Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1998-08-24
2001-08-07
Graser, Jennifer E. (Department: 1645)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S130100, C424S139100, C424S141100, C424S142100, C424S191100, C530S387100, C530S388100, C530S388600, C435S004000, C435S007100, C435S007220, C435S007920
Reexamination Certificate
active
06270767
ABSTRACT:
FIELD OF THE INVENTION
The subject of the present invention is a new genetic material encoding a new protein recognized by anti-
Trypanosoma cruzi
antisera, and it relates to the use of said gene and protein, especially for diagnostic, pharmaceutical and therapeutic purposes.
BACKGROUND OF THE INVENTION
Trypanosoma cruzi
is a flagellate protozoal 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 muscular cells, the glial cells and the 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 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 was therefore becoming essential to have available diagnostic tests which make it possible to determine the presence of the parasite in individuals. Various serological tests include direct agglutination, indirect immunofluorescence (IIF), complement fixation tests (CFR), ELISA tests (Enzyme Linked Immunosorbent Assay). The
Trypanosoma cruzi
antigens used for the serological tests are obtained from a total lysate of the noninfectious stage of the parasite or from partially purified protein fractions. 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
Trypanosoma rangeli
, a nonpathogenic parasite, and the family Leishmania. Another disadvantage of these techniques is the absence of determination of the disease phase which would allow a treatment from the onset of the acute phase.
In order to solve these various problems, it was envisaged to produce a serological diagnostic kit composed of recombinant proteins which would be 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 EcoR1 site localized in the lacZ gene, under the control of the lac promoter. The product obtained is a recombinant protein used 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. 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 for the first time a new genetic material encoding a new protein, recognized by anti-
Trypanosoma cruzi
antisera, which makes it possible to overcome the abovementioned 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
This gene was called Tc 100 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 in the identifier SEQ ID NO:1, or a sequence complementary or antisense or equivalent to said sequence identified in 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 better still at least 85% homology with said sequence.
Nucleotide sequence is understood to mean either a DNA strand or its complementary strand, or an RNA strand or its antisense strand or their corresponding complementary DNAs. The DNA sequence as represented in the identifier SEQ ID NO:1 corresponds to the messenger RNA sequence, it being understood that the thymine (T) in the DNA is replaced by a uracil (U) in the RNA.
According to the invention, two nucleotide sequences are said to be equivalent in relation to each other, or in relation to a reference sequence if, functionally, the corresponding biopolymers can play essentially the same role, without being identical, with respect to the application or use considered, or in the technique in which they are involved; two sequences obtained because of the natural variability, especially spontaneous mutation, of the species from which they were identified, or because of induced variability, as well as homologous sequences, homology being defined below, are especially equivalent.
Variability is understood to mean any spontaneous or induced modification of a sequence, especially by substitution and/or insertion and/or deletion of nucleotides and/or of nucleotide fragments, and/or extension and/or shortening of the sequence at at least one of the ends; a nonnatural variability may result from the genetic engineering techniques used; this variability may result in modifications of any starting sequence, considered as reference, and capable of being expressed by a degree of homology relative to the said reference sequence.
Homology characterizes the degree of identity of two nucleotide
Jolivet Michel
Lesenechal Mylene
Paranhos-Baccala Glaucia
Bio Merieux
Graser Jennifer E.
Oliff & Berridg,e PLC
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