Antigenic peptide sequence of echinococcus granulosus and diagno

Details Antigenic peptide sequence of echinococcus granulosus and diagno Antigenic peptide sequence of echinococcus granulosus and diagno

1993-03-10

1996-07-30

Parr, Margaret

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435 722, 435975, 436518, 436536, 436811, 530324, 530806, G01N 33569, G01N 33543, C07K14;435543

Patent

active

055410782

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to an immunogenic peptide sequence of Echinococcus granulosus, to a DNA sequence coding for this peptide sequence and also to the diagnostic and therapeutic applications of the peptide sequence.


BACKGROUND OF THE INVENTION

Unilocular echinococcosis or hydatidosis is a parasitic disease due to the development in man and also livestock of the larval form of a small cestode, Echinococcus granulosus. The dog represents the reservoir of the adult parasite. This parasitosis is a genuine scourge in breeding countries in Africa, in Latin America and in Australia, as well as the countries bordering the Mediterranean basin, where there are economic repercussions in both the medical and the veterinary field. While almost no diagnostic test is at present available for animals, medical diagnosis is often intricate on account of the non-specificity of the symptoms observed. On the other hand, several standard immunological methods, such as haemagglutination and the complement-fixation reaction, have been performed for the detection of hydatidosis. Hydatid fluid, used as a source of antigens, contains, in addition to the products of metabolism of the parasite common to other helminths, several components of the host (CAPRON A. et al. (1), RUSSI S. et al. (2), BEN-ISMAEL R. et al. (3). This gives rise to the problem of false-positive reactions. However, the almost constant presence of antibodies that precipitate an antigen designated antigen 5 has been shown by the immunoelectrophoresis technique (CAPRON A. et al. (4) and (5)). Since then, this test has remained one of the best immunological reference tests for any epidemiological study of hydatidosis. Antigen 5, which is highly immunogenic, has formed the subject of several studies showing that it is a thermolabile lipoprotein capable of binding to concanavalin A, of relative molecular mass approximately 60 kDa (ORIOL R. et al. (6), BOUT D. et al. (7), PIANTELLI M. (8)). Analysed under reducing conditions, this antigen dissociates into two subunits of 37 and 22 Dka. Using more sensitive techniques such as ELISA, the diagnostic potential of purified antigen 5 has been amply confirmed. Anti-antigen 5 antibodies have also been detected in sera of patients or animals infected with E. multilocularis or with other cestodes such as Taenia hydatigena, T. ovis and T. solium. However, their level remains low. These few observed false-positive reactions have been attributed in part to the presence of a phosphorylcholine epitope on antigen 5.
The current problem is to be able to have at one's disposal specific parasitic antigens which are well standardised and in sufficient quantity for the diagnostic tests. This is the standpoint from which the studies currently being carried out on hydatidosis, and which are based on monoclonal antibody and genetic engineering techniques, are endeavouring to isolate and identify parasitic components possessing a high degree of specificity.
The objective of the present invention is to provide a parasitic antigen specific to hydatidosis.


SUMMARY OF THE INVENTION

The inventors have characterised an IgG.sub.1 isotype monoclonal antibody, designated EG 02 154/12, directed towards a protein epitope carried by antigen 5 and specific to E. Granulosus.
This monoclonal antibody has made it possible to isolate, from a complementary DNA library obtained from mRNA of E. Granulosus protoscolex, clones designated Eg 6 and Eg 14, corresponding to peptide sequences which were shown to be recognised by the monoclonal antibody EG 02 154/12.
The subject of the present invention is consequently an immunogenic peptide sequence of antigen 5 of Echinococcus granulosus, characterised in that it is recognised both by sera of patients suffering from hydatidosis and by an IgG.sub.1 isotype monoclonal antibody produced by the hybridoma EG 02 154/12 deposited at the CNCM on 25 Jun. 1990 under No. 1-957.
The subject of the present invention is also a DNA sequence comprising the nucleotide sequence designated EG 6, (SEQ ID No: 15) ##STR1##
The

REFERENCES:
Biological Abstracts, vol. 88, No. 11, 1989, AN-120220, B. Hamrioui, et a "Production of Anti-Hydatid Monoclonal Antibodies".
Molecular and Biochemical Parasitology, vol. 20, 1986, pp. 133-142, G. DiFelice, et al., "Purification and Partial Characterization of the Major Antigen of Echinococcus Granulosus (Antigen 5) with Monoclonal Antibodies".
Molecular and Biochemical Parasitology, vol. 45, Apr. 1991, pp. 233-240, B. Facon, et al., "Molecular Cloning of an Echinococcus Granulosus Protein Expressing an Immunogenic Epitope of Antigen 5".
Molecular and Biochemical Parasitology, vol. 33, 1989, pp. 171-182, L. Hemmings, et al., "The Isolation, by Differental Antibody Screening, of Echinococcus Multilocularis Antigen Gene Clones With Potential for Immunodiagnosis".
Molecular and Biochemical Parasitology, vol. 36, 1989, pp. 287-290, M. W. Lightowlers, et al., "Amino Acid Sequence Homology Between Cyclophilin and a cDNA-Cloned Antigen of Echinococcus Granulosus".
Pozzouli et al, 1975. Isolation of the most immunoreactive antigens of Echinococcus granulosus from shepp hydatid fluid. J Immunol 115:1459-1463.
Chamekh et al, 1990. Use of a monoclonal antibody specific for a protein epitope of Echinococcus granulosus antigen 5 in a competitive antibody radioimmunoassay for diagnosis of hydatid disease. J Immunological Meth 134:129-137.
Heath et al, 1992. Echinococcus granulosus in sheep: transfer from ewe to lamb of `Arc 5` antibodies and oncosphere-killing activity, but not protection. Int J. Parasitol 22:1017-1021.
Bellanti, 1971. Immunolgy. W. B. Saunders, Philadelphia. pp. 484-487.

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