The opposing effect of acute and chronic Toxoplasma gondii infection on tumor development.

BACKGROUND: The interplay between Toxoplasma gondii infection and tumor development is intriguing and not yet fully understood. Some studies showed that T. gondii reversed tumor immune suppression, while some reported the opposite, stating that T. gondii infection promoted tumor growth. METHODS: We created three mouse models to investigate the interplay between T. gondii and tumor. Model I aimed to study the effect of tumor growth on T. gondii infection by measuring cyst number and size. Models II and III were used to investigate the effect of different stages of T. gondii infection on tumor development via flow cytometry and bioluminescent imaging. Mouse strains (Kunming, BALB/c, and C57BL/6J) with varying susceptibilities to tumors were used in the study. RESULTS: The size and number of brain cysts in the tumor-infected group were significantly higher, indicating that tumor presence promotes T. gondii growth in the brain. Acute T. gondii infection, before or after tumor cell introduction, decreased tumor growth manifested by reduced bioluminescent signal and tumor size and weight. In the tumor microenvironment, CD4+ and CD8+ T cell number, including their subpopulations (cytotoxic CD8+ T cells and Th1 cells) had a time-dependent increase in the group with acute T. gondii infection compared with the group without infection. However, in the peripheral blood, the increase of T cells, including cytotoxic CD8+ T cells and Th1 cells, persisted 25 days after Lewis lung carcinoma (LLC) cell injection in the group with acute T. gondii. Chronic T. gondii infection enhanced tumor growth as reflected by increase in tumor size and weight. The LLC group with chronic T. gondii infection exhibited decreased percentages of cytotoxic CD8+ T cells and Th1 cells 25 days post-LLC injection as compared with the LLC group without T. gondii infection. At week 4 post-LLC injection, chronic T. gondii infection increased tumor formation rate [odds ratio (OR) 1.71] in both KM and BALB/c mice. CONCLUSIONS: Our research elucidates the dynamics between T. gondii infection and tumorigenesis. Tumor-induced immune suppression promoted T. gondii replication in the brain. Acute and chronic T. gondii infection had opposing effects on tumor development.

Atg5 deficiency in macrophages protects against kidney fibrosis via the CCR6-CCL20 axis.

BACKGROUND: Autophagy is a lysosome-dependent degradation pathway that regulates macrophage activation, differentiation, and polarization. Autophagy related 5 (Atg5) is a key protein involved in phagocytic membrane elongation in autophagic vesicles that forms a complex with Atg12 and Atg16L1. Alterations in Atg5 are related to both acute and chronic kidney diseases in experimental models. However, the role of macrophage-expressed Atg5 in acute kidney injury remains unclear. METHODS: Using a myeloid cell-specific Atg5 knockout (MΦ atg5-/-) mouse, we established renal ischemia/reperfusion and unilateral ureteral obstruction models to evaluate the role of macrophage Atg5 in renal macrophage migration and fibrosis. RESULTS: Based on changes in the serum urea nitrogen and creatinine levels, Atg5 deletion had a minimal effect on renal function in the early stages after mild injury; however, MΦ atg5-/- mice had reduced renal fibrosis and reduced macrophage recruitment after 4 weeks of ischemia/reperfusion injury and 2 weeks of unilateral ureteral obstruction injury. Atg5 deficiency impaired the CCL20-CCR6 axis after severe ischemic kidneys. Chemotactic responses of bone marrow-derived monocytes (BMDMs) from MΦ atg5-/- mice to CCL20 were significantly attenuated compared with those of wild-type BMDMs, and this might be caused by the inhibition of PI3K, AKT, and ERK1/2 activation. CONCLUSIONS: Our data indicate that Atg5 deficiency decreased macrophage migration by impairing the CCL20-CCR6 axis and inhibited M2 polarization, thereby improving kidney fibrosis.

Unlocking the role of EIF5A: A potential diagnostic marker regulating the cell cycle and showing negative correlation with immune infiltration in lung adenocarcinoma.

BACKGROUND: Despite EIF5A upregulation related to tumor progression in LUAD (lung adenocarcinoma), the underlying mechanisms remain elusive. In addition, there are few comprehensive analyses of EIF5A in LUAD. METHODS: We investigated the EIF5A expression level in LUAD patients using data from the TCGA and GEO databases. We employed qRT-PCR and western blot to verify EIF5A expression in cell lines, while immunohistochemistry was utilized for clinical sample analysis. We analyzed EIF5A expression in tumor-infiltrating immune cells using the TISCH database and assessed its association with immune infiltration in LUAD using the "ESTIMATE" R package. Bioinformatics approaches were developed to discover the EIF5A-related genes and explore EIF5A potential mechanisms in LUAD. Proliferation ability was verified through CCK-8, clone formation, and EdU assays, while flow cytometry assessed apoptosis and cell cycle. Western blot was used to detect the expression of pathway-related proteins. RESULTS: EIF5A was significantly upregulated in LUAD. Moreover, we constructed a MAZ-hsa-miR-424-3p-EIF5A transcriptional network. We explored the potential mechanism of EIF5A in LUAD and further investigated the cAMP signaling pathway and the cell cycle. Finally, we proved that EIF5A silencing induced G1/S Cell Cycle arrest, promoted apoptosis, and inhibited proliferation via the cAMP/PKA/CREB signaling pathway. CONCLUSION: EIF5A serves as a prognostic biomarker with a negative correlation to immune infiltrates in LUAD. It regulated the cell cycle in LUAD by inhibiting the cAMP/PKA/CREB signaling pathway.

Multiomics and bioinformatics identify differentially expressed effectors in the brain of Toxoplasma gondii infected masked palm civet.

Introduction: The masked palm civet (Paguma larvata) serves as a reservoir in transmitting pathogens, such as Toxoplasma gondii, to humans. However, the pathogenesis of T. gondii infection in masked palm civets has not been explored. We studied the molecular changes in the brain tissue of masked palm civets chronically infected with T. gondii ME49. Methods: The differentially expressed proteins in the brain tissue were investigated using iTRAQ and bioinformatics. Results: A total of 268 differential proteins were identified, of which 111 were upregulated and 157 were downregulated. KEGG analysis identified pathways including PI3K-Akt signaling pathway, proteoglycans in cancer, carbon metabolism, T-cell receptor signaling pathway. Combing transcriptomic and proteomics data, we identified 24 genes that were differentially expressed on both mRNA and protein levels. The top four upregulated proteins were REEP3, REEP4, TEP1, and EEPD1, which was confirmed by western blot and immunohistochemistry. KEGG analysis of these 24 genes identified signaling cascades that were associated with small cell lung cancer, breast cancer, Toll-like receptor signaling pathway, Wnt signaling pathways among others. To understand the mechanism of the observed alteration, we conducted immune infiltration analysis using TIMER databases which identified immune cells that are associated with the upregulation of these proteins. Protein network analysis identified 44 proteins that were in close relation to all four proteins. These proteins were significantly enriched in immunoregulation and cancer pathways including PI3K-Akt signaling pathway, Notch signaling pathway, chemokine signaling pathway, cell cycle, breast cancer, and prostate cancer. Bioinformatics utilizing two cancer databases (TCGA and GEPIA) revealed that the four genes were upregulated in many cancer types including glioblastoma (GBM). In addition, higher expression of REEP3 and EEPD1 was associated with better prognosis, while higher expression of REEP4 and TEP1 was associated with poor prognosis in GBM patients. Discussion: We identified the differentially expressed genes in the brain of T. gondii infected masked palm civets. These genes were associated with various cellular signaling pathways including those that are immune- and cancer-related.

Four Chemotherapeutic Compounds That Limit Blood-Brain-Barrier Invasion by Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii, an intracellular protozoan parasite, exists in the host brain as cysts, which can result in Toxoplasmic Encephalitis (TE) and neurological diseases. However, few studies have been conducted on TE, particularly on how to prevent it. Previous proteomics studies have showed that the expression of C3 in rat brains was up-regulated after T. gondii infection. METHODS: In this study, we used T. gondii to infect mice and bEnd 3 cells to confirm the relation between T. gondii and the expression of C3. BEnd3 cells membrane proteins which directly interacted with C3a were screened by pull down. Finally, animal behavior experiments were conducted to compare the differences in the inhibitory ability of TE by four chemotherapeutic compounds (SB290157, CVF, NSC23766, and Anxa1). RESULTS: All chemotherapeutic compounds in this study can inhibit TE and cognitive behavior in the host. However, Anxa 1 is the most suitable material to inhibit mice TE. CONCLUSION: T. gondii infection promotes TE by promoting host C3 production. Anxa1 was selected as the most appropriate material to prevent TE among four chemotherapeutic compounds closely related to C3.

Unveiling of brain transcriptome of masked palm civet (Paguma larvata) with chronic infection of Toxoplasma gondii.

BACKGROUND: The aim of this study was to gain an understanding of the transcriptomic changes that occur in a wild species when infected with Toxoplasma gondii. The masked palm civet, an artifically domesticated animal, was used as the model of a wild species. Transcriptome analysis was used to study alterations in gene expression in the domesticated masked palm civet after chronic infection with T. gondii. METHODS: Masked palm civets were infected with 105 T. gondii cysts and their brain tissue collected after 4 months of infection. RNA sequencing (RNA-Seq) was used to gain insight into the spectrum of genes that were differentially expressed due to infection. Quantitative reverse-transcription PCR (qRT-PCR) was also used to validate the level of expression of a set of differentially expressed genes (DEGs) obtained by sequencing. RESULTS: DEGs were screened from the sequencing results and analyzed. A total of 2808 DEGs were detected, of which 860 were upregulated and 1948 were downregulated. RNA-Seq results were confirmed by qRT-PCR. DEGs were mainly enriched in cellular process and metabolic process based on gene ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that transcriptional changes in the brain of infected masked palm civets evolved over the course of infection and that DEGs were mainly enriched in the signal transduction, immune system processes, transport and catabolic pathways. Finally, 10 essential driving genes were identified from the immune signaling pathway. CONCLUSIONS: This study revealed novel host genes which may provide target genes for the development of new therapeutics and detection methods for T. gondii infection in wild animals.

A Transcriptome Analysis: Various Reasons of Adverse Pregnancy Outcomes Caused by Acute Toxoplasma gondii Infection.

BACKGROUND: Toxoplasma gondii (T. gondii) is an obligate intracellular parasite, which can affect the pregnancy outcomes in infected females by damaging the uterus, and the intrauterine environment as well as and the hypothalamus resulting in hormonal imbalance. However, the molecular mechanisms underlying the parasite-induced poor pregnancy outcomes and the key genes regulating these mechanisms remain unclear. Therefore, this study aimed to analyze the gene expression in the mouse's uterus following experimentally-induced acute infection with T. gondii RH strain. Three groups of female mice were intraperitoneally injected with tachyzoites as follow; 3 days before pregnancy (FBD6), after pregnancy (FAD6), and after implantation (FID8) as the experimental groups. Another corresponding three groups served as control, were injected with normal saline at the same time. Transcriptome analysis of the total RNA extracted from both infected and non-infected mouse uterus samples was performed using RNA sequencing (RNA-Seq). RESULTS: The three experimental groups (FBD6, FAD6, and FID8) had a total of 4,561, 2,345, and 2,997 differentially expressed genes (DEGs) compared to the controls. The significantly upregulated and downregulated DEGs were 2,571 and 1,990 genes in FBD6, 1,042 and 1,303 genes in FAD6 and 1,162 and 1,835 genes in FID8 group, respectively. The analysis of GO annotation, and KEGG pathway showed that DEGs were mainly involved in anatomical structure development, transport, cell differentiation, embryo development, hormone biosynthetic process, signal transduction, immune system process, phagosome, pathways in cancer, and cytokine-cytokine receptor interaction pathways. CONCLUSION: T. gondii infection can induce global transcriptomic changes in the uterus that may cause pregnancy hypertension, destruct the intrauterine environment, and hinder the normal development of placenta and embryo. Our results may help to understand the molecular mechanisms of the acute T. gondii infection, which could promote the development of new therapeutics or prophylactics for toxoplasmosis in pregnancy.

Toxoplasma gondii in four captive kangaroos (Macropus spp.) in China: Isolation of a strain of a new genotype from an eastern grey kangaroo (Macropus giganteus).

Marsupials are highly susceptible to Toxoplasma gondii infection. Here, we report T. gondii infection in four kangaroos from a zoo in China. Kangaroos were imported into China in 2000 and were since bred in zoo. In 2017-2018, four kangaroos died due to respiratory system disease or injury. The bodies were submitted to the laboratory to test for T. gondii infection. Antibodies to T. gondii were found in 75% (3/4) of the kangaroos via the modified agglutination test with the cut-off 1:25. Cysts were observed in the histopathological sections of tongue and diaphragm or squashes of fresh myocardium in two kangaroos. These cysts were confirmed as T. gondii by immunohistochemical staining and molecular biological analysis. One viable T. gondii strain was isolated from one kangaroo and designated as TgRooCHn1. DNA from T. gondii tachyzoites obtained from cell culture was characterized by 10 PCR-RFLP markers and the virulence genes ROP5 and ROP18. The genotype of this isolate did not match with any known genotypes; it was designated as ToxoDB#292. The virulence of TgRooCHn1 (104 tachyzoites) was non-lethal to mice, and it formed tissue cysts. To our knowledge, the present study is the first isolation of ToxoDB#292 strain from kangaroo. Improvemets for captive settings were initiated, including greater attention being paied to birds and stray cats, fed frozen meat for carnivores.

Immuno-efficacy of DNA vaccines encoding PLP1 and ROP18 against experimental Toxoplasma gondii infection in mice.

We constructed a new plasmid pIRESneo/ROP18/PLP1 that was injected intramuscularly into Kunming mice to evaluate its immune efficacy. The immunized mice exhibited significantly increased serum IgG2a levels, lymphocyte counts and Th1-type cytokine (IL-2, IL-12 and IFN-γ) levels. Moreover, the immunized mice exhibited longer survival times (44.7 ± 2.1 days for ROP18/PLP1 and 47.2 ± 2.9 days for ROP18/PLP1 + IL-18) and lower brain cyst burden (68.9% for ROP18/PLP1 and 72.4% for ROP18/PLP1 + IL-18) than control mice after T. gondii challenge. Our results demonstrate that the multiple-gene DNA vaccine including both ROP18 and PLP1 elicits greater protection against T. gondii challenge and stronger immunogenicity than single-gene vaccines.

Evaluation of protective efficacy of three novel H3N2 canine influenza vaccines.

Canine influenza virus (CIV) has the potential risk to spread in different areas and dog types. Thus, there is a growing need to develop an effective vaccine to control CIV disease. Here, we developed three vaccine candidates: 1) a recombinant pVAX1 vector expressing H3N2 CIV hemagglutinin (pVAX1-HA); 2) a live attenuated canine adenovirus type 2 expressing H3N2 CIV hemagglutinin (rCAV2-HA); and 3) an inactivated H3N2 CIV (A/canine/Guangdong/01/2006 (H3N2)). Mice received an initial intramuscular immunization that followed two booster injections at 2 and 4 weeks post-vaccination (wpv). The splenic lymphocytes were collected to assess the immune responses at 6 wpv. The protective efficacy was evaluated by challenging H3N2 CIV after vaccination (at 6 wpv). Our results demonstrated that all three vaccine candidates elicited cytokine and antibody responses in mice. The rCAV2-HA vaccine and the inactivated vaccine generated efficient protective efficacy in mice, whereas limited protection was provided by the pVAX1-HA DNA vaccine. Therefore, both the rCAV2-HA live recombinant virus and the inactivated CIV could be used as potential novel vaccines against H3N2CIV. This study provides guidance for choosing the most appropriate vaccine for the prevention and control of CIV disease.

Recombinant canine adenovirus type-2 expressing TgROP16 provides partial protection against acute Toxoplasma gondii infection in mice.

We previously demonstrated that the survival time of BALB/c mice challenged with Toxoplasma gondii RH strain was prolonged by immunising the mice with a eukaryotic vector expressing the protein ROP16 of T. gondii. Building upon previous findings, we are exploring improved vaccination strategies to enhance protection. In this work, a novel recombinant canine adenovirus type 2 expressing ROP16 (CAV-2-ROP16) of T. gondii was constructed and identified to express ROP16 in Madin-Darby canine kidney cells (MDCK) cells by western blot (WB) and indirect immunofluorescence (IFA) assays. Intramuscular immunisation of BALB/c mice with CAV-2-ROP16 was performed to evaluate the humoral and cellular immune responses. This vaccination triggered significant humoral and cellular responses, including ROP16-stimulated lymphoproliferation (P<0.05). Compared to control groups, the CAV-2-ROP16 immunised mice had high production of IFN-γ, IL-2 and IL-12 (P<0.05), with a predominance of IgG2a production, but not IL-10 (P>0.05), revealing that a predominant Th1-type response had developed. The cell-mediated cytotoxic activity with high levels of IFN-γ and TNF-α was significantly increased in both CD4+ and CD8+ T-cell compartments in the mice immunised with CAV-2-ROP16 (P<0.05), compared to three control groups. In addition, when immunised mice were challenged with the RH strain of T. gondii, they showed a significantly increased survival rate (25%) 80days post infection compared with control mice that all died within seven days (P<0.05). The 25% protection rate elicited by the recombinant virus CAV-2-ROP16 has not been achieved in the field of anti-T. gondii vaccination until now. Our work presents the successful use of recombinant virus CAV-2-ROP16 in vaccination protocols to protect against intraperitoneal challenge with the virulent RH strain of T. gondii. This system was shown to be extremely efficient in eliciting humoral and cellular immune responses that led to a significant improvement in survival time in mice.

Protective efficacy of recombinant canine adenovirus type-2 expressing TgROP18 (CAV-2-ROP18) against acute and chronic Toxoplasma gondii infection in mice.

BACKGROUND: The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune responses against the parasite, as well as a valuable tool for vaccine development. We have previously prolonged the survival time of mice challenged with the RH strain of T. gondii by immunizing the mice with a eukaryotic vector expressing the protein ROP18 of T. gondii. We are now looking for ways to improve this vaccination strategy and enhance protection. METHODS: In this study, we constructed and characterized a novel recombinant canine adenovirus type 2 expressing ROP18 (CAV-2-ROP18) of T. gondii by cytopathic effect (CPE) and indirect immunofluorescence assay (IFA) following transfection into MDCK cells. Intramuscular immunization of Kunming mice with CAV-2-ROP18 was carried out to evaluate humoral and cellular immune responses. RESULTS: The vaccination of experimental mice with CAV-2-ROP18 elicited antibody production against ROP18, including high levels of a mixed IgG1/IgG2a and significant production of IFN-γ or IL-2, and displayed a significant bias towards a helper T cell type 1 (Th1) profile. Furthermore, the presence of T. gondii-specific IFN-γ-production and TNF-α-production T cells was elicited in both CD4+ and CD8+ T cell compartments. Significantly higher survival rates (40%) occurred in the experimental group, and a reduction in brain cyst burden was detected in vaccinated mice. CONCLUSION: These results demonstrate the potential use of a CAV vector harboring the ROP18 gene in the development of a vaccine against acute and chronic toxoplasmosis.

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.

Protective efficacy of a Toxoplasma gondii rhoptry protein 13 plasmid DNA vaccine in mice.

Toxoplasma gondii is an obligate intracellular parasite infecting humans and other warm-blooded animals, resulting in serious public health problems and economic losses worldwide. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In the present study, a DNA vaccine expressing rhoptry protein 13 (ROP13) of T. gondii inserted into eukaryotic expression vector pVAX I was constructed, and the immune protection it induced in Kunming mice was evaluated. Kunming mice were immunized intramuscularly with pVAX-ROP13 and/or with interleukin-18 (IL-18). Then, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged with the virulent T. gondii RH strain (type I) and the cyst-forming PRU strain (type II). The results showed that pVAX-ROP13 alone or with pVAX/IL-18 induced a high level of specific anti-T. gondii antibodies and specific lymphocyte proliferative responses. Coinjection of pVAX/IL-18 significantly increased the production of gamma interferon (IFN-γ), IL-2, IL-4, and IL-10. Further, challenge experiments showed that coimmunization of pVAX-ROP13 with pVAX/IL-18 significantly (P < 0.05) increased survival time (32.3 ± 2.7 days) compared with pVAX-ROP13 alone (24.9 ± 2.3 days). Immunized mice challenged with T. gondii cysts (strain PRU) had a significant reduction in the number of brain cysts, suggesting that ROP13 could trigger a strong humoral and cellular response against T. gondii cyst infection and that it is a potential vaccine candidate against toxoplasmosis, which provided the foundation for further development of effective vaccines against T. gondii.

Saikosaponin a and its epimer saikosaponin d exhibit anti-inflammatory activity by suppressing activation of NF-κB signaling pathway.

Saikosaponin a (SSa) and its epimer saikosaponin d (SSd) are major triterpenoid saponin derivatives from Radix bupleuri (RB), which has been long used in Chinese traditional medicine for treatment of various inflammation-related diseases. In the present study, the anti-inflammatory activity, as well as the underlying mechanism, of SSa and SSd was investigated in lipopolysaccharide (LPS)-induced RAW264.7 cells. Our results demonstrated that both SSa and SSd significantly inhibited the expression of inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW264.7 cells, and finally resulted in the reduction of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). In addition, LPS-induced production of major pro-inflammatory cytokines: the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), was suppressed in a dose-dependent manner by the treatment of SSa or SSd in RAW264.7 cells. Further analysis revealed that both SSa and SSd could inhibit translocation of nuclear factor-κB (NF-κB) from the cytoplasm to the nucleus in the LPS-induced RAW264.7 cells. Furthermore, SSa and SSd exhibited significant anti-inflammatory activity in two different murine models of acute inflammation, carrageenan-induced paw edema in rats and acetic acid-induced vascular permeability in mice. In conclusion, SSa and SSd showed potent anti-inflammatory activity through inhibitory effects on NF-κB activation and thereby on iNOS, COX-2 and pro-inflammatory cytokines.

Ascaris suum enolase is a potential vaccine candidate against ascariasis.

Ascariasis caused by Ascaris is the most common parasite problem in humans and pigs worldwide. No vaccines are available for the prevention of Ascaris infections. In the present study, the gene encoding Ascaris suum enolase (As-enol-1) was amplified, cloned and sequenced. Amino acid sequence alignment indicated that As-enol-1 was highly conserved between different nematodes and shared the highest identity (87%) with enolase from Anisakis simplex s.l. The recombinant pVAX-Enol was successfully expressed in Marc-145 cells. The ability of the pVAX-Enol for inducing immune protective responses against challenge infection with A. suum L3 was evaluated in Kunming mice. The immune response was evaluated by lymphoproliferative assay, cytokine and antibody measurements, and the reduction rate of recovery larvae. The results showed that the mice immunized with pVAX-Enol developed a high level of specific antibody responses against A. suum, 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 blank controls, respectively. There was a 61.13% reduction (P<0.05) in larvae recovery compared with that in the blank control group. Our data indicated that A. suum enolase is a potential vaccine candidate against A. suum infection.

Vaccination with a DNA vaccine coding for perforin-like protein 1 and MIC6 induces significant protective immunity against Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is tightly related to microneme protein 6 (MIC6) and T. gondii perforin-like protein 1 (TgPLP1). In this study, we constructed a DNA vaccine expressing a TgPLP1/MIC6 fusion protein using the pIRESneo vector, and we evaluated the immune response induced by this vaccine in Kunming mice. Levels of IgG antibody, gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-12, IL-4, and IL-10 were examined. Five mice were chosen randomly from every group (vaccinated groups or the nonvaccinated control group) and were challenged intragastrically with 80 cysts of T. gondii strain PRU (genotype II) in order to observe mortality daily. To analyze protection against a less-virulent challenge, eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. The brain parasite load was evaluated 6 weeks after infection. The results demonstrated that immunization with pIRESneo/MIC6/PLP1 resulted in the lowest brain cyst count and prolonged the survival time of immunized mice. The levels of Toxoplasma-specific IgG, IFN-γ, IL-2, and IL-12 increased significantly, and the numbers of cysts in brains decreased more obviously, in the group immunized with plasmid pIRESneo/MIC6/PLP1 than in the other groups (P < 0.05). Compared with pIRESneo/MIC6/PLP1, coimmunization with pIRESneo/MIC6/PLP1 and adjuvant murine IL-18 promoted cellular and humoral immune responses but did not contribute significantly to cyst reduction (65.43% versus 61.60%) or the survival of immunized mice (45.0 ± 2.9 days versus 42.8 ± 2.9 days) (P > 0.05). Furthermore, the study also showed that the immune efficacy induced by pIRESneo/MIC6/PLP1 was better than that induced by pVAX/PLP1 or pVAX/MIC6 alone.

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.

Toxoplasma gondii: protective immunity against experimental toxoplasmosis induced by a DNA vaccine encoding the perforin-like protein 1.

Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1×10(3) tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P<0.05) increased survival time (12.7±1.2days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3±0.9days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.

Protective immunity induced by Toxoplasma gondii rhoptry protein 16 against toxoplasmosis in mice.

Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 production compared with results for other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with pVAX-ROP16 showed a significantly (P < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice, which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggers a strong humoral and cellular response against T. gondii and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.