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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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Agglutination test:  E. histolytica trophozoites in HT  medium after 1 h of incubation  (A); with isotype-matched control antibody BIP 1 after 1 h of  incubation (B); with supernatant  of antibody EH5 after 15 min of  incubation (C); the same after 1 h  of incubation (D); with protein  G–purified antibody EH5 after  15 min of incubation (E); and the  same after 1 h of incubation (F).
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Figure 4: Agglutination test: E. histolytica trophozoites in HT medium after 1 h of incubation (A); with isotype-matched control antibody BIP 1 after 1 h of incubation (B); with supernatant of antibody EH5 after 15 min of incubation (C); the same after 1 h of incubation (D); with protein G–purified antibody EH5 after 15 min of incubation (E); and the same after 1 h of incubation (F).

Mentions: If the EH5 antigen, or at least a significant portion of it, was able to bind to Con A, which is known to agglutinate preferentially pathogenic amebas (39, 40), then the EH5 antibody should also be able to agglutinate E. histolytica trophozoites. To test this, E. histolytica SFL-3 trophozoites (2– 4 × 104/well) were used in an agglutination experiment (Fig. 4) along the lines described for the agglutination by an antibody against a 96-kD antigen (36). Fig. 4 A shows the amebas 1 h after addition of only HT hybridoma medium, whereas B shows the results after addition of isotype-matched control antibody BIP 1. Fig. 4, C and D, shows the amebas 15 and 60 min after addition of antibody EH5 supernatant, whereas E and F show the effect of purified antibody EH5 after 15 and 60 min. The agglutination process began ∼15 min after addition of antibody EH5, and after 1 h the amebas were strongly agglutinated. No agglutination was observed when isolated monomeric Fab fragments from antibody EH5 were added (data not shown).


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)

Agglutination test:  E. histolytica trophozoites in HT  medium after 1 h of incubation  (A); with isotype-matched control antibody BIP 1 after 1 h of  incubation (B); with supernatant  of antibody EH5 after 15 min of  incubation (C); the same after 1 h  of incubation (D); with protein  G–purified antibody EH5 after  15 min of incubation (E); and the  same after 1 h of incubation (F).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Agglutination test: E. histolytica trophozoites in HT medium after 1 h of incubation (A); with isotype-matched control antibody BIP 1 after 1 h of incubation (B); with supernatant of antibody EH5 after 15 min of incubation (C); the same after 1 h of incubation (D); with protein G–purified antibody EH5 after 15 min of incubation (E); and the same after 1 h of incubation (F).
Mentions: If the EH5 antigen, or at least a significant portion of it, was able to bind to Con A, which is known to agglutinate preferentially pathogenic amebas (39, 40), then the EH5 antibody should also be able to agglutinate E. histolytica trophozoites. To test this, E. histolytica SFL-3 trophozoites (2– 4 × 104/well) were used in an agglutination experiment (Fig. 4) along the lines described for the agglutination by an antibody against a 96-kD antigen (36). Fig. 4 A shows the amebas 1 h after addition of only HT hybridoma medium, whereas B shows the results after addition of isotype-matched control antibody BIP 1. Fig. 4, C and D, shows the amebas 15 and 60 min after addition of antibody EH5 supernatant, whereas E and F show the effect of purified antibody EH5 after 15 and 60 min. The agglutination process began ∼15 min after addition of antibody EH5, and after 1 h the amebas were strongly agglutinated. No agglutination was observed when isolated monomeric Fab fragments from antibody EH5 were added (data not shown).

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