Induction of antigen-specific parasiticidal cytotoxic T cell splenocytes by a major membrane protein (P30) of Toxoplasma gondii

Infection with Toxoplasma gondii has become a major cause of morbidity in patients with AIDS. To investigate the mechanisms responsible for immune responses to toxoplasma Ag we used a highly purified membrane protein (P30) of T. gondii to stimulate an in vitro Ag-specific cytotoxic T cell response. P30 immune mouse splenocytes reduced extracellular T. gondii plaque-forming units by more than 50% when incubated at an E/T ratio of 10:1 or greater. By using a [3H]uracil radioisotope release assay, the effect of the immune splenocytes was determined to be a direct parasite lytic mechanism. The immune splenocytes were P30 Ag specific and of the Thy 1.2, Lyt2,3+ (CD4-, CD8+) phenotype, specific for mouse cytotoxic T cells. Opsonization of the parasites with monoclonal P30-reactive mAb did not enhance parasiticidal activity. Culture supernatants obtained during the 2-h cytotoxic assay were not parasiticidal, and anti-asialo-GM1 antibody plus C did not destroy the parasiticidal activity of the P30 responder cells. Accordingly, we have identified an Ag-specific subset of CD4-, CD8+, P30 responder T cells that are directly parasiticidal to extracellular T. gondii, and that exhibit cytotoxicity independent of antibody opsonization, lymphokine secretion, NK cell activity, and, apparently, MHC involvement as well.     

An unexpected response to vaccination with a purified major membrane tachyzoite antigen (P30) of Toxoplasma gondii

A purified Toxoplasma gondii tachyzoite membrane protein (P30) and a monoclonal antibody directed against this antigen were used to immunize mice. The P30 protein has an apparent m.w. of 30,000 and is the major radioiodinated tachyzoite membrane antigen identified by human and mouse antitoxoplasma antisera. Polyclonal and monoclonal antibody to the P30 antigen are parasiticidal in the presence of human serum. A series of mice were immunized with affinity column-purified P30 protein. This produced a dose-dependent, antigen-specific IgG and IgM response. The mice were challenged with the less virulent C strain tachyzoite. Immunized mice showed a statistically significant increase in mortality over nonimmunized control mice. In addition, vaccinated mice had an increased number of intracerebral tissue cysts when compared with the control group. Similar results were obtained with passive transfer immunization by using monoclonal antibody directed against the P30 antigen. Immunofluorescence assay of brain tissue cyst bradyzoites revealed a total absence of P30 antigen. Bradyzoites were also deficient in another major tachyzoite antigen of approximate m.w. 22,000 (P22). Mouse antibradyzoite serum absorbed with tachyzoites recognized bradyzoites but failed to identify tachyzoites. This suggests that there are stage-specific bradyzoite antigens of Toxoplasma gondii.     

Molecular weight determination of two genetically linked cell surface murine antigens: ThB and Ly-6

Various murine tumor lines were screened by FACS analysis for the surface antigens ThB and Ly-6.2. Positive cell lines were used for immunoprecipitation studies. A monoclonal ThB-specific antibody immunoprecipitated a unique acidic protein of approximately 16 000 daltons from several positive tumors and from concanavalin A (Con-A) and LPS activated splenic lymphocytes. Monoclonal Ly-6.2-specific antibody was used to immunoprecipitate a 33 500 dalton protein that was shown to exist in four similarly sized forms with different basic charges. In the course of these studies, the apparent molecular weight of the surface antigen T 30, immunoprecipitated with a monoclonal T 30-specific antibody from the cell line EL4, was found to be approximately 25 000 daltons.     

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.     

Characterization of a new gene WX2 in Toxoplasma gondii

Using hybridization techniques, we prepared the monoclonal antibody （Mab） 7C3-C3 against Toxoplasma gondii. The protection tests showed that the protein （Mab7C3-C3） inhibited the invasion and proliferation of T. gondii RH strain in HeLa cells. The passive transfer test indicated that the antibody significantly prolonged the survival time of the challenged mice. It was also shown that the antibody could be used for the detection of the circulating antigen of T. gondii. After immunoscreening the T. gondii tachyzoite cDNA library with Mab7C3-C3, a new gene wx2 of T. gondii was obtained. Immunofluorescence analysis showed that the WX2 protein was located on the membrane of the parasite. Nucleotide sequence comparison showed 28% identity to the calcium channel α-IE unit and shared with the surface antigen related sequence in some conservative residues. However, no match was found in protein databases. Therefore, it was an unknown gene in T. gondii encoding a functional protein on the membrane of T. gondii. Because it has been shown to have a partial protective effect against T. gondii infection and is released as a circulating antigen, it could be a candidate molecule for vaccine or a novel target for new drugs.     

Purification and Characterization of Neuron-Specific Surface Antigen Defined by Monoclonal Antibody BM88

Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N-Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.     

Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplasma gondii P30 antigen.

The first part of this work presents the sequence of the first 20 NH2 terminus residues obtained from P30, the major surface Ag of Toxoplasma gondii, purified by HPLC. A synthetic peptide (P30 48-67) has been prepared both in linear form and as a multiple antigenic peptide (MAP) construct. Immunization of mice and rats with the P30 48-67 MAP in the presence of IFA induces high levels of IgG antibodies that recognize both the linear peptide and the MAP construct in ELISA, and P30 in Western blots of NP-40-extracted tachyzoite Ag. Because these sera are negative in immunofluorescence assays with whole tachyzoites, it seems that IgG antibodies induced by P30 48-67 MAP, although recognizing the denatured structure, are unable to recognize the native protein. However, the protective effect of both constructs has then been studied in mice and nude rats. Whereas immunization of mice with the monomeric peptide does not confer any protection against oral infection with 1200 cysts of T. gondii 76K strain (mortality within 11 days), 40% of mice immunized with the MAP construct survived up to 75 days after infection. Nude rats were passively transferred with 5 x 10(4) T lymphocytes from P30 48-67 MAP-immunized Fischer rats before infection with 5 x 10(4) RH strain tachyzoites. They survived up to 40 days after infection and raised an intense IgG antibody response against P30, whereas nude rats transferred with control lymphocytes died within 21 days. This shows that immunization with P30 48-67 MAP also induces an efficient T cell immune response. The present work confirms the recently demonstrated role of P30 in protective immunity and shows the interest of peptide octameric constructions as inducers of partially protective immune responses in toxoplasmosis, as already demonstrated in schistosomiasis.     

Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.     

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin

Thrombospondin, a 450-kDa glycoprotein composed of three disulphide linked chains, is located in human blood platelet alpha-granules and is released from platelets upon stimulation. This glycoprotein is thought to play a major role in platelet aggregation. The aim of this study was to characterize two monoclonal antibodies (P10 and P12) directed against human blood platelet thrombospondin. When the released material obtained after stimulation of platelets with thrombin in the presence of 2 mM calcium was immediately treated with EDTA, labelled with 125I and incubated with monoclonal antibodies P10 and P12, both immunoprecipitated a major labelled protein band with a molecular mass of 160 kDa and a weaker band at 146 kDa, as analysed on reduced dodecyl sulphate/polyacrylamide gels. The major band corresponds in molecular mass to the thrombospondin subunits. If, however, the released material was left in the presence of Ca2+ for 48 h, then the main band was at 130 kDa and in addition one minor protein band (75 kDa) was immunoprecipitated by P10 whereas P12 recognized two minor protein bands (75 and 60 kDa). When P10 and P12 were incubated with 125I-labelled platelet releasates treated for 48 h at 4 degrees C with 10mM EDTA, three major protein bands (160, 146 and 130 kDa) were immunoprecipitated in addition to the minor bands mentioned above. These results indicate that thrombospondin is probably degraded by the endogenous platelet calcium-dependent protease. Investigation of tryptic peptide fragments of thrombospondin isolated by fast protein liquid chromatography showed that 125I-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas 125I-labelled P12 only recognized a 400-kDa fragment. Competition studies involving solid-phase antibody binding and double antibody sandwich assays showed that P10 and P12 were directed against different determinants of thrombospondin. Purified thrombospondin, isolated in the presence of calcium, either directly or after treatment with EDTA, haemagglutinated trypsinized, formaldehyde-fixed sheep erythrocytes identically. The haemagglutination activity of EDTA-treated thrombospondin was inhibited by P10 and enhanced by P12. On the other hand, P10 and P12, despite their binding to calcium-treated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies P10 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation.     

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.     

Use of recombinant antigens for detection of Toxoplasma gondii infection in swine

Five recombinant Toxoplasma gondii antigens, designated B427, C51, C55, V22, and MBP30 were assessed for their potential use in an enzyme-linked immunoassay (EIA) for detection of T. gondii infection in swine. The antigens were evaluated with sera from young pigs that had been fed 1-10,000 T. gondii oocysts of the VEG or GT-1 strains. Results were compared with an EIA using a native T. gondii antigen extract. All 5 recombinant antigens, as well as native antigen, detected antibody responses as soon as 3 wk after infection in pigs inoculated with 1 or 10 oocysts of the VEG strain. This antibody response persisted, at varying levels, for 14 wk when the experiment was terminated. All antigens also detected antibody responses in pigs 4 wk after inoculation with 10,000 oocysts of the GT-1 strain. The antibody response recognized by native antigen remained high through 51 wk after inoculation. However, there was considerable animal-to-animal variation in responses to the individual recombinant antigens. Only antigens C51 and MBP30 consistently detected a positive antibody response over the entire 51-wk course of the experiment. These results suggest that these antigens might be useful for the serological detection of T. gondii infection in pigs.     

Monoclonal antibodies against the mercaptoethanol-sensitive structure of a cell-cell adhesion protein of Polysphondylium pallidum

Monoclonal antibodies were prepared against a putative cell-cell adhesion glycoprotein, with an apparent molecular mass of 64,000 (gp64), of the cellular slime mold, Polysphondylium pallidum. Five monoclonal antibodies obtained by means of an enzyme-linked immunoadsorbent assay did not bind to the antigens which were subjected to gel electrophoresis and blotting method in the presence of a reducing agent, but they did bind specifically to the antigens prepared in unreducing conditions of samples and then processed by the same blotting method. To solubilize gp64 in a sodium dodecyl sulfate (SDS)-sample buffer without mercaptoethanol (heated) or SDS-sample buffer with 2-mercaptoethanol (nonheated) was critical for the antibody binding onto gp64 on a membrane. Hence the antibodies seem to bind to a surface portion(s) of the localized protein structure folded up by disulfide cross-linkages. One of the antibodies obtained blocked cell-cell adhesion by about 20%.     

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha-32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha-32P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.     

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.     

Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites

Babesia bigemina is one of several tick-borne hemoparasitic diseases of cattle that are inadequately controlled and cause substantial livestock production losses in tropical and subtropical climates. Recovery from acute babesiosis is associated with development of protective immunity against subsequent challenge with both homologous and heterologous parasites. Viable and infectious merozoites, the intraerythrocytic stage of B. bigemina responsible for clinical disease, were separated from contaminating host cells by density gradient centrifugation. Monoclonal antibodies developed against gradient-separated merozoites were screened for surface reactivity against live merozoites in an immunofluorescent binding assay. Surface-reactive antibodies immunoprecipitated five major biosynthetically radiolabeled merozoite proteins with relative m.w. of 72,000, 58,000, 55,000, 45,000, and 36,000 in SDS-PAGE. Two additional proteins immunoprecipitated with the 45,000 m.w. protein were unreactive with monoclonal antibody in western blots and are apparently part of a membrane complex co-precipitated by this antibody. In contrast, additional proteins of m.w. of 36,000, 35,000, and 33,000, immunoprecipitated with the 58,000 protein, all contain the surface-exposed epitope bound by monoclonal antibody. Immune serum from an animal that had recovered from infection with a Mexico isolate of B. bigemina immunoprecipitated five radiolabeled proteins from the Mexico isolate that co-migrated in SDS-PAGE with major proteins precipitated by surface-reactive monoclonal antibodies. In addition, antibodies against a Kenya isolate of B. bigemina immunoprecipitated the same co-migrating proteins from radio-labeled Mexico isolate, demonstrating epitope conservation between surface proteins of geographically different isolates. The identification of proteins with epitopes exposed on the surface of live merozoites and accessible to antibody provides candidates to be tested as protective immunogens in cattle.     

Bovine Herpesvirus 1 Glycoprotein M Forms a Disulfide-Linked Heterodimer with the UL49.5 Protein

Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U_L10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U_L10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U_L49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448–1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U_L49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.     

Antigen-specific (p30) mouse CD8+ T cells are cytotoxic against Toxoplasma gondii-infected peritoneal macrophages.

The importance of CD8+ T cells in immunity against Toxoplasma gondii is now well recognized. The mechanism by which these CD8+ T cells are able to confer this immunity is not yet understood. To examine the Ag specificity of this response, immune splenocytes from mice immunized with p30, a major surface parasite Ag, were evaluated for their ability to lyse peritoneal macrophages infected with three different strains of T. gondii. Macrophages infected with either the RH or P wild-type strain tachyzoites were lysed at varying E:T ratios by nylon wool nonadherent immune splenocytes whereas macrophages infected with a p30-deficient mutant (B mutant) of the P strain were not. The gene encoding p30 for the wild type and B mutant were amplified by the polymerase chain reaction. This revealed a nonsense mutation in the B mutant such that its primary translation product is predicted to be about two-thirds the size of the wild-type p30 molecule. mAb depletion studies indicate that the cytotoxic effect of the immune splenocytes is mediated by the CD8+ T cell population. Peritoneal macrophages infected with the three different strains (RH, P wild type, B mutant) from mice genetically restricted were not lysed by the immune CD8+ effector cell population. A cloned line (C3) of p30 Ag-specific CD8+ T cells exhibited significant cytotoxicity against syngeneic peritoneal macrophages infected with either the RH or P strain tachyzoites. There was no macrophage lysis observed by these CD8+ effector cells of either syngeneic macrophages infected with the B mutant or nonsyngeneic macrophages infected with the three different tachyzoite strains.     

A purified parasite antigen (p30) mediates CD8+ T cell immunity against fatal Toxoplasma gondii infection in mice

Induction of protective immunity against acute and chronic toxoplasmosis can be achieved using p30, the major membrane and excreted/secreted protein of Toxoplasma gondii. This protein, when administered to outbred mice in the presence of the saponin Quil A, is able to induce almost 100% protection against acute infection without evidence of intracerebral cyst development. Adoptive transfer of immune splenocytes from immunized inbred A/J mice conferred a significant level (p less than 0.001) of protection against subsequent challenge. Phenotypic analysis in outbred as well as two different strains of inbred mice (A/J and C57BL/6) demonstrated that CD8+ T cells are selectively stimulated by this immunization protocol. T cell depletion studies using specific mAb directed at either CD3+ or CD8+ T cell phenotype, followed by adoptive transfer, failed to confer protective immunity, whereas CD4+ depletion had no effect. These cytotoxic CD8+ T cells produced high titers of both IFN-gamma and IL-2. Moreover, these CD8+ T cells were directly parasiticidal against radiolabeled extracellular T. gondii, further supporting the critical immune function of these p30 Ag-specific CD8+ T cells in host immunity against T. gondii infection.