Soluble Tachyzoite Antigen Research Articles

Toxoplasma gondii in CD36 -/- mice shows lethal infection and poor immunization with probable macrophage immune defects.

Experimental toxoplasmosis is an excellent model for adaptive immune response. Gamma-irradiated tachyzoites or soluble tachyzoite antigen extracts (STag) induce protection against experimental toxoplasmosis in mice. Scavenger receptors recognize irradiated proteins, promote their entry into cells, and lead to antigen presentation. CD36 is a specific scavenger receptor involved in intracellular transport of free fatty acid (FFA), cellular recycling, and intracellular trafficking in lipid rafts outside the lysosomal pathways. CD36 is also associated with an altered immune response, as CD36-/- mice presented some immune defects in the cyst-forming Toxoplasma gondii. We studied T. gondii infection in CD36-/- mice, naïve or immunized, with irradiated T. gondii STags by investigating protection, antibody production, and primed macrophage transplantation. CD36-/- mice presented no resistance against the viable RH tachyzoites, even after immunization with gamma-irradiated STags that protected wild-type mice. The animals presented poor humoral responses to both immunogens despite adequate levels of serum immunoglobulins. CD36-/- mice failed to induce protection against virulent T. gondii infection with inadequate antibody production or an innate response. Irradiated antigens failed to induce antibodies in CD36-/- mice and only produced adequate levels of immunoglobulin G when transplanted with irradiated STag-primed wild-type macrophages. The CD36 pathway is necessary for humoral response against the irradiated antigen; however, several other pathways are also involved in mounting a humoral response against any antigen. CD36 is a multipurpose molecule for FFA and lipid transport, as well as for the immune response, and gamma radiation mimics the innate response by targeting irradiated antigens of this pathway.

Inflammasome Activation Dampens Type I IFN Signaling to Strengthen Anti-Toxoplasma Immunity.

Innate immunity acts as the first line of defense against pathogen invasion. During Toxoplasma gondii infection, multiple innate immune sensors are activated by invading microbes or pathogen-associated molecular patterns (PAMPs). However, how inflammasome is activated and its regulatory mechanisms during T. gondii infection remain elusive. Here, we showed that the infection of PRU, a lethal type II T. gondii strain, activates inflammasome at the early stage of infection. PRU tachyzoites, RNA and soluble tachyzoite antigen (STAg) mainly triggered the NLRP3 inflammasome, while PRU genomic DNA (gDNA) specially activated the AIM2 inflammasome. Furthermore, mice deficient in AIM2, NLRP3, or caspase-1/11 were more susceptible to T. gondii PRU infection, and the ablation of inflammasome signaling impaired antitoxoplasmosis immune responses by enhancing type I interferon (IFN-I) production. Blockage of IFN-I receptor fulfilled inflammasome-deficient mice competent immune responses as WT mice. Moreover, we have identified that the suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator induced by inflammasome-activated IL-1β signaling and inhibits IFN-I production by targeting interferon regulatory factor 3 (IRF3). In general, our study defines a novel protective role of inflammasome activation during toxoplasmosis and identifies a critical regulatory mechanism of the cross talk between inflammasome and IFN-I signaling for understanding infectious diseases. IMPORTANCE As a key component of innate immunity, inflammasome is critical for host antitoxoplasmosis immunity, but the underlying mechanisms are still elusive. In this study, we found that inflammasome signaling was activated by PAMPs of T. gondii, which generated a protective immunity against T. gondii invasion by suppressing type I interferon (IFN-I) production. Mechanically, inflammasome-coupled IL-1β signaling triggered the expression of negative regulator SOCS1, which bound to IRF3 to inhibit IFN-I production. The role of IFN-I in anti-T. gondii immunity is little studied and controversial, and here we also found IFN-I is harmful to host antitoxoplasmosis immunity by using knockout mice and recombinant proteins. In general, our study identifies a protective role of inflammasomes to the host during T. gondii infection and a novel mechanism by which inflammasome suppresses IFN-I signaling in antitoxoplasmosis immunity, which will likely provide new insights into therapeutic targets for toxoplasmosis and highlight the cross talk between innate immune signaling in infectious diseases prevention.

Rescue of maternal immune activation-induced behavioral abnormalities in adult mouse offspring by pathogen-activated maternal Treg cells.

Maternal immune activation (MIA) induced by lipopolysaccharides or polyinosinic:polycytidylic acid injections can induce behavioral abnormalities in adult mouse offspring. Here, we used the soluble tachyzoite antigen from Toxoplasma gondii, a parasite that infects approximately two billion people, to induce MIA in mice. The adult male offspring showed autism-relevant behaviors and abnormal brain microstructure, along with a pro-inflammatory T-cell immune profile in the periphery and upregulation of interleukin-6 in brain astrocytes. We show that adoptive transfer of regulatory T (Treg) cells largely reversed these MIA-induced phenotypes. Notably, pathogen-activated maternal Treg cells showed greater rescue efficacy than those from control donors. Single-cell RNA sequencing identified and characterized a unique group of pathogen-activated Treg cells that constitute 32.6% of the pathogen-activated maternal Treg population. Our study establishes a new preclinical parasite-mimicking MIA model and suggests therapeutic potential of adoptive Treg cell transfer in neuropsychiatric disorders associated with immune alterations.

C5aR1 Activation Drives Early IFN-γ Production to Control Experimental Toxoplasma gondii Infection

Toxoplasma gondii (T. gondii) is a parasite infecting animals and humans. In intermediate hosts, such as humans or rodents, rapidly replicating tachyzoites drive vigorous innate and adaptive immune responses resulting in bradyzoites that survive within tissue cysts. Activation of the innate immune system is critical during the early phase of infection to limit pathogen growth and to instruct parasite-specific adaptive immunity. In rodents, dendritic cells (DCs) sense T. gondii through TLR11/12, leading to IL-12 production, which activates NK cells to produce IFN-γ as an essential mechanism for early parasite control. Further, C3 can bind to T. gondii resulting in limited complement activation. Here, we determined the role of C5a/C5aR1 axis activation for the early innate immune response in a mouse model of peritoneal T. gondii infection. We found that C5ar1−/− animals suffered from significantly higher weight loss, disease severity, mortality, and parasite burden in the brain than wild type control animals. Severe infection in C5ar1−/− mice was associated with diminished serum concentrations of IL-12, IL-27, and IFN-γ. Importantly, the serum levels of pro-inflammatory cytokines, including IL-1α, IL-6, and TNF-α, as well as several CXC and CC chemokines, were decreased in comparison to wt animals, whereas anti-inflammatory IL-10 was elevated. The defect in IFN-γ production was associated with diminished Ifng mRNA expression in the spleen and the brain, reduced frequency of IFN-γ+ NK cells in the spleen, and decreased Nos2 expression in the brain of C5ar1−/− mice. Mechanistically, DCs from the spleen of C5ar1−/− mice produced significantly less IL-12 in response to soluble tachyzoite antigen (STAg) stimulation in vivo and in vitro. Our findings suggest a model in which the C5a/C5aR1 axis promotes IL-12 induction in splenic DCs that is critical for IFN-γ production from NK cells and subsequent iNOS expression in the brain as a critical mechanism to control acute T. gondii infection.

Toxoplasma gondii Soluble Tachyzoite Antigen Triggers Protective Mechanisms against Fatal Intestinal Pathology in Oral Infection of C57BL/6 Mice

Toxoplasma gondii induces a potent IL-12 response early in infection that results in IFN-γ-dependent control of parasite growth. It was previously shown that T. gondii soluble tachyzoite antigen (STAg) injected 48 hr before intraperitoneal infection reduces lipoxin A4 and 5-lipoxygenase (5-LO)-dependent systemic IL-12 and IFN-γ production as well as hepatic immunopathology. This study investigated the ability of STAg-pretreatment to control the fatal intestinal pathology that develops in C57BL/6 mice orally infected with 100 T. gondii cysts. STAg-pretreatment prolonged the animals’ survival by decreasing tissue parasitism and pathology, mainly in the ilea. Protection was associated with decreases in the systemic IFN-γ levels and IFN-γ and TNF message levels in the ilea and with increased TGF-β production in this tissue, but protection was independent of 5-LO and IL-4. STAg-pretreatment decreased CD4+ T cell, NK cell, CD11b+ monocyte and CD11b+CD11c+ dendritic cell numbers in the lamina propria and increased CD8+ T cells in the intestinal epithelial compartment. In parallel, decreases were observed in iNOS and IL-17 expression in this organ. These results demonstrate that pretreatment with STAg can induce the recruitment of protective CD8+ T cells to the intraepithelial compartment and decrease proinflammatory immune mechanisms that promote intestinal pathology in T. gondii infection.

Kinetics of IL-23 and IL-12 Secretion in Response to Toxoplasma gondii Antigens from THP-1 Monocytic Cells

IL-23 and IL-12 are structurally similar and critical for the generation of efficient cellular immune responses. Toxoplasma gondii induces a strong cell-mediated immune response. However, little is known about IL-23 secretion profiles in T. gondii-infected immune cells in connection with IL-12. We compared the patterns of IL-23 and IL-12 production by THP-1 human monocytic cells in response to stimulation with live or heat-killed T. gondii tachyzoites, or with equivalent quantities of either T. gondii excretory/secretory proteins (ESP) or soluble tachyzoite antigen (STAg). IL-23 and IL-12 were significantly increased from 6 hr after stimulation with T. gondii antigens, and their secretions were increased with parasite dose-dependent manner. IL-23 concentrations were significantly higher than those of IL-12 at the same multiplicity of infection. IL-23 secretion induced by live parasites was significantly higher than that by heat-killed parasites, ESP, or STAg, whereas IL-12 secretion by live parasite was similar to those of ESP or STAg. However, the lowest levels of both cytokines were at stimulation with heat-killed parasites. These data indicate that IL-23 secretion patterns by stimulation with various kinds of T. gondii antigens at THP-1 monocytic cells are similar to those of IL-12, even though the levels of IL-23 induction were significantly higher than those of IL-12. The detailed kinetics induced by each T. gondii antigen were different from each other.

The Soluble Tachyzoite Antigen of Toxoplasma gondii Has a Protective Effect on Mouse Allografts

BackgroundInfection with some types of parasites can significantly prolong allograft survival in mice. It is unknown whether the soluble tachyzoite antigen (STAg) from Toxoplasma gondii has the same effect and by what mechanism it acts. MethodsA mouse model of cardiac and skin allograft transplantation was established between BALB/c (H-2d) and C57BL/6(H-2b) mice. T gondii STAg was prepared, and 5 μg was administered subcutaneously to recipient mice 4 days before transplantation. The graft status was checked daily, and histologic and immunohistochemical assays were used to evaluate rejection. The serum cytokine levels from the recipient mice were analyzed by Luminex. ResultThe administration of 5 μg STAg 4 days before transplantation significantly prolonged the survival time of the heart and skin allografts to 85.17 ± 14.06 and 24.17 ± 2.32 days, respectively. Immunohistochemical staining showed that the CD4+ and CD8+ T lymphocytes were markedly reduced in the allografts at day 7 posttransplantation. Notably, interleukin (IL)-12, IL-2, and IL-17 levels were significantly reduced in the serum of mice treated with STAg compared with untreated mice 7 days after transplantation. In contrast, the levels of the antiinflammatory cytokine IL-10 were elevated. ConclusionA single administration of STAg before transplantation can significantly prolong the allograft survival time, which is accompanied by impaired lymphocyte infiltration and a reduced Th1 response.

Toxoplasma gondii: Proteomic analysis of antigenicity of soluble tachyzoite antigen

The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. The tachyzoite of T. gondii is the main life-cycle stage that is responsible for toxoplasmosis. Study of the antigenicity of soluble tachyzoite antigen (STAg) is important for discovery of protective antigens which will aid in the detection and prevention of toxoplasmosis. At present, no complete proteome map of T. gondii STAg is established, although a large-scale whole proteomic analysis of tachyzoites is underway. In this study, 1227 protein spots of T. gondii soluble tachyzoite antigen (STAg) were fractionated by 2-dimensional electrophoresis (2-DE) at pH range 3–10. By mass spectrometry (MS) analysis, among the separated 1227 protein spots, 426 were identified by searching the Swissport and NCBI nr databases. Two hundred and thirty of these identified spots (230/426, 54%) were demonstrated to be T. gondii protein by MS. Of the 21 Toxoplasma protein spots identified by Western blot with rabbit anti- T. gondii serum, 16 had immunoregulatory functions and five had immune defense functions. Due to multiple spots for a single protein, these 16 spots represented 11 proteins: a putative protein disulfide isomerase (PDI), heat shock protein 60 (Hsp60), a pyruvate kinase (PK), a putative glutamate dehydrogenase (GDH), a coronin, a heat shock protein 70 (Hsp70), a protein kinase C receptor 1 (RACK1), a malate dehydrogenase (MDH), a major surface antigen 1 (SAG1), an uridine phosphorylase (UPase) and a peroxiredoxin (Prx). Among the identified 11 proteins, except that the antigenicity and immunogenicity of the SAG1 has been reported and antigenicity of Hsp70 has been disputed, the remaining antigenic proteins were first identified in this study. In conclusion, we obtained nine novel types of immunogenic proteins that might be potential candidates of vaccine development for toxoplasmosis, which we will confirm in later studies.

The TNF-Family Receptor DR3 is Essential for Diverse T Cell-Mediated Inflammatory Diseases

DR3 (TRAMP, LARD, WSL-1, TNFRSF25) is a death-domain-containing tumor necrosis factor (TNF)-family receptor primarily expressed on T cells. TL1A, the TNF-family ligand for DR3, can costimulate T cells, but the physiological function of TL1A-DR3 interactions in immune responses is not known. Using DR3-deficient mice, we identified DR3 as the receptor responsible for TL1A-induced T cell costimulation and dendritic cells as the likely source for TL1A during T cell activation. Despite its role in costimulation, DR3 was not required for in vivo T cell priming, for polarization into T helper 1 (Th1), Th2, or Th17 effector cell subtypes, or for effective control of infection with Toxoplasma gondii. Instead, DR3 expression was required on T cells for immunopathology, local T cell accumulation, and cytokine production in Experimental Autoimmune Encephalomyelitis (EAE) and allergic lung inflammation, disease models that depend on distinct effector T cell subsets. DR3 could be an attractive therapeutic target for T cell-mediated autoimmune and allergic diseases.

Exogenous Pathogen and Plant 15-Lipoxygenase Initiate Endogenous Lipoxin A4 Biosynthesis

Lipoxin A4 (LXA4) is a potent endogenous lipoxygenase-derived eicosanoid with antiinflammatory and proresolving properties. Supraphysiological levels of LXA4 are generated during infection by Toxoplasma gondii, which in turn reduces interleukin (IL) 12 production by dendritic cells, thus dampening Th1-type cell-mediated immune responses and host immunopathology. In the present work, we sought evidence for the structural basis of T. gondii's ability to activate LXA4 biosynthesis. Proteomic analysis of T. gondii extract (soluble tachyzoite antigen [STAg]), which preserves the immunosuppressive and antiinflammatory activity of the parasite, yielded several peptide matches to known plant lipoxygenases. Hence, we incubated STAg itself with arachidonic acid and found using LC-UV-MS-MS–based lipidomics that STAg produced both 15-HETE and 5,15-diHETE, indicating that T. gondii carries 15-lipoxygenase activity. In addition, T. gondii tachyzoites (the rapidly multiplying and invasive stage of the parasite) generated LXA4 when provided with arachidonic acid. Local administration of a plant (soybean) lipoxygenase itself reduced neutrophilic infiltration in murine peritonitis, demonstrating that 15-lipoxygenase possesses antiinflammatory properties. Administration of plant 15-lipoxygenase generated endogenous LXA4 and mimicked the suppression of IL-12 production by splenic dendritic cells observed after T. gondii infection or STAg administration. Together, these results indicate that 15-lipoxygenase expressed by a pathogen as well as exogenously administered 15-lipoxygenase can interact with host biosynthetic circuits for endogenous “stop signals” that divert the host immune response and limit acute inflammation.

Mechanism of entry determines the ability of Toxoplasma gondii to inhibit macrophage proinflammatory cytokine production.

Macrophages (Mphi) infected with tachyzoites of the opportunistic protozoan Toxoplasma gondii are blocked in production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) in response to lipopolysaccharide (LPS) triggering, and this is associated with parasite-induced inhibition of NFkappaB translocation. Here, we demonstrate a requirement for active invasion in the ability of the parasite to mediate suppression. Neither soluble tachyzoite antigen nor secreted products were suppressive, and heat-inactivated, antibody-coated tachyzoites, which efficiently entered the cell through receptor-mediated uptake, failed to inhibit LPS responses. Cytochalasin D, a drug blocking tachyzoite invasion of, but not adherence to, Mphi, severely curtailed Toxoplasma-induced suppression. In addition, parasite-induced nonresponsiveness, as measured by TNF-alpha production, was reversed by treating infected cells with the toxoplasmastatic drugs pyrimethamine and 6-thioxanthine prior to LPS stimulation. A divergence in IL-12 and TNF-alpha responses was found during extended incubation of tachyzoites and Mphi in that 24 h of incubation of infected Mphi resulted in IL-12, but not TNF-alpha, secretion, and production of the latter cytokine remained suppressed when these cells were subjected to LPS triggering. Our results demonstrate that active invasion and survival of the parasite within the parasitophorous vacuole are required to induce and maintain Mphi cytokine-specific nonresponsiveness to LPS. They also show that the effects of Toxoplasma on IL-12 and TNF-alpha production are nonidentical, with the parasite exerting a longer-lasting suppression of the latter.

Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes.

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.

Discrimination between Patients with Acquired Toxoplasmosis and Congenital Toxoplasmosis on the Basis of the Immune Response to Parasite Antigens

Many persons infected with Toxoplasma gondii develop ocular lesions. Immunologic parameters in the response to T. gondii were evaluated in infected persons with and without ocular lesions and in noninfected controls. Subjects were divided into groups on the basis of presence of serum antibodies to T. gondii, presence of ocular lesions, and clinical history. Production of interleukin-2 and interferon-gamma by peripheral blood mononuclear cells from patients with probable congenital toxoplasmosis was decreased, compared with that in persons with presumed acquired infection. Cell proliferation and delayed-type skin reaction induced by soluble toxoplasma tachyzoite antigen followed the same pattern. Asymptomatic persons showed high levels of interleukin-12 and interferon-gamma, whereas persons with ocular lesions had high interleukin-1 and tumor necrosis factor-alpha responses toward soluble toxoplasma tachyzoite antigen. These data suggest that patients with ocular disease due to congenital infection show tolerance toward the parasite. Furthermore, susceptibility to ocular lesions after acquired toxoplasmosis is associated with high levels of interleukin-1 and tumor necrosis factor-alpha, whereas resistance is associated with high levels of interleukin-12 and interferon-gamma.

Partially protective vaccination permits the development of latency in a normally virulent strain of Toxoplasma gondii.

The virulent RH strain of Toxoplasma gondii is acutely lethal in mice and fails to establish chronic infection. Vaccination of BALB/c mice with a soluble tachyzoite antigen preparation, STAg, in combination with the immunostimulatory cytokine interleukin-12 results in partial protection against RH lethal challenge. Nevertheless, brain tissue obtained from surviving, vaccinated mice as late as 1 year after RH infection contained latent parasite forms as demonstrated by subinoculation into naive recipients. The tachyzoites arising in the subinoculated animals were genetically indistinguishable from the original RH inoculum. Microscopic examination revealed that the persistent parasite forms present in the brains of vaccinated and challenged mice have a tissue cyst-like morphology and express the bradyzoite antigen BAG-1 but not the tachyzoite-specific antigen SAG-2 but are different from the cysts formed by avirulent T. gondii strains in that the internal parasite stages display ultrastructural features intermediate between tachyzoites and bradyzoites. Moreover, the zoites within the RH tissue cysts are clearly distinct from conventional bradyzoites in their sensitivity to pepsin-HCl digestion. In contrast to the observations made with partially resistant STAg/interleukin-12-vaccinated animals, no latent forms could be detected in brain tissue after RH challenge of mice immunized with a live attenuated tachyzoite vaccine which confers total protection against this parasite isolate. The above findings demonstrate the potential of a virulent T. gondii strain to generate latent parasite stages, a process which may be promoted under conditions of incomplete vaccination.

Biochemical characterization and protein kinase C dependency of monokine-inducing activities of Toxoplasma gondii.

Previous reports have indicated that the early induction of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), IL-1beta, and IL-10 is crucial for the establishment and regulation of host cell-mediated immunity to the intracellular protozoan parasite Toxoplasma gondii. In this study, we demonstrate that a soluble tachyzoite extract (soluble tachyzoite antigen) can trigger the expression of these four monokines by murine inflammatory macrophages. Further characterization revealed that the parasite molecules in soluble tachyzoite antigen responsible for monokine induction are heat stable at 100 degree C but differ in sensitivity to protease digestion. Thus, the tachyzoite factors that stimulate TNF-alpha and IL-to expression were found to be more resistant to treatment with proteinase K than those responsible for IL-12 and IL-10 induction. Similarly, while the factors responsible for the induction of all four monokines were found to be sensitive to periodate oxidation, the TNF-alpha-stimulating activity was partially resistant to treatment with the compound at a low concentration (1 mM). A further dichotomy in monokine induction signals was inferred from experiments with isoquinoline sulfonamide protein kinase inhibitors. The latter work suggested that the pathways for TNF-alpha and IL-1beta are protein kinase C dependent, while expression of IL-12 and expression of IL-10 share distinct signal transduction mechanisms involving other kinases. Together, these data argue that monokine induction by T. gondii is mediated by glycoproteins that may belong to distinct groups in terms of their biochemical properties and intracellular signaling pathways.

Different roles for interleukin-4 during the course of Toxoplasma gondii infection

The course of Toxoplasma gondii infection from initiation of disease perorally until day 28 postinfection was compared between interleukin-4 (IL-4) gene knockout (IL-4-/-) mice and their wild-type (IL-4+/+) counterparts on a disease-susceptible genetic background. The rate of mortality was significantly greater in mice deficient in Il-4 than in the immunocompetent controls. Although levels of T. gondii-specific spleen cell proliferation measured in vitro were similar between groups at all time points examined throughout infection, the quantities of cytokines released into the culture supernatant differed. Culture supernatants from spleen cells derived from IL-4-deficient mice contained significantly more gamma interferon than those derived from IL-4+/+ mice at day 7 postinfection. Conversely, IL-10 production was significantly greater from the spleen cells derived from wild-type mice at day 28 postinfection. Splenocytes from both groups of mice had a marked inhibition of proliferation in response to soluble tachyzoite antigen as well as reduced proliferation in response to concanavalin A between days 7 and 14 postinfection and marked proliferation on days 21 and 28 postinfection. At day 28 postinfection, histological examination of the brains indicated that IL-4+/+ mice had more severe pathological changes and more cysts than IL-4-/- mice. In addition, although many nonencysted single organisms were present in IL-4+/+ mice within both necrotic lesions and microglial nodules, few nonencysted parasites were found, and no necrotic lesions were present in IL-4-deficient animals. These results suggest that the observed reduction in mortality during the early acute phases of infection may be due to the down-regulatory effects of Il-4 or associated Th2-derived products on proinflammatory cytokines such as gamma interferon. However, the long-term effects of IL-4 are detrimental, possibly because of the ability of this cytokine to inhibit proinflammatory antiparasitic products. This may explain the increased parasite multiplication with cysts observed in the brains of IL-4+/+ mice.

Antibody responses to Toxoplasma gondii antigen in human peripheral blood lymphocyte‐reconstituted severe‐combined immunodeficient mice reproduce the immunological status of the lymphocyte donor

These studies describe the production of specific antibodies in human peripheral blood lymphocyte-reconstituted severe-combined immunodeficient (PBL-SCID) mice following vaccination with antigen from the protozoan parasite Toxoplasma gondii. To determine the effect of previous exposure of the lymphocyte donor to antigen, human-PBL-SCID animals were created by transferring peripheral blood lymphocytes from either a single T. gondii-seronegative or a single seropositive donor. These reconstituted animals were subsequently inoculated with T. gondii soluble tachyzoite antigen (STAg) entrapped within non-ionic surfactant vesicles as an immunological adjuvant. Animals were bled at pre-determined time points post-vaccination and the expression of human anti-STAg antibodies in the plasma determined by enzyme-linked immunosorbent assay. Human antibodies specific for STAg were readily inducible in both groups of reconstituted animals, although the pattern of isotype production differed markedly between groups. The response in animals reconstituted with lymphocytes from the T. gondii-seronegative donor consisted primarily of IgM and subsequently of IgG (predominantly IgG1). In animals reconstituted with lymphocytes from the seropositive donor, no parasite-specific IgM could be demonstrated. The detectable response to STAg consisted entirely of human antibodies of the IgG isotype (IgG1), indicative of a memory-type response. These results mimicked exactly the antibody responses that would be expected had the lymphocyte donors been directly challenged with either the antigen or the live infectious agent, demonstrating that the immune system within these animals is functional and reproducible with regard to both the primary and secondary responses of the human donors.

Toxoplasma gondii possesses a superantigen activity that selectively expands murine T cell receptor V beta 5-bearing CD8+ lymphocytes.

To investigate early immune responses to the intracellular parasite Toxoplasma gondii, we examined the capacity of nonimmune splenocytes to respond in vitro to intact tachyzoites and soluble tachyzoite antigen (Ag). Both types of stimuli induced high levels of proliferation as well as interferon gamma (IFN-gamma) secretion. Based on several key criteria, the response appeared to be driven by a superantigen present in the parasite. Thus, stimulation of C57BL/6 spleen cells with T. gondii resulted in a preferential threefold expansion of a T cell population expressing the V beta 5 chain of the T cell receptor, and a survey of different inbred mouse strains revealed an inverse correlation between Ag-induced proliferation and genetic deletion of V beta 5. Moreover, proliferation was induced using irradiated Ag-pulsed and infected splenic adherent cells, and was blocked by a major histocompatibility complex class II-specific monoclonal antibody. Furthermore, paraformaldehyde-fixed IAb-, IAk-, and IEk-transfected fibroblast lines were able to specifically bind T. gondii Ag and drive proliferation of T lymphocytes, demonstrating that the response can be mediated by allogeneic class II molecules, and that it does not require cellular Ag processing. It is interesting to note that after 1 wk of culture with Ag, up to 70% of the expanded V beta 5-expressing cells were CD8+. These results provide the first description of a superantigen activity in a protozoan pathogen. In the case of T. gondii, superantigen-driven expansion of IFN-gamma-secreting CD8+ lymphocytes may play a role in the development of the dominant IFN-gamma-dependent, cell-mediated immunity characteristic of infection with this parasite.

Congenital toxoplasmosis in the Balb/c mouse: prevention of vertical disease transmission and fetal death by vaccination

Vertical disease transmission only occurs in Balb/c mice infected with Toxoplasma gondii for the first time during pregnancy. This is similar to the situation in humans, where a previous infection with T. gondii tends to give life-long immunity against reinfection and fetal disease transmission. The Balb/c mouse therefore provides a suitable model to study the effectiveness of T. gondii vaccine candidates. A soluble tachyzoite antigen (STAg) preparation was used to vaccinate female Balb/c mice. STAg was inoculated subcutaneously into Balb/c mice in phosphate-buffered saline (PBS), emulsified in Freund's complete adjuvant (FCA), or entrapped within non-ionic surfactant vesicles (NISV). While all inocula induced cellular immunity as measured by parasite-specific spleen cell proliferation in vitro, the highest mean proliferative values were observed in spleens from mice where NISV had been used as the adjuvant and the lowest values were observed where FCA had been used. More importantly, cultures from the NISV/STAg vaccinated mice produced significantly more γ-interferon than the other experimental groups. This vaccine formulation was therefore identified as that most likely to induce protective immunity against toxoplasmosis. Mice were inoculated subcutaneously with either NISV/STAg or STAg in PBS 4 and 2 weeks before mating and infected orally with 20 tissue cysts of T. gondii on day 12 of pregnancy. The incidence of fetal infection and death in these mice and non-vaccinated infected dams was compared. Of 84 pups born to 14 non-vaccinated dams 45 were viable, of which 18 were found to be infected on reaching maturity. All 67 of the pups born to the 11 dams vaccinated with STAg/NISV survived into maturity, of which a mere nine were infected. These studies emphasize that, given appropriate antigen and adjuvant preparations for vaccination, sufficient immunity can be generated in Balb/c dams to severely limit the likelihood of T. gondii-induced abortion and congenital disease.