Monoclonal Antibodies to Toxoplasma Cell Membrane Surface Antigens Protect Mice from Toxoplasmosis1,2

Groups of mice were given an intraperitoneal injection of one of six monoclonal antibodies to Toxoplasma gondii, a mixture of equal amounts of five monoclonal antibodies to T. gondii, or the murine myeloma protein MOPC 21, and challenged with either a highly virulent or moderately virulent parasite strain. Two monoclonal antibodies (FMC 19 and FMC 22) conferred total protection against the moderately virulent challenge, with all mice surviving, whereas 90% of control mice died. FMC 19 and FMC 22 also conferred significant protection against the highly virulent challenge as indicated by a prolonged mean time to death (MTD) of immunized compared with control groups of mice. One monoclonal antibody (FMC 23) and the mixture of five antibodies gave significant protection against the moderately virulent challenge only. Passive immunization with dilutions of FMC 22 antibody indicated that the lowest serum titer needed to confer significant protection to mice against a moderately virulent Toxoplasma challenge was 1/640. Mice challenged with highly virulent tachyzoites that had been preincubated with FMC 22 had a significantly longer MTD than mice challenged with highly virulent tachyzoites that had been preincubated with MOPC 21 or phosphate buffered saline, pH 7.2 (PBS). Immunoprecipitation and autoradiography of radiolabeled tachyzoites confirmed that FMC 19 was directed against a 35,000 molecular weight (mol. wt.) antigen and FMC 22 was directed against a 14,000 mol. wt. fraction. The potential for use of single antigens as protective immunogens in preventing toxoplasmosis is raised.     

Molecular weight analysis of soluble antigens from Toxoplasma gondii

Ultrasonicated Toxoplasma gondii (RH strain) tachyzoites were fractionated into a water-soluble and a deoxycholate-soluble fraction. Polyclonal immune mouse serum was prepared by challenging chronically-infected mice with viable RH strain tachyzoites. The parasite fractions were labelled with 125I, and the radio-labelled antigens were precipitated by the immune mouse serum or a monoclonal anti-Toxoplasma antibody (FMC 20), that reacts only in the indirect hemagglutination antibody test. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitates showed that the water-soluble fraction contained 10 antigenic polypeptides, and the deoxycholate-soluble fraction contained seven antigenic peptides. The FMC 20 reacted against a 98,000-dalton antigen that was present in the water-soluble fraction only.     

Effects of specific monoclonal antibodies to dense granular proteins on the invasion of Toxoplasma gondii in vitro and in vivo

Although some reports have been published on the protective effect of antibodies to Toxoplasma gondii surface membrane proteins, few address the inhibitory activity of antibodies to dense granular proteins (GRA proteins). Therefore, we performed a series of experiments to evaluate the inhibitory effects of monoclonal antibodies (mAbs) to GRA proteins (GRA2, 28 kDa; GRA6, 32 kDa) and surface membrane protein (SAG1, 30 kDa) on the invasion of T. gondii tachyzoites. Passive immunization of mice with one of three mAbs following challenge with a lethal dose of tachyzoites significantly increased survival compared with results for mice treated with control ascites. The survival times of mice challenged with tachyzoites pretreated with anti-GRA6 or anti-SAG1 mAb were significantly increased. Mice that received tachyzoites pretreated with both mAb and complement had longer survival times than those that received tachyzoites pretreated with mAb alone. Invasion of tachyzoites into fibroblasts and macrophages was significantly inhibited in the anti-GRA2, anti-GRA6 or anti-SAG1 mAb pretreated group. Pretreatment with mAb and complement inhibited invasion of tachyzoites in both fibroblasts and macrophages. These results suggest that specific antibodies to dense-granule molecules may be useful for controlling infection with T. gondii.     

Epitope-selected monospecific antibodies to recombinant antigens from Toxoplasma gondii reacted with dense granules of tachyzoites.

Epitope-selected monospecific antibodies were applied to investigate the localization of antigenic molecules in Toxoplasma gondii by immunoelectron microscopy. Eighty cDNA clones encoding antigenic polypeptides were immunoscreened from lambda gt11 expression library with T. gondii infected mouse sera. Twenty different clones with no crossreactivity were selected from eighty clones. Monospecific antibodies to antigens derived from respective cDNA clones extracted from infected mouse sera by the epitope selection method were used in Western blot analysis and immunoelectron microscopy. Eleven antigens were detected with epitope-selected antibodies in lysates of T. gondii tachyzoites. Five of the antigens with molecular weights of 60, 40, 35, 28, and 27 KD were localized in the dense granules. Monospecific antibodies purified by the epitope selection method were useful for investigating the localization of antigens without preparation of a monoclonal antibody from a hybridoma.     

Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies.

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, can infect a broad host range and is regarded as an important cause of bovine abortion worldwide. In the present study, four antigens of N. caninum were partially characterized using monoclonal antibodies. Immunofluorescence of viable tachyzoites as well as the immunoprecipitation of antigens extracted from tachyzoites previously labeled by surface biotinylation revealed that three of these antigens with apparent molecular weights of 40, 38, and 19 kDa are located in the outer surface membrane of this parasite stage. Further evidence for the surface localization of the 38-kDa antigen was obtained by immunoelectron microscopy. In addition to the surface molecules, an antigen located in dense granules and in the tubular network of the parasitophorous vacuole was detected by another monoclonal antibody. When tachyzoite antigens separated under nonreducing conditions were probed on Western blots, this antibody reacted mainly with a 33-kDa antigen. Immunohistochemical analysis of infected tissue sections indicated that the 33-kDa dense granule antigen is present in both tachyzoites and bradyzoites, while the 38-kDa surface antigen from tachyzoites seems to be absent in bradyzoites.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Analysis of the humoral responses of Toxoplasma gondii-infected cats using immunofluorescent assays with tachyzoite, bradyzoite, and gametogenic stages

We investigated levels of Toxoplasma gondii specific antibodies present in sera, intestinal secretions, and fecal extracts obtained from cats following primary and challenge infections. Antibodies specific to T. gondii tachyzoites, bradyzoites, sporozoites, and enteroepithelial stages were detected by indirect immunofluorescence assay. Enteroepithelial stage-specific antibodies were detected in serum as early as 2 wk after infection, whereas antibodies from intestinal secretions did not appear until 3 wk following infection. The T. gondii-specific IgG and IgA antibodies were present in serum, but only specific IgA antibodies were detected in the intestinal secretions. Serum IgG bound to tachyzoites, bradyzoites, sporozoites, and enteroepithelial stages of T. gondii, whereas serum IgA bound strongly to enteroepithelial stages but only weakly to tachyzoites and bradyzoites. IgA from intestinal secretions bound to antigens on all enteroepithelial stages and the distal tips of sporozoites and bradyzoites but did not bind to tachyzoites. IgA present in fecal extracts also bound to enteroepithelial stages of T. gondii. Toxoplasma gondii infection in cats induces the production of antibodies that bind with all forms of the parasite, including the enteroepithelial stages. Comparison of the staining patterns of T. gondii stages for serum and intestinal secretion IgA indicated differences. Thus, the intestinal antibody immune response may be uniquely focused on the intestinal stages relative to the circulating antibodies, resulting in a compartmentalization of the humoral response.     

Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites

Since we had previously demonstrated the protective role played by Toxoplasma excreted-secreted antigens, the aim of the present work was to produce monoclonal antibodies directed against these antigens in order to determine if their localization in the parasite is compatible with a mechanism of excretion or secretion. Western immunoblotting analysis revealed three monoclonal antibodies (TG17-179, TG17-43, and TG17-113) raised against excreted-secreted antigens of 28.5, 27, and 21 kDa, respectively. The TG17-179 which reacts with antigens isolated by Concanavalin A affinity chromatography is directed against a glycosylated 28.5-kDa component. Colloidal immunogold labeling showed the ultrastructural localization of the 21-, 27-, and 28.5-kDa antigens in the matrix of the dense granules of tachyzoites and associated with the microvilli network of the parasitophorous vacuole, after host cell invasion. These observations suggest the following mechanism of Toxoplasma secretion: secreted antigens are first stored in tachyzoite-dense granules and are then released inside the parasitophorous vacuole. Among the secretory molecules characterized here, the native 27-kDa antigen recognized by TG17-43 is a calcium-binding protein found to be intermixed with the 21- and 28.5-kDa antigens inside the dense granules and hence could play a role in the packaging of secretory products. In addition, the 21- and 28.5-kDa antigens were also located beneath the parasite plasma-lemma. This particular location could reflect a transient step characteristic of T. gondii secretion.     

Tachyzoite-induced life cycle of Toxoplasma gondii in cats

The tachyzoite-induced cycle of Toxoplasma gondii was studied in 46 cats. Tachyzoites of the M-7741 or Me-49 strain of T. gondii were administered orally to cats by pouring into the mouth or by stomach tube, or by intraintestinal inoculation. Ten weaned cats that had been inoculated with tachyzoites directly in the intestine were killed 1, 3, 6, 9, 12, 15, 18, or 25 days later, and their tissues were studied histologically and bioassayed in mice. Toxoplasma gondii was demonstrable in the blood of 8 cats and in other tissues of all these 10. Four out of five 1- to 8-day-old cats fed tachyzoites by stomach tube became infected with T. gondii, and 1 became ill because of toxoplasmosis. All 19 weaned cats fed tachyzoites (poured into the mouth) became infected, and 6 died of acute toxoplasmosis 9-15 days after being fed T. gondii. Six out of 12 weaned cats fed tachyzoites by stomach tube became infected but were asymptomatic. Overall, 12 out of 26 cats observed for 19 days or more shed oocysts with a prepatent period (pp) of 19 days or more, with the sole exception of 1 cat that shed oocysts with a pp of 5 days. Enteroepithelial stages of T. gondii were not found in any cat before oocysts were shed. Cats shed up to 360 million oocysts in a day, and oocysts were shed for 4-6 days.     

Selection and identification of dense granule antigen GRA3 by Toxoplasma gondii whole genome phage display

Toxoplasma gondii is a ubiquitous, unicellular, eukaryotic parasite with a complex intracellular life cycle capable of invading and chronically infecting a wide variety of vertebrate host species, including man. Although normally opportunistic in healthy adults, it is a lethal pathogen in immunocompromised humans, particularly in AIDS patients. We present the application of a genomic phage display as a tool for the direct identification of antigens with potential value in diagnosis and/or as subunit vaccine components. Using a polycosmid cloning strategy, we constructed a large phagemid display library (>10(9) independent clones) of mixed short genomic restriction fragments (< or = 500 bp) of T. gondii genomic DNA (80 Mbp genome size) fused to gene III of the filamentous phage M13. Biopanning of the library with monoclonal Toxoplasma antibodies resulted in the isolation and identification of an epitope of GRA3, an antigen located in the dense granules of T. gondii tachyzoites. The reactivity of the phage displaying the GRA3 epitope with the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. These results demonstrate the accessibility of midsized eukaryotic genomes to display technology and the feasibility to screen these whole genome display libraries with antibodies for isolating novel antigenic determinants.     

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite

Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein–protein or protein–carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii .     

Prevalence of Toxoplasma gondii antibodies in sera of domestic pigs and some wild game species from Zimbabwe

Serum samples of domestic pigs (Sus scrofa), elands (Taurotragus oryx), sable antelopes (Hippotragus niger), warthogs (Phacochoerus aethiopicus), bushpigs (Koiropotamus [Potamochoerus] koiropotamus), white rhinos (Ceratotherium simus), African buffalos (Syncerus caffer), wildebeest (Connochaetas taurinus), and African elephants (Loxodonta africana) from Zimbabwe were tested for Toxoplasma gondii IgG antibodies by the modified agglutination test (MAT) with whole formalized tachyzoites and mercaptoethanol. Sera were diluted at 1:25, 1:50, and 1:500 for MAT testing. Sera with antibodies in a 1:25 dilution were considered to have T. gondii infection. Toxoplasma gondii antibodies were found in 9.3% of 97 domestic pigs, 36.8% of 19 elands, 11.9% of 67 sables, 0 of 3 warthogs, 0 of 3 bushpigs, 50% of 2 white rhinos, 5.6% of 18 buffalos, 14.5% of 69 wildebeest, and 10.5% of 19 elephants examined.     

Use of monoclonal antibodies for flow cytometric detection of intracellular Toxoplasma gondii in murine splenic lymphocytes

Various monoclonal antibodies (mAbs) against Toxoplasma gondii RH tachyzoites were used for flow cytometric detection of intracellular parasites in murine splenic lymphocytes. Tg110 and Tg563 (reacting with the major surface protein SAG1), Tg505 (with another surface protein SAG2), Tg695 and Tg786 (with rhoptry proteins), Tg507, Tg621, and Tg317 (with dense granule proteins), Tg536 (with a microneme protein), and Tg685 (with a cytosol antigen) were the mAbs used. After an in vitro infection of lymphocytes with tachyzoites and reactions with the different mAbs, flow cytometry was performed using an indirect immunofluorescent technique. The proportions of whole infected lymphocytes and of each infected lymphocyte phenotype, CD4+ T cells, CD8+ T cells, and B cells, were determined, and their fluorescent intensities were quantified. The best reaction was seen when Tg110 or Tg695 was used as the mAbs. The results suggest that mAbs against surface or rhoptry proteins are highly useful for the flow cytometric detection of intracellular T. gondii in host cells.     

Toxoplasma gondii: redistribution of monoclonal antibodies on tachyzoites during host cell invasion

Immunodetection of protein P30, a major surface antigen of Toxoplasma gondii tachyzoites, by a specific monoclonal antibody has demonstrated a homogenous distribution of this antigen on the surface of intra- and extracellular tachyzoites at all stages of their endodyogenic development. On living zoites, no redistribution of anti-P30 was obtained, contrasting with the capping obtained with antiserum to T. gondii. Upon invasion of a host cell, however, most of the coat of anti-P30 was shed from preincubated zoites at the level of the moving junction governing the entry of the parasite into the host cell.     

Detection of Toxoplasma gondii by polymerase chain reaction in sera of acutely infected mice

The presence of Toxoplasma gondii DNA was detected in sera of acutely infected mice by polymerase chain reaction. Adult mice were inoculated intraperitoneally with 5 x 10(3) T. gondii RH strain tachyzoites. Five mice were killed every 3 hr from 3 to 21 hr post infection (PI) and every day from 1 to 7 days PI. Toxoplasma gondii DNA was first detected in 60% of the infected mice 18 hr PI and in 100% of the animals 21 hr PI and from 1 to 7 days PI. No mice survived longer than 7 days.     

Vero cell surface proteoglycan interaction with the microneme protein NcMIC(3) mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii

Neospora caninum and Toxoplasma gondii are characterised by a very low host cell specificity, thus they are able to infect a wide range of different cells in vivo and in vitro. Infection of the host cell by tachyzoites is a process which is preceded by adhesion onto the host cell surface. The receptors on the host cell surface which would allow N. caninum to establish a physical interaction have not been investigated so far. Here we report the role of host cell surface proteoglycans as receptors for the adhesion of N. caninum tachyzoites to Vero cell monolayers. We found that N. caninum tachyzoites, similar to T. gondii tachyzoites, can bind to sulphated proteoglycans which naturally occur on the surface of mammalian cells, including heparin/heparan sulphate, chondroitin sulphates, as well as to the artificially sulphated glycosaminoglycan dextran sulphate. Although removal of heparan sulphate from the host cell surface results in decreased adhesion of T. gondii tachyzoites, binding of N. caninum tachyzoites is not affected by this treatment. Conversely, enzymatic removal of chondroitin sulphate A, B and C decreases N. caninum adhesion but does not affect T. gondii binding to Vero cells. Thus, T. gondii and N. caninum tachyzoites exhibit differential adhesive properties with regard to host cell surface glycosaminoglycans. Additional experiments employing Triton X-100 solubilised NcSRS2 and NcMIC3 showed that NcSRS2 binds to the host cell surface, but not through those sulphated glycosaminoglycans investigated in this study. In contrast, NcMIC3 binding to the host cell surface is dramatically influenced by these modifications. Further experiments showed that the NcMIC3 adhesive motif comprised of four consecutive epidermal growth factor-like domains expressed as a recombinant protein exhibits a high binding activity for sulphated glycosaminoglycans. These results suggest that host cell surface proteoglycan interaction of N. caninum differs from that observed for T. gondii, and that the epidermal growth factor-like adhesive motif in NcMIC3 could be involved in this process.     

Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens.

The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with 3H-fatty acids, [3H]ethanolamine, and [3H]carbohydrates. Treatment of 3H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.     

Laboratory passage and characterization of an isolate of Toxoplasma gondii from an ocular patient in Korea

Toxoplasma gondii tachyzoites were isolated from the blood of an ocular patient, and have been successfully passaged in the laboratory, for over a year, by peritoneal inoculation in mice. The isolated parasite was designated the Korean Isolate-1 (KI-1) and its characteristics were compared with those of the RH strain, a wellknown virulent strain originating from a child who suffered from encephalitis. The morphology, pathogenicity, infectivity and cell culture characteristics of the KI-1 were similar to those of the RH strain. Both RH and KI-1 antigens were detected by an anti-T. gondii monoclonal antibody (mAb), Tg563, against the major surface protein SAG1 (30 kDa), whereas no reaction was observed against an anti-Neospora caninum mAb, 12B4. The KI-1 was confirmed as an isolate of T. gondii. A long-term laboratory maintenance and characterization of a local T. gondii isolate is reported for the first time in the Republic of Korea.     

Fatal toxoplasmosis in a bald eagle (Haliaeetus leucocephalus)

Toxoplasma gondii tachyzoites were identified in the myocardium of a bald eagle (Haliaeetus leucocephalus) that died of necrotizing myocarditis. The diagnosis was confirmed by immunohistochemical staining with T. gondii-specific polyclonal antibodies. This is a new host record for T. gondii.