Comparative compositions of cell surface glycoconjugates isolated from Trypanosoma cruzi epimastigotes.

Cell surface glycoconjugates of epimastigotes of Trypanosoma cruzi have been isolated and analyzed to give their amino acid and carbohydrate compositions. Those which have been investigated are a complex of three closely associated glycoproteins, GP24, GP31, GP37, and a lipopeptidophosphoglycan. The GP24-GP31-GP37 complex has an unusual amino acid composition with very low levels of hydrophobic amino acids, it contains 56% (w/w) carbohydrate, with mannose, galactose and glucosamine (presumably N-acetyl) being present in approximately equal quantities. The lipopeptidophosphoglycan also has low levels of hydrophobic amino acids and contains equal levels of mannose and galactose together with lesser amounts of (N-acetyl) glucosamine. The glycoconjugates are contrasted and compared with two other previously characterised cell surface glycoproteins (GP25 and GP72) from T. cruzi.

Crystal structure at 1.95 A resolution of the breast tumour-specific antibody SM3 complexed with its peptide epitope reveals novel hypervariable loop recognition.

The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

Antibody-dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores.

Using flow cytometry (FCM), an assay has been developed for the determination of antibody-dependent cell-mediated cytotoxicity (ADCC). Target cells were labelled with a membrane dye, PKH-26, to allow discrimination when incubated with effector cells and antibody. Post-incubation, cell death within the PKH-26+ target cell population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3). This ADCC method allows analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the assay is accurate and reproducible with the potential to be a useful tool for evaluating the therapeutic potential of antibodies and antibody-based reagents.

Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide.

A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Sequence homology of surface membrane proteins of Babesia rodhaini.

Four surface membrane proteins of Babesia rodhaini have previously been shown to induce a degree of protective immunity, and to carry both unique and cross-reactive determinants. cDNA clones for two of the genes coding for these proteins have been isolated and used as probes to isolate a single large genomic DNA fragment which contained all four genes. DNA sequence of two of the genes and their predicted amino acid sequences confirmed that the proteins had hydrophobic sequences at their N- and C-termini, an observation consistent with their proposed cell surface location. Homologies in both amino acid and nucleotide sequences were found at the 3'and at the 5' ends, but considerable sequence variations existed elsewhere in the genes and their products. The genes coding for these four proteins were tandemly arranged along a single relatively short length of chromosome, and such structures, because of their sequence homologies, probably could have arisen by gene duplication. The extensive variation suggested that there may be a functional need for these proteins to be different or capable of varying, although computer analysis implied that the extent of this variation may be constrained by structural requirements. This variation could be indicative of a role for these proteins in the host-parasite relationship or immune evasion.

An antigenic determinant common to both mouse red blood cells and several membrane proteins of the parasitic protozoa Babesia rodhaini.

Four membrane proteins of Babesia rodhaini have been identified by monoclonal antibodies. These proteins are differentiated by serological criteria and by tryptic peptide maps but contain common antigenic determinants. In vitro translation of parasite polyadenylated RNA followed by immunoprecipitation demonstrates that these common determinants are located within the primary sequence of the protein and do not result from post-translational modification. Part of the common amino acid sequence of these proteins is also present within a protein found in mouse red blood cells. Such common structures could potentially play an important role in the host-parasite relationship. The presence of sequence homology within a group of proteins from Babesia rodhaini could be further evidence for the atypical genetic rearrangements proposed to take place within Babesia species.

Cell surface antigens of Trypanosoma cruzi: use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein.

A monoclonal antibody was produced by cell fusion from mice immunized with Trypanosoma cruzi epimastigotes. The antibody was epimastigote specific but not strain specific; the antibody bound to Y, Peru and Tulahuén epimastigotes but did not bind to Y amastigotes or trypomastigotes. The antigen recognised by the monoclonal antibody was a 72 000 molecular weight cell surface glycoprotein which represented only 0.04% of the whole cell protein. Analysis of the glycoprotein purified by antibody affinity chromatography revealed a carbohydrate content of 52% by weight which was composed of glucosamine, mannose, galactose, glucose and three different pentoses: fucose, xylose and ribose. Immunisation with the purified glycoprotein did not protect mice from a lethal infection of T. cruzi.

Cloning and expression of a trypomastigote-specific 85-kilodalton surface antigen gene from Trypanosoma cruzi.

An 85-kDa trypomastigote-specific surface antigen gene from Trypanosoma cruzi has been identified by screening a genomic library in lambda gt10 with trypomastigote and epimastigote cDNA. The 1.3-kb genomic clone (pTt34) hybridizes to a single trypomastigote mRNA of 3.7 kb and to multiple bands in genomic Southern blots. Dot-blot experiments show that there are 5-10 copies of this sequence per haploid genome, and these are arranged in a non-tandem manner. pTt34 has been expressed as an anthranilate synthetase fusion protein in Escherichia coli, and inclusion bodies have been used to raise antiserum in rabbits. This antiserum immunoprecipitates a cell surface trypomastigote-specific protein of 85 kDa. The DNA and predicted amino acid sequences of pTt34 are given. Four further clones obtained from a PvuII/HpaI partial genomic library in pUC13 have extended the sequence of the 3' end of pTt34; each of these clones has regions of sequence divergence and each could represent a different member of the gene family.

Expression of HLA system antigens on placenta.

The levels of HLA-A and -B antigens expressed by placenta have been assessed relative to parental and other A and B antigen types that were not shared by the foetus. A purified preparation of placenta plasma membrane was used to estimate the antigen activities. The results indicate that maternally and paternally inherited A and B antigen activities and beta2-microglobulin are expressed to similar extents but at much lower levels than in spleen lymphocytes (less than 5%). The possibility that the amounts detected were caused by contamination with blood or maternal tissue was ruled out. The low levels of A and B antigens may account for the lack of a cellular immune response to the other polymorphic cell surface antigens of the trophoblast. No evidence was obtained for the expression of a significant level of Ia antigens.

Receptors and recognition mechanisms of Trypanosoma cruzi.

The current state of knowledge on receptor and recognition interactions which take place during the life-cycle of Trypanosoma cruzi is reviewed. Evidence suggests that carbohydrate plays a central role in these recognition mechanisms. Lectin-sugar interactions appear to be involved in uptake of the parasite by host cells including macrophages and a protein on the surface of trypomastigote which binds N-acetyl glucosamine on the host cell has been implicated in host cell invasion. Sugars on a 72,000 molecular weight glycoprotein on epimastigotes have also been implicated in colonization of the gut of the insect vector and in control of the morphological changes which take place in the insect gut.

Cell surface glycoproteins of Trypanosoma cruzi: protective immunity in mice and antibody levels in human chagasic sera.

Two cell surface glycoproteins from Trypanosoma cruzi have been compared for their ability to protect mice against an acute lethal infection. One of these, a 90,000 glycoprotein found on all stages of the parasite, protected against both bloodstream and metacyclic trypomastigote challenge. A 72,000 glycoprotein found only on insect-derived stages of T. cruzi protected only against a metacyclic trypomastigote challenge. Antibody against both of these glycoproteins was present in human chagasic sera.

Monoclonal antibodies demonstrate similarity of surface antigens on different clones of Schistosoma mansoni schistosomula.

Previous work has demonstrated wide variation in the susceptibility of different clones of Schistosoma mansoni to resistance in mice induced by a small schistosome infection. Eight monoclonal antibodies directed against individual surface antigens of juvenile schistosomes (schistosomula) were used to determine the distribution of these antigens among clones of schistosomula. All eight antigens were detected on the surface membranes of the 32 clones of schistosomula tested. This result implies that polymorphism of schistosomula surface antigens is very unlikely to be the cause of the variation in susceptibility of different clones to resistance in mice. Absence of heterogeneity among surface antigens of schistosomula may facilitate experimental vaccination aimed against these putative target antigens.

Immune responses in advanced colorectal cancer following repeated intradermal vaccination with the anti-CEA murine monoclonal antibody, PR1A3: results of a phase I study.

BACKGROUND AND AIMS: The aim was to determine the toxicity, clinical and immune responses to the murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, PR1A3, in patients with advanced colorectal cancer. MATERIALS AND METHODS: Fifteen patients with advanced colorectal cancer received either 0.5-, 1.0- or 5.0-mg doses of PR1A3 mixed with 10% w/v Alum adjuvant (Superfos Biosector, Denmark) intradermally at 4-week intervals for 3 months. Patient serum was assessed for anti-idiotypic (Ab2), anti-anti-idiotypic (Ab3) and human anti-mouse antibody (HAMA) reactivity. Peripheral blood mononuclear cell (PBMC) proliferation with phytohaemagglutinin (PHA), CEA and PR1A3, stimulated IL-2, IL-4 and IFN-gamma levels and PR1A3-stimulated IL-2 receptor expression during immunotherapy were determined. Comparisons were made with 16 age-matched controls without malignant disease. RESULTS: Hyperimmune sera from 12 of the 15 patients showed Ab2 reactivity with no detectable Ab3 responses. Strong HAMA reactivity was recorded in 7 of the 15 cases with no adverse clinical effect. Delayed-type hypersensitivity (DTH) responses developed in 12 of the 15 patients. Pre-treatment PBMC proliferation with PHA was subnormal in each patient compared with controls, becoming normal (or supranormal) in all patients during immunisation (P<0.001). PBMC proliferation with CEA and PR1A3 increased during immunotherapy (P<0.001) along with stimulated production of IL-2, IFN-gamma and IL-2 receptor expression. Progressive disease was observed in 14 of the 15 patients with minimal toxicity. CONCLUSION: PR1A3 generated limited idiotypic responses but robust DTH reactivity in most patients. In vitro PBMC proliferation with mitogens and recall antigens is greatly increased during the course of immunisation, with a shift in stimulated cytokine profile.

Studies on gastric mucoproteins. The production of radioactive mucoproteins by pig gastric mucosal scrapings in vitro.

1. Optimum conditions, including the effect of media of different pH values, were determined for the incorporation of radioactive precursors into mucoproteins by pig gastric mucosa in vitro. 2. Mucosal scrapings incorporated radioactivity from [U-(14)C]-glucose and from [G-(3)H]threonine or [G-(3)H]serine solely into the carbohydrate and protein portions respectively of the mucoprotein molecules. 3. Of the radioactive mucoprotein 22% was water-soluble and up to 80% of the remainder was soluble in other solvents. 4. Pronase was the most successful proteolytic enzyme tested for making the mucoprotein water-soluble, up to 94% dissolving after digestion. 5. The Pronase digestion products of the mucoproteins were separated from protein by equilibrium-density-gradient centrifugation in a CsCl gradient. 6. These Pronase-digested mucoproteins were further fractionated on Sepharose 4B and the isolated fractions analysed by chemical and sedimentation-velocity methods. 7. Pronase digestion and solvent extraction of mucosal scrapings labelled with (14)C in the carbohydrate and (3)H in the protein showed that one type of mucoprotein was the only non-diffusible biosynthetic product of the scrapings in vitro, and that this mucoprotein was the only mucoprotein constituent of the water-soluble and water-insoluble mucus.

Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa.

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with (14)C-labelled carbohydrate or with (3)H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-(14)C]glucose or [G-(3)H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s(0) (25,w) values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B-C, which were chemically, biosynthetically and immunologically very similar.

Genetically engineered vaccines: problems and promises.

Determinants of important protective antigens from a variety of viral, bacterial and parasite pathogens have been successfully cloned and expressed in novel hosts using recombinant DNA techniques. The use of these cloned determinants in the development of new vaccines, including immunologically defined subunit vaccines, genetically defined live attenuated vaccines, and synthetic peptide vaccines is described.

Quantitative differences in the expression of a 72,000 molecular weight cell surface glycoprotein (GP72) in Trypanosoma cruzi zymodemes.

A high affinity monoclonal antibody, 8G2 B9, was used to assess the expression of a 72,000 m.w. glycoprotein ( GP72 ) in isoenzyme-typed T. cruzi strains ( zymodemes ). Western blotting analysis of T. cruzi clones showed that 8G2 B9 bound strongly to GP72 and also suggested that this antigen was absent or weakly detectable in T. cruzi zymodeme 1 (Z1) strains. Purified 8G2 B9 was radiolabeled with 125I and used in an inhibition radioimmune binding assay to compare the quantities of GP72 in different zymodemes . Ninety-six T. cruzi strains were assayed, of which 36 were Z1, 36 were Z2, five were Z3 , and 19 were Z2 (heterozygous). Most (64%) Z1 strains lacked detectable GP72 , whereas this antigen was always detected Z2 and Z2 (heterozygous) strains. There was an 18-fold difference between geometric mean values for the quantities of GP72 (expressed as nanograms per milligram total cell protein) in Z1 and Z2 strains (Z1, 36 ng/mg; Z2, 639 ng/mg; p less than 0.001). There were also significant differences between the geometric mean values for Z2 and Z2 (heterozygous) strains, i.e., 639 ng/mg and 1648 ng/mg, respectively (p less than 0.001). GP72 was detected in four of the Z3 strains in quantities ranging from 740 to 3640 ng/mg. The absolute amounts of antigen in GP72 -positive strains were low, comprising less than 1% of the total cell protein. The specificities of two other anti- GP72 monoclonal antibodies, 7C6 D7 and WIC 29.26, were compared with 8G2 B9. Both antibodies completely inhibited the binding of 8G2 B9 to GP72 in solid phase immunoassays, suggesting that they reacted with the same antigenic determinants. The results show that monoclonal antibody-based assessments of the expression of GP72 correlate with zymodeme classification, and they also suggest that the monoclonal antibodies recognize major antigenic determinants on GP72 . It should be possible to use 8G2 B9 as an immunologic marker to additionally investigate the clinical significance of T. cruzi zymodemes and the biologic significance of GP72 .