Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis

Toxoplasma gondii, the pathogen of toxoplasmosis, can infect most mammals and birds. The high incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. We constructed a DNA cocktail, containing plasmids encoding the full-length SAG1 and ROP2 genes of T. gondii and evaluated its immune response and protective efficacy in comparison with single-gene vaccines and control groups. We immunized BALB/c mice intramuscularly three times. DNA cocktail elicited IgG and IFN-γ, TNF-α and IL-2 greater than single-gene plasmids and increased survival time against a lethal challenge with the highly virulent T. gondii RH strain. The current study shows that pc-SAG1+ pc-ROP2 as a cocktail DNA vaccine produces higher Th1 immune response than single-gene plasmids and cocktail DNA is effective to prime an enhanced and balanced specific immunity.     

Toxoplasma gondii: Vaccination with a DNA vaccine encoding T- and B-cell epitopes of SAG1, GRA2, GRA7 and ROP16 elicits protection against acute toxoplasmosis in mice

Toxoplasma gondii (T. gondii) is an obligate, intracellular, protozoan parasite that infects large variety of warm-blooded animals including humans, livestock, and marine mammals, and causes the disease toxoplasmosis. Although T. gondii infection rates differ significantly from country to country, it still has a high morbidity and mortality. In these circumstances, developing an effective vaccine against T. gondii is urgently needed for preventing and treating toxoplasmosis. The aim of this study was to construct a multi-epitopes DNA vaccine and evaluate the immune protective efficacy against acute toxoplasmosis in mice. Therefore, twelve T- and B-cell epitopes from SAG1, GRA2, GRA7 and ROP16 of T. gondii were predicted by bioinformatics analysis, and then a multi-epitopes DNA vaccine was constructed. Mice immunized with the multi-epitopes DNA vaccine gained higher levels of IgG titers and IgG2a subclass titers, significant production of gamma interferon (IFN-γ), percentage of T lymphocyte subsets, and longer survival times against the acute infection of T. gondii compared with those of mice administered with empty plasmid and those in control groups. Furthermore, a genetic adjuvant pEGFP-RANTES (pRANTES) could enhance the efficacy of the multi-epitopes DNA vaccine associating with humoral and cellular (Th1, CD8⁺ T cell) immune responses. Above all, the DNA vaccine and the genetic adjuvant revealed in this study might be new candidates for further vaccine development against T. gondii infection.     

Evaluation of Immunogenicity of Cocktail DNA Vaccine Containing Plasmids Encoding Complete GRA5, SAG1, and ROP2 Antigens of Toxoplasma gondii in BALB/C Mice

Background:

Severe and fatal complications of toxoplasmosis urge development of effective vaccines against the disease. The current study was performed to evaluate cocktail DNA vaccine containing plasmids encoding GRA5, SAG1, and ROP2 genes of Toxoplasma gondii in BALB/c mice in Tarbiat Modares University in 2012.

Methods:

The plasmids containing complete GRA5, SAG1, and ROP2 genes were mass extracted and then the recombinant plasmids were administered via intramuscular injections according to immunized mice three times with three-week intervals. Then splenocytes were cultured, and proliferation as well as cytokine assays were carried out. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis.

Results:

The results of cytokine assay for INF-γ were higher in the mice that received the cocktail DNA containing recombinant plasmids. Evaluation of proliferation of splenocytes using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay indicated induction of cellular response. Measurement of total IgG and the isotypes of IgG1 and IgG2a showed that the cocktail DNA stimulated IgG and IgG2a production in comparison with the control groups (P<0.05). Furthermore, the survival rate of mice in the groups that received the cocktail DNA was significantly higher than that in the control groups (P<0.05).

Conclusion:

Administration of the cocktail DNA vaccine led to production of higher levels of IFN-γ, confirmed by secretion of IgG2a, and the immune response was shifted toward Th1. Thus, the cocktail DNA containing the recombinant plasmids can be an appropriate candidate for immunization against toxoplasmosis.     

Toxoplasma gondii microneme protein 6 (MIC6) is a potential vaccine candidate against toxoplasmosis in mice

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. Microneme proteins which are responsible for adhesion and invasion have been implicated as vaccine candidates. In this study, we constructed a DNA vaccine expressing microneme protein 6 (MIC6) of T. gondii, and evaluated the immune response it induced in Kunming mice. The gene sequence encoding MIC6 was inserted into the eukaryotic expression vector pVAXI. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the group immunized with pVAX-MIC6 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or phosphate-buffered saline, respectively. These results demonstrate that pVAX-MIC6 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with the pVAX-MIC6 showed an increased survival time (13.3 ± 1.2 days) compared with control mice died within 7 days of challenge. Our data demonstrate, for the first time, that MIC6 triggered a strong humoral and cellular response against T. gondii, and that the antigen is a potential vaccine candidate against toxoplasmosis, worth further development.     

Protective immunity induced by a DNA vaccine expressing eIF4A of Toxoplasma gondii against acute toxoplasmosis in mice

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting humans, mammals and birds. Eukaryotic translation initiation factor (eIF4A) is a newly identified protein associated with tachyzoite virulence. To evaluate the protective efficacy of T. gondii eIF4A, a DNA vaccine (pVAX-eIF4A) encoding T. gondii eIF4A (Tg-eIF4A) gene was constructed. The expression ability of this recombinant DNA plasmid was examined in Marc145 cells by IFA. Then, Kunming mice were intramuscularly immunized with pVAX-eIF4A and followed by challenge infection with the highly virulent T. gondii RH strain. The results showed that vaccination with pVAX-eIF4A elicited specific humoral responses, with high IgG antibody titers and specific lymphocyte proliferative responses. The cellular immune response was associated with significant production of IFN-γ, IL-2 in Kunming mice, and a mixed IgG1/IgG2a response with predominance of IgG2a production, indicating that a Th1 type response was elicited after immunization with pVAX-eIF4A. In addition, the increase of the percentage of CD8+ T cells in lymphoid in mice suggested the activation of MHC class I restricted antigen presentation pathways. After lethal challenge, the mice vaccinated with the pVAX-eIF4A showed a significantly prolonged survival time (23.0 ± 5.5 days) compared with control mice which died within 7 days of challenge (P < 0.05). These results demonstrate that pVAX-eIF4A could elicit strong humoral, Th1-type cellular immune responses and increase survival time of immunized mice, suggesting that eIF4A is a promising vaccine candidate against acute T. gondii infection in mice.     

The virulence-related rhoptry protein 5 (ROP5) of Toxoplasma Gondii is a novel vaccine candidate against toxoplasmosis in mice

Infections with the intracellular protozoan parasite Toxoplasma gondii pose a serious public health problem and are of great economic importance worldwide. The parasite rhoptry protein 5 (ROP5) has been implicated as a major virulence factor that reduces the accumulation of immunity-related GTPases (IRG) in parasitophorous vacuole membrane (PVM), which maintains PVM integrity and evades IFNγ-mediated killing by intracellular parasites. To study the immunoprotective value of ROP5, BALB/c mice were immunized with a recombinant form of the protein administered alone or in combination with another promising vaccine antigen, rSAG1. All mice vaccinated with the recombinant antigens developed a high level of specific antibody responses against soluble tachyzoite antigens (STAg), a statistically significant increase of the splenocyte proliferation response, and significant levels of IFN-γ and IL-2 production. In contrast to rSAG1, which only stimulated the release of IFN-γ and IL-2, rROP5 induced the specific production of IL-10, the Th2-type cytokine, in addition to IFN-γ and IL-2. These results demonstrated that rROP5 could induce significant cellular and humoral (Th1/Th2) immune responses. Moreover, mice immunized with rROP5 displayed a prolonged survival time against a lethal challenge with the T. gondii RH strain. Additionally, vaccination with the mixture of rROP5 + rSAG1 resulted in higher levels of T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge compared to immunization with rROP5 or rSAG1 alone. Our studies show that recombinant ROP5 antigen may be a promising vaccine candidate against toxoplasmosis. To our knowledge, this is the first report to evaluate the immunoprotective value of ROP5.     

Protective effect against toxoplasmosis in mice induced by DNA immunization with gene encoding Toxoplasma gondii ROP18

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting mammals and birds including humans. Rhoptry protein 18 has been implicated as an important virulence factor. In this study, we constructed a DNA vaccine expressing rhoptry protein 18 (ROP18) of T. gondii, and evaluated the immune response and protective immunity in Kunming mice. The gene sequence encoding ROP18 was inserted into the eukaryotic expression vector pVAX I. Intramuscular immunization of mice with pVAX-ROP18 elicited specific humoral responses and stimulated lymphoproliferation (P < 0.05). The cellular immune response was associated with the production of IFN-γ, indicating that a Th1 type response was elicited, which was confirmed by the production of large amounts of IgG2a (P < 0.05). By the expression of the CD69, an activation marker of CD4+ and CD8+ T cells, we found that pVAX-ROP18 enhanced the activation of CD4+ and CD8+ T cells in lymphoid in mice. After lethal challenge, the mice immunized with the pVAX-ROP18 showed a significantly increased survival time (27.9 ± 15.1 days) compared with control mice which died within 7 days of challenge (P < 0.05). Our results show for the first time, that a ROP18 vaccine construct can enhance the T. gondii-specific CTL. Th1 responses and increased survival suggested that ROP18 is a promising vaccine candidate against infection with T. gondii.     

Th1-type epitopes-based cocktail PDDV attenuates hepatic fibrosis in C57BL/6 mice with chronic Schistosoma japonicum infection

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, which is characterized by a marked egg-induced CD4⁺ T-cell programmed granulomatous inflammation and cumulative fibrosis. Here PDDV (peptide–DNA dual vaccine), a widely used non-viral gene delivery system, was applied. The cocktail PDDV, based on four Th1-type epitope peptides identified from Schistosoma japonicum vaccine candidates and CpG ODN1826, could induce dominant Th1-type response in C57BL/6J mice (P < 0.05). The histopathological staging and collagen assessment for fibrosis showed that the cocktail PDDV presented an obvious down-regulation effect on hepatic fibrosis caused by chronic S. japonicum infection (P < 0.05), and IFN-γ, IL-4 and IL-13 mRNAs in liver detected by RT-PCR also showed that the cocktail PDDV represented the ability to up-regulate Th1-type responses, which paralleled with a decrease expression of α-SMA (P < 0.05) and the up-regulated MMP9/TIMP1 balance (P < 0.05) when compared to the control groups. Therefore, it is indicated that the cocktail PDDV can significantly attenuate hepatic fibrosis, in parallel with the decreased HSCs activation and the up-regulated MMP9/TIMP1 balance in favor of matrix degradation, which may be partially dependent on the increased Th1 response to restore the Th1/Th2 balance.     

Protective and antifecundity effects of Sm-p80-based DNA vaccine formulation against Schistosoma mansoni in a nonhuman primate model

Schistosomiasis is an important parasitic disease for which there is no available vaccine. We have focused on a functionally important antigen of Schistosoma mansoni, Sm-p80, as a vaccine candidate because of its consistent immunogenicity, protective potential and antifecundity effect observed in murine models; and for its pivotal role in the immune evasion process. In the present study we report that an Sm-p80-based DNA vaccine formulation confers 38% reduction in worm burden in a nonhuman primate model, the baboon (Papio anubis). Animals immunized with Sm-p80–pcDNA3 exhibited a decrease in egg production by 32%. Sm-p80 DNA elicited specific immune responses that include IgG; its subtypes IgG1 and IgG2; and IgM in vaccinated animals. Peripheral blood mononuclear cells (PBMCs) from immunized animals when stimulated in vitro with Sm-p80 produced appreciably more Th1 response enhancing cytokines (IL-2, IFN-γ) than Th2 response enhancing cytokines (IL-4, IL-10). PBMCs produced appreciably more spot-forming units for INF-γ than for IL-4 in enzyme-linked immunosorbent spot (ELISPOT) assays. Overall it appears that even though a mixed (Th1/Th2) type of humoral antibody response was generated following immunization with Sm-p80; the dominant protective immune response is Th1 type. These data reinforce the potential of Sm-p80 as an excellent vaccine candidate for schistosomiasis.     

Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant

Riemerella anatipestifer, a Gram-negative bacillus, causes septicemia that can result in high mortality for ducklings. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant outer membrane protein A (rOmpA) and plasmid constructs containing CpG oligodeoxynucleotides (ODN). Results showed that CpG ODN enhanced both humoral and cell-mediated immunity elicited by rOmpA as early as two weeks after primary immunization. When compared to ducks immunized with rOmpA, ducks immunized with rOmpA + CpG ODN showed higher levels (p < 0.05) of antibody titer, T cell proliferation, and percentages of CD4⁺ and CD8⁺ T cell in peripheral blood mononuclear cells (PBMCs). The relative fold inductions of mRNA expression of Th1-type (IFN-γ and IL-12), and Th2-type (IL-6) cytokines in PBMCs isolated from ducks immunized with rOmpA + CpG ODN were significantly higher than those of the rOmpA group. Homologous challenge result showed that the rOmpA + CpG ODN vaccine reduced the pathological score by 90% in comparison with the saline control. In conclusion, our study found that CpG ODN can enhance both humoral and cellular immunity elicited by a rOmpA vaccine. The rOmpA + CpG ODN vaccine can be further developed as a subunit vaccine against R. anatipestifer.     

Construction and immunogenicity of pseudotype baculovirus expressing Toxoplasma gondii SAG1 protein in BALB/c mice model

Toxoplasma gondii is a protozoan parasite causing toxoplasmosis to almost one-third of population all over the world. One of the most efficient ways to control this disease is immunization. However, so far, there is no effective vaccine available against this pathogen. Recently, a baculovirus pseudotype with vesicular stomatitis virus G protein (Bac-VSV–G) was found to efficiently transduce and express transgenes on mammalian cells, so it was considered as an excellent expressing vector. In this study, the value of Bac-VSV–G in delivering T. gondii antigen was investigated. T. gondii SAG1 gene was cloned into Bac-VSV–G, and recombinant baculovirus BV-G-SAG1 was obtained. Indirect immunofluorescence test showed BV-G-SAG1 was efficiently transduced and expressed in pig kidney cells. Then BALB/c mice were immunized with BV-G-SAG1 at different doses (1 × 10⁸, 1 × 10⁹, and 1 × 10¹⁰ PFU/mouse) and challenged with T. gondii RH strain tachyzoites after immunization. The levels of specific T. gondii antibody, interferon (IFN)-γ, IL-4, IL-10 expression and release, and the survival rate of treated mice were evaluated. Compared with the mice immunized with DNA vaccine (pcDNA/SAG1) encoding the same gene, BV-G-SAG1 induced higher levels of specific T. gondii antibody and (IFN)-γ expression with dose-dependent manner and the survival rate of mice with BV-G-SAG1 was significantly improved. These results indicated that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop new generation of vaccines against T. gondii infection.     

Characterization of a multi-epitope peptide with selective MHC-binding capabilities encapsulated in PLGA nanoparticles as a novel vaccine candidate against Toxoplasma gondii infection

No effective human vaccine against Toxoplasma gondii (T. gondii) has yet been developed; however, a protective vaccine using immunogenic peptides in a safe delivery vehicle system offers promise. Here, we employed bioinformatics to design a multimeric recombinant T. gondii vaccine using predicted T and B cell epitopes of SAG1, AMA1, ROP2, and GRA4 proteins based on their binding capabilities to common major histocompatibility complex (MHC) molecules. Furthermore, we encapsulated the expressed protein in poly lactic-co-glycolic acid (PLGA) nanoparticles as a delivery vehicle and also used alum as an adjuvant to determine the vaccine potency of this multimeric antigen. BALB/c mice were vaccinated and then challenged with T. gondii RH strain, and the survival rate and cytokine profiles were studied. Mice vaccinated with the multi-epitope-based vaccine, both with and without PLGA, had greater Th1 immune responses, survival rates, specific antibody titers, and IFN-γ and IL-2 levels than controls, while the alum-adsorbed vaccine stimulated a Th2-type humoral immune response.     

Evaluation of protective effect of pVAX-TgMIC13 plasmid against acute and chronic Toxoplasma gondii infection in a murine model

Toxoplasma gondii is a significant zoonotic parasite which can cause congenital infection and abortion in warm-blooded animals and humans. Microneme protein 13 (MIC13) plays an important role in attachment and penetration of the host cell by T. gondii. In this study, a DNA vaccine expressing mic13 of T. gondii was constructed and its protective efficacy was evaluated in Kunming L615^H2k mice. Immunization with pVAX-TgMIC13 induced a strong immune responses demonstrated by significant lymphocyte proliferation, cytokine production and antibody responses. Immunized mice showed increased survival time (21.3 ± 11.3 days) and reduced number of cysts in brain of mice (57.14%) after challenge with tachyzoites of the virulent T. gondii RH strain and cysts of the T. gondii PRU strain, respectively, demonstrating that T. gondii MIC13 is a potential vaccine candidate, worth being included in future vaccine development against acute and chronic T. gondii infection.     

Identification of T. gondii epitopes,adjuvants, and host genetic factors that influence protection of mice and humans

Toxoplasma gondii is an intracellular parasite that causes severe neurologic and ocular disease in immune-compromised and congenitally infected individuals. There is no vaccine protective against human toxoplasmosis. Herein, immunization of L^d mice with HF10 (HPGSVNEFDF) with palmitic acid moieties or a monophosphoryl lipid A derivative elicited potent IFN-γ production from L^d-restricted CD8⁺ T cells in vitro and protected mice. CD8⁺ T cell peptide epitopes from T. gondii dense granule proteins GRA 3, 6, 7, and Sag 1, immunogenic in humans for HLA-A02⁺, HLA-A03⁺, and HLA-B07⁺ cells were identified. Since peptide repertoire presented by MHC class I molecules to CD8⁺ T cells is shaped by endoplasmic reticulum-associated aminopeptidase (ERAAP), polymorphisms in the human ERAAP gene ERAP1 were studied and associate with susceptibility to human congenital toxoplasmosis (p < 0.05). These results have important implications for vaccine development.     

Candida skin test reagent as a novel adjuvant for a human papillomavirus peptide-based therapeutic vaccine

A vaccine adjuvant that can effectively promote cell-mediated immunity is currently not available. Because of the ability of a Candida skin test reagent injection to induce common wart regression, our group is using it as a novel adjuvant in a clinical trial of a peptide-based human papillomavirus therapeutic vaccine. The goal of this current study was to investigate the mechanisms of how Candida enhances the vaccine immune responses. Maturation effects on Langerhans cells, capacity to proliferate T-cells, expression of cytokines and pattern recognition receptors by Langerhans cells, and ability to induce Th1, Th2, and Th17 responses were investigated in healthy subjects. The vaccine, human papillomavirus peptides with Candida, demonstrated partial maturation effects on Langerhans cells indicated by significantly up-regulated CD40 (p = 0.00007) and CD80 (p < 0.00001) levels, and showed T-cell proliferative capacity (p < 0.00001) when presented by Langerhans cells in vitro. Interestingly, the maturation effects were due to the peptides while Candida was responsible for the T-cell proliferation. The cytokine profile (IL-1β, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ and TNF-α) of Langerhans cells treated with the vaccine or Candida alone showed that IL-12p40 mRNA was most frequently induced, and IL-12p70 protein was detected in the supernatants. The presence of pattern recognition receptors known to associate with Candida albicans (DC-SIGN, dectin-1, dectin-2, galectin-3, mincle, mannose receptor, Toll-like receptors-1, 2, 4, 6 and 9) were demonstrated in all subjects. On the other hand, the induction of Th1 response demonstrated by IFN-γ secretion by CD4 cells stimulated with the vaccine or Candida pulsed Langerhans cells was demonstrated only in one subject. In summary, the Langerhans cell maturation effects of the vaccine were due to the peptides while the T-cell proliferative capacity was derived from Candida, and the most frequently induced cytokine was IL-12.     

Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II

The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P < 0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II + alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.     

A novel gonorrhea vaccine composed of MetQ lipoprotein formulated with CpG shortens experimental murine infection

Bacterial surface lipoproteins are emerging as attractive vaccine candidates due to their biological importance and the feasibility of their large-scale production for vaccine manufacturing. The global prevalence of gonorrhea, resistance to antibiotics, and serious consequences to reproductive and neonatal health necessitate development of effective vaccines. Reverse vaccinology identified the surface-displayed L-methionine binding lipoprotein MetQ (NGO2139) and its homolog GNA1946 (NMB1946) as gonococcal and meningococcal vaccine candidates, respectively. Here, we assessed the suitability of MetQ for inclusion in a gonorrhea vaccine by examining MetQ conservation, its function in Neisseria gonorrhoeae (Ng) pathogenesis, and its ability to induce protective immune responses using a female murine model of lower genital tract infection. In-depth bioinformatics, phylogenetics and mapping the most prevalent Ng polymorphic amino acids to the GNA1946 crystal structure revealed remarkable MetQ conservation: ~97% Ng isolates worldwide possess a single MetQ variant. Mice immunized with rMetQ-CpG (n = 40), a vaccine containing a tag-free version of MetQ formulated with CpG, exhibited robust, antigen-specific antibody responses in serum and at the vaginal mucosae including IgA. Consistent with the activity of CpG as a Th1-stimulating adjuvant, the serum IgG1/IgG2a ratio of 0.38 suggested a Th1 bias. Combined data from two independent challenge experiments demonstrated that rMetQ-CpG immunized mice cleared infection faster than control animals (vehicle, p < 0.0001; CpG, p = 0.002) and had lower Ng burden (vehicle, p = 0.03; CpG, p < 0.0001). We conclude rMetQ-CpG induces a protective immune response that accelerates bacterial clearance from the murine lower genital tract and represents an attractive component of a gonorrhea subunit vaccine.     

Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis

The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response against RON4 could lead to more encouraging results.     

DNA vaccine encoding the Toxoplasma gondii bradyzoite-specific surface antigens SAG2CDX protect BALB/c mice against type II parasite infection

The surface antigens SAG2C, SAG2D, and SAG2X, which expressed specifically on bradyzoite stage of Toxoplasma gondii, have been demonstrated to be important for persistence of cyst in the brain. In this study, DNA vaccines expressing SAG2C, SAG2D, and SAG2X of T. gondii were constructed and their protective efficacy were evaluated in BALB/c mice. Mice vaccinated with pVAX1-SAG2C (pSAG2C), pVAX1-2D (pSAG2D) or pVAX1-2X (pSAG2C) showed higher levels of serum IgG antibodies and lymphocyte proliferation response compared to PBS and pVAX1 treated mice (p < 0.05). The immune response was characterized by a strong Th1 response and increased cytokine production of IL-2 and IFN-γ. Vaccinated mice displayed significant protection against the challenge with the cyst of T. gondii genotype II strain of PRU (cyst-forming in mouse). A significant reduction in the brain cyst burden was detected in the mice immunized with pSAG2C (72%), pSAG2D (23%), pSAG2X (69%) alone and even more reduction rate, 77%, was achieved in the combination group compared to PBS treated mice. The results implied that immunization with DNA vaccines expressing SAG2C, SAG2D, and SAG2X, and, in particular, a combination of all three DNA plasmids, could effectively protect the mice against T. gondii chronic infection.