FI71165C - REFERENCE TO A FRAMEWORK FOR TOXOPLASMA ANTIGENS AND PROTECTION OF TOXOPLASMA IMMUNITIES - Google Patents

Description

71 1 65 ^ Method for the preparation of toxoplasma antigen for the study of toxoplasma immunity For the preparation of toxoplasma immunity for toxoplasma antimony for the detection of toxoplasma immunity 5

The invention relates to a method for the preparation of toxoplasma antigen for the study of toxoplasmaimmune.cytes, which method comprises inoculating a cell culture in a suitable medium with Toxoplasma gondii and recovering a culture medium from which the cells and toxoplasmas have been removed and which contains toxoplasma exoantigens.

Toxoplasmosis is a disease to which the entire population appears to be becoming increasingly susceptible. It is particularly severe in fetuses, and suppression of haem-15 toxoplasmic immunity before marriage has now been made mandatory in France, for example.

Immunity can be tested by serological tests, but for reasons of simplicity and cost, efforts have been made to use skin tests to detect slowed hypersensitivity.

20

As early as 1948, J.K. Frenkel has proposed toxoplasma antigens, termed "Toxoplasmines," to detect delayed hypersensitivity (Proceedings of the Society for Experimental Biology and Medicine 1948, 68, 634-639). These antigens were formed from crude powder of parasites in the peritoneal fluid of the peritoneal fluid of animals such as mice or hamsters, with 0.25% phenol being used as a preservative to prevent possible contamination of the reagent during storage. This type of reagent is then marketed and widely used (see W.F. Mc 30 CULLOCH - Public Health Reports 1963, 78 No. 8, 689-698 "and J.L.

KRAHENBUHL - Infection and Immunity 1971, 3, No. 2, 260-267). However, such a preparation has not met current safety standards due to the possible presence of contaminants in the inflammatory fluid of mice.

Since then, attempts have been made to prepare antigen preparations by culturing parasites using Toxoplasma gondii monkey kidney cell lines 35 2 71165 1. The antigen has been obtained by cooling and thawing the recovered and parasites (see S.D. CHAPARAS et al. - Proceedings of the Society for Experimental Biology and Medicine 1966, 121, 734-739). With these antigenic preparations used in the application of the injection-induced intradermal reaction, e.g. using a reagent prepared by J.K. However, based on Frenkel's work, the disadvantage is that they contain numerous particles from parasites.

Subsequently, it has been possible to demonstrate from cultures using cell lines derived from human diploid cells or cell lines derived from monkey kidney cells that toxoplasmas release exo antigens in the culture medium (PT DESGEORGES, X. RILLIAULT, P. AMBR0I-SE- THOMAS and M. BOUTTAZ, Lyon Medical 30, 06, 80-243, 12, 737-740). It has thus been shown advantageous to use such toxoplasma exo-anti-15 genes to study toxoplasmic immunity. The advantage of these antigens is, in fact, that they contain virtually no finely divided materials derived from toxoplasmas and that they do not contain contaminants derived from cells. However, the antigenic preparations thus described are not suitable for use in the field of human medicine.

It is therefore an object of the invention to provide such a toxoplasma antigen for the study of toxoplasmic immunity, which antigen is of high purity and does not contain contaminants which could pose a high risk of sensitization in humans.

Another object of the invention is to provide an antigen which can be used to study toxoplasma immunity by means of multipuncture experiments.

30

It is a further object of the invention to provide an antigen which can also be used in serological tests.

It is a further object of the invention to provide an antigen which does not pose a risk of virulence.

The method according to the invention for the preparation of toxoplasma antigen for the expression of toxoplasma in humans or animals is characterized in that the culture is carried out in the presence of toxoplasmas in a medium from which the animal serum, in particular calf serum, has been removed, to remove toxoplasmas and wall waste by sterilizing filtration, that any virus particles present in the soluble antigen obtained after filtration can be deactivated, preferably with formalin, and that the soluble antigen is concentrated after deactivation.

10 One HSR unit is defined as the amount of antigen that, when injected subcutaneously in a dose of 0.1 ml into a specially sensitized guinea pig, gives a reaction of 100 mm in 18 hours or 24 hours.

The concentration of antigen is preferably on the order of two hundred times that of the initial yield.

In cell culture, cell lines derived from primary kidney cells of various animal species, such as monkey, pig, chicken chicks, can be used, but cell lines derived from either human diplold cells or animal heteroploid cells are preferably used.

Preferably, one of the MRC5 cell lines described by J.P. JACOBS et al.

25 - Nature 1970, 227, No. 5254, 168-170, and is available, for example, from the National Institute for Medical Research Holly Hill - London NW3 - United Kingdom.

Non-tumorigenic cell lines, such as the VERO cell line described by B.J. MONTAGNON et al. Intern. Symp. in Reassessment of Inactivated Poliomyelitis Vaccine, Bilthoven 1980, Develop. Biol. Standard, 47, pp. 55-64 (S. Karger, Basel 1981).

In a particularly preferred embodiment of the invention, a sufficiently high concentration of at least 190-fold and preferably about 200-fold is performed to give an antigen with a titer of at least .160 4 L in the ELISA assay, which bevels approximately 500 HSR Merieux units, after which the antigen is suspended in glycerol so that this antigen can be used by a scratching method, in particular by means of a nitite puncture method.

5

The ELISA titer of antigen is the inverse of the dilution value corresponding to 50% of the maximum optical density determined using a plate containing excess antigen. The activity of the unknown antigen is expressed relative to the activity of the standard antigen circulated under the same conditions. Calculate the relative potency equal to the antilogarithm of the difference between the ELISA titer of the unknown antigen and the standard antigen. The final titre of the test antigen is obtained by multiplying its relative potency by the standard titre, the result already being expressed in units per milliliter.

The cell culture is preferably continued after inoculation for about 6 days at 37 ° C.

The filtration step may preferably include a first sterilizing filtration, followed by ultrafiltration dialysis and then a new sterilizing filtration.

The deactivation is preferably performed with formalin in a ratio of 1: 1000 for about 24 hours at room temperature and with constant stirring.

According to a preferred embodiment, the concentrated antigen is thus suspended in glycerol or another carrier so that it can be used by the scratching method, in particular by applying a multipunk-30 lure.

Other advantages and features of the invention will become apparent upon reading the following description, given by way of example.

35 Perform MRC 5 cell culture in EAGLE BME medium supplemented with 10% calf serum. This culture is carried out in a flask with a surface of about 500 cm.

5,71165 1 Culture is performed at 37 ° C for 3 days at an initial cell concentration of 20 million flasks in EAGLE BME medium with an additional 10% calf serum. On the third day, the medium is replaced with EAGLE BME medium supplemented with 2% calf serum. Bottles 5 are again kept at 37C for 4 days. The cell layer is then washed three times with physiological saline after removal of the medium and the cells are again placed in EAGLE BME medium without calf serum. Twenty-four hours after these washes, the BME medium is again replaced without calf serum before parasites 10 are added. Toxoplasmas maintained in Swiss-type female mice are inoculated in an amount of 2.4 x 10 toxoplasm per milliliter of culture medium.

The culture is then performed for six days at 37C.

15

After the sixth day, the supernatant is harvested and subjected to the first sterilizing filtration (pre-filter AP 20 -AW 0.6-sterile Millipore filters 0.22).

20% formalin is then added and the solution is kept at room temperature with constant stirring for 24 hours. This deactivation step is followed by ultrafiltration dialysis and at least about 200-fold concentration with Amicon material (column and cells) using a PM 10 membrane.

The concentrate is then further sterilized by re-filtration through successive Millipore filters (AP 20.8 ... 0.45, 0.22).

At this stage, the resulting antigen solution has a delayed hypersensitivity titer in guinea pigs of at least 500 20% toxoplasmic HSR Merieux units.

The purified soluble antigen, concentrated and deactivated, is then serologically titrated by an "in vitro" ELISA, after which its delayed hypersensitivity activity is calibrated by titration "in vivo" in guinea pigs pre-sensitized to Toxoplasma 6 71 1 65 1 gondii '. n specialty.

Finally, the antigen is suspended in 70% glycerol. This product is intended to be applied to the tips of a multipoint puncture site.

5

Preclinical studies have shown the following results:

Guinea Pigs 10 _____________ r ------ T -----------------------

Dilution pure thirty-thirty-hundred-

Antigens part part nesosa part

Test group X 5.5 mm 4.2 2.5 0 0 15 ______________________________________________________

Experimental group Y5.8tnm 4.2 3.2 2.8 1 20 25 30 35 7 71 1 65 1 In humans

Ser. Antigen X Antigen X Antigen X

5 pure 1/3 1/10 T.L. p.i. 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h target 160 4.5 4.5 3.5 0 3 2 2 2 2 JO A u.i. mn mn mn mn tnn mn mn mn target 80 0 33.50 0 0 0 0 0 B u.i. mn mn mn mn mn mn mn 15 items 8 0 2 2.5 - C u.i. mn mn

item 0000000000 D

20 ______________________________________________

Explanation of abbreviations in the table above:. : serology 25 - T.L. : reading times - p.i. : by immunofluorescence - u.i. : international units 30 35

Claims (10)

1 Pentent requirements A method of producing a toxoplasmic antigen for detecting toxoplasmic immunity, inoculating Toxoplasma gondii in a cell culture in a suitable medium, and utilizing the culture medium liberated from cells and toxoplasms and containing toxoplasmic cells exo-antigens, characterized in that the culture is carried out in the presence of toxoplasms in a culture medium liberated from animal serum, such as in particular calf serum, to eliminate the remnants of the toxoplasms and the walls derived from the cells or toxoplasms by sterilizing filtration; that the virus particles possibly present in the soluble antigen after filtration are deactivated, advantageously with formalin, and that the soluble antigen is concentrated after deactivation. 15 2. A method according to claim 1, characterized in that at least a two-fold concentration is carried out. 3. A method according to any one of claims 1 or 2, characterized in that a cell line derived from human diploid cells is used, and in particular the cell line MRC 5. Method according to any one of claims 1 or 2, characterized in that a cell line of heteroploid animal cells is used, and in particular the cell line VERO. 5. A method according to any one of claims 1 or 2, characterized in that a culture of primary cells is used. A method according to any one of claims 1-5, characterized in that the antigen is suspended in glycerol. Process according to any of claims 1-6, characterized in that the cell culture is carried out after inoculation for 6 days at 37 ° C. 35 Method according to any of claims 1-7, characterized