Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages.

By using stage-specific monoclonal antibodies, an in vitro model has been developed to analyze the kinetics of expression of stage-specific antigens during the conversion process between tachyzoites and bradyzoites of Toxoplasma gondii. Following infection of murine macrophages with bradyzoites, the expression of bradyzoite-specific antigens declined, whereas the expression of tachyzoite-specific antigens increased during the first 72 h postinfection. Conversely, in gamma interferon-treated murine macrophages infected with tachyzoites, the inhibitory effect of gamma interferon on replication of parasites was accompanied by the induction of bradyzoite-specific antigens.     

Strain-specific antigens of Toxoplasma gondii.

A detailed immunological assessment of strain-specific antigens of Toxoplasma gondii has not been reported. We developed rabbit antisera against three strains of toxoplasma obtained from divergent sources. These strains included the frequently studied laboratory strain RH, strain C, obtained from a naturally infected kitten, and strain P, which is maintained by passage in mice. The rabbit antisera were used to identify unique strain-specific and commonly shared tachyzoite antigens by radioiodination followed by immunoprecipitation and Western blot analysis. Both qualitative and quantitative differences of a number of the major tachyzoite antigens were found in these assays. A parasite plaque reduction assay using parasiticidal monoclonal antibody showed marked differences in the ability to kill these three different tachyzoite subtypes, further supporting antigenic variation among T. gondii strains.     

Identification of stage-specific antigens of Toxoplasma gondii.

An immunologic evaluation of the surface antigens of the three major life-cycle stages of Toxoplasma gondii was performed. Mouse antisera were raised against these stages, which included the oocyst-sporozoite (feline-excreted stage), bradyzoite (chronic tissue cyst stage), and tachyzoite (invasive stage). The antisera were used in an enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to demonstrate the presence of stage-specific antigens. These antigens were of various molecular weights and were specific to each stage investigated. Cross-reaction studies showed that the mouse antisera recognized commonly shared antigens to at least two of the three stages. A panel of monoclonal antibodies identified specific immune epitopes unique to each of the stages investigated. These studies further support the hypothesis that stage-specific antigens are present in T. gondii.     

Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion.

Stage conversion between tachyzoites and bradyzoites of Toxoplasma gondii was investigated in vitro by using murine bone marrow-derived macrophages (BMMs) as host cells. Following infection of untreated BMMs with tachyzoites, spontaneous expression of bradyzoite-specific antigens (Bsa) occurred at low frequency with Toxoplasma strain-dependent ratios from 0.03 to 2%. As previously described for peritoneal macrophages, activation of tachyzoite-infected BMMs with gamma interferon (IFN-gamma) or lipopolysaccharide resulted in the induction of Bsa. When bradyzoites were used for infection, prolonged expression of Bsa could be observed in IFN-gamma-activated BMMs. The induction of Bsa expression seemed to be closely linked to parasite multiplication and increased to maximal values of 50 to 70% in intermediately activated macrophages with nitric oxide (NO) levels that allowed reduced parasite replication. Identical results in stage conversion were obtained when sodium nitroprusside was used as a source of exogenous NO, indicating that NO might be a molecular trigger of stage conversion. NO is reactive with iron-sulfur centers in proteins, thereby inhibiting proteins involved in the mitochondrial respiratory chain. Using oligomycin and antimycin A as inhibitors of mitochondrial function, growth inhibition of parasites and induction of Bsa were obtained. Since microglia are the functional correlates of macrophages in the central nervous system and inhibit T. gondii upon activation with IFN-gamma, a similar mechanism might be involved during cyst development in the brain.     

Cell free synthesis of Toxoplasma gondii antigens

Functionally active poly(A)-containing mRNA was isolated from tachyzoites of the RH strain of Toxoplasma gondii. The T. gondii mRNA was capable of directing the synthesis of proteins in a wheat germ in vitro translation system, but not in a cell free system derived from rabbit reticulocyte lysate. Efficient translation in the wheat germ system required spermine and exogenous tRNA. Amino acid incorporation was maximal at 110 mM K+ and 1.8 mM Mg2+. Tachyzoite antigens synthesized in vitro were immunoprecipitated with T. gondii antibodies from rabbits, mice and humans. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated polypeptides yielded patterns that differed according to antibody source, but all T. gondii antibody preparations reacted with a translation product with an apparent molecular weight of 24 000.     

Detection of Toxoplasma gondii antigens by a dot-immunobinding technique.

A sensitive assay for the detection of antigens of Toxoplasma gondii by spotting samples directly onto nitrocellulose paper was developed. The sensitivity ranged from 10 to 40 pg of antigen diluted in phosphate-buffered saline and 40 to 130 pg of antigen diluted in normal mouse serum, normal human serum, or human cerebrospinal fluid. T. gondii antigen in serum samples taken from mice infected with T. gondii was detectable by day 2 of infection. Antigen was also detectable in cerebrospinal fluid samples taken from four of six infants congenitally infected with T. gondii and in serum samples from two of these infants.     

Identification of novel bradyzoite-specific Toxoplasma gondii genes with domains for protein-protein interactions by suppression subtractive hybridization

By using suppression subtractive hybridization we identified five so far uncharacterized stage specific genes in Toxoplasma gondii, which are induced during tachyzoite-to-bradyzoite differentiation. The mRNA level of a putative zinc-finger protein was increased 23-fold in bradyzoites, while the remaining four genes displayed induction levels >100-fold. Two of these genes predict proteins with domains for protein-protein interactions. One protein (ANK1) contains both, a TPR-domain and an ankyrin motif, which consists of seven repeats. ANK1 was shown by epitope tagging experiments to be localized in the cytosol. In a fraction of parasites, the myc-tagged fusion protein was additionally localized in the nucleus, which is in agreement with the presence of a bipartite nuclear targeting sequence in ANK1. The identification of bradyzoite-specific proteins with TPR- and ankyrin-domains supports the concept that during stage conversion a variety of proteins which are involved in protein-protein interactions are induced, thereby assisting the rebuilding of the proteome.     

Monoclonal antibodies identify new Toxoplasma gondii soluble antigens

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.     

Characterization of microneme proteins of Toxoplasma gondii

Three microneme proteins of Toxoplasma gondii have been characterized using 3 monoclonal antibodies and a recombinant protein specific antiserum. In all cases, apical labeling of tachyzoites and bradyzoites was observed by indirect immunofluorescence assay. Immunogold localization on ultrathin sections of bradyzoites or tachyzoites showed a specific labeling of micronemes. The following proteins were characterized using 2-dimensional gel electrophoresis and Western immunoblotting: Mic 1 (60 kDa, Pi 6.5), Mic 2 (120 kDa, Pi 5) and Mic 3 (90 kDa, Pi 6.75). The 90-kDa protein (Mic 3) is a heterodimer of two 38-kDa polypeptides (Pi 6.7 and 6.75 respectively) linked by disulfide bridges. Metabolic labeling and immunoprecipitation assays showed that at least one of the 38-kDa polypeptides was processed from a 40-kDa precursor. No processing was observed during the biosynthesis of the 120- and 60-kDa polypeptides.     

Use of acute-stage-specific antigens of Toxoplasma gondii for serodiagnosis of acute toxoplasmosis.

Antisera to acetone-fixed tachyzoites were prepared by immunizing rabbits intravenously with the fixed organisms. Immunoblots in which purified immunoglobulin G (IgG) of the antisera was used recognized four antigens of a supernatant (after centrifugation at 10,000 x g for 30 min) fraction of sonicated tachyzoites. A purified toxoplasma antigen fraction, referred to as acute-stage-specific (AC) antigen, that contained mainly three antigens was obtained by affinity chromatography by using the purified IgG. One antigen had a molecular weight of approximately 52,000, and two antigens had molecular weights of approximately 6,000. An enzyme-linked immunosorbent assay (AC-ELISA) in which this purified antigen fraction, obtained by affinity chromatography, was used for detection of IgG antibody detected early acute infection in sera of patients with toxoplasmic lymphadenopathy. Ninety-two percent of sera obtained within 2 months after onset of lymphadenopathy were positive in the AC-ELISA, whereas only 9% of sera obtained later than 5 months after onset of the clinical illness were positive. The AC-ELISA appears highly sensitive and specific for diagnosis of acute toxoplasmosis.     

Detection of circulating antigens in sera of rabbits infected with Toxoplasma gondii.

Experiments were performed to investigate the usefulness of an enzyme-linked immunosorbent assay utilizing avidin-biotin interaction as a diagnostic tool for detection of Toxoplasma antigen in blood. The lower limit of sensitivity of the assay by this method was ca. 4 ng/ml, and standard assays provided a linear plot of antigen concentration over a range up to 250 ng/ml. In rabbits inoculated subcutaneously with trophozoites of the RH strain, Toxoplasma antigen became demonstrable in the circulation 3 days after injection, before emergence of antibody in serum and development of parasitemia. Analysis of the antigen in serum from infected rabbits by high-permeation liquid gel chromatography suggested the occurrence of antigens of four different molecular weights, greater than or equal to 400,000, 220,000, 130,000, and 45,000. Of these antigens, those of molecular weights 220,000 and 130,000 showed a conspicuous elevation with time after infection.     

Recognition of tachyzoite and bradyzoite antigens of Toxoplasma gondii by infected hosts.

Western immunoblots of tachyzoite and bradyzoite antigens of Toxoplasma gondii were probed with antisera from rabbits and mice at intervals between 2 and 8 weeks after infection and with human antisera with various titers of antibody. With rabbit and mouse antisera, two groups of antigens with molecular masses of 54 to 63 kDa (designated 58.5-kDa antigens) and 26 to 29 kDa (designated 27.5-kDa antigens) were demonstrated commonly for both stages, while those antisera reacted strongly with tachyzoite (but not bradyzoite) antigens with molecular masses of 29 to 54 kDa. Tachyzoite antigens of 21.5, 26.5, 31, 38, 40, 49, and 58 kDa reacted with antisera 2 to 4 weeks after infection, while bradyzoite antigens of 27, 51, 220, and 290 kDa reacted with antisera obtained 4 or more weeks after infection. The 58.5-kDa antigens of both stages reacted primarily with human antisera that had low titers of anti-T. gondii antibodies. Human (as well as rabbit and mouse) sera with high antibody titers reacted with the 27.5-kDa antigens as well as the 58.5-kDa antigens, but the reactivity of the 27.5- to 58.5-kDa antigens of tachyzoites was greater than that of bradyzoites.     

Serological reactivity against cyst and tachyzoite antigens of Toxoplasma gondii determined by FAST-ELISA.

AIMS--To obtain quantitative data on the human serological response to Toxoplasma gondii tachyzoite and bradyzoite antigens. METHODS--Serum samples from 30 patients who had positive antibody titres against T gondii and from 14 who were seronegative, together with sequential serum samples from four infected individuals, were screened by FAST-ELISA. RESULTS--Serum samples from the 30 seropositive patients showed high IgG and IgM titres against the T gondii tachyzoite antigen but very low responses to cyst antigen. This result was borne out in sequential serum samples from patients with toxoplasmosis. CONCLUSION--Antibody recognition of the cystic stage of T gondii is low, implying that either this stage is poorly immunogenic or that the antigen load is low.     

Role of lymphocyte blastogenesis to Toxoplasma gondii antigens in containment of chronic, latent T. gondii infection in humans.

Lymphocyte blastogenic transformation in response to Toxoplasma lysate antigen was markedly impaired in six of eight patients with chronic, latent Toxoplasma gondii infection and treated Hodgkin's disease. None of these patients with serum antibody to T. gondii measured by the Sabin Feldman Dye test and impaired lymphocyte transformation to T. gondii antigens had clinical or serologic evidence of disseminated, active infection with T. gondii. Partial depletion of adherent mononuclear leukocytes improved the impaired lymphocyte transformation of three of six patients; treatment of cultures from all patients with indomethacin improved their blastogenic transformation but culture with normal heterologous serum did not. These studies indicate that lymphocyte blastogenic response to T. gondii antigens is impaired in some patients with chronic, latent T. gondii infection and treated Hodgkin's disease but that this impairment of lymphocyte function is not sufficient to cause reactivation of chronic, latent T. gondii infection.     

Antigen specific lymphocyte transformation induced by secreted antigens from Toxoplasma gondii

Secreted (TSA) and water lysed (WLA) antigens derived from cell culture of the RH strain of Toxoplasma gondii have been used to induce antigen specific mitogenesis of lymphocytes from patients with symptomatic and asymptomatic toxoplasmosis. Lymphocyte responsiveness to WLA was similar to previous reports, with about 50% of patients showing a false negative reaction. Responses to TSA however were highly specific, with no false negative reactions. This increased specificity was not due to an increased response against TSA by patients' lymphocytes (P less than 0.001), but a lower TSA response by uninfected subjects' lymphocytes (P greater than 0.1) compared with WLA in both cases. In a minority of both infected and uninfected subjects, there was a low but detectable response to antigens secreted by the host cell line (HCA), and this was directly compared to their responses against TSA. There was at least a 10-fold increase in the patients' responses to TSA when compared with HCA (P less than 0.001), whereas there was no significant difference between the uninfected subjects' responses to these antigens (P greater than 0.1). Preliminary observations have suggested that TSA is distinct from other defined secreted antigens as both heat treatment and solid phase immunosorption did not have any noticeable effect on TSA-induced mitogenesis.     

Characterization of a family of rhoptry proteins of Toxoplasma gondii

A monoclonal antibody (McAb 4A7) raised against a rhoptry enriched subcellular fraction of Toxoplasma gondii tachyzoites reacted in immunofluorescence studies with rod-like organelles located in the anterior part of the organisms and gave specific labeling of rhoptries in immunoelectron microscopy. On immunoblots, two major proteins of 55 and 60 kDa were identified by McAb 4A7. Similar results were obtained both by immunodetection and immunoblotting with tachyzoites, bradyzoites and sporozoites. Pulse chase analysis of [35S]methionine labeled tachyzoites demonstrated that the 55 and 60 kDa rhoptry proteins derived from a 66-68 kDa doublet which was processed approximately 30 min after biosynthesis. Two other monoclonal antibodies (McAb 2F8, McAb 2H3) respectively specific for rhoptry proteins of 55 kDa having a 66 kDa precursor and 60 kDa having a 68 kDa precursor were also obtained; we suggest that they recognize separately the two components of the 55-60 kDa rhoptry protein family of Toxoplasma.     

Characterization and in vitro translation of Toxoplasma gondii ribonucleic acid

RNA was extracted from purified tachyzoites of Toxoplasma gondii (RH strain) by sequential centrifugation in guanidine hydrochloride, urea, and lithium chloride. The subunits of the RNA were characterized by denaturing and non-denaturing electrophoresis in agarose gels. Poly(A)⁺-RNA, purified by oligo(dT)-cellulose affinity chromatography, was translated in a rabbit reticulocyte lysate assay and the products were immunoprecipitated with an experimentally infected mouse serum and a naturally infected human serum. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, fluorography of the polypeptides confirmed that the mRNA translated specific parasite antigens.     

Toxoplasma gondii antigens recognized by sequential samples of serum obtained from congenitally infected infants.

The Sabin-Feldman dye test, the immunoglobulin M (IgM) immunosorbent agglutination assay, and the immunoblot technique were used to study the evolution of the antibody response to Toxoplasma gondii and to examine antigens of the organism recognized by antibodies in the sera of 12 congenitally infected infants and 7 mothers. In the sera of eight infants, a significant rise was noted in the dye test titers, while the serum of only one infant demonstrated a late increase in the IgM immunosorbent agglutination assay titer. In each infant and mother, antigens with approximate masses of 35,000 and 115,000 daltons were strongly recognized by IgG antibodies. An antigen(s) with an approximate mass of 4,000 daltons was recognized by IgM antibodies in the sera of each of the mothers but was recognized by antibodies in the sera of only two of the infants.     

Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis.