Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-06-30
2001-01-09
Minnifield, Nita (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S007100, C435S007200, C435S007220, C435S007920, C435S961000, C435S968000, C435S973000, C435S971000, C435S975000
Reexamination Certificate
active
06172192
ABSTRACT:
The present invention relates to isolated and pure
Toxoplasma gondii
antigenic fragments, recombinant polypeptides, nucleic acids encoding them, primers and probes derived from the same, as well as the use of these polypeptides, nucleic acids, primers and probes in methods and kits for the diagnosis and prevention of
T. gondii
infection in mammals (humans and animals).
Toxoplasma gondii
is an ubiquitous intracellular protozoan parasite which infects mammals and birds. Although toxoplasmosis is in general clinically asymptomatic in healthy individuals, it may cause severe complications in pregnant women and immunocompromised patients. If primary infection occurs during pregnancy, transplacental transmission can lead to abortion or neonatal malformations (for reviews see Remington and Krahenbuhl, 1982; Hughes, 1985). In AIDS patients, Toxoplasma is recognized as a major opportunistic pathogen. In such immunodeficient individuals, rupture of cysts which persist in the tissues of the host after a primary infection and release of proliferative forms of the parasite may cause severe disseminated toxoplasmosis and/or encephalitis. In approximately 30 percent of Toxoplasma-antibody-positive patients with AIDS, toxoplasmic encephalitis will develop due to reactivation of their latent infection.
The fetus and the newborn are very sensitive to toxoplasmosis. Infection of the mother during pregnancy and transmission to the fetus can lead to miscarriage, birth of abnormal children (especially with ocular and cerebral lesions), or birth of apparently normal children who will develop grave sequaelae months or years later (blindness, mental retardation). Current estimates indicate that 0.1 to 0.9% of newborns are afflicted with congenital toxoplasmosis.
Besides its negative impact on human health, the parasite is also detrimental in sheep and pig farming since abortions resulting from the infection lead to relatively important economic losses (Beverly, 1976).
In the absence of efficient profylactic measures, there is a high necessity for early, sensitive and specific diagnosis.
In response to infection, immunocompetent hosts mount an immune response, which involves both humoral and cellular components (Remington and Krahenbuhl, 1982). The immune response confers protection against subsequent infection of the host: women showing serological evidence of previous infection before the onset of pregnancy are at no risk of transmitting toxoplasmosis to their fetus. In-vitro and in-vivo studies have indicated that cell-mediated immunity plays an essential role in protection and have identified interferon-gamma (IFN-gamma) as the major mediator of resistance (Frenkel, 1967; Nathan et al., 1984; Pfefferkorn, 1984; Sethi et al., 1985; Suzuki and Remington, 1988; Suzuki et al., 1988; Suzuki and Remington, 1990; Gazinelli et al., 1991).
The humoral component underlies the methods generally used in toxoplasmosis diagnosis. Like in other infectious diseases IgM class antibodies appear before IgG class antibodies. This difference is used to know whether a pregnant woman has an acute or chronic phase infection, only the former being dangerous for fetal transmission. However, in a number of cases IgM class antibodies remain high for a longer than usual time period, making a differential diagnosis difficult.
The reference tests in Toxoplasma diagnosis are the Toxoplasma lysis test (TLT) and the immunofluorescence test (IF). The detection of antibodies against Toxoplasma is most often carried out by an Enzyme Linked Immunoassay (ELISA) or tests based on the same principle. The specificity and sensitivity of these tests is not always optimal and depend on the quality of the antigen preparation used. Most often the antigen used is a fraction of a total cell lysate, lacking sufficient specificity.
The use of a selection of well characterised
Toxoplasma gondii
recombinant antigens could furnish better tools for a Toxoplasma diagnostic assay.
Several antigens of Toxoplasma have already been cloned and expressed as recombinant antigens. Several excreted/secreted antigens have been shown to be recognized by sera from patients: GRA1 (23 kDa) (Cesbron-Delauw et al, 1989; EP-A-0 346 430), GRA2 (28.5 or 28 kDa) (Mercier et al, 1993; S. F. Parmley et al, 1993; WO 93/25689), GRA6 (Lecordier et al, 1995), as well as some other cellular antigens, like SAG2 (P22) (Parmley et al, 1992), SAG1 (P30) (Kim et al, 1994), ROP2 (54 kDa) (Van Gelder et al, 1993) and a number of
T. gondii
antigenic fragments as described e.g. in EP-A-0 431 541 (Behringwerke).
A vaccine for controlling this infectious agent would be of great value and the feasibility of its development is suggested by the fact that primary infection with Toxoplasma results in specific and long lasting immunity against reinfection (Remington and Kranenbuhl, 1982). However, no effective and safe vaccine is currently available against toxoplasmosis in humans. A live temperature-sensitive mutant of the highly virulent RH strain, can induce protective immunity in mice and hamsters. This mutant, names ts-4 does not persist in the host, as it cannot form bradyzoites and cysts (Waldeland and Frenkel, 1983; McLeod et al., 1988; Suzuki and Remington, 1990). Since 1988, a live vaccine is available for sheep. It consists of
T. gondii
tachyzoites of the S48 incomplete strain grown on tissue culture (Toxovax, Ministry of Agriculture and Fisheries, New Zealand). By passing through laboratory mice the strain lost the ability to develop bradyzoites (cysts). This vaccine protects naive sheep against an infect with
T. gondii
(Buxton, 1991, 1993).
The aim of the present invention is to provide new polypeptides and peptides useful in the diagnosis and/or profylaxis of
Toxoplasma gondii
infection in mammals.
It is more particularly an aim of the invention to provide polypeptides and peptides useful in the serodiagnosis of
T. gondii
infection, and possibly enabling discrimination between chronic and acute infection.
It is in addition an aim of the present invention to provide polypeptides and peptides useful in diagnostic assay for
T. gondii
infection based on the cellular immune response of the infected host.
It is moreover an aim of the present invention to provide for polypeptides and peptides useful in a vaccine preparation against
T. gondii
infection.
It is a specific aim of the present invention to provide purified and isolated Tg20 antigenic fragments.
More specifically, it is an aim of the invention to provide recombinant Tg20 antigenic fragments.
It is moreover an aim of the invention to provide the amino acid sequence of Tg20 antigenic fragments, and nucleic acid sequences coding for the same.
It is moreover an aim of the invention to provide for monoclonal and polyclonal antibodies specifically reacting with antigenic fragments of the Tg20 protein.
It is also an aim of the present invention to provide for primers specifically amplifying Tg20 nucleic acid sequences, as well as probes specifically hybridizing with Tg20 nucleic acid sequences.
It is another aim of the invention to provide for diagnostic methods and/or kits for
T. gondii
infection, using the above-mentioned Tg20 polypeptides, peptides, antibodies, primers and/or probes as one of the active principles.
It is in particular an aim of the present invention to provide for a serodiagnostic method or kit for
T. gondii
infection, whereby the active principle comprises the Tg20 polypeptides or peptides of the invention in combination with other
T. gondii
antigens, more particularly, in combination with the Tg34 antigen (=Rop2 antigen), or fragments thereof, as described by Van Gelder et al. (1993).
It is finally an aim of the present invention to provide for a vaccine composition for providing protective immunity against
T. gondii
infection in mammals, more particularly in humans and/or domesticated animals.
All of the above-mentioned aims have been achieved by the following embodiments of the invention.
As described in more detail in the examples section, the current invention describes the
Jacobs Dirk
Saman Eric
Van Heuverswyn Hugo
Innogenetics N.V.
Minnifield Nita
Nixon & Vanderhye P.C.
LandOfFree
Toxoplasma gondii antigen Tg20 does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Toxoplasma gondii antigen Tg20, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Toxoplasma gondii antigen Tg20 will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2512286