Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-10-02
2002-04-16
Graser, Jennifer E. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S243000, C435S252300, C536S023700, C424S273100
Reexamination Certificate
active
06372897
ABSTRACT:
The present invention relates to a polypeptide, capable of being substituted for a
Toxoplasma gondii
antigen, a polynucleotide whose expression corresponds to said polypeptide, and their use for diagnostic and therapeutic purposes.
BACKGROUND OF THE INVENTION
Toxoplasmosis is an infectious disease caused by a
Toxoplasma gondii
protozoal parasite, a member of the class Sporozoa, and of the order Coccidia.
Toxoplasma gondii
is an intracellular parasite which reproduces in a wide variety of cell types in its hosts, which are mammals.
This parasite, which is very widespread geographically, is an important pathogenic agent, not only in human medicine, but also in veterinary medicine.
In man, two forms of the parasite have been described: the “tachyzoite”, which is the multiplicative form encountered during the acute phase of the disease and the “bradyzoite”, a resistant form which persists encysted in the nervous tissues, and which is probably responsible for maintaining a durable immunity to reinfection.
In humans, toxoplasmosis is most often asymptomatic and most often passes unnoticed without any consequences. There are however cases for which a Toxoplasma infection or a reactivation of a previously acquired infection can generate serious disorders for the so-called at risk individuals who are pregnant women and immunodepressed and immunosuppressed subjects. This organism has multiple replication sites. Thus, it may be responsible for severe cerebral and ocular impairments when its replication site is the cells of the central nervous system and the cells of the reticuloendothelial system. Pregnant women represent high-risk subjects, since a Toxoplasma infection, especially during the first few months of pregnancy, may be responsible for serious fetal and neonatal complications if maternal treatment is not undertaken early and pursued assiduously. In particular, newborns contaminated via the transplacental route are subject to serious ocular and cerebral disorders which are even fatal in certain cases. Immunodepressed patients and particularly AIDS patients are subject to serious Toxoplasmosis due most often to reactivations of previous infections, although dissemination following a primary infection cannot be completely excluded (see FARKASH et al., J. Neurology, Neurosurgery and Psychiatry 1986, 49, 744-748 and Luft et al., J. Am. Med. Ass. 1984, 252, 913-917).
It is therefore essential to have available diagnostic tests which make it possible to determine the presence of the parasite, especially in pregnant women, either by detecting specific antibodies or by detecting Toxoplasma antigens in the subject.
DESCRIPTION OF THE PRIOR ART
HUGUES in “Current topics in Microbiology and Immunology” (Vol. 120, 1985, SPRINGER Ed., pages 105-139) has listed a number of commercially available serodiagnostic tests such as the SABIN and FELDMAN staining test, standardized by BEVERLY and BEATTLE in 1958 and perfected by FELDMAN and LAMB (1966), WALDELAND (1976) and BALFOUR et al. (1982); the REMINGTON (1968) test for the detection of antibodies by immunofluorescence, optimized in 1975 by KARIM and LUDLAM; the hemagglutination tests; the ELISA test for the detection of antibodies specific for Toxoplasma, by the isolation of IgM in situ on a microplate described in 1983 by WIELARRD et al.
The different tests used are based on the formation of immune complexes between the
Toxoplasma gondii
antigens and their specific antibodies. One of the critical points therefore consists in the characterization of the
Toxoplasma gondii
major antigens, which induce a specific immune response and are capable of being used in serological detection tests.
Some authors have shown that most of the monoclonal antibodies directed against the
Toxoplasma gondii
surface recognize a protein of 27 to 35 KDa, called P30 (Kasper et al.; 1983, J. Immunol. 130, 2407-2412). Several studies have demonstrated that this P30 protein is a major surface antigen which can be used for the production of vaccines or in diagnostic tests, especially in immunoassays. Moreover, Boothroyd et al. (see Patent Application WO 89/08700) have identified and obtained the genetic material encoding the
Toxoplasma gondii
P30 protein and suggested the use of the gene for the production of recombinant protein, peptides and antibodies. This gene has been cloned (Burg et al., 1988, J. Immunol., 141, 3584-3591). Analysis of the sequence shows a potential N-glycosylation site, a secretory signal positioned at the N-terminal end which is cleaved in the mature P30 protein and a highly hydrophobic C-terminal region which is also cleaved and replaced by a glycolipid which allows membrane anchorage of the P30 protein.
There remains, however, a problem which consists in producing sufficient quantities of the P30 antigen. Indeed, three approaches are currently available, with the aim of obtaining antigens and parts of P30 antigens. which can be used especially in diagnostic procedures.
The first consists in performing cultures of the
Toxoplasma gondii
parasite in a large number of infected mice, in collecting the natural
Toxoplasma gondii
antigens, and especially P30, via the peritoneal route and in purifying them. However, this technique has many disadvantages such as the cost, the need of having available experienced staff and a large number of animals, the complexity of the technique or the difficulties linked with the extraction which is not very efficient.
The second approach, derived from the preceding one, involves the in vitro culture of the parasite in cell cultures (Grimwood et al. 1979, Experimental Parasitology, 48, 282-286). However, this approach does not make it possible to overcome the disadvantages of cost or extraction mentioned above.
The third approach refers to genetic recombination technologies and consists in introducing and cloning into a prokaryotic, eukaryotic or viral genome all or part of the gene encoding the P30 protein, in expressing said gene in a host cell and in extracting the recombinant P30 protein obtained. This technique offers many advantages but suffers from a low level of expression of the protein. Moreover, Burg et al. (1988, Journal of Immunology, 141, 3584-3591) has shown that the natural P30 protein is a highly structured antigen exhibiting conformational constraints. The conformation of the P30 protein is a critical element for its recognition by specific antibodies and results especially from the presence of disulphide bridges in the protein. Now, it is difficult, through genetic recombination, to perfectly control the post-translational modifications to which the synthesized proteins are subjected. Consequently, it is difficult to obtain, by genetic recombination, a P30 protein or a part of said protein which is appropriately matured so that it adopts a conformation which is sufficiently close to that of the natural P30 protein to allow specific recognition by antibodies directed against said protein.
In accordance with the document WO-A-89/12683, polypeptides are described which are capable of reacting with. an anti-
Toxoplasma gondii
P30 protein antibody and which possess a peptide sequence chosen from the sequence SEQ ID No.15, fragments of SEQ ID No.15, SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18, which are identified at the end of the description. These polypeptides were determined from the peptide sequence of a
Toxoplasma gondii
protein, and consist of the sequence of the latter or of the antigenic fragments of the latter. Given the low level of homology of said sequences with that of the P30 protein, it can be stated that the peptide sequence of the protein at the origin of these polypeptides is not that of the P30 protein. These polypeptides pose the problem of their specificity; indeed, they have antigenic properties toward anti-P30 antibodies, but they probably have the same properties toward antibodies directed against the protein from which they were defined.
Surprisingly, it has been shown that a polypeptide synthesized by the chemical route or by genetic recombination methods, whic
Bio Merieux
Graser Jennifer E.
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