FR2808278A1 - PRION PROTEINS AND THEIR USES - Google Patents

Abstract

The invention concerns isolated human or animal prion proteins PrP<c> or PrP<sc> capable of being fixed to DNA or RNA, in particular viral DNA or RNA and preferably to DNA or RNA of human or animal retrovirus. The invention also concerns the biological uses of said products.

Description

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PRION PROTEINS AND THEIR USES
Prions are unconventional transmissible agents responsible for subacute spongiform encephalitis. The diseases associated with these agents have been called prion diseases, referring to a protein called prion protein (PrP).

Prion diseases are fatal neurodegenerative diseases such as Creutzfeld-Jacob (CJD), Gerstmann-StrausslerScheinker (GSS), fatal familial insomnia (FFI) and Kuru's disease in humans; bovine spongiform encephalitis (BSE) and sheep scrapie in animals.

Prion diseases can be infectious (Kuru disease, iatrogenic Creutzfeldt-Jakob disease (CJD), (BSE)), sporadic (Creutzfeldt-Jakob disease (CJD)) and genetic (familial Creutzfeldt-Jakob (familial CJD), Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI).

- Familial CJD: in this disease, a PrP mutation is linked to a heterozygous familial variant (codon 129 Met / Val). A mixture of wild and mutated form exists (Silvestini MC et al., Nat Med 1997 3: 521).

The phenotypic heterogeneity of Creutzfeldt-Jakob disease (sCJD) is well documented but there is not yet a systematic classification of disease variants (Parchi P et al., Ann Neurol 1999,46: 224).

- GSS is a rare inherited disorder due to different mutations in the human PrP gene which codes for PrP and which manifests itself, among other things, by the mutation of an amino acid P102L (Manson et al., Embo J 1999,18: 6855). The disease is characterized by the accumulation of abundant deposits of an abnormal PrPsc protein in the gray matter of the brain.

- Fatal familial insomnia is another disorder caused by the mutation of codon 178 of the PrP gene (Keohane C, Clin Exp Pathol 1999,47: 125). Codon 129 is especially involved in the susceptibility to this disease. Attempts have been made to correlate neuropathology with the genotype of codon 129 and the type of PrP to establish a molecular classification (Mikol J, BiomedPharmacother 1999,53: 19).

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All of these diseases result in progressive degeneration of the brain leading to death. The main characteristics of prion diseases are: astrocytosis, neuronal loss, inconsistent spongiosis and gliosis, the presence of plaques of amyloid fibrils and different deposits of anti-PrP antibodies.

The human PrP protein is encoded by a single gene located on chromosome 20 and is present in many vertebrates and invertebrates. 32 PrP sequences have been described (Kutznetsov IB et al., Febs Lett 1997,412: 429; Kaluz S et al., Gene 1997,199: 283). PrP is made up of 253 amino acids with N-terminal repeats and two potential glycosylation sites. The mature human protein is a hydrophobic protein consisting of amino acid residues 23-231 and has an apparent molecular weight of 19-33-35 kDa. The three-dimensional structure of the recombinant cellular normal PrP protein (amino acids 23-230) is composed of a globular domain (residues 125-228) and a flexible N-terminal tail; the globular domain contains 3 a-helices and a short anti-parallel p-sheet (Zahn R et al., PNAS 2000, 97: 145). The a-helices have certain properties of hydrophobicity, of charge distribution which make them unique among the alpha helices generally encountered. Despite intense research over the past three decades, no nucleic acid has been found in prions, suggesting that they are agents different from viruses and viroids. The glycoprotein is present in various cells, and is in particular expressed constitutively on the surface of neurons (Sakaguchi S et al., Nature 1996, 380: 528), glial cells, follicular dendritic cells (Brown KL et al., Nat Med 1999.5: 1308). It is attached to the cell membrane by a glycosylphosphatidylinositole anchor sequence. Observations suggest the existence of cell-type specific PrP glycoforms which may determine the cellular susceptibility to infection by the prion agent (Dodelet VC et al., Blood 1998,91: 1556).

Several studies suggest that the PrP protein can adopt two stable conformations, the so-called normal conformation (PrPc) sensitive to proteases which can be recycled and degraded, and a so-called abnormal form.

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séquences primaire et secondaire entre les protéines PrPc et PrPsc (Dormont D, Rev Neurol 1998, 154 : 142 ; Riek R et al., PNAS 1998, 95 : 11667). Par contre, elles auraient des structures tertiaires différentes. La conformation native et normale de PrPc est composée d'un domaine N-terminal pauvrement structuré avec des hélices a en C-terminal tandis que la conformation anormale PrPsc est

composée de feuillets p à la place d'hélices a. La séquence 121-231 de PrPsc contient deux feuillets p antiparallèles et 3 hélices a. Ce domaine contient la plupart des mutations ponctuelles qui ont été liées à l'apparition de maladies familiales à prions (Riek R et al., Nature 1996,382 : 180). Grâce à sa structure en feuillets ss, la protéine PrPsc présente la capacité de former des dépôts d'agrégats fibrillaires, cette structure étant acquise avant la formation de

multimères. Le modèle de p-nucléation propose que la PrPsc s'organise en agrégats avec un core hydrophile qui consiste en un arrangement des composants de l'hélice a de type feuillets P (Morrissey MP et al.,PNAS 1999, 96:11293). D'une façon générale, les protéines qui possèdent dans leur structure des éléments répétitifs peuvent adopter des conformations riches en feuillets ; d'où des mutations protéiques qui induisent ces éléments répétitifs entraînent un changement de structure protéique qui peut aboutir à la formation

d'agrégats (Liu JJ et al., Nature 1999, 400 : 573). Les protéines PrP*0 peuvent s'agréger mais également se fixer à la PrPc. Cette fixation sélective supporte l'idée que PrPc sert de ligand critique et/ou de récepteur pour PrPsc et inversement (Horiuchi M et al., Embo J 1999,18 : 3193). (PrPsc) partially resistant to proteinase K. There is no difference in

primary and secondary sequences between the PrPc and PrPsc proteins (Dormont D, Rev Neurol 1998, 154: 142; Riek R et al., PNAS 1998, 95: 11667). On the other hand, they would have different tertiary structures. The native and normal conformation of PrPc is composed of a poorly structured N-terminal domain with C-terminal a-helices while the anomalous PrPsc conformation is

composed of p-sheets instead of a-helices. PrPsc sequence 121-231 contains two antiparallel p-sheets and 3 a-helices. This domain contains most of the point mutations which have been linked to the development of familial prion diseases (Riek R et al., Nature 1996,382: 180). Thanks to its ss-layered structure, the PrPsc protein has the ability to form deposits of fibrillar aggregates, this structure being acquired before the formation of

multimers. The p-nucleation model proposes that PrPsc organizes into aggregates with a hydrophilic core which consists of an arrangement of the components of the α-helix of the P-sheet type (Morrissey MP et al., PNAS 1999, 96: 11293). In general, proteins which have repetitive elements in their structure can adopt conformations rich in layers; hence protein mutations which induce these repeating elements lead to a change in protein structure which can lead to the formation

aggregates (Liu JJ et al., Nature 1999, 400: 573). PrP * 0 proteins can aggregate but also bind to PrPc. This selective binding supports the idea that PrPc serves as a critical ligand and / or receptor for PrPsc and vice versa (Horiuchi M et al., Embo J 1999,18: 3193).

The abnormal PrPsc protein could be formed from the normal form PrPc, thanks to a trans-conformation process (Liautard JP, J Soc Biol 1999,193: 311; Brockes JP, Curr Opin Neurobiol 1999,9: 571). But what initiates this conversion remains unexplained. There are different factors that influence and / or promote this conversion. The primary and secondary structures of the protein could for example influence the conversion with the presence or absence of mutations and the conservation of the disulfide bridge (C178C213) which stabilizes the conformation of the protein by connecting the helices to C-

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terminals, respectively (Herrmann LM et al., Neuroreport 1998,9: 2457; Chen SG et al., Nat Med 1997, 3: 1009). Any mutation affecting this disulfide bridge causes severe abnormalities in the intracellular routing of the protein (Yanai A. et al., Febbs Lett 1999, 460: 11). The topology and the glycosylation conditions of the protein acquired in the endoplasmic reticulum could also be involved in a phenomenon of neurodegeneration (Ma J et al., Nat Cell Biol 1999,1: 358; Hegde RS et al., Science 1998,279 : 827). The regulation of the topology of PrP in the endoplasmic reticulum takes place through trans factors which can function as Molecular Switch to give proteins different conformations with different functional consequences (Hegde RS et al., Mol Cell 1998 2: 85 ). The trans factors can be lipids and / or enzymes. Sphingolipids and especially sphingomyelin play an essential role in the post-translational mechanisms of PrP which lead to PrPsc; the level of PrPsc seems to be inversely correlated with that of sphingomyelins (Naslavsky N et al., J Biol Chem 1999,274: 20763; Morillas M. et al., J Biol Chem 1999,274: 36859). Enzymes, inter alia, N-acetylglucosaminyltransferase III (GnTIII) show differences in enzymatic activity vis-à-vis the PrPC protein in the endoplasmic reticulum, resulting in the formation of PrPsc. In the cells in which the PrPsc proteins would be produced, the intracellular glycosylation machinery would be disrupted (Rudd PM et al., PNAS 1999,96: 13044).

biologique de PrPc reste inconnue. Il a été décrit que PrP9c possède une neurotoxicité ex vivo pour des neurones et des cellules de la microglie en The normal cellular protein PrP is highly conserved in vertebrates and invertebrates but its physiological and / or pathological role is still unknown. Given its ubiquitous character in the animal kingdom, its pathological role is extremely difficult to characterize and demonstrate. Throughout the bibliography, many functions are discussed, but none is really demonstrated or has any biological relevance in relation to a pathology. We can, for example, hypothesize that the loss of normal function of the protein contributes to the development of a pathology, although this has never been demonstrated. Likewise, the function

Biological results of PrPc remain unknown. PrP9c has been described to possess ex vivo neurotoxicity to neurons and microglia cells by

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culture. The amino acid sequence which has the optimum toxicity corresponds to AGAAAAGA; it is necessary but not sufficient to induce neurotoxicity, and necessary to bind to neurons (Brown DR, Mol Cell neuroxci 2000, 15: 66). This peptide induces neuronal apoptosis through various mechanisms, in particular by increasing the flow of calcium (Brown DR et al., Eur J Neuroxci 1997, 9: 1162; Herms JW et al., Glia 1997, 21: 253).

Since the physiological and pathological functions of the PrPc and PrPsc proteins are unknown, the mechanisms of so-called prion diseases therefore still remain an enigma for the scientific community.

It is now accepted that the diseases associated with PrP result directly from the accumulation of an abnormal form of the protein in nervous tissue and particularly in the brain (Griffith JS Nature 1967, 215:
1043, Prusiner SB Biochemistry 1982,21: 6942). Neurodegeneration requires the deposition of the abnormal isoform of the PrPsc protein. Certain inherited mutations also appear to cause neurodegeneration in the absence of the abnormal isoform, by promoting the synthesis of a transmembrane form of PrP (CtmPrP) whose accumulation kinetics are very closely linked to the course of the disease (Hegde RS et al., Nature 1999, 402: 822). Different groups that produced mice not expressing PrP have provided experimental evidence that indicates that mice never develop the disease after injection of extracts from the brains of patients where PrPsc accumulates and that prior expression is required. and / or the presence of PrPsc in the brain (Borchelt DR et al., Chem Biol 1996,3: 619).

The transmission of prion diseases can be genetic and / or infectious. In infectious transmission, the mechanism of spread in the brain is not clear. The diseases are probably transmitted by mouth or other peripheral routes. It is believed that prions are transmissible (Prusiner SB, PNAS 1998.95: 13363), are present at peripheral sites and can be transported to cells of nervous tissues (neurons and microglia cells) (Aguzzi A et al. ., Cell Mol Life Sci 1997,53: 485) by hematogenous propagation or by neuronal propagation, or axonal transport (Peurin JM et al., Neuroreport 1999 10: 723; Baker CA et al.

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al. J. Virol. 1999 73: 5089; Rodolfo K et al., Neuroreport. 1999, 10: 3639;
Race R et al., J Virol 2000 74: 828; Ma et al. Nat; Med. 1998.4: 1429).

Some authors believe that the disease is transmitted by PrP alone.

They are convinced that the infectious pathogen is abnormal PrP that enters healthy brain and could attach to normal PrP and change its shape or could activate enzymes that alter the structure of PrP. Thus, the PrPsc would be said to be infectious by itself. The brain which does not have PrPc would not be damaged by an exogenous PrPsc (Brandner S et al., Nature 1996, 379: 339). But other researchers believe that this hypothesis is implausible and that the acquisition of resistance to proteases by PrP is not sufficient to explain the spread of the infection (Dormont J. et al., Science
1998; Hill AF et al., J Gen Virol 1999, 80: 11) because there are different strains of probably genetic prions and it has never been demonstrated that proteins could be carriers of genetic information.

Other teams hypothesize that the diseases associated with PrP could require the presence of other factors (Manuelidis L Ann NY Acad Sci 1994,724: 259, Manuelidis L PNAS 1995,92: 5124). These teams working on the subject have evoked the participation of IAP-type retroviral particles in the transmissibility of prion diseases, but on the one hand the use of animal models has helped to implicate contaminations and on the other hand, a role of these elements has never been demonstrated and even less explained
The present inventors have now surprisingly shown that PrP (PrP or PrPsc), of human or animal origin was capable of binding to nucleic acids (DNA, (cDNA or genomic DNA) or RNA (mRNA or tRNA)) , preferably viral or retroviral, but also to itself or to the core protein of a human or animal retrovirus, and that it has all the properties of the proteins of the core of retroviruses, which bind to viral nucleic acids. The proteins of the nucleocapsid of retroviruses are chaperone molecules which perform the essential functions in the formation of viruses, the reverse transcription of the retroviral genome and its stability (Darlix JL et al. J. Mol. Biol., (1995) 254: 523 ).

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More specifically, PrP (PrPc or PrPsc) is capable of binding to RNAs (transfer, structural), in particular to viral or retroviral RNAs and also to single and double-stranded DNAs as well as to cDNAs and at the nucleocapsid.

The inventors have shown in particular that human PrP (huPrP) interacts strongly with RNA and DNA by forming nucleoprotein complexes, which resemble the complexes formed between the NCp7 nucleocapsid protein of HIV-1 and the DNA of HIV-1 ( Darlix JL et al. J. Mol. Biol., (1995) 254: 523; Darlix JL et al. Acad. Sci. III (1993) 316: 763; Braat C et al.

Embo. J. (1989) 8: To examine the biological significance of these PrP - nucleic acid complexes, the inventors compared the consequences of this binding with those of the binding between nucleic acids and NCp7, which is essential for retroviral replication (Darlix JL et al. J Mol Biol 1995, 254: 523).

Like the nucleocapsid protein of HIV-1 (NCp7): - huPrP, by binding to RNAs, facilitates the dimerization of HIV RNA and the hybridization of the primer tRNA at the site of initiation of reverse transcription (PBS ); - huPrP chaperones the minus (-) and plus (+) DNA strand transfers that take place during the synthesis of proviral DNA by reverse transcriptase (RT) and which are essential to generate Long Terminal Repeat (LTR) proviral DNA; - huPrP binds to the NCp7 nucleocapsid of HIV-1.

These results suggest that at least one of the major functions of PrP is to bind to viral nucleic acids and act as a retroviral core, as shown by the inventors, and that one of the natural functions of PrP is linked to nucleic acid metabolism.

In parallel, the inventors have shown the presence of huPrP in highly purified virions of HIV-1, of SIV, of the measles virus, produced in vitro by infected cells or in vivo in tissues. The presence of this protein in the viral particles suggests that the protein is packaged in the particles. The inventors formulated and verified this hypothesis by showing

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peut convertir la PrP' en PrPsc. Les travaux des inventeurs montrent que la PrP, est véhiculée par une particule rétrovirale et apportent une explication nouvelle, cohérente et innovante à la transmissibilité ou transmission des protéines PrP, et donc des maladies à prions. that a PrP, expressed in cells infected with a retrovirus is encapsidated in the new retroviral particles formed by interacting with the nucleocapsid or the retroviral nucleic acid. Thus, the new retroviral particles formed can in turn infect other cells, from the same organism or from a different organism, and release PrP in the newly infected cell, ensuring the transmission of an infection. It is in fact accepted that PrP and, preferably PrPsc, is the agent responsible for prion diseases, when it is present in human or animal tissue and

can convert PrP 'to PrPsc. The work of the inventors shows that PrP is conveyed by a retroviral particle and provides a new, coherent and innovative explanation for the transmissibility or transmission of PrP proteins, and therefore of prion diseases.

The inventors have shown that the overexpression of PrP in various cell lines leads to a strong attenuation of the production of virions and of the retroviral infectivity, in particular of the HIV and SIV retroviruses produced by the cells of said cell lines.

These results suggest at least that another of the major functions of huPrP is to interfere with the replication and infectivity of retroviruses. Thus, PrP can be considered as the body's defense system against any retroviral infection.

Also, the present invention relates to an isolated PrP (PrPc or PrPsc), human or animal, capable of binding to a nucleic acid (DNA or RNA), in particular viral, and preferably retroviral, such as to RNA. or to the RNA of retroviruses HIV-1 (group M, group 0, non-M group not 0), HIV-2, enveloped viruses, such as the measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses.

A subject of the invention is in particular the forms of huPrP of 19 kDa corresponding to huPrP (27-30) and of 10 kDa corresponding to residues 100 to 200. It relates in particular to proteins corresponding to the unstructured N-terminal part of these. proteins, ranging from position 23 to 145 on the sequence given in Figure 8.

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According to another aspect, the invention relates to analogs of NCp7 characterized in that it is an isolated PrP, or its fragments as defined above.

By binding to nucleic acids (viral RNAs), PrP of human or animal origin facilitates the dimerization of the viral RNA and the hybridization of the primer tRNA at the site of initiation of reverse transcription (PBS); directs the transfers of DNA (-) and DNA (+) strands which take place during the synthesis of proviral DNA by reverse transcriptase and which are essential for generating the LTRs of the provirus.

A subject of the invention is therefore also isolated nucleoprotein complexes, comprising a human or animal PrP (PrPc or PrPsc) associated with DNA or with RNA, in particular viral, and preferably with human or animal retroviruses.

The inventors have also shown that PrP (PrP or PrPsc) was capable of binding to a retroviral core protein. A subject of the invention is therefore also a prion protein (PrPc or PrPsc), human or animal, capable of binding to itself or to a core protein of a human or animal retrovirus, in particular to the NCp7 core of the HIV-1 retrovirus and the isolated protein complexes thus formed.

The invention also relates to the use of a PrP (PrPc or PrPsc modified, that is to say modified so that it does not induce a so-called prion disease), or of a fragment of at least one of said proteins as antiviral agent, optionally in interaction with a ligand capable of binding to said PrP or to its fragments to promote its anti-viral activity, in particular a protein or a polypeptide (such as PrP itself or a protein capable of associating with PrP or a fragment of said proteins or even an antibody directed against a virus), a nucleic acid (in particular a viral and preferably retroviral nucleic acid), a chemical molecule or a drug, in particular an agent anti-viral, such as AZT (Azido Deoxythymidine), DDI (DiDeoxylnosine), DDC (DiDeoxyiCytosine), a vector, that is to say a macromolecular complex making it possible to deliver a therapeutically active substance, such as a liposome, a viral vector and all others

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molecules capable of acting as an anti-viral agent; as well as the use of a nucleic acid encoding at least a fragment of at least one of said proteins designated above by the generic term PrP, as an anti-viral agent, as well as the therapeutic and / or prophylactic compositions comprising, inter alia, one of the aforementioned anti-viral agents. In these therapeutic and / or prophylactic compositions, the anti-viral agent, used in a therapeutically effective amount, is optionally with an adjuvant and / or a diluent and / or an excipient and / or a pharmaceutically acceptable vehicle.

The term “pharmaceutically acceptable vehicle” is understood to mean the carriers and vehicles which can be administered to humans or to an animal, as described, for example, in Remington's Pharmaceutical Sciences 16th ed., Mack Publishing Co.

The pharmaceutically acceptable carrier is preferably isotonic, hypotonic or exhibits low hypertonicity and has relatively low ionic strength. The definitions of pharmaceutically acceptable excipients and adjuvants are also given in Remington's Pharmaceutical Sciences supra.

The same is true for the other therapeutic and / or prophylactic compositions according to the invention. In fact, the inventors have shown that the overexpression of a recombinant human PrP, in different cell lines infected with the HIV virus or with the SIV virus, resulted in a decrease in the production of retroviral particles and a strong attenuation of infectivity. viral particles in vitro within these cultures.

The term PrP is understood in the description and the claims to mean any normal or pathological molecule, mutated or not, of physiological conformation or not, belonging to the family of prion proteins, of whatever origin, this definition including proteins which present a percentage of homology or identity with respect to a given reference protein sequence when they have the properties of binding to nucleic acids and to a core protein, as demonstrated in the examples. The definitions of PrP (PrPc and PrPsc) include proteins

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purified natural, recombinant proteins, synthetic polypeptides, or fragments of said proteins insofar as they have the properties mentioned above, these proteins or protein fragments being obtained by conventional techniques well known to those skilled in the art. Included in these fragments are the forms of 19 and 10 kDa mentioned above and the N-terminal domain of the proteins and fragments.

The sequences of said PrP proteins (PrPc and PrPsc) of human or animal origin are listed in FIG. 8, as well as the nucleic sequences encoding said proteins. These proteins included in a therapeutic composition will then be administered in vivo to humans or animals to bind to viral nucleic acids, as described above, and thus reduce the production of infectious viral particles and lower the infectivity of said particles. retrovirals.

The invention therefore relates to a therapeutic and / or prophylactic composition which comprises, inter alia, (i) an expression cassette functional in a cell derived from a prokaryotic or eukaryotic organism allowing the expression of DNA or of the RNA or a fragment of DNA or RNA, encoding all or part of a modified PrPc or PrPsc protein and, optionally allowing the expression of DNA or RNA, or of a fragment of DNA or RNA, encoding all or part of a ligand of said modified PrPc or PrPsc, placed under the control of the elements necessary for its expression; (ii) a vector comprising such an expression cassette or (iii) a cell derived from a prokaryotic or eukaryotic organism comprising an expression cassette or a vector, as defined above.

Numerous expression cassettes for the production of proteins of interest in prokaryotic or eukaryotic cells are described in the literature, in particular in eukaryotic cells. In general, a promoter region is located in the 5 ′ flanking region of the genes and comprises the set of elements necessary for the transcription of the DNA fragment placed under their control. A promoter region may also consist of an assembly of elements of various origins which are functional in the host cell, such as, for example, a minimal promoter region comprising

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the TATA box and the transcription initiation site and the regions located upstream of the TATA box which make it possible to obtain an appropriate level of transcription.

By cell derived from a eukaryotic organism is meant in particular cells derived from yeasts (for example derived from Schizosaccharomyces pombe or Saccharomyces cerevisiae), human or induced stem cells, HeLa cells, 293 cells, COS cells, Vero cells and CHO cells.

The invention also relates to an expression cassette functional in a viral vector functional for transducing cells of a eukaryotic organism allowing the expression of DNA or RNA, or of a fragment of DNA or of RNA. RNA, encoding all or part of a modified PrPc or PrPsc protein, placed under the control of the elements necessary for its expression and allowing the replication of a recombinant viral nucleic acid, in particular retroviral, such as the nucleic acids of HIV viruses -1 group M, group 0, non-M group not 0, HIV-2 virus, SIV, enveloped viruses such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses; a vector comprising said expression cassette; a transformed cell, derived from a prokaryotic or eukaryotic organism, comprising an expression and replication cassette or a vector, as defined above; said cells being chosen in particular from eukaryotic 293 cells, HeLa and HeLa P4 cells, human or animal stem cells; as well as a therapeutic and / or prophylactic composition, comprising, inter alia, such a transformed cell, such a vector or an expression cassette as defined above; as well as their uses as anti-viral agents.

Another object of the invention is a method for detecting and / or quantifying an infection by a virus, in particular a human or animal retrovirus, according to which: (i) a human or animal biological sample is taken, such as 'a biological fluid, a cell or a tissue fragment, (ii) a PrPc or PrPsc is detected in said biological sample, in free form or associated with DNA or RNA, preferably viral and in particular

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retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of HIV-1, (iii) said PrPc or PrPsc of step (ii) is detected, and (iv) if desired, the concentration of said PrPc or PrPsc of step (ii) relative to a reference concentration.

According to this method, said PrPc or PrPsc is detected and optionally quantified in free form or associated with DNA or with RNA, preferably viral and in particular retroviral, or associated with a retroviral nucleocapsid protein and in particular with the NCp7 nucleocapsid of HIV-1, using at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPc or PrPsc in free form or directed against at least a first epitope of said PrPc or PrPsc associated with DNA or RNA, preferably viral or retroviral, or associated with a retroviral core protein, in particular the NCp7 core of the HIV-1 retrovirus. Optionally, a second antibody is used, preferably a monoclonal antibody directed against a second epitope, different from said first epitope, from said PrPc or PrPsc in free or associated form.

Indeed, during a viral or retroviral infection, the expressed or overexpressed PrP can not only bind to viral or retroviral nucleic acids but also to cellular nucleic acids.

The method is preferably a so-called sandwich-type method, using two polyclonal antibodies or two monoclonal antibodies or a polyclonal antibody and a monoclonal antibody, preferably two monoclonal antibodies, one of which is fixed on a solid phase (capture antibody). and the other of which is labeled with any suitable marker (detection antibody). The first and second antibodies as defined above are indifferently a capture or de detection antibody. Furthermore, the term “first and second antibody” also encompasses the antibody fragments and the anticomplex antibodies specific for the protein / core protein complex, for the protein / nucleic acid complex and directed against this complex.

The invention also relates to a kit for the detection and / or quantification of an infection by a virus, in particular a human retrovirus or

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moins un premier épitope de ladite PrPc ou PrP'c sous forme libre ou dirigé contre au moins un premier épitope de ladite PrPc ou PrPsc associée à de l'ADN ou à de l'ARN, de préférence viral, en particulier rétroviral ou associée à une protéine de nucléocapside rétrovirale, en particulier à la nucléocapside NCp7 de HIV-1, et éventuellement un deuxième anticorps, de préférence un anticorps monoclonal dirigé contre un deuxième épitope, différent dudit premier épitope, de ladite PrPc ou PrPsc sous forme libre ou associée à de l'ADN ou à de l'ARN viral, en particulier rétroviral, ou associée à une protéine de nucléocapside rétrovirale, en particulier à la nucléocapside NCp7 de HIV-1 . Les anticorps du kit répondent aux définition données ci-dessus pour le procédé. animal, characterized in that it comprises, inter alia, at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against the

at least one first epitope of said PrPc or PrP'c in free form or directed against at least a first epitope of said PrPc or PrPsc associated with DNA or RNA, preferably viral, in particular retroviral or associated with a retroviral nucleocapsid protein, in particular to the NCp7 nucleocapsid of HIV-1, and optionally a second antibody, preferably a monoclonal antibody directed against a second epitope, different from said first epitope, from said PrPc or PrPsc in free form or associated with viral DNA or RNA, in particular retroviral, or associated with a retroviral core protein, in particular with the NCp7 core of HIV-1. The antibodies in the kit meet the definitions given above for the method.

The production of polyclonal and monoclonal antibodies is part of the general knowledge of those skilled in the art. By way of reference, mention may be made of Kohler G. and Milstein C. (1975): Continuous culture of fused cells secreting antibody of predefined specificity, Nature 256: 495-497 and Galfre G. et al. (1977) Nature, 266: for the production of monoclonal antibodies and Roda A., Bolelli G.F.: Production of high-titer antibody to bile acids, Journal of Steroid Biochemistry, Vol. 13, pp 449-454 (1980) for the production of polyclonal antibodies. Antibodies can also be produced by immunizing mice or rabbits with the HBVm virus particles. For the production of monoclonal antibodies, the immunogen can be coupled to Lymphet Keyhole hemocyanin (KLH peptide) as a carrier for immunization or to serum albumin (SA peptide). Animals are injected with immunogen using Freund's complete adjuvant. The sera and the hybridoma culture supernatants obtained from the immunized animals are analyzed for their specificity and their selectivity using standard techniques, such as, for example, ELISA or Western Blot tests. The hybridomas producing the most specific and sensitive antibodies are selected. Monoclonal antibodies can also be produced in vitro by cell culture of the hybridomas produced or by recovery of ascitic fluid, after intraperitoneal injection of the hybridomas in mice. Whatever the mode of

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production, supernatant or ascites, the antibodies are then purified. The purification methods used are essentially filtration on ion exchange gel and exclusion chromatography or affinity chromatography (protein A or G). A sufficient number of antibodies are screened in functional assays to identify the best performing antibodies. The in vitro production of antibodies, antibody fragments or antibody derivatives, such as chimeric antibodies produced by genetic engineering is well known to those skilled in the art.

More particularly, by antibody fragment is meant the fragments F (ab) 2, Fab ', sFv (Blazar et al., 1997, Journal of Immunology 159: 5821-5833 and Bird et al., 1988, Science 242: d. 'a native antibody and by derivative is meant, inter alia, a chimeric derivative of a native antibody (see for example Arakawa et al., 1996, J. Biochem 120: 657-662 and Chaudray et al., 1989, Nature 339 : 394-397), such as a humanized antibody.

The monoclonal or polyclonal antibody, or fragment of said antibodies, thus obtained is used in a diagnostic method or is incorporated into a diagnostic composition or kit.

The invention also relates to isolated viral particles, in particular particles of a human or animal retrovirus, such as the HIV-1 virus of group M, of group 0, of non-M group or of O, the HIV-2 virus, the SIV virus, the enveloped viruses, such as the measles virus, the Rhabdoviruses, the Filoviruses and the Paramyxoviruses, and comprising a PrPc protein or a PrPsc protein, as defined above.

Another object of the invention is a method for detecting and / or quantifying a prion disease according to which: (i) a human or animal biological sample is taken, such as a biological fluid, a cell or a fragment tissue, (ii) viral particles, in particular retroviral, are isolated or extracted and purified from said biological sample, (iii) said viral particles are lysed so as to obtain a lysate, and (iv) a PrP, of preferably PrPsc, in said lysate, in free form or associated with DNA or viral RNA, in particular retroviral, or associated with

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a retroviral core protein, in particular the NCp7 core of HIV-1, and (v) if desired, said PrP, preferably said PrPsc in free or associated form is quantified; said viral particles corresponding to the definition given above.

According to this method, said PrPsc is detected and optionally quantified in free form or associated with DNA or with viral RNA, in particular retroviral, or associated with a retroviral core protein, in particular with the retroviral nucleocapsid of HIV -1, using at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPsc in free form or directed against at least a first epitope of said PrPc or PrPsc associated with DNA or with viral RNA, in particular retroviral or associated with a retroviral core protein, in particular with the retroviral core of HIV-1 and, optionally, a second antibody is used, preferably a monoclonal antibody directed against a second retroviral epitope, different from said first epitope, from said PrPsc in free or associated form.

Preferably, the method is a so-called sandwich-type method using two antibodies, either polyclonal or monoclonal, or a monoclonal antibody and a polyclonal antibody.

A kit for the detection of a prion disease will comprise, inter alia, at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of a PrPsc in free form or directed against the at least one first epitope of said PrPsc associated with DNA or with RNA, in particular viral and preferably retroviral or associated with a retroviral core protein, in particular with the retroviral nucleocapsid of HIV-1 and optionally a second antibody, preferably a monoclonal antibody directed against a second epitope, different from said first epitope, from said PrPc or PrPsc in free or associated form.

The polyclonal or monoclonal antibodies described above meet the definition given above for the method and the kit of

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detection and / or quantification of a virus infection and include, among others, antibody fragments and anti-complex antibodies.

The method and kit of the invention can be used, inter alia, for the detection of PrPsc in animals whose carcasses are intended to prepare feed for cattle.

In general, the methods of the invention for the detection and / or the quantification of a viral infection or the demonstration of a prion disease use protocols well known to those skilled in the art, such as ELISA tests, Western blots, immunofluorescence or other appropriate technologies.

The invention also relates to a composition for therapeutic and / or prophylactic use, characterized in that it comprises, inter alia, a therapeutic agent capable of preventing the transmission of a PrP protein, preferably PrPsc, by a viral particle, in particular a retroviral particle as defined above, optionally in interaction with an anti-viral agent, such as a chemical molecule, a drug, a ligand capable of binding to said PrP (preferably PrPsc), a nucleic acid, a viral vector, said anti-viral agents corresponding to the definitions given above.

By transmission or transmissibility is meant the transmission from one cell to another cell of the same organism or of different organisms.

Furthermore, the invention relates to a composition for therapeutic and / or prophylactic use which comprises, inter alia, a therapeutic agent capable of preventing the encapsidation of a PrP protein (preferably PrPsc) in a viral particle, in particular a retroviral particle as defined above or a therapeutic agent capable of preventing the binding of a PrP protein, possibly interacting with an antiviral agent, such as a chemical molecule, a drug, a ligand capable of binding said PrP ( preferably PrPsc), a nucleic acid, a viral vector; these meet the definitions given above.

Finally, the invention relates to a composition for therapeutic and / or prophylactic use which comprises, inter alia, a therapeutic agent capable of

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prevent the binding of a PrP protein (preferably PrPsc) to a viral nucleic acid, in particular retroviral, possibly in interaction with an anti-viral agent, such as a chemical molecule, a drug, a ligand capable of bind to said PrP (preferably PrPsc), a nucleic acid, a viral vector which meet the definitions given above. It is in fact presumed that it is by binding to viral nucleic acids or to the nucleocapsid that the protein predominantly enters the viral particles.

The therapeutic agent is, for example, a molecule capable of preventing the encapsidation of PrP and / or its interaction with nucleic acids, preferably viral, and / or nucleoproteins and therefore of inhibiting or reducing the transmissibility of this protein associated with a so-called prion disease; in particular a ligand capable of binding to said PrP, preferably PrPsc, in particular a polyclonal or monoclonal antibody or an antibody fragment and preferably an antibody or monoclonal antibody fragment, or is a nucleic acid encoding an antibody polyclonal or monoclonal or for an antibody fragment and preferably for an antibody or a monoclonal antibody fragment, optionally in combination with one or more anti-viral agent (s). The definition of therapeutic agent also comprises a nucleic acid or a vector encoding a ligand, as defined above or a cell transformed or transduced by said nucleic acid or vector.

The therapeutic agent is also an anti-viral molecule, that is to say a chemical molecule, a drug, a protein, a nucleic acid, an expression vector containing a nucleic acid encoding an anti-viral molecule, a cell transformed or transduced by said vector. By drug or chemical molecule is meant in particular anti-viral agents, such as A.Z.T (Azido Deoxythymidine), D.D.I (DiDeoxylnosine), D. D.C (DiDeoxyiCytosine), as well as any other molecules capable of acting as an anti-viral agent.

The invention also relates to a composition for therapeutic and / or prophylactic use for the treatment and / or prevention of a prion disease which comprises an expression cassette functional in a viral vector functional for transducing cells from an organism. eukaryote allowing the expression of DNA or of a DNA fragment encoding all or

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part of a modified PrPc or PrPsc protein, placed under the control of the elements necessary for its expression and allowing the replication of a viral nucleic acid, in particular recombinant retroviral, such as the nucleic acids of viruses
HIV-1 group M, group 0, non-M group not 0, HIV-2 virus, SIV, enveloped viruses, such as measles virus, Rhabdoviruses,
Filoviruses and Paramyxoviruses or a vector comprising such an expression cassette or a transformed cell, derived from a eukaryotic organism, comprising an expression and replication cassette or a vector, in particular a cell chosen from eukaryotic cells 293, HeLa and HeLa P4 cells, human or animal stem cells.

The invention relates to the use of a therapeutic and / or prophylactic composition as defined above or of a therapeutic agent as defined above, for the treatment and / or prevention of a prion disease.

Such therapeutic and / or prophylactic compositions are used for the treatment or prevention of prion disease. These compositions are for example in injectable form to be administered into a tissue, a fluid or an organism carrying the agent responsible for the disease or to be injected as a preventive measure and the invention relates to their use for the treatment or prevention of disease. 'a prion disease. These compositions may further comprise an adjuvant and / or a diluent and / or a pharmaceutically acceptable excipient and / or a vehicle.

By pharmaceutically acceptable vehicle is meant the carriers and vehicles administrable to humans or to an animal, as described for example in Remington's Pharmaceutical Sciences 16th ed., Mack Publishing Co. The pharmaceutically acceptable vehicle is preferably isotonic, hypotonic or exhibits low hypertonicity and has relatively low ionic strength. The definitions of pharmaceutically acceptable excipients and adjuvants are also given in Remington's Pharmaceutical Sciences supra.

The invention therefore also relates to a method for demonstrating an overexpression of the PrP protein (PrPc or PrPsc) and the reduction of a

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viral infection in vitro, in particular a retroviral infection by a human or animal retrovirus according to which: (i) a cell is transformed with a nucleic acid encoding a retrovirus and a nucleic acid encoding a PrPc or PrPsc of human or animal origin , in particular a plasmid, (ii) a cell culture of said cell is carried out in an appropriate culture medium, the production of virions is detected in the culture supernatant of said cell culture and, (iii) said production of virion is correlated with virion production carried out under the same conditions but in the absence of PrPc or PrPsc. In particular, the cell is chosen in particular from 293 cells, HeLa and HeLa P4 cells, human or animal stem cells, and the retroviral nucleic acid is the nucleic acid of a retrovirus chosen from the HIV-1 viruses of group M, group 0 or group not M, not 0, HIV-2 virus, SIV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses.

The invention also relates to a method for detecting and / or quantifying the expression of a PrPc or PrPsc protein and the expression of at least one viral protein, preferably retroviral and in particular of a retrovirus chosen from among HIV-1 viruses of group M, group 0 or non-M, non-0 group, HIV-2 virus, SIV virus, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as Measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses, according to which at least a first ligand is used, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPc or of said PrPsc, under free form or associated with DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of the HIV-1 retrovirus, and at least a second ligand, in particular an antibody and preferably one year monoclonal antibody directed against at least a first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from HIV-1 viruses of group M, of group 0 or of group not M, not 0, the virus HIV-2, SIV virus, BIV virus, virus

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VISNA, CAEV virus, enveloped viruses, such as measles virus,
Rhabdoviruses, Filoviruses and Paramyxoviruses.

In a particular embodiment, a third ligand is used, in particular an antibody and preferably a monoclonal antibody directed against at least one second epitope, different from the first epitope, from said PrPc or from said PrPsc, in free form or associated with de DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of the HIV-1 retrovirus, and / or at least a fourth ligand, in particular an antibody and preferably a monoclonal antibody directed against at least one second epitope, different from the first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from the HIV-1 viruses of group M, of group 0 or group not M, not 0, HIV-2 virus, SIV virus, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses.

A kit for detecting and / or quantifying the expression of a PrPc or PrPsc protein and the expression of at least one viral protein, preferably retroviral and in particular of a retrovirus chosen from HIV-viruses. 1 of group M, group O or group not M, not 0, HIV-2 virus, SIV virus, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as measles virus , Rhabdoviruses, Filoviruses and Paramyxoviruses, comprises at least a first ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPc or of said PrPsc, in free form or associated with l 'DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of the HIV-1 retrovirus, and at least one second ligand, in particular a antibody and preferably a monoclonal antibody directed against at least a first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from the HIV-1 viruses of group M, of group 0 or of non-M, non-0 group, the HIV-2 virus, the virus SIV, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and

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Paramyxoviruses and in a preferred embodiment also comprises a third ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a second epitope, different from the first epitope, from said PrPc or from said PrPsc, in free form or associated with DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of the HIV-1 retrovirus, and / or at least one fourth ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a second epitope, different from the first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from group HIV-1 viruses M, group O or group not M, not 0, HIV-2 virus, SIV virus, BIV virus, VISNA virus, CAEV virus, BIV virus, VISNA virus, CAEV virus, viruses enveloped, such as virus measles, Rhabdoviruses, Filoviruses and Paramyxoviruses.

The aforementioned antibodies correspond to the preceding definitions and include, inter alia, anti-complex antibodies.

Finally, the invention relates to a method of therapeutic treatment of a viral infection, according to which a PrPc or PrPsc protein as defined above is administered to a human being or to an animal as defined above as an anti-viral agent or a therapeutic composition consisting of a functional expression cassette of said proteins or of their fragments, or a vector comprising such an expression cassette or else a cell comprising such an expression cassette or such a vector. as well as a treatment of a prion disease, according to which humans or animals are administered a composition comprising a therapeutic agent capable of preventing the transmission of a PrP protein (preferably PrPsc) by a viral particle. and preferably retroviral, or a composition comprises a therapeutic agent capable of preventing the encapsidation of a PrP protein (preferably PrPsc) in a viral particle (preferably retroviral) or a composition capable of preventing the binding of a protein. PrP protein (preferably PrPsc) to a viral or retroviral nucleic acid in a viral or retroviral particle.

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Definitions:
Nucleic acids: The molecules or sequences of nucleic acids
DNA or RNA consist of a fragment of DNA and / or RNA, double or single stranded, linear or circular, natural and isolated or synthetic, corresponding to a precise sequence of nucleotides, modified or not, and making it possible to define a fragment or region of a nucleic acid selected from the group consisting of cDNA; genomic DNA; Plasmid DNA; a messenger RNA. Nucleic acid sequences are deduced from the amino acid sequence of proteins or their fragments using the genetic code. Due to the degeneration of the genetic code, the invention also encompasses equivalent or homologous sequences. These defined sequences allow a person skilled in the art to define the suitable molecules himself. He can thus define and use nucleic acid molecules complementary to DNA and / or RNA sequences encoding the molecules of interest of the invention or of their fragment (s).

Vector: Said nucleic acid can comprise at least two sequences, identical or different, exhibiting a transcriptional promoter activity and / or at least two genes, identical or different, located in relation to each other in a contiguous, distant manner, in the same direction or in the reverse direction, provided that the function of transcriptional promoter or the transcription of said genes is not affected. In this type of nucleic acid construct, it is possible to introduce neutral nucleic sequences or introns which do not interfere with transcription and are spliced before the translation step. Such sequences and their uses are described in the literature (see for example PCT patent application WO 94/29471). The nucleic acids which can be used according to the present invention can also be nucleic acids modified so that it is not possible for them to integrate into the genome of the target cell, or nucleic acids stabilized using agents, such as, for example, spermine, which as such has no effect on the efficiency of transfection.

The nucleic acid sequence is preferably a naked DNA or RNA sequence, ie free of any compound facilitating its introduction into the cells (transfer of nucleic acid sequence). However, in order to promote

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its introduction into the target cells and in order to obtain the genetically modified cells of the invention, this nucleic acid sequence can be in the form of a vector, and more particularly in the form of a viral vector, such as for example an adenoviral or retroviral vector, a vector derived from a poxvirus, in particular derived from vaccinia virus or Modified Virus Ankara (MVA) or from a non-viral vector such as, for example, a vector consisting of at least a nucleic acid sequence complexed or conjugated to at least one molecule or carrier substance selected from the group consisting of a cationic amphiphilic, in particular a cationic lipid, a cationic or neutral polymer, a practical polar compound in particular chosen from propylene glycol, the polyethylene glycol, glycerol, ethanol, 1methyl L-2-pyrrolidone or their derivatives, and an aprotic polar compound chosen in particular from dimethylsulfoxide (DMSO), diethylsulfoxide, di-npropylsulfoxide, dimethylsulfone, sulfonate, diomethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or their derivatives. The literature provides a large number of examples of such viral and non-viral vectors.

Such vectors may further and preferably comprise targeting elements capable of directing the transfer of nucleic acid sequence to certain cell types or certain tissues. They can also make it possible to direct the transfer of an active substance to certain preferred intracellular compartments such as the nucleus, the mitochondria or the peroxisomes, for example. They can also be elements facilitating the penetration into the interior of the cell or the lysis of intracellular compartments.

Such targeting elements are widely described in the literature. They may for example be all or part of lectins, peptides, in particular the peptide JTS-1 (see PCT patent application WO 94/40958), oligonucleotides, lipids, hormones, vitamins, etc. antibody antigens, specific ligands for membrane receptors, ligands capable of reacting with an antiligand, fusogenic peptides, nuclear localization peptides, or a composition of such compounds. These nucleic acid sequences and / or vectors make it possible to target the cells either by use of a targeting molecule

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introduced into or onto the vector, as described above, or by use of a particular property of the cell.

Cellular transformation in vitro: Thus, in a particular case, a cell can be modified in vitro by at least (i) a nucleic acid sequence or a fragment of a nucleic acid sequence or an association of nucleic acid sequences corresponding to fragments of nucleic acids derived from the same gene or from different genes, said nucleic acid sequence (s) being deduced (s) DNA and RNA sequences encoding all or part of the molecules of interest, from a fragment of the molecule, of an antibody specific for the molecule which will be expressed on the surface of said mammalian cell or secreted by the cell; by at least (ii) a vector comprising at least one nucleic acid as described in (i).

More particularly, said cell is a mammalian cell and originates either from the mammal to be treated or from a mammal other than that to be treated. In the latter case, it should be noted that said cell will have undergone treatment making it compatible with the mammal to be treated. These cells are established in cell lines and are preferably CMHII + or CMHIIinducible, such as lymphocytes, monocytes, astrocytes, oligodendrocytes.

Transformation of cells in vivo and gene therapy: Cells can be transformed in vivo after injection of vectors comprising at least one gene encoding a molecule of the invention and / or for a therapeutic agent described in the present invention, for the preparation of a composition for therapeutic and / or prophylactic use called gene therapy and said composition. The vector comprises at least elements ensuring the expression of a gene in vivo.

By elements ensuring the expression of a gene in vivo, reference is made in particular to the elements necessary for ensuring the expression of said gene after its transfer into a target cell. These are in particular promoter sequences and / or effective regulatory sequences in said cell, and optionally the sequences required to allow expression on the surface of target cells of said polypeptide. The promoter used can be a

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viral, ubiquitous or tissue-specific promoter or alternatively a synthetic promoter. By way of example, mention will be made of promoters such as promoters of RSV (Rous Sarcoma Virus), MPSV, SV40 (Simian Virus), CMV (Cytomegalovirus) or vaccinia virus, promoters of the gene encoding creatinine muscle kinase, for actin. It is also possible to choose a promoter sequence specific for a given cell type, or which can be activated under defined conditions. The literature provides a great deal of information relating to such promoter sequences.

The composition comprises at least one vector containing a gene encoding a molecule of the invention and / or for a therapeutic agent as described, capable of being introduced into a target cell in vivo and of expressing the gene in vivo. The advantage is based on the possibility of maintaining a basal level of molecules expressed in the treated patient over the long term. Vectors or nucleic acids comprising the genes which code for the therapeutic agents are injected. These vectors and nucleic acids must be transported to the target cells and transform or transfect these cells in which they are to be expressed in vivo.

According to one embodiment, gene therapy is used so as to target the cells. For this, it is obvious that the cells to be targeted for transformation with a vector are, among others, cells infected with a virus, in particular a retrovirus, and / or cells expressing the PrP protein.

Numerous tools have been developed for introducing various heterologous genes and / or vectors into cells, in particular mammalian cells. These techniques can be divided into two categories: the first category involves physical techniques such as microinjection, electroporation or particle bombardment. The second category is based on the use of techniques in molecular and cellular biology with which the gene is transferred with a biological or synthetic vector which facilitates the introduction of the material into the cell in vivo. Today, the most effective vectors are viral vectors, in particular adenovirals and retrovirals. These viruses have natural properties to cross plasma membranes, prevent the degradation of their genetic material and

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introduce their genome into the nucleus of the cell. These viruses have been widely studied and some are already used experimentally in human applications in vaccination, in immunotherapy, or to compensate for genetic deficiencies. However, this viral approach has limitations, in particular due to the restricted cloning capacity in these viral genomes, the risk of disseminating the viral particles produced in the organism and the environment, the risk of artefactual mutagenesis by insertion into the host cell, in the case of retroviruses, and the possibility of inducing a strong inflammatory immune response in vivo during treatment, which limits the number of possible injections (Mc
Coy et al., 1995 Human Gene Therapy 6: 1553-1560; Yang et al., 1996
Immunity 1: 433-422). Other alternative systems to these viral vectors exist. The use of non-viral methods such as co-precipitation with calcium phosphate, the use of receptors which mimic viral systems (for a summary see Cotten and Wagner 1993, Current
Opinionin Biotechnology, 4: 705-710), or the use of polymers such as polyamidoamines (Haensler and Szoka 1993, Bioconjugate Chem 4: 372-379).

Other non-viral techniques are based on the use of liposomes, the efficiency of which for the introduction of biological macromolecules such as DNA, RNA, proteins or active pharmaceutical substances has been widely described in the literature. Teams have proposed the use of cationic lipids having a strong affinity for cell membranes and / or nucleic acids. In fact, it has been shown that a nucleic acid molecule itself can cross the plasma membrane of certain cells in vivo (WO 90/11092), the efficiency being dependent in particular on the polyanionic nature of the nucleic acid. As early as 1989 (Felgner et al., Nature 337: 387-388) cationic lipids have been proposed to facilitate the introduction of large anionic molecules, which neutralizes the negative charges of these molecules and promotes their introduction into cells. Different teams have developed such cationic lipids: DOTMA (Felgner et al., 1987, PNAS 84: 7413-7417), DOGS or TransfectamTM (Behr et al., 1989, PNAS 86: 6982-6986), DMRIE and DORIE (Felgner et al., 1993 Methods 5: 67-75), DC-CHOL (Gao and Huang 1991, BBRC 179: 280-285), DOTAPTM (McLachlan et al., 1995 Gene

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Therapy 2: 674-622) or LipofectamineTM, and the other molecules described in patents WO91 16024, WO9514651, WO9405624. Other groups have developed cationic polymers which facilitate the transfer of macromolecules, in particular anionic macromolecules, into cells.

Patent WO95 / 24221 describes the use of dendritic polymers, document WO96 / 02655 describes the use of polyethyleneimine or polypropyleneimine and documents US-A-5,595,897 and FR 2,719,316 the use of polylysine conjugates.

Depending on what it is desired to obtain in vivo for a targeted transformation towards a given cell type, it is obvious that the vector used must be capable of being itself targeted, as described above.

Drugs: These substances are administered in effective amounts.

The substances can be small synthetic or natural molecules, derivatives of the proteins identified in this invention, lipids, glycolipids, chemical compounds, etc. Small molecules can be screened and identified in large quantities using combinatorial libraries. chemical.

Injection: The biological material defined in the present invention can be administered in vivo, in particular in injectable form. It is possible to envisage injection by epidermal, intravenous, intra-arterial, intramuscular, intracerebral route by syringe or any other equivalent means, and according to another embodiment by oral administration or any other means well known to those skilled in the art. and applicable to the present invention. The administration can take place as a single or repeated dose, once or several times after a certain defined time interval. The most suitable route of administration and dosage vary depending on various parameters such as for example the individual or the disease to be treated, the stage and / or the course of the disease, or the nucleic acid. and / or the protein and / or the peptide and / or the molecule and / or the cell to be transferred and / or the target organ / tissue.

Figures:

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- Figure 1 shows that huPrP has chaperone molecule activity for nucleic acids.

Figure 1-A: DNA oligonucleotides corresponding to the HIV Tar (+) and Tar (-) 57 nucleotide sequences were used. The Tar (-) DNA was labeled with P32 and the tests were carried out with 0.1 pmole of the two Tar (+) and Tar (-) DNAs. nucleic acids were extracted by phenol-chloroform protocol well known to those skilled in the art and analyzed on a 10% native gel.

Lane 1 corresponds to the control of the hybridization reaction at 65 C for
30 minutes ; lane 2 corresponds to the hybridization reaction at 37 C; lanes 3 to 7 correspond to hybridization directed by NcP7; lanes 8 to 12 correspond to hybridization directed by huPrP
The numbers indicate the molecular relationships between protein and nucleotide of
1:19 to 1:24. The horizontal arrow shows the DNA Tar double stranded. The gel was exposed for 3 hours.

Figure 1-B:
The Tar (-) oligonucleotide radiolabeled with P32 was hybridized with an HIV-1 3 'RNA of 627 nucleotides. The nucleic acids were extracted by phenolchloroform protocol and separated by electrophoresis on a native 1% agarose gel.

The Tar (-) oligonucleotide and the viral RNA: Tar (-) complexes are indicated by an arrow.

Lanes 1 to 5 correspond to controls without proteins; lanes 2 to 4 correspond to hybridization directed by NcP7; andlanes 6-8 correspond to hybridization directed by huPrP.

The numbers indicate the protein: nucleotide molecular ratios - Figure 2 shows that huPrP inhibits the initiation of reverse transcription.

Figure 2-A: HIV-1 5 'RNA was used as template RNA. The reverse transcription was carried out in the presence of dCTP radiolabeled with P32 (Amersham) and the cDNA products were analyzed on 10% PAGE-UREE gel. The protein: nucleotide molecular ratios are shown in the figure. The arrow shows the direction of

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electrophoresis and the size of the markers (in nucleotides) are shown on the left.

Lanes 1 to 5 correspond to the reaction without proteins; lanes 2 to 4 correspond to the reaction with NCp7; andlanes 6 to 8 correspond to the reaction with huPrP.

Figure 2-B: The diagram of this figure provides a possible explanation for the inhibition of the initiation of reverse transcription.

- Figure 3 shows that huPrP causes the dimerization of the HIV-1 RNA and the hybridization of the primer tARNLys, 3 at the PBS site.

The HIV-1 5 'RNA and the tRNA, 3 radiolabeled with P3Z were incubated with or without NCp7 or huPrP and the reactions were carried out. The molecular ratios between protein: nucleotide are shown in the figure. The arrow shows the direction of electrophoresis migration and the size of the markers (in nucleotides) is indicated on the left. HIV-1 monomer and RNA dimer, and tRNALys3 are shown on the right.

Lanes 1 and 5 correspond to the reaction without protein; lanes 2 to 4 correspond to the reaction with NCp7; and lanes 6-8 correspond to the reaction with huPrP.

It should be noted that NCp7 causes multimeric RNA formation while huPrP does not.

- Figure 4 shows that huPrP directs the initiation of reverse transcription of HIV-1 RNA.

L'ARN HIV-1 5' et l'ARNtys,3 radiomarqué au P3z ont été incubés avec ou sans NCp7 ou huPrP et la transcriptase inverse de HIV p66-p51 a été ajoutée en même temps avec les dNTPs pour permettre l'initiation de la transcriptase inverse. Les réactions ont été réalisés et les ADNc simple brin ont été analysés. Figure 4-A:

The HIV-1 5 'RNA and the tRNA, 3 radiolabeled with P3z were incubated with or without NCp7 or huPrP and the reverse transcriptase of HIV p66-p51 was added at the same time with the dNTPs to allow the initiation of reverse transcriptase. The reactions were performed and the single stranded cDNAs were analyzed.

The molecular ratios between protein: nucleotide are shown in the figure.

The arrow shows the direction of electrophoresis migration and the size of the markers (in nucleotides) is shown on the left. Single stranded cDNA and tRNALys, 3 primer are shown on the right.

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Lanes 1 and 5 correspond to the reaction without protein; lanes 2 to 4 correspond to the reaction with NCp7; and lanes 6-8 correspond to the reaction with huPrP.

Figure 4-B:
This figure is a diagram showing the initiation of reverse transcription and the synthesis of single-stranded (-) cDNA.

- Figure 5 shows the transfers of DNA strand HIV-1 by huPrP.

To examine the strand transfer of the HIV-1 (-) DNA, of the template 3 'and 5' HIV-1 RNA and a DNA complementary to the PBS site radiolabeled to P32 (Darlix et al.,
1993; Allain et al., 1994) were used. For the transfer of the + DNA strand, HIV-1 5 'RNA, Lys, 3 tRNA, a P32 radiolabeled + strand primer and a DNA template acceptor were used (Auxilien et al., 1999). The reactions were performed and the produced cDNAs were analyzed. The molecular ratios between protein and nucleotide are shown in the figure. The arrow shows the direction of electrophoresis migration and the size of the markers (in nucleotides) is indicated on the left.

Figure 5-A: less strand.

Lanes 1 and 5 correspond to the reaction without protein; lanes 2 to 4 correspond to the reaction with NCp7; and lanes 6-8 correspond to the reaction with huPrP.

Note that the minus strand transfer is less efficient with huPrP (compared to lanes 4 and 8). Addition of NCp7 and huPrP results in a transfer of more than 90%. The cDNA transfer product and single stranded cDNA are shown on the right.

Figure 5-B: This figure is a diagram to show the transfer of single stranded (-) cDNA from the 5 'end of the genome to its 3' end.

Figure 5-C: more strand.

Lane 1 corresponds to the reaction without protein; lanes 2 to 4 correspond to the reaction with NCp7; and lanes 5-7 correspond to reaction with huPrP. The transfer product and single-stranded (+) DNA are shown on the right. The DNA products at the top of the gel correspond to the self-primed products.

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Note that the amount of transfer product is large with huPrP (lane 7).

Figure 5-D: This figure is a diagram to show the transfer of single stranded (+) DNA from the 3 'end of the genome to its acceptor which replaces the minus strand DNA.

- Figure 6 shows the direct interactions between huPrP and the HIV-1 nucleoprotein in vitro.

The reverse transcriptase of HIV-1, integrase, NCp7 and huPrP (23-231) were separated by electrophoresis on a 13% SDS-PAGE gel and electro-transferred onto a nylon membrane. The proteins were labeled with S35 radiolabeled huPrP (TNTsystem, Promega). The proteins are: Lanes 1 (2 g RTp66-p51), 2 (1 g Inp32), 3 (1 g Vpr), 4 and 5: and 1 g NCp7), 6 and 7 (0. 5 and 1 g huPrP) . The kDA protein markers are shown on the right. Autoradiography was performed over two days.

- Figure 7 shows that huPrP is incorporated into the HIV-1 and SIV virions.

Highly purified HIV-1, SUVmac and HTLV-1 virions were donated by.

Arthur (NCI, USA). The virion proteins were separated on 13% SDS-PAGE gel and electro-transferred onto a membrane which was then hybridized with 3F4 monoclonal antibodies.

Figure 7-A: Staining of the gel with Coomassie blue.

Figure 7-B: Western blot.

Track 1, 10 Ng HTLV-1. Track 2, 6 nu SIVmac. Lanes 3 and 4, 5 and 10 µg HIV-1, respectively. Protein markers in kDa are shown on the left. The arrow indicates huPrP23-231. Similar results were obtained with a polyclonal antibody.

FIG. 8 represents the alignment of the nucleotide sequences of PrPs originating, respectively, from mouse, sheep, pig, human, hamster, goat and bovine.

FIG. 9 represents the alignment of the amino acid sequences of PrPs originating, respectively, from sheep, goat, bovine, pig, human, mouse and hamster.

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Examples:
I. Materials and methods: - 1.1. Recombinant proteins, enzymes, RNAs and DNA plasmids.

The recombinant huPrP protein was produced in E. coli. It corresponds to the mature form PrPc and is composed of amino acids 23 to 231. It was purified on a chromatography column and recovered by removing any contaminating trace of nucleases, as described by Swietnicki et al. 1998, J Biol
Chem 273: 31048.

The NCp7 nucleocapsid of HIV-1 was synthesized with the fmoc / opfp chemical method and purified by high pressure liquid chromatography as described by de Rocquigny et al., 1992 PNAS 89: 6472; Cornille et al., 1999 J Pept Res 54: The proteins are resuspended in 30 mM HEPES, 30 mM NaCl, 0.1 mM ZnCl2 buffer, pH 6.5 at a concentration of 1 mg / ml.

- The DNA oligonucleotides Tar (-) and Tar (+), 57 nucleotides long and corresponding to the HIV-1 pNL4 sequence. 3, were produced by the company Eurogentec (Belgium).

- The 5 'RNA of HIV-1 corresponding to nucleotides 1-415 was produced in vitro, as previously described by Barat et al., Embo J 1989,8: 3285. The 3' RNA of HIV-1 (positions 8583-9208 of the HIV-1 genome) was prepared with a polyA tail by transcription, as described by Darlix et al., 1993, Reports Acad Sci., Life Sciences 316: 763 The synthetic tRNA Lys, 3 was produced in vitro using T7 RNA polymerase (Barat et al., 1993 J Mol Biol 231: 185). The reverse transcriptase of HIV-1 (RT p66 / p51) and the Inp32 integrase of HIV-1 were purified from E. coli as described by Carteau et al., 1997 J Virol 71: 6225. The reverse transcriptase of MoMuLV virus purified from E. coli was purchased from Gibco BRL.

- PNL4. 3 of HIV-1 was used to transform 293T or Hela cells under the conditions described by Ottmann et al., J Virol 69: 1778. The plasmid pSIV-TGP which was constructed is derived from the SIVmac251 genome (Genbank: number d 'accession M19499) in which a GFP expression cassette controlled by the cytomegalovirus early promoter was inserted in place of the 5' env region and with a cytomegalovirus pCMV early promoter which replaces U3 in the

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part LTR5 '. The DNA plasmid encoding the huPrP protein comes from the laboratory of Pr D. Dormont (Fontenay). All the plasmids were amplified in E. coli 1035 (RecA-) and purified by affinity chromatography (protocol
Qiagen).

- 1.2. Virions.

Highly purified HIV-1 MN virions (lots P3592 and P3620, SIVmac239 (lot
3654) and HTLV-1 (lot P3571) which were used for the study, were produced by differential centrifugation as described by Dr. Larry Arthur (SAIC
Frederick, Frederick MD, USA) - 1.3. Hybridization tests between nucleic acids.

The DNA oligonucleotides corresponding to the Tar (+) and Tar (-) sequences of HIV-
1 of 57 nucleotides were used. The DNA of HIV-1 Tar (-) was labeled 5 ′ with P32 and the hybridization tests were carried out with 0.1 pmole of the two DNA oligonucleotides Tar (-) and Tar (+). Incubations in the presence of huPrP or NCp7 were carried out for 5 minutes at 37 C in 10 l of a solution containing 20 mM Tris-HCl, 30 mM NaCl, 0.2 mM MgCl2, 5 mM DTT and 0.01 mM ZnCl2 at pH 7. 5. The tests were stopped with 0.5% SDS and EDTA.
10 mM. The nucleic acids were extracted in the presence of phenol / chloroform and analyzed on a native 10% polyacrylamide gel.

Reactions with HIV-1 RNA (P32 labeled tRNA) or P32 labeled Tar (-) DNA and huPrP or NCp7 were performed for 10 minutes at 37 C in 10 µl of 20mM Tris-HCl, NaCl 30 mM, 0.2 mM MgCl2, 5 mM DTT, 0.01 mM ZnCl2 at pH 7.5 in the presence of Rnasin 5 units (Promega), 1.5 pmol of RNA, 3 pmol of tRNA and huPrP or NCp7 at quantities making it possible to have the ratios indicated above in the corresponding examples. The reactions were stopped in the presence of SDS-EDTA (0.5% / 5 mM), treated with proteinase K (2 g) for 10 minutes at room temperature. The nucleic acids were extracted by the phenol / chloroform-based technique, analyzed on 1.3% agarose gel in 50 mM Tris Borate buffer, pH 8.3 and visualized after staining with ethidium bromide followed by fixation of the gel. at 5% TCA, a

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drying and autoradiography. A mixture of marker DNA with a molecular weight between 0.16 and 1.77 Kb was used to determine the sizes of the DNA sequences visualized on gel. The percentage of primers hybridized to HIV-1 RNA was determined by performing a densitometric scan of the autoradiography.

- 1.4. Reverse transcription protocols.

The reactions were schematically carried out as described previously by Barat et al., 1989; Darlix et al., 1993; Allain et al., 1994 Embo J 13: After 5 minutes at 30 C in 10 NI of buffer (see the detailed protocols described), the reaction volume was increased to 25 l by adding 2 pmoles of Reverse Transcriptase (RT) of HIV-1 or MoMuLV, 0.25 mM each dNTP, 60 mM NaCl and 2.5 mM MgCl2. After 20 minutes at 37 C, the incubation was stopped and the products were analyzed as those produced by nucleic acid hybridization (see above), except that, after the phenol / chloroform extraction, the products were nucleic acids were precipitated with ethanol, recovered by centrifugation, dissolved in formamide, denatured to
95 ° C. for 2 minutes and analyzed on 8% polyacrylamide gel in the presence of 7M urea and 50 mM Tris-Borate at pH 8. 3. The Hinf DNA markers labeled with P32 (Promega) were used for the size determination on gel. The levels of cDNA synthesized by reverse transcriptase were quantified on a densitometric scan.

For the (-) strand transfer tests, the reactions were carried out with 1.5 pmoles of 5 'and 3' RNA and under the conditions described above (Allain et al., 1994).

For the transfer of the (+) strand, the reaction conditions are exactly those described by Auxilien et al., 1999 J Biol Mol 274: 4412.

- 1.5. Far Western protocol and Dot-blot analysis.

The NCp7 proteins of HIV-1, VPR, RTp66-p51 and Inp32 such as the huPrP protein (23-231) were separated on 13% polyacrylamide gel in the presence of SDS and electro-adsorbed on cellulose membranes in TrisGlycine buffer. containing 30% methanol. The membranes were saturated with a

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Hepes binding buffer (HBB: 25 mM Hepes, 25 mM NaCl, 5 mM MgCl2 and 0.01 mM ZnCl2 at pH 7. 9) containing 5% milk powder (w / v) and NP-40 0.05 % for 4 hours at 20 C (Lener et al., 1998 J Biol Chem 273: 33781).

After washing with HBB buffer, the membrane was hybridized for 2 hours at 4 ° C. with the huPrP protein synthesized in a rabbit reticulocyte system (TNT system, Promega) and radiolabeled with S35. The membrane was then washed extensively with BPS buffer, dried and autoradiographed. According to another method, the huPrP protein at 100 ng / ml was adsorbed on the membrane for 2 hours at 4 C; the membrane was washed extensively in PBS and incubated with anti-PrP polyclonal antibodies to detect the huPrP-NCp7 complexes. The membrane was then washed extensively in PBS buffer again and the detection was carried out with the ECL system marketed by Amersham.

For Dot blot analyzes, the viral proteins and the huPrP protein were adsorbed on a nitrocellulose membrane. The membrane was dried and the proteins cross-linked with it by UV irradiation (0.1 J / cm2). The membrane was saturated with HBB in the presence of 5% milk powder for 4 hours, washed with HBB and hybridized for 4 hours at 4 ° C. with the huPrP protein radiolabeled with S35 then washed extensively in PBS buffer. The membrane was then dried and autoradiographed.

- 1.6. Cell culture, DNA transfection and viral infection.

The HeLa, HeLa P4 and 293 cells were cultured at 37 C in a 5% CO2 atmosphere in Dubelcco's modified Eagle's medium (life technologies-Gibco-BRL) supplemented with 10% fetal calf serum, penicillin (100 International Units / ml), streptomycin (100 g / ml) and 2mM L-glutamine.

Cell transfections were performed using a protocol based on the use of calcium phosphate as previously described by Chen C et al., 1987 Mol Cell Biol 7: 2745. To produce SIV virus in the presence or absence of huPrP protein, the cells were transfected with 3 g of pSIVTGP, 1.2 g of pVSV-G with 3 g of pDNA-huPrPhe. Other reports from

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plasmids such as 1 g of pSIV-TGP, 0.3 g of pVSV-G and 4 g of pDNAhuPrPhe were also used. Similar experimental conditions were used with HIV-1 pNL4.3 except that pVSV-G was not added.

The supernatants containing the viruses were collected 48 hours after the transfections, filtered and used to infect the cells. The amounts of virus produced in the presence or absence of huPrP by cell transfection were measured by Elisa test (SIV Cap28 or HIV Cap24). Identical amounts of virus were used to infect 293 cells (SIV) or HeLa-P4 cells (HIV-1).

Two days after infection, viral expression was followed two days later by LacZ expression in HeLa-P4 (HIV-1) cells or by expression of GFP by FACS analysis.

The production of HIV-1 or SIV gag proteins in cells or in viral particles was monitored by Western analyzes using anticapsid (anti-CA) antibodies and the ECL detection technique marketed by Amersham (Tanchou et al. , 1998, J Virol 72: 4442), or by ELISA (p24). HuPrP in 293 cells or in HIV-1, SIV-mac, or HTLV-1 virions was detected by Western analysis using either a 3F4 monoclonal antibody or a polyclonal antibody. Monoclonal antibodies directed against the N-terminus, the central domain or the C-terminus of huPrP have also been used.

II. Results 11.1 Binding of huPrP to retroviral nucleic acids Like the NCp7 nucleocapsid of HIV-1, huPrP binds both to RNAs (transfer RNA, structural RNAs such as the leader RNA of HIV-1) and to double DNA strand. In addition, the binding of huPrP or NCp7 to nucleic acids results in the formation of high molecular weight complexes which can sediment by centrifugation.

The chaperone activity of huPrP was examined using DNA and RNA in parallel, and comparing it with that described for NCp7 of HIV-1 (Darlix et al., (1995) J. Mol. Biol. 254,523. -537 and Gabus et al. (1998) EMBO J. 17,4873-

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4880). It is in fact known that NCp7 promotes the hybridization of nucleic acid sequences such as the highly structured Tar (-) and Tar (+) sequences of HIV-1.

As the NCp7 nucleocapsid of HIV-1: - huPrP causes hybridization which leads to double-stranded TAR sequences, in a dose-dependent manner and under physiological conditions (Figure 1A, lanes 4-7 for HIV-1 NCp7 and lanes 9 -12 for huPrP).

- huPrP causes the hybridization of Tar (-) to the Tar (+) sequence of the 3 'RNA, in a dose-dependent manner (FIG. 1 B lanes 3-4 for NCp7 and 7-8 for huPrP).

Complementary nucleic acid sequences derived from viruses other than HIV-1 or from different cells were also used and led to the same results (FIG. 1).

These tests clearly indicate that huPrP has a nucleic acid hybridization activity analogous to that of the NCp7 protein of HIV-1 and more generally to that of the nucleocapsids of retroviruses. The core protein (NC) is believed to hybridize directly to nucleic acids, possibly facilitating intermolecular interactions while preventing intramolecular folding. This is illustrated by the fact that NCp7 and other retroviral NC proteins can inhibit the reverse transcription started at the 3 'end of RNA, a process called' Self Priming '(Lapadat et al, 1997) (Fig 2 track 1). As shown in Figure 2, the two proteins huPrP and NCp7 can inhibit Self Priming of reverse transcription (lanes 2-4 for NCp7 and lanes 6-8 for huPrP) (Figure 2).

11.2 Role of huPrP in retroviral RNA dimerization and primer tRNA hybridization at the reverse transcription initiation site.

In the viral particle, the retroviral genome is in dimeric RNA form and has a reverse transcriptase initiation site (Primer Binding Site PBS) which binds the primer tRNA. The dimerization of the genomic RNA and the binding of the primer tRNA to PBS are induced by the retroviral nucleocapsid as is known among others with NCp7 and the genome of HIV-1 (Darlix JL et al., J Mol Biol 1995,254: 523). An HIV-1 model system has been developed and used in vitro

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to i) examine the dimerization of the viral RNA and ii) the binding of the primer tRNA to the PBS site (Lapadat et al., 1997; Gabus et al., Embo J 1998 17: NCp7 is capable of dimerizing HIV-1 RNA and lead to tRNA binding
Lys, 3 primer at the PBS site (Figure 3, lanes 2-4).

Like NCp7, huPrP is responsible for the dimerization of HIV-1 RNA and for binding of tRNA Lys, 3 primer to the PBS site, in a dose dependent manner (Figure 3, lanes 6 to 8). This sighting had never been described until today.

The same HIV-1 model system was developed and used in vitro to initiate the reverse transcription of retroviral RNA into cDNA from the primer tRNA attached to the PBS site.

This step was reproduced in vitro from RNA / tRNA complexes by adding the reverse transcriptase (RT) and the NCp7 nucleocapsid to the medium. This process requires NCp7 (comparison of tracks 1 and 2-4).

- Like NCp7, the huPrP protein is also capable of allowing the initiation of reverse transcription by RT (Figure 4A, lanes 7-8; Figure 4B) and is necessary for performing this step.

11.3 Role of huPrP in DNA strand transfer during viral synthesis in vitro.

After being synthesized, the stong-stop cDNA strand (ss-cDNA) is transferred to the 3 'end of the retroviral RNA which is necessary for the complete synthesis of the DNA strand (see Figure 5B). This step, known as DNA strand transfer, is necessary to complete the synthesis of the DNA- strand, and requires that i) the R sequences be present at each end of the retroviral genome, ii) reverse transcriptase (RT) with its RNaseH activity and iii) the core protein (NC) (Darlix JL et al., 1995 J Mol Biol 254: 523). Using an in vitro HIV-1 model system composed of 5 'and 3' RNAs which mimic the 5 'and 3' ends of the HIV-1 RNA genome, it was verified that the presence of NCp7 is critical for strand transfer. (Figure 5A lanes 2-4) and shown for the first time that, like NCp7, the huPrP protein is able to induce DNA - strand transfer in vitro (lanes 6-8). However this transfer is less

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effective than with NCp7 because DNA intermediates accumulate along with whole single-stranded DNAs (lanes 7-8).

The second obligatory strand transfer for LTR DNA synthesis takes place after initiation of the DNA plus strand (Figure 5B). Using another model system
HIV-1 in vitro, it was found that the viral PBS sequence, the tRNA primer with the modified bases, the reverse transcriptase with its RNaseH activity and the NCp7 are necessary for the transfer of the + strand (Figure 6, lanes 2-4 ) and showed for the first time that huPrP was able to replace NCp7 in the + strand transfer (Figure 6, lanes 2-4) (Auxilien et al., (1999) J. Biol. Chem. 274: This observation did also never been described so far (Figures 5 and 6).

All of these results indicate very clearly that the cellular protein huPrP and viral NCp7 both have the same nucleic acid chaperone functions that are absolutely necessary for the replication and synthesis of the retroviral genome. They are key molecules for the initiation of reverse transcription of retroviral RNA and for the DNA strand transfer step leading to the synthesis of retroviral DNA. huPrP and NCp7 can also complement each other in vitro, suggesting that they may interact.

II.4 Binding of huPrP to NC proteins.

Very pure proteins huPrP or huPrP radiolabelled with isotopic sulfur (S35) were used to study the binding of huPrP with the protein NCp7, in Western blot experiments (Figure 7). In parallel, the binding of huPrP to other viral proteins, binding nucleic acids like Vpr, integrase (IN) and RTp66-p51, was also studied.

It has very clearly been verified that huPrP can bind to itself (lanes 6-7) to form complexes which are observed in vivo in the brain of patients.

It has also been shown for the first time that, like NCp7, huPrP can bind to NCp7 (lanes 4-5), but not to the other proteins RTp66-p51, IN and Vpr (lanes 1-3). Under the same experimental conditions NCp7 interacts with itself and with the RT of HIV-1 (Tanchou V. et al. J Mol Biol 1995,252: 563,

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Lener D. et al. Febs Lett 1995, 361: 85). Other tests have been carried out using the proteins of the matrix and the capsid of HIV-1 and of HTLV-1 which show that there is no interaction between the protein huPrP and other proteins of the capsid. and the HIV-1 and HTLV-1 matrix. huPrP binds specifically to the nucleocapsids of retroviruses, and in particular to NCp7 of HIV-1. This observation has never been described until today. The results are shown in Figures 7 and 8.

The interactions that take place between huPrP and HIV-1 NCp7 in vitro suggest that huPrP could be incorporated into HIV-1 virions.

11.5 Presence of huPrP in retroviral particles.

The presence of huPrP in retroviral particles was studied by Western blot analysis using i) highly purified particles of HIV-1, SIV and HTLV virions given by the National Cancer Institute (L. Arthur, NCI, USA) and ii) a polyclonal (Demaimay et al. 1997 J Virol 71: 9685) or anti-PrP monoclonal 3F4 antibody (Kascsak et al., 1987 J Virol 61: 3688). Two major forms of huPrP were found at 19 and 10 kDa and a minor form at 32 kDa in the two batches of HIV-1 virions purified and used (FIG. 8, lanes 3 and 4). Three major forms of huPrP were found at 19, 16 and 10 kDa and a minor form at 32 kDa in the purified virions of SIV (lane 2). At the same time, huPrP was not found in the HTLV particles (lane 1). The amount of total huPrP in the HIV and SIV particles was quantified by ELISA and compared with that of the capsid protein (CA). The results show that approximately 150 molecules of huPrP are present per viral particle, taking into account that approximately 2,500 molecules of capsid protein (protein-CA) are present per virion.

11.6 Influence of huPrP on the production of viral particles and on the infectivity of virions.

The presence of different forms of huPrP in HIV-1 and SIV-mac virions suggests that this cellular protein could influence virus production and viral infectivity.

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The influence of the overexpression of huPrP on the production and on the infectivity of
HIV-1 has been examined in cell culture. 293 T cells (Didier Thono CHU
Geneva, Switzerland) were used to verify this hypothesis because they physiologically express a low level of huPrP protein, as shown by Western blot and immunocytochemistry analyzes. These cells were co-transfected with plasmids encoding HIV-1 and huPrP, respectively.

Two days after transfection, the HIV-1 virions produced in the presence or absence of huPrP were collected and the production of virions was analyzed by
ELISA and Western blot. Virions produced under conditions of overexpression of huPrP were found in mature form. The infectivity of HIV-1 was monitored by detecting the expression of LacZ in Hela-P4 cells. The results show that for a huPrP: HIV-1 DNA ratio of 1: 1 the production of viral particles was reduced by about three-fold and the guest infection of the virus was reduced by about three-fold. For a huPrP: HIV-1 DNA ratio of 5: 1, viral production was reduced by about five times and virus infectivity was decreased by 5 to 6 times for a single round of replication. These results are shown in the table below.

To co-express SIV and huPrP, the 293 cells were transfected with three recombinant plasmids which express i) SIVmac with the GFP protein instead of env, ii) VSV-G to pseudo-type the SIVmac virions and iii) the protein huPrP, respectively. The rate of transfection of the cells was verified by FACS analysis and the amount of virions produced SIVmac was measured, in the presence or absence of huPrP, using an ELISA test measuring CAp28. The infectivity of the recombinant SIVmac viruses produced in the presence or absence of huPrP was analyzed by FACS of cells expressing GFP, two or three days after infection with SIVmac pseudotyped by VSV-G. The results of five sets of experiments carried out with two different ratios of huPrP: SIV show that huPrP has a negative influence on both virion production and viral infectivity. The results are shown in the table below. For a huPrP: SIV ratio of 1: 1, the amount of viral particles produced is about 2 to 3 times less and the infectivity is decreased by 3 times for a single

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replication cycle. For a ratio of huPrP: SIV equal to 1: 5, the viral infectivity is reduced by 5 to 10 times.

Board

<tb>
<tb> DNA <SEP> Production <SEP> (protein-CA) <SEP> Infectivity
<tb> HIV-1 <SEP> 100 <SEP> 100
<tb> huPrP <SEP>: HIV-1 <SEP> (1 <SEP>: 1) <SEP> 35 +/- 5 <SEP> 35 +/- 5
<tb> huPrP <SEP>: HIV-1 <SEP> (5 <SEP>: 1) <SEP> 20 +/- 4 <SEP> 15 +/- 4
<tb> SIVmac / VSV-G <SEP> 100 <SEP> 100
<tb> huPrP <SEP>: SIV-1 <SEP> (1 <SEP>: 1) <SEP> 30 +/- 5 <SEP> 35 +/- 5
<tb> huPrP <SEP>: SIV-1 <SEP> (5 <SEP>: 1) <SEP> - <SEP> 15 +/- 3
<tb>
The numbers given correspond to the average values of at least five sets of experiments. In both cases, lentivirus infectivity was approximately 106 infectious units per ml of medium. All controls were arbitrarily set at 100%.

The inventors have therefore shown very clearly for the first time that huPrP has a negative influence on the production of virions by the same cells and on the infectivity of the virus.

The decrease in retroviral production and virion infectivity can be explained by competition between the nucleocapsid of the retrovirus infecting the cell and the cellular protein huPrP to bind to the nucleic acids of the retrovirus and induce replication and genome synthesis. viral. The overexpression of huPrP promotes binding of the protein with the retroviral genome to the detriment of binding of the genome with the own viral nucleocapsid. The replication of the viral genome would be disturbed and less efficient with huPrP compared to NCp7, even if these two proteins have the same functions. The particles newly produced by the cell overexpressing huPrP

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themselves contain the protein huPrP. The protein can enter a new cell during retroviral infection and increase the concentration of this protein in the cell, again allowing a negative influence of this protein on the production of new viral particles and their infectivity.

We could understand in this way how a protein can be responsible for the spread of the disease. In fact this spread would be due to the protein, mediated by a retroviral infection (or other viruses).

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Bibliographical references on the nucleic and protein sequences of PrP and NCp7.

PrP. Huang Z et al., PNAS 1994 91: 7139 # Lehmann S et al., J Biol Chem 1995 270: 24589 # Puckett C et al. Am J Hum Genet 1991 49: 320 # Kretzschmar HA et al., DNA 1986 5: 315 # Lee IY et al., Genome Res 1998 8: 1022 # Liao YC et al., Science 1986 233: 364 NCp7 # Huang Y and al., J Virol 1997 71: 4378 # Schmalzbauer E et al., J Virol 1996 70: 771 # Morellet N et al., Embo J 1992 11: 3059

Claims (23)

1 PrPc or PrPsc prion protein, human or animal isolated, characterized in that it is capable of binding to DNA or to RNA, in particular to DNA or to viral RNA and preferably to the DNA or to the RNA of human or animal retroviruses 2 Protein according to claim 1, characterized in that it is capable of binding to the RNA or to the DNA of a virus , such as group M, group 0, non-M group, non-0 HIV-1 viruses, HIV-2 virus, SIV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxovirus 3 PrPc or PrPsc prion protein, human or animal, characterized in that it is capable of binding to a core protein of a human or animal retrovirus 4 Protein according to claim 3, characterized in that ' it is capable in particular of binding to the NCp7 nucleocapsid of the retrovirus HIV-1 5 Analogue of Ncp7, characterized in that it is a protein according to any one of the r claims 1 to 4. 6 Isolated nucleoprotein complex which comprises a prion protein, PrPc or PrPsc, human or animal, associated with DNA or RNA, in particular with viral DNA or RNA and preferably with DNA or RNA from human or animal retroviruses / A nucleoprotein complex according to claim 6, wherein the prion protein is human or animal PrPc or PrPsc, associated with RNA or DNA from 'a virus, such as group M, group 0, non-M group HIV-1 viruses <Desc / Clms Page number 47> no 0, HIV-2 virus, SIV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses. 8 Isolated protein complex which comprises a prion protein, PrPc and / or PrPsc, human or animal, associated with themselves or with each other and / or with a core protein of a human or animal retrovirus 9 Protein complex according to claim 8 , in which the PrPc or PrPsc is associated in particular with the NCp7 nucleocapsid of the HIV-1 retrovirus. 10. PrPc or PrPsc prion protein according to any one of claims 1 to 4, in modified form or in the form of a fragment of at least one of these proteins, for use as an antiviral agent, optionally in interaction. with a ligand capable of binding to said PrPc or PrPsc, such as a protein, a polypeptide, a nucleic acid, a chemical molecule, a drug, a vector 1 Protein or protein fragment according to claim 10, characterized in that 'he or she consists of a protein or a fragment of natural protein, isolated or of a protein or a fragment of a recombinant protein or of a synthetic polypeptide or polypeptide fragment 12 Therapeutic and / or prophylactic composition characterized in that it comprises, an effective amount of an anti-viral agent according to claims 10 or 11, optionally with an adjuvant and / or a diluent and / or a pharmaceutically acceptable excipient and / or vehicle. 13 Nucleic acid encoding a PrPc or a modified PrPsc or for at least a fragment of at least one of these proteins according to claim 10 or 11, for use as an anti-viral agent. <Desc / Clms Page number 48> 14 Nucleic acid according to claim 13, characterized in that it codes for a protein or a fragment of a protein, natural or recombinant, or in that it is the product of the transcription of a nucleic acid or of a nucleic acid fragment according to claim 13 15 Therapeutic and / or prophylactic composition, characterized in that it comprises a therapeutically effective amount of an anti-viral agent according to claims 13 or 14 16. Therapeutic composition and / or prophylactic, characterized in that it comprises, an expression cassette functional in a cell derived from a prokaryotic or eukaryotic organism allowing the expression of DNA or RNA, or of a DNA fragment or of RNA, encoding all or part of a PrPc protein or of a modified PrPsc according to claim 13, and optionally allowing the expression of DNA or of RNA, or of a DNA fragment or of RNA, encoding all or part of a ligand of said PrPc or PrPsc modif iée, placed under the control of the elements necessary for its or their expression. 17 Therapeutic and / or prophylactic composition, characterized in that it comprises a vector comprising an expression cassette according to claim 16. 18 Therapeutic and / or prophylactic composition, characterized in that it comprises a cell derived from a prokaryotic or eukaryotic organism comprising an expression cassette according to claim 16 or a vector according to claim 17. Use of an expression cassette according to claim 16, or a vector according to claim 17 or a cell according to claim 18, for the manufacture of an anti-viral drug. <Desc / Clms Page number 49> Expression cassette functional in a viral vector functional for transducing cells derived from a eukaryotic organism allowing expression of DNA or of a DNA fragment encoding all or part of a modified PrPc or PrPsc protein according to claim 13, placed under the control of the elements necessary for its expression and allowing the replication of a viral nucleic acid, in particular recombinant retroviral, such as the nucleic acids of HIV-1 viruses of group M, of group 0, of non-M non-0 group, HIV-2 virus, SIV, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses PrP21 Vector comprising an expression cassette according to claim 20. 22 Transformed cell, derived from a eukaryotic organism, comprising an expression and replication cassette according to claim 20 or a vector according to claim 21. 23 Cell according to claim 22, characterized in that it is in particular chosen from eukaryotic 293 cells, HeLa and HeLa P4 cells, human or animal stem cells 24 Therapeutic and / or prophylactic composition, characterized in that it comprises , a cell according to claims 22 and 23, or a vector according to claim 21 or an expression cassette according to claim 20 25. Use of a cell according to claims 22 and 23, or a vector according to claim 21 or a An expression cassette according to claim 20 for the manufacture of an anti-viral medicament. 26 Method for detecting and / or quantifying an infection by a virus, in particular a human or animal retrovirus, according to which. (i) a human or animal biological sample is taken, such as a biological fluid, a cell or a tissue fragment, <Desc / Clms Page number 50> (n) said PrPc or PrPsc is detected in said biological sample, in free form or associated with DNA or with RNA, in particular viral and preferably retroviral, or alternatively associated with a nucleocapsid protein of a retrovirus, in particular the NCp7 nucleocapsid of HIV-1, (ni) said PrPc or PrPsc of step (ii) is detected, and dv) if desired, the concentration of said PrPc or PrPsc of step (ii) is quantified relative to a reference concentration 27 Method according to claim 26, according to which said PrPc or PrPsc is detected and optionally quantified in free form or associated with DNA or RNA, in particular viral, preferably retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of the HIV-1 retrovirus, using at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPc or PrPsc in free or di against at least a first epitope of said PrPc or PrPsc associated with DNA or RNA, preferably viral and in particular retroviral, or with a retroviral core protein 28 Method according to claim 27, according to which one uses a second antibody, preferably a monoclonal antibody directed against a second epitope of said PrPc or PrPsc in free or associated form. 29 Kit for the detection of an infection by a virus, in particular a human or animal retrovirus, characterized in that it comprises at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPc or PrPsc, in free form, or directed against at least a first epitope of said PrPc or PrPsc associated with DNA or RNA, in particular viral, preferably retroviral or with a core protein of a retrovirus, in particular the NCp7 of HIV-1, and optionally a second antibody, preferably a monoclonal antibody directed against a second epitope of said PrPc or PrPsc in free or associated form, different PrPdudit first epitope <Desc / Clms Page number 51> Isolated viral particle, in particular particle of a human or animal retrovirus, such as group M, group 0, non-M group, non-0 HIV-1 virus, HIV-2 virus, SIV virus, viruses. enveloped, such as measles virus,

Habdoviruses, Filoviruses and Paramyxoviruses, and comprising a PrP 'protein or a PrPsc protein as defined in claim 1 or in claim 3 31 A method of detecting and / or quantifying a prion disease according to which (i) a human or animal biological sample is taken, such as a biological fluid, a cell or a tissue fragment, (II) viral particles, in particular retroviral particles, are isolated or extracted and purified from said biological sample, ( iii) said viral particles are lysed so as to obtain a lysate, and (iv) a PrP, preferably PrPsc is detected in said lysate, in free form or associated with viral DNA or RNA, preferably retroviral, or associated with a retroviral core protein, in particular the NCp7 core of HIV-1, and (v) if desired, said PrP, preferably said PrPsc, is quantified in free or associated form. 32 The method of claim 31, wherein said viral particles are particles as defined in claim 30. 33 The method of claims 31 and 32, wherein said PrP is detected and optionally quantified, preferably said PrPsc, in free form or associated with DNA or with viral RNA, preferably retroviral, or associated with a viral DNA or RNA. core protein of a retrovirus, in particular the ICp7 core i of HIV-1, using at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPsc in free form or directed against at least a first epitope of said <Desc / Clms Page number 52> PrPc or PrPsc associated with DNA or with RNA, in particular with DNA or with viral RNA, and preferably retroviral or with a nucleocapsid of a retrovirus. 34 The method of claim 33, wherein a second antibody is used, preferably a monoclonal antibody directed against a second epitope of said PrP, preferably said PrPsc in free or associated form, afferent of said first epitope 35 Kit for detection and / or the quantification of a prion disease, characterized in that it comprises, inter alia, at least one ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of a PrP, preferably a PrPsc, in free form or associated with DNA or with RNA, in particular with DNA or with viral RNA, and preferably retroviral or associated with a core protein of a retrovirus , in particular the NCp7 nucleocapsid of HIV-1, directed against at least a first epitope of said PrP, preferably PrPsc and optionally a second antibody, preferably a monoclonal antibody directed against a second epitope, different from said first epitope, of said PrP, preferably PrPsc in free form or associated with DNA or RNA, in particular viral, and preferably retroviral or associated with a retroviral nucleocapsid, in particular with the NCp7 nucleocapsid of HIV- 1. 36 Composition for therapeutic and / or prophylactic use for the treatment and / or prevention of a prion disease, characterized in that it comprises a therapeutic agent consisting of a ligand capable of binding to said PrP, preferably PrPsc , in particular a polyclonal or monoclonal antibody or an antibody fragment and preferably an antibody or monoclonal antibody fragment, or consisting of a nucleic acid encoding a polyclonal or monoclonal antibody or for an antibody fragment and preferably for an antibody or a fragment of a monoclonal antibody or the therapeutic agent is an anti-viral molecule <Desc / Clms Page number 53> 37 Composition for therapeutic and / or prophylactic use for the treatment and / or prevention of a prion disease, characterized in that it comprises a drug chosen from A.Z.T (Azido Deoxythymidine), D D.l. (DiDeoxylnosme) 38 Use of a therapeutic and / or prophylactic composition according to any one of claims 36 to 37, for the manufacture of a medicament for the treatment and / or prevention of a prion disease 39 Use of a therapeutic and / or prophylactic composition according to claim 24, for the manufacture of a medicament for the treatment and / or prevention of prion disease. 40 Method for demonstrating an overexpression of the PrPc protein or of the PrPsc protein and the reduction of a viral infection in vitro, in particular a retroviral infection by a human or animal retrovirus according to which (I) a cell is transformed with a nucleic acid encoding a retrovirus and a nucleic acid encoding a PrPc or PrPsc of human or animal origin, in particular a plasmid, (n) a cell culture of said cell is carried out in an appropriate culture medium, the production is detected vinons in the culture supernatant of said cell culture and, (ni) correlating said virion production with virion production carried out under the same conditions but in the absence of PrPc or PrPsc. 41 The method of claim 40, wherein the cell is chosen in particular from 293 cells, HeLa and HeLa P4 cells, human or animal stem cells and the retroviral nucleic acid is the nucleic acid of a retrovirus chosen from among the following. HIV-1 group M, group 0 or non-M group, non-0 virus, HIV-2 virus, SIV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses <Desc / Clms Page number 54> 42 Method for detecting and / or quantifying the expression of a PrPc or PrPsc protein and the expression of at least one viral protein, preferably retroviral and in particular of a retrovirus chosen from HIV-1 viruses of group M, of group 0 or of group not M, not 0, the HIV-2 virus, the SIV virus, the BIV virus, the VISNA virus, the CAEV virus, enveloped viruses, such as the measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses, according to which at least a first ligand is used, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPc or of said PrPsc, in free form or associated with DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of retrovirus 1-IIV-1, and at least one second ligand , in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from HIV-1 viruses of group M, of group 0 or of non-M, non-0 group, the HIV-2 virus , SIV virus, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxovirus 43 Method according to claim 42, according to which a third ligand, in particular an antibody and preferably a monoclonal antibody directed against at least one second epitope, different from the first epitope, from said PrPc or from said PrPsc, in free form or associated with DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of retrovirus I-IIV-1, and / or at least a fourth ligand, in particular an antibody and preferably a monoclonal antibody directed against at least one second epitope, different from the first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from HIV-1 viruses of group M, of group 0 or of non-M, non-0 group, the HIV- virus 2, SIV virus, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as measles virus, Rhabdovirus, Filovirus and Paramyxovirus <Desc / Clms Page number 55> 44 Kit for the detection and / or quantification of the expression of a PrPc or PrPsc protein and of the expression of at least one viral protein, preferably retroviral and in particular of a retrovirus chosen from HIV-viruses. 1 of group M, group 0 or group not M, not 0, HIV-2 virus, SIV virus, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as glow virus , Rhabdoviruses, Filoviruses and Paramyxoviruses, comprising at least a first ligand, in particular an antibody and preferably a monoclonal antibody directed against at least a first epitope of said PrPc or of said PrPsc, in free form or associated with l 'DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of the HIV-1 retrovirus, and at least one second ligand, in particular a antibody and preferably a monoclonal antibody directed against me ns a first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from HIV-1 viruses of group M, of group 0 or of non-M, non-0 group, the HIV-2 virus, the SIV virus, BIV virus, VISNA virus, CAEV virus, enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses. 45 Kit according to claim 44, comprising a third ligand, in particular an antibody and preferably a monoclonal antibody dinged against at least one second epitope, different from the first epitope, from said PrPc or from said PrPsc, in free form or associated with de DNA or RNA, preferably viral and in particular retroviral, or associated with a core protein of a retrovirus, in particular the NCp7 core of the HIV-1 retrovirus, and / or at least a fourth ligand, in particular an antibody and preferably a monoclonal antibody directed against at least one second epitope, different from the first epitope of a viral protein, preferably retroviral and in particular of a retrovirus chosen from the HIV-1 viruses of group M, of group 0 or group not M, not 0, HIV-2 virus, SIV virus, BIV virus, VISNA virus, CAEV virus enveloped viruses, such as measles virus, Rhabdoviruses, Filoviruses and Paramyxoviruses.