FR2715939A1 - Two pathogenic or infectious agents associated with multiple sclerosis - Google Patents

Abstract

Compsn. (A) comprises 2 pathogenic and/or infectious agents, isolated or purified, associated with multiple sclerosis i.e. (1) agent (Ia) that is a human virus with reverse transcriptase activity and of the endogenous retroviral family, or its variant and (2) agent (Ib) or its variant. (Ia) and (Ib) are derived from the same viral strain, i.e. POL-2 (ECACC V92072202) or MS7PG (ECACC V93010816), or their variants. Alternatively (Ia) and (Ib) are produced by the same cell line, i.e. PLI-2 (ECACC 92072201) or LM7PC (ECACC 93010817) or other similar infected cell cultures.

Description

Multiple sclerosis (MS) is a disease

  demyelinating of the central nervous system (CNS) the cause of which is still unknown.

  Numerous studies have supported the hypothesis of a viral etiology of the disease, but none of the known viruses tested has proven to be the causative agent sought: a review of the viruses sought for years in MS has been made by E. Norrby (Prog. Med. Virol., 1978; 24, 1-39) and RT Johnson (in 'Handbook of clinical neurology, 47 Demyelinating diseases ". Vinken P. and Bruyn GW, eds. Amsterdam, Elsevier science Publishing, 1985, 319-336). At the same time, the possibility of an exogenous and / or infectious factor is suggested by the existence of localized epidemics or "clusters" of MS as was observed in the Feroes Islands between 1943 and 1960 (Cook 1980), Sardinia (Rosati, 1988), Norway (Riisse, 1991), as well as migrant studies (Elian, 1990). Of all the exogenous factors suggested, viruses have been studied most often and a viral etiology is conventionally mentioned.

  The observation, in MS, of phenomena assimilable to an autoimmunity reaction has led to an "essential" etiological autoimmune hypothesis (see: Lisak RP, Zweiman B. New Engl. J. Med. 1977; 297 , 850-853, and, Lassmann H. and Wisniewski HM Arch.

  Neurol. 1979; 36, 490-497). However, this autoimmunity directed against certain components of the CNS has been shown to be unspecific for MS and frequent in CNS inflammations, whether or not associated with an infection, as has been shown by Hirayama M. et al.

  (Neurology 1986; 36, 276-8), Kenneth G. Warren et al.

  (Annals of Neurology 1986; 20, 20-25), Suzumura A. et al. (Journal of Neuroimmunology 1986; 11, 137-47), and, Tourtelotte W. et al. (Journal of Neurochemistry 1986; 46, 1086-93). In addition, as noted by E.J. Field (The Lancet 1989; I, 1272.) none of the immunosuppressive therapies has produced decisive results against MS. It now seems likely that the "autoimmune" manifestations are induced by a viral mechanism: co-sensitization to viral determinants associated with molecules of cellular origin, phenomena of molecular mimicry - as described by Fujinami RS and Oldstone MBA (in "Molecular mimicry: Cross-reactivity between microbes and host proteins as a cause of autoimmunity". Oldstone MBA, ed .. Current Topics in Microbiology and Immunology, vol. 145, Berlin, Springer-Verlag, 1989) - or, according to P. Rudge (Journal of Neurology, Neurosurgery and Psychiatry, 1991 54, 853-855), by expression of retroviral superantigens.

  Research has supported a hypothesis that a retrovirus is the cause of the disease: the recent discovery by A. Gessain et al. (J. Infect.

  Disease 1988; 1226-1234), of neurological syndromes associated with the HTLV-I virus, originally known as an agent for adult T leukemia, has led many authors, such as H. Koprowski et al. (Nature 1985; 318, 154), M. Ohta et al. (J. Imunol. 1986; 137, 3440), E. P. Reddy et al. (Science 1989; 243, 529), S.J. Greenberg et al. (Proc. Natl. Acad. Sci. USA 1989; 86, 2878), J.H.

  Richardson et al. (Science 1989; 246, 821), S.L. Hauser et al. (Nature 1986; 322, 176) and A. Karpas et al.

  (Nature 1986; 322, 177), to seek an implication of this human retrovirus in MS, however without success or with results suggesting cross-reactions.

  In addition, there is an animal model very close to MS, induced by a retrovirus: the MAEDI-VISNA sheep virus. It is known that natural infection by this virus can cause two types of disease in this animal: Maedi, interstitial lymphocytic pneumonia which can occur during primary infection with this retrovirus and late demyelinating neurological pathology generally following a phase of prolonged latency, the Visna. The pathophysiology of natural Visna agrees with most clinical and biological data on MS, as reported by Johnson RT (Rev. Infect. Dis. 1985; 7, 66-67), Narayan O. and Cork LC (Rev. Infect. Dis. 1985; 7, 89-98), and Nathanson N. et al. (Rev. Infect. Dis.

  1985; 7, 75-82). Experimental infection of sheep by intra-ventricular inoculation of neurovirulent strains of the VISNA virus has made it possible to establish the responsibility of this virus in the genesis of this demyelinating disease of sheep. As explained by Nathanson N. et al. (Rev. Infect. Dis. 1985; 7, 75-82), Hoffman P.M. and Panitch H.S. (in, "Handbook of clinical neurology, 12: Viral diseases" R.R. Mc Kendall, ed.

  Elsevier science Publishing, Amsterdam, 1989, p 453-466), and A. Haase (Nature 1986; 322, 130-136), it differs from natural infection by its exacerbated neuropathological consequences, but remains close to MS. It is moreover notable that, in all the work carried out on this subject by the aforementioned authors, in particular, the Visna virus is regularly found in the choroid plexus cells of the brain which constitute a site of latency and occasional replication of the provirus. Visna; the location of these cells at the blood / cerebrospinal fluid (CSF) interface certainly explains this phenomenon.

  Recently, a retrovirus, different from known human retroviruses, has been isolated from MS patients by H. Perron et al. (Res. Virol. 1989; 140, 551-561 / in: "Current concepts in multiple sclerosis" Wiethôlter et al., Eds. Amsterdam, Elsevier, 1991, p. 111-116 / The Lancet 1991; 337, 862-863 ). The authors were also able to show that this retrovirus could be transmitted in vitro, that MS patients produced antibodies capable of recognizing proteins associated with the infection of leptomeningeal cells by this retrovirus and that the expression of the latter could be strongly stimulated by the immediate-early genes of certain herpesviruses (Perron et al. Herpes simplex virus ICPO and ICP4 immediate-early proteins strongly enhance expression of a retrovirus harboured by a leptomeningeal cell-line from multiple sclerosis. 1993. J. Gen. Virol . 74; 65-72).

  All these results plead in favor of the role in MS of at least one unknown retrovirus or of a virus having a detectable reverse transcriptase activity according to the method published by H. Perron et al. (Res. Virol.

  1989; 140, 551-561) and qualified as an "LM7 type RT" activity.

  Recently, the Applicant's work has made it possible to obtain two continuous lines of cells infected with natural isolates originating from two different patients suffering from MS, by a culture method as described in patent application No. WO 93/20188. These two lines derived from human plexus-choroid cells, designated LM7PC and PLI-2 were deposited with the ECACC on July 22, 1992 and January 8, 1993, under the numbers 92072201 and 93010817, in accordance with the provisions of the Budapest Treaty. . In addition, viral isolates with RT activity of LM7 type have also been deposited with the ECACC under the global name of "strains". The "strain" or isolate hosted by the PLI-2 line, named POL-2, was deposited on July 22, 1992 under the number V92072202. The "strain" or isolate hosted by the LM7PC line, called MS7PG, and was deposited on January 8, 1993 under the number V93010816.

  From the above cultures and isolates, characterized by biological and morphological criteria, we then set out to characterize the nucleic material associated with the viral particles produced in these cultures.

  Thus the objects of the present invention are the following: - a virus, having a reverse transcriptase activity, associated with multiple sclerosis, contained in a viral strain having a reverse transcriptase activity, chosen from the strains respectively named POL-2, deposited 22.07.1992 with ECACC under the access number V92072202, and MS7PG filed on 08.01.1993, with ECACC under the access number V93010816, other than the human virus, possessing reverse transcriptase activity, and of which at least part of the POL sequence has homology with the same POL region of the endogenous virus ERV9 or HSERV9, contained in each of the same strains, and among the variant strains consisting of viruses comprising at least one antigen which is recognized by at least one antibody directed against at least one corresponding antigen of one or the other of said viruses of the above-mentioned viral strains POL-2 and MS7PG, other than the huma virus in above.

  - a virus, possessing reverse transcriptase activity, associated with multiple sclerosis, produced by a cell line chosen from the cell lines respectively named PLI-2 deposited on 07.22.1992 with the ECACC under the access number 92072201 and LM7PC deposited on 08.01.93 with the ECACC under the access number 93010817, other than the human virus, possessing reverse transcriptase activity, and of which at least part of the POL sequence has homology with the same POL region of endogenous ERV9 or HSERV9 virus, produced by each of the same lines, and among infected cell cultures capable of producing a virus comprising at least one antigen which is recognized by at least one antibody directed against at least one corresponding antigen of one or more other virus produced by the aforementioned PLI-2 and LM7PC lines, other than the aforementioned human virus.

  a virus whose genome comprises a nucleotide sequence having at least 50%, and preferably at least 70% of homology with a nucleotide sequence SEQ ID NO1, described in FIG. 1, or its complementary sequence.

  - A virus whose genome codes for a peptide sequence having at least 50%, and preferably at least 70% homology, with a peptide sequence coded by the nucleotide sequence SEQ ID N01 or its complementary sequence.

  - A nucleotide fragment comprising a nucleotide sequence having at least 50%, and preferably at least 70% of homology with SEQ ID N01 or its complementary sequence.

  - A preferred fragment of the invention consisting of a nucleotide sequence having at least 50%, and preferably at least 70% homology, with the nucleotide sequence SEQ ID N01 or its complementary sequence.

  - an RNA or DNA and in particular a replication vector, comprising a fragment of the invention.

  a specific primer for the amplification by polymerization of an RNA or of a DNA specific for a virus associated with multiple sclerosis, comprising a nucleotide sequence having at least 50%, and preferably at least 70% of homology, with at least part of a fragment of the invention.

  - a preferred primer having from 10 to 30 nucleotides.

  a probe capable of hybridizing specifically with RNA or DNA specific for a virus associated with multiple sclerosis, comprising a nucleotide sequence having at least 50%, and preferably at least 70% homology, with at least at least part of a fragment of the invention.

  - a preferred probe having at least 10 nucleotides.

  a use of a probe of the invention or of a primer of the invention for detecting and / or identifying, in a biological sample, a virus associated with multiple sclerosis.

  - An antisense therapeutic composition in particular for the treatment of multiple sclerosis, comprising at least one nucleotide sequence of the invention.

  a method for detecting and / or identifying a virus associated with multiple sclerosis, in a biological sample, characterized in that an RNA and / or a DNA specific to said virus is exposed, or their complementary RNA and / or DNA, at least one probe of the invention.

  - A preferred method of the invention characterized in that, before exposing the RNA and / or DNA or their DNA and / or complementary DNA and / or RNA to the probe, said RNA and / or said DNA are hybridized with at least one primer of the invention and said RNA and / or DNA are amplified.

  - A peptide encoded by the nucleotide sequence of the genome of a virus associated with multiple sclerosis, and encoded by at least part of a nucleotide fragment of the invention.

  - a protein comprising a peptide of the invention.

  an oligopeptide comprising at least five contiguous amino acids of the peptide of the invention.

  - A diagnostic, and / or therapeutic and / or prophylactic composition, comprising at least one peptide of the invention, or at least one protein of the invention or at least one oligopeptide of the invention.

  - A diagnostic, and / or therapeutic and / or prophylactic composition, characterized in that it comprises a ligand specific to a peptide of the invention, or to a protein of the invention, or to an oligopeptide of the invention.

  Before detailing the invention, various terms used in the description and the claims are now defined: - according to the invention, a nucleotide fragment or an oligonucleotide is a chain of monomers, characterized by the informational sequence of natural nucleic acids, susceptible to hybridize to a nucleotide fragment under predetermined conditions, the sequence being able to contain monomers of different structures and to be obtained from a natural nucleic acid molecule and / or by genetic recombination and / or by chemical synthesis, - Thus, a monomer can be a natural nucleotide of nucleic acid whose constituent elements are a sugar, a phosphate group and a nitrogenous base; in RNA the sugar is ribose, in DNA the sugar is deoxy-2-ribose; depending on whether it is DNA or RNA, the nitrogen base is selected from adenine, guanine, uracil, cytosine, thymine; or a nucleotide modified in at least one of the three constituent elements; for example, the modification can take place at the base level, generating modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine , bromo-5-deoxyuridine and any other modified base promoting hybridization, at the sugar level, namely the replacement of at least one deoxyribose with a polyamide (PE Nielsen et al, Science, 254, 1497-1500 (1991) ), at the phosphate group, for example its replacement with esters chosen in particular from diphosphate, alkyl and arylphosphonate and phosphorothioate esters, - by "informational sequence" means any ordered sequence of monomers, the chemical nature of which and the order in a reference direction constitute information of the same quality as that of natural nucleic acids, by hybridization is understood the process during which, under appropriate conditions, two nucleotide fragments , having sufficiently complementary sequences, are paired to form a double strand, a probe is a nucleotide fragment comprising at least 10 monomers, advantageously from 10 to monomers, having a specificity of hybridization under determined conditions. A probe can be used for diagnostic purposes such as capture and / or detection probes or for therapy purposes, - the capture probe can be immobilized on a solid support by any suitable means, i.e. - say directly or indirectly, for example by covalence or passive adsorption, - the detection probe is marked by means of a marker chosen from radioactive isotopes, enzymes especially chosen from peroxidase and alkaline phosphatase and those capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, nucleotide base analogs, and biotin, - the probes used for diagnostic purposes of the invention can be used in all known hybridization techniques and in particular the so-called "DOT-BLOT" techniques (MANIATIS et al, Molecular Cloning, Cold Spring Harbor, 1982), "SOUT HERN BLOT "[SOUTHERN. E.M., J. Mol. Biol., 98, 503 (1975), "NORTHEN BLOT" which is a technique identical to the "SOUTHERN BLOT" technique but which uses RNA as target, the SANDWICH technique (DUNN AR, HASSEL JA, Cell, 12, 23 (1977)]; advantageously, the SANDWICH technique is used in the present invention comprising a specific capture probe and / or a specific detection probe, it being understood that the capture probe and the detection probe must have a nucleotide sequence at less partially different, - another application of the invention is a therapy probe, said probe being capable of hybridizing in vivo on RNA and / or on DNA to block the phenomena of translation and / or transcription , - a primer is a probe comprising from 10 to 30 monomers, having a specificity of hybridization under conditions determined for the initiation of an enzymatic polymerization for example in an amplification technique such as PCR (Poly merase Chain Reaction), in an elongation process, such as sequencing, in a reverse transcription method or the like, - homology characterizes the degree of similarity of two compared nucleotide fragments.

  Since a virus possessing reverse transcriptase enzymatic activity can be genetically characterized in the form of both RNA and DNA, both DNA and viral RNA will be used to characterize the sequences relating to a virus having such reverse transcriptase activity.

  The invention will be better understood on reading the detailed description which follows, made with reference to the appended figures in which: FIG. 1 represents the sequence of MSRV-2A type obtained from LM7 cultures according to the protocol of Shih et al. . (J. Virol. 1989; 63, 64-75), - Figure 2, corresponds to the alignment of a protein sequence MSRV-2A obtained from the LM7 culture with known retroviral protein sequences; the amino acids conserved between the amplification product (Trad pol SHIH) and certain retroviruses are underlined in the alignment. This sequence is very far from all the retroviruses known to date, in particular from HTLV, in particular by the presence of a deletion in the 5 ′ region, - Figure 3, gives an example of consensus for sequences of MSRV-2B , - Figure 4 shows the definition of a consensus sequence of MSRV-2B type according to Fig 4A, a definition of a functional reading frame according to Fig 4B, a repetition of the primer 3 'according to Fig 4C and a sequence included between VLPQG and YVDD according to Fig 4D, - Figure 5, represents a comparative analysis of nucleic acid (Fig 5A) and protein sequences (Fig 5B), of MSRV-2 type obtained from LM7, LM7PC and PLI-2 cultures and lymphocytes B of a patient with MS, according to the original or modified protocol of Shih et al., With LigT4 corresponding to a sequence obtained from the lines, according to the modified protocol, LBpatDuT4 corresponding to a sequence obtained from the lines, according to the modified protocol and polSHIH corresponding to a sequence obtained from the lines, according to the original protocol, - Figure 6, corresponds to an alignment of a protein sequence MSRV-2B (with one or two primers 3 ') with known retroviral protein sequences; the amino acids conserved (1st line) between the MSRV-2B consensus and certain retroviruses are underlined in the alignment; this sequence is very far from all the retroviruses known to date, in particular from HTLV, in particular by the presence of a deletion in the 5 ′ region and of an active site YVDD and not YMDD, - Figure 7, is a representation of reverse transcriptase activity in sucrose fractions taken from a virion purification gradient produced in B lymphocytes, in culture, in a patient suffering from MS (reverse transcriptase activity (RT) in dpm (disintegrations per minute) ) is represented on the ordinate. The numbers of the fractions are represented on the abscissa), - FIG. 8, is a representation of the reverse transcriptase activity, as in FIG. 7, but obtained from a culture of B lymphocytes of a control free from MS, and - Figure 9, illustrates the consensus and majority sequences of MSRV-2B obtained from B lymphocytes of a patient suffering from MS, according to Fig 9A, with: * MAJ corresponding to the sequence m ajoritaire in which the bases kept in all cases are represented by ATGC, the majority bases are represented by atgc and the bases deleted in relation to consensus are represented by -, * VAR corresponding to the majority sequence (variation) in which the bases kept with respect to the consensus are represented by., the minority bases are represented by atgc and the deleted bases with respect to the consensus are represented by - and * MIN corresponding to the minority sequences (exception) in which the bases preserved with respect to the majority sequence are represented by and the deleted bases with respect to the consensus by as well as the translation of the majority sequence, according to Fig 9B, in which the legend is as follows: an: nucleic acids: - in bold, characters of small sizes: the restriction sites primers - in bold, capital letters, 3 'end of primers - in s line, the majority nucleic sequence coding prot: protein sequence: - underlined: amino acids coded by the primers EXAMPLE 1: OBTAINING CLONES, DEFINING A MSRV-2A FAMILY, BY AMPLIFICATION OF POL REGIONS

  PRESERVED RETROVIRUSES ON A VIRION PREPARATION

FROM LM7 LINE.

  The molecular approach consisted in using a PCR technique making it possible to amplify a relatively conserved region of the pol gene of exogenous and endogenous retroviruses, but also of viruses coding for an enzyme with reverse transcriptase activity such as in particular the hepatitis B virus. , which was published by Shih et al. (Shih A., Misra R., and Rush M.G. Detection of multiple, novel reverse transcriptase coding sequences in human nucleic acids: relation to primate retroviruses. J. Virol. 1989; 63, 64-75). This technique was used on the RNA extracted from a preparation of purified virions, obtained according to the protocol described below, from the supernatants of the original LM7 culture (Perron et al.

  Res. Virol. 1989; 140, 551-561) kept frozen at -800C since then: culture supernatants are collected twice a week, pre-centrifuged at 10,000 rpm for 30 minutes to remove cell debris, and then frozen at -80 C or used as for the next steps.

  The fresh or thawed supernatants are centrifuged on a cushion of PBS-glycerol 30% at 100,000 g (or 30,000 rpm in an LKB-HITACHI type 45 T rotor) for 2 h at 4 C. After removal of the supernatant, the pellet sedimented is taken up in a small volume of PBS and constitutes the fraction of concentrated, but not purified, virions. The concentrated virus is then deposited on a sucrose gradient in a sterile PBS buffer (15 to 50% w / w) and ultracentrifuged at 35,000 rpm (100,000 g) for 12 h at +4 C in a bucket rotor. 10 fractions are collected and 20 g1 are taken from each fraction after homogenization to assay the reverse transcriptase activity according to the technique described by H. Perron et al. (Res. Virol. 1989; 140, 551-561). The fractions containing the LM7 RT activity peak are then diluted in sterile PBS buffer and ultracentrifuged for one hour at 35,000 rpm (1,000,000 g) to sediment the viral particles. The pellet of purified virions thus obtained is then taken up in a small volume of a buffer suitable for the subsequent use which will be made of it (Ex. Guanidium Thiocyanate buffer for the extraction of RNAs; sterile PBS for storage at -80 ° C. ).

  Prior to the PCR reaction, the sample RNA was transcribed into complementary DNA (cDNA) using the "cDNA synthesis system plus" kit (Amersham), according to the manufacturer's instructions and based on a value. approximate to a log near the amount of RNA present in our sample. After PCR amplification according to the technique of Shih et al., The nucleic material obtained was deposited on a gel with 2% agarose and the band visualized under ultraviolet light after labeling with ethidium bromide in a molecular weight region of approximately 100 base pairs were cut and the nucleic acids contained, extracted according to the usual protocol (Sambrook J., Fritsch EF, and Maniatis T., Molecular cloning, a laboratory manual. Cold spring harbor laboratory press, 1989), then clones using the TA cloning KIT (British Biotechnology) and the procedures recommended in the protocols attached to the kit, then sequenced, as described below: the products resulting from the purification on agarose gel are resuspended in 10 ml of water distilled. One of the properties of the Taq polymerase consists in adding an adenine to the 3 'end of each of the two DNA strands, the DNA obtained was directly inserted into a plasmid using the TA CloningTM kit (British Biotechnology) . The 2 μl of DNA solution were mixed with 5 μl of sterile distilled water, 1 ml of 10 times concentrated ligation buffer "10X LIGATION BUFFER", 2 μl of "IpCRTM VECTOR" (25 ng / ml) and 1 μl of "TA DNA LIGASE". This mixture was incubated overnight at 12 C. The following steps were carried out in accordance with the instructions of the TA Cloning kit (British Biotechnology). At the end of the procedure, the white colonies of recombinant bacteria (white) were subcultured to be cultured and allow the extraction of the plasmids incorporated according to the so-called "miniprep" procedure (Sambrook J., Fritsch EF, and Maniatis T. , Molecular cloning, a laboratory manual. Cold spring harbor laboratory press, 1989). The plasmid of each recombinant colony was cut with an appropriate restriction enzyme and analyzed on agarose gel. The plasmids having an insert detected under UV light after labeling the gel with ethidium bromide, were selected for sequencing the insert, after hybridization with a primer complementary to the Sp6 promoter present on the cloning plasmid of the TA Cloning kit. The reaction prior to sequencing was then carried out according to the method recommended for the use of the sequencing kit "Prism ready reaction kit dye deoxyterminator cycle sequencing kit" (Applied Biosystems, ref. 401384) and the automatic sequencing was carried out with the "automatic sequencer" model 373 A Applied Biosystems according to the manufacturer's instructions.

  The sequences obtained were then analyzed using Mac Vector and Geneworks software on a Genebank computer database, for the nucleic sequences, and Swiss Prot, for the amino acid sequences deduced from the reading frames highlighted in the sequences. nucleic acids. Analysis of the sequences obtained from a viral sample from thawed LM7 supernatants purified at the peak of reverse transcriptase activity on a sucrose gradient, highlighted three categories of sequences. A first category, corresponding to artefactual associations of the nucleic primers used for PCR amplification, a second category corresponding to sequences without a consistent open reading frame and a third category corresponding to retroviral sequences in the "pol" region expected by the primers used, and having at least one substantial open reading frame.

  In this third category, we could distinguish sequences strictly homologous to murine leukaemia virus strains which were found by the same methodological approach, in control tubes bringing together only water and the reverse transcriptase enzyme from Moloney-murine leukaemia virus used for the synthesis of cDNA. It was obvious that these sequences came from nucleic contaminants associated with this murine retrovirus enzyme used for this reaction step. We could also distinguish sequences represented by a number of clones ranging from one to three, and strictly homologous to known human endogenous retroviruses. These same sequences were detected in non-MS control samples, treated under the same conditions. Finally, we could also distinguish a sequence represented by a majority population of clones (approximately 42% of the clones), relative to the individual representativeness of the other sequences (always less than 5%, even% for a small number), and having partial homologies with known retroviruses in the expected "pol" region. The interrogation of the Genebank database updated to date (version 79, November 93) did not reveal any identical sequence or with significant homologies.

  This sequence is shown in Figure 1.

  It has an open reading frame in phase with the two PCR primers found at the ends but it is shorter than all of the known retroviral sequences in the region expected between these primers. A "deletion" of 45 base pairs (15 amino acids) is observed there following the sequence of the "upstream" primer, as presented in FIG. 2, while the sequences preceding the "downstream" primer " are presented.

  However, the reading frame is open and uninterrupted over the entire sequence including the primers and the deduced amino acid sequence has significant homology with the corresponding region of known retroviruses, as is presented in FIG. 2. In the internal sequence of the primers PCR, amino acids E, R, Q, P and D, normally fairly well conserved in this pol region of retroviruses and viruses with known reverse transcriptase activity (Shih et al. J. Virol.

  1989; 63, 64-75) are found preserved in the correct positions in the reading frame of our original sequence.

  Given that this sequence is sufficiently divergent from the retroviral sequences already described in the databases, it can be argued that it is a sequence belonging to a new virus which we will name MSRV-2A. This virus is a priori similar, according to the analysis of the sequences obtained, to a retrovirus, but, given the technique used to obtain this sequence, it could also be a RNA virus whose genome codes for an enzyme which incidentally has reverse transcriptase activity as is the case, for example, for the hepatitis B virus, HBV (Shih et al. J. Virol. 1989; 63, 64-75).

  EXAMPLE 2 OBTAINING CLONES DEFINING AN MSRV-2B FAMILY BY "NICHEATED" PCR AMPLIFICATION

  POL REGIONS PRESERVED RETROVIRUSES ON PREPARATIONS

  VIRIES FROM LM7 AND PLI-2 LINES.

  A PCR technique derived from the technique published by Shih et al. has been used. This technique makes it possible, by treatment of all the components of the reaction medium with DNase, to remove all traces of contaminating DNA. At the same time, by using different but overlapping primers in two successive series of PCR amplification cycles, it makes it possible to increase the chances of amplifying a cDNA resulting from a quantity of RNA initially low and further reduced in the sample by the parasitic action of DNAse on RNA; this, all the more so the DNase is used under conditions of excess activity which make it possible to eliminate all traces of contaminating DNA before inactivation of this enzyme remaining in the sample by heating at 85 ° C. for 10 minutes. Furthermore, this variant of the PCR technique described by Shih et al, was used on virion fractions, purified, as described in Example 1, as previously for the frozen supernatants of LM7 cultures, this time from the lymphoblastoid line. B spontaneous from a new case of MS, from the PLI-2 culture (ECACC n 92072201) and from the LM7PC culture (ECACC n 93010817), the latter two cultures having been obtained according to the methods which were the subject of the patent n WO 93/20188.

  After cloning with the TA cloning kit of the products amplified by this technique and analysis of the sequence using the automatic sequencer according to what is described in Example 1, the sequences were analyzed using the Geneworks software, on the latest version (79, November 93) to date of the Genebank database.

  This technique was first applied to two fractions of virions purified on sucrose from the "POL-2" isolate produced by the PLI-2 line on the one hand, and from the MS7PG isolate produced by the LM7PC line on the other hand. The cloned and sequenced sequences from these two samples correspond to four categories. A first category of sequence (type 1) corresponds to sequences amplified from retroviral nucleic material contaminating the reverse transcriptase of MoMuLV used for the step of synthesis of complementary DNA, as has already been reported previously. The second category (type 2), found in the majority of clones (55% of clones from POL-2 isolates from PLI-2 cultures, and 67% of clones from MS7PG isolates from LM7PC cultures) corresponds to a family of similar but different "pol" sequences from the endogenous human retrovirus called ERV-9 or HSERV-9. The third category (type 3) corresponds to sequences very highly homologous to the sequence attributed to the virus named MSRV-2A and obtained previously from the supernatants of culture LM7 (see example 1) by the unmodified technique of shih et al. However, the proportion of clones in this category is lower than that previously found on the viral isolate from the LM7 culture (5% of the clones from the POL-2 isolates from the PLI-2 culture, and, 6% of the clones from MS7PG isolates from LM7PC cultures). The fourth category corresponds to various sequences generally found in one copy, as well as one of endogenous retroviral type, however never found by other approaches or in other samples from culture expressing reverse transcriptase activity of LM7 type.

  The third category of sequences obtained in this experiment is identical to the "pol" sequences of the MSRV-2A type obtained previously, apart from a few mutations which can be explained by normal variability of the viral genome and by errors of the reverse transcriptase enzymes and taq-polymerase used in this technique.

  However, during this second approach, using a modification of the technique described by Shih et al., An a priori artefactual duplication of the downstream primer ("primer 3 ') was observed. This may stem from the configuration of the overlapping primers used for the two series of PCR amplification cycles ("semi-nested" technique). The consensus sequence of these clones is presented in FIG. 3. The corresponding functional reading frame, a representation of the duplication of the downstream primer (3 ′ primer) as well as the amino acid sequence visualizing the internal motif of the regions conserved in the most of the retroviruses (VLPQG and YVDD) are presented in FIG. 4. A comparison of the amino acid sequences of MSRV-2 type obtained during this experiment and during the previous one shows a notable difference in the region commonly cloned, namely the replacement of the methionine M found in the region of the PCR primer with a valine V, thus substituting the YVDD motif for the YMDD motif (see FIG. 5).

  The analysis in the databases of this last sequence, obtained by this modified technique on separate viral samples, does not change the fact that no significant homology is found with sequences already listed in the Genebank database . However, a certain analogy of the amino acid sequence with the equivalent region of known retroviruses exists, as is presented in FIG. 6. This analogy involves, as in the clones resulting from the previous technique, a deletion following the primer upstream. This sequence is, here too, compatible with an unknown retroviral genome or with an RNA viral genome (transcribed into cDNA in our protocol) coding for an enzyme having reverse transcriptase activity. In fact, the hepatitis B and cauliflower mosaic viruses (CaMV) which are not retroviruses but which have a polymerase with reverse transcriptase activity have been able to be amplified according to the technique of Shih et al. In addition, as also shown in FIG. 6, CaMV (clone CaMV QG-YM) has a deletion in this region but, unlike our sequence of MSRV-2 type, it is located on the side of the downstream primer (3 'primer).

  EXAMPLE 3 OBTAINING CLONES DEFINING AN MSRV-2B FAMILY BY "NICHEATED" PCR AMPLIFICATION

  POL REGIONS PRESERVED RETROVIRUSES ON PREPARATIONS

  OF B LYMPHOCYTES FROM A NEW CASE OF MS.

  Finally, the same PCR technique modified according to the technique of Shih et al. was used to amplify and sequence the RNA nucleic material present in a fraction of virions purified at the peak of “LM7 type” reverse transcriptase activity on a sucrose gradient, according to the protocols described in Example 1, from a spontaneous lymphoblastoid line, obtained by self-immortalization in culture of B lymphocytes of an MS patient seropositive for Epstein-Barr virus (EBV) after cultivation of blood lymphoid cells in an appropriate culture medium containing an appropriate concentration of cyclosporine A. A representation of the reverse transcriptase activity in the sucrose fractions taken from a gradient of purification of the virions produced by this line (Duc.) is presented in FIG. 7. Similarly, the culture supernatants of a line B obtained under the same conditions from a control free from multiple sclerosis were treated under the same conditions and the dosag e of the reverse transcriptase activity in the fractions of the sucrose gradient was negative everywhere (background noise) as illustrated in FIG. 8. Fraction 3 of the gradient corresponding to the B line of MS and the same fraction without activity reverse transcriptase of the non-MS control gradient, were analyzed by the same RT-PCR technique as previously, derived from Shih et al., followed by the same cloning and sequencing steps, as described in Example 1.

  The analysis of the recombinant clones selected at random provided four categories of sequences: a first category of sequence (type 1), corresponds to sequences amplified from retroviral nucleic material contaminating the reverse transcriptase of MoMuLV used for the synthesis step complementary DNA.

  The second category (type 2) corresponds to a family of "pol" sequences which are close but different from the endogenous human retrovirus called ERV9 or HSERV-9.

  The fourth category corresponds to various sequences generally found in one copy, as well as one of endogenous retroviral type, however never found by other approaches or in other samples from culture expressing reverse transcriptase activity of LM7 type. The results are presented in the appended table. It is quite remarkable that the MSRV-2 type sequences are found in the only material associated with a peak of “LM7 type” reverse transcriptase activity originating from the B lymphoblastoid lineage of MS. These sequences were not found with the material of the control B lymphoblastoid line (non-MS) in 26 randomly selected recombinant clones. Only contaminating sequences of the Mo-MuLV type and sequences without any particular retroviral analogy were found in this control. The difference in results is obviously highly significant (chi-2, p <O, 001). The analysis of the MSRV-2 type sequences obtained from line B of this MS patient is presented in FIG. 9. The analysis of the MSRV-2 type sequences obtained in all the fractions of purified virions originating respectively from the cultures LM7, LM7PC, PLI-2 and of the B lymphoblastoid line of SEP (Duc.), By the two PCR techniques derived from the technique published by Shih et al., Is presented in FIG. 5. It can be seen there that the sequence in nucleic acid is very conserved there, apart from a few mutations, and that the only variation in amino acid sequence observed in these clones concerns methionine or valine of the 3 'primer site YMDD / YVDD.

BOARD

  BREAKDOWN OF SEQUENCES OBTAINED BY PCR

  MODIFIED FROM SHIH AND COLL_ FROM CULTURE- OF LYMPHOCYTES B OF A PATIENT WITH SCLEROSIS

  IN PLATES VERSUS A PATIENT CONTROLLED

  TYPE I TYPE 2 TYPE 3 TYPE 4 MSRV-2B Lympho B 0 Il 4 8 MS patient 0% 48% 17% 35% Lympho B 5 0 0 21 non-MS patient 19% 0% 0% 81%

Claims (20)

  1 / Virus, having a reverse transcriptase activity, associated with multiple sclerosis, contained in a viral strain having a reverse transcriptase activity, chosen from the strains respectively named POL-2, deposited on 07.29.1992 with the ECACC under the access number V92072202, and MS7PG deposited on 08.01.1993, with ECACC under access number V93010816, other than the human virus, possessing reverse transcriptase activity, and at least part of the POL sequence of which is present a homogy with the same POL region of the endogenous virus ERV9 or HSERV9, contained in each of the same strains, and among the variant strains consisting of viruses comprising at least one antigen which is recognized by at least one antibody directed against at least one corresponding antigen of one or other of said viruses of the above-mentioned viral strains POL-2 and MS7PG, other than the aforementioned human virus.   2 / Virus, having a reverse transcriptase activity, associated with multiple sclerosis, produced by a cell line chosen from the cell lines respectively named PLI-2 deposited on 07.22.1992 with the ECACC under the access number 92072201 and LM7PC deposited on 08. 01.93 with the ECACC under the access number 93010817, other than the human virus, possessing a reverse transcriptase activity, and of which at least part of the POL sequence presents a homogy with the same POL region of the endogenous ERV9 or HSERV9 virus, produced by each of the same lines, and from infected cell cultures capable of producing a virus comprising at least one antigen which is recognized by at least one antibody directed against at least one corresponding antigen of one or more other virus produced by the aforementioned PLI-2 and LM7PC lines, other than the aforementioned human virus.   3 / Virus characterized in that its genome comprises a nucleotide sequence having at least 50%, and preferably at least 70% of homology with a nucleotide sequence SEQ ID N01 or its complementary sequence.   4 / Virus characterized in that its genome codes for a peptide sequence having at least 50%, and preferably at least 70% homology with a peptide sequence coded by the nucleotide sequence SEQ ID N01 or its complementary sequence.   5 / Nucleotide fragment, characterized in that it comprises a nucleotide sequence having at least%, and preferably at least 70% of homology with SEQ ID N01 or its complementary sequence.   6 / Nucleotide fragment, characterized in that it consists of a nucleotide sequence having at least%, and preferably at least 70% of homology with the nucleotide sequence SEQ ID NO1 or its complementary sequence.   7 / RNA or DNA and in particular a replication vector, comprising a fragment according to claim 5 or 6.   8 / Specific primer for the amplification by polymerization of an RNA or of a DNA specific for a virus associated with multiple sclerosis, characterized in that it comprises a nucleotide sequence having at least 50%, and preferably at least 70% homology with at least part of a fragment according to claim 5 or 6.   9 / A primer according to claim 8, characterized in that it has from 10 to 30 nucleotides.   10 / Probe capable of hybridizing specifically with an RNA or DNA specific for a virus associated with multiple sclerosis, characterized in that it comprises a nucleotide sequence having at least 50%, and preferably at least 70% d homology with at least part of a fragment according to claim 5 or 6.   11 / A probe according to claim 14, characterized in that it has at least 10 nucleotides.   12 / Use of a probe according to claim or 11 or a primer according to claim 8 or 9, for detecting and / or identifying, in a biological sample, a virus associated with multiple sclerosis.   13 / Antisense therapeutic composition in particular for the treatment of multiple sclerosis, characterized in that it comprises at least one nucleotide sequence according to claim 10 or 11.   14 / Method for detecting and / or identifying a virus associated with multiple sclerosis, in a biological sample, characterized in that an RNA and / or a DNA specific to said virus is exposed, or their complementary RNA and / or DNA, at least one probe according to claim 10 or 11.   15 / A method according to claim 14 characterized in that before exposing the RNA and / or DNA or their DNA and / or complementary RNA, to the probe, said RNA and / or said DNA are hybridized with at least one primer according to claim 8 or 9 and amplifying said RNA and / or DNA.   16 / Peptide encoded by the nucleotide sequence of the genome of a virus associated with multiple sclerosis, characterized in that it is encoded by at least part of a nucleotide fragment according to claim 5 or 6.   17 / Protein characterized in that it comprises a peptide according to claim 16.   18 / Oligopeptide characterized in that it comprises at least five contiguous amino acids of the peptide according to claim 16.   19 / A diagnostic, and / or therapeutic and / or prophylactic composition, characterized in that it comprises at least one peptide according to claim 16, or at least one protein according to claim 17 or at least one oligopeptide according to claim 18.   20 / A diagnostic, and / or therapeutic and / or prophylactic composition, characterized in that it comprises a ligand specific to a peptide according to claim 16, or to a protein according to claim 17, or to an oliqopeptide according to claim 18.