ABSTRACT
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Protozoan/therapeutic use , Antigens, Protozoan/immunology , Entamoeba histolytica/immunology , Entamoebiasis/prevention & control , Glycosphingolipids/immunology , Membrane Proteins/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/metabolism , Antibodies, Protozoan/biosynthesis , Antibodies, Protozoan/metabolism , Antigens, Protozoan/chemistry , Antigens, Protozoan/ultrastructure , Binding Sites, Antibody , Concanavalin A/metabolism , Entamoeba histolytica/ultrastructure , Entamoebiasis/immunology , Entamoebiasis/parasitology , Female , Fluorescent Antibody Technique, Indirect , Glycosphingolipids/chemistry , Immunization, Passive , Immunoblotting , Membrane Proteins/chemistry , Mice , Mice, Inbred BALB C , Mice, SCID , Microscopy, Confocal , Microscopy, Electron , Protein Binding/immunology , Protozoan Proteins/immunologyABSTRACT
Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones from Trichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which contained T. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of the T. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists like Entamoeba histolytica, Trypanosoma cruzi, and Leishmania infantum.
Subject(s)
Histones/genetics , Phylogeny , Trichomonas vaginalis/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA, Complementary/genetics , Genome , Histones/physiology , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Trichomonas vaginalis/physiologyABSTRACT
Immunogenic properties of TVGRGDPHQ nonapeptide which is correspondent to the region 1094-1102 of B. pertussis filamentous hemagglutinin (FHA) were studied. The conjugate of bovine serum albumin with nonapeptide was used for immunization of BALB/c and CBA mice. Antisera of the both lines of mice cross-reacted with a number of antigens, but using affinity chromatography peptide and FHA specific antibodies were extracted. Affinity purified rabbit antibodies to TVGRGPHQ which recognize FHA were also obtained. Therefore the antibodies to the peptide which placed RGD-containing region responsible for macrophage CR3-integrin interaction are capable to distinguish the native antigen. Thus these data are an additional evidence for the nonapeptide use as a component of synthetic vaccine against whooping-cough.
Subject(s)
Bordetella pertussis/chemistry , Oligopeptides/immunology , Serum Albumin, Bovine/chemistry , Amino Acid Sequence , Animals , Antibody Specificity , Cross Reactions , Hemagglutinins , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Molecular Sequence Data , Oligopeptides/chemistryABSTRACT
Some biological properties of polyreactive antibodies (PRABs) from the serum of mice, rabbit and cattle are investigated. Optimal parameters of the activation process are determined. It is shown that solution of KSCN (potassium rhodanide) more effectively impedes interaction of PRABs with the antigen on the immunological plate than interaction between specific antibodies. Dynamics of binding of PRABs and affine-purified antibodies to an immobilized antigen is analyzed. The competitive ELISA shows the absence of specificity of PRABs and their less avidity in comparison with the usual antibodies. It is supposed that PRABs play an essential role in the immune system of animals and man.
