Protective efficacy induced by DNA prime and recombinant protein boost vaccination with Toxoplasma gondii GRA14 in mice.

Toxoplasma gondii, the etiological agent of toxoplasmosis, can cause severe or lethal damages in both animals and man. So, tends to develop a more effective vaccine to prevent this disease is extremely needed and would be so prominent. The novel dense granule antigen 14 (GRA14) has been identified as a potential vaccine candidate against T. gondii infection. The aim of this study was evaluation of protective immunity induced by prime/boost vaccination strategy of GRA14 antigen with calcium phosphate (CaPNs) or Aluminum hydroxide (Alum) nano-adjuvants in BALB/c mice. The finding showed that immunization with the prime-boost strategy using plasmid DNA (pcGRA14) and recombinant protein (rGRA14) with nano-adjuvants significantly elicited levels of specific IgG antibodies and cytokines against T. gondii infection. Given that, there were the high levels of total IgG, IgG2a, IFN-γ in mice of rGRA14-CaPNs and pcGRA14 + rGRA14-CaPNs groups, which indicating a Th-1 type response. While immunization of mice with Alum based rGRA14 and pcGRA14 + rGRA14 elicited specific IgG1 and IL-4 levels, which was confirmed a Th-2 type response. Mice immunized with DNA prime-protein boost vaccine with nano-adjuvants produce more vigorous specific lymphoproliferative responses than mice immunized with other antigen formulations. In addition, the CaPNs-based prime-boost vaccine of pcGRA14 + rGRA14 showed the longest survival time in mice and the lowest parasitic load in their brain tissue compared to the other groups. The results obtained in this study show that the use of GRA14 based DNA prime-protein boost vaccination regime with CaPNs can dramatically enhanced both humoral and cellular immune responses. Therefore, this strategy can provide a promising approach to the development of an effective vaccine against T. gondii infection in the future.

Immunological evaluation of a DNA cocktail vaccine with co-delivery of calcium phosphate nanoparticles (CaPNs) against the Toxoplasma gondii RH strain in BALB/c mice.

Many recent studies have been conducted to evaluate protective immunity mediated by DNA vaccines against toxoplasmosis. Cocktail DNA vaccines showed better immune responses compared to single vaccines. The objective of the current study was to evaluate the protective efficacy of rhomboid 4 (ROM4) and cocktail DNA vaccines (ROM4 + GRA14) of the Toxoplasma gondii RH strain with or without coated calcium phosphate nanoparticles (CaPNs) as the adjuvant to improve the immunogenicity against the T. gondii RH strain in BALB/c mice. Cocktail DNA vaccines of pcROM4 + pcGRA14 of the T. gondii RH strain were constructed. CaPNs were synthesized and the cocktail DNA vaccine was coated with the adjuvant of CaPNs. Immunogenicity and the protective effects of cocktail DNA vaccines with or without CaPNs against lethal challenge were evaluated in BALB/c mice. pcROM4 and cocktail DNA vaccine coated with CaPNs significantly enhanced cellular and humoral immune responses against Toxoplasma compared to pcROM4 and cocktail DNA vaccine without CaPNs (p < 0.05). These findings indicate that the survival time of immunized mice after challenge with the RH strain of T. gondii was increased compared to that of controls and the DNA vaccine provided significant protection in mice (p < 0.05). The CaPN-based cocktail DNA vaccine of pcROM4 + pcGRA14 showed the longest survival time compared to the other groups. Co-immunization with CaPN-based cocktail DNA vaccine (pcROM4 + pcGRA14) boosted immune responses and increased the protective efficacy against acute toxoplasmosis in BALB/c mice compared to both single gene and bivalent DNA vaccine without nano-adjuvants.

Ginseng Stem-and-Leaf Saponin (GSLS)-Enhanced Protective Immune Responses Induced by Toxoplasma gondii Heat Shocked Protein 70 (HSP70) Against Toxoplasmosis in Mice.

Toxoplasma gondii is an obligate intracellular protozoan parasite and is able to infect birds and mammals including humans. In order to find effective antigen-adjuvant combinations that can boost the immunogenicity and protection of antigen vaccines against toxoplasmosis, we examined the protective efficacy in mice immunized with recombinant protein HSP70 when co-administered with ginseng stem-and-leaf saponins (GSLS) isolated from Panax ginseng . All immunized mice produced significantly high levels of specific antibodies against rTgHSP70, and splenocytes from mice presented strong proliferative immune responses. Vaccinated mice displayed a significantly increased percentage of CD4+ and CD8+ T cells, indicating a strong immune response was triggered. The cellular and humoral immune responses were enhanced, which could be reflected of the increased mRNA levels of IFN-γ and IL-4, respectively. Immunization with rTgHSP70 and GSLS prolonged survival time of the treated mice compared to the controls, which died within 6 days after challenge with the virulent T. gondii RH strain. Our data demonstrate that by addition with GSLS, rTgHSP70 induced a strong immune response and provided partial protection against T. gondii ; therefore GSLS could be used as a promising vaccine adjuvant against acute toxoplasmosis.

[Effects of atovaquone and astragalus combination on the treatment and IL-2, IL-12, IFN-γ levels on mouse models of acute toxoplasmosis]. / Atovakuon ve astragalus kombinasyonunun akut toksoplazmozlu fare modeli tedavisi ve IL-2, IL-12, IFN-γ düzeyleri üzerindeki etkileri.

Reactivation of Toxoplasma gondii infections and serious clinical manifestations such as encephalitis may develop in immunocompromised subjects and AIDS patients. Different protocols are used for the treatment of toxoplasmosis in high-risk patient groups, however life-long prophylactic therapy against reactivation risk in AIDS patients may lead to several undesired results. Atovaquone is an effective antiprotozoal agent against toxoplasmosis with minor side effects. On the other hand, Astragalus membranaceus root extract (AmE) has been shown to have immunomodulatory and antimicrobial activities, empowering immunity by enhancing proliferation and activation of phagocytic cells mainly macrophages, and inducing Th1 type immune response. The aim of this study was to investigate the effectiveness of atovaquone alone and in combination with AmE, in the treatment of toxoplasmosis, and on the levels of IL-2, IL-12 and IFN-γ in experimentally infected mice with T.gondii. For this purpose, four experimental groups, each consisting of eight BALB/c mice, were set with the approval of Ethics Committee for the Animal Experiments. All the mice were infected with 0.5 ml of a suspension containing 2 x 104/ml trophozoites prepared from T.gondii RH strain by intraperitoneal injection. Twenty-four hours after the infection, atovaquone (100 mg/kg/day) was given to atovaquone group, AmE (0.075 mg/g) to astragalus group and atovaquone (100 mg/kg/day) plus AmE (0.075 mg/g) to Atovaquone + Astragalus (Ato + Astra) group by oral gavage. The mice in the fourth group, which was the control group, were all infected but untreated. The above administrations were carried out for seven days. On the 8th day peritoneal fluids of mice were collected under anaesthesia and trophozoite numbers per 1 ml were detected by counting on the Thoma slide. In addition, the heart bloods of mice were drawn and IL-2, IL-12, IFN-γ levels were determined in serum samples by using commercial ELISA kits (eBioscience, Austria). The mean number of trophozoites in Ato + Astra group was found significantly lower than the number of trophozoites in the other three groups (p< 0.05). The number of trophozoites in the atovaquone and astragalus groups were found significantly lower than the number of trophozoites in the control group (p< 0.05). There was a significant increase in IL-2 levels of astragalus group compared with the other three groups, in addition when IL-2 levels of Ato + Astra group were compared with ones in other three groups, a significant decrease was noticed (p< 0.05). There was a definite increase in IL-12 levels of atovaquone, astragalus and the control groups compared to those in Ato + Astra group (p< 0.05). A significant increase was found in IFN-γ levels in atovaquone and Ato + Astra groups compared with those in the control group (p< 0.05). Within the reach of our literature survey, this study was the first research in which the effectiveness of the combination of atovaquone and AmE was investigated in the treatment of acute toxoplasmosis. The results of our study suggested that there might be a synergy between atovaquone and AmE in the treatment of acute toxoplasmosis. In case these results are supported by further studies, atovaquone and AmE combination may have a potential to be used for therapy in immunocompromized patients such as AIDS patients who have a risk for toxoplasmosis.

Immunoglobulin and cytokine changes induced following immunization with a DNA vaccine encoding Toxoplasma gondii selenium-dependent glutathione reductase protein.

Toxoplasma gondii Glutathione Reductase (TgGR) plays important role during the survival of the parasite. In this investigation, immunological changes and protection efficiency of this protein delivered as a DNA vaccine (pTgGR) have been evaluated. Mice were immunized with pTgGR, followed by challenge with virulent T. gondii RH strain, 2 weeks after the booster immunization. Compared to the control groups pVAX1, PBS and Blank groups, the results showed that pTgGR stimulated specific humoral response defined by significant titers of total IgG, subclasses IgG1 and IgG2a, classes IgA and IgM, but not IgE. Analysis of IFN-γ, IL-4, IL-17 and TGF-ß1 cytokines after immunization and compared with the control groups showed significant increments in pTgGR group. Additionally, T lymphocytes subpopulation CD4(+) T was positively recruited with significant percentage detected, while subset CD8(+) appeared not to be involved in response to this antigen. Vaccinated mice showed a significantly longer survival time, 15 days, in contrast with control groups which died within 8-10 days after challenge. These results demonstrated that TgGR could induce significant humoral and cell mediated responses leading to a considerable level of resistance against toxoplasmosis infection.

Prevalence and risk factors for anti-Toxoplasma gondii antibodies in goats of the Seridó Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil.

The prevalence and risk factors for anti-Toxoplasma gondii antibodies were investigated in goats of the Seridó Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil. Three hundred and sixty-six blood samples from goats collected by jugular venopuncture were used. For the serologic diagnosis of Toxoplasma gondii infection, the indirect fluorescent-antibody test (IFAT) with cut-off value 1:64 was carried out. The prevalence of anti-T. gondii antibodies was 30.6% [95% CI=25.9-35.6%] with titers ranging from 1:64 to 1:16,384. The multivariate logistic regression analysis showed that the risk factors associated to anti-T. gondii antibodies were presence of cats in the herd, extensive/semi-intensive management systems and lack of mineral supplementation.

TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection.

To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.

Effect of zinc and melatonin supplementation on cellular immunity in rats with toxoplasmosis.

The effects of zinc (Zn) and/or melatonin supplementation on cellular immunity were investigated in rats infested with Toxoplasma gondii. Fifty Sprague-Dawley male rats were used for this study. All animals were fed a normal diet, ad libitum, containing 97 mg Zn/kg. They were divided into five experimental groups, as follows. Group I (n = 10) received intraperitoneal injections of zinc sulfate at a dose of 3 mg/kg/d for 3 wk. Group II (n = 10) received intraperitoneal injections of melatonin at a dose of 3 mg/kg/d for 3 wk. Group III (n = 10) received intraperitoneal injections of zinc sulfate (3 mg/kg/d) and melatonin (3 mg/kg/d) for 3 wk. Group IV (n = 10) was infested controls. Group V (n = 10) was healthy controls. There were no differences in the percentage of CD3+ lymphocytes among all groups. For groups I-III, the CD4+ and CD8+ ratios were higher than those of the groups IV and V controls (p<0.01). Similarly, the total lymphocyte ratios in groups I-III were higher than those of infested and healthy controls (p<0.01). The total lymphocyte ratios in group III were significantly higher than those of groups I and II (p<0.01). The plasma Zn levels in the supplemented groups were significantly higher than those of control groups IV and V (p<0.01). These results suggest that melatonin and/or Zn supplementation may activate cellular immunity by stimulating CD4+ and CD8+ production in infected rats with T. gondii.

Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production.

The cytokines IL-4 and IL-13 inhibit the production of NO from activated macrophages through an unresolved molecular mechanism. We show here that IL-4 and IL-13 regulate NO production through depletion of arginine, the substrate of inducible NO synthase (iNOS). Inhibition of NO production from murine macrophages stimulated with LPS and IFN-gamma by IL-4 or IL-13 was dependent on Stat6, cell density in the cultures, and pretreatment for at least 6 h. IL-4/IL-13 did not interfere with the expression or activity of iNOS but up-regulated arginase I (the liver isoform of arginase) in a Stat6-dependent manner. Addition of exogenous arginine completely restored NO production in IL-4-treated macrophages. Furthermore, impaired killing of the intracellular pathogen Toxoplasma gondii in IL-4-treated macrophages was overcome by supplementing L-arginine. The simple system of regulated substrate competition between arginase and iNOS has implications for understanding the physiological regulation of NO production.

Phenotype and functions of brain dendritic cells emerging during chronic infection of mice with Toxoplasma gondii.

During chronic infection of mice with Toxoplasma gondii, gene message for IL-12p40, CD86, and the potassium channel Kv1.3 was detected in brain mononuclear cells, suggesting the presence of dendritic cells (DC) in the CNS. Consistently, cells bearing the DC markers CD11c and 33D1 were localized at inflammatory sites in the infected brain. The number of isolated CD11c+ brain cells increased until peak inflammation. The cells exhibited the surface phenotype of myeloid DC by coexpressing 33D1 and F4/80, little DEC-205, and no CD8alpha. These brain DC were mature, as indicated by high-level expression of MHC class II, CD40, CD54, CD80, and CD86. They triggered Ag-specific and primary allogeneic T cell responses at very low APC/T cell ratios. Among mononuclear cells from encephalitic brain, DC were the main producers of IL-12. Evidence for a parasite-dependent development of DC from CNS progenitors was obtained in vitro: after inoculation of primary brain cell culture with T. gondii, IL-12-secreting dendriform cells emerged, and DC marker genes were expressed. Different stimuli elicited the generation and maturation of brain DC: neutralization of parasite-induced GM-CSF prevented outgrowth of dendriform cells and concomitant release of IL-12. IL-12 production was up-regulated by external IFN-gamma but was stopped by inhibiting parasite replication. Consistently, DC isolated from GM-CSF-treated brain cell culture were activated to secrete IL-12 by exposure to parasite lysate. In sum, these results demonstrate T. gondii-induced expansion and functional maturation of DC in the CNS and, thus, highlight a mechanism that may contribute to the chronicity of the host response.

Coccidial infection in immunosuppressed mice: prophylaxis and treatment with dehydroepiandrosterone.

Cryptosporidiosis and toxoplasmosis are diseases caused by opportunistic coccidial parasites that can lead to life-threatening infection in immunocompromised patients. We evaluated dehydroepiandrosterone as prophylaxis and therapy in immunosuppressed mice infected with Cryptosporidium parvum and avirulent Toxoplasma gondii. Mice were infected with either Cryptosporidium oocysts or Toxoplasma cysts. Assessment was by mortality rates, parasitic counts and electron microscopic studies. Mortality rates were significantly reduced in all treated groups. A significant reduction in the cryptosporidial oocyst count in stool and intestinal villi and in Toxoplasma cysts in the brains of infected mice was observed in all the groups. The effect of the drug was greater when given prior to infection.

Clinical and serologic evaluation of two llamas (Lama glama) infected with Toxoplasma gondii during gestation.

Two pregnant llamas (Lama glama) infected with Toxoplasma gondii and their offspring were evaluated clinically and serologically. Llama 1 was inoculated orally with 1,000 infective occysts of the P89 strain of T. gondii at 82 days of gestation (DOG). Llama 2 became naturally infected with T. gondii between 26 and 119 DOG. Both llamas remained clinically normal and delivered healthy offspring. Sera collected from both llamas during pregnancy and from their offspring before and after colostral ingestion were evaluated for antibodies to T. gondii by the modified agglutination test (MAT), latex agglutination test (LAT), indirect hemagglutination test (IHAT), and the Sabin-Feldman dye test (DT). In llama 1, MAT antibody titers were < 1:20, 1:320, 1:1,280, 1:640, and 1:80 at 82, 97, 109, 132, and 152 DOG, respectively. The MAT titers in naturally infected llama 2 were < 1:32, 1:320-1:640, and 1:1,280 at 26, 119-200, and 346 DOG, respectively. In both llamas, antibody titers in the DT were of similar magnitude as the MAT, but titers in the LAT and IHAT were inconsistent. Antibodies to T. gondii were not detected in precolostral sera obtained from offspring of both llamas suggesting there was no fetal T. gondii infection.

Inhibitory effect of interferon-gamma activated ovine umbilical vein endothelial cells on the intracellular replication of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that is a major cause of abortion and neonatal mortality in sheep. In congenital toxoplasmosis, T gondii first invades the umbilical vein endothelial cells and are then disseminated throughout the fetus. Treatment of ovine umbilical vein endothelial cells with bovine recombinant gamma-interferon (IFN-gamma) blocked the growth of T gondii. Growth of the parasite was measured by 3H-uracil incorporation 18 h after the onset of the infection and by microscopic enumeration of parallel cultures. This assay revealed that when the cells were pretreated with IFN-gamma in concentrations ranging from 0.15-1,250 U/mL, a high degree of inhibition of T gondii replication was observed with the effect being dose-dependent. Maximum activation was achieved by incubating with 625 U/mL IFN-gamma and no activity was present at 0.15 U/mL. This technique could be of relevance as a first line of defense against congenital ovine Toxoplasma infection. Inhibition of T gondii replication is due to a different mechanism from that existing in mouse macrophages and human fibroblasts. L-Arginine-dependent production of reactive nitrogen and oxygen intermediates was not responsible for the inhibition of T gondii replication. Supplements of five amino acids were able to overcome the inhibition partially but significantly. The mechanism of the inhibition remains to be elucidated.

The effect of zinc supplementation in experimentally induced Toxoplasma gondii infection.

The effect of zinc added diet on cellular immunity in toxoplasmosis was investigated on 50 male wistar albino rats. Total lymphocyte CB8 count were found higher in the infected group when compared to the control group (P < 0.001). The relative increase of CD8 was found to be responsible for the decrease in ratio. In the infected group, there was a slight decrease in CD4 count but it was statistically insignificant. While no significant differences in serum zinc level were observed between the groups, there was a positive correlation with CD8 count in infected group (r = 0.005, P < 0.05). As a result, zinc added diet in toxoplasmosis stimulated the cellular immunity, increased CD8 and total lymphocytes.

Transfer of antibodies to kittens from mother cats chronically infected with Toxoplasma gondii.

By indirect immunofluorescence assay, anti-Toxoplasma gondii antibody levels were examined in fetuses and kittens born from chronically infected cats. Titer of anti-T. gondii IgG in sera of kittens born from infected cats was significantly high on the seventh day post-birth, and decreased to a serologically non-detectable level at 8-12 weeks post-birth under continuous suckling of maternal milk. Littermates nursed by a non-infected cat showed a faster rate of IgG antibody depletion. In sera of fetuses obtained from infected cats, anti-T. gondii IgG titer was lower than that of offspring born from infected cats. Anti-T. gondii IgM titer was non-detectable in sera of all kittens and fetuses. Kittens born from infected cats inoculated with T. gondii oocysts on Day 35 after birth shed oocysts and showed a transient increase of anti-T. gondii IgM titer. Findings in this study suggest that anti-T. gondii antibody IgG in kittens is transferred mainly via colostrum and the kittens that receive maternal anti-T. gondii antibodies develop inadequate resistance to T. gondii infection.

Effects of immobilization stress on the pathogenesis of acute murine toxoplasmosis.

Stress modulates a variety of immune responses. We investigated the effects of immobilization stress on the pathogenesis of acute murine toxoplasmosis, an infection in which cell-mediated immunity is of major importance in host defense. Repetitive overnight immobilization beginning 3 days prior to infection enhanced (p less than 0.05) the mortality of mice infected with a virulent strain (C56) of Toxoplasma gondii (77% vs 15% mortality in restrained and control mice, respectively). Daily immobilization for 14 days prior to infection abrogated (p less than 0.05) the lethal effect of immobilization, suggesting an adaptive mechanism. To explore the effect of immobilization with a less virulent strain, the Me49 strain of T. gondii was studied. Acute infection with T. gondii Me49 resulted in anorexia and weight loss, while spleen size and respiratory burst activity of peritoneal exudate cells were enhanced (p less than 0.01). Immobilization (twice daily for 2 h) did not significantly alter survival or other clinical features of acute T. gondii infection. In addition, immobilization suppressed (p less than 0.05) phorbol myristate acetate-stimulated release of superoxide anion by peritoneal exudate cells in healthy naive mice, but not in infected mice. These findings indicate that immobilization stress can alter the pathogenesis of acute T. gondii infection in healthy mice, but the effect of this stress paradigm will be influenced, in part, by the timing of the immobilization and the virulence of the strain of T. gondii.