WO2010026346A1 - Clone cellulaire stable exprimant un prion - Google Patents
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- WO2010026346A1 WO2010026346A1 PCT/FR2009/051673 FR2009051673W WO2010026346A1 WO 2010026346 A1 WO2010026346 A1 WO 2010026346A1 FR 2009051673 W FR2009051673 W FR 2009051673W WO 2010026346 A1 WO2010026346 A1 WO 2010026346A1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2828—Prion diseases
Definitions
- 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.
- TSEs Transmissible spongiform encephalopathies
- CJD central nervous system
- NCTA unconventional transmissible agents
- PrPsc 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).
- 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.
- inactivation processes conventionally used such as the Tween-TNBP solvent / detergent treatment
- cryoprecipitable plasma proteins factor VIII, von Willebrand factor, etc.
- plasma coagulation proteins 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.
- 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.
- an infectious titer 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.
- 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.
- PrPsc pathological protein
- PrP normal protein
- PMCA Protein misfolding cyclic amplification
- 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.
- 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.
- infectious homogenate e.g., scrapie strain 127-S
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 Dondel 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.
- ATNC unconventional transmissible agent
- 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.
- ATNC a pathological conformational protein
- 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.
- 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.
- 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.
- GPI phosphatidyl glycolipid
- 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.
- telomeres 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.
- 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.
- 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.
- 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.
- TID50 is meant the infectious dose which causes the infection of 50% of the inoculations tested. These doses are preferably measured using the TCIA method.
- uWB Transverse-blot unit
- 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
- 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 .
- the ATNC is also referred to as "infectious agent”.
- sample means any source of material that may be contaminated by a NCTA.
- 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.
- it is a biological sample, for example a biological fluid or tissue or tissue extract.
- 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.
- a biological fluid it may be blood, lymph, urine or milk, this list not being limiting.
- 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.
- ATNC unconventional transmissible agent
- 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 test NCTA, or with an infectious material containing IATNC as reference material, for example extracts of brains from infected animals. by a test NCTA, or with an infectious material containing IATNC as reference material, for example extracts of brains from infected animals. by a test NCTA, or with an infectious material containing IATNC as reference material, for example extracts of brains from infected animals. by a test NCTA, or with an infectious material containing IATNC as reference material, for example extracts of brains from infected animals. by a test NCTA, or with an infectious material containing IATNC as reference material, for example extracts of brains from infected animals. by a test NCTA, or with an infectious material containing IATNC as reference material, for example extracts of brains from
- 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.
- 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.
- the step of culturing cell clones potentially infected with the biological product is required for the replication of the ATNC, thus to amplify an insufficient amount of ATNC initially to be detected.
- 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).
- DMEM Dulbecco's Modified Eagle's Medium
- Ham- F12 Life Technologies, Cergy Pontoise, France
- 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.
- 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.
- 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.
- the unconverted non-pathological form is not detected by the detection system, as will be explained later.
- 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.
- 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) .
- 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.
- 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.
- 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.
- 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.
- 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
- 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).
- PrPc non-pathological soluble normal form of PrP
- digestion with proteinase K 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.
- an antibody specifically recognizing the pathological form and not the non-pathological form can itself be marked to make detection easier.
- an antibody may be for example the antibody
- 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).
- the method of calculating the title is the method of
- 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).
- the estimated standard deviation is calculated using the following formula:
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 .
- 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.
- 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.
- infected cell lysate stocks can be prepared for ??
- 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.
- 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.
- 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.
- 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 graphy 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.
- 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.
- 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
- 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).
- the cells were brought into contact with 150 ⁇ l 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
- Cell pellet samples were thawed and taken up in 60 ⁇ l of PBS.
- the cells were lysed by sonication for 15 "using a sonic bath (power: 15W) .20 ⁇ l 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.
- the volume of sample actually deposited in the gel track was 4.35 ⁇ l.
- 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.
- 3.10 6 cells taken up in 60 ⁇ l of PBS corresponded to 3.10 6 cell / 0.06ml or 50.10 6 cell./ml.
- the signal observed on the track corresponding to the "undiluted" sample in the dilution range came from 4.35 ⁇ l of the cell pellet suspension taken up in PBS and undiluted either 0.22 ⁇ 10 6 cells.
- 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.
- 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 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.
- MovS6 used for in vitro titration.
- 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).
- PrPsc 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.
- MOI multiplicity of infection
- 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 °
- Table 6 Title (logio) of the PrPsc measured in the lysate of infected cells.
- 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.
- 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).
- TCIA tissue culture infectivity assay
- the production of PrPsc declined over time since a significant loss of productivity was observed between the 6 th and 23 rd passage postinoculation .
- 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.
- the cells were brought into contact with 150 ⁇ l 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 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.
- 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).
- MovS6-4 clone unlike MovS6, is stable in particular as regards its permissiveness to infection by a prion strain.
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Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2009289107A AU2009289107A1 (en) | 2008-09-05 | 2009-09-04 | Stable cell clone expressing a prion |
CA2735774A CA2735774A1 (fr) | 2008-09-05 | 2009-09-04 | Clone cellulaire stable exprimant un prion |
EP09741370A EP2329273A1 (fr) | 2008-09-05 | 2009-09-04 | Clone cellulaire stable exprimant un prion |
CN2009801348584A CN102203615A (zh) | 2008-09-05 | 2009-09-04 | 表达朊病毒的稳定细胞克隆 |
US13/062,475 US20110165613A1 (en) | 2008-09-05 | 2009-09-04 | Stable clone cell expressing a prion |
BRPI0917877A BRPI0917877A2 (pt) | 2008-09-05 | 2009-09-04 | clone celular expressando um prion e métodos in vitro de detecção e/ou titulação da infecciosidade de um agente transmissível não convencional (atnc), de avaliação e/ou de controle de um processo de obtenção ou de tratamento de um produto biológico ou de um material capaz de ser contaminado por um atnc, de avaliação e/ou de controle de procedimento de descontaminação de um produto biológico ou de um material, de avaliação de um composto capaz de modular a infecciosidade de um produto biológico infeccioso e de um diagnóstico de uma encefalopatia espongiforme transmissível em um indivíduo humano ou animal não humano |
JP2011525600A JP2012501638A (ja) | 2008-09-05 | 2009-09-04 | プリオンを発現する安定な細胞クローン |
IL211492A IL211492A0 (en) | 2008-09-05 | 2011-03-01 | Stable cell clone expressing a prion |
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FR0855985A FR2935711A1 (fr) | 2008-09-05 | 2008-09-05 | Clone cellulaire stable exprimant un prion |
FR0855985 | 2008-09-05 |
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US (1) | US20110165613A1 (fr) |
EP (1) | EP2329273A1 (fr) |
JP (1) | JP2012501638A (fr) |
KR (1) | KR20110069801A (fr) |
CN (1) | CN102203615A (fr) |
AR (1) | AR073494A1 (fr) |
AU (1) | AU2009289107A1 (fr) |
BR (1) | BRPI0917877A2 (fr) |
CA (1) | CA2735774A1 (fr) |
FR (1) | FR2935711A1 (fr) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2732827A1 (fr) | 2012-11-20 | 2014-05-21 | Franklab | Procédé de détermination de l'aptitude d'une formulation à inactiver un ATNC |
WO2014184317A1 (fr) | 2013-05-15 | 2014-11-20 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Procédé d'inactivation d'une protéine prion |
Citations (3)
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WO2002004954A2 (fr) * | 2000-07-07 | 2002-01-17 | Applied Research Systems Ars Holding N.V. | Diagnostic precoce de maladies conformationelles |
FR2859222A1 (fr) * | 2003-08-25 | 2005-03-04 | Lab Francais Du Fractionnement | Methode d'evaluation et/ou de controle d'un procede d'obtention d'un produit biologique susceptible d'etre contamine par un agent transmissible non conventionnel (atnc) |
WO2006117483A2 (fr) * | 2005-05-04 | 2006-11-09 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Materiel infectieux synthetique et standardise de type prion et ses utilisations en tant qu’inoculum infectant |
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US6277970B1 (en) * | 1999-05-11 | 2001-08-21 | The Regents Of The University Of California | PRP-like gene |
US20040048237A1 (en) * | 2002-05-15 | 2004-03-11 | Lindquist Susan L. | Mammalian prion proteins and transgenic mice expressing them |
US7727728B2 (en) * | 2003-08-25 | 2010-06-01 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Method for the in vitro titration of an NCTA application thereof in a method for the evaluation and/or monitoring of a biological product production method |
FR2929290B1 (fr) * | 2008-03-27 | 2010-05-07 | Lfb Biotechnologies | Methode de detection et/ou de titrage in vitro d'un agent transmissible non conventionnel |
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- 2009-09-04 US US13/062,475 patent/US20110165613A1/en not_active Abandoned
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WO2002004954A2 (fr) * | 2000-07-07 | 2002-01-17 | Applied Research Systems Ars Holding N.V. | Diagnostic precoce de maladies conformationelles |
FR2859222A1 (fr) * | 2003-08-25 | 2005-03-04 | Lab Francais Du Fractionnement | Methode d'evaluation et/ou de controle d'un procede d'obtention d'un produit biologique susceptible d'etre contamine par un agent transmissible non conventionnel (atnc) |
WO2006117483A2 (fr) * | 2005-05-04 | 2006-11-09 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Materiel infectieux synthetique et standardise de type prion et ses utilisations en tant qu’inoculum infectant |
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SABORIO GABRIELA P ET AL: "Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding", NATURE, NATURE PUBLISHING GROUP, LONDON, UK, vol. 411, no. 6839, 1 January 2001 (2001-01-01), pages 810 - 813, XP002201227, ISSN: 0028-0836 * |
SOTO C ET AL: "Cyclic amplification of protein misfolding: application to prion-related disorders and beyond", TRENDS IN NEUROSCIENCE, ELSEVIER, AMSTERDAM, NL, vol. 25, no. 8, 1 August 2002 (2002-08-01), pages 390 - 394, XP004371962, ISSN: 0166-2236 * |
ZHANG W ET AL: "The In Vitro Bioassay Systems for the Amplification and Detection of Abnormal Prion PrP<Sc> in Blood and Tissues", TRANSFUSION MEDICINE REVIEWS, GRUNE AND STRATTON, ORLANDO, FL, US, vol. 22, no. 3, 1 July 2008 (2008-07-01), pages 234 - 242, XP022757098, ISSN: 0887-7963, [retrieved on 20080620] * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2732827A1 (fr) | 2012-11-20 | 2014-05-21 | Franklab | Procédé de détermination de l'aptitude d'une formulation à inactiver un ATNC |
WO2014184317A1 (fr) | 2013-05-15 | 2014-11-20 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Procédé d'inactivation d'une protéine prion |
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KR20110069801A (ko) | 2011-06-23 |
AU2009289107A1 (en) | 2010-03-11 |
EP2329273A1 (fr) | 2011-06-08 |
BRPI0917877A2 (pt) | 2015-11-24 |
CA2735774A1 (fr) | 2010-03-11 |
IL211492A0 (en) | 2011-05-31 |
FR2935711A1 (fr) | 2010-03-12 |
AR073494A1 (es) | 2010-11-10 |
US20110165613A1 (en) | 2011-07-07 |
JP2012501638A (ja) | 2012-01-26 |
CN102203615A (zh) | 2011-09-28 |
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