Toxoplasma gondii antigens, the preparation thereof and the use thereof

The present invention relates to the identification of Toxoplasma gondii antigens and the preparation thereof by genetic engineering. A cDNA expression gene bank of this parasite was prepared. Recombinant clones which are of diagnostic interest were identified using a high-titer rabbit anti-Toxoplasma gondii serum, and isolated.

The present invention relates to the identification of Toxoplasma gondii 
antigens and the preparation thereof by genetic engineering. A cDNA 
expression gene bank of this parasite was prepared. Recombinant clones 
which are of diagnostic interest were identified using a high-titer rabbit 
anti-Toxoplasma gondii serum, and isolated. 
Toxoplasma gondii (T. gondii) is a obligatory intra-cellular single-cell 
parasite which is categorized as a coccidium. The parasite has a 
relatively wide range of hosts and can, in addition to very many mammals, 
also infect man. In the latter case there are two forms which differ from 
each other physiologically: "tachyzoites" reproduce asexually in a number 
of different cell types. This form is found exclusively in the acute stage 
of the infection. "Bradyzoites", in contrast, persist in cells of the 
cardiac and skeletal muscles and in cells of the central nervous system in 
encapsulated form and are responsible for a persistent immunity to 
reinfection. It is estimated that globally there are 500 million people 
who are chronically infected by T. gondii. 
In healthy adults, a T. gondii infection normally has no symptoms with the 
exception of a slight swelling of the lymph nodes. During pregnancy and in 
immunosuppressed patients, however, an infection with this parasite may 
present particular problems. Thus there is the risk of an intra-uterine 
transfer of these parasites in pregnant women who have not acquired a 
protection from T. gondii by immunity. This leads to the infection of the 
fetus and may result in deformities of the child or the expulsion of the 
fetus. 
Immunosuppressed patients frequently acquire an acute T. gondii infection 
as a result of the reactivation of enzysted "bradyzoites". In most cases 
this leads to cerebral toxoplasmosis (encephalitis), which may, under 
certain circumstances, be lethal. In addition to cerebral toxoplasmosis, 
T. gondii has also been mentioned as causative agent of eye diseases 
(chorioretinitis). These cases, too, are infections which can be blamed on 
a reactivation of "bradyzoites". 
The clinical picture of toxoplasmosis often causes difficulties concerning 
differential diagnosis to the clinician so that the support by laboratory 
analyses in establishing the diagnosis is sought. The detection of 
antibodies and the determination of the titer or of the dynamics of the 
titer have therefore become essential tools for diagnosing toxoplasmosis. 
Methods for determining toxoplasma-specific immunoglobulins of the G and M 
class, such as indirect immunofluorescence (IF), complement fixation 
reaction (CF), indirect hemagglutination (IHA), latex agglutination (LA) 
and enzyme-linked immunoassay (ELISA) are very common in the field of 
serodiagnosis but often have faults. For example these test methods vary 
very greatly as regard specificity and sensitivity. These differences are 
primarily caused by the preparation of the antigen which is used for the 
serological test. In most cases total cell antigen which contains a high 
proportion of unspecific cell components and is held responsible for the 
occurrence of false positive test results, is prepared. In addition, 
obtaining the antigens from infected mice holds the risk of infection for 
the person working in the laboratory. 
In view of the specificity and sensitivity of a diagnostic of this type, it 
would thus be desirable to employ defined immunoreactive antigens which 
should additionally make it possible to distinguish between IgG- and 
IgM-specific anti-T. gondii antibodies. 
A number of antigens of diagnostic interest have been described for T. 
gondii in the literature. For example Hughes describes in a review (Curr. 
Top. Microbiol. (1985), 120: 105-139) four major antigens which are 
potentially suitable for detecting anti-T. gondii antibodies of the IgG 
class, having molecular weights of 45, 32, 27 and 21 kilodalton (kD). 
Handman et al. (Immunol. (1980), 40: 579-588) and Potasman et al. (J. 
Infect. Diseases (1986), 154: 650-657) analyzed sera taken throughout the 
course of the disease of acutely infected T. gondii patients using Western 
blots and demonstrated that a 35 kD membrane antigen reacts with IgG 
antibody at a very early stage. Decoster et al. (Clinic. Exper. Immunol. 
(1988), 73: 376-382) describe four antigens of diagnostic interest, which, 
in contrast to the 35 kD antigen, can be isolated from the culture medium 
and have been termed "excreted-secreted antigens" (ES antigens) and which 
have molecular weights of 105, 97, 66 and 28.5 kD. IgG antibodies which 
react with antigens of 105, 97 and 28.5 kD seem to be good markers for a 
chronic toxoplasmosis. Similarly to the 35 kD antigen, the 97 kD antigen 
and the 66 kD antigen are recognized at a very early stage by IgM 
antibodies of acutely infected patients. It has to be pointed out that 
these antigens have not been sufficiently characterized by giving a 
molecular weight after electrophoretic fractionation because there usually 
are several proteins within one molecular weight range. 
A 6 kD antigen is a further marker for acute toxoplasmosis (Ehrlich et al., 
(1983), Infect. Immun. 41: 683-690). In IgM Western blots, this antigen 
reacts relatively strongly. To date there are only very few data which 
might reveal the nature of this antigen. 
Only very few T. gondii antigens have been biochemically characterized so 
far. The main surface protein P30 is an exception. This antigen is a 
glycoprotein which is anchored in the membrane via a glycolipid (Nagel et 
al., (1989), J. Biol. Chem. 264: 5569-5576). The diagnostic importance of 
this antigen is controversial since P30 also reacts with unspecific 
antibodies of the IgG class (Potasman et al., (1986), J. Clin. Microbiol. 
24: 1050-1054). 
The isolation and purification of individual antigens for the use in 
serodiagnosis often involves a considerable amount of work. Both the 
molecular weight data and the classification of the immunoreactivity of an 
antigen can substantially differ from case to case in conventionally 
purified antigen. Cloning and expressing such antigens and investigating 
the structure of the corresponding genes might not only improve the yield 
of purified antigen but should also contribute to the serological 
characterization and therefore to the investigation of the diagnostic 
relevance of the antigen. So far the structure of the genes of two 
immunologically interesting T. gondii antigens has been investigated. The 
complete nucleotide sequences of these antigens, which are P30 (Burg et 
al., (1988), J. Immunol. 141: 3584-3591) and a 28 kD antigen (Prince et 
al., (1989), Mol. Biochem. Parasitol. 34: 3-14), are known. 
The object of the present invention is to prepare by genetic engineering 
defined antigens of T. gondii, which are suitable for diagnosis and 
prevention. It has been possible to successfully identify suitable T. 
gondii gene products from a lambda gt11 cDNA expression gene bank using a 
high-titer rabbit anti-T. gondii serum. Partial nucleic acid sequences, 
and aminoacid sequences derived therefrom, of 8 clones (F2, F28, F29, F34, 
F45, F61, F74 and F76) have been determined. All the abovementioned clones 
react in Western blots with human anti-T. gondii IgG sera. The clones F34, 
F61 and F76 additionally react with specific antibodies of the IgM class. 
The partial nucleotide sequences are listed in Tab. 1-8 and, as far as 
they are apparent, also the translational reading frames (in Tab. 1-6). 
F61 (Tab. 1) is assigned to a protein having a molecular weight of 66 kD. 
F34 (Tab. 2) belongs to a protein of about 68 kD. 
F29 (Tab. 3) belongs to a protein of about 30 kD. 
F28 (Tab. 4) belongs to a protein of about 28 kD. 
F2 (Tab. 5) belongs to a protein of about 30 kD. 
F76 (Tab. 6) belongs to a protein of about 35 kD. 
F45 (Tab. 7) belongs to a protein of about 29 kD. 
F74 (Tab. 8) belongs to a protein of about 64 kD. 
With the aid of the partial sequences mentioned it is readily possible to 
clone the complete genes for the abovementioned partial sequences. 
The partial sequences depicted in the Tables 1, 2 and 6 were accordingly 
used to complete the coding cDNA regions of the genes belonging thereto. 
For this purpose, the cDNAs F61, F34 and F76 were radiolabeled and used as 
probes for screening the cDNA gene bank. The sequence from Table 1, F61, 
was used to isolate the cDNA of the P66 protein. The sequence from Tab. 2, 
F34, was used for the isolation of the cDNA of the P68 protein. For the 
isolation of the cDNA of the P35 protein, the sequence from Tab. 6, F76, 
was used. Recombinant clones having homologies to these sequences were 
isolated and characterized structurally by sequencing the inserted T. 
gondii-specific cDNA regions. The nucleotide sequences of the complete 
ranges of the structural genes of the P35, P66 and P68 proteins are 
depicted in the Tables 9-11. 
Immunologically reactive partial regions (immunogenic parts) are 
representatively described for P35, P66 and P68 in the Examples 6 and 7. 
Other immunogenic protein regions are tested or determined in an analogous 
way. 
The invention therefore relates to 
(a) the isolated inserted DNA sequences of the abovementioned clones, 
including the transcription products thereof and the remaining sequences 
to complete the particular structural genes, 
(b) DNA structures and vectors which contain, completely or in part, these 
sequences, 
(c) prokaryotic or eukaryotic cells which have been transformed with DNA of 
this type, 
(d) the polypeptides expressed by transformed cells of this type, or 
immunogenic parts thereof including the use thereof for diagnosis and 
therapy or prevention, 
(e) the amino-acid sequences (AS) belonging thereto, 
(f) antibodies against the polypeptides under (d), including the use 
thereof for the diagnosis and therapy or prevention of T. gondii 
infections, and 
(g) processes for the preparation by genetic engineering of the 
polypeptides mentioned under (d) or of immunogenic parts thereof.