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Toxoplasmosis is a significant global zoonosis with devastating impacts, and an effective vaccine against toxoplasmosis for humans has not yet been developed. In this study, we designed and formulated a novel DNA vaccine encoding the inhibitor of STAT1 transcriptional activity (IST) of T. gondii utilizing the eukaryotic expression vector pEGFP-N1 for the first time, with CL264 being a molecular adjuvant. Following intramuscular injection of the vaccine into mice, the levels of antibodies and cytokines were assessed to evaluate the immune response. Additionally, mice were challenged with highly virulent RH-strain tachyzoites of T. gondii, and their survival time was observed. The results show that the levels of IgG in serum, the ratio of IgG2a/IgG1 and the levels of IFN-γ in splenocytes of mice were significantly higher in the pEGFP-TgIST group and the pEGFP-TgIST + CL264 group than in the control group. In addition, the proportion of CD4+/CD8+ T cells was higher in mice immunized with either the pEGFP-TgIST group (p < 0.001) or the pEGFP-TgIST + CL264 group (p < 0.05) compared to the three control groups. Notably, TgIST-immunized mice exhibited prolonged survival times after T. gondii RH strain infection (p < 0.05). Our findings collectively demonstrate that the TgIST DNA vaccine elicits a significant humoral and cellular immune response and offers partial protection against acute T. gondii infection in the immunized mice, which suggests that TgIST holds potential as a candidate for further development as a DNA vaccine. [ABSTRACT FROM AUTHOR]

Abstract Background Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. Methods In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×104 tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. Results Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P4 tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P Conclusions The study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.

There is no effective protective vaccine against human toxoplasmosis, which is a potential threat to nearly a third of the world population. Vaccines based on virus-like particles (VLPs) have been highly successful in humans for many years, but have rarely been applied against Toxoplasma gondii infection. In this study, we inserted a B cell epitope (SAG182−102 or SAG1301−320), a CD8+ cell epitope (HF10 or ROP7), and a CD4+ cell epitope (AS15) of T. gondii into a truncated HBcΔ(amino acids1–149) particle to construct four chimeric VLP vaccine formulations, i.e., HBcΔH82, HBcΔH301, HBcΔ R82, and HBcΔ R301. When these chimeric HBc particles were expressed in Escherichia coli , they showed icosahedral morphology similar to that of the original VLPs and were evaluated as vaccine formulations against acute and chronic toxoplasmosis in a mouse model (BALB/c mice (H-2d). All these chimeric HBc VLPs induced strong humoral and cellular immune responses with high IgG antibody titers and interferon(IFN)-γ production. Only the mice immunized with HBcΔH82 showed prolonged survival time (15.6 ± 3.8 vs. 5.6 ± 0.8 days) against acute infection with RH tachyzoites and decrease in brain parasite load (1,454 ± 239 vs. 2,091 ± 263) against chronic infection with Prugniuad cysts, as compared to the findings for the control group. These findings suggest that HBc VLPs would act as an effective carrier for delivering effective multiple antigenic epitopes and would be beneficial for developing a safe and long-acting vaccine against toxoplasmosis. [ABSTRACT FROM AUTHOR]

Multiple antigenic peptide (MAP) vaccines have advantages over traditional Toxoplasma gondii vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both in vitro, via cell culture, and through in vivo mouse immunization. In vitro, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). In vivo, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against T. gondii. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is defined as an obligate intracellular apicomplexan parasite and influences approximatelyone-third of the human all over the world. ROP54 protein is expressed in the rhoptry of Toxoplasma gondii. In the present study, we used SMART software to analyzethe secondary structure of ROP54. The 3D model of ROP54 protein was constructed and analyzed using SWISS-MODEL server and VMD software. The structure results fully showed that ROP54 proteinis an importantmember from the ROP family. Moreover, DNAMAN software and Epitope Database online service were used to analyze liner-B cell epitopes and Th-cell epitopes of the protein. The bioinformatics prediction of ROP54 protein could provide positive information on treatment and vaccine for toxoplasmosis. Furthermore, ROP54 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-ROP54) was constructed in the following study. After identification of enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result suggested that the recombinant plasmid could transcribe successfully in HEK 293-T cell. [ABSTRACT FROM AUTHOR]

Background: Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects all warm-blooded animals including humans and causes toxoplasmosis. An effective vaccine could be an ideal choice for preventing and controlling toxoplasmosis. T. gondii Superoxide dismutase (TgSOD) might participate in affecting the intracellular growth of both bradyzoite and tachyzoite forms. In the present study, the TgSOD gene was used to construct a DNA vaccine (pEGFP-SOD).Methods: TgSOD gene was amplified and inserted into eukaryotic vector pEGFP-C1 and formed the DNA vaccine pEGFP-SOD. Then the BALB/c mice were immunized intramuscularly with the DNA vaccine and those injected with pEGFP-C1, PBS or nothing were treated as controls. Four weeks after the last immunization, all mouse groups followed by challenging intraperitoneally with tachyzoites of T. gondii ME49 strain.Results: Results showed higher levels of total IgG, IgG2α in the sera and interferon gamma (IFN-γ) in the splenocytes from pEGFP-SOD inoculated mice than those unvaccinated, or inoculated with either empty plasmid vector or PBS. The proportions of CD4+ T cells and CD8+ T cells in the spleen from pEGFP-SOD inoculated mice were significantly (p < 0.05) increased compared to control groups. In addition, the survival time of mice immunized with pEGFP-SOD was significantly prolonged as compared to the controls (p < 0.05) although all the mice died.Conclusion: The present study revealed that the DNA vaccine triggered strong humoral and cellular immune responses, and aroused partial protective immunity against acute T. gondii infection in BALB/c mice. The collective data suggests the SOD may be a potential vaccine candidate for further development. [ABSTRACT FROM AUTHOR]

Background: Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with a broad host range including most warm-blooded animals, including humans. T. gondii surface antigen 1 (SAG1) is a well-characterized T. gondii antigen. T. gondii expresses five nonmitochondrial rhomboid intramembrane proteases, TgROM1-5. TgROM4 is uniformly distributed on the surface of T. gondii and involved in regulating MIC2, MIC3, MIC6, and AMA1 during T. gondii invasion of host cells. Bioinformatics have predicted ROM4 B-cell and T-cell epitopes. Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing attention in T. gondii vaccine research. In this study, we used a DNA prime-peptide boost vaccination regimen to assess the protective efficacy of various vaccination strategies using TgROM4.Methods: We identified a polypeptide (YALLGALIPYCVEYWKSIPR) using a bioinformatics approach, and immunized mice using a DNA-prime and polypeptide-boost regimen. BALB/c mice were randomly divided into six groups, including three experimental groups (peptide, pROM4 and pROM4/peptide) and three control groups (PBS, pEGFP-C1 and pSAG1). Mice were then immunized intramuscularly four times. After immunization, IgG and cytokine productions were determined using enzyme-linked immunosorbent assays. The survival time of mice was evaluated after challenge with tachyzoites of T. gondii RH strain. Additionally, the number of cysts in the brain was determined after intragastric challenge with cysts of T. gondii PRU strain.Results: Mice vaccinated with different immunization regimens (peptide, pROM4 and pROM4/peptide) elicited specific humoral and cellular responses, with high levels of IgG, IgG2a, and interferon (IFN)-γ. Moreover, IgG, IgG2a and IFN-γ levels were highest in the pROM4/peptide group. Immunized mice, especially those in the pROM4/peptide group, had prolonged survival times after challenge with tachyzoites and reduced numbers of brain cysts after infection compared with negative controls.Conclusion: A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among immunization regimens, and may be a promising approach to increase the efficacy of DNA immunization. [ABSTRACT FROM AUTHOR]

Background: A widely prevalent disease, toxoplasmosis poses serious health threats to both humans and animals; therefore, development of an ideal DNA vaccine against Toxoplasma gondii is needed eagerly. The purpose of the present study is to assess the protective efficacy of a DNA vaccine encoding the T. gondii toxofilin gene (pEGFP-toxofilin). In addition, toxofilin DNA vaccine combined with the individual adjuvants, alum or monophosphoryl lipid A (MPLA), or a mixture of alum-MPLA adjuvant were screened for their ability to enhance antibody responses.Methods: Using bioinformatics, we analyzed the gene and amino acid sequences of the toxofilin protein, recognizing and identifying several potential linear B and T helper (Th)-1 cell epitopes. BALB/c mice were immunized three times with either toxofilin DNA vaccine alone or in combination with the adjuvants such as alum, MPLA or an alum-MPLA mixture. The systemic immune response was evaluated by cytokine, the percentage of CD4 (+) and CD8 (+) T cells and specific antibody measurement. Two weeks after the last immunization, protective efficacy was evaluated by challenging intraperitoneally with 1 × 104 tachyzoites of T. gondii or intragastrically with 20 cysts of T. gondii PRU strain.Results: All experimentally immunized mice developed strong humoral and cellular immune responses compared with the control groups. Moreover, by comparison with the non-adjuvant toxofilin DNA vaccine group, adding alum adjuvant to toxofilin DNA vaccine resulted in an increase in humoral response and a skewed Th2 response. However, the MPLA adjuvant with toxofilin DNA vaccine induced significantly enhanced humoral and Th1-biased immune responses. Importantly, the co-administration of alum-MPLA adjuvant in combination with the toxofilin DNA vaccine shifted the Th2 immune response to a Th1 response compared with the alum-toxofilin group, and elicited the strongest humoral and Th1 responses among all the groups. At the same time, a longer survival time and less cyst amounts against T. gondii infection were also observed in the alum-MPLA-toxofilin group in comparison with single or no adjuvant groups.Conclusions: Toxoplasma gondii toxofilin is a promising vaccine candidate that warrants further development. Co-administration of the alum-MPLA adjuvant mixture with DNA vaccine could effectively enhance immunogenicity and strongly skew the cellular immune response towards a Th1 phenotype. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an obligate intracellular apicomplexan parasite, and can infect warmblooded animals and humans all over the world. In the past years, ROP family genes encoding particular proteins of T. gondii had made a great contribution to toxoplasmosis. In this study, we used multiple bioinformatics approaches to predict the physical and chemical characteristics, transmembrane domain, epitope, and topological structure of the rhoptry protein 48 (ROP48). The results indicated that ROP48 protein was mainly located in the membrane and had several positive linear-B cell epitopes and Th-cell epitopes, which suggested that ROP48 is a potential DNA vaccine candidate against toxoplasmosis. Then the PCR product amplified from the ROP48 cDNA was inserted into a pEASY-T1 vector to build a recombinant cloning plasmid. After sequencing, ROP48 was subcloned into a eukaryotic expression plasmid pEGFP-C1 to obtain pEGFP-C1-ROP48 (pROP48). After identification by PCR and restriction enzyme digestion, the recombinant plasmid pROP48 was transfected into HEK 293-T cell and identified by RT-PCR. The results showed that the eukaryotic expression plasmid pROP48 was constructed and transfected to the cells of HEK 293-T successfully. Western blotting showed that the expressed proteins can be recognized by anti-STAg mouse sera. [ABSTRACT FROM AUTHOR]

Abstract: Toxoplasma gondii is an obligate intracellular protozoan that infects mammals and birds. Human infection during pregnancy may cause severe damage to the fetus. Reactivation of latent infection in immunocompromised patients can cause life-threatening encephalitis. T. gondii strains are highly diverse but only a few lineages (Type I, II and III) are widely spread. In mouse model, Type I strains are highly virulent, whereas Type II and III strains are intermediately or non virulent. It is not clear how much quantitative difference exists in proteomic profiles among these distinct T. gondii lineages. In the present study, the proteomic profiles of T. gondii tachyzoites from these lineages were investigated by two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) technologies. A total of 2321 protein spots were detected. Overall, the GT1 strain of Type I lineage and the strain PTG of Type II lineage have highly similar proteomic profiles and both are different from that of the CTG strain of Type III lineage. Eighty-four protein spots were differentially expressed by greater than 1.5-fold in relative abundance and 10 of them were identified to 7 T. gondii proteins in existing database. Investigation of the quantitative differences in proteomics among distinct T. gondii strains should facilitate our understanding of difference in biological processes and pathogenesis of distinct T. gondii genotypes, which will provide basic information to determine treatment regimen for different manifestation of toxoplasmosis. [Copyright &y& Elsevier]

Abstract: A fast, sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of Toxoplasma gondii (T. gondii) in pork was developed. In this study, we used a conserved sequence of 18s rRNA of Toxoplasma gondii to design primers for RT-LAMP test. The amplication was able to finish in 60min under isothermal condition at 63°C by employing a set of six primers. The assay showed higher sensitivity than RT-PCR using T. gondii RNA as template. The RT-LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. Furthermore, the assay correctly detected T. gondii from contaminated pork, and had the detect limit of 1 tachyzoite in 1g pork. This is the first report of a study which applied the RT-LAMP method to detect T. gondii from pork. As RT-LAMP requires very basic instruments and the results can be obtained by visual observation, this technique provides a simple and reliable tool for inspecting food which are T. gondii-contaminated. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Highlights • Mice showed neurobehavioral disturbances during the behavioral testing after T. gondii infection. • Histopathology revealed the activation of astrocytes and microglia, apoptosis of neurons and decreases in synapses in the brain sections of T. gondii infected mice. • Activation of NF-κB signaling pathways induced by the over productions of cytokines and chemokines were detected in the brain sections. • Excessive activation of inflammation in the brain and dysregulation of the neurotransmitters could potentially explain the neurobehavioral disorders in the infected hosts. Abstract Toxoplasma gondii is a protozoan parasite that can cause a latent infection in the central nervous system, leading to neurobehavioral abnormalities in the host. However, the mechanism underlying these changes remains relatively unexplored. In this study, we detected behavioral changes, pathological injury, secretion of neurotransmitters and related signal pathway in mice infected by T. gondii using behavioral test, histopathology, immunofluorescence staining, western blotting, HPLC and real time PCR. Mice showed neurobehavioral disturbances two months after infection with T. gondii. Histopathology revealed the activation of astrocytes and microglia, apoptosis of neurons and decreases in synapses in the brain of infected mice. Excessive secretion of cytokines and chemokines was detected in the brains of mice infected by T. gondii compared to uninfected mice. Furthermore, T. gondii infection led to abnormalities in neurotransmitters and the activation of NF-κB and dopamine (DA) signaling pathways in the infected mice. In conclusion, excessive activation of the inflammation in the brain could induce neuronal apoptosis in mice chronically infected with T. gondii. Dysregulation of the dopaminergic neurotransmitter could provide an explanation of neurobehavioral disorders in infected hosts. [ABSTRACT FROM AUTHOR]

Infections with the protozoan parasite Toxoplasma gondii , which are common around the world, can lead to congenital infections in humans. T. gondii surface antigen protein 5B (SAG5B) and SAG5C are potential stimulators of humoral and cellular immune responses. In this study, a multi-antigenic DNA vaccine constructed to express T. gondii SAG5B and SAG5C proteins simultaneously was used to immunize BALB/c mice to evaluate the protective efficacy of the vaccine. IgG antibody and gamma interferon (IFN-γ) cytokine production in the pSAG5B/SAG5C DNA vaccine group were significantly higher (0.853 ± 0.103 and 915.2 ± 106.9, respectively) than in the single DNA vaccine groups (pSAG5B, 0.667 ± 0.109 and 598.3 ± 74.9, respectively; pSAG5C, 0.696 ± 0.092 and 623.7 ± 95.5, respectively). After a lethal challenge with 1 × 10 4 RH strain tachyzoites, the survival time of the mice (17 days) immunized with pSAG5B/SAG5C was longer than that of the single-gene-immunized mice (12 days) or the control mice (6 days). Moreover, after intragastric infection with 20 T. gondii PRU (low virulence) strain cysts, the number of brain cysts in the pSAG5B/SAG5C-vaccinated mice was only 25% of the number for the PBS-injected mice. Our findings indicate that, in comparison with the other mouse groups, the multi-antigenic DNA vaccine (pSAG5B/SAG5C) significantly induced immune responses and improved the protection against challenge with T. gondii in the host animals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bioinformatics approaches were used to identify B-cell epitopes and T-cell epitopes on SAG5A protein. Compared to SAG1, SAG5A with good B-cell epitopes and T-cell epitopes had a potentiality to become a more successful vaccine against Toxoplasma gondii. Thereafter, SAG5A DNA vaccine was constructed successfully and was injected into mice with peptide to evaluate the immunoprotection. Compared to the control groups, the vaccine (DNA/peptide) could induce more effective cellular and humoral immune responses in immunized mice. Furthermore, a significant reduction of brain cyst was detected in the mice vaccinated with peptide (732 ± 160), pSAG5A (815 ± 197), or pSAG5A/peptide (436 ± 174) compared by the mice injected by PBS (1260 ± 241) or pEGFP-C1 (1350 ± 268). The number of cysts in brains was 35% reduced in the mice immunized with DNA/peptide than in the control mice treated by PBS. The results indicated that the DNA vaccine encoding SAG5A significantly induced immune responses and enhanced protection against cysts of PRU strain, especially with the help of peptide. [ABSTRACT FROM AUTHOR]

Abstract: Toxoplasma gondii is an obligate intracellular protozoan parasite, infecting a large variety of animals and human beings. In recent years, the study of DNA vaccine against T. gondii has made a great progress; however, few vaccines have completely controlled toxoplasmosis. Thus people started to look for more effective antigenic proteins. Here we report a novel T. gondii protein termed immune mapped protein 1 (TgIMP1). We used multiple bioinformatics approaches to predict the physical and chemical characters, signal peptide, transmembrane domain, epitope, topological structure and function of the protein, and we theoretically determined that the TgIMP1 has multiple epitopes, and with immunogenicity, suggesting that the TgIMP1 may be a vaccine candidate against toxoplasmosis. Then the gene coding TgIMP1 was obtained by PCR and connected with cloning vector. Recombinant plasmid was identified by PCR, double digestion and sequencing analysis. Then the TgIMP1 gene was directly inserted into the eukaryotic expression vector pBudCE4.1, so that the recombinant eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed. After identification by PCR and restriction enzyme digestion, the recombinant plasmid pBudCE4.1-TgIMP1 was transfected into cells of HFF, and then identified by RT-PCR. The results showed that the eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed and was transfected to the HFF cells successfully. [Copyright &y& Elsevier]

Abstract: Effective vaccines against Toxoplasma gondii may contribute to preventing and controlling the spread of toxoplasmosis, which is important for improving outcomes of infections in humans and livestock animals. The dense granule antigen 7 (GRA7) of T. gondii might be an immunodominant antigen for a vaccine candidate. In the present study, a further exploration of its vaccine effect, a heterologous prime-boost vaccination strategy with a recombinant eukaryotic plasmid pEGFP-GRA7 and a recombinant protein GRA7 expressed from a prokaryotic plasmid pET30-GRA7, was performed in BALB/c mice. The data reveal that a DNA prime-protein boost vaccination induces both humoral and cellular immune responses against T. gondii associated with high levels of total IgG, IgG2a isotype and gamma interferon (IFN-γ). Challenge experiments further show that the DNA prime-protein boost vaccination significantly increases survival rate (60%), compared with controls in which all died within 8 days of challenge. Therefore, the DNA prime-protein boost vaccination based on GRA7 might be a promising regimen for further development of an effective vaccine against T. gondii. [Copyright &y& Elsevier]

Abstract: SAG1 codes for the stage-specific major surface antigen P30 of Toxoplasma gondii (T. gondii) tachyzoites. Six tandemly repeated, conserved 27bp cassettes in the region from −231 to −70bp were previously confirmed to be essential for high-level expression of SAG1 and serve as a positioning element directing the initiation of transcription. We demonstrate here that an element located between +19 and +28bp is necessary for SAG1 gene expression by using deletion mutagenesis analysis and electrophoresis mobility shift assay (EMSA). This will provide an insight into the regulatory mechanisms of SAG1 gene expression. [Copyright &y& Elsevier]