AU2922399A - Diagnosis of multiple sclerosis and other demyelinating diseases - Google Patents

Description

WO 99/53103 'L li/vYv/ouUvy 1 DIAGNOSIS OF MULTIPLE SCLEROSIS AND OTHER DEMYELINATING DISEASES FIELD OF INVENTION 5 The present invention relates to a method of diagnosing demyelinating diseases, e.g. multiple sclerosis (MS) and chronic myelopathy, comprising detecting, in a sample ma terial derived from a patient suspected of suffering from such a disease, the presence of an RGH virus nucleic acid (SEQ ID NO:1) or a subsequence or variant of said virus in a retroviral particle or an encapsidated virion-like particle by use of at least one 10 probe which specifically recognizes SEQ ID NO:1 or a subsequence or variant thereof, or by use of at least one primer which is capable of generating a nucleic acid fragment (SEQ ID NO:1) or a subsequence or variant thereof. There is also provided diagnostic agents for the diagnosis, subgrouping or monitoring of multiple sclerosis by the detec tion of the presence of SEQ ID NO:1 or a subsequence or variant thereof. The inven 15 tion further relates to a method of diagnosing, subgrouping or monitoring multiple scle rosis comprising detecting the presence of an antibody reacting specifically with an epitope of a viral particle or an encapsidated virion-like particle containing SEQ ID NO:1 or a protein or a peptide encoded by SEQ ID NO:1 or by a subsequence or vari ant of said sequence. 20 Within the scope of the invention is the combination of said methods and diagnostic agents with methods and diagnostic agents for the detection of other retroviruses such as an MSRV virus. 25 GENERAL BACKGROUND For several decades, the unexplained etiology of MS has been one of the great scien tific challenges in medicine. MS is a demyelinating inflammatory disease affecting the 30 central nervous system. There is considerable evidence that MS is immune-mediated, although the antigen or antigens against which the immune response is directed have not yet been identified. MS is usually considered to be an autoimmune disease, but intensive research for several decades has not pinpointed the auto-antigen(s) in ques tion. The extensive epidemiological studies of MS clearly indicate that, as well as ge- WO 99/53103 PCT/DK99/00199 2 netic factors, environmental agent(s) are involved, most probably virus(es). Virus(es) can initiate an autoimmune disease in various ways and can directly elicit the immune mediated reaction causing the demyelination. MS also shares both clinical and histo pathological features with retrovirus-mediated neurological diseases in animals and 5 human beings. Currently, the MS diagnosis is also troublesome. Ever since Chargot recognized the characteristic clinical and pathologic features of the disease about a hundred years ago, no single test has been found consistently positive in 100% of clinically definite 10 MS. Presently, the MS diagnosis is usually based on three criteria: - the appearance of lesions on magnetic resonance scans (MRI) - the appearance of oligoclonal bands (OB) in analyses of IgG in the cerebrospinal fluid 15 - indications of demyelination in multimodality sensory evoked response studies (EP). During recent years, several groups have performed research in order to develop inter alia diagnostic assays to be used in the diagnosis of multiple sclerosis, see e.g. WO 20 93/07259, WO 95/21256 and WO 97/06260. However, none of these patent applica tions have provided diagnostic assays with sufficiently high sensitivity and specificity to make them useful for clinical use. Thus, a reasonably simple, straightforward diagnostic assay with a high sensitivity and 25 specificity would be a vast improvement on the present situation. SUMMARY OF THE INVENTION 30 Accordingly, the present invention relates in a first aspect to methods of diagnosing demyelinating diseases including multiple sclerosis, subgrouping patients having a de myelinating disease including multiple sclerosis and/or monitoring the stage or activity of demyelinating disease including multiple sclerosis, the methods comprising detect ing, in a retroviral particle or an encapsidated virion-like particle present in a sample de- WO 99/53103 'CI/DI)99/UUIv9 3 rived from a patient suspected of suffering from demyelinating disease including multiple sclerosis, the presence of SEQ ID NO:1 or a subsequence or variant thereof. As used herein, the expressions "retroviral particle" and "encapsidated virion-like particle" include any kind of nucleic acid and/or peptide/protein fragment of such particles that are useful in 5 the methods and diagnostic agents of the invention. In a further aspect the invention pertains to a diagnostic agent for the diagnosis, sub grouping or monitoring of demyelinating diseases including multiple sclerosis by the detection, in a sample material, of the presence of SEQ ID NO:1 or a subsequence or 10 variant thereof, the diagnostic agent comprising a probe which is capable of hybridiz ing specifically with SEQ ID NO:1 or with a subsequence or variant thereof that has a sequence identity with a similar part of SEQ ID NO:1 of at least 80%. There is also provided a diagnostic agent for the diagnosis, subgrouping or monitoring of 15 demyelinating disease including multiple sclerosis by detection of the presence, in a sam ple material derived from a patient suspected of suffering from demyelinating disease in cluding multiple sclerosis, of SEQ ID NO:1 or a subsequence or variant thereof, said agent comprising at least one primer that is capable of generating a nucleic acid fragment, the nucleotide sequence of which has a sequence identity with a similar part of SEQ ID NO:1 20 of at least 80%. In yet other aspects of the invention there are provided a method of diagnosing, sub grouping or monitoring demyelinating disease including multiple sclerosis, the method comprising detecting the presence of a protein or a peptide encoded by SEQ ID NO:1 25 or by a subsequence or variant thereof, and a diagnostic agent for the diagnosis, sub grouping or monitoring of demyelinating disease including multiple sclerosis by detec tion of a peptide encoded by SEQ ID particle containing SEQ ID NO:1 or a peptide en coded by SEQ ID NO:1 or by NO:1 or by a subsequence or variant of said sequence, said diagnostic agent comprising an antibody reacting specifically with an epitope of a 30 protein or a peptide encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence.There is also provided a method of diagnosing, subgrouping or monitoring demyelinating diseases including multiple sclerosis, the method comprising detecting the presence of an antibody reacting specifically with an epitope of a viral particle containing SEQ ID NO:1 or a peptide encoded by SEQ ID NO:1 or by a subsequence or WO 99/53103 PCIT/DK99/001U99 4 variant of said sequence and a diagnostic agent for such a method that comprises an epitope of a viral a subsequence or variant of said sequence. In a still further aspect, there is provided a method of isolating and/or purifying from a 5 sample material a retroviral particle or an encapsidated virion-like particle, the method comprising contacting the sample material with an antibody reacting specifically with an epitope of a protein or a peptide encoded by SEQ ID NO:1 or by a subsequence or variant thereof. 10 In further useful aspects, the invention pertains to the use of an isolated and/or puri fied RGH virus particle or encapsidated virion-like particle, or a fragment hereof, as a diagnostic agent, to an isolated and/or purified RGH virus particle or encapsidated vi rion-like particle, or a fragment hereof, for use as a medicament and to the use of an isolated and/or purified RGH virus particle or encapsidated virion-like particle, or a 15 fragment hereof, in the manufacturing of a medicament for the treatment or preven tion of a demyelinating disease e.g by vaccination. DETAILED DISCLOSURE OF THE INVENTION 20 The present invention is based upon the finding that in a majority of multiple sclerosis patients investigated so far, nucleotide sequences related to a known endogenous ret rovirus, RGH, have been identified in samples of retroviral particles or encapsidated virion-like particles. Noteworthy, all MS patients with active disease at the time of 25 sampling were RGH-positive. No similar sequences have been found in similar samples from healthy blood donors nor from patients with other autoimmune diseases. This virus, which has previously been described by Hirose et al., 1993, as two variants, RGH-1 (SEQ ID NO:2) and RGH-2 (SEQ ID NO:1), has not previously been associated with multiple sclerosis. The sequences found in the experiments described in Example 30 2 are 81%-95% identical with RGH-2. Within the scope of the invention are subse quences and variants of RGH-2 (SEQ ID NO:1). When in the present specification and claims reference is made to RGH, the reference is meant to include all subsequences or variants which have a sequence identity with a similar part of SEQ ID NO:1 of at WO 99/53103 UI/DIL99YY/UU199 5 least 80% as described and defined in the following. RGH-1 is an example of such a variant. The experiments were designed so as to detect only virion-like encapsidated RGH 5 RNA. Any RGH sequence which is co-packaged in a virion may also be detected. Thus, if a patient is infected with another retrovirus such as a Human Immunodefi ciency Virus (HIV) or a Human T-cell Leukemia Virus (HTLV), then the RGH sequences may be co-packed without being an indication of the presence of multiple sclerosis. In cases where such an origin of the detected RGH sequence is suspected, it will be 10 necessary to further investigate whether the patient in question is in fact infected with another retrovirus and thus that the presence of the RGH sequences may be the result of the co-packing instead of being of diagnostic significance with respect to multiple sclerosis. The invention provides methods and diagnostic agents useful in that re spect. 15 In one aspect, the present invention relates to a method of diagnosing a demyelinating disease including multiple sclerosis, the method comprising detecting in a retroviral particle or an encapsidated viral-like particle present in a sample material derived from a patient suspected of suffering from a demyelinating disease such as multiple sclero 20 sis, the presence of RGH nucleic acid or a subsequence or variant thereof. In the pres ent context, the expression "sample material" includes any tissue or body fluid mate rial from which the retroviral particles or encapsidated virion-like particles may be iso lated or purified, including, but not limited to, a cell free serum sample or a sample of plasma from heparinized, EDTA-treated or citrate-treated blood, a sample of cerebro 25 spinal fluid and supernatants of cell cultures such as of mononuclear cells from hepa rinized, EDTA-treated or citrate-treated blood. The retroviral particles can be isolated and/or purified based upon e.g. size, density, charge or specific surface determinants. Examples of such methods are centrifugation in gradient media such as self-forming Optiprep gradients or antibody-based affinity isolation or purification methods. 30 As used herein, the term "diagnosing" is not meant as an absolute term implying that the diagnosis of a demyelinating disease such as multiple sclerosis can be based solely upon the detection of the presence of SEQ ID NO:1 or a subsequence or variant thereof in the retroviral particles or encapsidated viral-like particles, but rather that the WO 99/53103 PC'I'/DK99/UU199 6 presence or absence of such sequences can supplement other signs of the disease and thereby aid in the diagnosis of multiple sclerosis or any other demyelinating disease that can be diagnosed by the method of the invention. Based upon the results in Ex ample 2 it appears that a positive result is highly indicative of the person having multi 5 ple sclerosis. Actually, the present results indicate a diagnostic specificity of 100% whereas the diagnostic sensitivity is 24/33 = 72.7%. It is contemplated that when run as a routine analysis, the diagnostic specificity of the method according to the in vention will be in the range of 80% to 100%, and the diagnostic sensitivity in the range of 50% to 100%, such as 66% to 90%, e.g. 75%-80%. The invention thus in 10 its broadest aspect relates to a method of diagnosing multiple sclerosis which has a diagnostic sensitivity in the range of 50% to 100%, such as 66% to 90%, e.g. 75% 80%. In general, the method is performed on samples of plasma, serum, cerebrospinal fluid 15 or any other body samples from a person suspected of having a demyelinating disease including multiple sclerosis in order to aid in the differential diagnosis towards other diseases with similar symptoms. However, it may also be performed on samples from persons not suspected of having multiple sclerosis or other demyelinating disease, i.e. for epidemiological studies. It is possible that samples from apparently non-MS indi 20 viduals may turn out to be positive. It is likely that such a result is indicative of a per son who may later develop multiple sclerosis or indicative of the person being infected with another retrovirus, the presence of the RGH sequence being a result of co packaging. However, it cannot be ruled out that such a result may constitute a truly false positive indicating that the diagnostic specificity is not 100%. In any event, the 25 method will be within the scope of the present invention. It is possible that the subpopulation of the patients suffering from multiple sclerosis or other demyelinating disease which has a negative test result represents a subgroup having a common pathogenetic cause of their disease which is different from the sub 30 population with the positive result which may indicate that these two subgroups should be given different medical treatments as this finding may indicate a difference in the effect of the various treatments and/or indicate a different prognosis. Therefore, it may be of medical interest to determine, for an individual multiple sclerosis patient, whether he or she is RGH positive or RGH negative. Accordingly, the invention relates WO 99/53103 'I/)K9/UU199 7 in a further aspect to a method of subgrouping patients having multiple sclerosis com prising detecting the presence of SEQ ID NO:1 or a subsequence or variant thereof in a retroviral particle or an encapsidated virion-like particle, the particle being isolated or purified as described above. 5 By the term "subsequence" is meant a sequence which comprises at least 100, 150, 200, 300 or 400 nucleotides. By the term "variant" is meant a sequence which is not identical to SEQ ID NO:1, but which has a sequence identity of at least 80%, 85%, 90%, 95%, 98%, 99% or 99.5%. These two terms may be combined, i.e. the inven 10 tion comprises detection of a variant of SEQ ID NO:1 which has a sequence identity of at least 80% with a subsequence of SEQ ID NO:1 of at least 100, 150, 200, 300 or 400 nucleotides. The region of the RGH sequence which is most unique for RGH com prises the gag, po/ and env regions as well as the intervening sequences between gag and pol and between pol and env which comprises nucleotides 500 to 8214 of SEQ ID 15 NO:1. As used herein, the term "sequence identity" indicates that the sequence is compared with a similar part of SEQ ID NO:1. The best possible alignment of the sequences is made and then the degree of identity is calculated. A computer program such as GCG, 20 Wiconson Package, Version 9.1, Genetics Computer Group (GCG), 575 Science Drive, Madison, Wiconsin, USA 53711 using the defaults proposed in the program Bestfit with respect to gap weight (50), length weight (3), average match 10,000, average mismatch (-9,000) may be useful in this respect. A DNA sequence variant of RGH is defined as having a sequence identity with a similar part of SEQ ID NO:1 of at least 25 80%. When the term "nucleotide" is used in the present specification, it includes DNA and RNA as well as combinations of DNA and RNA, PNA, and combinations of PNA with DNA or RNA and similar embodiments such as described in WO 97/06260. 30 The calculation of amino acid sequence identity is preferably based on the sequence of a mature protein or peptide, i.e. without taking any possible leader sequence or modi fications such as phosphorylations or glycosylations of the amino acids into considera tion. The definition of sequence identity is as described above with respect to nucleo- WO 99/53103 tI' UlKY99/UU199 8 tides and the preferred range of sequence identity is the same. Preferably, the length of the peptides will be in the range of 7 to 100 amino acids, such as 10 to 30 amino acids. 5 The presence of SEQ ID NO:1, subsequences or variants thereof may be detected by use of at least one probe which specifically recognizes SEQ ID NO:1 or a subsequence or variant thereof, or by use of at least one primer that is capable of generating a nu cleic acid fragment (SEQ ID NO:1) or a subsequence or variant thereof. The design of such probes or primers and the principles are well known in the art, see Dieffenbach 10 et al., 1993. Suitable oligonucleotides which are useful as primers or probes are selected on the ba sis of the RGH sequence with the aid of e.g. OLIGO (Rychlik and Rhoads, 1989) and hybridization conditions which are likely to lead to the hybridization of the selected 15 oligonucleotides with SEQ ID NO:1 or a subsequence or variant. The oligonucleotides usually have a length of 6-100 bp, such as 10-30 bp. After nucleic acid amplification such as reverse transcription PCR (RT-PCR) has been performed, an amplification product is identified e.g. by gel electrophoresis and the 20 specificity of the product is confirmed e.g. by dideoxy sequencing so that it is con firmed that the product has sequence identity with a similar part of SEQ ID NO:1 of at least 80%, such as at least 85%, 90%, 95%, 98%, 99% or 99.5%. Alternative methods of nucleic acid amplification could be the ligase chain reaction (LCR) or nu cleic acid sequenced based amplification (NASBA) (Barany, 1991; Romano et al., 25 1997). If the sequence is likely to be almost identical to RGH, hybridization to a long probe may suffice, or the specificity of the amplification product could be confirmed by di gestion with a selection of restriction enzymes known to have sites within the ex 30 pected sequence of the amplification product followed by e.g. agarose or acrylamide gel electrophoresis. After such an initial validation procedure, those primers which specifically recognize SEQ ID NO:1 or a subsequence or variant thereof under the chosen hybridization con- Wo 99/SIU.3 I/ILYY/UUIW 9 ditions will be considered useful in the method of the invention. Examples of useful probes or primers are SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 as described herein as well as the primers described in Hirose et al., 1993. 5 Although the invention is not limited thereto, it is presently preferred that the primers or probes are at least 20 nucleotides long and at least 90% identical to a similar re gion on SEQ ID NO:1 and the hybridization conditions are as described in Example 1. 10 Within the scope of this invention is a diagnostic agent for the diagnosis, subgrouping or monitoring of multiple sclerosis or other demyelinating disease by the detection, in a sample material, of the presence of SEQ ID NO:1 or a subsequence or variant thereof which comprises a probe capable of hybridizing specifically with SEQ ID NO:1 or a subsequence or variant thereof, or at least one primer capable of generating a nu 15 cleic acid fragment (SEQ ID NO:1) or a subsequence or variant thereof. By the term "a diagnostic agent" is meant a means for detecting the presence of a nucleotide se quence, an epitope, a protein, a peptide or an antibody as the case may be. The fun damental principles as well as the more specific details with respect to the various dif ferent kinds of diagnostic agents are well known to the person skilled in the art of 20 producing diagnostic agents. In general, the nucleotide sequence, epitope, peptide or antibody will be bound to a carrier or support as described below, and e.g. antibodies from the sample binding to the nucleotide sequence or epitope can be detected using a secondary antibody capa 25 ble of binding to the first bound antibody and provided with a label as described be low. The substance used as label may be selected from any substance which is in itself de tectable or which may be reacted with another substance to produce a detectable 30 product. Thus, the label may be selected from radioactive isotopes, enzymes, chromo phores, fluorescent or chemiluminescent substances, and complexing agents.

WO 99/53103 re iv/vuUIvy 10 Examples of enzymes useful as labels are j-galactosidase, urease, glucose oxidase, carbonic anhydrase, peroxidases (e.g. horseradish peroxidase), phosphatases (e.g. al kaline or acid phosphatase), glucose-6-phosphate dehydrogenase and ribonuclease. 5 Enzymes are not in themselves detectable, but must be combined with a substrate to catalyze a reaction the end product of which is detectable. Thus, a substrate may be added to the reaction mixture resulting in a coloured, fluorescent or chemiluminescent product or in a colour change or in a change in the intensity of the colour, fluore scence or chemiluminescence. Examples of substrates which are useful in the present 10 method as substrates for the enzymes mentioned above are H 2 0 2 , p-nitrophenylphos phate, lactose, urea, P-D-glucose, CO 2 , RNA, starch or malate. The substrate may be combined with e.g. a chromophore which is either a donor or acceptor. Fluorescent substances which may be used as labels for the detection of the compo nents as used according to the invention may be 4-methylumbelli-eryl-phosphate, 15 4-methylumbelliferyl-D-galactopyranoside, and 3-(p-hydroxyphenyl) propionic acid. These substances may be detected by means of a fluorescence spectrophotometer. Chemiluminescent substances may be peroxidase/eosin/EDTA, isoluminol/EDTA/H 2 0 2 and a substrate therefor. 20 Chromophores may be o-phenylenediamine or similar compounds. These substances may be detected by means of a spectrophotometer. Radioactive isotopes may be any detectable and in a laboratory acceptable isotope, e.g. 1251, 1311 3 H, 3 2 P, 3 5S or 14 C. The radioactivity may be measured in a 25 gamma-counter or a scintillation counter or by autoradiography followed by densito metry. Complexing agents may be Protein A, Protein G (which forms a complex with immuno globulins), biotin (which forms a complex with avidin and streptavidin), and lectin 30 (which forms a complex with carbohydrate determinants, e.g. receptors). In this case, the complex is not in itself directly detectable, necessitating labelling of the substance with which the complexing agent forms a complex. The marking may be performed with any of the labelling substances described above.

WO 99/53103 I l !i /iUu I y 11 As indicated above, this type of diagnostic agent normally comprises the nucleotide sequence or epitope bound to a carrier or support so that appropriate washing or other treatments as appropriate may be performed without risk of appreciable loss of the bound nucleotide sequence or epitope. The carrier or support is normally solid, and the 5 nucleotide sequence or epitope is bound to the carrier or support by any suitable mode of binding, such as hydrogen bonding, hydrophobic bonding, van der Waals' forces, covalent bonding, etc. In an embodiment of the invention, the nucleotide sequence or epitope of the inven 10 tion may be indirectly coupled to a solid support via a bridging compound or "linker". The linker, which is designed to link the solid support and the nucleotide sequence or the epitope, may be hydrazide, Protein A, glutaraldehyde, carbodiimide, or lysine. The solid support employed is e.g. a polymer or it may be a matrix coated with a 15 polymer. The matrix may be of any suitable solid material, e.g. glass, paper or plastic. The polymer may be a plastic, cellulose such as specially treated paper, nitrocellulose paper or cyanogenbromide-activated paper, silicone, silica, or a polysaccharide such as agarose or dextran. Examples of suitable plastics are latex, a polystyrene, polyvinyl chloride, polyurethane, polyacrylamide, polyvinylacetate and any suitable copolymer 20 thereof. Examples of silicone polymers include siloxane. The solid support may be in the form of a tray, a plate such as a mitrotiter plate, e.g. a thin layer or, preferably, strip, film, threads, solid particles such as beads, including Protein A-coated bacteria, or paper. 25 In a further aspect, the invention relates to a diagnostic agent which comprises an an tibody as defined above, preferably a monoclonal antibody. The diagnostic agent may comprise the antibody coupled to a carrier or support. Alternatively, the diagnostic agent may be in the form of a test kit comprising an antibody as defined above in a 30 container. The diagnostic agent may be used in the diagnosis of multiple sclerosis, comprising contacting a sample of a body fluid, such as a blood sample or a sample of cerebrospinal fluid, with a diagnostic agent comprising an antibody as defined above, and determining the presence of any nucleotide sequence or epitope from the sample binding to the antibody. Optionally, the sample may have been purified by appropriate WO 99/53103 PCT/DK99/00199 12 means prior to contact with the diagnostic agent in order to increase the relative amount of nucleotide sequence or epitope present in the biological sample. The diagnostic agent may be one which is suited for use in an agglutination assay in 5 which solid particles to which the antibody is coupled agglutinate in the presence of a nucleotide sequence or an epitope of the invention in the serum sample subjected to testing. In this type of testing, no labelling of antibody is necessary. For most uses it is, however, preferred that the antibody is bound to a carrier or support, using, e.g., the techniques described above in connection with diagnostic agents based on a nu 10 cleotide sequence or epitope, and that the binding of nucleotide sequence or epitope from the sample to the diagnostic agent is detected using a secondary antibody which is capable of binding to the thus bound nucleotide sequence or epitope, the second antibody being provided with a label for the detection of bound secondary antibody. The substance used as label may be selected from any substance which is in itself de 15 tectable or which may be reacted with another substance to produce a detectable product. Thus, the label may be selected from radioactive isotopes, enzymes, chromo phores, fluorescent or chemiluminescent substances, and complexing agents, all of which are described in greater detail above. 20 In one preferred embodiment of the invention it is preferred that the antibody used in the diagnostic agent is a monoclonal antibody as this generally provides a higher pre cision and accuracy of the assay, at the same time possibly requiring less time to per form. Furthermore, a mixture of two or more different monoclonal antibodies may be employed as this may increase the detection limit and sensitivity of the test. The 25 monoclonal antibody may be obtained by the method described below. Antibodies possessing high avidity such as polyclonal antibodies may be selected for catching techniques. The antibody used in the present method is preferably in substantially pure form puri 30 fied according to suitable techniques well known in the art in order to improve the precision and/or accuracy of the assays of the invention. The invention also encompasses novel probes and primers which are useful in said diagnostic agent, e.g. long probes as described above having a length of at least 100 WO 99/53103 PCTI/DK99/U00199 13 bp. When used for diagnostic or similar purposes the probe will usually be marked by means of e.g. a radioactive isotope, an enzyme such as peroxidase or alkaline phos phatase or be detectable by any other means. 5 Detection of virion-associated MSRV-RNA in serum of patients with multiple sclerosis has recently been published by Garson et al., 1998. In this paper it is indicated that it is presently not known whether MSRV itself is endogenous or exogenous. Although the present inventors do not want to be bound by any specific theory, it is possible that multiple retroviruses, e.g. RGH and MSRV virus, may be present simultaneously 10 or consecutively in multiple sclerosis patients. One possibility is that a general activa tion of endogenous retroviruses takes place in the disease process, another that MSRV stimulates the activation of RGH virus or that RGH stimulates the activation of MSRV virus. 15 It is thus not unlikely that RGH virus as well as MSRV virus may be present at the same time which may represent a means for increasing inter alia the diagnostic speci ficity and sensitivity of the test. Furthermore, the testing of the same samples for the presence of both viruses may represent a means for further investigating the patho genesis of these viruses. If a general activation takes place it is likely that all the pa 20 tients will be positive for both retroviruses at substantially the same time provided that for both retroviruses viral material is present in sufficient amounts to be detectable. Consequently, under these circumstances the diagnostic sensitivity of a test will be increased, if both viruses are searched for. 25 If, on the other hand, MSRV stimulates the activation of RGH virus it is likely that some of the patients positive for MSRV virus will be negative for RGH virus at the first investigation but at a later point in time change to be positive for RGH virus. Also, some of the patients tested positive for RGH virus will be negative for MSRV virus in dicating that this virus is no longer present in detectable amounts. Such findings may 30 be useful in establishing the stage of the disease process and also with respect to de termining the kind of medical treatment which should be given to the individual patient and/or determining the prognosis of the individual patient. Based upon the present re sults it seems that the presence of SEQ ID NO:1 or a subsequence or variant thereof WO 99/53103 PCT/DK99/00199 14 indicates an active stage of disease. It is concievable that the same applies if is as sumed that RGH stimulates the activation of MSRV virus. In a further embodiment, the invention relates to a method of monitoring the stage or 5 activity of multiple sclerosis or any other demyelinating disease, the method com prising detecting the presence of SEQ ID NO:1 or a subsequence or variant thereof in a retroviral particle or an encapsidated virion-like particle that is present in a sample ma terial derived from a patient suffering from multiple sclerosis or another demyelinating disease. 10 Within the scope of the present invention is a method of detecting SEQ ID NO:1 or a subsequence or variant thereof which method further comprises detecting the pres ence of one or more non-RGH retrovirus(es), such as an HIV, HTLV, MSRV or HERV virus, or a subsequence or variant of said retroviruses, in a retroviral particle, e.g. a 15 particle isolated or purified from cell free plasma samples as described above e.g. by means of affinity purification. This method may also be useful for subgrouping or monitoring patients having a demyelinating disease including multiple sclerosis. One embodiment of the invention provides a diagnostic agent as described above 20 which comprises further sets of primers or probes, the individual set of probes each being capable of hybridizing specifically with one other retrovirus, such as an HIV, HTLV, MSRV or another retrovirus, or which individual set of primers is capable of generating a nucleic acid fragment of a sequence coding for an HIV, HTLV, MSRV or another retrovirus or a subsequence or variant thereof. Examples of such primers and 25 probes are well known within the art. A preferred embodiment is a method wherein an MSRV virus is detected, said method being accomplished by use of at least one probe which specifically recognizes an MSRV virus or a subsequence or variant thereof, or a primer which is capable of gen 30 erating an MSRV nucleic acid fragment or a subsequence or variant thereof. The terms "subsequence" and "variant" are defined as outlined above with respect to RGH. A number of different MSRV virus sequences which may be useful in the generation of probes and primers as well as specific probes and primers for the detection of MSRV WO 99/53103 PCT/DK99/00199 15 virus are described in WO 97/06260 which is hereby incorporated by reference. A useful sequence which is present in a retroviral particle from at least some patients infected by the MSRV virus and/or in whom the MSRV virus has been reactivated, can be selected from the group consisting of nucleotides having sequences identical, par 5 tially or in total, or equivalent to the sequence defined in SEQ ID NO: 1, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 88, SEQ ID NO: 89 the sequences of the gene pol of MSRV, the gene env of MSRV, the gene gag of MSRV and the sequences defined in 10 paragraphs (d)-(g) of claim 13 of WO 97/06260. Under the circumstances where the RGH nucleotide sequence(s) is not a co-packaging phenomenon, it is likely that the sequence codes for at least one protein or peptide. In these cases an antibody which binds specifically to such a protein or peptide may be 15 raised in an individual or may be produced in an animal by conventional methods well known to the person skilled in the art. Within the scope of the invention are mono clonal as well as polyclonal antibodies raised against such RGH-encoded antigens. It may be possible to detect a protein or a peptide encoded by SEQ ID NO:1 or a subse quence or variant of said sequence in plasma e.g. by means of an antibody reacting 20 specifically with an epitope of such a protein or peptide. Within the scope of the in vention is a peptide encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence, an antibody reacting specifically with such a peptide as well as a method of detecting such a peptide in plasma and a diagnostic agent comprising such an anti body. 25 Purification of a protein or a peptide encoded by SEQ ID NO:1 or a subsequence or variant of said sequence from plasma samples may be a useful means of purification. Examples of procedures which may be useful for the purification include: (i) immunoprecipitation or affinity chromatography with antibodies, (ii) affinity chro 30 matography with a suitable ligand, (iii) other chromatography procedures such as gel filtration, ion exchange or high performance liquid chromatography or derivatives of any of the above, (iv) electrophoretic procedures such as polyacrylamide gel electro phoresis, denaturating polyacrylamide gel electrophoresis, agarose gel electrophoresis WO 99/53103 PCT/DK99/00199 16 and isoelectric focusing, (v) any other specific solubilization and/or purification tech niques. The invention further relates to a method of diagnosing, subgrouping or monitoring 5 multiple sclerosis or any other demyelinating disease, the method comprising detecting the presence of an antibody reacting specifically with an epitope of one or more pro teins encoded by SEQ ID NO:1 or a subsequence or variant of said sequence. Apart from being useful in the diagnosis of multiple sclerosis, the presence of e.g. an in crease in the antibody titer may be indicative of an activation or reactivation of the 10 disease process which may have implications with respect to treatment or prognosis. Possible protein sequences of RGH are known from Hirose et al., 1993. Based upon the diagnostic significance of the presence of an RGH nucleotide sequence in a retrovi ral particle, it is likely that it will also be possible to detect in plasma or serum samples 15 or in samples of cerebrospinal fluid an antibody reacting specifically with an epitope of one or more proteins encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence and thereby provide or aid the diagnosis, subgrouping or monitoring of mul tiple sclerosis. 20 Epitopes of a protein or a peptide encoded by SEQ ID NO:1 or a subsequence or vari ant of said sequence may be deduced by methods well known in the art, cf. WO 97/06260. The epitope may constitute a consecutive subsequence of a protein or of a peptide encoded by SEQ ID NO:1 or a subsequence or variant hereof, or be an epitope which is present in a more spatial configuration e.g. including amino acids which are 25 positioned with some distance apart in the linear outline of the sequence of a protein or a peptide. The epitope can also - entirely or partially - consist of compounds associ ated with one or more of the amino acids, should they be modified in various ways, e.g. by glycosylation. The peptide comprising an epitope can, e.g., be synthesized by the conventional Merrifield synthesis technique and may be coated or bound to a suit 30 able carrier in order to provide a diagnostic agent useful for the detection of an anti body against an epitope of a peptide encoded by SEQ ID NO:1. Experiments for de tecting an antibody reacting specifically with an epitope of a peptide encoded by SEQ ID NO:1 or a subsequence or variant of said sequence are outlined in Example 4.

WO 99/53103 PCT/DK99/0U0199 17 Within the scope of the invention is a diagnostic agent for the diagnosis, subgrouping or monitoring of multiple sclerosis or any ohter demyelinating disease by the detection of an antibody reacting specifically with an epitope of a protein or a peptide encoded by SEQ ID NO:1 or a subsequence or variant of said sequence as well as such a diag 5 nostic agent which further comprises an epitope from one or more other retroviruses, such as an HIV, HTLV, MSRV or HERV virus epitope. A useful epitope which is recognized by at least some sera from patients infected by the MSRV virus and/or in whom the MSRV virus has been reactivated, can be selected 10 from the group consisting of peptides encoded by any open reading frame found in MSRV or subsequences or variants thereof. As examples such peptides may have se quences identical, partially or in total, or equivalent to the sequence defined in the se quences SEQ ID NO: 39, SEQ ID NO:41 to SEQ ID NO: 44, SEQ ID NO: 63 and SEQ ID NO: 87 in WO 97/06260. 15 The combined test of the presence of an antibody reacting specifically with an epitope from an RGH protein or peptide and an antibody reacting specifically with an epitope of a peptide from at least one other retrovirus may provide supplemental information which is useful in the diagnosis of multiple sclerosis or another demyelinating disease, 20 the determination of disease stage, selection of medical treatment and the determina tion of the prognosis of the individual patient as outlined above with respect to diag nostic agents comprising probes or primers. Further, the presence of e.g. an increase in the antibody titer against MSRV may be indicative of an activation or reactivation of the disease process which may have implications with respect to treatment or progno 25 sis. It is concievable that an isolated and/or purified RGH virus particle is useful as a diagnostic agent for the purposes as described herein. Accordingly, in an interesting aspect of the in vention, there is provided the use of such particles as a diagnostic agent. 30 Further concievable uses of an isolated and/or purified RGH virus particle according to the invention is its use as a medicament e.g. for the treatment or prevention of demyelinating diseases including multiple sclerosis or its use in the manufacturing of a medicament for the treatment of such diseases.

WU 99/WSiU Yl I/lVWUUIY 18 The invention is further illustrated in the following, non-limiting examples. EXAMPLE 1 5 Establishment of RGH-producing cell lines from peripheral blood from MS patients The following is a description of variants of the human endogenous retrovirus RGH as the retrovirus produced by spontaneously formed B-lymphoblastoid cell lines from peri 10 pheral blood mononuclear cells from several MS patients. Spontaneously formed B-lymphoblastoid cell lines from peripheral blood mononuclear cells from MS patients produce type C retrovirus-like particles in addition to the ex pression of Epstein-Barr virus (EBV) proteins and occasionally mature EBV particles 15 (Sommerlund et al., 1993; Munch et al., 1995). The retrovirus particles possess reverse transcriptase activity and share a few anti genic determinants with HTLV-1, but are distinct from the known retroviruses at the antigenic level (Christensen et al., 1997). The B-lymphoblastoid cell lines were estab 20 lished from venous blood from MS patients. The cell lines described in Table 1 arose spontaneously after long-term cultivation. The MS patients were from the Neurology Dept., Aarhus University Hospital; the Dermatology Dept., Marselisborg Hospital, Aarhus; the MS Hospital in Ry or the Dept. of Internal Medicine, Middelfart Hospital (all in Denmark). All blood samples were obtained with informed consent. Blood sam 25 pies were obtained by drawing blood into Venoject VT100H tubes containing 150 u.s.p.u. sodium heparine (Meda, Denmark). The heparinized blood was subjected to Ficoll-lsopaque density gradient centrifugation. For establishing long-term cultured, spontaneously transformed cell lines, mononuclear cells were isolated, seeded at 20 x 106 cells/5 ml and cultivated in RPMI 1640 (Seromed) supplemented with 200 i.u./ml 30 penicillin (Leo), 0.2 mg/ml streptomycin (Rosco), 290 mg/mI glutamine (Sigma), 10 mM HEPES (Bioproduct) and 10% heat inactivated human serum (serum pool ob tained from the blood bank at Skejby Hospital, Denmark) in Falcon Primaria bottles. After an initial period, the cells were subcultured three times a week at a density of 0.5 x 106 cells/ml. The cell lines arose spontaneously after long-term cultivation (more WO 99/53103 PCT/DK99/U0199 19 than two months) and are continuously growing. The established cell lines were sup plemented with 10% heat inactivated fetal calf serum (Whitaker) instead of human serum. The human origin of the line was confirmed by RFLP mapping and HLA-DQb typing. The cell lines were tested for mycoplasmal contamination and found negative 5 by repeated analyses (standard Hoechst stain). Table 1 MS B-lymphoblastoid cell lines 10 Patient Age, Diagnosis Duration, No. Sex years years 15 MS1533 M 30 Chronic progressive 7 myelopathy MS1844 F 40 Benign MS 11 20 MS1845 M 33 Relapsing-Remitting MS 2 MS1851 F 24 Primary progressive MS 2 25 Expression of RGH variants in retroviral particles in B-lymphoblastoid cell lines estab lished from MS patients 30 The retrovirus produced by the spontaneously formed B-lymphoblastoid cell lines from peripheral blood mononuclear cells from several MS patients can be identified at the nucleic acid level by concentration and purification of the retroviral particles, isolation of the genomic RNA in the retroviral particles, RT-PCR with the genomic RNA as tem plate and subsequent sequencing of the obtained amplicons. Retroviral particles were 35 purified from cell line supernatants by ultracentrifugation in self-forming Optiprep gra dients.

WU 99/53I1Ui Pt I/IKY/UUI 99 20 1.3-2 litres of suspension cultures were centrifuged at 4oC for 30 minutes at 2500 x g. The cell-free supernatant was aspirated, layered on a cushion of 4 ml of 50% Optiprep in NaCI/Hepes (Nycomed) in 60 ml tubes and ultracentrifuged at 4 0 C for 2 hours at 45,000 x g (Beckmann ultracentrifuge). Optiprep is an iodinated, nonionic 5 density gradient medium (Nycomed Pharma, Norway) which, in contrast to sucrose, maintains intact retroviral particles. The cushion and proximal overlaying retrovirus-containing medium was mixed, filtered through a 0.45 gm filter (Acrodisc 32, Gelman Sciences) to remove any cellular de 10 bris, adjusted to 20% Optiprep and centrifuged in 11.2 ml Optiseal (Beckmann) tubes in a Beckmann NVTi rotor at 364,000 x g for 3.5 hours. 400-500 pl fractions (12 drops) were collected after centrifugation. RNA was purified from the 5-6 fractions containing RT activity. Poly-A RNA was purified by lysing these fractions directly in 5 volumes of lysis-binding buffer (Dynal, Norway), adding 100 pl of beads/sample 15 (mRNA Direct kit, Dynal, Norway) and following the manufacturer's instructions. If not used directly, the RNA-coupled beads were stored in 80% EtOH at -80 0 C. Before use, each RNA sample was treated in a 20 tl final volume with amplification grade DNAse (Life Technologies) according to the manufacturer's instructions. After cooling on ice, the buffer was exchanged with 10 pl of DEPC-treated ddH 2 0, and 2 pl aliquots were 20 used for each cDNA synthesis. Using RT-PCR on retroviral RNA templates isolated from cell culture supernatants as described above, with subsequent sequencing of the cloned amplicons, several frag ments with homology to RGH were identified (Hirose et al., 1993) in the retroviral par 25 ticles produced by the cell lines indicated in Table 1 (MS1533, MS1844, MS1845 and MS1851). RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer) according to the manufacturer's suggestions, except that 1 unit of Taq polymerase was used per 30 reaction. All relevant controls were included. First strand cDNA synthesis was primed with random hexamers, in conjunction with the bead-oligo-dT complexed RNA tem plate.

WO 99/53103 r lim~WuuIy 21 Primer sets for the PCR amplification of gag and env were as follows: 5'-CTTTTATTACCCAATCTGCTCCCGAYAT-3' (SEQ ID NO:3) 5 5'-TTAGTGGTGGACAGTCTCTTTTCCARTG-3' (SEQ ID NO:4) (45 0 C), (Medstrand et al., 1992) 5'-ATTTTATTACCCAATCTGCTCCAAACAT-3' (SEQ ID NO:5) 10 5'-AGGTGAGTTGAACAGTCTGATTTTTA-3' (SEQ ID NO:6) (50 0 C) 5'-CGTTTACATATCACTCCCTTCCTAGTCTCTGT-3' (SEQ ID NO:7) 5'-GCATTAACCTTGACTATGTCTTTAGCTCCAG-3' (SEQ ID NO:8) (50oC). 15 5'-GATCCTCCCCACTGGGTTCACCATT-3' (SEQ ID NO:9) 5'-GGAAGTATTGGAGGGTGCCCTGCC-3' (SEQ ID NO: 10) (60oC). 20 All reactions were run in a Perkin Elmer DNA Thermocycler. Conditions for first strand synthesis: 5 minutes at room temperature, 45 minutes at 42oC, and 5 minutes at 95 0 C, followed by cooling to 4oC. PCR: one cycle for 2 minutes at 95 0 C was followed by 45/50 cycles with annealing temperatures as indicated above: 1 minute at 941C, 2 minutes annealing, 3 minutes at 72 0 C with 10 seconds extension. This was followed 25 by a final extension for 7 minutes at 72oC. PCR products were analyzed by agarose gel electrophoresis, and each product was cloned in pUC using the SureClone Ligation kit (Pharmacia) and sequenced using the ABI Prism kit (Applied Biosystems) on an automatic sequenator (ABI 377). 30 5-30 clones from each product were analyzed for each positive RT-PCR. Clones were sequenced in both orientations and appeared to be variants of RGH-2.

WU /,bblUJ YCI/lJiK9/UUI 9 22 EXAMPLE 2 Expression of RGH variants in retroviral particles in peripheral blood from MS patients 5 The retrovirus produced by the spontaneously formed B-lymphoblastoid cell lines from peripheral blood mononuclear cells from MS patients can also be identified at the nu cleic acid level in clinical samples, i.e. peripheral blood from a large proportion of MS patients, by concentration and purification of the retroviral particles in the plasma or serum fraction, isolation of the genomic RNA in the retroviral particles, RT-PCR with 10 the genomic RNA as template, and subsequent sequencing of the obtained amplicons. RGH sequence variants at particle level were demonstrated to be associated with MS in vivo by performing RT-PCR analysis on clinical specimens, i.e. blood samples from MS patients, from patients with autoimmune diseases, and from healthy controls. 15 RNA extracted from particles isolated from plasma was assayed by RT-PCR with sub sequent sequencing of the cloned amplicons. All blood samples were obtained with informed consent from either healthy volunteers or from patients at the Neurology Dept., Aarhus University Hospital; the Dermatology 20 Dept., Marselisborg Hospital, Aarhus; the MS Hospital in Ry; or the Dept. of Internal Medicine, Middelfart Hospital (all in Denmark). Blood samples were drawn at the re spective clinics into VT-100 SCPDA Venoject tubes containing 1.4 ml of CPD-adenine solution (Meda, Denmark) and delivered by hand immediately after drawing. To avoid contamination from nucleic acids, the samples were processed immediately without 25 any freeze-thawing procedures (plasma obtained by standard separation is not entirely cell-free and freeze-thawing will disrupt remaining cells and release intracellular nucleic acids). All samples were processed in the inventors' laboratory. Cell- and debris-free plasma 30 samples were obtained from the citrate blood samples (40 ml of blood -+-> appr. 10 ml of plasma). After the initial separation by centrifugation at room temperature for 15 min. at 500 x g, the plasma was aspirated and subjected to another centrifugation at 4oC for 30 minutes at 1000 x g to pellet any remaining cells. The cell-free plasma was aspirated and samples layered on 50% Optiprep, ultracentrifuged and filtered as de- WO 99/53103 PCT/DK99/U00199 23 scribed in Example 1. The retrovirus/Optiprep mixture was directly lysed in 5 volumes of lysis-binding buffer (Dynal, Norway) and poly-A RNA was purified on 50 I of beads/sample using the mRNA-Direct kit (Dynal, Norway) as described in Example 1. 5 If not used directly, the RNA-coupled beads were stored in 80% EtOH at -80 0 C. Be fore use, each RNA sample was treated in a 20 gl final volume with amplification grade DNAse (Life Technologies) according to the manufacturer's instructions. After cooling on ice, the buffer was exchanged with 10 gl of DEPC-treated ddH 2 0, and 2 p1 aliquots were used for each cDNA synthesis. 10 The RT-PCR conditions were as described for RT-PCR in Example 1. PCR products were analysed by agarose gel electrophoresis, and each product was cloned in pUC using the SureClone Ligation kit (Pharmacia) and sequenced using the ABI Prism kit (Applied Biosystems) on an automatic sequenator (ABI 377). 5-30 clones from each 15 product were analyzed for each positive RT-PCR. The results of the analyses, including characterization of the patients included in the study, are shown in Table 2. RGH sequence variants in particles were thus demonstrated in cell-free plasma sam ples from 24 of 33 MS patients. The sequences were absent in all of 29 cell-free 20 plasma samples from patients with autoimmune diseases as well as in all of 20 cell free plasma samples from healthy controls. The expression of RGH sequence variants in particles is thus specific for MS. All MS patients with active disease at the time of sampling were RGH-positive. 25 A large subgroup of MS cases is thus associated with the production in the blood of retroviral particles containing RGH sequence variants.

WU 99I51Ui PCT'/DK99/00199 24 Table 2 All MS patients had clinically definite MS, the MS type and disease activity is indi cated. Chronic progressive MS with no stationary phase, and exacerbations in relaps ing/remitting MS were defined as active MS. RR: relapsing-remitting, SP: secondary 5 progressive, PP: primary progressive, SLE: systemic lupus erythematosus. RGH positive by RT-PCR on plasma Type No. of patients Sex Age, years Diagnosis Duration, years Multiple 6 F 16 - 50 RR (2 active) 1 - 11 Sclerosis 2 M 50 - 52 9 - 15 n = 24 5 F 34 - 56 SP (2 active) 4 - 20 7 M 40 - 72 (2 active) 9 - >20 4 F 33 -47 PP (3 active) 3 -7 RGH negative by RT-PCR on plasma Multiple 1 F 35 RR 10 Sclerosis 1 M 30 1 n=9 1 F 34 SP 15 3 M 40-50 9-15 2 F 31-36 PP 2- 5 1 M 36 6 Healthy 13 F 24 - 53 Control 7 M 27 - 34 n = 20 Patient 3 F 56 - 62 Diabetes mellitus Control 5 M 50 - 63 n = 29 1 M 68 Hairy cell leukemia 1 F 42 Prurigo nodularis Hide 2 F 35 - 61 Colitis ulcerosa 2 M 31 -32 1 F 52 SLE 1 F 55 Arteritis temporalis 1 F 53 Rheumatoid arthritis 4 M 47 -61 1 F 63 Thyreotoxicosis 1 M 55 1 M 47 Sarcoidosis -1 M 50 Collagenosis 1 F 58 Polymyositis 1 F 76 Myelomatosis I M 58 Leukemia/lymphoma 1 F 72 Polvnivalgia rheumatoides WU 99/IU30 PLCI/UK99/UUI199 25 EXAMPLE 3 Characterization of expressed RGH sequence variants 5 In Example 1 and Example 2 expressed RGH retroviral sequences could be identified in retroviral particles produced by spontaneously formed B-lymphoblastoid cell lines from peripheral blood mononuclear cells from several MS patients (Example 1) and in clini cal samples, i.e. the particulate fraction of plasma from peripheral blood from a large proportion of MS patients (Example 2) by RT-PCR with gag and env primers on geno 10 mic RNA from retroviral particles. RGH belongs to the RTVL-H/HERV-H (priming of transcription with tRNAHis) family of endogenous retroviruses. This family of type-C retroviruses is related to the onco retroviruses human T-cell leukemia virus (HTLV-1/-2), to bovine leucosis virus (BLV), 15 and to ERV-9. A genomic RTVL-H clone which potentially encodes functional proteins has been described (Wilkinson et al., 1991). RGH particles as such have not been reported previously. Two RGH clones have been described: RGH-1 and RGH-2. The published RGH-1 clone is 4869 bp long and en 20 compasses a partial pol and an env 3'LTR region and thus lacks gag, whereas the pub lished RGH-2 clone is 8715 bp long and includes the complete coding potential: 5'LTR gag-pol-env 3'LTR. They are present in about 100 copies/haploid genome (Hirose et al., 1993). The majority of cDNA clones isolated from MS patient material are closely related to the full-length clone RGH-2 as they contain gag sequences. 25 Each and every one of the genomic RGH copies in each and every individual person's genome represents a DNA sequence variant of RGH as defined above, with the poten tial of being transcribed to an RNA sequence variant. 30 Examples of RGH sequence variants are listed in Table 3. The sequence variants were obtained using the RT-PCR strategy outlined in Example 1 and Example 2 (Table 3). Examples of expressed RGH sequence variants are listed in Table 3 below.

WO 99/53103 T/ILY/UUJ1y 26 Table 3 SEQ ID NO:11 CCCAATGCAA CTAATCCCAA ATCTTCCTCC TTTCCCTCCC GCCTGTCCCC TCAGTACCAA CCCCAAGCGT CGCTGAGTCT TTCTAATCTT CCTTTTCTAC AGACCCATCT GACCTCTCCC CTCCTCACCA GGTCGAGCTA GGTCCCAATT CTTCTTCAGC CTCCACTCCT CCACCCTATA ATCCTTTTAT CACCTCCCCT CCTCACACCT GGTCCAGCTT ACAGTTTTCGT TCTGCGACTA GCCTTCCCCC ACCTGCCCAG CAATTTCCTC TTAAAAAGGT GG SEQ ID NO:12 TCCAGTGCAA CTCATCCCAA ATGTTCCTTC TTTCCCTCCC ATCTGTCCCC TCAGTACCAA CCCCAAGCGT CACTGAGTCT TTCTAATCTT CCTTTTGTAC AGACCCATCT GACCTCTCCC TTCCTCCCCA GGTCGTCCTC GCCAGGCCGA GCTAGGTCCC AATTCTTCCT CAGCCTCCAT TCCTCCACGC TGTAATCTTT TTATCACCTC CCCTCCTCAC ACCTGGTCCG GCTTACAGTT TCGTTCTGTG ACTAGCCCTC CCCCACCTGC CCAGCAATTT ACTCTTAAAA AGGCGG SEQ ID NO:13 CCCGACATTA AATAAAGCTC CAAAAATTAG ATTCCAGCCC GGAAACCCCA CAACAGGACT TAACCTCACC TTCAAGGTGT ACAATAATAG AGAGGAGTCT CAATTCTTGC CTCTGCTGTG AGAGAAACCC CAGCCACATC TCCAGCACAC AAGAACTTCA AAATGCCT SEQ ID NO:14 CCAAACATTA AATAAAACTC CAAAAATTAA ATTCCGGCCC TCAAACCCCA CAACAAGGCT TAATTAACCT CACCTTCAAG GTGTTCAATA ACAGAGTAGA GGCAACCAAG TAGCAATGTA TTTCTAAGTT GCAATTCCTT ACCTCCACTG TAAGACAAAC CCCAGCCACA TCTCCAGCAC ACAAGAACTC CAAATGCCCG SEQ ID NO:15 CCAGAATAAA GCTGTGTCCA TCGGACAGCC AGCCTAATCC CTCCTCTTCC TCCTGGAAGT CACAAGTATT CTCCCCTACT TCCCTTAAAC TCACTCGTAT TTCTGAAGAA CAGTAATAGC CCTTATGAGC CTAATACATC CCTTCATTCT ATTAGATCTG TTCGTCCTTA CCCTACTTTT TGCAACAGGG CTTTACGAAG TCACCCTNAC CACTTGGCCT GATCCCCAAA AAACTAGTCA ACCCTACTAT CTTCTGTCTA GTCATACTCC TATTCTCCAT TCTCAANTAC TTATAAATGC CCTACTCTTG TTTCCACTGC TGGTTTANAC TGTTTCTTCA AGCCATCACA GCTGATATCT CTTGGTGCTA TCCCGCAACT GCCACTCTTA ACTCCCTCTT AGAGTGGATA GATGATCTTT GC SEQ ID NO: 16 CCAGAATAAA GCTCTGTCCA TCAGACAGCC AGCCTAATCC CTCCTCTTCC TCCTGGAAGT CGCAAGTACT CTCCCCTACT TCCCTTAAAC TCACTCATAT TTCTGAAGAA CAGTAATAAC CCTTATGAGC CTAATACATC CCTTCATTCT GTTAGGTCTA TTCGTCCTTA CCCTACTTTT TGCAACGGGG CTTTATGACG TCACCCCCAC CACTTAGGCT GAGCCCCAAA AAACTGTCA TCCCTACTAT TTTCTGTCTA GTCATACTCC TATTCTCTGC TCTTAACTAC TTATAAATGC CCTACTCTTG TTTACACTGC CGGTTTACAC TGTTTCTTCA AGCCATCACA GCTGATATCT CTTGGTGCTA TCCCCAAACC ACTACTCTTA TTCCCTCTTA GAGTGGGTAG ATGATCTTTG

C

WO 99/53103 PI'I/DKY99/UUI99 27 EXAMPLE 4 The present invention also relates to diagnosis and prognosis by immunological means 5 such as characterization of the RGH-associated antibody response or characterization of RGH-associated antigens in samples from patients as well as other immunological aspects such as production of RGH-specific antibodies and RGH encoded peptide anti gens for diagnostic and purification purposes. 10 RGH encoded antigens RGH encoded peptide antigens can be acquired by different means, e.g. by chemical synthesis of peptides deduced from the nucleotide sequence or by purification from different sources such as e.g. various expression systems based on RGH nucleic acid 15 sequences or by purification from patient samples or patient derived cell cultures. As an example, Optiprep-purified RGH particles from 800 ml supernatant from various cell lines are separated by 4-20% SDS-PAGE, transferred to a membrane, cut in strips and screened against a panel of sera from MS patients as well as controls thus providing a means for selecting suitable epitopes for further investigation. 20 Having defined peptides with an amino acid sequence derived from the known se quences of RGH, e.g. the gag and env regions, RGH subsequences or sequence vari ants, the peptide comprising an epitope can be chemically synthesized, e.g. by the conventional Merrifield synthesis technique. 25 Peptides or proteins may be produced in commercially available prokaryotic or eukary otic expression systems. Typical prokaryotic expression systems are based on E. coil whereas typical eukaryotic systems are based on fungal cells including yeast cells, or other eukaryotic cells such as e.g. baculovirus-controlled expression in insect cells. A 30 prokaryotic expression system (GST Gene Fusion System, Pharmacia Biotech) is de scribed in the following: Based on the sequence of RGH, RGH subsequence or sequen ce variant: two complementary oligonucleotides with suitable cloning sites at the 5' and 3' end are synthesized. The oligonucleotides typically correspond to sequences encoding 10 - 30 amino acids. The oligonucleotides are annealed by denaturation. The WO 99/53103 PCT/)K99/U0199 28 annealed linear double-stranded DNA fragment produced in this way is processed with the appropriate restriction enzymes and cloned in an appropriate vector. The frag ment/vector clone is transformed into a suitable E. coli host and expression is induced by adding IPTG to a growing culture. The expressed peptide/protein has a glutathione 5 tag, which enables the purification of the expressed peptide/protein on a glutathione sepharose column. The tag is subsequently removed by protease digestion. RGH-derived antigens may be purified by different methods from patient or cell culture samples e.g. from retroviral particles purified according to the methods outlined in Ex 10 amples 1 and 2. The RGH-derived antigens can be purified by e.g size-dependent separation, charge-dependent separation, separation based on hydrophobicity or affin ity chromatography and using methods based on antibodies e.g. as outlined in Harris and Angal, 1990 and Harlow and Lane, 1988. 15 The purified RGH-derived peptide or protein is useful for e.g. generation of antibodies and as a binding component in an ELISA. Characterization of the RGH-associated antibody response 20 The RGH-associated antibody response is most easily characterized e.g. by using RGH derived peptides purified as outlined above. Serologic reactivity can e.g. be character ized as follows: a) Serologic reactivity characterized by a standard peptide ELISA: Sera can be ob 25 tained by standard procedures and the peptide may be coupled to a carrier as outlined in Harlow and Lane, 1988, or used for passive coating of microtiter plates. Free bind ing sites are then blocked and the immuno-reactivity of the sera is determined in a standard indirect ELISA. All sera are assayed as ten-fold dilution series in duplicate. 30 An RGH-associated antibody response was demonstrated to be associated with MS by performing peptide ELISAs on clinical specimens, i.e. blood samples from MS patients, from patients with autoimmune diseases and from healthy controls.

WO 99/53103 l'CI/DK99/U199 29 All serum samples were obtained from blood samples as described in Example 2, ex cept for the type of Venoject tubes, which in this case was VT-100UX Venoject with out additions. After the initial separation by centrifugation at room temperature for 15 min. at 500 x g, the serum was aspirated, aliquoted into 1 ml portions and stored at 5 70 0 C. The peptides were synthesized by Genosys Biotechnologies (Europe) Ltd., London Road, Pampisford, Cambridge, UK, at more than 80% purity and used for passive coating of FluoroNunc MaxiSorp microtiter trays (NalgeNunc International, Life Tech 10 nologies, Denmark) at a concentration of 0.1 gg or 1 tg/100 pl/well in coating buffer (15 mM Na 2

CO

3 , 35 mM NaHCO 3 , 0.2% NaN 3 , pH 9.6). The plates were incubated in a wet chamber at room temperature for 48 hrs, washed 3 times with 200 pl/well TBS Tween (20 mM Tris-HCI, 137 mM NaCI, 0.05% Tween 20) and incubated with 100 pl serum/well for 24 hrs at 4 0 C. The sera were diluted at 1:20, 1:25, 1:250 and 1:500 15 in TBS-Tween. The sera were aspirated and the plates were washed three times in TBS-Tween before adding 100 Il bionylated rabbit anti-human IgG (Dako E0482)/well diluted 1:1000 in TBS-Tween. The plates were washed 3 times with TBS-Tween and 100 dl/well of Eu-labelled streptavidin (1244-363; LKB, Wallac, Turku, Finland) was added at 1:1000 dilution in TBS-Tween supplemented with 25 pM EDTA. 200 ptl/well 20 of 10 jM diethylene-triaminepentaacetic acid (LKB, Wallac), 0.1% Triton X-100, 15 gM 2-naphtoyltrifluoroacetone in 0.1 M acetate, pH 3.2, was added. After gently shaking the plates for 5 minutes, the fluorescence was measured using a time resolved plate fluorometer (LKB, Wallac). 25 Examples of RGH-encoded synthetic peptide antigens used in the ELISA are shown in Table 4 below: Table 4. RGH-encoded synthetic peptide antigens SEQ ID NO:17 GLDLLTAEKGGLCIFLNE SEQ ID NO: 18 STHLAATSRAPGTLAQG SEQ ID NO: 19 QPGLVYDNIKKLKERDQKL SEQ ID NO:20 VLQAKVDSLAAVVLQNCR WO 99/53103 PLCI/DK99/UU0199 30 SEQ ID NO:21 YKKPSPVHGSFGSNPET SEQ ID NO:22 FITQSAAPNIKIKLQKLNSGP SEQ ID NO:23 LQNRCGLDLLTAEKGGLCI SEQ ID NO:24 DNIKKLKERDQKLANQAS Table 5 summarizes the results for the peptides shown in Table 4. Table 5. Testing of peptide antigens shown in Table 4 5 Equal numbers of MS sera and control sera were tested. MS: sera from MS patients; C: sera from healthy controls and from control patients with other autoimmune dis eases; n: number of sera; DF: dilution factor; MS:C: average fluorescence of MS sera relative to control sera; ER Elisa ratio: individual readings relative to mean of controls; 10 MS:C > 1 and/or ER > 1 indicates a higher MS than control response. DF n MS:C ER(C) > 1 ER(MS) > 1 SEQ ID NO:17 1:20 40 0.9 36% 30% 1:200 56 0.8 SEQ ID NO:18 1:25 50 1.2 33% 39% 1:250 11 1.5 SEQ ID NO:19 1:25 50 1.3 37% 55% 1:250 11 1.5 SEQ ID NO:20 1:25 50 1.1 27% 37% 1:250 11 1.4 SEQ ID NO:21 1:25 40 1.1 28% 32% 1:250 40 1.0 SEQ ID NO:22 1:25 40 1.2 30% 45% 1:250 40 1.2 SEQ ID NO:23 1:25 40 1.1 28% 23% 1:500 40 1.0 SEQ ID NO:24 1:25 40 1.2 21% 35% 1:500 40 1.1 WO 99/53103 ru In/IJK y/ouuIvy 31 b) In the experiments outlined in Example 3, the RGH sequence variants were found in particles from plasma samples from all patients with active disease at the time of sampling. This indicates that the production of RGH into the bloodstream is associated with disease activity. The activity of the MS disease may be monitored by following 5 the changes in RGH production either by establishing a quantitative RT-PCR in essence as outlined in Example 3 but with the addition of either an extra, competing template or by the addition of primers amplifying a household transcript (as outlined in PCR pro tocols, a guide to methods and applications, edited by Micael A. Innes, D. H. Gelfand, J. J. Sninsky, T. J. White, Academic Press, Inc. Harcourt Brace Jovanovich, Publish 10 ers, 1990) or by following the changes in seroreactivity towards retroviral antigens (both IgM and IgG). An MS attack or severe progression is thus reflected in an in crease in RGH transcripts or by an increase in serologic reactivity towards RGH vari ants. 15 c) Serologic reactivity characterized by SDS-PAGE followed by Western blotting: Antibody reactivities towards RGH-derived antigens can be examined by standard Western blotting procedures. It is also possible to perform inhibition studies by prein cubation of antibody and antigen before application to the blot. 20 Characterization of RGH-associated antigens in samples from patients RGH-derived antigens can be purified, identified, and characterized from patient sam ples, e.g. by methods as outlined above. Specifically, the antigens can be identified by 25 using methods based on antibodies, e.g. RGH-specific antibodies, e.g. derived as out lined below. Ultimately, the purified RGH-derived antigens can be characterized by protein sequencing, e.g. as outlined in Harris and Angal, 1990. RGH-specific antibodies 30 The source of RGH-specific antibodies can be e.g. patient serum samples and serum samples from immunized animals as well as ascitic fluid from animals inoculated by hybridoma cells or growth medium from cultured hybridoma cells. The antibody sources all contain various amounts of impurities which can be removed by different WO 99/53103 YUI/LlK99/UU i 99 32 protein purification methods as described above, e.g. by affinity chromotography using RGH-derived peptides. Useful standard techniques for immunization of animals and for production of hybridoma cells is described in e.g. Hudson and Hay, 1980. Antibody reactivity may be characterized as outlined above.

WO 99/53103 P LUI/IDK99/UUIYY99 33 REFERENCES Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase; 1991. Proc. Nat/. Acad. Sci. 88:189-193 5 Christensen T, Dissing Sorensen P, Riemann H, Hansen HJ and Moller-Larsen A. Expression of sequence variants of endogenous retrovirus RGH in particle form in multiple sclerosis. The Lancet, 1998, 352:1033. 10 Christensen T, Jensen AW, Munch M, Haahr S, Dissing Sorensen P, Riemann H, Hansen HJ and Meller-Larsen A. Characterization of retrovirus from multiple sclerosis patients. Acta Neurol Scand 1997; Suppl 169:49-58 Christensen T, T6njes RR, Zur Megede J, Boller K and Meller-Larsen A. Reverse 15 transcriptase activity and particle production in B lymphoblastoid cell lines established from lymphocytes of patients with multiple sclerosis. Aids Research and Human Retroviruses 1999, 15:285-291. Dieffenbach CW, Lowe TMJ and Dveksler GS. General concepts for primer design. 20 PCR Methods and Applications. 1993; 3(3):S30-S37 Garson et al., The Lancet, 1998, 351:33. Ed Harlow and David Lane: Antibodies, a laboratory manual. Cold Spring Harbor 25 Laboratory 1988 Harris and Angal. Protein purification methods, a practical approach. IRL PRESS 1990 Hirose Y, Takamatsu M, Harada F. Presence of env genes in members of the RTVL-H 30 family of human endogenous retrovirus-like elements. Virology 1993; 192:52-61 Hudson L and Hay FC. Practical Immunology, Blackwell Scientific Publications 1980 Medstrand P, Lindeskog M and Blomberg J. Expression of human endogenous WO 99/53103 PCT/DK99/U00199 34 retroviral sequences in peripheral blood mononuclear cells of healthy individuals. J Gen Virol 1992; 73:2463) Munch M, Moller-Larsen A, Christensen T, Morling N, Hansen HJ, Haahr S. B 5 lymphoblastoid cell lines established from multiple sclerosis patients and a healthy control producing a putative new human retrovirus and Epstein-Barr virus. Multiple Sclerosis 1995; 1:78-81 Moller-Larsen A and Christensen T. Isolation of retrovirus from multiple sclerosis 10 patients in self-generated lodixanol gradients. J. Virol. Methods 1998, 73:151-161. Romano JW, Williams KG, Shurtliff RN, Ginnochio C and Kaplan M. NASBA technology: isothermal RNA amplification in qualitative and quantitative diagnostics. 1997; Imrnmunol Invest Jan; 26:15-28 15 Rychlik W and Rhoads RE. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Research 1989;17:8543-8551 20 Sommerlund M, Pallesen G, Moller-Larsen A, Hansen HJ, Haahr S. Retrovirus-like particles in an Epstein-Barr virus-producing cell line derived from a patient with chronic progressive myelopathy. Acta Neurol Scand 1993; 87:71-76 Wilkinson DA, Goodchild NL, Saxton TM, Wood S, Mager DL. Evidence for a 25 functional subclass of the RTVL-H family of human endogenous retrovirus-like sequences. J. Virol 1991; 67:2981-2989

Claims (27)

1. A method of diagnosing a demyelinating disease including multiple sclerosis, the method comprising detecting, in a retroviral particle or an encapsidated virion-like particle present 5 in a sample derived from a patient suspected of suffering from multiple sclerosis, the pres ence of SEQ ID NO:1 or a subsequence or variant thereof.

2. A method of subgrouping patients having a demyelinating disease including multiple sclerosis, said method comprising detecting, in a retroviral particle or an encapsidated vi 10 rion-like particle derived from a patient suspected of suffering from a demyelinating disease including multiple sclerosis, the presence of SEQ ID NO:1 or a subsequence or variant thereof.

3. A method of monitoring the stage or activity of a demyelinating disease including multi 15 ple sclerosis, the method comprising detecting, in a retroviral particle or an encapsidated virion-like particle derived from a patient suspected of suffering from a demyelinating dis ease including multiple sclerosis, the presence of SEQ ID NO:1 or a subsequence or vari ant thereof. 20

4. A method according to any of claims 1-3 wherein the detection is accomplished by using at least one probe that specifically recognizes SEQ ID NO:1 or a subsequence or variant thereof that has a sequence identity with a similar part of SEQ ID NO: I of at least 80%.

5. A method according to any of claims 1-3 wherein the detection is accomplished by using 25 at least one primer that is capable of generating a nucleic acid fragment, the nucleotide se quence of which has a sequence identity with a similar part of SEQ ID NO:1 of at least 80%.

6. A method according to any of claims 1-3 which further comprises the detection of the 30 presence in a retroviral particle of one or more other retroviral nucleotide sequences in cluding a nucleotide sequence selected from the group consisting of a sequence derived from a Human Immunodeficiency Virus (HIV), a sequence derived from a Human T-cell Leukemia Virus (HTLV), a sequence derived from a Multiple Sclerosis Retrovirus (MSRV) and a sequence derived from Human Endogenous Retrovirus-K (HERV-K). 35 WO 99/53103 PCT/DK99/00199 36

7. A method according to claim 6 wherein the detection of the nucleotide sequence of the other retrovirus or retroviruses is accomplished by using one or more probes or sets of primers which is useful for recognizing a nucleotide sequence specific for one retrovirus. 5

8. A diagnostic agent for the diagnosis, subgrouping or monitoring of a demyelinating dis ease including multiple sclerosis by the detection of the presence, in a sample material de rived from a patient suspected of suffering from demyelinating disease including multiple sclerosis, of SEQ ID NO:1 or a subsequence or variant thereof, said agent comprising a probe that is capable of hybridizing specifically with SEQ ID NO:1 or with a subsequence or 10 variant thereof that has a sequence identity with a similar part of SEQ ID NO:1 of at least 80%.

9. A diagnostic agent for the diagnosis, subgrouping or monitoring of a demyelinating dis ease including multiple sclerosis by detection of the presence, in a sample material derived 1 5 from a patient suspected of suffering from demyelinating disease including multiple sclero sis, of SEQ ID NO:1 or a subsequence or variant thereof, said agent comprising at least one primer that is capable of generating a nucleic acid fragment, the nucleotide sequence of which has a sequence identity with a similar part of SEQ ID NO:1 of at least 80%. 20

10. A diagnostic agent according to claim 8 and 9.

11. A diagnostic agent according to any of claims 8-10 which further comprises a probe or primer which is useful for the detection of the presence in a retroviral particle or an encap sidated virion-like particle of one or more other retroviral nucleotide sequences including a 25 nucleotide sequence selected from the group consisting of a sequence derived from a Hu man Immunodeficiency Virus (HIV), a sequence derived from a Human T-cell Leukemia Virus (HTLV), a sequence derived from a Multiple Sclerosis Retrovirus (MSRV) and a se quence derived from Human Endogenous Retrovirus-K (HERV-K). 30

12. A method of diagnosing, subgrouping or monitoring demyelinating disease including multiple sclerosis, the method comprising detecting the presence, in a sample material de rived from a patient suspected of suffering from demyelinating disease including multiple sclerosis, of a protein or a peptide encoded by SEQ ID NO:1 or by a subsequence or vari ant of said sequence. 35 WO 99/53103 PCT/DK99/00199 37

13. A method according to claim 12 which further comprises detecting the presence of a protein or peptide encoded by one or more other retroviral nucleotide sequences including a nucleotide sequence selected from the group consisting of a sequence derived from a Human Immunodeficiency Virus (HIV), a sequence derived from a Human T-cell Leukemia 5 Virus (HTLV), a sequence derived from a Multiple Sclerosis Retrovirus (MSRV) and a se quence derived from Human Endogenous Retrovirus-K (HERV-K).

14. A diagnostic agent for the diagnosis, subgrouping or monitoring of demyelinating dis ease including multiple sclerosis by detection, in a sample material derived from a patient 10 suspected of suffering from demyelinating disease including multiple sclerosis, of a protein or a peptide encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence, said diagnostic agent comprising an antibody reacting specifically with an epitope of a pep tide encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence.

15 15. A diagnostic agent according to claim 14 which further comprises an antibody useful for detecting an epitope selected from the group consisting of an epitope of a Human Immu nodeficiency Virus (HIV), an epitope of a Human T-cell Leukemia Virus (HTLV), an epitope of a Multiple Sclerosis Retrovirus (MSRV) and an epitope of a Human Endogenous Retro virus-K (HERV-K). 20 .

16. A method of diagnosing, subgrouping or monitoring demyelinating disease including multiple sclerosis, the method comprising detecting, in a sample material derived from a patient suspected of suffering from demyelinating disease including multiple sclerosis, the presence of an antibody reacting specifically with an epitope of a protein or a peptide en 25 coded by SEQ ID NO:1 or by a subsequence or variant of said sequence.

17. A method according to claim 16 which further comprises detecting the presence of an antibody useful for detecting an epitope selected from the group consisting of an epitope of a Human Immunodeficiency Virus (HIV), an epitope of a Human T-cell Leukemia Virus 30 (HTLV), an epitope of a Multiple Sclerosis Retrovirus (MSRV) and an epitope of a Human Endogenous Retrovirus-K (HERV-K).

18. A diagnostic agent for the diagnosis, subgrouping or monitoring of demyelinating dis ease including multiple sclerosis by the detection, in a sample material derived from a pa 35 tient suspected of suffering from demyelinating disease including multiple sclerosis, of an WO 99/53103 PCT/DK99/00199 38 antibody reacting specifically with an epitope of a protein or a peptide encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence, said diagnostic agent comprising an epitope of a peptide encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence. 5

19. A diagnostic agent according to claim 18 which further comprises an epitope selected from the group consisting of an epitope of a Human Immunodeficiency Virus (HIV), an epi tope of a Human T-cell Leukemia Virus (HTLV), an epitope of a Multiple Sclerosis Retrovi rus (MSRV) and an epitope of a Human Endogenous Retrovirus-K (HERV-K). 10

20. A method of isolating or purifying, from a sample material, a retroviral particle or an en capsidated virion-like particle, the method comprising contacting said sample material with an antibody reacting specifically with an epitope of a protein or a peptide encoded by SEQ ID NO:1 or by a subsequence or variant thereof. 15

21. A method according to any of claims 1-7 comprising that, prior to the detection, in a ret roviral particle or an encapsidated virion-like particle, of the presence of SEQ ID NO:1 or a subsequence or variant thereof, said retroviral particle or encapsidated virion-like particle is isolated or purified using the method of claim 20. 20

22. A diagnostic agent according to any of claims 8-11 for use in a method according to claim 21.

23. A method according to claim 12 or 13 comprising that, prior to the detection of a pro 25 tein or a peptide encoded by SEQ ID NO:1 or by a subsequence or variant thereof, a retro viral particle or an encapsidated virion-like particle comprising said said sequence, subse quence or variant, is isolated or purified using the method of claim 20.

24. A diagnostic agent according to claim 14 or 15 for use in a method according to claim 30 23.

25. Use of an isolated and/or purified RGH virus particle or encapsidated virion-like parti cle, or a fragment hereof, as a diagnostic agent. WO 99/53103 PCT/DK99/00199 39

26. An isolated and/or purified RGH virus particle or encapsidated virion-like particle, or a fragment hereof, for use as a medicament.

27. Use of an isolated and/or purified RGH virus particle or encapsidated virion-like parti 5 cle, or a fragment hereof, in the manufacturing of a medicament for the treatment of a de myelinating disease including multiple sclerosis. 10