WO2013056841A1

WO2013056841A1 - A method for diagnosing tse

Info

Publication number: WO2013056841A1
Application number: PCT/EP2012/004377
Authority: WO
Inventors: Olivier ANDREOLETTI
Original assignee: Inra (Institut National De La Recherche Agronomique)
Priority date: 2011-10-19
Filing date: 2012-10-19
Publication date: 2013-04-25

Abstract

The present invention relates to a method for diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt- Jakob disease (CJD), and wherein said method comprises the steps of a) mixing a biological sample from said subject with a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171, derivative or fragment thereof to make a reaction mixture; b) incubating said reaction mixture in conditions allowing the amplification of the PrP^Sc protein present in the biological sample; c) disrupting the reaction mixture; d) repeating steps (b) and (c) one or more times; and e) detecting the PrP^Sc protein in the amplified reaction mixture; and the associated kit.

Description

A METHOD FOR DIAGNOSING TSE

[0001 ] The present International patent application claims the priority of European patent application n° 1 1008416.7 filed on October 19, 201 1, which is incorporated herein by reference. Field of the Invention

[0002] The present invention relates to Transmissible Spongiform Encephalopathies (TSE), more specifically to the diagnosis of a TSE that is not scrapie.

Background

[0003] Prion diseases, which are also called Transmissible Spongiform Encephalopathies (TSEs), comprise a group of fatal infectious neurodegenerative diseases that include Creutzfeldt- Jakob disease (CJD), kuru, Gerstmann-Strauss!er Sheinker syndrome (GSS), fatal familial insomnia (FFI) and sporadic fatal insomnia (sFI) in humans and scrapie, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in animals. These diseases are characterized by brain vacuolation, astrogliosis, neuronal apoptosis and the accumulation of the misfolded prion protein (PrP^Sc) in the central nervous system. TSEs have asymptomatic incubation periods of months to years. Attempts at TSE risk reduction have led to profound changes in the production and trade of agricultural goods, medicines, cosmetics, and biotechnology products. [0004] The hallmark event of prion disease is the formation of a misfolded protein called PrP^Sc, which result from post-translational conversion of an host normal protein, termed PrP^c. The conversion seems to involve a conformational change whereby the a-helical content of the normal protein diminishes and the amount of β-sheet increases. The structural changes are followed by alterations in the biochemical properties: PrP^c is soluble in non-denaturing detergents, PrP^Sc is insoluble; PrP^c is readily digested by proteases, while PrP^Sc is partially resistant, resulting in the formation of a N-terminally truncated fragment.

[0005] Formation of PrP^Sc is not only the most likely cause of the disease, but also is the best known marker. Detection of PrP^Sc in tissues and cells correlates widely with the disease and with the presence of TSE infectivity.

[0006] Thus, the identification of PrP^Sc on human or animal tissues is considered key for TSE diagnosis. Nevertheless, there is actually no accurate premortem diagnosis for TSEs.

[0007] For human diseases, diagnosis is based mainly on clinical examination and the disease is considered possible, or probable, depending upon the degree to which the clinical symptoms fit the standard guidelines. Definitive diagnosis can only be made postmortem by brain histological analysis of spongiform changes, astrogliosis and amyloid plaques (although these plaques are not consistently seen in all TSEs). Although brain biopsy has been used to establish a definitive diagnosis, it is strongly discouraged because it is invasive and costly. Moreover, a brain biopsy sometimes produces a false-negative result, because the tissue sample has been taken from an unaffected area of the brain. [0008] The serious consequences of BSE epidemics motivated the European Community to implement a system to evaluate and validate biochemical tests aimed at rapidly detecting infected animals. Since postmortem identification of sick cattle by histological analysis of the brain is time consuming, new tests were developed to enable processing multiple samples in just a few hours, so that commercialization of the animals could be withheld until results were available. 9 different tests using blind samples from BSE- infected and normal cattle were evaluated. Using sensitivity and specificity as criteria, 5 post-mortem test using obex (Central nervous system) as tissue target were approved by the European Community for TSE detection.

[0009] However, the sensitivity of these tests enables detection of infected individuals in the late phase of the disease evolution.

[00010] This limitation is problematic since prion infectivity accumulates in various peripheral tissues during the early phase following contamination. In particular low infectivity level can be present in the blood of infected individuals which, in humans, is associated to a risk of CJD transmission through blood transfusion as confirmed by several cases in patients. Because of the risk of iatrogenic CJD transmission, the development of a sensitive and presymptomatic blood test for CJD is a top priority. Nevertheless, the only method enabling to reliably detect prions in blood remains the infectivity detection by bioassay. This approach is limited for widespread use by the length of incubation periods (several months to years), incurred cost and ethical issues.

[0001 1 ] Recently a more rapid prion detection method was developed based on the ability of prions to replicate in vitro in substrates containing PrP^c. This technique termed protein misfolding cyclic amplification (PMCA) involved mixing samples with such substrate, incubating the mixture, breaking PrPSc aggregates, then performing repeated incubation and desegregation steps, see International Patent Application PCT WO 02/04954. In vitro amplified prion was then detectable by classical biochemical PrP^Sc detection like Western blot or ELISA assays. This technique offered the significant advantage of rapid results, however, still was not believed reliable for diagnosis (specificity problem and potentially insufficient sensitivity) and certainly not as reproducible. Subsequent modification of this assay by other researchers has shown that prion can be continually replicated such as in the International patent application WO 2009/015091. However, improvements in the performances (sensitivity/specificity, capacity to detect TSE agents considered in their diversity) are still needed for diagnostic tests. Thus, currently there continues to be a need for a rapid method for the detection of prion that is sensitive/specific enough to detect low level prion contamination, especially in blood samples.

Summary of the Invention

[00012] The present invention is based on the discovery by the present inventors that

[00013] (i) Ovis aries and Capra hircus (and closely related species) PrP isoforms of harboring Glutamine at the residue 171 as for instance ARQ (Accession number CAEOO 186), VRQ (Accession number CAEOO 190.1) and AHQ (Accession number ABG78479) isoforms ;

[00014] (ii) recombinant full length or fragment of these PrP isoforms; and

[00015] (iii) chimeric protein containing residues 160 to 1 80 of these PrP isoform; [00016] enable to amplify human, primate, murine, hamster, bovine PrP ^c in blood samples with high sensitivity/specificity and reproducibility.

[00017] If it was known that heterologous conversion was possible, this conversion was also known to clearly less efficient as compared to the homologous one as demonstrated by example in RAYMOND et al. {Nature, vol.388, p:285-288, 1997). Consequently, nothing in the prior art suggests that heterologous conversion using specific isoforms of PrP can be used in a method of amplification and detection of PrP^Sc in blood samples with a higher sensitivity and equivalent or greater specificity as compared to homologous conversion.

[00018] Consequently, in one aspect the present invention relates to a method for diagnosing a Transmissible Spongiform encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), and wherein said method comprises the steps of:

[00019] (a) mixing a biological sample from said subject with a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 , a derivative or a fragment thereof to make a reaction mixture;

[00020] (b) incubating said reaction mixture in conditions allowing the amplification of the

PrP^Sc protein present in the biological sample;

[00021 ] (c) disrupting the reaction mixture, preferably by sonication by high energy agitation of microbeads;

[00022] (d) repeating steps (b) and (c) one or more times; and

[00023] (e) detecting the PrP^Sc protein in the amplified reaction mixture. [00024] In a preferred embodiment, said caprinae PrP protein isoform having a glutamine amino acid residue at its position 171 is an ovine or a caprine PrP^c protein isoform having a glutamine amino acid residue at its position 171.

[00025] Advantageously, the ovine PrP^c protein isoform having a glutamine amino acid residue at its position 171 is selected in the group comprising the ARQ Ovis aries PrP^c protein isoform having the sequence SEQ id n° l (Accession number CAE00186), the VRQ Ovis aries PrP^c protein isoform having the sequence SEQ id n°2 (Accession number CAE00190) and the AHQ Ovis aries PrP^c protein isoform having the sequence SEQ id n°3 (Accession number ABG78479).

[00026] Preferably, said ovine PrP^c protein isoform is the Ovis aries PrP^c protein isoform having the sequence SEQ id n°l (Accession number CAE00186).

[00027] In a still preferred embodiment, said biological sample is a blood sample or any product derived thereof, such as a buffy coat or a White blood cell sample.

[00028] In another preferred embodiment, the caprinae PrP^c protein isoform derivative or fragment is from a tissue homogenate, cell lysate or preferably from a source overexpressing said protein isoform derivative or fragment thereof, and most preferably said caprinae PrP protein isoform, derivative or fragment is from a brain homogenate.

[00029] In still another preferred embodiment, the step b) of incubating the reaction mixture in conditions allowing the amplification of the PrP^Sc protein present in the biological sample is done by maintaining the reaction mixture at a temperature comprised between 36 and 45°C, preferably between 38 and 41 °C, and most preferably between 39 and 40°C. [00030] In still another preferred embodiment, the step c) of disrupting the reaction mixture is done by sonication.

[00031] Still preferably, and for the step d), the steps b) and c) are repeated at least 10 times, preferably at least 50 times and most preferably at least 90 times.

[00032] In still another preferred embodiment, said subject is a human, and said TSE is CJD such as sporadic (sCJD), familial (fCJD), iatrogenic (iCJD), and variant form of CJD (vCJD), preferably said CJD is vCJD.

[00033] In still another preferred embodiment, said subject is a bovine, and said TSE is BSE.

[00034] In a second aspect, the resent invention relates to a kit for diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), wherein said kit comprises a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 , derivative or fragment thereof.

Brief Description of the Drawings

[00035] Figure 1 shows v-CJD amplification using Wild typegoat PrP^c, ovine ARQ PrP^c, or ovine AHQ PrP as substrate.

[00036] Figure 2 shows the vCJD/BSE agent amplification by PMCA using brain from transgenic mice expressing different species PrP sequence as substrate.

[00037] Figure 3 shows the PrP^res detection in PMCA reactions seeded with white blood cells (WBC) from BSE infected and healthy sheep. [00038] Figure 4 shows the vCJD agent detection in the blood of experimentally infected primates.

[00039] Figure 5 shows PrP^res in PMCA reactions seeded with blood samples from vCJD infected and healthy primates.

[00040] Figure 6 shows PrP^res in PMCA reactions seeded with WBC from a vCJD affected patient and healthy controls.

[00041] Figure 7 shows the results in White blood cells prepared from one v-CJD affected patients after 2 rounds of amplification

Detailed Description

[00042] The first aspect of the invention relates to a method for diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), and wherein said method comprises the steps of:

[00043] (a) mixing a biological sample from said subject with a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 , a derivative or a fragment thereof to make a reaction mixture;

[00044] (b) incubating said reaction mixture in conditions allowing the amplification of the

PrP^Sc protein present in the biological sample;

[00045] (c) disrupting the reaction mixture, preferably by sonication by high energy agitation of microbeads

[00046] (d) repeating steps (b) and (c) one or more times; and [00047] (e) detecting the PrP ^c protein in the amplified reaction mixture.

[00048] In animals the most common TSE is scrapie, but the most famous and dangerous disease is the recently discovered BSE, which affects cattle and is known in the world over by its lay term "mad cow disease."

[00049] As used herein, the term subject refers to a mammal except caprinae, preferably except ovine.

[00050] The method provides sensitivity and reproducibility that is sufficiently high to detect or diagnose disease prior to the onset of clinical symptoms.

[0005 1 ] Consequently the subject may be healthy, but the method of the invention is particularly useful for testing a subject thought to develop TSE.

[00052] In a preferred embodiment, said TSE is BSE (classical, 1-Type, H-type) and said subject is a bovine.

[00053] In humans the most common TSE is CJD, which occurs worldwide with an incidence of 0.5 to 1.5 new cases per one million people each year.

[00054] In other preferred embodiment, said TSE is CJD and said subject is a human.

[00055] Three different forms of CJD have been traditionally recognized: sporadic (sCJD; 85% of cases), familial (fCJD; 10%), and iatrogenic (iCJD; -5%). However, in 1996, a new variant form of CJD (vCJD) emerged in the UK, which has been associated with the exposure to BSE contaminated products. In contrast with typical cases of sCJD, vCJD affects young patients with an average age of 27 years old, and is a relatively long illness (14 months compared with 4.5 months for sCJD). Because of insufficient information available about the incubation time and the levels of exposure to contaminated cattle food products it is impossible to make well-founded predictions about the potential future incidence of vCJD.

[00056] Advantageously, said CJD is selected among sporadic (sCJD), familial (fCJD), iatrogenic (iCJD), and variant form of CJD (vCJD).

[00057] The clinical diagnosis of sCJD is actually based on a combination of rapidly progressive multifocal dementia with pyramidal and extrapyramidal signs, myoclonus, and visual or cerebellar signs, associated with a characteristic periodic electroencephalogram (EEG). A key feature for diagnosing sCJD, and distinguishing it from Alzheimer's disease and other dementias, is the rapid progression of clinical symptoms and the short duration of the disease, which is often less than 2 years.

[00058] The clinical manifestations of fCJD and of sCJD are comparable, except that the disease onset occurs earlier than in sCJD. Family histories of inherited CJD or genetic screening for mutations in the PrP gene are used to establish fCJD diagnosis, although lack of family history does not excludes an inherited origin.

[00059] Variant CJD appears initially as a progressive neuropsychiatric disorder characterized by symptoms of anxiety, depression, apathy, withdrawal and delusions. This is combined with persistent painful sensory symptoms and is followed by ataxia, myoclonus, and dementia. Variant CJD is differentiated from sCJD by the duration of illness (usually longer than 6 months) and EEG analysis (vCJD does not show the atypical pattern observed in sCJD). A high bilateral pulvinar signal noted during MR! is usually used to help diagnose vCJD. in addition, a tonsil biopsy may be used to help diagnose vCJD, based on a number of cases of vCJD have been shown to test positive for PrP^Sc staining in lymphoid tissue (such as tonsil and appendix). However, because of the invasive nature of this test, it should be performed only in patients who fulfill the clinical criteria of vCJD where the MRI of the brain does not show the characteristic pulvinar sign.

[00060] Still advantageously, said CJD is vCJD.

[00061 ] As used herein, the term "biological sample" refers to a sample from tissues including, but not limited to, blood, lymph nodes, brain, spinal cord, tonsils, spleen, skin, muscles, appendix, olfactory epithelium, cerebrospinal fluid, urine, milk, intestines, tears and/or saliva.

[00062] Advantageously, said biological sample refers to a blood sample or any product derived thereof.

[00063] Still advantageously said blood sample comprises a volume of 100 μΐ or less from one subject, preferably 50 μΐ or less, and still preferably 10 μΐ or less from one subject.

[00064] When the blood sample refer to a plasma sample, said sample corresponds to a plasma volume of 5ml or less from one subject, preferably a volume of 2.5 ml or less and more preferably of 1 ml or less.

[00065] As used herein a product derived from a blood sample refers to a product comprising blood components such as plasma or plasma derived products, red cells, platelets, or leukocytes, etc.

[00066] Preferably, said product derived from a blood sample refers to a buffy coat or a white blood cell sample.

[00067] As an example, said buffy coat sample is obtained as disclosed in the examples. Preferably, said buffy coat sample is diluted more than ten folds, preferably more than twenty folds, and preferably at least fifty folds. In fact, the inventors have established that a minimum dilution of this buffy coat sample fosters PrP^Sc protein amplification.

[00068] As an example, said White blood cell sample is obtained as disclosed in the examples.

[00069] Still preferably, said product derived from a blood sample refers to a plasma sample. Said plasma sample may be further processed, as an example, ultracentrifuged (i.e. about lOO OOOg and for at least 10 minutes, preferably one hour) so as to purified microvesicles for said plasma.

[00070] The method of the invention may further comprise a previous step of concentration of the PrP^Sc protein of the biological sample. For example protein may be concentrated by phosphotungstic acid (PTA) precipitation, or binding to ligands, shown to interact specifically to PrP^Sc, such as conformational antibodies, certain nucleic acids, plasminogen or various short peptides (SOTO & CASTILLA, Nature Med., vol.10, p:S63-S67, 2004). It is also contemplated that samples may be fractionated. For example, the fraction that is insoluble in mild detergent could be harvested, a procedure that would increase the concentration of prion within the sample (WO 02/04954).

[00071 ] The skilled person can simply identify the caprinae PrP protein isoforms having a glutamine amino acid residue at its position 171.

[00072] As an example, one can cite the Ovis aries PrP^c protein isoform having the sequence SEQ id n° l corresponds to the ARQ isoform (Alal 36-Argl 54-Gln l 71 ) of sheep PrP (Accession number: CAE00186), the Ovis aries PrP^c protein isoform having the sequence SEQ id n°2 corresponds to the VRQ isoform (Val l 36-Argl 54-Glnl 71 ) of sheep PrP (Accession number: CAE00190) and Ovis aries PrP^c protein isoform having the sequence SEQ id n°3 corresponds to the AHQ isoform (Alal36-Hisl 54-Glnl71) of sheep PrP (Accession number: ABG78479).

[00073] Preferably, said ovine PrP^c protein isoform is the Ovis aries PrP^c protein isoform having the sequence SEQ id n° 1.

[00074] As used herein, the term "derivative" refers to a polypeptide having an identity of at least 85%, preferably at least 95% and most preferably at least 95% with the complete sequence of a mammalian PrP^c protein isoform and an identity of at least 95%, preferably at least 99% and most preferably 100% of identity with the amino acids 160 to 180 of a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n° l , SEQ id n°2 or SEQ id n°3.

[00075] As used herein, "percentage of identity" between two amino acids sequences, means the percentage of identical amino-acids, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acids sequences. As used herein, "best alignment" or "optimal alignment", means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two amino acids sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity. The best sequences alignment to perform comparison can be realized, beside by a manual way, by using the global homology algorithm developed by SMITH and WATERMAN (Ad. App. Math., vol.2, p: 482, 1981), by using the local homology algorithm developed by NEDDLEMAN and WUNSCH (J. Mol. Biol, vol.48, p:443, 1970), by using the method of similarities developed by PEARSON and LIPMAN (Proc. Natl. Acad. Sci. USA, vol.85, p:2444, 1988), by using computer softwares using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), by using the MUSCLE multiple alignment algorithms (Edgar, Robert C, Nucleic Acids Research, vol. 32, p: 1792, 2004 ). To get the best local alignment, one can preferably used BLAST software, with the BLOSUM 62 matrix, or the PAM 30 matrix. The identity percentage between two sequences of amino acids is determined by comparing these two sequences optimally aligned, the amino acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.

[00076] As used herein, the term "fragment" refers to a polypeptide comprising the amino acids 25 to 233 of a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n°l , 2 or 3 corresponding to the full length maturated PrP protein, but also to a polypeptide comprising the amino acids 90-23 1 of a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n° 1 , 2 or 3, such as 80-231 or 70-231 , preferably 60-23 1 , such as 50-231 or 40-231 , and more preferably 30-231 of a caprinae PrP protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n° l , 2 or 3.

[00077] As detailed above, a variety of sources may be used to obtain said PrP^c protein isoform, derivative or fragment thereof for use in the method of the invention, preferably said source is a cell lysate.

[00078] For instance, the protein may be the endogenous protein isoform expressed in caprinae cells such as ovine cells, and these cells used to make a lysate that provides the corresponding protein isoform. The lysate may be from tissue culture cells, or extracted from whole organisms, organs or tissues. As an example, the source of the protein isoform may be from ovine brain homogenates.

[00079] Advantageously, the source of said PrP^c protein isoform, derivative or fragment thereof may be from cells made or engineered to over-express it. For instance, cells may be transformed with a nucleic acid vector that expresses said PrP protein isoform, derivative or fragment thereof. These cells may comprise mammalian cells, bacterial cells, yeast cell, insect cells, whole organisms, such as transgenic mice, or other cells that may be a useful source of the non-pathogenic protein. Preferably, the protein isoform's source may be a brain homogenate.

[00080] Any of the wide variety of vectors known to those of skill in the art could be used to over express the PrP^c protein isoform, derivative or fragment thereof. For example, plasmids or viral vectors may be used. It is well understood to these of skill in the art that these vectors may be introduced into cells by a variety of methods including, but not limited to, transfection (e.g., by liposome, calcium phosphate, electroporation, particle bombardment, etc.), transformation, and viral transduction.

[00081] Still advantageously, the source of said PrP^c protein isoform, derivative or fragment thereof is a transgenic mammal such as mouse over-expressing it, such as the mouse Tg ARQ disclosed in the example, preferably a brain homogenate obtained from said transgenic mammal.

[00082] Such a brain homogenate can be simply obtained by methods well known from the skilled person.

[00083] As an example, such brain homogenate can be obtained by (i) low speed homogenization (e.g. using ULTRATURAX) of brain tissue in 4°C PBS pH 7-7.65 + 0.1% TRITON® X100+ 150 mM NaCl (10% Weight/vol) , (ii) lying on ice for 15 to 45 min, (iii) filtration through a 50μπι mesh disposable cell strainer or low speed centrifugation 100 to 500 g for 1 min. The obtained homogenate is then aliquoted and snap frozen at -80°C before use.

[00084] For instance in the case of PrP^c, it has been shown that the majority of the protein localizes to the membrane in structures known as "lipid-rafts." Thus, partial purification of PrP^c can be achieved by enriching the lysate for lipid-rafts. Methods for this enrichment typically rely on the resistance of lipid-raft structures to mild detergent, such as ice-cold TRITON® X-100, and are well known to those in the art.

[00085] Generally, "purified" will refer to a caprinae ovine PrP^c protein isoform composition that has been subjected to fractionation or isolation to remove various other protein or peptide components, and which composition substantially retains caprinae PrP^c protein isoform, as may be assessed, for example, by Western blot to detect said protein isoform.

[00086] To purify non-pathogenic protein from natural or recombinant composition the composition will be subjected to fractionation to remove various other components from the composition. Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PTA, PEG, antibodies and the like, or by heat denaturation followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.

[00087] It may in some cases be preferable that the caprinae PrP^c protein isoform, derivative or fragment thereof be deglycosylated. For example, caprinae PrP^c protein isoform, derivative or fragment thereof may be treated with peptide N- glycosidase F (NEW ENGLAND BIOLABS) according to the manufacturer's instructions. In this case, incubation for about 2h at 37°C results in significant deglycosylation of the protein.

[00088] In some cases it may be preferable that the recombinant protein be fused with additional amino acid sequence. For example over expressed protein may be tagged for purification or to facilitate detection of the protein in a sample. Some possible fusion proteins that could be generated include histidine tags, Glutathione S-transferase (GST), Maltose binding protein (MBP)), green fluorescent protein (GFP), Flag and myc tagged PrP. These additional sequences may be used to aid in purification and/or detection of the recombinant protein and in some cases may then be removed by protease cleavage. For example coding sequence for a specific protease cleavage site may be inserted between the non-pathogenic protein coding sequence and the purification tag coding sequence. One example for such a sequence is the cleavage site for thrombin. Thus fusion proteins may be cleaved with the protease to free the non-pathogenic protein from the purification tag.

[00089] In the case where the caprinae PrP^c protein isoform, derivative or fragment thereof is highly purified the reaction mixture may further comprise additional factors or cell lysate to provide secondary factors important for conversion. For example in the case of PrP , brain homogenate from PrP null mice may be ideal or polyanions such as RNA or synthetic polyanions (DELEAULT et al, Proc. Natl. Acad. Sci. USA., vol.l04(23), p:9741 -6, 2007; and International Patent Application PCT WO 2007/082173).

[00090] As used herein, the expression "reaction mixture" refers to a composition minimally comprising a biological sample and the caprinae PrP^c protein isoform, derivative or fragment thereof.

[00091 ] In some embodiments, the reaction mixture further comprises a "conversion buffer" that is favorable for prion replication. As an example, said conversion buffer comprises a salt solution and detergents such as the buffer comprising l x phosphate buffered saline (PBS) with 150 mM additional NaCl, 0.5% TRITONX®-100 and a protease inhibitor cocktail.

[00092] The conversion buffer may further comprise a metal chelator. This is of particular advantage since Cu²⁺ and to some extent Zn²⁺ interferes with the amplification PrP^Sc. As an example, the metal chelator is EDTA. [00093] The reaction mixture may also comprise additional elements, for example, one or more buffers, salts, detergents (e.g. digitonin, saponin or SDS), lipids, or proteins or polypeptides (e.g. Plasminogen).

[00094] It is preferable that the reaction mixture be kept in a sealed environment to prevent evaporation and cross contamination. For example amplification may be carried out while reaction mixture is maintained in a sealed plastic enclosure.

[00095] The step (b) of incubating the reaction mixture in conditions allowing the amplification of the PrP^Sc protein present in the biological sample is done by maintaining the reaction mixture at a temperature comprised between 36 and 45°C, preferably between 38 and 41°C, and most preferably between 39 and 40°C

[00096] Said temperature enable the conversion of the caprinae PrP^c protein isoform or fragment thereof by the PrP^Sc protein present in the biological sample.

[00097] In certain embodiments the temperature of the reaction mixture is monitored and/or controlled by a programmable thermostat. For example the sample may be placed in an automated thermocycler thus allowing the temperature of the reaction mixture to be programmed over the course of amplification. It is also contemplated that incubation of the reaction mixture could be performed over a range of time periods. For example the reaction mixture may be incubated at step (b) for about one minute to about 10 hours, preferably for about 10 minutes to 1 hour and most preferably for about 30 minutes.

[00098] It is also contemplated that the incubation time may be varied through out the amplification. For example the incubation time may be increased or decreased by an increment of time after each amplification step. [00099] In step (c), the term "disrupting" refers to any method by which molecular complexes in reaction mixture may be break out.

[000100] Exemplary disaggregation methods include treatment with solvents, modification of pH, temperature, ionic strength, or by physical method such as sonication or homogenization.

[000101] In a preferred embodiment, the step c) of disrupting the reaction mixture is done by high energy agitation of microbeads contained in the reaction mixture, which high energy agitation is realized by sonication. To prevent contamination it is preferred that the sonication apparatus not come in direct contact with the samples.

[000102] As an example, this step of sonication may be performed with a commercially available sonication apparatus such as MISON1X 3000 or MISONIX 4000.

[000103] The sonication apparatus may be automated and capable of programmed operation thus allowing high throughput sample amplification.

[000104] As an example of microbeads which can be used at this step and which have to be introduced in the reaction mixture, one can cite iron, glass and silica microbeads, preferably said microbeads have diameters ranging from 0.3 to 2.5 mm, most preferably silica beads displaying 0.8 to 1.2 mm diameter.

[000105] For example, said sonication could comprise a pulse or a serial of pulses of about 5 to 60 seconds, preferably 10 to 50 seconds, as an example of 30 seconds. As still another example, said sonication could comprise one or several pulses at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% potency. [000106] Advantageously, it is contemplated that, for the step d), the steps b) and c) could be repeated at least 10 times, preferably at least 50 times and most preferably at least 90 times.

[000107] The method of the invention may include the step d') of mixing, in an alternative step a), the obtained amplified reaction mixture with a caprinae PrP protein isoform having a glutamine amino acid residue at its position 171 , a derivative or a fragment thereof to obtain a new reaction mixture, which new reaction mixture is subjected to new steps b), c) and d).

[000108] Advantageously, said step d') is repeated one or more times.

[000109] Still advantageously, it is contemplated that, said step d' may be repeated one time, two times, three times or , four times

[0001 10] It is envisioned that in some embodiments of the present invention, the total duration of the steps a) to d) is of three days or less, preferably of two days or less, and more preferably of one day or less. This may be preferable since in some cases the PrP^c protein isoform or other cofactors may have a limited stability and extended incubation may result in an eventual fall-off of the conversion rate. In particular it has been shown that PrP conversion rates drop after about 48 hours of incubation.

[0001 1 1] The step e) of detecting the PrP^Sc protein in the amplified reaction mixture can be done by both direct and indirect assays known to those of skill in the art such as Western Blot, Immunoassays like ELISA, animal bioassays, cellular infectivity assays and spectroscopic assays. [0001 12] For methods in which PrP ⁰ is directly detected, discrimination between the newly-formed PrP^Sc from remaining PrP^c usually is required. This typically is accomplished based on the different natures of PrP^Sc versus PrP^c. For instance, PrP^Sc typically is highly insoluble and resistant to protease treatment. Therefore, in the case of PrP^Sc and PrP^c, separation can be by, for instance, protease treatment.

[0001 13] When PrP^Sc and PrP^c are separated by protease treatment, reaction mixtures are incubated with, for example, Proteinase (PK). An exemplary protease treatment includes digestion of the PrP^c protein in the reaction mixture with 1 -20 μg/ml of PK for about 1 hour at 37° C. Reactions with PK can be stopped prior to assessment of prion levels by addition of PMSF or electrophoresis sample buffer. Depending on the nature of the sample, incubation at 37° C with 1 -50 μg/ml of PK generally is sufficient to remove PrP^c protein.

[0001 14] PrP^Sc also can be separated from the PrP^c protein by the use of ligands that specifically bind and precipitate the misfolded form of the protein, including conformational antibodies, certain nucleic acids, plasminogen, PTA and/or various peptide fragments.

[0001 15] The PrP^Sc protein can be then directly detected by Western blot or by immunoassays such as ELISA using anti-PrP antibody, for example the 3F4 monoclonal antibody. If an ELISA assay is used, this assay may be a two-site immunometric sandwich ELISA.

[0001 16] Typical Western blot procedures begin with separating proteins by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The proteins are then electroblotted onto a membrane, such as nitrocellulose or PVDF and probed, under conditions effective to allow immune complex (antigen/antibody) formation, with an anti-PrP protein antibody. Exemplary antibodies for detection of prion protein include the 3F4 monoclonal antibody, the monoclonal antibody D13 (directed against residues 96-106 (PERETZ et al, Nature, vol.412, p: 739-743, 2001), the polyclonal antibodies Rl 8 (directed against residues 142-154), and R20 (directed against C-terminal residues 218-232) (CAUGHEY et al, J Virol, vol.65, p:6597-6603, 1991). Following complex formation, the membrane is washed to remove non-complexed material. An exemplary washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. The immunoreactive bands are visualized by a variety of assays known to those in the art. For example, the enhanced chemoluminesence assay (AMERSHAM) can be used. If desired, PrP^Sc protein concentration can be estimated by Western blot followed by densitometric analysis, and comparison to Western blots of samples for which the concentration of PrP protein is known. For example, this can be accomplished by scanning data into a computer followed by analysis with quantitation software. To obtain a reliable and robust quantification, several different dilutions of the sample generally are analyzed in the same gel.

[0001 17] As described above, immunoassays in their most simple and direct sense are binding assays. Specific non-limiting immunoassays of use include various types of enzyme linked immunosorbent assays (ELISAs), immunochromatographic strip assays, radioimmunoassays (RIA), and specifically conformation-dependent immunoassays. In one exemplary ELISA, anti-PrP antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a reaction mixture suspected of containing prion protein antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound prion protein can be detected. Detection generally is achieved by the addition of another anti-PrP antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection also can be achieved by the addition of a second anti- PrP antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label. In another exemplary ELISA, the reaction mixture suspected of containing the prion protein antigen is immobilized onto the well surface and then contacted with the anti-PrP antibodies. After binding and washing to remove non-specifically bound immune complexes, the bound anti-prion antibodies are detected. Where the initial anti-PrP antibodies are linked to a detectable label, the immune complexes can be detected directly. Again, the immune complexes can be detected using a second antibody that has binding affinity for the first anti-PrP antibody, with the second antibody being linked to a detectable label. Another ELISA in which protein of the reaction mix is immobilized involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against PrP protein are added to the wells, allowed to bind, and detected by means of their label. The amount of PrP^Sc protein antigen in a given reaction mix is then determined by mixing it with the labeled antibodies against PrP before or during incubation with coated wells. The presence of PrP^Sc protein in the sample acts to reduce the amount of antibody against prion available for binding to the well and thus reduces the ultimate signal. Thus, the amount of PrP^Sc in the sample can be quantified. Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below. In coating a plate with either antigen or antibody, one generally incubates the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antibodies. These include bovine serum albumin, casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, and thus reduces the background caused by nonspecific binding of antibodies onto the surface.

[0001 18] It is customary to use a secondary or tertiary detection means rather than a direct procedure with ELISAs, though this is not always the case. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand. "Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin, milk proteins, and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. "Suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about lto 2 to 4 hours, at temperatures preferably on the order of 25° C to 27° C, or can be overnight at about 4° C or so. Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. An exemplary washing procedure includes washing with a solution such as PBS/Tween or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined. To provide a detecting means, the second or third antibody generally will have an associated label to allow detection. In some examples, this is an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, the first or second immune complex is contacted and incubated with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (for instance, incubation for two hours at room temperature in a PBS-containing solution such as PBS-Tween). After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, for instance, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid) and H₂0₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, for instance, using a visible spectra spectrophotometer.

[0001 19] In certain embodiments, the caprinae PrP^c protein isoform, derivative or fragment thereof can be labeled to enable high sensitivity of direct detection of protein that is converted into PrP^Sc. For example, the caprinae PrP^c protein isoform, derivative or fragment thereof can be radioactively labeled, epitope tagged, or fluorescently labeled. The label can be detected directly or indirectly. Radioactive labels include, but are not limited to ^,251, ³²P, ³³P, and ³⁵S.

[000120] In still another embodiment, the amplified PrP^Sc protein may be also directly detected by animal bioassays, wherein test animal are inoculated with the reaction mixture and assessed for clinical symptoms.

[000121] Animals (Syrian Golden hamsters) 4- to 6-weeks old are anesthetized and injected stereotaxically in the right hippocampus with about 1 μΐ of the reaction mixture. This may be accomplished using a computerized perfusion machine that delivers the sample into the brain at a given rate, for example 0.1 μΐ/min. The onset of clinical disease is measured by scoring the animals twice a week using the following scale:

[000122] 1. Normal animal;

[000123] 2. Mild behavioral abnormalities including hyperactivity and hypersensitivity to noise;

[000124] 3. Moderate behavioral problems including tremor of the head, ataxia, wobbling gait, head bobbing, irritability and aggressiveness; [000125] 4. Severe behavioral abnormalities including all of the above plus jerks of the head and body and spontaneous backrolls;

[000126] 5. Terminal stage of the disease in which the animal lies in the cage and is no longer able to stand up.

[000127] Animals scoring level 4 during two consecutive weeks are considered sick and are sacrificed. Sacrifice may be by exposition to carbon dioxide to avoid excessive pain. Brains and other tissues are extracted and analyzed histologically by methods that are well known in the art. For instance one hemisphere is fixed in 10% formaldehyde solution, cut in sections and embedded in paraffin. Serial sections (~6 μπι thick) from each block are stained with hematoxylin-eosin, using standard protocols or incubated with antibodies recognizing PrP, in some cases incubation with an antibody to the glial fibrillary acidic protein may be used as a control. Immunoreactions are developed, for example using the peroxidase-antiperoxidase methods. In this case antibody specificity is verified by absorption. In some cases biochemical examination for PrP^Sc using Western blot analysis may also be used, in some case both histologic and biochemical analyses may be undertaken, by using one brain hemisphere for each.

[000128] Amplified PrP^Sc protein may be also be detected by functional assays, such as by their ability to infect certain mammalian cells in culture (cellular infectivity assay; KLOHN et al., Proc. Natl. Acad. Sci. USA, vol.100 (20), p: l 1666-1 1671 , 2003). In this assay, susceptible mouse neuroblastoma N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split three times. The proportion of PrP^Sc-containing cells is determined with automated counting equipment. In certain applications the number of prion containing cells may also be determined by flow cytometry. The dose-response to infection is linear over two logs of prion concentrations. The cell assay was claimed to be as sensitive as the mouse bioassay, 10 times faster, 2 orders of magnitude less expensive and suitable for automation by use of robots.

[000129] Finally, amplified PrP^Sc protein may be also detected by indirect methods such as spectroscopic assays, including multispectral ultraviolet fluoroscopy, confocal dual-color fluorescence correlation spectroscopy, Fourier-transformed infrared spectroscopy or capillary electrophoresis, and Fluorescence Resonance Energy Transfer (FRET).

[000130] As an example, one class of dyes which have been developed to give large and different Stokes shifts, based on the Fluorescence Resonance Energy Transfer (FRET) mechanism and used in the simultaneous detection of differently labeled samples in a mixture, are the ET (Energy Transfer) dyes. These ET dyes include a complex molecular structure consisting of a donor fluorophore and an acceptor fluorophore as well as a labeling function to allow their conjugation to biomolecules of interests. Upon excitation of the donor fluorophore, the energy absorbed by the donor is transferred by the FRET mechanism to the acceptor fluorophore and causes it to fluoresce. Different acceptors can be used with a single donor to form a set of ET dyes so that when the set is excited at one single donor frequency, various emissions can be observed depending on the choice of the acceptors. Upon quantification of these different emissions, changes in the folding of a labeled protein may be rapidly determined. Some exemplary dyes that may be used comprise BODIPY FL, fluorescein, tetmethylrhodamine, IAEDANS, EDANS or DABCYL. Other dyes have also been used for FRET for examples dyes disclosed in U.S. Patents 5,688,648, 6, 150, 107, 6,008,373 and 5,863,727 and in PCT publications WO 00/13026, and WO 01/19841.

[000131] In still another preferred embodiment, the method of the invention further comprises a step f of inactivating residual PrP^Sc protein.

[000132] Said residual PrP^Sc protein may be inactivated by various methods known to those in the art, such as treatment with a concentrated base or treatment at high temperature, for example, treatment with 2N NaOH for 1 hour and/or autoclaving at 134°C for 18 min. This would eliminate the danger of prion as biohazardous waste and also help to minimize contamination that could occur when testing multiple samples.

[000133] In a preferred embodiment, the method of the invention is using no-powdered gloves. In fact, the inventors established that the talc or the corn starch inhibit the PrP^Sc protein amplification.

[000134] In a second aspect the present invention relates to a kit for the method described previously of diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae , preferably not an ovine or a caprine, and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD) wherein said kit comprises a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 , derivative or fragment thereof.

[000135] In one embodiment, the kit is made up of instructions for carrying out the method described herein for diagnosing a TSE in a subject. The instructions can be provided in any intelligible form through a tangible medium, such as printed on paper, computer readable media, or the like.

[000136] Kits for implementing methods of the invention described herein are specifically contemplated. In some embodiments, there are kits for amplification and detection of PrP^Sc in a biological sample. In these embodiments, a kit can comprise, in suitable container means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more of the following: 1) a conversion buffer; 2) PrP^c protein; 3) decontamination solution; 4) a positive control, PrP^Sc containing sample; 5) a negative control sample, not containing PrP^Sc; or 6) reagents for detection of PrP^Sc.

[000137] The kit may further comprise reagents for expressing or purifying the caprinae PrP^c protein isoform, derivative or fragment thereof. The kit may also comprise reagents that may be used to label the said protein isoform, with for example, radio isotopes or fluorophors.

[000138] Reagents for the detection of PrP^Sc can comprise one or more of the following: pre coated microtiter plates for ELISA and/or CDI detection of prion; tissue culture cells in which PrP^Sc can replicate; or antibodies for use in ELISA, CDI or Western blot detection methods.

[000139] Additionally, kits of the invention may contain one or more of the following: protease free water; copper salts for inhibiting PrP^Sc replication; EDTA solutions for enhancing PrP^Sc replication; Proteinase K for the separation of PrP^Sc from PrP^c protein; fractionation buffers for the separation of PrP^Sc from PrP^c, modified, or labeled proteins (increase sensitivity of detection); or conversion factors (enhance efficiency of amplification). In certain embodiments the conversion buffer may be supplied in a "ready for amplification format" where it is allocated in a microtiter plate such that the sample and PrP^c protein isoform may be added to first well, and subjected to amplification.

[000140] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, plate, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing proteins, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[000141] When components of the kit are provided in one and/or more liquid solutions, the liquid solution is typically an aqueous solution that is sterile and proteinase free. In some cases proteinatious compositions may be lyophilized to prevent degradation and/or the kit or components thereof may be stored at a low temperature (i.e. less than about 4°C). When reagents and/or components are provided as a dry powder and/or tablets, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. [000142] In the following, the invention is described in more detail with reference to amino acid sequences, nucleic acid sequences and the examples. Yet, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.

EXAMPLES

[000143] Material & Methods

[000144] 1) Transgenic mice expressing sheep isoforms of PrP), human PrP and bovine PrP

[000145] The transgenic mice expressing sheep isoforms were established following the construction method described for the transgenic mice expressing the VRQ sheep isoform in VILOTTE et al. (J. Virol, vol.75(13), p:5977-5984, 2001 ).

[000146] The transgenic mice expressing bovine PrP isoform were established following the method disclosed in CASTILLA et al. {Arch. Virol, vol.148, p:677-691 , 2003).

[000147] The transgenic mice expressing human PrP (Meti₂₉) and murine PrP isoform were established following the method disclosed in PADILLA et al. (PloS Pathog., vol.7 (3), p :el001319, 201 1).

[000148] 2) Brain homogenates

[000149] 6 to 10 week old Mice are euthanized and perfused (intra-cardiac) with PBS pH 7.4 /EDTA 5mMol (40-60 ml per mice). The brains were then harvested and snap frozen in liquid nitrogen. [000150] 10% brain homogenate is prepared by low speed homogenization (e.g. using disposable ULTRATURAX - 3 MIN) of brain tissue in 4°C PBS pH 7-7.65 + 0.1% Triton X100+ 150 mM NaCl (10% Weight/vol) , (ii) lying on ice for 15 to 45 min, (iii) filtration through a 50μιη mesh disposable cell strainer or low speed centrifugation 100 to 500 g for 1 min. The obtained homogenate is then aliquoted and snap frozen at -80°C before use. This material constitutes the substrate for PrPSc amplification.

[000151] 3) Sheep oral inoculation with BSE

[000152] TSE free sheep were produced in the DEFRA 'TSE free flock' which is a unique source able to provide animals that can be considered free from classical scrapie (SIMMONS et al , BMC Vet. Res., vol.5, p:8, 2009) .The animals included in our experiments were imported into France and housed, in a dedicated scrapie free facilities before their use in experiments. In all cases, PrP genotype was obtained by sequencing the Exon 3 of the Prnp gene.

[000153] Four ARQ/ARQ sheep (6-10 month old) were orally challenged with 5g equivalent of brain material (1% brain homogenate in glucose). Animals were then observed until the occurrence of clinical signs and euthanased when exhibiting locomotor signs of the disease that impaired their feeding capacities. White blood cells (WBC) from age and breed match un-inoculated TSE free ARQ/ARQ (n=60) were obtained by osmotic lysis of buffy coat (one volume) with AC solution (one volume) (NH₄C1 0, 15 M, HCO₃ 1 mM, Na₂EDTA 0, 1 mM, pH 7.4) for 5 min RT. WBC were washed 3 times with 50 mL of PBS before being pelleted and stored at -80°C

[000154] 4) Control and vCJD infected primates [000155] Captive-bred 2.5 years-old male cynomolgus macaques {Macaca fascicularis) were provided by NOVEPRIM (Mauritius). Primates were checked for the absence of common primate pathogens before importation, and handled in accordance to national guidelines. Animals were housed in level-3 animal care facilities. One animal (Macaque 6) was transfused with 40 mL of blood from a vCJD-infected primate sampled at the terminal stage of the disease. The other primates were intravenously inoculated with clarified supernatants (obtained by centrifugation at 1 ,500 g for 10 minutes after extensive sonication) derived from 10 or 100 mg of brains from BSE- or vCJD-infected primates. Primate blood samples were drawn into sodium citrate and fractionated by centrifugation at 2,000 g for 13 minutes according to the techniques classically applied in human transfusion. WBCs were obtained by osmotic lysis of buffy coat (one volume) with EASY- LYSE (DAKO, 9 volumes) for 10 minutes RT. WBCs were washed three times with 50 mL of PBS. All samples were encoded before dispatch and tested blind. Animals were handled under anesthesia to limit stress, and euthanasia was performed for ethical reasons when animals lost autonomy

[000156] 5) Blood sample preparation

[000157] Blood is collected on anticoagulant (like Citrate sodium/EDTA/Heparinate). 5 ml whole blood is centrifuged at 3600 rpm 10 min room temperature. Plasma is removed and buffy coat is collected using a disposable hard bulb pipette. Buffy coat are then mixed volume/volume with ACK solution (NH4CL 0, 15 M, KHC03 I mM, Na2EDTA 0, 1 mM, pH 7.4) for 5 min RT or with other red cell lysis solution . The obtained white blood cells are washed twice with PBS. Cell numeration performed using Mallasez's cell or automatic cell counter. Desired amount of cells (Typically 10⁷ cells) are then resuspended in 200μί of 4°C PBS pH 7.4 + 150mMoL NaCl+ 0.1 TRITON®X100 and homogenized at high speed (For example using Precess 48). Samples are then spin down 15000g -20 sec. Sample can be stored at -80°C or used freshly.

Γ0001581 6) PrP⁵' amplification usins PMC A

[000159] Desired amount of WBC or Buffy coat were re suspended in 4°C PBS pH 7.4 + 150mMoL NaCl+ 0.1 TRITON XI 00 and homogenized at high speed. Samples were then spin down 15000g for 20 seconds and then stored at -80°C or used freshly. 7μ1 of the blood sample are mixed with 63μί of substrate in 0.2mL ultrathin wall PCR tube or 96 well PGR plate. In each tube or well 5 to 15 silica microbeads (1.00 mm diameter) were added beforehand. The tubes are then sealed hermetically with appropriate caps or film. Tubes- Plate are placed on MISONIX 4000 microplate horn containing 200-240 mL of deionized water (water level in the horn is at the same level than the sample level in the reaction tubes). The horn is then sealed with hood to avoid evaporation. The horn is at 39.5°C in a way ensuring that after energy burst the temperature in the horn returns to nominal level within 20-40 seconds. The samples are then submitted to 96 cycles of 30 sec sonication at potency 70% - followed by 29 min 30 incubation period. After 96 cycles, 7μΙ, of the amplicon are collected and mixed with fresh substrate and new amplification round (96 cycles) can be performed. The amplification is typically stopped after 3 to 6 rounds and PrPSc detection is carried out on amplicons.

[000160] 7) Abnormal PrP Western-blot (WB) [000161] PK resistant abnormal PrP extraction (PrP^res) and Western blot were performed as previously described in ANDREOLETTI et al. (PlosS Pathog., vol.7, el001285, 2011), using a commercial extraction kit (BIORAD). For PMCA products the equivalent of 20μί, of reactions were loaded on each lane. PrP immunodetection was performed using either Sha31 monoclonal antibody (0,06 μg per mL, epitope: YEDRYYRE (SEQ id n°4), amino acid 145-152)

[000162] Results

[000163] 1) Detection of PrP^Sc protein in human brain sample with high sensitivity

[000164] Amplification of human CJD was tested by PCMA using WT goat (source Transgenic mice expressing goat WT PrP^c: "TgGo WT substrate"), ovine AHQ (source Transgenic mice over-expressing AHQ ovine PrP^c: "Tg AHQ substrate"), or ovine ARQ

C C

PrP (source Transgenic mice over-expressing ARQ ovine PrP : "Tg ARQ substrate") as substrates.

[000165] The Figure 1 shows v-CJD amplification (dilution series of human brain in PBS) using caprinae WT or ovine ARQ or AHQ PrP^c as substrate. T+: positive control, 0: negative Human brain, 10^"3 to 10 ^s v-CJD brain dilution from 10^"3 to 10^"8 in PBS. Amplification was performed under the same run.

[000166] The results demonstrate the capacity of caprinae PrP to amplify human v-CJD prion.

[000167] 2) Detection of PrP^Sc protein using 0171 caprinae PrP^c

[000168] The amplification capacity of Q 171 caprinae PrP^c (Tg over-expressing mice) as substrate as compared to PrP^c from other species was testedFor that purpose, brain material from different transgenic mouse lines expressing ovine (Ai3₆Ri₅₄Qi₇i and Vi₃₆Ri₅₄Qi₇i variants), bovine, human (Meti₂9 variant) and murine PrP^c were used to prepare substrates. Reactions were then seeded with ten-fold dilution series of brain homogenate from vCJD/BSE-affected human, primate, porcine, bovine and sheep. Unseeded reactions were included as specificity control. PMCA reactions were then submitted to 2 to 6 amplification rounds each constituted with 96 cycles (30s sonication-30 minutes incubation at 39.5°C) in a M1SONIX 4000 sonicator. After each round, (i) reaction products (1 volume) were mixed with fresh substrate (9 volumes) to seed the following round while (ii) a part of the same product was analysed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE, SEQ id n°4).

[000169] The figure 2 shows the vCJD/BSE agent amplification by PMCA using brain from transgenic mice expressing different species PrP sequence as substrate. PMCA reactions were seeded with ten-fold dilution series of vCJD/BSE brain material ( 10^" to 10^"9) from different species (human, cynomologus macaque, bovine, sheep and porcine). PMCA substrates were prepared using brain from transgenic mice over-expressing either human (methionine 129 variant-□), bovine (V), murine (o) or sheep (VRQ variant:A,

ARQ variant: A) Prion protein.

[000170] The results show that all the tested substrates allowed the amplification of vCJD/BSE, but displayed dramatically different detection limits. Whatever the origin (species) of the BSE/vCJD agent, the ovine PrP substrates (ARQ and VRQ) provided surprisingly the best detection performances, i.e. positive for reactions seeded with a 10^"6 to 10^"8 dilution of the original brain homogenates (figure 2). In opposition with the common knowledge, all these results support the view that the homology of PrP amino-acid sequence between the seed and the substrate is not a crucial determinant for vCJD/BSE agent PMCA amplification. Strikingly, the capacity of human PrP substrate to amplify the vCJD/BSE agent varied greatly according the infectious source species. Human vCJD, porcine BSE and sheep BSE prions were amplified using human PrP as a substrate but in contrast vCJD/BSE in cattle and primates were barely or not amplified

[000171] The results also show that, after six PMCA rounds, no P resistant abnormal PrP (PrP^res) could be detected by Western blot (WB) in un-seeded reactions or in those seeded with healthy brain material (figure 2). The results have further shown that no PrP^res was detected in reactions seeded with brain material from Alzheimer affected patients (data not shown). For all vCJD/BSE agent source /substrate combination, the results have shown that the PrP^res WB pattern (Glycoprofile and mobility) observed after PMCA amplification was indistinguishable from that observed in the brains of the transgenic mouse line used to prepare the PMCA substrate (data not shown). These results suggest that whatever the substrate, bona fide vCJD/BSE prions were amplified.

[000172] Finally, the results have demonstrated the surprising greater capacity of Q171 caprinae PrP^c to amplify heterologous vCJD/BSE agents.

[000173] 3) Detection of PrP^Sc protein in sheep blood sample with high sensitivity and specificity

[000174] In order to establish the capacity of the assay to detect endogenous vCJD/BSE agent in the blood, white blood cells (WBCs) from 4 sheep orally challenged with BSE and 60 healthy control sheep were tested using the ovine ARQ substrate. [000175] In that experiment, the BSE infected sheep had developed disease 20 months post inoculation (mpi). Blood was collected in these animals at 0, 2, 6, 12, 16 and 20 (clinical onset) months post inoculation (mpi). White blood cells (WBC) were obtained by osmotic lysis of the red blood cells. WBCs were then homogenized in PMCA buffer and homogenates were used to seed PMCA reactions in which the brain of transgenic mice that expressed the ARQ variant of the ovine PrP was used as substrate. Each sample was used to seed 4 independent reactions (two different runs onto 2 different sonicators). Five successive PMCA amplification rounds (R) were applied. The resulting PMCA products were analysed by Western Blot (WB) for the presence of abnormal P resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE, SEQ id n°4). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control).

[000176] The table I shows the PrP^res detection in Protein Misfolding Cyclic Amplification (PMCA) reactions seeded with white blood cells from ARQ/ARQ sheep orally inoculated with BSE agent, collected at different time points of the incubation period. After each amplification round, the number of PrP^res positive replicates (as assessed by Western Blot) is indicated in the table. (-) indicated that all 4 replicates were negative.

Table I

[000177] The Figure 3 A shows the Western Blot of PMCA products (3 rounds) from WBC prepared from BSE orally challenged sheep at different time points (indicated as month post inoculation: mpi). [000178] The figure 3B shows the Western Blot of PMCA products (6 rounds) from WBC from BSE affected sheep (3 different individuals) and from healthy controls.

[000179] The results show that for all the symptomatic sheep, reactions seeded with WBC were positive after two PMCA rounds with a typical BSE PrP^res WB pattern. After four rounds, reactions seeded with WBCs collected at 6 mpi in some animals and at 12, 16 and 20 mpi in all animals were positive (table 1 and figure 3A). The WBC from the 60 TSE- free controls remained negative after 6 PMCA rounds (figure 3B).

[000180] The results thus show that the use of Q171 caprinae PrP^c enable to detect the vCJD/BSE agent in a blood sample with high sensitivity and specificity.

[000181] 4) Detection of PrP^Sc protein in primate blood sample with high sensitivity and specificity

[000182] The promising results obtained in sheep enabled us to test blood samples collected in vCJD-infected primates experiments considered to be the closest to infection in humans.

[000183] Eight cynomologus macaques were intravenously challenged with vCJD brain homogenate or with a blood from a vCJD affected macaque for the macaque 6. At different time points of the incubation period, blood was collected and buffy coat prepared. Buffy coat (BC-n=33) and WBC (n=14) obtained by red cell lysis of BC from the vCJD-infected (n=8) and control (n=15) cynomologus macaques were tested. The WBC or BC samples were used (as homogenates 1/100 diluted in PMCA buffer except Table 2, wherein dilution 1/10, 1/50 or 1 /100 were used) to seed PMCA reactions in which brain homogenate from ovine PrP transgenic mouse (ARQ variant) was used as substrate. Each sample was submitted to successive rounds of amplification (i.e. up to 6) each constituted with 96 cycles (30s sonication-30 minutes incubation at 39.5°C) in a MISONIX 4000 sonicator. PMCA products were analysed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYY E, SEQ id n°4). On each gel a classical scrapie isolate (PK digested) was used as positive controls (WB control). Samples were received encoded and tested blind.

[000184] The figure 4 indicates for each macaque, the clinical onset (din) and time to euthanasia of the animals are indicated (upper label on arrows) as months post inoculation (mpi). The time point corresponding to blood samples (months post inoculation) that were tested and the results of PrP^res WB detection in PMCA reactions are indicated (under arrow). No positive WB result was observed before the third PMCA round. No additional positive result was observed after 5 PMCA rounds

[000185]

[000186] The figure 5 A shows the Western Blot of buffy coat BC samples (collected between 2005 and 2010) at different time points of the incubation period (indicated as mpi) in a vCJD inoculated primate (intravenous route, macaque 6 in figure 4). The animal developed clinical signs at 46 mpi and was euthanized at 58 mpi.

[000187] The figure 5 B shows the Western Blot of buffy coat from four different unchallenged cynomolgus macaques (cont) and 5 different vCJD affected primates (see figure 4) [000188] The figure 5 C shows the Western Blot of White blood cells and of Buffy Coat prepared from the same blood sample collected in a clinically affected primate (macaque 6, 38 mpi). WBC and BC (both 1/50 diluted) were used to seed PMCA reactions.

[000189] The table II shows the PrP^res detection results in PMCA reactions seeded with White blood cells (WBC) or Buffy coat (BC) from Cynomologus macaques intravenously challenged with (i) blood from a vCJD affected macaque (macaque 6) or with (ii) human vCJD brain homogenate (macaque 8). WBC and BC were homogenized and then diluted 1/10, 1/50 and 1/100 in PMCA buffer before seeding PMCA reactions for 5 successive rounds (R) of amplification.

Table II

[000190] The results show that after 4 PMCA rounds, blood from all the clinically affected primates was positive (figure 4, 5 A). All the reactions seeded with BC or WBC (n= 17) from unchallenged primates remained negative after 6 PMCA rounds (figure 5A). The results also show that, in four vCJD-infected primates (macaques 2, 4, 6 and 7), the reactions seeded with BC collected from 10 mpi to 14 mpi (three years before clinical onset) were positive after five PMCA rounds (figures 4 and 5B). These data indicate that vCJD infection can be detected in the early preclinical stage in primates

[000191] The comparison of PMCA reactions seeded with BC and WBC prepared from the same blood samples indicated the presence of amplification inhibitor(s) in the BC (figure 5C and table II). To limit inhibition, BC had to be diluted at least fifty-fold before being processed (table II).

[000192] 5) Detection of PrP^Sc protein in human blood sample with high sensitivity and specificity

[000193] These results allowed access to WBC from a French vCJD affected patient, which was tested by PCMA using brain homogenate from ovine ARQ PrP^c transgenic mouse as substrate. As a specificity control, the same amplification was also realized on White blood cells from 135 healthy humans. All samples were processed in the same round. PMCA products were analysed by Western Blot (WB) for the presence of abnormal P resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE, SEQ id n°4). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control).

[000194] The figure 6 A shows the Western Blot of PMCA reaction unseeded (no seed), or seeded with WBC from the vCJD affected patient (vCJD-WBC) or with WBC from 6 different (among the 135 tested) healthy human control (H-WBC). [000195] The results show that, after 6 PMC A rounds, no PrP^res was detected in reactions seeded with WBC from human healthy controls (n=135) (figure 6A). In contrast, two PMCA rounds were sufficient to detect PrP^res in reactions seeded with vCJD affected patient's WBC. To model the capacity of this assay to detect lower amount of blood vCJD agent (as expected in patients at preclinical stage) a ten-fold dilution series of WBC from the same vCJD patient was made.

[000196] The figure 7 shows the results of a two rounds amplification in White blood cells prepared from one v-CJD affected patients (BC+) and a dilution series of v-CJD brain material.. 1 : WB positive control, 2: negative Human brain, 3 to 6 v-CJD brain dilution from 10^"6 to 10^"9 in PBS, 8 and 9 empty. 10: v CJD WBC cells equivalent to 30μΙ, of whole blood

[000197] The results illustrate the good sensitivity and specificity of the methodology.

[000198] 6) High detection sensitivity of PrP^Sc protein in human blood

[000199] The detection limit of PrP^Sc protein in human blood using ovine Q171 PrP^c as substrate was tested on a dilution series of White blood cells (From 1/10 to 1/10 000) from a v-CJD affected patient.

[000200] The figure 6 B shows the Western Blot of ten-fold dilution series of WBC homogenate from the vCJD affected patient (Hu vCJD WBC), which were submitted to three PMCA amplification rounds. The equivalent whole blood amount used to seed the reactions is indicated in the figure. Each dilution was tested in duplicate. WBCs from healthy patients (H-WBC) were used as amplification specificity control After three amplification rounds, PrP^res could be detected in one out two replicates seeded with WBC material equivalent to 0.05 μΐ, of starting whole blood.

[000201 ] The results illustrate the very high sensitivity of the methodology. Starting material corresponded to less than ImL of blood meaning that WBC in the last positive dilution corresponds to less than 1 μΐ, of starting blood.

[000202] In one other experiment, WBC from the vCJD patient were mixed with WBC from either eleven (8 different pools), twenty-three (4 different pools), forty-seven (2 different pools) or ninety-five (1 pool) healthy donors and subjected to 3 rounds of PMCA reactions. The PMCA products were then tested as previously. The WBC homogenates used to prepare pools were equivalent to 50μί of starting whole blood. Reactions seeded with WBC from healthy controls (H-WBC) were included as controls

[000203] The figure 6 C shows the Western Blot obtained of WBC homogenate from the vCJD affected patient (Hu vCJD WBC) alone, or after pooling with WBC from 1 1 (pi 2), 23 (p24), 47 (p48) or 95 (p96) healthy controls.

[000204] The results show that after three PMCA rounds, reactions seeded using pool constituted with up to forty-seven healthy donors plus the v-CJD affected patient's WBC were PrP^res positive. All the reactions seeded with pools containing healthy donors were negative.

[000205] Finally, the method of the invention was very sensitive..

Claims

A method for diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), and wherein said method comprises the steps of:

a) mixing a biological sample from said subject with a caprinae PrP protein isoform having a glutamine amino acid residue at its position 171, a derivative or , a fragment thereof, to make a reaction mixture;

b) incubating said reaction mixture in conditions allowing the amplification of the PrP^Sc protein;

c) disrupting the reaction mixture;

d) repeating steps (b) and (c) one or more times; and

e) detecting the PrP^Sc protein in the amplified reaction mixture.

The method of claim 1 , wherein the caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 is an ovine or a caprine protein isoform having a glutamine amino acid residue at its position 171 .

The method of claim 1 , wherein the caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 is selected in the group comprising the ARQ Ovis aries PrP^c protein isoform having the sequence SEQ id n° l , the VRQ Ovis aries PrP^c protein isoform having the sequence SEQ id n°2 and the AHQ Ovis aries PrP^c protein isoform having the sequence SEQ id n°3; preferably said protein isoform is the ARQ Ovis aries PrP^c protein isoform having the sequence SEQ id n° l .

4. The method of any one of claims 1 to 3, wherein the biological sample is a blood sample or any product derived thereof, preferably said biological sample is a buffy coat sample or a White blood cells sample.

5. The method of any one of claims 1 to 4, wherein the caprinae PrP^c protein isoform, derivative or fragment thereof is from recombinant origin (including bacteria, yeast or cell) or a cell lysate, preferably from cells overexpressing said protein isoform or fragments thereof, and most preferably said cell lysate is a brain homogenate.

6. The method of any one of claims 1 to 5, wherein the step b) of incubating the reaction mixture in conditions allowing the amplification of the PrP^Sc protein present in the biological sample is done by maintaining the reaction mixture at a temperature comprised between 36 and 45°C, preferably between 38 and 41 °C, and most preferably between 39 and 40°C.

7. The method of any one of claims 1 to 6, wherein the step c) of disrupting the reaction mixture is done by sonication.

8. The method of claim 7, wherein the sonication corresponds to one or a serial of pulses of 5 to 60 seconds, preferably 10 to 50 seconds.

9. The method of any one of claims 1 to 8, wherein for the step d), the steps b) and c) are repeated at least 10 times, preferably at least 50 times and most preferably at least 90 times.

10. The method of any one of claims 1 to 9, wherein the total duration of the steps a) to d) is of three days or less, preferably of two day or less, and most preferably of one day or less.

1 1. The method of any one of claims 1 to 10, wherein the step e) of detecting the PrP^Sc protein in the amplified reaction mixture is done by a method selected in the group comprising Western Blot, Immunoassays like ELISA, animal bioassays, cellular infectivity assays and spectroscopic assays.

12. The method of any one of claims 1 to 8, wherein said subject is a human, and said TSE is CJD such as sporadic (sCJD), familial (fCJD), iatrogenic (iCJD), and variant form of CJD (vCJD), preferably said CJD is vCJD.

13. The method of any one of claims 1 to 8, wherein said subject is a bovine, and said TSE is BSE.

14. The method of anyone of claims 1 to 13, wherein said method further comprises the step f) of inactivating residual PrP^Sc protein.

15. A kit for diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), wherein said kit comprises a caprinae PrP^c protein isoform having a glutamine amino acid residue at its position 171 , derivative or fragment thereof.