rTgHSP70 dissolved in 100μL of Alum adjuvant (Alhydrogel 2%)
ROP18 (both protein and DNA vaccine)	76 K strain of T. gondii (type II) for experimental infection	Drosophila Schneider 2 cells (S2 cells) expression vector pMT/BiP/V5-HisA the DNA vaccine plasmid was cloned in Escherichia coli DH5α	His tag	HisTrap HP affinity column	Immunized either with ROP18S2 (15 μg) emulsified in Montanide™ ISA 71 combined with 10 μg poly I:C by subcutaneous injection (or with ROP18S2 (15 μg) plus 1 μg cholera toxin intranasally For DNA immunization, mice received three intramuscular injections at 2-week intervals of pROP18 (100 μg) without or with adjuvant, IL-12-containing plasmid (pIL12) at 100 μg/mouse
GRA2 and GRA5	Challenge by T. gondii virulent RH strain	Escherichia coli, BL21 (DE3) pLysS	Not mentioned	Affinity purification	Subcutaneously with final protein dose of 10 mg
ROP2 + ROP4 + SAG1 + MAG1	Low-virulence T. gondii DX strain for challenge highly virulent RH and BK strains of T. gondii as a source of native antigen and DNA template for cloning	E. coli BL21(DE3) pJET1.2/blunt vector pHis expression vector	His tag	Purified by Ni(2 +) affinity chromatography on His-Bind columns	Subcutaneous injection 30 μg of each antigen supplemented with 10 μg of MPL and Alhydrogel® 2%
GRA2	The gene sequence of gene sequence of RH strain strain	Escherichia coli pUET1 expression plasmid	His tag	Ni2+-nitrilotriacetic acid resin	Injected subcutaneously in their hind footpad of 20 μg of GRA2 in a volume of 50 μl
rROP2 (administered intranasally)	RH strain for obtaining DNA to amplify rop2 gene and ME-49 strain was used for tissue cyst production for challenge	Escherichia coli DH5-α Escherichia coli Rosetta 2 pTrcHis	His tag	Ni–NTA Superflow resin	Intranasal 100 µg of rROP2 plus Quil-A (20 µg) (100 µl of final solution was administrated in each animal per nostril)

Elevated IgG1 and IgG2a in sera

Increased in vitro proliferation and IL-4 and IFN-g production in splenocyte following incubation with Ag

Increased survival time of immunized mice

The C57BL/6 lower brain cyst burden (only in rGRA4 or combination of both)

In C3H mice Lower brain cyst burden in immunizationby both Ags or their combinations gra4 gene DNA vaccine lower brain cyst burden (like rGRA4 protein + alum) humoral response All immunized C57BL/6 and C3H mice showed high IgG titers against rGRA4 and rROP2 splenocytes: specific proliferative response (all immunized groups)

Cytokine: change only in immunized C57BL6 mice ROP2 stimulation: high IL-4

Gra4 stimulation high IFN-g

Boosted Twice with the same dose for the peptide vaccine

For the DNA vaccine boosted twice with the same dose (100 μg) of DNA or 2.5 μg of rROP2-SAG1 peptide mixed with equally volume of Freund’s incomplete adjuvant (FIA)

Specific IgG (IgG1 twice the IgG2a) in the serum of group immunized by hybrid protein (but not DNA)

Ag-specific proliferation of splenocyes was increaseed especially in the group immunized by hybrid protein

IFN-g production was increased in the immunized groups (pro and DNA)

High titers of immunoglobulin in the serum

Recombinant SAG1 vaccination provided protection against maternofetal transmission

In the mice received combination vaccine:

Significant reduction in brain cyst load

Increased IgG1 (higher) and IgG2a specific to both Ags

Elevated Ag-specific proliferation and IFN-g and IL-2 production in splenocytes

The best result by the following combination: “rROP2 and rROP4” + “rGRA4” or “rSAG1”

immunization by trivalent vaccines induced significant reduction in the brain cyst number vaccination resulted in antigen-specific proliferative response in splenocytes elevated IFN-g production (not by rROP2 + rGRA4 + rSAG1 mixture) elevated IL-2 production (by all three mixtures) All recombinant proteins used in the vaccines induced a IgG1 and IgG2a with (higher IgG1)

Freund’s complete adjuvant (1st week)

Freund’s incomplete adjuvant (2nd week)

Reduced number of brain cyst Increased survival time

Specific serum IgG antibody

Elevated percentage of CD4 + cells in the spleen

Strong IgG response high ratio of IgG2a to IgG1 rGRA2 immunization induce high levels of IFN-g and IL-2 (Th1 cytokines)

Reduced number of brain cysts (in rGRA2 immunized or combination)

Yes

ROP5 alone or in combination with rSAG1

High levels of IgG (Both IgG1 and IgG2a); A predominance of IgG2a over IgG1 single rSAG1 group, (rSAG1 elicited aTh1-type response)

High splenocyte proliferation after specific Ag activation (the highest rROP5 + rSAG1, followed by rROP5) The highest IFN-g and IL-2 concentrations were detected in the group of rROP5 + rSAG1

rROP5 + rSAG1 or rROP5 induces mice immunized with rROP5 + rSAG1 or rROP5 produced specific amounts of IL-10 (and low levels of IL-4): a mixed Th1/Th2 response in rROP5 + rSAG1 or rROP5 groups

Increased survival time in all the immunized groups (the most prolonged: rROP5 + rSAG1)

Elevated IgG

rTgPI-1 induced significant production of both isotypes IgG1 and IgG2a

rROP2 predominantly IgG1 (Th2)

rGRA4 1 elicited only IgG1 (low levels)

Elevated IFN-γ, IL-2 (cellular immune response)

Mucosal response: IgA after immunization by combination of the 3 antigens MLN lymphocytes significantly proliferated in vitro after stimulation

Cellular response:

rTgPI-1 + rGRA4 or rTgPI-1 + rROP2 + rGRA4 induced a response with a mixed Th1/Th2 profile while splenocytes from mice immunized with rTgPI-1 + rROP2 showed no cytokine secretion

Reduction of cerebral parasitism and less cyst numbers

Induces high antibody titers (high IgG1) Immunization did not alter the production of IL-2, IL-4, IL-6, IL-10, IL-17a, IFN-g, nor TNF enhanced Nitric oxide (NO) production by peritoneal macrophages

Increased Number of iNOS + Cells in the Brain

Significant brain cyst reduction in intranasal group

Higher IgG in the subcutaneous immunized group

Both IN and SC Predominantly IgG1 over IgG2a indicating predominant Th2 responses

Splenocytes produced IFN-g and IL-2 following antigen stimulation indicating cell-mediated immunity induced by vaccine. IL-5 and IL-10 (non-significantly), which are in favor of Th2 responses, both produced significantly by splenocytes after activation. Both routs induced a mixed Th1/Th2 type cellular immune response

DNA vaccine group: high IgG response (IgG2a higher than IgG1––- > Th1 predominant) Splenocytes produced IFN-g and IL-2 following antigen stimulation with no increase in IL-5 or IL-10

Strong humoral immune response

Both IgG2a and IgG1 (Th1 andTh2 responses)

Increased Ag-specific proliferation of T lymphocytes

Higher level of IFN-g and IL-2 compared, In contrast, relatively low levels of IL-4and IL-10 (indicative of skewed responses to Th1 responses)

IgG and IgA levels above cut-off level only in rROP2

Immunized group fewer oocysts shedding however not statistically significant

Vaccine design based on DNA genome of antigens

The DNA vaccines are a new strategy to prevent infectious diseases to help reduce the antibiotics use and diseases spread. The DNA vaccines can be used in oral or injective forms to induce rapid immunization against a diverse range of diseases that are hard to be overcome by antibiotics or traditional vaccines. A typical DNA vaccine is composed of a plasmid with a strong viral promoter and the gene of interest that is expressed and induces specific immune responses. The advantages of DNA vaccines include stability, cost effectiveness, and safety. However, the hurdle in DNA vaccine development against parasites is the complexity of the parasitic diseases.

The GRA and SAG antigens that are parasitic secretory and surface antigens are suitable candidate antigens to design DNA vaccines for immunity against toxoplasmosis. It has been demonstrated that DNA vaccination with the sequences of GRA1, GRA7, and ROP2 proteins can induce protection against infection with different virulent T. gondii strains in C3H mice but not in BALB/c and C57BL/6 mice. Furthermore, immunization of sheep with a DNA vaccine containing the GRA1, GRA4, GRA6, and GRA7 sequences formulated in liposome showed a significant immune response against T. gondii [54]. Table 2 lists the studies of antigens used as candidate DNA vaccines. Including SAG1 antigen from ME49, VEG strains [55], SAG1, ROP16, GRA14,MIC8,ROP54, Toxoplasma gondii calcium-dependent protein kinase 2 (TgCDPK2), T. gondii Myc regulation 1 (MYR1), Perforin-like proteins (PLP)1, ROP18, GRA2, GRA5, GRA17, GRA23, GRA7, ROP2, TgHSP60, ROP21, TgHSP-40,GRA16, Rhomboid 4(ROM4), ROP35,GRA8, GRA4, GRA24, GRA25, MIC6, SAG5-D for RH [42, 55–77] and ROP35, GRA8, ROP19, GRA24, GRA25, MIC6 for PRU strain [72, 74, 75].

Table 2

Toxoplasma antigens based on DNA vaccines studies

Antigen	Toxoplasma strain		Plasmid and the cloning organism		Processing and purification method		Route of injection and the location and dose
SAG1 DNA	Mice challenged with 80 tissue cysts of ME49 strain Rats with VEG strain oocysts resulted (brain cysts) highly virulent RH strain tachyzoites for mice challenge and also to derive the SAG1 cDNA clone		pCMVInt expression vector		By double-banding on CsCl		Intramuscular Hindquarters 100 mg of pCMVToxo or pCMVInt in 100 mL of PBS with 25% sucrose + 50 mg of pGM-CSF
ROP16 DNA	Highly virulent RH strain of T. gondii The DNA sequence of the Ag from T. gondii RH strain		pVAX➔ pGEM		Not mentioned		Intramuscularly (i.m.) with 100 µg of plasmid DNA suspended in 100 µl sterile PBS, 100 µl in each thigh skeletal muscle,
MIC8	Challenge by RH strain of T. gondii genomic DNA of T. gondii RH strain		pGEM-T easy vector➔ pVAX vector		Not mentioned		Intramascular, pVAXMIC8 plasmid or empty pVAX I vector into each anterior tibial muscle (final plasmid concentration, 100 μg/100 μl)
GRA4	Oral challenge by 76 K T. gondii RH strain cysts		MLPIX➔ pcDNA3 expression vector		All the plasmids were purified from transformed E. coli DH5a by anion exchange chromatography		The tibialis anterior (TA) muscles of both hind legs each 50 mg pGRA4 in 50 ml PBS
GRA14 DNA (boosted by recombinant protein)	Highly virulent RH strain of T. gondii for challenge		Cloning vector pTG19-T➔ eukaryotic expression vector pcDNA3		Plasmids were purified by EndoFree Plasmid Giga Kit		Intramuscularly with 100 μg pcGRA14 into thigh skeletal muscle the first time
ROP54	Intraperitoneally T. gondii RH (acute infection) and oral Prugniaud (PRU) strains tissue cists for chronic		pVAX I plasmid		Not mentioned		Route, location, and dose 100 μg of plasmid DNA dissolved in 100 μl sterile PBS intramuscular injection into the quadriceps
TgCDPK2	T. gondii RH strain (type I) for the DNA sequence and challenge intraperitoneally		pMD18-T➔ vectorpVAX I vector		Anion exchange chromatography		Bilateral intramuscular injection into the quadriceps with 100 mL (1 mg/mL)
MYR1 DNA	T. gondii RH strain for the DNA sequence and challenge		pMD19-T ➔vector pVAX1 vector		End o-free plasmid giga kit		Subcutaneous injection with 100 mL of sterile PBS containing 100 mg pVAX1-MYR1
PLP1 and ROP18 DNA	The RH (used to produce the PLP1 and ROP18 clones) 59 and PRU strains (used to challenge mice with tissue cysts)		PIRESneo➔ pVAX		Not mentioned		Eight groups of mice (30 mice/group) were vaccinated intramuscularly with 100 μg of 82 plasmid dissolved in 100 μl of PBS
GRA2 and GRA5 DNA (separate groups, not in combination)	The virulent T. gondii RH strain for lethal challenge		pcDNA 3.1C		Not mentioned		Intramuscular at tibialis anterior muscle of both leg with 100 μL (50 μL in each leg) of PBS containing 100 μg of pcGRA2, or 100 μg of pcGRA5
GRA17 and GRA23 DNA	Challenge infection with the highly virulent RH strain of T. gondii DNA sequence from RH strain of T. gondii		pMD18-T➔ pVAX		Not mentioned		Intramuscular injections 100 ml (100 mg) of pVAX-TgGRA17, pVAXTgGRA23, and pVAX-TgGRA17 + pVAX-TgGRA23
GRA7 and ROP2 DNA (each alone or in combination)	DNA sequence from the T. gondii RH strain Challenge by		pTOPO➔ pcDNA3.1 plasmid Mass replication in (E. coli), strain TOP10		Mass replication was extracted from the bacteria using endotoxin-free plasmid extraction kit		Intramuscularly with 100 μg of plasmid DNA in 100 μl PBS, 50 μl in each thigh skeletal muscle
tgHSP60	Two T. gondii strains RH and PRU for acute and chronic disease challenge, respectively		pMD18-T vector➔ pVAX		Not mentioned		Thigh muscle, 100 μg pVAX-HSP60
ROP21 DNA	Challenged with tachyzoite cells of RH T. gondii and cysts of T. gondii PRU strain		pMD19-T vector ➔ pVAX plasmid Escherichia coli DH5a		Purification by A commercial kit (TianGen, Beijing, China)		Intramuscular injection containing 100 mg of recombined plasmids
GRA14 DNA	RH strain tachyzoites for challenge and DNA sequence		pTG19-T➔ pcDNA3		End o-free plasmid mega kit		Intramuscularly (anterior tibial muscle).Concentration 100 µg/100 µl plasmid DNA
TgHSP-40	DNA sequence from T. gondii RH tachyzoites challenge		pMD18-T linear vector➔ pVAX1		Not mentioned		intramuscularly (i.m.) 100 µL of PBS containing 100 µg pVAX1-HSP40
SAG1 DNA	Virulent T. gondii RH for challenged DNA sequence from tachyzoites of T. gondii RH		pTZ57 R/T cloning vector➔ pVAX1 E. coli DH5α		Anion exchange chromatography (end-free plasmid mega kit, Qiagen)		Intramuscular, each thigh skeletal muscle 100 μg of plasmid DNA, and different adjuvants suspended in 100 μl sterile PBS
SAG1 and SAG3 DNA	T. gondii, strain RH		pcDNA3 E. coli, strain TOP10		Plasmid purification kit (Qiagen)		Intramuscularly with 100 μg of plasmid DNA suspended in 100 μL sterile PBS bilateral biceps
GRA16 DNA	Challenge by T. gondii RH (acute) and PRU (chronic) strain		pVAX E. coli DH5α cells		Anion exchange chromatography (EndoFree Plasmid Giga Kit, Qiagen		Intramuscularly injected with pVAXGRA16 plasmids 100 µl (1 μg/μl)
ROM4 DNA (alone or in combination with a peptide derived from its gene) Or SAG1 DNA as control	Challenge by T. gondii RH and PRU strain		pEASY-T1 vector pEGFP-C1 expression plasmid Escherichia coli DH5α		Endotoxin-free mega kit following the manufacturer’s instructions (Qiagen)		Intramuscular route, location,and dose
ROP35 DNA	Virulent T. gondii RH strain and PRU strain for challenge		pMD19-T vectorpVAXI vector E. coli DH5α cells		A commercial kit (TianGen, Beijing, China) to isolate the plasmid and to eliminate endotoxin contamination		Intramuscular injections containing 100 μg of recombined plasmids (1 μg/ μl)
GRA8 DNA	DNA sequence from RH strain tachyzoites Challenged with highly virulent T. gondii GFP-RH strain		pGEM-T Easy vectorression plasmid pEGFP-C1HEK-293 T cell pDsRed2-N1 vector Escherichia coli DH5α		Endotoxin-Free mega kit according to the manufacturer’s instructions (Qiagen)		50 μg pDsRed2-GRA8 into the tibialis anterior muscles of both hind legs (100 μg/per mouse)
ROP19 DNA	DNA sequence from T. gondii PRU strain tachyzoites Challenge by T. gondii strain PRU cysts		pEASY-T1 vector ➔ expression plasmid pEGFP-C1 HEK-293 T cells		The endotoxin-free mega plasmid kit (Qiagen)		Intramuscullar, buttocks injection 100 µl
TgGRA24, TgGRA25 and TgMIC6 DNA	DNA sequence from T. gondii RH strain challenge with the T. gondii RH and Pru strains		pMD-18 T Vector➔ pVAX I vector		Not mentioned		Intramuscular injection into the quadriceps, 100 µL (1 µg/µL) of pVAXGRA24
SAG5D DNA	DNA sequence from T. gondii (RH strain) Challenge by T. gondii RH strain		pEASY-T1 vector➔ pEGFP-C1 HEK 293-T cells		Endotoxin-free mega kit according to the manufacturer’s instructions (Qiagen)		Intramuscularly, pEGFP-C1-SAG5D 100 μg/ each (1 µg/µl)

SAG1 specific AbA Th1 dominant response (in contrast to the peptide vaccine)

Splenocytes produce IL-2 and IFN-g but not IL-4 following specific Ag re-stimulation Increased survival and reduced brain cyst

In the pVAX-ROP16 group: Significant anti-rop16 Ab production splenocytes significant proliferative response and high degree of CTL activity higher IFN-g, IL-2, IL-4, and IL-10 (cell-mediated immunity) and lower pro-inflammatory cytokines IL-6 and IL-12

Increased survival time

In the VAXMIC8 group high levels of specific IgG splenocytes proliferative response to MIC8 significant increase in IFN-γ, IL-2, IL-4, and IL-10

Increased survival time

In the pGRA4 or pGRA4 + pGM-CSF group: strong antibody response significant proliferative response of splenocytes

Increased IFN-g and IL-10 (& low amounts of IL-2) (a modulated involvement of Th1 response)

pGRA4 mixed pIL-12-, which intensifies the Th1 response, dramatically decreases the survival rate

Increased survival time in immunized mice

CaPNs adjuvanted DNA prime-protein boost vaccination induce both humoral and Th1 type cellular immune responses and high levels of total IgG, IgG2a isotype and IFN-γ (a Th-1 type response) and reduced brain parasitic load

Alum adjuvanted DNA

prime-protein boost vaccination:

predominance of IgG1 over IgG2a and increased IL-4 (a Th-2 type response)

Twice at

2-week intervals the same dose

In the pVAX-TgCDPK2 plasmids immunized mice: significant IgG response higher levels of IgG1 and IgG2a (elevated IgG2a/ IgG1)➔ mixed Th1/Th2, with a predominant Th1

higher proliferation of splenocytes

increased % of CD4 + and CD8 + T cells in the splenocytes

Increased IFN-g, IL-12(p70) and IL-10 but not IL-4 in spleen cell cultures longer survival time of the mice

In the group immunized with pVAX1-MYR1:

Specific IgG and IgG Isotypes (high IgG2a at first)

Th1 response at 2 weeks after vaccination and a mixed Th1/Th2 immune response at 6 weeks after vaccination

Higher proliferation of splenocytes

significantly higher levels of IFN-g, IL-12, and IL-10 but not IL-4 higher levels of CD4 + and CD8 + T cells in the splenocytes

Increased Expression of p65 and

T-bet in spleen lymphocytes mRNA

Increase in CTL activity

Increased survival time

In both the vaccinated groups: predominant Th1-like response➔ cellular-mediated immune response with significantly higher levels of interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-10

Increased splenocyte Ag-specific proliferation slightly prolonged survival

No elevation of IgG was detected

In the vaccinated groups after specific Ag re-stimulation: Increased IFN-γ levels

And decreased IL-4 expression level

Increased spleen lymphocyte proliferation

Significant high levels of IgG in the serum

Predominance of the levels of IgG2a over IgG1 increased survival time

In the HSP60 DNA-immunized mice: increase of CD3+CD4+ and CD3+CD8+ T cells in spleen increased levels of IL-2, IL-4, IL-10, IL-12p70, and IFN-γ

Increased proliferation of splenocytes

higher levels of specific antibodies in sera

Increased survival time (in the acute infection)

Decreased brain cyst (in the chronic infection)

Specific pathogen-free

(SPF) grade Kunming mice

In the pVAX-ROP21 vaccinated animals:

increased levels of IgG, IgG1, and IgG2a (IgG2a predominant)

IFN-g was significantly increased (while no significant changes were detected in IL-2, IL-4, and IL-10)

prolonged survival time (virulent T. gondii RH strain challenge)

The number and size of brain cyst

decreased

Increased levels of level of IgG1 and IgG2a (IgG2a predominance)

Increased proliferation of splenocytes

Increased IFN-g levels

Increased survival time

Reduced tissue parasite load

In immunized mice:

increase in T lymphocyte subclasses (CD3+CD4+ T and

CD3+CD8+ T lymphocytes) in splenic tissues

reduction in the parasite cyst

burden in the brain

Pru strain–infected mice

No difference in survival time in challenge with the virulent RH strain

No difference in the level of antibodies, lymphocyte proliferation and concentration of cytokines (IFN-g, IL-2, IL-4, IL-10, and IL-12p70)

FliC of Salmonella typhimurium plasmid (Toll-like receptor 5 agonist)

and

(alum and saponin)

The pVAX1-SAG1 + pVAX1-fliC group (compared to both traditional adjuvants and controls):

Higher IgG with a predominance of IgG2a over IgG2b and IgG1

higher levels of IFN-γ, IL-12 and IL-10 cytokines and low levels of IL-4 production

higher splenocyte proliferation response

increased survival time

Cocktail

DNA group:

higher total IgG and the isotypes of IgG1 and IgG2a

higher levels of IFN-γ (the immune response was shifted toward Th1)

increase antigen-specific lymphocyte proliferation of splenocytes

increased survival time and rate

The pVAX-GRA16 group:

higher levels of specific IgG antibody

high Ag-specific proliferation of spleen lymphocytes

increased levels of IFN-γ, IL-2, IL-4, and IL-10 cytokines

higher percentages of CD4 + and CD8 + T cells

reduced numbers of tissue cysts

no change in the survival time

Specific pathogen-free (SPF) grade inbred

Kunming mice

Boosted three times 2-week intervals

(in the pROM4/peptide group the first 2 times by plasmid and the second 2 times by peptide)

The vaccinated groups: high levels of IgG, IgG2a (predominant), and interferon (IFN)-γ, IL-12, and IL-2. (IgG, IgG2a, and IFN-γ, IL-12, and IL-2 levels were highest in the pROM4/peptide group)

Prolonged survival times and reduced numbers of brain cysts (especially those in the pROM4/peptide group)

In the pVAX-TgROP35 group:

Higher IgG (both IgG2a and IgG1)

IFN-γ, IL-2, and IL-10 levels were significantly increased, while there were no significant differences in IL-4 expression

increased survival time

Reduced brain cysts number and size

Higher IgG (both IgG2a and IgG1 increased predominant IgG2a)

Higher splenocyte proliferation

Increased IL-10, IL-12 (p70), IFN-γ, and TNF-α but not IL-4

Increased survival time

Higher levels of IgG antibodies

higher levels of IFN-g

reduced brain cysts

In the immunized groups (more apparently in the multi-antigenic groups):

Increased IgG titer

higher IgG2a to IgG1 ratio

Increased IL-2, IFN-g, IL-12 and IL-23 levels (but not IL-4 and IL-10)

Increased percentages of CD3+

CD4+CD8− and CD3+CD8+CD4− T lymphocytes

Increased survival time

Increased spleen lymphocytes proliferation

Decreased brain cyst

Boosted twice at 2-week intervals

pEGFP-C1-SAG5D 100 μg/

each (1 µg/µl) with α-GalCer at the 3rd time

Alpha-Galactosylceramide

(α-GalCer) (2 μg/mouse)

In both pEGFPC1-

SAG5D or α-GalCer/pEGFP-C1-SAG5D groups:

increase of IgG (IgG2a over IgG1)

higher level of IFN-γ

higher IL-4 (only in α-GalCer-treated groups)

longer survival time

Vaccine design based on nanoparticles of recombinant antigens

Nanoparticles can be used to improve delivery of subunit vaccine in order to increase the immunogenicity of the pathogen proteins used in the vaccine design [78]. Furthermore, virus-like particles (VLPs) or nanoparticles have been used to design recombinant vaccines with promising safety and efficacy both in preclinical and clinical studies. VLPs display the antigens in a repetitive high-density manner similar to the proteins of viral surface proteins, which contribute to strong T-cell and B-cell immune responses against the vaccine antigens [34]. Nanoparticles can play an adjuvant role in the vaccine formulation and improve the humoral and cellular immune responses. Among different type of nanoparticles, the calcium phosphate nanoparticle (CaPN) is a well-known member that has been used for many years as a delivery system in DNA vaccines and is approved to be utilized as the adjuvant.

Much research has been done on nanoparticles to design vaccine against T. gondii. Table 3 summarized the results of several different studies on nanoparticle vaccines, including the type of particle used for the SAG1, 2 and GRA1 antigen were muramyl dipeptide (MDP) microparticle [79], for MIC16 was yeast Saccharomyces cerevisiae EBY100 strain [68], and for ROP2, ROP18, MIC8, MIC3, ROP9, SAG2, SAG1ROP18, SAG1, SAG1, and AMA1 antigens were Mycobacterium bovis, Poly (lactideco—glycolide)(PLGA), virus-like particles (derived from baculovirus + influenza matrix protein 1), recombinant adenoviruses, PLGA, virus-like particle, polymeric nanospheres, virus-like particle, and virus-like particle respectively [80-88].

Table 3

Particulate vaccines (nanoparticle, viral and quasi-viral, microparticle, bacterial or yeast) Toxoplasma

Antigen	Toxoplasma strain	Particle type	Route of injection and the location and dose	Booster
rSAG1, rSAG2 and rGRA1	N/A	Muramyl dipeptide (MDP) microparticle	Intramuscularly in the dorsal neck region 300 µg Ag in 1 ml PBS	Boosted once, 6 weeks after the initial one
Microneme protein 16 (TgMIC16)	Challenged by virulent T. gondii RH strain tachyzoites	Yeast S. cerevisiae EBY100 strain (containing pCTCON2 plasmid)	Intraperitoneally with heat-killed transfected yeast or orally with live transfected yeast 100 µl (4 × 107 cells)	Boosted twice at weeks 2 and 4 after the initial one
ROP2	Protein sequence from T. gondii RH strain challenge by T. gondii RH strain	M. bovis BCG, sub-strain Pasteur pMV262 vector	Subcutaneous 0.1 ml (107 cfu/ml) BCG/ pMV262-ROP2	Boosted once after 4 weeks by the same dose
ROP18	N/A	Poly (lactideco-glycolide) (PLGA) nanoparticle	Intraperitoneally (ip) with 10 μg rROP18	Bossted twice in 2-week intervals
Microneme protein 8 (MIC8)	Challenge by highly virulent T. gondii (RH) (oral challenge)	Virus-like particles (derived from baculovirus + influenza matrix protein 1 {M1})	Intranasal immunization (IN), intramuscular immunization (IM) with 75 μg of total MIC8 VLP protein per mouse	Boosted once 4 weeks later
MIC3, ROP9, and SAG2	Challenge by lethal T. gondii RH strain	Recombinant adenoviruses	50 µL purified recombinant adenoviruses (109 PFU) intramuscular injection at 2-week intervals	Boosted once, 2 weeks later
rSAG1	Tachyzoites of T. gondii RH strain for challenge	PLGA	Subcutaneously (s.c) immunized in the right-hind footpad 20 μg of rSAG1-adsorbed PLGA nanoparticles and rSAG1-encapsulated PLGA nanoparticle	Once, 3 weeks later by the same dose
Rhoptry protein 18 (ROP18) and microneme protein 8 (MIC8)	Intraperitoneally (IP) with tachyzoites of GT1 strain or orally challenged with T. gondii ME49 strain	Virus-like particle	Intranasally (IN) with 60 mg of ROP 18VLPs or MIC8 VLPs or a mixture of 30 mg ROP18 VLPs and 30 mg MIC8 VLPs (combination VLP vaccine)	Once, 4 weeks later by the same protocol
SAG1	N/A	Polymeric nanospheres	Intraperitoneal (i.p.) injections 10 mg rSAG1 protein + montanide or rSAG1 + PLGA intranasally	Boosted twice with 2-week intervals
SAG1	Challenged by T. gondii RH	Virus-like particle	Intramuscular administration with SAG1-VLPs (120 µg)	Boosted once after 4 weeks
Apical membrane antigen 1 (AMA1)	Challenged with T. gondii ME49	Virus-like particle	Intranasally immunized with 100 μg of VLPs	Boosted once after a 4-week interval

Increased IFN-g in the rGRA1

Immunization with recombinant proteins rSAG1, rSAG2 and rGRA1 alone or as a cocktail vaccine elicited IgG2 and a weak IgG1 response

In both intraperitoneally orally vaccinated groups:

Higher serum Ab concentration

(dominant IgG2a over IgG1)

Higher lymphocyte proliferative response

Higher percentage of CD4 + and CD8 + T cells

increased levels of IL-2 and IFN-g (but not IL-4 or IL-10)

increased survival time

Increased survival time

Elevated total Ab (humoral immunity)

Increased IFN-g and IL-2 production

Higher percentage of CD4 + cells (cellular immunity)

Both adjuvant group and PLGA demonstrated elevated IgG

IgA levels was significantly higher in PLGA + ROP18 group

In the PLGA group the IgG2a was dominant while in the adjuvant group the IgG1 was dominant

IN mice group showed higher levels of T. gondii-specific

IgG antibody response compared to IM mice group

IN group (IgG1 predominance {Th2})

IN induced higher levels of systemic and mucosal antibody responses

IM group no effective Ab response

Higher CD4 T cell, CD8 T cell and germinal center B cells in both IN and IM groups

Following parasite challenge higher levels of IFN-γ and IL-6 were detected in Naïve and IM groups compared to IN group (IN group reduced inflammatory

reaction but higher humoral)

100% survival of IN group and 60% survival of IM group, 100% mortality in the control group

In the mice immunized with the recombinant adenoviruses group: extremely significantly higher T. gondii-specific IgG antibody

Levels

Increased production of IL-6, TNF-a, IL-22, IFN-g, IL-17A and IL-10

Increased T lymphocytes (and activated Th lymphocytes) percentage in the spleen

Elevated survival time

Increased survival time in rSAG1 loaded PLGA groups

Total serum IgG and high IgG2a/IgG1 ratio in rSAG1 loaded PLGA groups

higher amounts of IFN-γ (but unchanged IL-10) in rSAG1 loaded PLGA groups after in vitro Ag re-stimulation of spleen cells

All three vaccine groups showed similar levels of IgG antibody responses which were significantly increased after boost immunization (combined ROP18 VLPs + MIC8 VLPs vaccine immunization showed a higher level of IgA antibody responses)

higher levels of CD4 + T cells, CD8 + T cells, and memory phenotypic T cells (combination T. gondii VLP immunization induces higher T cell responses after challenge)

Combination T. gondii ROP18 and MIC8 VLP immunization attenuates apoptotic cellular response after challenge

Combination VLP vaccine immune sera exhibit higher activity of controlling parasite loads in vivo

Combination VLP vaccination reduces pro-inflammatory cytokine (IFN-g and IL-6)

responses after challenge

Combined VLP vaccines improved protection against challenge infection with T. gondii via an oral or IP route

Both adjuvant group and PLGA demonstrated elevated IgG

IgA levels was significantly higher in PLGA + ROP18 group

In the PLGA group the IgG2a was dominant while in the adjuvant group the IgG1 was dominant

SAG1-VLP immunization: Significant increase of the antibody (IgG, IgG1, IgG2a, and IgA) levels (IgG1 predominance) not only decreases the production of cytokines (IL-4, IL-12, and IFN-g) associated with the infection of pathogens in the host, but also effectively inhibits the inflammatory cytokines (IL-1

, IL-6, and TNF-a) after T. gondii infection

The survival rates of the immunized infection group were significantly increased compared to the non-immunized infection group

In the AMA1 VLPs-immunized group:

higher levels of T. gondii-specific IgG and IgA

higher germinal center B cell populations

smaller cysts and lower cyst counts were detected from the brain, reduced body weight loss, higher survival rate

Discussion

In recent years, progress has been made in designing a potential vaccine against T. gondii. Studies have also been performed using different types of T. gondii antigens, including recombinant vaccines, DNA vaccines, subunit vaccines, attenuated live vaccines, and nanoparticle vaccines [20]. Accordingly, significant advances have been made in characterization and isolation of antigens, gene cloning, antigen expression, and immunological methods. In addition to the prevention strategies, new options are now needed to develop effective vaccines as a way to prevent the toxoplasmosis [29]. Most of the T. gondii antigens are important for the virulence and immunogenicity of the parasite. However, future studies should focus on the quality and quantity of antigens and identify potential candidate antigens against T. gondii infection. In addition, more extensive studies are needed to identify recombinant vaccines, DNA vaccine performance, and evaluate recombinant nanoparticle vaccines. Many of the vaccine strategies against toxoplasmosis have been experiments in animal models; nevertheless, these experiments only resulted in relative protection against T. gondii infection.

Vaccines designed with recombinant antigens rely on the defined antigens to induce a host-specific immune system against pathogenic microorganisms, which can be expressed by plasmids in the bacterial and yeast hosts or delivered by viral vectors [89]. Recombinant antigen vaccines have advantages over classic methods. One of the problems with using live vaccines is that if the host’s immune system is defective, it may cause the tachyzoite to return from an attenuated form to an active invasive form [29]. Another disadvantage is the complexity of obtaining sufficient amounts of purified immunogenic components of the antigen by the classical methods. The quality and effect of vaccination against T. gondii using recombinant antigens is very important, especially in pregnant women who may be at risk for the first time [6, 18, 20]. In the preparation of recombinant antigens, when the antigen is purified well, it can have far fewer side effects than raw antigens or live vaccines [90-93].

Studies on rodent animal models have shown that DNA vaccines can effectively induce both humoral and T-cell responses against a wide variety of candidate vaccine antigens [94]. Nevertheless, for unclear reasons, in primates and human’s poor immune, responses have been observed to parasite DNA vaccines. Various adjuvants including cytokines and CpG oligonucleotides have been studied to improve the immune responses in these large animals [95].

Monomeric linear protein that can assemble into a nanoparticle is a new method for inducing immune responses against peptide epitopes of antigens from an infectious agent. As most of the infectious agents invade the host through mucosal surfaces, researchers are interested to design vaccines with the ability to mimic this aspect of the pathogens to induce an effective immune response; therefore, a better understanding of the mechanisms that the pathogens use to interact with cells and the biological fluids is required to design vaccines with adequate efficiency. Monomeric linear protein based nanoparticles vaccine against toxoplasmosis were in a study and effectively elicited T-cell-dependent cellular immune cells responses [35]. As the group SAG antigens are highly expressed on the Toxoplasma gondii tachyzoite, they are considered as one of the main candidates for toxoplasmosis vaccine design. The SAG1 is a 30 KDa beta-glycoprotein that can be extracted from tachyzoites and sporozoites of the T. gondii [96].

The SAG1 is the most immunogenic structure in tachyzoites and the first structure of the parasite that interact with the host cells. The gene encoding this protein is a single-copy gene and contains no introns. It is believed that SAG1 is the most promising candidate to develop an effective vaccine against T. gondii because it stimulates both the cellular and humoral immune responses [6, 20, 37, 79, 96]. According to the results of previous studies use of vaccines based on recombinant forms of the antigen and nanoparticle-based vaccines can induce stable specific immunity in hosts, including pregnant women (at risk of primary infection) and immunocompromised patients [79]. Previous studies indicate that the use of GRA1, GRA2, GRA6, GRA5, GRA4, and GRA7 antigens are good candidate for the design and production of DNA vaccines. For example, the GRA7 antigen is an acidic 29 KDa protein and comprise about 0.5% of all the T. gondii proteins. The Gra7 gene is composed of 1.3 Kbps and has no introns. The GRA7 antigen is present in the parasitophorous vacuole in host cells infected by tachyzoites and also in the cytoplasm of host cells infected by bradyzoites. GRA7 is expressed in all of the stages of T. gondii infection and is a considerable candidate for the vaccine design [91, 93]. It can effectively induce both cellular and humoral immune responses against the T. gondii [29]. Another member of this family is GRA4 that is a 40 KDa protein secreted into the parasitophorous vacuole by the parasite [40, 41]. This protein strongly interacts with the milk IgA and to a lesser degree with the intestinal mucosal layer IgA [24]. The amino acid sequences 297–345 in the GRA4 are called C protein and can interact with the milk and intestinal mucosal IgA and serum IgG in mice infected by T. gondii and also serum IgG in human and sheep [24]. The GRA4 stimulates the mucosal T lymphocytes in BALB/C and CBA/J mice strains. The GRA4 can induce mucosal and systemic immune responses in mice after T. gondii ingestion [24, 29, 41]. Furthermore, GRA14 is a 47 KDa protein with 409 amino acids. The gene encoding this protein is consisting of 1227 bps. The GRA14 is present in the membranes of parasitophorous vacuole and intravacuolar network. This protein has a unique topology that is not seen in other proteins [29, 58, 97]. Due to the unique topology and its long length inside the vacuole system, this protein probably is a potential strong inducer of the immune responses. ROP proteins are the largest family of T. gondii serine-threonine kinases [24]. The evaluation of previous studies demonstrates that the ROP18 is the most interesting member of the ROP family in recombinant, nanoparticle, and DNA vaccine development studies [24, 64, 98]. It is probably due to the pre-formed presence of the antigen inside the rhoptry and as it is secreted into the parasitophorous vacuole during invasion to the host cells [64, 98, 99]. This antigen is one of the key virulence factors of T. gondii that protects the parasite from the host immune responses by its kinase activity [98]. The amino acids 243 to 539 are involved in the protein kinase activity of the protein. Another effect of this kinase activity is enhancing the parasite replication inside the host cells [99]. Previous studies have shown that GRA1, SAG1, SAG2, MIC1, MAG1, ROP18, GRA6, and GRA2 antigens are highly immunogenic. In addition, these antigens have been shown to stimulate specific antibodies in the host body or cytokines in vitro in the culture medium of splenocyte cells [6, 24, 29, 92]. ROP, GRA, and SAG antigens are the strongest candidates for the vaccine because they have been shown to contain relatively long antigenic fragments and regions, especially ROP, which appears to be a more suitable candidate than the other two antigens [6, 20]. It has also been shown that this antigen can elicit a strong protective immune response. DNA immunization of BALB/c mice with homogeneous mixtures of plasmids encoding short micronemic antigen fragments has been shown to enhance protective immunity, leading to an 85% reduction in the burden of T. gondii cysts [55].