EP2329273A1

EP2329273A1 - Stable cell clone expressing a prion

Info

Publication number: EP2329273A1
Application number: EP09741370A
Authority: EP
Inventors: Bruno You
Original assignee: Institut National de la Recherche Agronomique INRA; LFB Biotechnologies SAS
Current assignee: Institut National de la Recherche Agronomique INRA; LFB Biotechnologies SAS
Priority date: 2008-09-05
Filing date: 2009-09-04
Publication date: 2011-06-08
Also published as: JP2012501638A; IL211492A0; CA2735774A1; AU2009289107A1; CN102203615A; US20110165613A1; WO2010026346A1; BRPI0917877A2; AR073494A1; KR20110069801A; FR2935711A1

Abstract

The invention relates to a cell clone from the MovS6 cell line, said cell clone expressing a prion protein PrP and being capable of supporting the replication or the propagation of the pathological form PrPsc of said Prp, characterized in that it has a titer stability to at least the sixth passage in a marker for infection by a non-conventional transmissible agent (NCTA).

Description

Stable cell clone expressing a prion

The present invention relates to a cell clone derived from the MovS6 line expressing a PrP prion protein and capable of supporting the replication or propagation of the PrPsc pathological form of said PrP, as well as its application, in particular to an evaluation method and / or in vitro control of the effectiveness of a process for obtaining or treating a biological product or a method of evaluation and / or in vitro control of a decontamination procedure or an evaluation method or screening of compounds with an activity modulating the infectivity associated with "unconventional transmissible agents" ATNC.

State of the art:

Transmissible spongiform encephalopathies (TSEs) are groups of genetic or acquired diseases characterized by degeneration of the central nervous system (CNS). The most common form in humans is Creutzfeldt-Jakob disease (CJD), but there are also TSEs in many mammals (including scrapie and bovine spongiform encephalopathy). The etiologic agent of these diseases is classified as "unconventional transmissible agents" (NCTA). The signature of the disease is the presence of an extracellular protein, called prion protein (PrP), which is transformed during the disease into an insoluble form, resistant to proteases such as proteinase K, and which accumulates in the body. central nervous system. This abnormal pathological form of PrP, called PrPsc, is co-purified with infectivity and its accumulation precedes the appearance of histological lesions. It results from a modification of the conformation of the prion protein PrP. There has been no evidence of altering the expression of the gene encoding PrP or altering its translation (Prusiner, Biochemistry 1992; 31: 12277-88).

The data currently available do not demonstrate that the transmissible agent responsible for TSEs is in an infective form in blood derivatives (Brown et al., 2001, Semin Hematol., 38 (4 Suppl 9): 2-6). However, we can not conclude from its absence, this uncertainty resulting, on the one hand, from the probable very low concentration in the blood and, on the other hand, from the very long period of clinically silent incubation characteristic of these diseases, which precedes the appearance of clinical signs.

In addition, the exceptional resistance of NCTAs prevents the use of inactivation processes conventionally used, such as the Tween-TNBP solvent / detergent treatment, which have proved their effectiveness in reducing the viral load of blood derivatives. , such as cryoprecipitable plasma proteins (factor VIII, von Willebrand factor, etc.). Since obtaining or processing biological products, such as plasma coagulation proteins, must incorporate viral elimination / inactivation steps for therapeutic use, the blood-derived pharmaceutical industry seeks today to evaluate the theoretical risk of transmission of variant CJD by blood products.

Currently, the method of titration of the NCTI-related infectious disease conventionally used uses an in vivo titration method in golden hamster, by intracerebral injection of different dilutions of a product to be tested charged with NCTA. Depending on the number of affected animals in the different groups corresponding to the dilutions made, one can calculate an infectious titer and establish the reduction factor of a given method from an untreated reference. However, this method has the disadvantage of being long (about one year), expensive and not compatible with a development on an industrial scale which we want to quickly know the effectiveness vis-à-vis the elimination of the prion.

In addition, it is often necessary to introduce a step of concentration of the infectious agent so as to increase the sensitivity of the titration methods. All the procedures for the concentration of infectious agents responsible for the TSE now pass through PrPsc purification.

Other methods have been proposed for the in vitro titration of infectious agents of TSEs. Techniques for detecting PrPsc by Western-Blot (Mac Gregor, Transfusion J. Medicine 2001; 11, 3-14) or by ELISA generally require prior digestion of the sample to be analyzed by Proteinase K, or denaturation by chaotropic agents to distinguish the pathological protein (PrPsc) from the normal protein (PrP).

Another assay method has recently been developed based on the use of PrPsc-specific antibodies not recognizing PrP (Korth et al, Nature 1997 Nov6, 390 (6655): 74-7).

US Pat. No. 6,150,583 for its part describes the production of transgenic animals expressing a PrP labeled with a heterologous epitope exposed or not on the surface of the prion protein, according to the conformation of the latter.

The process called "PMCA" (Protein misfolding cyclic amplification), described in the document Saborio et al. (Nature, 2001, 411, 810-3) provides for contacting pathological forms from tissue or fluid from a contaminated animal with non-pathological form of ATNC protein to convert the latter in a pathological form. The PMCA method, although still in development, is at least as sensitive as the bioassay. Nevertheless, it provides no evidence on the infectivity of the PrPsc detected and is difficult to implement with plasma matrices. In addition, unreliable reproducibility and false positive results have been reported.

WO 2005/022148 discloses an in vitro titration method, called "tissue culture infectivity assay" (TCIA), of an ATNC in a biological product, by means of bringing into contact stable transgenic cells supporting replication said ATNC with the biological product, and then culturing these cells during one or more passages to amplify the amount of ATNC present in the biological product by replicating the ATNC. Specifically, TCIA involves contacting successive dilutions of an infectious homogenate (e.g., scrapie strain 127-S) with cells in multi-well plate culture, 5 wells per dilution. The number of positive wells is then evaluated by detection of PrPsc (indissociable marker of infectivity) after 8 to 10 passages, and the titre determined by the Spearman - Karber method. Reproducibility of titres obtained from the same infectious sample (brain homogenate) shows the feasibility of such an approach. However, the cell line used in this in vitro assay method, the MovS6 line, consists of a heterogeneous population of cells. Eventually, this heterogeneity is likely to alter the stability of the line and consequently the reproducibility of the TCIA process.

There is therefore still a strong need to have one or more stable clones from the MovS6 line to improve the stability of the TCIA process over time.

Summary of the invention

The invention now provides a novel cell clone derived from the MovS6 line expressing a PrP prion protein and capable of supporting the replication or propagation of the PrPsc pathological form of said PrP, characterized in that it exhibits a stability of its production of PrPsc at least until 6 ^th passage, preferably over at least between the 6 ^th and the 100 ^th passage.

The inventors have in fact demonstrated that it is possible to have clone strongly responding to the infection and showing a PrPsc production stability, that is to say without significant variation in the level of PrPsc production.

Preferably, the cell lysate derived from infected culture of this clone, has, from 6 weeks after infection, a titre in marker of infection greater than or equal to 3 log 10 TCID 50 per million cells, preferably higher or equal to 4 log 10 TCID 50 per million cells, preferably greater than or equal to 6 log 10 TCID 50 per million cells, preferably greater than 7 log 10 TCID 50 per million cells. In a particular embodiment of the invention, the cell lysate of an infected culture has, from 6 weeks, a titre in marker of the infection greater than 3 log 10 TCID 50 / million cells, preferably between 4, 5 log ₁₀ TCID ₅₀ and 6 log ₁₀ TCID ₅₀ per million cells.

In a particular embodiment of the invention, the cell lysate of an infected culture has, from 6 weeks has a marker titre of infection greater than 3 logio TCID50 per million cells, preferably between 4 , 5 logio TCID50 and 5.5 logio TCID50 per million cells.

In another embodiment, the cell lysate derived from infected culture of this clone, has, from 6 weeks after infection, a titre in marker of the infection greater than or equal to 2 logio units Western Blot / million of cells (uWB / million cells), preferably greater than or equal to 3 log 10 uWB / million cells.

Particularly preferably, the cell clone of the invention is designated MovS6-4 in the experimental results set out below, and was deposited on February 22, 2008 under number CNCM 1-3922 in the National Collection of Cultures of Microorganisms ( CNCM, Pasteur Institute, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, FRANCE).

The invention also relates to an in vitro method for detecting and / or titrating the infectivity of an unconventional transmissible agent (ATNC) whose marker is a pathological conformation protein, in a sample, comprising the steps of: ) contacting said sample with a cell clone as described above, ii) culturing said cell clones to amplify the amount of ATC present in said sample by replicating said ATNC, iii) determining the presence and / or amount of the ATNC in the sample, the step ii) of culture being carried out during one or more passages.

Advantageously, step (iii) comprises the following steps: a) contacting the NCTA thus amplified at the end of step ii) with a source of substrate for PrP and / or ATNC, b) incubating the reaction medium allowing the transformation of the non-pathological conformer of the PrP in pathological conformation and / or amplification of the ATNC, c) disaggregate aggregates possibly formed in step i) or ii), d) determine the presence and / or quantity of the NCTA in the sample, steps (a) to (c) constituting a cycle of operations that is repeated at least twice before step (d).

Another subject of the invention is an in vitro method of evaluation and / or control of a process for obtaining or treating a biological product or material likely to be contaminated by an NCTA, method wherein to said biological or material product, (A) upstream and (B) downstream of said process, a titration method as defined above, and that the two values (A) and (B) title obtained.

The invention also relates to an in vitro method for evaluating and / or controlling a procedure for decontaminating a biological product or a material, in which method it is applied to said biological or material product, (A) upstream and (B) downstream of said procedure, a titration method as defined above, and that the two obtained values (A) and (B) of title are compared.

Yet another subject of the invention is an in vitro method for evaluating a compound capable of inhibiting the infectivity of an infectious biological product, in which method is applied to said infectious biological product, (A) in the presence and (B) in the absence of said compound to be evaluated, a titration method as defined above, and in that the two obtained values (A) and (B) of title are compared.

Yet another subject of the invention is an in vitro method for diagnosing a transmissible spongiform encephalopathy in a human or non-human animal, comprising detecting the presence in a biological sample of said subject of a non-transmissible transmissible agent. conventional (ATNC) which is a pathological conformational protein, by the method as defined above. The cell clone of the invention allows the stable production over time and a synthetic and standardized infectious material of the prion type, immediately available since it has been detected in the cytosol (lysate) and in the infected culture supernatant.

Legend of the Figure:

The figure is an autoradiogram showing the detection of PrPsc in the cell lysate of 13 clones (numbered 1 to 13) with PM: molecular weight indicator.

Detailed description of the invention

Definitions

The term "MovS6 line" denotes a cell line expressing the PrP prion protein, derived from cell isolation made from a transgenic mouse dorsal root ganglion overexpressing the ovine PrP gene. The transgenic mouse overexpressing the ovine PrP gene from which the MovS6 cell line originates is derived from a cross between a tg301 mouse overexpressing the ovine PrP gene (Vilotte et al (2001), J. Virol, Vol. 75, p5977-5984) and a prpO / 0 mouse expressing the SV40 T-tag antigen (Schwarz et al (1991), Bio Cell 73: 7-14). The manufacture of the MovS6 cell line is described in Archer et al. (2004), J. Virol., P. 482-490. The MovS6 cell line is a heterogeneous cell population, not only in terms of functional properties, but also biological properties. Indeed, the dorsal root ganglion from which it was isolated brings together different cell populations, including glial cells, different types of neurons, and so on.

In the context of the invention, the expression "cell clone derived from the MovS6 line" represents the set of daughter cells derived by cell division from a single parent cell belonging to the MovS6 line, and having a genetic inheritance identical to that of the mother cell. The isolation of the clones can be done using a technique known to those skilled in the art, for example by limiting dilution or flow cytometer, this list is not limiting.

A "Prion Prion Protein" is usually a plasma membrane-anchored sialoglycoprotein by a phosphatidyl glycolipid (GPI), naturally occurring in cells and involved in their normal functioning. The normal form of the protein, i.e. non-pathological, is generally called Prpc. It is involved in the development of the nervous system in the embryo. In adults, it is expressed primarily in the brain and spinal cord (neurons and glia). It is involved in the processes of cell differentiation and adhesion. It would also have an antioxidant protective role and vis-à-vis programmed cell death (apoptosis). This protein also has a role in the folding of other proteins.

The "PrP sc pathological form of PrP" generally refers to an isoform of the non-pathological protein and represents the marker of prion diseases. This pathological form, associated with physicochemical properties to the prion protein which result notably in a greater resistance to the usual means of disinfection and sterilization (heat, chemicals, enzymes, etc.). The pathological prion protein thus acquires self-aggregating abilities and can thus form deposits, especially in the brain, causing neuronal death. The agent responsible for the replication or propagation of the pathological prion protein would be the pathological prion protein itself as able to "propagate or multiply" in an "exponential" manner, by deforming the healthy prion proteins into pathological prion proteins. The so-called "pathological" form of PrP is therefore the form of the protein whose conformation is correlated with the appearance of an EST in the infected human or non-human animal.

By "capable of supporting the replication or propagation of the PrPsc pathological form of said PrP" is meant the ability of the cellular clone of the invention to produce pathological prion proteins from non-pathological prion proteins transformed into pathological proteins in the presence of NCTA. The pathological form of the produced prion protein is detected in the cytosol (lysate) or in the culture supernatant. All or part of this cytosolic pathological form can be recovered in the cell lysate.

The "marker titre of NCTA infection" is determined by the infectivity dose in a sample that allows infection in 50% of the inoculation trials. The infectivity dose is visualized by the titre of a marker of infection, for example PrP. The marker titre of the infection may be determined by the titration method described in WO 04/02179.

The marker titre of the infection may also be determined by methods well known to those skilled in the art. For example, serial dilutions of the material to be titrated are injected intracerebrally, this at the rate of a dilution of the material per group of animals. Depending on the incubation time of the disease in each group of animals and the number of animals affected, an infective titre can be deduced from it by statistical methods known to those skilled in the art, for example, and preferably, the method. of Karber or that of Spearman-Karber.

By "marker titre stability of infection" is meant an infectious agent productivity (PrPsc) that does not decay significantly over time, i.e. no significant loss of productivity can be measured . A significant loss is a decrease in infectious agent productivity greater than or equal to 1 log uWB / ml.

A "passage" is generally the transplantation of all or part of the cells of a culture dish into another box containing new culture medium. Each passage makes it possible to control the size of a cell population and to provide a quantity of substrate adapted to its size.

In a particular embodiment, the cell clone of the invention is characterized in that it has a stability of the title as a marker of infection by a transmissible agent. unconventional (NCTA) at least until 6 ^th passage, preferably over at least between the 6 ^th and the 100 ^th passage. Preferably the cell clone of the invention has a stability of the title infection marker with an NCTA until at least 7 ^th passage, more preferably at least up to 8 ^th passage, more preferably at least until at 9 ^th passage, more preferably at least until 10 ^th passage, more preferably at least until H ^th passage, more preferably at least until 12 ^th passage, more preferably at least until 13 ^th passage, more preferably at least until 14 ^th passage, more preferably at least until 22 ^th passage, more preferably at least until 23 ^th passage, more preferably at least until ^45th passage, more preferably at least up to 46 ^th passage, more preferably at least up to 47 ^th passage, more preferably at least up to 48 ^th passage, more preferably at least up to 49 ^th passage, more preferably at least up to 50 ^th passage.

By "TCID50" is meant the infectious dose which causes the infection of 50% of the inoculations tested. These doses are preferably measured using the TCIA method.

By "uWB" (Western-blot unit) is meant an amount of PrPsc detectable by Western blotting.

Generally, there is a difference in sensitivity between Western-blot and TCIA titration methods. Considering this difference, a titre in marker of the infection greater than or equal to 2 logio units Western blot / million cells (uWB / million cells), preferably greater than or equal to 3 logio uWB / million cells is equivalent to a titre in infectivity greater than or equal to 3.5 log10, preferably greater than or equal to 5 logio TdD ₅ o / ml

In the context of the invention, the term "ATNC" represents any unconventional transmissible agent, such as those responsible in humans for familial or sporadic CJD, Kuru disease or variant CJD, or those officials in natural TSE animals, such as ovine scrapie, bovine or feline spongiform encephalopathy, chronic wasting of deer or spongiform encephalopathy of mink, or EST strains experimentally adapted to laboratory animals . In the context of the invention, the ATNC is also referred to as "infectious agent".

"Sample" means any source of material that may be contaminated by a NCTA. Such a source of material may for example be a liquid, a food product, a beverage, a cosmetic product or a product resulting from genetic engineering, a molecule capable of modulating the infectivity of an NCTA, this list not being limiting. . Preferably it is a biological sample, for example a biological fluid or tissue or tissue extract. Such a tissue may be a tissue of the brain, vertebral column, or tonscillary tissue, this list not being limiting. The sample may also be a composition derived from a human or animal source, such as growth hormones or cell extracts, such as pituitary extracts. Such a composition can indeed be contaminated by an NCTA. In the case of a biological fluid, it may be blood, lymph, urine or milk, this list not being limiting. Preferably, the sample is a blood product or a derivative, for example a plasma derivative or a plasma protein concentrate.

The cell clone of the invention may advantageously be used in an in vitro method for detecting and / or assaying the infectivity of an unconventional transmissible agent (ATNC) whose marker is a pathological conformational protein, in a sample.

Contacting the cell clone with the test sample:

The cell clone of the invention is contacted with the sample that may be infected with a test NCTA, or with an infectious material containing IATNC as reference material, for example extracts of brains from infected animals. by a

ATNC, such as a sheep prion. The bringing into contact is carried out according to known methods those skilled in the art (see WO / 2005/022148 or also for example Archer et al., Journal of Virology, Jan. 2004, pp. 482-490),

The cell clone is then cultured for one or more passages to allow the ATNC to replicate or propagate.

Advantageously, the cell clone can be contacted with at least one dilution of the sample susceptible to be infected by the ATNC in a biologically acceptable aqueous solution, in particular with several dilutions, especially with serial dilutions or successive. These dilutions make it possible to refine the quantification of the infectivity in the sample tested.

Several replicates of clones can be brought into contact with the same dilution of the biological product, in order to allow a finer statistical resolution of the results.

Culture of cell clones (step H)):

The step of culturing cell clones potentially infected with the biological product, according to any technique known to those skilled in the art, is required for the replication of the ATNC, thus to amplify an insufficient amount of ATNC initially to be detected.

In an exemplary embodiment, the cell cloning step ii) performed to amplify the amount of ATNC present in said biological sample by replication of the ATNC is carried out in DMEM (Dulbecco's Modified Eagle's Medium) + Ham- F12 (Life Technologies, Cergy Pontoise, France) (4: 1) supplemented with glutamine and fetal calf serum (5% final). The cells are incubated at 37 ° C. under 5% CO ₂ . A passage of the cells (with a ratio of re-ratio of: 1 to 10, ie 1 cell out of 10 put back in culture) is carried out every week.

The product resulting from the culturing of cellular clones contains PrP in its pathological form, the quantity of which is greater than the quantity initially present in the biological sample, if it contains one. PrP in its pathological form and NCTA accumulate within the infected cells, and are excreted in the culture medium or exposed on the surface of the cells.

The cultivation step ii) can be carried out during one or more passages, for example between 2 and 10 passages, preferably between 4 and 10 passages.

Determination of the presence and / or amount of deprion or ATNC (step Hi)):

The method may directly comprise a step of determining the presence and / or amount of prion or ATNC (step iii)), or being coupled to a contact with a source of substrate for PrP and / or NCTA.

Contacting a substrate source for PrP and / or ATNC (step a)):

Step ii) of the method of the invention may be followed by contacting the product resulting from culturing the cells with a substrate source for the non-pathological conformation of PrP and / or the ATNC amplified during step ii).

This source of substrate may be provided for example in the form of a product of animal origin, for example a healthy brain homogenate, or material derived from in vitro culture. This material may for example be derived from cells, such as MovS, N2A (Weissmann et al., (2003), PNAS vol.100 No. 20, p1666-1 1671), Rov for example, or even yeasts, fungi, or bacteria, this list not being limiting. This material derived from in vitro culture can be expressed in various cellular compartments, such as the extracellular compartment (for example the supernatant, the exosomes, this list not being limiting) and / or membrane and / or cytosolic compartments (cell lysate). for example). This step serves to allow in vitro amplification PrPsc and / or the ATNC harvested during step ii) accompanying the conversion of the non-pathological form of PrP (contained in the substrate ) in pathological form, a self-conversion of the non-pathological form being theoretically impossible. PrP under its pathological form thus initiates the transformation of the non-pathological form into a pathological form.

At the end of the conversion reaction, the unconverted non-pathological form is not detected by the detection system, as will be explained later.

Incubation of the reaction medium and / or amplification of VATNC (step b))

In a preferred embodiment, step (a) is followed by incubation of the cell culture product of step (ii) with a substrate source for PrP and / or ATNC for a period of time. sufficient to allow at least a portion of the proteins having a non-pathological form, to be transformed into a pathological form of the protein, and to enhance the NCTA. Preferably, each incubation step is carried out for a period of between 10 seconds and 4 hours, preferably between 20 minutes and 1 hour, and particularly preferably for 30 minutes.

The amplification medium is advantageously constituted by a buffer (PBS Ix), NaCl 15OmM, Triton 1%) supplemented with non-pathological PrP substrate (for example a volume 10 times greater than the volume of the sample to be amplified) .

Separation of aggregates:

It turns out that the proteins converted into pathological forms (PrP ^sc type) can aggregate with each other and with other particles of pathological form (PrP ^sc ), preventing the conversion of other non-pathological form proteins into pathological form. This is a drawback because the method could thus be slowed down by the small number of "conversion foci" present in the reaction medium.

Thus, step c) of the method of the invention consists in the disaggregation of the aggregates possibly formed during the preceding steps, so as to release the particles of PrP of pathological form so that they can convert other non-pathological proteins.

Many methods can be used to disaggregate aggregates during step c) of the method of the invention. By way of example, mention may be made of treatment with a solvent (such as sodium dodecyl sulphate, dimethyl sulphoxide, acetonitrile, guanidine, urea, trifluoroethanol, diluted trifuroacetic acid, diluted formic acid, this list is not limiting), modification of the physicochemical characteristics of the solution, such as pH, temperature, ionic strength, dielectric constant, as well as physical methods, such as sonication, irradiation with laser, freezing / thawing, autoclave incubation, high pressure, gentle homogenization, or other sources of irradiation, this list not being limiting. Preferably, sonication is used. Sonication is a method known to those skilled in the art, and often used in PrP ^sc purification methods, by making it possible to increase the solubility of the aggregates.

The disintegration is carried out for a time sufficient to allow at least a portion of the aggregates formed during the contacting of step iii) and the incubation / amplification. It is possible that not all aggregates are disaggregated when implementing a single disintegration step. In this case, the concentration of pathological proteins increases as the disintegration steps progress.

The duration of the disintegration step is readily determinable by those skilled in the art, and may depend on the method of disintegration chosen. Preferably, the duration of the disintegration step is between 1 second and 60 minutes, more preferably between 5 seconds and 30 minutes, and more particularly between 5 seconds and 30 seconds.

Advantageously, steps a) to c) are repeated at least twice, preferably between 5 and 100 times, preferably between 20 and 60 times. This cycle of steps iii) to v) repeated between 2 and 100 times constitutes a series of amplification cycles. A series can also be repeated several times. In this case, the new series will be initiated from a volume of the previous series in place of the original NCTA sample.

Protein detection:

Step d) of detecting PrP pathological proteins can be carried out by any method known to those skilled in the art. The specific detection of PrPsc can be carried out by a first step of separation of the two isoforms PrPc and PrPsc. This separation is carried out on the basis of the biochemical properties of PrPsc which make it possible to distinguish it from non-pathological proteins, in

particularly the fact that PrPsc is resistant to protease-based treatments and is less soluble or insoluble even in the presence of detergents. Thus, the first step after amplification is preferably the separation of the PrPc (non-pathological soluble normal form of PrP) from the sample, which can be effected for example by means of protease treatment, for example proteinase K or by centrifugation to separate soluble forms (PrPc) from insoluble forms (PrPsc).

In a particular embodiment, digestion with proteinase K (PK) is a step prior to Western Blot, because it leads to digestion mainly PrPc and little or no PrPsc. It is indeed a property of the PrPsc to be more resistant to PK compared to the PrPc. The subsequent detection step therefore no longer detects the non-pathological form of the protein because it has been digested by the protease.

The detection step can be carried out using the following methods: immunocytochemistry (for example by labeling cells or Facscan) or immunochemistry (as the Western blot or an ELISA test), immunoblot method after a step of SDS -PAGE, radioactivity test, fiuoresence test, electron microscopy, and turbidimetry test for aggregates, as well as structural tests including NMR (nuclear magnetic resonance), circular dichroism, Raman spectroscopy, UV absorption, a monoclonal antibody recognizing the pathological form of the protein, this list is not limiting.

It is also possible to detect the pathological form of the proteins by means of an antibody specifically recognizing the pathological form and not the non-pathological form. To make its detection easier, this antibody can itself be marked to make detection easier. Such an antibody may be for example the antibody

15B3 (Korth et al., 1997, Nature 1997 Nov6, 390 (6655): 74-7). The use of such an antibody is relevant for detecting by flow cytometry the prion protein present on the surface of living infected cells.

Titration of NCTAs:

The detection of the pathological form of PrP may be associated with a determination of the amount of PrPsc present in the sample.

The titration can be carried out using any titration method known to those skilled in the art. In particular, it can be performed on the model of the methods described in the documents WO2005022148 and WO2006117483 (in particular reference examples A and B).

The set of Western-blot results for the different dilutions and the different replicates can be analyzed by a statistical method known to those skilled in the art for establishing an infectious titer, such as the method of Spearman Karber (Schmidt NJ , Emmous RW, Diagnosis Procedures for Viral, Rickettsial and Chlaveydial Infection, 1989, 6th Edition).

In one embodiment of the invention, the method of calculating the title is the method of

Spearman - Karber. This method supposes the dilution of the sample to be tested according to a geometric progression, that is to say of constant reason between the successive dilutions, and sowing a constant volume (usually 0.150 ml) of each dilution in at least five wells. The most commonly used dilution factor is the decimal factor.

For the Spearman-Karber formula to be applicable, it is necessary to use a constant number of wells inoculated with each dilution, a constant dilution factor and a range of dilutions large enough to control both the dilutions on the one hand and others of which one hundred per cent of the wells will give a positive reaction, and the dilutions on either side of which one hundred per cent of the wells will give a negative reaction.

If one or more of these conditions are not satisfied, it is sometimes assumed that for a constant dilution factor, the higher or lower dilution following the last performed would have given the desired result. Such "manufacturing" of data is not based on any theoretical basis, but if applied with sufficient caution, it is not dangerous. However, it is best to repeat the titration with a more appropriate range of dilutions, which is essential if there are serious data gaps.

According to Spearman-Karber's formula:

Median dose logio = (Xo) - (d / 2) + dS (V ₁ Zn ₁ )

or:

Xo = logio of the reciprocal value of the lowest dilution at which all test inocula are positive.

d = log10 of the dilution factor, also referred to as "no dilution" (i.e. the difference between log dilution intervals).

n = number of test inocula used at each dilution.

R ₁ = number of positive inocula of test (on ni). S (T ₁ Zn ₁ ) = S (P) = sum of the proportion of positive tests starting at the lowest dilution and giving one hundred percent of positive results.

The summation begins at Xo dilution.

The estimated standard deviation is calculated using the following formula:

Log standard deviation = d * V (E (p * (l-p) / (ni-1)))

With p = X ₁ In ₁ = proportion of positive assays (i.e. inoculum cups with reaction) at each dilution.

The method of the invention makes it possible to quantify the infectivity associated with NCTAs over a range of 4 log, that is, 10,000 infectious units in vitro. This may, for example, make it possible to satisfy the criteria for validating the efficiency of the processes for obtaining biological products with respect to the elimination of NCTAs.

Applications of the method of detection or titration of NCTA infectivity:

The invention also relates to the application of the titration method according to the invention to a method of evaluation and / or in vitro control of a process for obtaining or treating a biological product likely to be contaminated. by an NCTA. This evaluation and / or control method is characterized in that a titration method according to the invention, as described above, is applied to the biological product, upstream and downstream of said process, and in that the we compare the two title values obtained. By comparison between the two measurements, the degree of elimination or the reduction factor of the NCTA is determined.

In particular, the titration method according to the invention has the capacity to be easily applicable to any type of process for obtaining or purifying biological products, in particular blood products, such as blood plasma derivatives, using for example, chromatography or nanofiltration, in particular the chromatographies described in documents EP 0 359 593 and WO 02/092632.

Thus, the implementation of the method of the invention makes it possible to evaluate and / or control the efficiency of a process (or a part of a process) for obtaining or treating, or even purifying of any biological product likely to be contaminated by an NCTA, in the elimination of this NCTA, by means of a titration using specific transgenic cell lines, promoting the replication of the ATNC, brought into contact with an infectious material or potentially infectious containing the ATNC to be tested. The amounts of NCTA are measured upstream and downstream of the process (or part of the process) whose performance with respect to the NCTA is to be assessed. By comparing the two measurements, the degree of elimination of the pathogen is determined. Thus, the implementation of the present method can be carried out during a process for obtaining a biological product or in the context of an elimination treatment of the NCTA according to obtaining the biological product.

The invention also relates to the application of the titration method according to the invention to a method of evaluation and / or in vitro control of a procedure for decontaminating a material. In this case, the ATNC titre of a biological product containing an NCTA is determined using the titration method of the invention. This infected biological product is then contacted with the material to be decontaminated and the decontamination procedure is applied to this material. Finally, the title of the biological product having undergone the decontamination procedure is again determined. The two titration measurements performed upstream and downstream of the decontamination procedure are compared to evaluate the effectiveness of the decontamination procedure. The material may, for example, be purification equipment, in particular a chromatography column, or be the sanitization of a chromatography column using sodium hydroxide.

The invention also relates to the application of the titration method according to the invention to a selection procedure and / or method for evaluating a compound making it possible to reduce the titre of the infectious material. In this case, the ATNC titre of a biological product containing an NCTA is determined using the titration method of the invention. We This infected biological product is then contacted with the test compound and the title of the biological product having undergone the decontamination procedure is determined again. The two titration measurements made upstream and downstream of the contacting of the sample with the test compound are compared.

The invention also relates to the application of the titration method according to the invention to a selection procedure and / or evaluation method of a compound capable of modulating the infectivity of an ATNC. In this case, the ATNC titre of a biological product containing an NCTA is determined, in the presence and then in the absence of the compound to be evaluated, by means of the titration method according to the invention. The methods for bringing the compound into contact are determined according to whether the action of the compound prevents the initiation of an infectious cycle or blocks an infectious cycle already initiated. In all cases, the title of the biological product is determined with and without treatment with the product to be tested. The two titration measurements performed are compared to evaluate the modulatory activity of the NCTA infectivity of the compound.

The invention also relates to the application of the titration method according to the invention to a method of identifying a compound for modulating the transformation of the non-pathological form into the pathological form of the ATNC, for example the transformation of PrP into PrP ^sc . In this case, the ATNC titre of a biological product containing an NCTA is determined using the titration method of the invention. This infected biological product is then contacted with the test compound and the titration method of the invention is applied to again determine the title of the biological product. The two titration measurements made upstream and downstream of the contact with the compound are compared to evaluate the effectiveness of the test compound.

Infectious and standardized material of the prion type produced by the cell clone

The cell lysate or culture supernatant produced by the cell clone of the invention can be standardized in infectious units per dose by methods known to those skilled in the art. For example, infected cell lysate stocks can be prepared for ??

contain 3 logio TCID50 per milliliter. The availability of standardized stocks then allows the strict comparison of separate test results.

In addition, it can be stabilized by freezing, drying, lyophilization, atomization, possibly in the presence of substances known to those skilled in the art and intended to prevent loss of infectious titer depending on the stabilization method used.

Advantageously, this cell lysate or culture supernatant may be used as an infecting inoculum for a method of evaluating and / or controlling a process for obtaining or treating a biological product that may be contaminated by an ATNC, in particular a method for purifying blood plasma derivatives, in particular still chromatographies or nanofiltration.

Advantageously, this cell lysate or culture supernatant may be used as an infecting inoculum for a method of evaluating and / or controlling a procedure for decontaminating material that may be contaminated by an NCTA.

Advantageously, this cell lysate or culture supernatant can be used as an infecting inoculum for a method for evaluating a compound that inhibits the infectivity of an NCTA.

Advantageously, the infectious material (that is to say the inoculum prepared from the cell clone of the invention) makes it possible to overcome the use of ovine or bovine brain collections infected with sheep scrapie or Bovine Spongiform Encephalopathy.

The infectious material can be used as an infecting inoculum in a method of evaluation and / or in vitro control of a procedure for decontaminating a material.

The material may, for example, be a purification material, in particular a chromato graphy column. The decontamination procedure may, for example, be the sanitization of a chromato graphie column using soda.

The invention also relates to the use of the infective material as an infecting inoculum for a method of evaluating a compound modulating the infectivity of an NCTA. In this case, the ability to modulate the infectivity of an ATNC according to the invention is tested in the presence or absence of the compound capable of modulating this infectivity the infectivity of an ATNC.

The invention also relates to the use of the infectious material as an infecting inoculum for a method of evaluating and / or controlling a procedure for confining infectious material, in particular a P3 procedure and equipment.

The method of the invention will be better understood using the additional description which follows, which does not limit the scope of the invention.

EXAMPLES

Materials and methods

1 ° Cells and infectious material:

MovS6 cells (Archer F. et al., 2004) were selected as replication-supporting cells for ATNCs, and Scrapie 127-S strain adapted for transgenic mice.

Tg301 has been used as a source of natural infectivity (Vilotte JL et al., 2001). The cells were cultured in DMEM / Ham F-12 (3: 1) supplemented with glutamine

(2mM final) and fetal calf serum (5% final).

The source of initial infectivity was a homogenate of mouse brains infected at 200 mg / ml (lot: LN-3326 under 6.17 log 10 uWB / ml).

2 ° Culture: The MovS6 cells were cultured to constitute, after 15 days of incubation, 2 primary libraries (coded: LC-19 and LC-21). An aliquot of LC-19 was thawed and cultured for 3 passages to make up 3 secondary libraries, coded: LC-46, LC-47 and LC-48. An LC-46 aliquot was thawed and cultured for 2 passages to form an LC-59 coded tertiary library.

3 ° Comparison of in vitro titration of a reference sample with 2 batches of cells

MovS6:

An aliquot of batch cells LC-46 and LC-59 was thawed and maintained in parallel for 2 passages. With each of these lots of cells, 24-well assay plates ( ² cm ² / well) were then inoculated in parallel at the rate of 100,000 cells per well. After 24 hours of incubation, serial dilutions of the homogenate of LN-3326 mouse brains were prepared with culture medium and not in a dilution of 10 by 10 (10 ^"1-10 ^'8). For each dilution of the inoculum, 5 wells of each of the cell lots were infected.

The cells were brought into contact with 150 μl of the different dilutions of the inoculum for 24 hours, then 1 ml of new culture medium was added. The cells were maintained in culture for 72 additional hours, until the first passage where all the cells were re-seeded in 10 cm ² wells (6-well plate format). When cell culture was continued, the culture medium was changed once a week and the cells were transplanted with a ratio of 1 in 10. The cells that were not started were stored at -80 ^° C. in the form of a dry pellet. These cell pellets kept at each passage, allowed to search the PrPsc produced by the cells. The detection of the PrPsc produced was carried out directly by Western-blot

4 ° Detection of PrPsc in cells:

Cell pellet samples were thawed and taken up in 60μl of PBS. The cells were lysed by sonication for 15 "using a sonic bath (power: 15W) .20μl of sonicated cell lysate were taken to be treated with Proteinase K. The product of the digestion was denatured and then analyzed by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE). Proteins migrated in the gel were transferred to a PVDF membrane by electrotransfer. The PrPsc present on the membranes was detected by incubation with the antibody 6H4 (Prionics) and then a secondary antibody labeled with alkaline phosphatase (goat antibody "anti-mouse antibody"). The labeled membranes were revealed by chemiluminescence. One sample was considered positive if the electrophoretic profile with the three forms of glycosylated PrPsc was visible on autoradiograms. Considering the dilutions of the sample in the different reagents necessary for the Western blot, the volume of sample actually deposited in the gel track was 4.35μl.

For each Western Blot analysis, negative controls (uninfected MovS6 cells) were processed in parallel with the samples. At each of the cell passages, all the wells of culture plates were tested. When all the replicates of cell cultures inoculated with a given dilution of the inoculum were found to be positive for the PrPsc during two successive passages, these cultures were no longer tested during the following passages.

5 ° Reproducibility of an in vitro titration:

To evaluate the reproducibility of the in vitro extraction system, the titration of the LN-3326 reference sample was repeated twice more with a new aliquot of the LC-46 cell batch for each assay. Assays were performed as described above.

6 ° Stability of PrPsc production by infected cell MovS6 (lot LC-46):

In order to evaluate the stability of the production of PrPsc by infected MovS6 cells, 5.10 ⁵ cells (lot LC-46) aged 4 passages after thawing, were seeded in 10 ml of 25 cm ² flask culture medium and inoculated with a PrPsc load of 2.21 μGiouWB, ie with a multiplicity of infection (MOI) of 1: 3100. The infected culture was maintained for 23 passages each week with a split ratio of 1: 10. At each passage, from the non-re-seeded cells, an aliquot of 3 × 10 ⁶ cells was stored at -80 ^° C. in the form of a dry pellet to allow at the end of the test, a Western-blot assay of the PrPsc in the cell lysates harvested at different passages.

PrPsc was assayed by limiting dilution of the aliquot and Western blot analysis as described in paragraph 4. The first dilution of the sample no longer showing specific PrPsc signals was considered to contain 1 Western Blot unit. The titer of the sample was then calculated as the inverse of the limiting dilution, taking into account the volume of the aliquot actually deposited on the electrophoresis gel. Thus 3.10 ⁶ cells taken up in 60μl of PBS corresponded to 3.10 ⁶ cell / 0.06ml or 50.10 ⁶ cell./ml. Under the Western Blot (WB) analysis conditions described in Section 4, the signal observed on the track corresponding to the "undiluted" sample in the dilution range, came from 4.35 μl of the cell pellet suspension taken up in PBS and undiluted either 0.22 × 10 ⁶ cells. For example if the first dilution at which the signal was no longer detected corresponds to the dilution l / 81 ^Similarly, the title PrPsc in the cell pellet was 81 uWB / 4,35μl is 81x1000 / 4.35 = 18620 uWB / ml is expressed in logarithm decimal 4,27 logio Uwb / ml, or 81 / 0.22 uWB / million cells, or 368 uWB / million cells is expressed in log logarithm 2.57 logio uWB / million cells.

Results

The results of titrations of the reference brain homogenate (lot LN-3326) with each of the MovS6 cell batches (LC-46 and LC-59) are summarized in Tables 1 and 2.

Table No. 1. Titration of the homogenate of infected mouse brains (LN-3326) with the LC-59 cell batch.

NT: Not tested

Table n ° 2. Titration of homogenate of brains from infected reference mice (LN-3326) with LC-46 cell batch.

NT: Not tested

The results of repeated titration of the LN-3326 reference sample are summarized in Tables 3 and 4 below: Table n ° 3. Repetition of Homogenate Titration of Infected Mouse Brains (LN-3326) with LC-46 Cell Lot

NT: Not tested

Table n ° 4. Repetition of Homogenate Titration of Infected Mouse Brains (LN-3326) with LC-46 Cell Lot

NT: Not tested In each of these titration assays, no trace of infectivity was observed in any of the replicates of cells inoculated with the uninfected brain homogenate sample (Neg. Control). Conversely increasing PrPsc production was observed with cells inoculated with the LN-3326 reference sample. These results confirm that the in vitro titration system makes it possible to demonstrate production of PrPsc de novo by infected cells.

The LN-3326 reference homogenate sample at the titration repeats with the LC-46 lot of cells shows an average infectious titer of 6.4 log ₁₀ TdD ₅ o / ml (range 6.3 to 6.5 logio TCID ₅ o / ml) .

The LN-3326 reference homogenate sample when titrated with the LC-33 cell batch

59 shows an infectious titer of 3.3 log 10 TCID 50 / ml.

The infectious titre of the reference sample, measured with the two batches of cells: LC46 and LC59, shows a difference of 3.1 log. This difference is significant.

This difference in title indicates that the infectious titer is dependent on the cell lot

MovS6 used for in vitro titration.

7. Stability of PrPsc Production by MovS6 Cells: The kinetics of the PrPsc titre measured in a constant amount of inoculated MovS6 cells is summarized in Table 5.

Table 5: Title in PrPsc measured in a lysate of infected cells

Legend: PI: post-inoculation

These data indicate a mean titre 5.5 log uWB / ml. The standard deviation (σ) is ± 0.6 logio uWB / ml. Variation of the PrPsc titre in the cell lysate over time, expressed by the standard deviation is greater than the variability of the Western blot titration method of ± 0.5 log. These data indicate a significant reduction of the title in the lysate of cells harvested between the 6 ^th passage and the 23 ^th passage after inoculation.

8 ° Under cloning:

An aliquot of LC-46 was thawed and cultured for 8 days. From this culture, seeding of 96-well plates was performed at the rate of 0.3 cell / well. After 37 days of culture, 13 clones were isolated and re-seeded (split ratio: 100%) on a 24-well plate ( ² cm ² / well). After 8 days of incubation, each of these cultures was divided into 2 lots. The first was maintained in a 25cm ² vial until a pre-bank (LC-82-1 to LC-82-13) was formed and the other was plated on a 24-well plate to become infected as follows: Each Clone culture was adjusted to 100,000 cells / well then inoculated with an identical PrPsc load of 1.9 log uWB. These cultures were maintained during 6 passages. At the end of this time, the cells of each culture were harvested and adjusted to a concentration of 10 ⁶ cells / ml. These cell suspensions were stored at -80 ^° C. in the form of a dry pellet.

8.1 ° Selection of clones:

To evaluate by Western Blot the intensity of the infection response of each of the clones, a dry pellet aliquot of each of the clones was thawed, sonicated, and analyzed by

Western blotting. Detection of PrPsc in the different clone lysates is shown on the autoradiogram (Figure).

These data show that the response to infection of each of the clones, measured 6 weeks after inoculation, is heterogeneous between the clones. The MovS6-13 and -10 clones have no specific PrPsc signal.

The clones MovS6- 2 - 3 - 5 - 7 - 8 - 9 - 12 present a specific signal of the PrPsc of medium intensity.

The MovS6- 1 - 6 - 11 clones show a signal specific to the high intensity PrPsc.

The MovS6-4 clone shows a specific signal of the PrPsc of very strong intensity. The corresponding uninfected cell aliquot (LC-82-4) was thawed and maintained in culture to form a primary library (LC-91) and 2 secondary libraries (LC-93 and LC-94).

8.2 ° Stability of the production of PrPsc by the cells of the clone MovS6-4:

In order to evaluate the stability of PrPsc production by infected MovS6-4 cells, 5.10 ⁵ cells aged 2 passages after thawing were seeded under 10 ml of 25 cm ² fiask culture medium and inoculated with an inoculum. PrPsc of 3.0 logiouWB, ie with a multiplicity of infection (MOI) of 1: 500. The infected culture was maintained for 37 passages each week with a split ratio of 1: 10. At each passage, from the non-re-seeded cells, an aliquot was stored at -80 ^° C. in the form of a dry pellet to make it possible to carry out, at the end of the assay, a Western-b lot assay of the PrPsc in cell lysates harvested at different passage. At the end of this test the PrPsc in the lysates was titrated by limit dilution and Western-blot as described in paragraph 6 °.

8.3 ° Comparison of in vitro titration of a reference sample with MovS6 and MovS6-4 cells:

An aliquot of MovS6-4 cells (LC-94) was thawed and maintained for 5 passages. 24-well assay plates ( ² cm ² / well) were then inoculated at a rate of 100,000 cells per well. After 24 hours of incubation, serial dilutions of the homogenate of LN-3326 mouse brains were prepared with culture medium and after a step of dilution from 10 to 10 (10 ^{~ ι} 10 ^'8). For each dilution of the inoculum, 5 wells of cells were infected. The cells were brought into contact with 150 μl of the different dilutions of the inoculum for 24 hours, then 1 ml of new culture medium was added. The cells were maintained in culture for 72 additional hours, until the first passage where all the cells were re-seeded in 10 cm ² wells (6-well plate format). When cell cultures were continued, the culture medium was changed once a week and the cells transplanted with a ratio of 1 in 10. The cells were not ensemmencées were kept at -80 ⁰ C as a dry pellet. These cell pellets kept at each passage, allowed to search the PrPsc produced by the cells. The detection of the PrPsc produced was carried out directly by Western-blotting as described in paragraph 4 °

Results

The kinetics of the PrPsc titer measured in a constant amount of inoculated MovS6-4 cells is summarized in Table 6.

Table 6: Title (logio) of the PrPsc measured in the lysate of infected cells.

These data indicate a mean titre 4.6 log uWB / ml. The standard deviation (σ) is ± 0.3 log uWB / ml. The variation in PrPsc titre in the cell lysate over time, expressed as the standard deviation, is less than the variability of the Western-blot assay method of ± 0.5 log. These data indicate stability of the title in the lysate of cells harvested between the 6 ^th passage and the 47 ^th passage after inoculation. Although these data indicate a Western-blot measured titre expressed in Westerne Blot (uWB) units, the infectious titer of infected cells (cell lysates) can also be considered to be stable over the study period. Given the difference in sensitivity between the assay methods by Western Blot and TCIA, infectivity between the 6 ^th and 47 ^th passage would be about 6.21ogio TCID ₅ o / mL. The results of the titrations of the reference brain homogenate (batch LN-3326) with the different batches of MovS6 cells and the MovS6-4 clone are summarized in Table 7.

Table n ° 7. Titration of the homogenate of infected mouse brains of reference (LN-3326) with the different batches of MovS6 cells (LC-46, LC-59) and the clone MovS6-4 (batch LC-94).

The LN-3326 reference homogenate sample at the repetitions of the titration with MovS6 cells (batch: LC-46) showed an average infectious titer of 6.4 log 10 TCID ₅ o / ml

(range 6.3 to 6.5 logio TdD ₅ o / ml) see tables 3 and 4.

The reference homogenate sample LN-3326 when titrated with MovS6-4 cells showed an infectious titer of 4.7 log 10 TdD ₅ / ml.

The infectious titre of the reference sample, measured with the two cells MovS6 (lot LC-46) and MovS6-4, shows a difference of 1.7 log. This difference is significant, it confirms that the infectious titre is dependent on the cell used for in vitro titration.

conclusions

During this study, 2 batches of cells of the MovS6 line, designated respectively; LC-46 and LC-59, were used. These 2 lots differ in age from their cells to inoculation. Lot LC-46 is aged 5 passes and a single freeze cycle

defrosting while batch LC-59 is 7 passes and 2 cycles of freezing / thawing.

The titration data of the reference brain homogenate sample (LN-3326) showed that the infectious titer of a sample is dependent on the MovS6 cell batch used. The titration system, tissue culture infectivity assay (TCIA), performed with The oldest cells (lot: LC-59) were characterized by a loss of sensitivity compared to the titration system performed with the older cells (batch: LC-46). Moreover, the data from this study show that after inoculation of MovS6 cells (batch LC-46), the production of PrPsc declined over time since a significant loss of productivity was observed between the 6 ^th and 23 ^rd passage postinoculation .

These two observations show an effect of cell age, expressed in number of passages and freeze / thaw cycles, on the biological properties of the MovS6 line. This age effect consists of a decrease in the permissiveness of cells to infection and a loss of PrPsc productivity by the infected cells.

Clones MovS6-l and MovS6-10 showed no PrPSc production to 6 ^th week after inoculation while a high production level was observed with MovS6-4 clone.

8.4 ° Comparison of the permissivity of MovS6 and MovS6-4 cells:

An aliquot of uninfected MovS6-4 cells (LC-82-4) was thawed and maintained in culture to form a primary library (LC-91).

Sample preparation before infection. An aliquot of MovS6-4 cells (LC-91) was thawed and maintained in culture to form four secondary libraries (LC-131 to LC-134).

An aliquot of MovS6-4 cells (LC-131) was thawed and maintained for 64 days and

10 passages, then frozen to form the tertiary bank (LC-139).

An aliquot of MovS6-4 cells (LC-139) was thawed and maintained for 66 days and 10 passages, then frozen to form the quaternary library (LC-143).

Comparison of in vitro titration of a reference sample with MovS6 cells and the MovS6-4 clone.

An aliquot of MovS6-4 cells (LC-143) was thawed and maintained for 2 passages. 24-well assay plates (2 cm / well) were then inoculated at a rate of 100,000 cells per well. After 24 hours of incubation, serial dilutions of the homogenate of LN-3326 mouse brains were prepared with culture medium and not in a dilution of 10 by 10 (10 ^{^"1-10" 8).} For each dilution of the inoculum, 5 wells of cells were infected.

The cells were brought into contact with 150 μl of the different dilutions of the inoculum for 24 hours, then 1 ml of new culture medium was added. The cells were maintained in culture for an additional 72 hours, until the first passage where all of the cells were reseeded in 10 cm ² wells (6-well plate format). During the continuation of the cell cultures, the culture medium was changed once a week and the cells were subcultured with a ratio of 1 in 10. The non-reseeded cells were stored at -80 ^° C. in the form of a dry pellet. These cell pellets kept at each passage, allowed to search the PrPsc produced by the cells. The detection of the PrPsc produced was carried out directly by Western blotting as described in paragraph 4 above.

The results of titrations of the reference brain homogenate (batch LN-3326) with different batches of cells and MovS6 MovS6-4 clone 6 ^th passage after inoculation is summarized in Table 8 below:

Table n ° 8. Titration of the reference infected mouse homogenate (LN-3326) with the different batches of MovS6 cells (LC-46 and LC-59) and the MovS6-4 clone (LC-143).

The so-called "young" MovS6 cells (Lot: LC-46) had, prior to the titration test, undergone a single freeze / thaw cycle and totaled 5 passages. With these cells the LN-3326 reference homogenate sample showed an infectious titer of 6.4 log ₁₀

TCID / ml. The so-called "aged" MovS6 cells (Lot: LC-59) had, before the titration test, undergone two cycles of freezing / thawing and totaled 7 passages. With these cells the LN-3326 reference homogenate sample showed an infectious titer of 3.3 log ₁₀

TCID / ml.

The so-called "aged" MovS6-4 cells (Lot: LC-143) have, before the titration test, undergone two cycles of freezing and three cycles of thawing and totaled 22 passages. With these cells the LN-3326 reference homogenate sample showed an infectious titer of

5.82 log TCID 50 / ml (range 5.72 to 5.92).

The titer of the reference brain homogenate sample (LN-3326) determined with the "young" MovS6 cells is not significantly different from the infectious titer determined with the "aged" MovS6-4 cells (the difference between the securities is below the difference threshold of 1 logio commonly considered as the threshold value for a significant difference).

The titer of the reference brain homogenate sample (LN-3326) determined with the "aged" MovS6 cells is significantly different from the infectious titer determined with the "aged" MovS6-4 cells.

Conclusion:

During this study, 2 batches of cells of the MovS6 line, designated respectively;

LC-46 and LC-59, were used. These 2 lots differ only in their "age" (expressed as the number of freeze / thaw cycles and the number of cell passages before inoculation).

The LC-46 lot is 5 passages and a single freeze / thaw cycle while the LC-59 lot is 7 passes and 2 freeze / thaw cycles. Titration data from the reference brain homogenate sample (LN-3326) showed that the infectious titer of a sample is age dependent on the MovS6 cell used.

The titration system, TCIA ("Tissue Culture Infectivity Assay"), performed with the aged cells (batch: LC-59), was characterized by a significant loss of sensitivity compared to the titration system performed with the older cells ( lot: LC-46).

These observations show an effect of cell age, expressed in number of passages and freezing / thawing cycles, on the biological properties of the MovS6 line. This effect of age consists of a decrease in cell permissiveness to infection in MovS6.

Furthermore, these data also show that after inoculation of the cells MovS6-4 (lot LC-143), these cells having undergone 2 cycles of freezing / thawing and additional thawing and totaling 22 passages, the infectious titre of a sample no. is not affected by the age of this cell. As a result, the age of MovS6-4 cells (LC-143) does not affect the sensitivity of the TCIA titration system.

The titration system, TCIA ("Tissue Culture Infectivity Assay"), performed with the "aged" MovS6-4 cells (batch: LC-143) was characterized by a stability of the sensitivity compared to the titration system performed with MovS6 "young" cells (lot: LC-46).

This observation shows that the effect of cell age, expressed as number of passages and freeze / thaw cycles, has no impact on cell permissiveness to infection in MovS6-4.

It can therefore be concluded that the MovS6-4 clone, unlike MovS6, is stable in particular as regards its permissiveness to infection by a prion strain.

Claims

claims

1. A cell clone derived from the MovS6 line, said cell clone expressing a PrP prion protein and being capable of supporting the replication or propagation of the PrPsc pathological form of said PrP, characterized in that it has a stability of the marker titre. of infection by an unconventional transmissible agent (UTA) at least until 6 ^th passage.

2. cell clone according to claim 1, characterized in that it has a stability of the title infection marker by an unconventional transmissible agent (UTA) over at least between the 6 ^th and the 100 ^th passage

3. Clone cell according to claim 1 or 2, characterized in that it has a stability of the title infection marker by an unconventional transmissible agent (UTA) at least until the 7 ^th passage.

4. Clone cell according to any one of claims 1 to 3, characterized in that it has a stability of the title infection marker by an unconventional transmissible agent (UTA) at least until 8 ^th passage.

5. Clone cell according to any one of claims 1 to 4, characterized in that it has a stability of the title infection marker by an unconventional transmissible agent (UTA) at least until the 10 ^th passage.

6. Clone cell according to any one of claims 1 to 5, characterized in that it has a stability of the title infection marker by an unconventional transmissible agent (UTA) at least until 14 ^th passage.

7. Cell clone according to any one of claims 1 to 6, characterized in that it has a stability of the title as a marker of infection with an unconventional transmissible agent (ATNC) at least until the 22 ^nd passage.

8. Cell clone according to any one of claims 1 to 7, characterized in that it has a stability of the title marker of infection with an unconventional transmissible agent (ATNC) at least until the 23 ^rd passage.

9. Cell clone according to any one of claims 1 to 8, characterized in that it has a marker stability of the marker of infection with a non-conventional transmissible agent (ATNC) at least until the 47 ^th passage.

10. Cell clone according to any one of claims 1 to 9, characterized in that the cell lysate or the culture supernatant of said cell clone has, from 6 weeks after infection, a marker titre of the infection. greater than or equal to 3 logio TCID50 per million cells, preferably greater than or equal to 4 logio TCID50 per million cells, preferably greater than or equal to 6 logio TCID50 per million cells, preferably greater than 7 logio TCID50 per million cells.

11. Cell clone according to any one of the preceding claims, filed February 22, 2008 under the deposit number CNCM 1-3922 to the National Collection of Cultures of Microorganisms.

12. In vitro method for detecting and / or titrating the infectivity of a non-conventional transmissible agent (NCTA) whose marker is a pathological conformation protein, in a sample, comprising the steps of: i) putting said sample in contact with a cell clone as described in one of claims 1 to 11, ii) cultivating said cell clone to amplify the amount of ATC present in said sample by replicating said ATNC, iii) determining the presence and / or amount of pathological protein and / or ATNC in the sample, the culturing step ii) being performed during one or more passages.

The method of claim 12, wherein step (iii) comprises the steps of: a) contacting the thus amplified ATNC at the end of step ii) with a substrate source for the pathological protein and / or the ATNC, b) incubate the reaction medium allowing the transformation of the non-pathological conformer into pathological conformation and / or the amplification of the ATNC, c) disaggregate the aggregates possibly formed during step i) or ii d) determine the presence and / or amount of pathological protein and / or ATNC in the sample, steps (a) through (c) constituting a cycle of operations which is repeated at least twice before step (d).

14. In vitro method for evaluation and / or control of a process for obtaining or treating a biological product or material likely to be contaminated by an NCTA, in which method it is applied to said biological product or material, (A) upstream and (B) downstream of said method, a titration method according to any one of claims 12 or 13, and in that the two values (A) and (B) are compared of title obtained.

15. In vitro method for evaluating and / or controlling a procedure for decontaminating a biological product or material, method in which it is applied to said biological or material product, (A) upstream and (B) downstream of said procedure, a titration method according to any one of claims 12 or 13, and in that the two obtained values (A) and (B) of title are compared.

16. In vitro method for evaluating a compound capable of modulating the infectivity of an infectious biological product, in which method it is applied to said biological product infectious, (A) in the presence and (B) in the absence of said compound to be evaluated, a titration method according to any one of claims 12 or 13, and in that the two values (A) and (B) are compared ) of title obtained.

17. In vitro method for diagnosing a transmissible spongiform encephalopathy in a human or non-human animal, comprising detecting the presence in a biological sample of said subject of a non-conventional transmissible agent (NCTA) which is a protein pathological conformation, by the method as defined in any one of claims 12 or 13.