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.     

Protective immune response against Toxoplasma gondii elicited by a recombinant DNA vaccine with a novel genetic adjuvant

Previous immunological studies from our laboratory have demonstrated the potential role of Toxoplasma gondii antigens SAG1 and GRA2 as vaccine candidates. To further evaluate the vaccine's effects, a series of recombinant DNA vaccines pVAX1-SAG1, pVAX1-GRA2 and pVAX1-SAG1-GRA2, termed pSAG1, pGRA2 and pSAG1-GRA2, respectively, were constructed. A plasmid pVAX1-S/PreS2, termed pSPreS2 encoding hepatitis B virus (HBV) surface antigen (HBsAg) S and PreS2 as a novel genetic adjuvant, was also constructed. The expression abilities of those DNA plasmids were examined in HFF cells by Western blotting. Then BALB/c mice were intramuscularly immunized with DNA plasmids and followed by challenging with the highly virulent T. gondii RH strain. The results demonstrated that the recombinant DNA vaccine pSAG1-GRA2 was capable of eliciting high levels of antibodies, a Th1 type of immune response with significant production of IFN-γ and low levels of IL-4 or IL-10 in BALB/c mice, and partial protection against the acute phase of toxoplasmosis as compared to pSAG1, pGRA2 and controls. In addition, the adjuvant pSPreS2 formulated with DNA vaccine induced a Th1 type of immune response and therefore might be a novel genetic adjuvant to DNA vaccine for further investigation.     

Toxoplasma gondii immune mapped protein-1 (TgIMP1) is a novel vaccine candidate against toxoplasmosis

Immune mapped protein1 (IMP1) is a new protective protein in apicomplexan parasites, and exists in Toxoplasma gondii. In the present study, a DNA vaccine expressing IMP1 of T. gondii was constructed and the immune response induced in BALB/c mice was evaluated. The coding sequence of IMP1 was inserted into the eukaryotic expression vector pcDNA 3.1(+), resulting a recombinant plasmid pcDNA-IMP1, which was used to immunize BALB/c mice intramuscularly. After immunization, the immune response was evaluated using lymphoproliferative assay, and cytokine and antibody measurements. The mice were challenged with tachyzoites of the virulent T. gondii RH strain 14th day after the last immunization to observe the survival time. The results showed that the group immunized with pcDNA-IMP1 developed a high level of specific antibody responses against Escherichia coli expressed recombinant TgIMP1, with high IgG antibody titers, predominance of IgG2a production, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production compared with the control groups. These results demonstrate that pcDNA-IMP1 could elicit strong humoral and Th1 immune responses. Immunized mice showed a significantly (15.8 ± 6 days) prolonged survival time compared with control mice, which died within 7 days of challenge infection. These results suggest that IMP1 is a promising vaccine candidate against toxoplasmosis.     

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.     

Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses

Recent studies have suggested that yeast cell wall components possess adjuvant activities. In the present study, heat-killed whole recombinant Hansenula polymorpha yeast expressing hepatitis B surface antigen (yeast-HBsAg) was generated, and the immune responses elicited by yeast-HBsAg were investigated in mice. The studies showed that yeast-HBsAg as well as yeast greatly promotes the accumulation of immune cells in mouse spleen and contributes to the maturation of dendritic cells (DCs). Yeast-HBsAg not only induces significantly higher antibody responses (including IgG, IgG1 and IgG2a), but also increases the IgG2a/IgG1 ratio, while alum combined with HBsAg (HBsAg + alum) only enhances antibody responses, but not the IgG2a/IgG1 ratio compared to HBsAg alone. Analysis of HBsAg-specific cytokines revealed that yeast-HBsAg is associated with production of both IFN-γ and IL-4, but neither IFN-γ nor IL-4 was detected in the HBsAg + alum-immunized group. Moreover, yeast-HBsAg induces potent HBsAg-specific lymphocyte proliferation and Cytotoxic T lymphocyte (CTL) responses. In conclusion, yeast-HBsAg enhances both HBsAg-specific Th1 and Th2 immune responses, while alum only enhances Th2 immune responses, suggesting that yeast-HBsAg may be an ideal candidate for an effective vaccine for the control of chronic hepatitis B virus (HBV) infection.     

Vaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals and birds, causing toxoplasmosis. Several types of vaccines against T. gondii have been developed, but these have limitations in terms of their safety and inadequate efficacy. T. gondii profilin (TgPF) is a potential immunodominant antigen for a candidate vaccine. In this study, we encapsulated TgPF in oligomannose-coated liposomes (OMLs) to evaluate the immune response induced by this vaccine. C57BL/6 mice were immunized with TgPF-OML three times at 14-day intervals and challenged with T. gondii. TgPF-OML increased the survival of the mice and reduced the parasite burden in their brains after T. gondii infection. Immunization with TgPF-OML also induced TgPF-specific interferon-γ production and IgG antibodies in mice. Our results demonstrate that OML-encapsulated TgPF triggers strong humoral and cellular responses against T. gondii, and that TgPF-OML is a candidate vaccine that warrants 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.     

MyD88-dependent protective immunity elicited by adenovirus 5 expressing the surface antigen 1 from Toxoplasma gondii is mediated by CD8(+) T lymphocytes

Toxoplasma gondii is an intracellular parasite widely spread around the world. The surface antigens (SAG) 1, 2 and 3 are the main proteins expressed on the surface of T. gondii tachyzoites. Replication-defective adenovirus serotype 5 (rAd5) is one of the most potent recombinant viral vectors for eliciting T cell-mediated immunity in mice and humans. Here we show that vaccination with rAd5 expressing SAG1 (AdSAG1), but neither SAG2 nor SAG3, induces protective immunity in the highly susceptible C57BL/6 mice challenged with T. gondii. Furthermore, we evaluated different immunological components involved on viral induced protective immunity. We observed that host protection elicited by AdSAG1 is highly dependent on IL-12, IFN-γ and CD8⁺ T lymphocytes. Importantly, the induction of protective immunity (T cell-derived IFN-γ) was also dependent on Myeloid Differentiation Factor 88 (MyD88), and thus, likely to involve Toll-like Receptors. We conclude that protective parasite specific-CD8⁺ T cells are elicited by a mechanism that involves MyD88-dependent induction of IL-12.     

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.     

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.     

Protection in mice immunized with a heterologous prime-boost regime using DNA and recombinant pseudorabies expressing TgSAG1 against Toxoplasma gondii challenge

An effective vaccine of animals can block transmission of Toxoplasma gondii to humans. In this study, mice have been protected against lethal T. gondii challenge by a prime-boost vaccination strategy using DNA vaccine pVAX/TgSAG1 and recombinant pseudorabies virus rPRV/TgSAG1, both expressing the major immunodominant surface antigen of T. gondii (TgSAG1). High levels of splenocyte proliferative responses and significant levels of IFN-γ resulted, with strong cytotoxic T lymphocyte (CTL) responses in vitro. After lethal challenge, prime-boost vaccinated mice showed an increased survival time (15.4 ± 5.0 days) and a 40% survival rate compared with controls who all died within 11 days of challenge. Results of the present study indicated that this novel immunization strategy is useful in enhancing immune protection in mice against lethal T. gondii infection, which would provide foundation for the development of effective vaccines against T. gondii.     

Production and Evaluation of Toxoplasma gondii Recombinant Surface Antigen 1 (SAG1) for Serodiagnosis of Acute and Chronic Toxoplasma Infection in Human Sera

Background

The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis.

Methods

This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii (RH Strain) was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D – thiogalactopyranoside (IPTG). A total of 204 sera were tested using a commercial IgG and IgM ELISA kit (Trinity, USA) as gold standard prior to testing them with the recombinant antigen.

Results

Tested sera were divided into the following groups:(a) The 74 T. gondii IgG positive (b) 70 T.gondii IgM positive (c) 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis (N=5), malaria (N=14), leishmaniasis (N=7),fasciolasis (N=4), sterengyloidiasis (N=1). Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (Com ELISA) were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively.

Conclusion

The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis.     

Construction of Neospora caninum stably expressing TgSAG1 and evaluation of its protective effects against Toxoplasma gondii infection in mice

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine.     

Identification of Setaria cervi heat shock protein70 by mass spectrometry and its evaluation as diagnostic marker for lymphatic filariasis

Using mass spectrometry and immunological approaches, a heat shock protein70 associated with lymphatic filariasis (LF) has been identified from a bovine filarial parasite Setaria cervi. A heat shock protein was detected in different life stages of S. cervi when exposed to an elevated temperature of 44 °C. A combination of ATP-agarose column chromatography and electro-elution was used for its purification from adult female extract. On closer examination, it migrated as a single band at 68 kDa on 10% SDS-PAGE. Peptide sequences TTPSYVAFTDTER, DSGAIAGLNVLR, IINEPTAAAIAYGLDK, NALESYAFNMK and LLSDFFSGK were obtained through MALDI-LC/MS analysis. Confirmation of peptides was accomplished by MASCOT database which showed substantial sequence homology with S. digitata, Wuchereria bancrofti, and Caenorhabditis elegans. Multiple sequence alignment using Clustal W showed 98% identity with W. bancrofti and only 28% with human HSP70. Furthermore, the antigenicity plot has shown that the highly antigenic amino acid residues are constituted within the conserved peptides. These observations suggest a plausible biological connection of ScHSP70 with the disease and its strong immunogenic nature. ScHSP70 showed antigenic cross-reactivity with IgG class of antibody in different categories of filarial sera. However, when IgG subclasses were tested, IgG4 showed high specificity and sensitivity with asymptomatic microfilaraemic sera.     

Protective immunity against Toxoplasma challenge in mice by coadministration of T. gondii antigens and Eimeria profilin-like protein as an adjuvant

Toll-like receptor (TLR) ligands are attractive adjuvant candidates in vaccine development. Eimeria tenella profilin-like protein has recently been shown to be a potent agonist of the innate immune system through its recognition by Toll-like receptor-11. In this report, we studied the systemic and mucosal adjuvant activity of Eimeria profilin-like protein within a vaccinal strategy against Toxoplasma gondii in mice. Using intraperitoneal (i.p.) immunization, we observed that coadministration of the recombinant Eimeria antigen (rEA) with T. gondii antigen (TAg) effectively elevates plasma levels of IL-12p70 and consequently induced both enhanced specific humoral and Th1 cellular immune responses. The co-administration of TAg plus rEA by i.p route significantly enhanced the protection against T. gondii infection (62% brain cyst reduction) in comparison with control mice and with mice immunized with TAg alone (only 36% brain cyst reduction). After intranasal immunization, humoral and cellular responses were weak. However mice immunized nasally with TAg plus rEA were significantly protected with 50% of brain cyst reduction, conversely TAg immunized mice did not present any brain cyst reduction.These results indicate that Eimeria profilin-like protein would serve as an efficacious systemic and mucosal adjuvant inducing protective immune response against chronical stage of T. gondii infection through TLR11 activation.     

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.     

Further analysis of protection induced by the MIC3 DNA vaccine against T. gondii: CD4 and CD8 T cells are the major effectors of the MIC3 DNA vaccine-induced protection,both Lectin-like and EGF-like domains of MIC3 conferred protection

The present study was conducted mainly to evaluate the contribution of the cellular and the humoral responses in protection conferred by the MIC3 DNA vaccine (pMIC3i) that was proved as a potent vaccine against toxoplasmosis. We performed the adoptive transfer of CD4⁺ and CD8⁺ T lymphocytes from pMIC3i immunized mice to naive ones and the role of humoral immunity was evaluated by in vitro invasion assays. We also constructed plasmids encoding the EGF-like domains and the Lectin-like domain of MIC3, to define which domains of MIC3 are involved in the protection. Furthermore, the adjuvant effect of the GM-CSF-expressing vector (granulocyte-macrophage colony-stimulating factor) required the precise temporal and spatial codelivery of GM-CSF with antigen, thus, we constructed a bicistronic plasmid expressing MIC3 and GM-CSF. In conclusion, the protection induced by pMIC3i was mainly mediated by CD4⁺ and CD8⁺ T lymphocytes and both EGF and Lectin domains of MIC3 conferred protection. Furthermore, the codelivery of GM-CSF by a bicistronic plasmid appeared to be a most effective way for enhancing the adjuvant properties of GM-CSF.     

Heat shock protein 70 from Trichinella spiralis induces protective immunity in BALB/c mice by activating dendritic cells

Trichinella spiralis heat shock protein 70 (Ts-Hsp70) is a protective antigen that induces partial protective immunity against T. spiralis infection in mice. To determine whether dendritic cells are involved in the mechanism responsible for the protection induced by Ts-Hsp70, mouse bone marrow-derived dendritic cells (DCs) were incubated with recombinant Ts-Hsp70 (rTs-Hsp70), and the DC-secreted cytokines and expressed surface markers were measured. The results demonstrated that rTs-Hsp70 activated DC maturation that was characterized by the secretion of IL-1β, IL-12p70, TNF-α, and IL-6 and the increased surface expression of CD11c, MHC II, CD40, CD80, and CD86. The rTs-Hsp70-activated DCs enabled the stimulation, proliferation and secretion of Th1/2 cytokines (i.e., INF-γ, IL-2, IL-4 and IL-6) in CD4⁺ T cells from T. spiralis-infected mice. The mice that received rTs-Hsp70-activated DCs exhibited a 38.4% reduction in muscle larvae upon larval challenge with T. spiralis compared to the group that received PBS-incubated DCs. This partial protection was correlated with Th1 and Th2 mixed anti-Ts-Hsp70-specific immune responses that included high titers of total IgG, IgG1 and IgG2a and increased levels of Th1/2 cytokines (i.e., IFN-γ, IL-2, IL-4, IL-6). These results indicate that the rTs-Hsp70-induced protective immunity was mediated by the activation of the DCs and that rTs-Hsp70-loaded DCs could be an alternative vaccine approach against trichinellosis.     

Synergy of mIL-21 and mIL-15 in enhancing DNA vaccine efficacy against acute and chronic Toxoplasma gondii infection in mice

The synergistic protective efficacy of murine interleukin 21 (mIL-21) and mIL-15 administrated with DNA vaccine against acute and chronic Toxoplasma gondii infection in mice was investigated using T. gondii MIC8 (TgMIC8) as a model. We cloned mIL-21 and mIL-15 from splenic tissues of Kunming mice, and constructed eukaryotic plasmid pVAX/mIL-15, pVAX/mIL-21, and pVAX/mIL-21/mIL-15, respectively. After immunizing with pVAX/TgMIC8 in the presence or absence of these cytokines, immune responses were analyzed using lymphoproliferative assay, cytokine and serum antibody measurements, flow cytometric surface markers on lymphocytes and protection against acute and chronic T. gondii infection. Mice receiving pVAX/TgMIC8 alone developed a strong humoral responses and Th1 type cellular immune responses, and showed an increase of CD4+ and CD8+ T cells compared with all the controls. Adding pVAX/mIL-21 to pVAX/TgMIC8 compared to pVAX/TgMIC8 resulted in only a slight increase in humoral and cellular immune responses, and this immune response was lower than that induced by the pVAX/mIL-15 combined with pVAX/TgMIC8. Co-administration of pVAX/mIL-21/mIL-15 combined with pVAX/TgMIC8 elicited the strongest humoral and cellular immune responses among all the groups, leading to significantly increased survival time against acute infection and the significant reduction of tissue cysts, compared to all the controls. Synergy of mIL-21 and mIL-15 can facilitate specific humoral as well as cellular immune responses elicited by DNA vaccine against acute and chronic T. gondii infection in mice.