Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Parasitic organism or component thereof or substance...
Reexamination Certificate
1997-12-10
2002-06-11
Graser, Jennifer E. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Parasitic organism or component thereof or substance...
C424S185100, C424S190100, C424S193100, C424S265100, C530S350000, C530S387100, C530S387200, C530S387900, C530S388600, C435S007100
Reexamination Certificate
active
06403103
ABSTRACT:
FIELD OF THE INVENTION
The subject of the present invention is a new genetic material encoding a new protein and its fragments and its antigenic determinant recognized by anti-
Trypanosoma cruzi
antisera, and it relates to promoter sequence and to the use of said gene and protein and/or antigenic determinant, especially for diagnostic, pharmaceutical and therapeutic purposes.
BACKGROUND OF THE INVENTION
Trypanosoma cruzi
is a flagellate protozoan parasite, a member of the order Kinetoplastida and of the family Trypanosomatidae, which is responsible for Chagas' disease which affects naturally millions of persons, mainly in Latin America.
In vertebrate hosts,
Trypanosoma cruzi
is present in two forms: one which is mobile by means of its flagellum or trypomastigote and which does not divide; the other is aflagellate, or intracellular amastigote, which multiplies by binary division.
Transmission of the protozoan in man occurs through hematophagous insects of the family Reduviidae, during a blood meal followed by dejections at the site of the bite. The vector insect thus releases the infectious metacyclic trypomastigote forms which will colonize many cell types through the blood circulation.
Trypanosoma cruzi
infects cardiac and skeletal muscle cells, glial cells, and cells of the mononuclear phagocytic system. After passive penetration into the host cell, the trypomastigote form of the parasite differentiates into the amastigote form, divides actively and then this is followed by a release of the trypomastigote forms, thereby causing a new cell invasion.
The vector insects will complete the parasitic cycle by ingesting, during a blood meal, the trypomastigote forms in the host. The latter differentiate into epimastigote forms in the vector's middle intestine and finally into the infectious metacyclic trypomastigote forms in the posterior intestine.
Two phases can be distinguished in the Chagas disease: the acute phase and the chronic phase. The acute phase occurs after a transfusional, congenital, or vectorial type contamination and lasts for a few weeks. It is characterized by a large number of parasites circulating in the blood and corresponds to an exponential division of the protozoan. The acute phase is most often asymptomatic. However, in infants contaminated by their mother, the acute phase, which is marked by an acute cardiopathy, may be critical. The chronic phase may extend over many years. In some individuals, this phase is asymptomatic. On the other hand, other patients have tissue lesions in the heart or digestive type manifestations. In any case, clinical diagnosis must always be confirmed by tests for the detection either of antibodies directed against the parasitic antigens, or of the parasite itself.
This disease is becoming a worldwide problem because of the contamination through blood transfusion. It has therefore become essential to have available diagnostic tests which make it possible to determine the presence of the parasite in individuals. Various serological tests are avalable, such as direct agglutination, indirect immunofluorescence (IIF), complement fixation tests (CFR), and ELISA tests (Enzyme Linked Immunosorbent Assay). The
Trypanosoma cruzi
antigens currently used for the serological tests are obtained from a total lysate or from partially purified protein fractions of the noninfectious stage of the parasite. However, these fractions do not allow antigens to be obtained in sufficient quantity and quality for the production of a reliable serological diagnostic test. Furthermore, the complexity of the parasite and the strain-to-strain antigenic polymorphism introduce an additional difficulty in the reproducibility of the different preparations. Finally, there are many risks of cross-reactivity with other protozoa, more particularly with the family Leishmania and
Trypanosoma rangeli,
a nonpathogenic parasite.
In order to solve these various problems, it was envisaged to produce a serological diagnostic kit composed of recombinant proteins which would be highly sensitive and specific for
Trypanosoma cruzi.
Various research groups have screened libraries for expression of
Trypanosoma cruzi
genomic DNA or complementary DNA in the vector &lgr;gt11, using sera from patients suffering from Chagas disease. The &lgr;gt11 phage allows the insertion of foreign DNA of a maximum size of 7 kb into the EcoRI site localized in the lacZ gene, under the control of the lac promoter. The product obtained is a recombinant protein fused with beta-galactosidase, which is inducible by IPTG (isopropyl beta-D-thiogalactoside).
Various
Trypanosoma cruzi
genes, encoding proteins recognized by the Chagasic sera were thus characterized (Moncayo and Luquetti, 1990 and Levin et al. (1991), FEMS Microbiol. Immunol. 89: 11-20). Among the recombinant antigens described, the H49 antigen may be mentioned (Paranhos et al., 1994 (1)). However, this antigen does not allow a serological detection sensitivity of 100% of the patients in the acute or chronic phase. It was therefore envisaged to combine the H49 antigen with the CRA antigen (Cytoplasmic Repetitive Antigen) (Lafaille et al., (1989) (2)) but still without solving this problem.
SUMMARY OF THE INVENTION
The present inventors have identified and obtained a new genetic material encoding a new protein, its fragments, and antigenic determinants recognized by anti-
Trypanosoma cruzi
antisera, which makes it possible to overcome the above-mentioned disadvantages. The genetic material may be used to produce proteins or polypeptides for the production of diagnostic tests, or for the preparation of vaccinal or pharmaceutical compositions, or may itself either be used as a probe, or for the determination of specific probes which can be used in nucleic acid hybridization tests for the detection of
Trypanosoma cruzi
infections. Likewise, the protein or any corresponding polypeptide may be used for the production of antibodies specific for the parasite, for diagnostic or passive protection purposes.
DETAILED DESCRIPTION OF THE INVENTION
The new genetic material is designated Tc100 and it encodes a protein designated PTc100 by the applicant. The new genetic material and protein have also been designated Tc40 and PTc40, respectively, by the applicant.
Consequently, the subject of the present invention is a DNA or RNA molecule consisting of at least one strand comprising a nucleotide sequence represented by the identifier SEQ ID No.1, or a sequence complementary or antisense or equivalent to said sequence identified by the identifier SEQ ID No.1, and especially a sequence having, for any succession of 100 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at least 85% homology with said sequence.
The invention moreover relates to DNA or RNA fragments whose nucleotide sequence is identical, complementary, antisense, or equivalent to any one of the following sequences:
that starting at nucleotide 1232 and ending at nucleotide 2207 of SEQ ID No.1,
that starting at nucleotide 1232 and ending at nucleotide 1825 of SEQ ID No.1,
and that starting at nucleotide 1266 and ending at nucleotide 2207, and especially the DNA or RNA fragments whose sequence has, for any succession of 30 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at least 85% homology with any one of said sequences.
The prefered DNA and RNA fragment being the 1232-1825 fragment of SEQ ID No.1, the invention relates to DNA or RNA fragments which hybridize with this fragment knowing that it is tolerated until 10% of error in bases' matching.
The DNA and RNA fragment could be of any length and be comprised in the sequence from 266 to 3010 of SEQ ID No.1, knowing that this sequence is highly specific for
Trypanosoma cruzi
. Any fragment which hybridizes with the DNA or RNA of this sequence is considered as been a specific equivalent nucleotide or deoxunucleotide sequence.
Nucleotide sequence is understood to mean either a DNA strand or its complementary strand, or an RNA strand or its antisense strand or
Jolivet Michel
Lesenechal Mylene
Mandrand Bernard
Paranhos-Baccala Glaucia
Bio Merieux
Graser Jennifer E.
Oliff & Berridg,e PLC
LandOfFree
Trypanosoma cruzi antigen, gene encoding therefore, and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Trypanosoma cruzi antigen, gene encoding therefore, and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Trypanosoma cruzi antigen, gene encoding therefore, and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2926588