Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-11-04
2002-07-16
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023740, C435S320100, C435S235100, C435S069100, C435S069300, C435S471000, C424S273100
Reexamination Certificate
active
06420540
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to peptides immunoreactive with antibodies to
Toxoplasma gondii
(to be referred to as
T. gondii
hereinafter), nucleic acid sequences encoding these peptides, recombinant vector molecules comprising these nucleic acid sequences, host cells transformed with the recombinant vector molecule, immunochemical reagents comprising the peptides or antibodies directed against the peptides, a test kit for the detection of
T. gondii
infections as well as a vaccine for the protection against
T. gondii
infections.
BACKGROUND SUMMARY OF THE INVENTION
T. gondii
is an intracellular protozoan parasite found throughout the world and capable of infecting all species of mammals and all types of cells within a given individual.
T. gondii
is classified as a coccidian with two life cycles, asexual and sexual.
In the asexual stage,
T. gondii
exists in different forms, for instance; tachyzoites, pseudocysts, bradyzoites, and oocysts. The tachyzoite is the obligate intracellular form of
T. gondii
which characterizes acute infection. The infective stage of
T. gondii
for human host cells is the pseudocyst. The pseudocyst has a diameter of 30-100 micrometers and contains hundreds to thousands of infectious units termed bradyzoites. Infection is frequently initiated by ingestion of pseudocysts present in raw or uncooked unfrozen meats. In addition, infection can occur by ingestion of oocysts in the faeces of cats experiencing active intestinal infection. Infections may also be acquired through blood or leukocyte transfusion, by organ transplantation or by transplacental transmission during pregnancy (Wilson et al., J. Exp. Med. 151, 328-346, 1980). The wall of either the pseudocyst or the oocyst is broken down in the small intestine by host digestive enzymes releasing the bradyzoites or sporozoites, respectively, which then penetrate the columnar epithelium. It is probable that bradyzoites or sporozoites reach the liver by the hematogenous route where they are ingested by Küpffer cells. Once inside a cell, the organisms are referred to as tachyzoites. Liver parenchymal cells also become infected (Kranenbühl & Remington, Immun. of Parasitic Infections; eds. Cohen, S., Warren, K. S. London: Blackwell Scientific Publications pp. 356-421, 1982; Remington & Kranenbühl, Immun. of Human Infection, part II, Edited by Nahmias, A. J., O'Reilly, R. J. New York, Plenum Medical Book Company, pp. 327-371, 1982).
Replication immediately follows entry into host cells. In the host, macrophages transport bradyzoites throughout the body. The bradyzoites survive and replicate within the macrophage parasitophorous vacuole by preventing the fusion of lysosomes with it. Replication results in the lysis of the host cell. Organisms are phagocytosed by new macrophages or other cell types and repeat the cycle.
The sexual stage of
T. gondii
occurs only in feline hosts (Miller et al., J. Parasitology, 58, p. 928-937, 1972). The intermediate host (e.g., mouse) becomes infected by ingesting either oocysts or pseudocysts. The mouse develops pseudocysts throughout its own tissues. The cat becomes infected when it eats infected meat (e.g., rodent tissues) containing the pseudocysts or ingests oocysts. Bradyzoites or sporozoites penetrate columnar epithelial cells and differentiate into merozoites. Following replication, merozoites rupture infected epithelial cells and infect adjacent ones. Some merozoites differentiate into pre-sex cells termed macrogametocytes (female form) and microgametocytes (male form). The microgametocytes fuse with macrogametocytes, forming zygotes termed oocysts (Dubey & Frenkel, J. Protozool. 19:155-177, 1972). Oocysts enter lumen of the small intestine and are defecated. Each oocyst sporulates,in the soil, producing eight infectious sporozoites, the infectious stage for the intermediate host.
The bradyzoite is the form that encysts approximately 8-10 days after acquisition in vivo and characterizes the chronic, latent phase of infection. Thus, the bradyzoite form is the only stage that has the ability to initiate the enteroepithelial cycle.
T. gondii
induces a mild or unapparent disease in healthy adults but causes a severe disease, toxoplasmosis, or even death in congenitally infected children and in immunocompromised patients. Primary infection of pregnant women occurs in European countries with frequencies between 0.2 and 1.0%. In approximately 40-50% of the cases, the unborn child is infected. Infection in the fetus during pregnancy will (in approximately 10% of the cases) lead to neonatal death or a severely multi- handicapped child, but in 90% of the cases the child will be born with an asymptomatic, latent infection (Desmonts and Couvreur, Ann. Pediatr. 1984, 31, 805-809; Alford et al. Bull. NY Acad. Med. 1974, 50, 160-181). Up to 85% of the patients with latent congenital toxoplasmosis will develop significant sequelae including one or more episodes of active retinochoroiditis. Other clinical symptoms are inflammation, lymphadenitis, encephalitis and fever.
In immunocompromised patients, especially in the case of AIDS,
T. gondii
causes a severe pathology. In approximately 30 percent of Toxoplasma-antibody positive patients with AIDS, toxoplasma encephalitis will develop due to reactivation of their latent infection. In immunocompetent humans,
T. gondii
infection induces a long-lasting protective immunity against reinfection (Remington & Krahenbühl, 1982)
SUMMARY OF THE INVENTION that could be attributed to the persistence of encysted parasites throughout the host life.
There are several strains of
T. gondii
known. One of the most important strains is the RH-strain which is highly virulent and originally isolated from human brain tissue.
Most investigations in the past regarding
T. gondii
were focused on the identification and molecular characterization of tachyzoite-specific antigens. More than 1000 different
T. gondii
-specific proteins have been identified. Surface proteins of
T. gondii
have been studied extensively. Of these surface-proteins the RH strain p30 surface protein is the most abundant; it constitutes approximately 5% of the total tachyzoite protein. The p30 surface protein is recognized intensively by human IgM, IgG, IgA and IgE antibodies (Decoster et al., Clin. Exp. Immunol., 73, 376-382, 1988; Godard et al., Infect. Immun., 58, 2446-2450, 1990) and is therefore useful for diagnostic purposes. Using monoclonal antibodies directed against p30, two immunocapture tests have been developed for the detection of anti-Toxoplasma IgM (Cesbron et al., J. Immunol. Methods, 83, 151-158, 1985) and IgA (Decoster et al., Lancet, ii, 1104-1106, 1988) antibodies.
The present invention provides new peptides, immunoreactive with antibodies to T. gondii, that can be used in diagnosing
T. gondii
infections in humans.
In particular the present invention provides peptides, immunoreactive with antibodies to
Toxoplasma gondii
, comprising part of the amino acid sequences as shown in SEQ ID No.: 1.
A preferred embodiment of the present invention are peptides comprising at least part of the amino acid sequences shown in SEQ ID No. 3 or 5.
Another preferred embodiment of the present invention are polypeptides comprising polymeric forms of said peptides.
The term “peptide” as used herein refers to a molecular chain of amino acids with a biological activity, and does not refer to a specific length of the product. Thus inter alia, proteins, oligopeptides, polypeptides and fusion-peptides as well as fusion-proteins are included.
The term “polypeptide” refers to dimeric, trimeric, . . . , polymeric forms of any length of a peptide according to the present invention.
The term “peptide” as used herein further refers to so-called functional variants, for example, acid addition salts, amides, esters, and specifically C-terminal esters, and N-acyl derivatives of the peptides according to the invention. These functional variants are considered to be part of the present invention. Also included are peptides which are modified in vivo or i
De Haard Johannes Jozef Wilhelmus
Koolen Marcus Josephus Marie
Akzo Nobel N.V.
Minnifield Nita
Myers Bigel Sibley & Sajovec P.A.
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