Molecular mimicry of carbohydrate and protein structures by hybridoma antibodies

A large proportion of tumour-associated antigens seem to be determined by carbohydrate structures. Advances in the study of the antigenicity of cell-surface carbohydrates have been hampered by the absence of advanced monoclonal hybridoma technology comparable to that available for the study of protein antigens. Monoclonal antibodies have been raised against a carbohydrate epitope (43–9F) that is associated with the proliferative features of squamous lung carcinomas. These were used in turn to generate anti-idiotype antibodies with homology to 43–9F. The method and its possible applications are described, together with a procedure to detect rare cell membrane variants within large populations.     

Amphotericin B-induced carbohydrate changes on the Trypanosoma cruzi surface membrane.

Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities. Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCl3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.     

Trypanosoma cruzi: description of a highly purified surface antigen defined by human antibodies

A glycoprotein of 25,000 daltons (G25) purified from T. cruzi extracts is recognized by serum antibodies of Chagas' disease patients. These human antibodies were isolated by affinity chromatography and were used to demonstrate that G25 antigenic determinants are i) represented at the parasite surface, and ii) are expressed in all developmental stages of the parasite's life cycle, as well as in several T. cruzi strains. This antigen-antibody system may be useful for the diagnosis of Chagas' disease because antibodies to radiolabeled G25 are found in the serum of 96.5% of 173 chagasic patients from different endemic areas, but are not found in the serum from other individuals. Taken collectively, the data suggest that antibodies to G25 define highly conserved determinants of the species T. cruzi. Moreover, its remarkable immunogenicity to infected humans offers an opportunity to investigate the role of specific immunologic responses in the pathogenicity of Chagas' disease.     

Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion

Null mutants of the Trypanosoma cruzi insect stage-specific glycoprotein GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromycin phosphotransferase genes were flanked by GP72 sequences. These plasmids were sequentially transfected into T. cruzi epimastigotes by electroporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No aberrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsewhere in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the normal attachment of the flagellum to the cell membrane of the parasite is lost.     

Characterization of epitopes on a variant surface glycoprotein from Trypanosoma congolense by six monoclonal antibodies

Monoclonal antibodies were isolated from mice immunized with variant surface glycoprotein of Trypanosoma congolense. Five out of the six monoclonals were able to detect epitopes at the cell surface in an indirect immunofluorescence analysis. One antibody did not react. Using protein-A-containing bacterial adsorbent all monoclonal antibodies precipitate glycosylated as well as non-glycosylated variant surface glycoprotein. Carbohydrate chains therefore do not appear to be part of the immunodeterminant structure recognized by the various monoclonal antibodies. Interaction of the monoclonal antibodies with protein fragments obtained by partial proteolysis with V8 protease from Staphylococcus aureus or papain allows the classification of the antibodies into three groups with different epitope specificity.     

Partial characterization of the cross-reacting determinant, a carbohydrate epitope shared by decay accelerating factor and the variant surface glycoprotein of the African Trypanosoma brucei

The variant surface glycoprotein (VSG) of the African trypanosome is anchored in the cell membrane by a complex glycan attached to phosphatidylinositol. The carboxyl terminal portion of VSG contains a cryptic carbohydrate epitope, the cross-reacting determinant (CRD), that is revealed only after removal of the diacylglycerol by phosphatidylinositol-specific phospholipase C (PIPLC) or VSG lipase. Recently, we have shown that after hydrolysis by PIPLC, decay-accelerating factor (DAF)--a mammalian phosphatidylinositol-anchored protein--also contains the CRD epitope. Using a two site immunoradiometric assay in which the capturing antibody is a monoclonal antibody to DAF and the revealing antibody is anti-CRD, we now show that sugar phosphates significantly inhibited the binding of anti-CRD antibody to DAF released by PIPLC. DL-myo-inositol 1,2-cyclic phosphate was the most potent inhibitor of binding (IC50 less than 10(-8) M). Other sugar phosphates, such as alpha-D-glucose-1-phosphate, which also possess adjacent hydroxyl and phosphate moieties in cis also inhibited binding at low concentrations (IC50 = 10(-5) to 10(-4) M). In contrast, sugar phosphates which do not possess adjacent hydroxyl and phosphate moieties in cis and simple sugars weakly inhibited binding (IC50 greater than 10(-3) M). These results suggest that myo-inositol 1,2-cyclic phosphate contributes significantly to the epitope recognized by the anti-CRD antibody and is consistent with analysis of the carboxyl terminus of VSG, which also suggested the presence of the cyclic inositol phosphate. In light of the recent findings that human serum contains a glycan-phosphatidyl-inositol-specific phospholipase D, which converts DAF from a hydrophobic to a hydrophilic form lacking the CRD, the observation that the phosphate is crucial for expression of the epitope may be relevant in understanding the origin of CRD-negative DAF in urine and plasma.     

Down-regulation of human B lymphocyte activities by a Trypanosoma cruzi membrane glycoprotein

The effects of purified AGC10, a Trypanosoma cruzi membrane glycoprotein, on normal human B lymphocytes were studied in this work. In the presence of AGC10, [3H]-thymidine uptake by human peripheral blood mononuclear cells stimulated with the B cell-specific mitogen SACI (killed Staphylococcus aureus Cowan I) was markedly decreased. This alteration was accompanied by others such as decreased expression of the CD122 and CD132 chains of the IL-2R complex. These inhibitory effects appeared to be somewhat selective, as expression of CD25, another IL-2R chain, was not affected by AGC10 and no significant modification occurred in the expression of the B-cell-specific marker CD19 or CD21. In contrast, AGC10 did reduce the levels of expression of CD86 and CD80, molecules known to play critical roles in B cell interactions with T lymphocytes. Fairly large subpopulations of, but not all, B lymphocytes had their expression of CD122(+), CD132(+), CD86(+) and CD80(+) reduced to undetectable levels in the presence of AGC10. However, the SACI-activated B cells that remained capable of expressing these molecules in the presence of AGC10 did so at normal levels. This was denoted by comparable mean fluorescence intensity values representing the expression of CD122, CD132, CD86 or CD80 molecules on the surface of SACI-stimulated CD19(+) cells cultured without or with AGC10. These results indicated that AGC10, derived from an organism that causes immunosuppression in infected hosts, down-regulates B cell activities and suggested that the relevant mechanism could involve the molecular alterations described above.     

Studies on the suppression of the immune response to a defined protein epitope by anti-idiotypic antibodies

We have previously reported that C3H.SW (CSW) and A/J mice immunized with the tobacco mosaic virus protein (TMVP) produce antibodies to a decapeptide epitope corresponding to amino acid residues 103-112 of the protein. In the C3H.SW (CSW) strain, the antibodies to the decapeptide contain major crossreactive idiotope, C10-IdX, which is found on a CSW-derived monoclonal antidecapeptide antibody, designated as C10. The in vivo administration of anti-C10 antibodies suppresses the response to the decapeptide epitope in CSW mice. The present communication describes experiments designed to elucidate several parameters responsible for the suppression produced by the in vivo administration of anti-C10. It was found that 50 micrograms of anti-C10 was required to suppress the response to the decapeptide in CSW mice when 2 weeks elapsed between administration of the anti-C10 and immunization with TMVP; however, only 10 ng was required when one injection of antigen instead of two was administered and when the interval between treatment with anti-C10 and immunization was extended to 6 weeks. This suggests that the anti-C10 induces alterations in the idiotypic network which are not yet fully developed after 2 weeks. Furthermore, experiments presented herein demonstrate that decapeptide-binding antibodies from A/J mice, which lack the C10-IdX, were also suppressed by pretreatment with anti-C10. Interestingly, unlike the case with the CSW strain, the titer to TMVP was decreased by the administration of anti-C10 to A/J mice which were subsequently immunized with TMVP. These findings suggest that the polyclonal anti-C10 contains antibodies to an idiotype which is a major component of the overall anti-TMVP response of A/J mice and may be important in the overall regulation of the anti-TMVP response.     

Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein

We have recently reported that Trypanosoma cruzi infection protects cardiomyocytes against apoptosis induced by growth factor deprivation. Cruzipain, a major parasite antigen, reproduced this survival effect by a Bcl-2-dependent mechanism. In this study, we have investigated the molecular mechanisms of cruzipain-induced cardiomyocyte protection. Neonatal BALB/c mouse cardiac myocytes were cultured under minimum serum conditions in the presence of cruzipain or T. cruzi (Tulahuen strain). Some cultures were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 or specific inhibitors of the mitogen-activated protein kinase (MAPK) family members such as the mitogen-activated protein kinase kinase (MEK1) inhibitor PD098059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 MAPK inhibitor SB203580. Inhibition of PI3K and MEK1 but not JNK or p38 MAPK increased the apoptotic rate of cardiomyocytes treated with cruzipain. Phosphorylation of Akt, a major target of PI3K, and ERK1/2, MEK1-targets, was achieved at 15 min and 5 min, respectively. In parallel, these kinases were strongly phosphorylated by T. cruzi infection. In cultures treated with cruzipain, cleavage of caspase 3 was considerably diminished after serum starvation; Bcl-2 overexpression was inhibited by PD098059 but not by Ly294002, whereas Bad phosphorylation and Bcl-xL expression were increased and differentially modulated by both inhibitors. The results suggest that cruzipain exerts its anti-apoptotic property in cardiac myocytes at least by PI3K/Akt and MEK1/ERK1/2 signaling pathways. We further identified a differential modulation of Bcl-2 family members by these two signaling pathways.     

Anti-idiotypic mimicry of a neutralizing epitope on the glycoprotein B complex of human cytomegalovirus.

Experiments were carried out to investigate the ability of rabbit anti-idiotype antibodies (Ab2), directed against an anti-human cytomegalovirus monoclonal antibody (Ab1), to induce neutralizing antibodies specific for the immunodominant glycoprotein B viral complex. Mice immunized with Ab2 produced anti-Ab2 (Ab3) that was both antigen and idiotype specific with regard to Ab1. We conclude that the Ab2 antibodies mimicked a neutralizing epitope and acted as a network antigen for inducing a specific anti-human cytomegalovirus antibody response in this experimental system.     

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus.

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.     

Molecular Comparison of the Mitochondrial and Cytoplasmic hsp 70 of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major

ABSTRACT. We compared the expression and localization of the mitochondrial and cytoplasmic hsp70 of the protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. The mitochondrial protein is encoded by multiple mRNA in all species, while the cytoplasmic protein is encoded by a single mRNA. In all three species, the mitochondrial hsp70 is concentrated in the kinetoplast, a submitochondrial structure that houses the unusual DNA (kDNA) that characterizes this group of organisms, while the cytoplasmic protein is distributed throughout the cell. These results suggest that, in all kinetoplastid species, mt-hsp70 has a specific function in kDNA biology, possibly in the processes of kDNA replication, RNA editing or kinetoplast structure.     

Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies.

Antibodies that lyse trypomastigotes in a complement-mediated reaction are believed to be the main participants in the protection against virulent Trypanosoma cruzi. Antibodies with a specificity for alpha-galactosyl-containing determinants--generally called antiGal--were studied to determine their role in the lysis of trypomastigote forms. The titers of antiGal markedly increase in Chagas's disease. In the present study we demonstrate binding of this antibody to T. cruzi and the complement-mediated lysis of trypomastigotes by antiGal. Lysis of metacyclic trypomastigotes by whole Chagasic (Ch) serum or isolated antiGal fractions was equally inhibited by alpha- but not by beta-galactosides. Most of the lytic power of the Ch antiGal as well as of the whole Ch serum was removed by absorption on Synsorb-linked Gal alpha 1, 3Gal beta 1, 4GlcNAc followed by rabbit erythrocyte absorption. The Ch antiGal had a lower affinity for melibiose bound to agarose than for the trisaccharide linked to Synsorb, and was several times more effective in the immunolysis of trypomastigotes than the corresponding antiGal from normal human serum. Lytic antibodies were partly absorbed by Serratia marcescens but not by Escherichia coli O111. A human volunteer immunized with an S. marcescens vaccine elicited a specific antiGal response that was lytic to trypomastigotes (70% lysis). We suggest that in vivo high-affinity antiGal antibody clones, as occur in Ch patients, may significantly contribute to the destruction of the parasite, whereas low-affinity antiGal clones are much less effective in the protection against T. cruzi infection.     

A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells.

When trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers alpha (2-3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans-sialidase does not utilize cytidine 5' monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied alpha (2-3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition.     

Antigen mimicry by anti-idiotypic antibodies: study of interactions between complementary surfaces in macromolecules.

Anti-idiotypic antibodies were obtained from New Zealand White rabbits injected with affinity-purified rabbit anti-TrpR antibodies. In gel mobility shift studies, such immunoglobulin preparations were shown to contain one or more species able to form specific complexes with DNA molecules bearing a trp operator. In competitive ELISA assays, the binding of anti-idiotypic antibodies to operator-bearing DNA was reversed by TrpR. The demonstration that the immune repertoire contains information for operator-specific DNA-binding proteins may be relevant to the etiology of certain autoimmune diseases.     

A superfamily of Trypanosoma cruzi surface antigens

Several genes o f Trypanosoma cruzi encode surface antigens that include an amino acid motif that is conserved among bacterial neurominidases. Oscar Campetella, Daniel Sdnchez, Juan Jose Cazzulo and Alberto Carlos Frasch here suggest grouping these gene families in a superfamily.     

Lectin receptors in Trypanosoma cruzi. An N-acetyl-D-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unaffected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since N-acetyl-D-glucosamine inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface N-acetyl-D-glucosamine-specific receptor activity.     

Trypanosoma cruzi: identification of a cell surface polysaccharide.

An immunogenic polysaccharide prepared by phenol-water extraction of Trypanosoma cruzi was characterized by polyacrylamide gel electrophoresis and molecular sieve chromatography. The polysaccharide was shown to be a cell surface constituent by adsorption of rabbit anti-polysaccharide serum with live culture forms of the protozoa. The cell surface localization of the antigen was visualized using fluorescein- and ferritin-conjugated antibodies.