Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-03-13
2002-04-16
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007920, C435S007930, C435S007940, C435S007950, C435S962000, C530S350000
Reexamination Certificate
active
06372443
ABSTRACT:
The subject of the present invention is a system for expressing a
Toxoplasma gondii
surface antigen, the antigen thus obtained and its use for diagnostic and/or therapeutic purposes.
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 inside its hosts, which are mammals.
This parasite, which is very widespread geographically, is an important pathogen, 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 persist 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 in 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 or immunosuppressed subjects. This organism has multiple replication sites. However, 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 foetal 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.
It was 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 antibodies which may be present in the individual, or by detecting the presence of Toxoplasma antigens in the subject.
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 detection of antibodies or antigens specific for toxoplasmosis. One of the critical points therefore consisted 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.
In this regard, Burg et al. (Abstract c85 J. Cell. Biochem., 1986, 1017, 145) have described the exploitation of an expression library in
E. coli
using the complementary DNA (cDNA) obtained from
Toxoplasma gondii
messenger RNAs and the isolation, from this bank, of sequences encoding the antigens present at the surface of the parasite, with the aid of polyclonal antisera directed against the purified surface antigenic proteins P30 and P22
Some authors have shown that P30 constitutes the major surface antigen (see Kasper et al., J. Immunol. 1983, 130, 2407-2412) and 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
Toxoplasma gondii
P30 protein and suggest 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 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 its membrane anchorage, and a potential N-glycosylation site.
There remains, however, a problem which consists in producing sufficient quantities of the P30 antigen both for the preparation of vaccines and for use in immunological tests. Kim et al. (Infection and Immunity, January 1994, 62, 203-209) have described the expression of a recombinant P30 with a conformation similar to that of the native protein in CHO cells without, however, succeeding in obtaining satisfactory expression levels.
There is, moreover, an advantage in producing a P30 which is secreted into the culture medium in order to facilitate its production and its use for the preparation of diagnostic tests or of pharmaceutical compositions.
Consequently, the subject of the present invention is an expression cassette which is functional in a cell derived from a nonmammalian eukaryotic organism allowing the expression of a DNA fragment encoding a
Toxoplasma gondii
P30 protein, placed under the control of the elements necessary for its expression, said P30 protein being secreted from said cell derived from a eukaryotic organism and recognized by human antisera.
In general, any cell derived from a nonmammalian eukaryotic organism can be used within the framework of the present invention and most particularly an insect cell or a cell derived from a lower eukaryotic organism. The term “cell derived from a lower eukaryotic organism” refers to a cell derived from a unicellular or pluricellular eukaryotic organism not possessing a mechanism which allows cell differentiation. Such cells are known to a person skilled in the art. There will be preferably used, however, a fungus, especially a unicellular fungus, or a yeast, especially of the strain Kluyveromyces, Pichia, Hasegawaea, Saccharomyces or Schizosaccharomyces and most particularly selected from the group consisting of
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae, Schizosaccharomyces octosporus
and
Hasegawaea japonicus
. Of course, these examples are not limiting. A large number of these cells are commercially available in collections such as ATCC (Rockville, Mass., USA) and AFRC (Agriculture and Food Research Council, Norfolk, UK).
For the purposes of the present invention, said cell may be of the wild type or mutant type. In this context, an auxotrophic mutant cell is particularly preferred which has lost the capacity to synthesize at least one metabolite essential for its growth, such that it can grow only in a medium supplemented with this specific metabolite or by complementation with a gene allowing its synthesis. Although a large number of auxotrophy mutations for various essential metabolites are currently known, there may be mentioned more particularly the mutations inhibiting the synthesis of arginine, of leucine and of uracil. Such mutations are described in the literature which is accessible to persons skilled in the art. A large number of auxotrophy mutations affecting various biosynthesis pathways can generally be generated by applying the following approach. Briefly, it is sufficient to cultivate culture the wild-type cells treated with the aid of a mutagen, at the same time in the presence and in the absence of the targeted essential metabolite and to search for the mutants which will multiply only in its presence contrary to the wild-type cells which can grow in both cases.
An expression cassette according to the invention is intended for the producti
Bissardon Odette
Jacobs Eric
Jolivet Michel
Mougin Bruno
Silvestre Nathalie
Burns Doane Swecker & Mathis L.L.P.
Navarro Mark
Transgene S.A.
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