FR2564731A1 - New exoantigen fractions of toxoplasma, method for preparing them and their use in testing for toxoplasma immunity in man by skin tests - Google Patents

Abstract

The new exoantigen fraction is obtained by culturing Toxoplasma gondii on diploid human cells, and possesses a molecular weight of the order of 350,000 daltons and an absorption of 5.7 OD units at 492 nm.

Description

 The present invention relates to new exo-antigenic fractions, their preparation process and their application to the research of toxoplasmic immunity in humans by skin tests.

 Toxoplasmosis is a parasitic disease caused by an intracellular sporozoan Toxoplasma gondii. This disease, which is transmitted from the mother to the fetus, is particularly serious for the latter, and is translated shortly after birth by hydrocephalus, convulsions, fever and atrophic and pigmentary choritinin. It can lead to a fatal outcome and leave nerve and eye damage.

 The diagnosis of toxoplasmosis is most often made on the basis of biological elements provided by biological tests, such as 1 / indirect immunofluorescence, 2 / the lysis test of SABIN and FELDMAN, 3 / the reaction indirect hemagglutination, 4 / the toxoplasma agglutination test, and 5 / various serological anti-reactions.

 These diagnostic methods are however of a long implementation and are expensive.

 So we quickly realized the interest in having toxoplasmic antigens that would allow the search for toxoplasmic immunity by skin tests.

Studies have been carried out in this direction by
DESGEORGES, and colt. Lyon Médical, 1980, 243: 737-740, which prepared exo-antigens in vitro by culturing Toxoplasma gondii on VERO cells (monkey kidney cells) in the presence of calf serum. The antigens prepared by DESGEORGES could not, however, be used in human clinics due to the presence of various contaminants provided by cell culture.

More recently, AMBROISE-THOMAS and Coll. Lyon
Médical, 1982, 248: 79-83, showed that the toxoplasmic exoantigens produced on human diploid cells MRC 5 could be used in vivo, as a means of detection of toxoplasmic immunity in human clinic.

As an extension of the work undertaken by
AMBROISE-THOMAS et al., The depositor sought to identify and isolate the exo-antigenic fractions with high antigenic activity and specificity1 and capable of being used in skin tests for the control of acquired anti-human immunity in humans -toxoplasmic.

 The present invention therefore relates to new exo-antigenic fractions obtained by culture of Toxoplasma gondii on human diploid cells and their use for the research of anti-toxoplasmic immunity in humans by intra-dermal tests.

 The present invention also relates to the process for the preparation of such fractions.

 It also relates to the application of these exo-antigenic fractions to the detection of toxoplasmic immunity in humans by skin tests.

 Finally, it relates to a new test substance for the detection in humans of toxoplasmic immunity.

The process for preparing the exogenous tigenic fractions in accordance with the instruction which consists successively - in cultivating Toxoplasma gondii on diploid cells
human, especially on cells known as
designation MRC 5. This line corresponds to cel
human embryonic pulmonary cells; she
is available from FLOW Lab., 99 Rue de la
Republic, 92800 Puteaux.

- collecting the supernatant, - extracting the exo-antigens from the supernatant, - concentrating the exo-antigens, is characterized in that after having concentrated the exoantigens - said exo-antigens are dialyzed against a solution
appropriate buffer, at a pH close to 9, - the said exo-antigens are subjected to fractionation, - the separate fractions are identified for their activity
antigenic, - the fractions rich in exo antigens are combined.

The culture supernatants are collected on the 6th day, subjected to sterilizing filtration (0.22%) and then treated with 5% formalin for 24 hours. They are then concentrated 200 times and undergo 10 dialysis cycles on the hollow fiber filtration columns (AMICON) with cutoff at 10,000 Daltons. The concentrated antigen is again filtered through a 0.22ss sterilizing filter. The fractionation is carried out by gel chromatography
G 200 (PHARMACIA) at a pH of 8.8 in 0.05 M Tris-glycine buffer, 0.01% NaN3, at a temperature of 40C and with a flow rate of 0.75 ml cm-2 x h-1 for a 1.6 x 60 cm column containing 98.5 of gel.

 The characterization of the exo-antigenic fractions was carried out using different biochemical and immunological techniques.

For the identification of exo-antigenic fractions and the determination of their molecular weight, use was made of immuno-enzymatic techniques commonly known as ELISA and GEDELISA tests, abbreviations of the English expressions "Enzyme Linked Immuno Sorbent Assai" and "Gel Electrophoresis Derivated of Enzyme Linked Immuno
Sorbent Assay ". These techniques are known to those skilled in the art who may refer to if necessary
AMBROISE-THOMAS et Coll. WHO Bulletin, 1978, 56 609-613 and DESGEORGES et al., Annals of clinical biology 1980, 38: 361-363 for details of execution.

 The applicant was thus able to detect the presence of six exo-antigenic fractions having molecular weights ranging from 15,000 to more than 660,000 daltons. The preferred exo-antigenic fraction is that having a molecular weight of the order of 325,000 daltons, which has very interesting pharmacological properties which make it particularly suitable for use in the search for toxoplasmic immunity in skin tests.

 This fraction is characterized by the fact that it faces an immune serum produced in an animal from this fraction an optical density (OD) of 5.7 measured by ELISA at 490 nm and a characteristic arc of precipitate in counter-electrophoresis and in two-dimensional immunoelectrophoresis.

 The exo-antigenic fractions according to the invention are essentially protein in nature, as shown by the fact that they are found to be practically inactivated when they are allowed to incubate with enzymes from the protease group such as trypsin.

They exhibit good resistance to deoxyribonuclease and ribonuclease.

 The exo-antigenic fractions according to the invention are also characterized by good heat stability.

 The invention will be better understood using the additional description which follows, referring to an example of preparation of exo-antigenic fractions and characterization of said fractions. It goes without saying that it should naturally be understood that this example is given solely by way of illustration of the invention and that it has no limiting character.

EXAMPLE
Preparation of toxoplasmic exo-antigenic fractions
Toxoplasma gondii strain RH of SABIN was maintained on white OF1 mice, by intraperitoneal injections. The in vitro cultures were carried out on MRC 5 cells in the presence of EAGLE BME medium at the rate of 1.5 × 10 8 parasites per 3.5 liters of culture. The supernatants were collected after 6 days and the antigens were extracted and concentrated according to the technique described by AMBROISE-THOtGS et Coll. Lyon Médical, 1982, 248: 79-83. The resulting antigen solution contained 0.108 mg protein nitrogen per ml.

Sample preparation
It was obtained under the same conditions as for toxoplasmic antigens, but from cultures of MRC 5 cells not inoculated with Toxoplasma gondii.

Preparation of control sera
We used, as positive control sera, a pool of very strong positive human sera in indirect immunofluorescence (IF Carried out with a counter-staining by Evans blue) and in counter-electrophoresis.

As for the negative control sera, they came from a set of non-immune subjects and whose serology in indirect immunofluorescence and in counterelectrophoresis was also negative. In addition, experimental sera from rabbits were used which were immunized by subcutaneous injection (8 injection points) of 1 ml of exo-antigens added to 1 ml of complete adjuvant.
FREUND. 15 days after (D 15), the injections were repeated under the same conditions, but using a mixture of exo-antigens, and incomplete adjuvant
FREUND. On days D 21, D 28 and D 35, the animals received an intramuscular injection of 1 ml of exo-antigens without adjuvant. On D 43, the animals were sacrificed and their sera were checked individually. indirect and double diffusion immunofluorescence, using the Ouchterlony technique, before being mixed.

Demonstration of toxoplasmic exo-antigenic fractions
The exo-antigenic fractions were demonstrated by the ELISA test.

 This test was carried out on microtiter plates, according to the method described by AMBROISE-THOMAS et al. in WHO Bulletin 1978, 56: 609-613, using ortho-phenylene diamine (OPD) 2 HC1 as chromogen (3 mg / ml) and as hydrogen peroxide at 30 Vol. (at 3 ttl / ml in phosphate-citrate buffer pH 5.15 0.075 M). The reaction was stopped after 10 min.

in the dark by adding 200 l of 2N sulfuric acid to each well. The reading was taken at 492 nm using a spectrophotometer.

 The toxoplasmic exo-antigenic fractions were detected until the dilution of 1/1600, which corresponds to approximately 67.5 ng of protein nitrogen / ml. At this dilution, the positive controls (extracts from parasitized cultures) have an optical density of 0.46 facing the pool of positive sera and 0.01, under the same experimental conditions, facing the negative sera. The non-parasitized culture extract, reported at the same dilution as the exo-antigens, gave no detectable O.D. to the pool of positive sera.

 The demonstration of the exo-antigenic fractions was also carried out by electrophoresis.

This was carried out according to the method described by
PINON and Coll. in American Journal of Tropical Medicine and Hygiene, 1978, 28: 318-324 on strips of cellulose acetate (Cellogel) (2.5 cm x 17 cm x 190 ssm) placed on racks and in special tanks (SEBA ). The buffer used was Tris-glycine pH 8.8 with an ionic strength of 0.05 M. The migration was carried out for 2 hours at 110 volts, after which it was stained with Coomassie blue at 0.2%, the excess dye being removed with a solution of mdthanol (500 months), acetic acid (100 ml) and distilled water (400 ml).

 We were thus able to highlight 6 precipitation bands opposite the reference serum. Selective staining demonstrated that these bands were protein in nature.

Determination of molecular weights of exo-antigenic fractions
The fractionation of the exo-antigens and the determination of their molecular weight were carried out by gel filtration under the following conditions.

A chromatographic column (1.6 × 60 cm) loaded with Sephadex G 200 gel of superfine quality (PHARMACIA), 0.05 M Tris-glycine buffer pH 8.8, 0.01% NaN3, height of the gel bed: 49 cm. Total volume: 98.5 ml. The column was calibrated with the following substances: ribonuclease, ovalbumin, aldolase, ferritin, chymotrypsinogen, albumin, catalase, thyroglobulin and dextran blue 2,000. 2 ml of exo-antigens previously dialyzed against the chromatography buffer were deposited in top of the column. Was eluted at a flow rate of 0.75 ml / hour and collected 160 fractions of 1 ml at 40C. These fractions were checked at 280 nm and in
ELISA.

 The dilution diagram obtained has been illustrated in FIG. 1 which shows the contents of the studied volumes (in ml) as measured by UV spectrophotometry (absorption at 492 nm). The molecular weights of known substances used as standards are plotted at the top of the graph on a line parallel to the abscissa axis.

 Six antigenic peaks have been isolated corresponding to molecular weights of the order of 15,000, 60,000, 175,000, 240,000, 340,000 and more than 600,000 daltons. The separation between the peaks at 175,000, 240,000 and 340,000 daltons was however not very good with this technique.

 Another method was used to determine the molecular weights of the exo-antigenic fractions, the GEDELISA technique. To this end, the procedure described by DESGEORGES & COLL was followed. in Annals of clinical biology, 1980, 38: 361-363, using a polyacrylamide gel PAA 4/30 (PHARtECIA). The following proteins were used as standard proteins: thyroglobulin, serritin, catalase, lactate, dehydrogenase and albumin. After electrophoresis, half of the gel was stained with Coomassie blue while the other half was frozen and cut into 2.5 mm thick transverse slices which were each placed in a cup of a baking sheet. microtitration, diluted and incubated overnight at + 40C, with mechanical shaking in the presence of 1 ml of carbonate-bicarbonate buffer pH 9.6.

The presence of antigenic proteins was detected by the ELISA test with the various control sera.

 We have in Fig. 2 represents the absorption curve at 492 nm, plotting the number of the fractions on the abscissa, the D.O. on the ordinate and at the top of the graph, on a straight line parallel to the abscissa axis the molecular weights of the standard proteins.

 By this technique, the molecular weights of the fractions detected are of the order of 64,000, - 83,000, 126,000, 322,500 and more than 1,400-000 daltons. We note the existence of a non-specific peak at 570,000 daltons.

With this technique, better resolution is obtained and some of the peaks detected correspond to those highlighted by chromatography: 64,500 and 60,000, 322,500 and 340,000. As for the other values, we observe that the antigenic fractions detected by GEDELISA are the monomers of those which have been isolated in gel filtration as shown by the agreement of the figures 2 x 83,000 = 166,000 (instead of 175,000 in gel filtration), 2 x 126,000 + 252,000 (instead of 240,000 in gelfiltration). The slight differences observed are explained by the fact that, during the electrophoresis constituting the first stage of GEDELISA, there is a migration by difference of potential, which does not exist in gelfiltration. We note that the fraction of 15,000 daltons detected by filtration on gel nc could by be found in GEDELISA since the lower limit of this technique is 50,000 daltons.

Biochemical nature of exo-antigens
The study of the biochemical nature of exo-antigens was carried out by enzymatic and chemical attack with trypsin, deoxyribonuclease, ribonuclease A, lipase and sodium periodate. Trypsin and ribonuclease A were used at concentrations of 2.5, 0.25, 0.025, 0.0025 and 0.00025 mg / ml.

In the case of dsoxyribonuclease, the concentrations of 1.0, 0.1, 0.01, 0.001 and 0.0001 mg / ml were used and for lipase 25, 2.5, 0.25, 0.025 and 0 .0025 mg / ml. The periodate was used in concentrations of 10, 1, 0.1, 0.01 and 0.001 mg / ml. The buffers used are those mentioned by BERGMEYER in Methods of enzymatic analysis, vol. 2, 2nd Edition, Academic Press
Ed. 1974 / with 0.05% Tween 20 added, except for the lipase buffer. The sodium periodate was dissolved in 0.05 l glycine. Each reagent (100 1 per well) was placed in a polystyrene microtiter plate previously sensitized with toxoplasmic exo-antigens or control preparations. After an incubation of one hour at 370C, an ELISA test was performed in front of the serum reference. We were thus able to highlight the essentially protein nature of exo-anti toxoplasmic genes. Indeed, after the treatment with trypsin carried out under the conditions described above, only 7% of residual antigenicity is found. Treatment with periodate allows 67% of antigenicity to persist, which shows that exo-antigens have a low carbohydrate character. The same applies to the lipid character with 73% residual antigenicity after the action of the lipase. Ribonuclease A and deoxyribonuclease have practically no action on exo-antigens (98% and 96% of residual antigenic activity, respectively).

Heat stability
The thermostability was studied at 600C and 800C, for times of 5, 10, 15, 20, 30, 40 and 50 min. Previously, the exo-antigen solutions were diluted to 1/200 in carbonate-bicarbonate buffer pH 3.6 0.05 M.

Residual antigenicity was detected by ELISA against the control sera.

 The toxoplasmic exo-antigens are partially thermolabile with a residual antigenicity of 45% and 15%, respectively after 50 min. heating at 600C and 80 C. Between 20 and 50 min. at 800C, the residual antigenic activity remains practically the same. However, it continues to vary at 600C in the same time interval.

Chromato-focusing
The technique of PHARMACIA Chromatofocusing with Polybuffer and PBE, Rahms & Lund, Ed, 1980 was used.

with PBEC TM 94 as gel, in a column (10 x 20 cm) for a total volume of 15 ml. The pH study area was varied from 7 to 4, using Poly buffer 74 HCl (1/8) pH 4.0 as elution buffer.
PHARMACIA. 250 1 ml fractions were collected. From fraction 200, the column was flowed with 1 M Na C1. Chromatography was carried out at + 40C with a flow rate of 20 ml / h. The plf of each fraction, its absorption at 280 nm and its reaction to the ELISA test were measured. For this last test, 100 Rl of carbonate-bicarbonate buffer pH 2.6 0.35 M was previously added to each fraction.

 Most of the antigenic activity is in the fold interval 4 h 5 and less than 5, as shown in Fig. 3 where the curve of variation of the D.O. at 492 nm and the curve of variation of the pH as a function of the volumes elected has been shown.

Pharmacological studies
The exo-antigenic fraction of molecular weight of the order of 325,000 daltons was subjected to safety controls in mice, cell cultures and bacteriological cultures (sterility checks).

Use for screening for toxoplasmic immunity
The exo-antigenic fraction can be used - as an intra-dermal injection; for that we realize
solution containing 1 biological unit (as it is
defined in guinea pigs, AMBROISE-THOMAS et Coll., Lyon
Medical, 1982, 248: 78-83), which is injected into a
0.2 ml volume. The result is read
after 72 hours or more by measuring the induration obtained
naked.

- in patch tests using rings
multipunctures. The antigenic fraction beforehand
titrated is mixed in a proportion of 70% with a
glycerol solution. This mixture is deposited on
multipuncture rings with a kitten of 9
spikes, as they have been described for several
other applications and in particular in screening for
anti-tuberculin immunity (KRAVITZ et al.

Pediatrics, 1968, 42: 465-470). The results are read
after 72 hours or more by measurement of induration.

Claims (8)

1. New toxoplasmic exo-antigenic fraction obtained by culture of Toxoplasma gondii on human diploid cells, characterized in that it has a molecular weight of the order of 325,000 daltons. 2. New toxoplasmic exo-antigenic fraction according to claim 1, characterized in that it has an absorption of 5.7 units of OD at 492 nm and in that it gives, when faced with an immune serum produced in an animal from said fraction, a characteristic precipitation band in two-dimensional immunoelectrophoresis. 3. New toxoplasmic exo-antigenic fraction according to claim 1, characterized in that it has a maximum antigenic activity at a pH below 5. 4. New exo-antigenic fraction according to any one of the preceding claims, characterized in that it has a residual activity of the order of 45% when said fraction is heated to 600C for 50 minutes. 5. Process for the preparation of exo-antigenic fractions according to claims 1 to 4, consisting successively - in cultivating Toxoplasma gondii on diploid cells  human, especially on cells known as  designation MRC 5, - to collect the supernatant, - to extract the exo-antigens from the supernatants, - to concentrate the exo-antigens,  characterized in that, after concentration of the exo-anti  genes, - exo-antigens are dialyzed against a buffer solution  appropriate, at a pH close to 9, - the said exo-antigens are subjected to fractionation, - the separate fractions are identified for their activity  antigenic, - the fractions rich in exo-antigens are reduced. 6. Method according to claim 5, characterized in that the fractionation is carried out by filtration on gel G 200 (PHARMACIA) at a pll 8.8, and at a temperature between 3 and 40 C. 7. Application of the exo-antigenic fraction defined in claims 1 to 4 to the research of toxoplasmic immunity in humans by skin tests. 8. New test substance for the detection of toxoplasmic immunity, characterized in that it consists of the exo-antigenic fraction as defined in claims 1 to 4, associated with a vehicle, in particular with glycerol.