Evaluation of protective immunity induced by a DNA vaccine encoding SAG2 and SRS2 against Toxoplasma gondii infection in mice.

Toxoplasma gondii is an important protozoan pathogen, which can cause severe diseases in the newborns and immunocompromised individuals. Developing an effective vaccine against Toxoplasma infection is a critically important global health priority. Immunofluorescence staining analysis revealed that TgSAG2 and TgSRS2 are membrane associated and displayed on the surface of the parasite. Immunizations with pBud-SAG2, pBud-SRS2 and pBud-SAG2-SRS2 DNA vaccines significantly increased the production of specific IgG antibodies. Immunization with pBud-SAG2-SRS2 elicited cellular immune response with higher concentrations of IFN-γ and IL-4 compared to the control group. Antigen-specific lymphocyte proliferations in the pBud-SRS2 and pBud-SAG2-SRS2 groups were significantly higher compared to that in the control group. Furthermore, 30 % of mice immunized with pBud-SAG2-SRS2 survived after the challenge infection with virulent T. gondii RH tachyzoites. This study revealed that immunization with pBud-SAG2-SRS2 induced potent immune responses, and has the potential as a promising vaccine candidate for the control of T. gondii infection.

Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.

BACKGROUND: Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity. METHODS: The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain. RESULTS: We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 µM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 µM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii, and genes involved in "ribosome biogenesis (GO:0042254)" and "ribosome (GO:0005840)" were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis. CONCLUSIONS: Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis.

[Construction and expression of an eukaryote vector of 14-3-3 protein in Toxoplasma gondii].

OBJECTIVE: To construct and express the eukaryotic expression vector of 14-3-3 protein of Toxoplasma gondii RH stain. METHODS: The structure and physicochemical property of 14-3-3 protein were predicted by bioinformatics analysis tools. The desired gene fragment was amplified from total RNA in T. gondii RH strain by RT-PCR, and sub-cloned into pcDNA3.0 to construct recombinant plasmid pcDNA3.0/14-3-3. After PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector pcDNA3.0/14-3-3 was transfected into HeLa cells and the target protein was detected by Western blotting. RESULTS: The prediction of its gene sequence and amino acid sequence suggested that the 14-3-3 protein was acid soluble protein with five conserved regions, existing as homo- or hetero-dimers. The amplified gene fragment was about 800 bp, and the inserted fragment in pcDNA3.0/14-3-3 was 801 bp by sequencing, which had 99% homology to the 14-3-3 gene sequence of T. gondii in GenBank (Accession No. AB012775.1). Western blotting showed that there was more 14-3-3 protein expressed in the pcDNA3.0/14-3-3-transfected HeLa cells than not-transfected and mock transfected cells. Its relative molecular mass (Air) was about 30 000. CONCLUSION: The eukaryotic expression vector pcDNA3.0/14-3-3 is constructed and expressed in eukaryotic cells.

Quantitative Peptidomics of Mouse Brain After Infection With Cyst-Forming Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite capable of establishing persistent infection within the host brain and inducing severe neuropathology. Peptides are important native molecules responsible for a wide range of biological functions within the central nervous system. However, peptidome profiling in host brain during T. gondii infection has never been investigated. Using a label-free peptidomics approach (LC-MS/MS), we identified a total of 2,735 endogenous peptides from acutely infected, chronically infected and control brain samples following T. gondii infection. Quantitative analysis revealed 478 and 344 significantly differentially expressed peptides (DEPs) in the acute and chronic infection stages, respectively. Functional analysis of DEPs by Gene Ontology suggested these DEPs mainly originated from cell part and took part in cellular process. We also identified three novel neuropeptides derived from the precursor protein cholecystokinin. These results demonstrated the usefulness of quantitative peptidomics in determining bioactive peptides and elucidating their functions in the regulation of behavior modification during T. gondii infection.

Chemotherapeutics for Toxoplasma gondii: Molecular Biotargets, Binding Modes, and Structure-Activity Relationship Investigations.

Toxoplasmosis, an infectious zoonotic disease caused by the apicomplexan parasite Toxoplasma gondii (T. gondii), is a major worldwide health problem. However, there are currently no effective options (chemotherapeutic drugs or prophylactic vaccines) for treating chronic latent toxoplasmosis infection. Accordingly, seeking more effective and safer chemotherapeutics for combating this disease remains a long-term and challenging objective. In this paper, we summarize possible molecular biotargets, with an emphasis on those that are druggable and promising, including, without limitation, calcium-dependent protein kinase 1, bifunctional thymidylate synthase-dihydrofolate reductase, and farnesyl diphosphate synthase. Meanwhile, as important components of medicinal chemistry, the binding modes and structure-activity relationship profiles of the corresponding inhibitors were also illuminated. We anticipate that this information will be helpful for further identification of more effective chemotherapeutic interventions to prevent and treat zoonotic infections caused by T. gondii.

Protective immunity induced in mice by multiantigenic DNA vaccine with genes encoding SAG1 and MIC8 of Toxoplasma gondii.

OBJECTIVE: To observe the immunoprotection induced by multiantigenic SAG1-MIC8 DNA vaccine of Toxoplasma gondii in C57BL/6J mice. METHODS: The sequences of genes encoding SAG1 and MIC8 protein were inserted into the eukaryotic expression vector pcDNA3.1 and the multiantigenic recombinant plasmid pcDNA3.1-SAG1-MIC8 was constructed. Then the recombinant plasmid was transfected into Hela cells to test its expression and the recombinant protein characterized by Western blotting 70 mice were divided into 5 groups randomly: PBS, pcDNA3.1, pcDNA3.1-SAG1, pcDNA3.1-MIC8 and pcDNA3.1-SAG1-MIC8. Each mouse was injected intra-muscularly by 100 microg recombinant plasmid for 3 times every two weeks. Mice were bled on day 0, 13, 27, 41, and 55. Four weeks after the final inoculation (on day 56), spleens from seven immunized mice per group were collected. Another seven immunized mice per group were intraperitoneally challenged with 1 x 10(4) tachyzoites of RH T. gondii and the survival time was observed. Serum IgG antibody and cytokines IFN-gamma and IL-4 were demonstrated by ELISA and the T lymphocyte proliferation assay were carried out with 3H-TdR incorporation. RESULTS: Western blotting showed that the mature protein extracts in Hela cells upon transfection with pcDNA3.1-SAG1 (Mr 34,000), pcDNA3.1-MIC8 (Mr 74,000) and pcDNA3.1-SAG1-MIC8 (Mr 109,000) were effectively expressed in cells. The results of IgG antibodies (on day 41 and 55), IgG2b, IgG2c, IFN-gamma (on day 55) and T lymphocyte proliferation assay (on day 56) were more obvious in mice immunized with pcDNA3.1-SAG1-MIC8 multiantigenic DNA vaccine than those in mice with single-gene plasmids (P < 0.05). There was no significant difference in IgG1 and IL-4 levels between vaccinated and control mice after the final inoculation (on day 55) (P > 0.05). The median survival time was 3, 4, 7, 7, and 10d, respectively, with considerable difference among the groups (P < 0.01). CONCLUSION: The multiantigenic DNA vaccine elicits a stronger immuno-protection in mice than the monovalent DNA vaccine.

[Effect of Escherichia coli heat-labile enterotoxin B subunit on SAG1-ROP2 compound gene vaccine of Toxoplasma gondii tachyzoite].

OBJECTIVE: To investigate the effect of Escherichia coil heat-labile enterotoxin B subunit (LTB) as a genetic adjuvant in enhancing the immune response induced by Toxoplasma gondii tachyzoite compound gene vaccine. METHODS: The eukaryotic expression plasmids of pcDNA3.1-SAG1-ROP2 and pEASY-E1-LTB were constructed. Eighty-eight BALB/c mice were randomly divided into four groups: PBS (group A), pcDNA3.1(B), pcDNA3.1-SAG1-ROP2 (C) and pcDNA3.1-SAG1-ROP2+pEASY-E1-LTB (D). Fifteen mice in each group were randomly selected, and intranasally immunized weekly with 20 microg plasmid or 20 microl PBS, respectively. Each mouse received four immunizations with the same dose of antigen. Two weeks after the final immunization, the antibodies and cytokines were detected, including the specific IgG and IgA antibodies in serum, sIgA in mucosa douche, IFN-gamma and IL-4 in splenocyte culture supernatant. The remaining mice in each group were immunized three times weekly with 20 microg plasmid or 20 microl PBS, respectively, and challenged by T. gondii tachyzoites at four weeks after the final vaccination (1 x 10(3) per mouse). The survival time of mice was recorded. RESULTS: The recombinant plasmids pEASY-E1-LTB were constructed. The specific IgG (0.626/- 0.100) and IgA antibodies (1.086 +/- 0.138) in serum, sIgA (0.886 +/- 0.164) in mucosa douche, cytokines IFN-gamma [(2017 +/- 266) pg/ml] and IL-4 [(203.31) pg/ml] in splenocyte culture supernatant in group D were all higher than those in other groups (P < 0.05). After challenged with T. gondii tachyzoites, the median survival time of mice in groups A, B, C, and D were 3, 4, 6, and 10 d, respectively. The survival time of mice in group D was longest (P < 0.05). CONCLUSION: E. coil heat-labile enterotoxin can enhance the immune response induced by the compound gene vaccine of T. gondii tachyzoites.

Analysis of the serum peptidome associated with Toxoplasma gondii infection.

Toxoplasma gondii is an obligate intracellular parasite that can cause severe disease in immunocompromised individuals and congenitally infected neonates. In order to determine whether serum peptide profile could reveal disease markers or allow determination of toxoplasmosis aggressiveness, mouse sera were collected from acutely infected, chronically infected and control subjects, and analyzed by a quantitative label-free pepdomics approach (LC-MS/MS). Six hundred and seven endogenous peptides were identified among all samples, with peptide profiling of difference that readily distinguished between acutely infected samples and other samples. Among these peptides detected in this study, 81 and 68 differentially expressed peptides (DEPs) were found in the acute and chronic infection stages, respectively. Through Gene Ontology analysis, most of the precursor proteins of these DEPs were associated with biological regulation and binding activity. These findings in this study will help in the search of peptide targets with a key role in disease diagnosis and create new opportunities for the development of better means for the prevention and control of toxoplasmosis. SIGNIFICANCE: Toxoplasma gondii is an unicellular parasite which infects humans and a wide range of warm-blooded animals. The serum peptidome contains a large set of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs. In the present study we quantified the effects of T. gondii infection on the serum peptidome to identify novel disease regulated secretory factors. We developed an optimized label-free LC-MS/MS method to analyze endogenous peptides during toxoplasmosis progression. This resulted in quantification of 607 unique peptides at both acute and chronic infection stages. Collectively, our deep peptidomic analysis of serum revealed that peptide variations were affected by disease development, and peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to pathophysiological perturbations such as T. gondii infection.

[Construction of monovalent and compound nucleic acid vaccines against Toxoplasma gondii with gene encoding p30].

OBJECTIVE: To construct a monovalent gene vaccine pcDNA3.1-p30 and a compound gene vaccine pcDNA3.1-p30-ROP2 and assess the protective effect of the two vaccines against Toxoplasma gondii. METHODS: The sequences encoding p30 and ROP2 were amplified from the genomic DNA of T. gondii RH strain by polymerase chain reaction (PCR) and inserted into eukaryotic vector pcDNA3.1 to construct pcDNA3.1-p30 and pcDNA3.1-p30-ROP2. Mice were injected with the recombinant plasmid to observe the immunoprotectivity of the nucleic acid vaccine by using ELISA for detection of total IgG and observing the survival time after tachyzoites challenge. RESULTS: The recombinant plasmids pcDNA3.1-p30 and pcDNA3.1-p30-ROP2 were constructed. Mice in pcDNA3.1-p30-ROP2 group showed higher IgG (P < 0.05) and survived longer than those in pcDNA3.1-p30 group (P < 0.01) after challenged with T. gondii. CONCLUSION: Compound vaccine of genes from different stages of T. gondii elicits stronger immunoprotectivity in mice than a single gene vaccine.

[Oral mixed DNA vaccine protects mice from infection of Toxoplasma gondii].

OBJECTIVE: To examine the immune response in BALB/c mice induced by oral mixed Toxoplasma gondii DNA vaccine delivered by live attenuated Salmonella typhimurium. METHODS: Gene fragments SAG1 and SAG2 were amplified from the genomic DNA of T. gondii RH strain by PCR and were subcloned into pcDNA3.1(+/-) eukaryotic expression vector. The positive recombinant plasmid was transformed into aroA- and aroD-attenuated Salmonella typhimurium BRD509 (BRD509/pSAG1/SAG2). After screened by PCR, restrictive enzyme digestion and sequencing, recombinant Salmonella strain was used to immunize BALB/c mice by i.g. route, three times at 2-week interval. Humoral and cellular responses were assayed using ELISA for determining antibody, IFN-gamma and IL-4. MTT assay was used to detect the proliferation activity of T lymphocytes and the activity of NK killers. FCM was also used to sort the lymphocyte subsets of spleen cells All mice were then infected with highly virulent RH tachyzoites of T. gondii intraperitoneally. RESULTS: Significant increase of specific IgG level was observed in immunized mice with a titer of 1:100. Proliferation activity of specific NK cells and T lymphocytes was highly enhanced in BRD509/ pSAG1/SAG2 vaccinated mice: the killing activity of NK cells was 85% +/- 7%, the proliferation SI of T lymphocytes was 2.83, which resulted in a 5 days longer survival time than mice in control group after challenge infection. CONCLUSION: The oral mixed DNA vaccine delivered by attenuated Salmonella typhimurium shows an immunoprotection against T. gondii in mice.

Multi-epitope DNA vaccine linked to the A2/B subunit of cholera toxin protect mice against Toxoplasma gondii.

The search for an effective vaccine against toxoplasmosis remains a challenging and elusive goal. Combination of epitopes from different stages of Toxoplasma gondii life cycle is an optimal strategy to overcome the antigen complexity of the parasite. Based on published epitope derived from several promising candidate vaccine antigens, we construct a DNA vaccine encoding multi-epitope of T. gondii and CpG motif, with or without the A2/B subunit of cholera toxin as a genetic adjuvant. The immunity induced by this vaccine in BALB/c mice and the protection afforded against challenge with the highly virulent RH strain of T. gondii is assessed. This vaccine was able to elicit a significant humoral and cellular immune response in vaccinated mice. Furthermore, CTXA2/B as a genetic adjuvant could enhance the magnitude of immune responses as well as increased survival rate in mice infected with the lethal RH tachyzoites. This study is the first report of a multi-epitope DNA construct strategy as a potential DNA vaccine against toxoplasmosis.