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Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

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.

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Sensitivity of the EH5 antigen towards mild acid digestion  (lanes A and B), and towards PI-PLC (lanes C and D) and binding to Con  A (lanes E and F). For lanes A–D, fractions were chromatographed  through phenyl-coupled Sepharose, eluted fractions were dotted onto nitrocellulose and reacted with antibody EH5, and bound antibody was detected with iodine-labeled sheep anti–mouse antibodies; lane A, mild acid  digestion; lane B, undigested control; lane C, PI-PLC digestion; lane D,  control incubated in PI-PLC buffer only. For lanes E and F, eluted fractions from immunoaffinity column (E) tested with antibody EH5, bound  antibodies detected with alkaline phosphatase–coupled anti–mouse antibodies; (F) tested with biotin-labeled Con A, bound Con A detected with  alkaline phosphatase–coupled streptavidin.
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Figure 3: Sensitivity of the EH5 antigen towards mild acid digestion (lanes A and B), and towards PI-PLC (lanes C and D) and binding to Con A (lanes E and F). For lanes A–D, fractions were chromatographed through phenyl-coupled Sepharose, eluted fractions were dotted onto nitrocellulose and reacted with antibody EH5, and bound antibody was detected with iodine-labeled sheep anti–mouse antibodies; lane A, mild acid digestion; lane B, undigested control; lane C, PI-PLC digestion; lane D, control incubated in PI-PLC buffer only. For lanes E and F, eluted fractions from immunoaffinity column (E) tested with antibody EH5, bound antibodies detected with alkaline phosphatase–coupled anti–mouse antibodies; (F) tested with biotin-labeled Con A, bound Con A detected with alkaline phosphatase–coupled streptavidin.

Mentions: To test whether these phosphates were forming acid-labile diester bonds, fraction E from E. histolytica SFL-3 trophozoites was digested for 8 min at 100°C in 40 mM trifluoroacetic acid. The products were analyzed by hydrophobic chromatography on phenyl-coupled Sepharose. The undigested EH5 antigen in fraction E eluted over a large range (Fig. 3, lane B), mild acid digestion reduced the binding of the EH5 antibody, and the partially digested EH5 antigen eluted only in the hydrophobic region (Fig. 3, lane A), in agreement with the degradation of the hydrophilic parts of the molecules.


Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Sensitivity of the EH5 antigen towards mild acid digestion  (lanes A and B), and towards PI-PLC (lanes C and D) and binding to Con  A (lanes E and F). For lanes A–D, fractions were chromatographed  through phenyl-coupled Sepharose, eluted fractions were dotted onto nitrocellulose and reacted with antibody EH5, and bound antibody was detected with iodine-labeled sheep anti–mouse antibodies; lane A, mild acid  digestion; lane B, undigested control; lane C, PI-PLC digestion; lane D,  control incubated in PI-PLC buffer only. For lanes E and F, eluted fractions from immunoaffinity column (E) tested with antibody EH5, bound  antibodies detected with alkaline phosphatase–coupled anti–mouse antibodies; (F) tested with biotin-labeled Con A, bound Con A detected with  alkaline phosphatase–coupled streptavidin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199119&req=5

Figure 3: Sensitivity of the EH5 antigen towards mild acid digestion (lanes A and B), and towards PI-PLC (lanes C and D) and binding to Con A (lanes E and F). For lanes A–D, fractions were chromatographed through phenyl-coupled Sepharose, eluted fractions were dotted onto nitrocellulose and reacted with antibody EH5, and bound antibody was detected with iodine-labeled sheep anti–mouse antibodies; lane A, mild acid digestion; lane B, undigested control; lane C, PI-PLC digestion; lane D, control incubated in PI-PLC buffer only. For lanes E and F, eluted fractions from immunoaffinity column (E) tested with antibody EH5, bound antibodies detected with alkaline phosphatase–coupled anti–mouse antibodies; (F) tested with biotin-labeled Con A, bound Con A detected with alkaline phosphatase–coupled streptavidin.
Mentions: To test whether these phosphates were forming acid-labile diester bonds, fraction E from E. histolytica SFL-3 trophozoites was digested for 8 min at 100°C in 40 mM trifluoroacetic acid. The products were analyzed by hydrophobic chromatography on phenyl-coupled Sepharose. The undigested EH5 antigen in fraction E eluted over a large range (Fig. 3, lane B), mild acid digestion reduced the binding of the EH5 antibody, and the partially digested EH5 antigen eluted only in the hydrophobic region (Fig. 3, lane A), in agreement with the degradation of the hydrophilic parts of the molecules.

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

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.

Show MeSH
Related in: MedlinePlus