EP1068361A1 - Diagnosis of multiple sclerosis and other demyelinating diseases - Google Patents

Abstract

Methods of diagnosing demyelinating disease including multiple sclerosis (MS), subgrouping patients suffering from such disease including MS and/or monitoring the stage of activity of demyelinating disease including MS based on the detection, in a retroviral particle or an encapsidated virion-like particle, of the RGH virus-derived SEQ ID NO:1 or a subsequence or variant hereof and diagnostic agents comprising probes capable of hybridizing to such sequences, subsequences or variants, antibodies reacting with epitopes of proteins or peptides encoded by sequences, subsequences or variant and/or such epitopes for such methods are provided.

Description

DIAGNOSIS OF MULTIPLE SCLEROSIS AND OTHER DEMYELINATING DISEASES

FIELD OF INVENTION

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 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 detection of the presence of SEQ ID NO: 1 or a subsequence or variant thereof . The inven- tion further relates to a method of diagnosing, subgrouping or monitoring multiple sclerosis 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 variant of said sequence.

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.

GENERAL BACKGROUND

For several decades, the unexplained etiology of MS has been one of the great scientific challenges in medicine. MS is a demyelinating inflammatory disease affecting the 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 question. The extensive epidemiological studies of MS clearly indicate that, as well as ge- 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 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 1 00% of clinically definite 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 - 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 93/07259, WO 95/21 256 and WO 97/06260. However, none of these patent applications 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 specificity would be a vast improvement on the present situation.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates in a first aspect to methods of diagnosing demyelinating diseases including multiple sclerosis, subgrouping patients having a demyelinating disease including multiple sclerosis and/or monitoring the stage or activity of demyelinating disease including multiple sclerosis, the methods comprising detecting, in a retroviral particle or an encapsidated virion-like particle present in a sample de- 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 the methods and diagnostic agents of the invention.

In a further aspect the invention pertains to a diagnostic agent for the diagnosis, subgrouping 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 variant thereof, the diagnostic agent comprising a probe which is capable of hybridizing 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 demyelinating disease including multiple sclerosis by detection of the presence, in a sample material derived 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 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%.

In yet other aspects of the invention there are provided a method of diagnosing, subgrouping 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 or by a subsequence or variant thereof, and a diagnostic agent for the diagnosis, subgrouping or monitoring of demyelinating disease including multiple sclerosis by detection of a peptide encoded by SEQ ID particle containing SEQ ID NO: 1 or a peptide encoded 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 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 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 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.

In further useful aspects, the invention pertains to the use of an isolated and/or purified 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 virion-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 fragment hereof, in the manufacturing of a medicament for the treatment or prevention of a demyelinating disease e.g by vaccination.

DETAILED DISCLOSURE OF THE INVENTION

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 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., 1 993, 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 2 are 81 %-95% identical with RGH-2. Within the scope of the invention are subsequences 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 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 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 Immunodeficiency 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 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 respect.

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- sis, the presence of RGH nucleic acid or a subsequence or variant thereof. In the present context, the expression "sample material" includes any tissue or body fluid material from which the retroviral particles or encapsidated virion-like particles may be isolated 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- spinal fluid and supernatants of cell cultures such as of mononuclear cells from heparinized, 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.

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 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 Example 2 it appears that a positive result is highly indicative of the person having multi- pie sclerosis. Actually, the present results indicate a diagnostic specificity of 1 00% 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 invention will be in the range of 80% to 1 00%, and the diagnostic sensitivity in the range of 50% to 1 00%, such as 66% to 90%, e.g. 75%-80% . The invention thus in 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 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- viduals may turn out to be positive. It is likely that such a result is indicative of a person 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 1 00% . In any event, the 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- 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 in a further aspect to a method of subgrouping patients having multiple sclerosis comprising 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.

By the term "subsequence" is meant a sequence which comprises at least 100, 1 50, 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- 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 1 00, 1 50, 200, 300 or 400 nucleotides. The region of the RGH sequence which is most unique for RGH comprises the gag, pol 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 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, Wiconson Package, Version 9.1 , Genetics Computer Group (GCG), 575 Science Drive, Madison, Wiconsin, USA 5371 1 using the defaults proposed in the program Bestfit with respect to gap weight (50), length weight (3), average match 1 0,000, 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 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.

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 modifications such as phosphorylations or glycosylations of the amino acids into consideration. The definition of sequence identity is as described above with respect to nucleo- 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 1 00 amino acids, such as 1 0 to 30 amino acids.

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 nucleic 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 et al., 1 993.

Suitable oligonucleotides which are useful as primers or probes are selected on the basis of the RGH sequence with the aid of e.g. OLIGO (Rychlik and Rhoads, 1 989) and hybridization conditions which are likely to lead to the hybridization of the selected oligonucleotides with SEQ ID NO: 1 or a subsequence or variant. The oligonucleotides usually have a length of 6-1 00 bp, such as 1 0-30 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 specificity of the product is confirmed e.g. by dideoxy sequencing so that it is confirmed 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 nucleic acid sequenced based amplification (NASBA) (Barany, 1 991 ; Romano et al., 1 997).

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 digestion with a selection of restriction enzymes known to have sites within the ex- 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- 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 N0:8, SEQ ID NO:9, SEQ ID NO: 1 0 as described herein as well as the primers described in Hirose et al., 1 993.

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 region on SEQ ID NO: 1 and the hybridization conditions are as described in Example 1 .

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- 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 sequence, an epitope, a protein, a peptide or an antibody as the case may be. The fundamental principles as well as the more specific details with respect to the various different kinds of diagnostic agents are well known to the person skilled in the art of 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- ble of binding to the first bound antibody and provided with a label as described below.

The substance used as label may be selected from any substance which is in itself detectable 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. 1 0

Examples of enzymes useful as labels are β-galactosidase, urease, glucose oxidase, carbonic anhydrase, peroxidases (e.g. horseradish peroxidase), phosphatases (e.g. alkaline or acid phosphatase), glucose-6-phosphate dehydrogenase and ribonuclease.

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, fluorescence or chemiluminescence. Examples of substrates which are useful in the present method as substrates for the enzymes mentioned above are H₂0₂, p-nitrophenylphos- phate, lactose, urea, β-D-glucose, C0₂, 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 components as used according to the invention may be 4-methylumbelli-eryl-phosphate, 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/H₂0₂ and a substrate therefor.

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. ¹²⁵l, ¹³¹l, ³H, ³²P, ³⁵S or ¹⁴C. The radioactivity may be measured in a 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 (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. 1 1

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 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- 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 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 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.

In a further aspect, the invention relates to a diagnostic agent which comprises an antibody 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 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 1 2

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 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- 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- 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.

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 precision and accuracy of the assay, at the same time possibly requiring less time to perform. 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 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- 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 1 00 1 3

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 phosphatase or be detectable by any other means.

Detection of virion-associated MSRV-RNA in serum of patients with multiple sclerosis has recently been published by Garson et al., 1 998. 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 or consecutively in multiple sclerosis patients. One possibility is that a general activation 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.

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 specificity 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- 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.

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 indicating that this virus is no longer present in detectable amounts. Such findings may be useful in establishing the stage of the disease process and also with respect to determining 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 results it seems that the presence of SEQ ID NQ: 1 or a subsequence or variant thereof 14

indicates an active stage of disease. It is concievable that the same applies if is assumed that RGH stimulates the activation of MSRV virus.

In a further embodiment, the invention relates to a method of monitoring the stage or activity of multiple sclerosis or any other demyelinating disease, the method comprising detecting the presence of SEQ ID N0: 1 or a subsequence or variant thereof in a retroviral particle or an encapsidated virion-like particle that is present in a sample material derived from a patient suffering from multiple sclerosis or another demyelinating disease.

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 presence 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 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 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 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- 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 1 5

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- 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 paragraphs (d)-(g) of claim 1 3 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 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 monoclonal 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 subsequence or variant of said sequence in plasma e.g. by means of an antibody reacting specifically with an epitope of such a protein or peptide. Within the scope of the invention is a peptide encoded by SEQ ID N0: 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 antibody.

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- 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 electrophoresis, denaturating polyacrylamide gel electrophoresis, agarose gel electrophoresis 1 6

and isoelectric focusing, (v) any other specific solubilization and/or purification techniques.

The invention further relates to a method of diagnosing, subgrouping or monitoring 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 proteins 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 increase in the antibody titer may be indicative of an activation or reactivation of the disease process which may have implications with respect to treatment or prognosis.

Possible protein sequences of RGH are known from Hirose et al., 1 993. Based upon the diagnostic significance of the presence of an RGH nucleotide sequence in a retroviral particle, it is likely that it will also be possible to detect in plasma or serum samples 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 multiple sclerosis.

Epitopes of a protein or a peptide encoded by SEQ ID NO: 1 or a subsequence or variant 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 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 associated 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- able carrier in order to provide a diagnostic agent useful for the detection of an antibody against an epitope of a peptide encoded by SEQ ID NO: 1 . Experiments for detecting 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. 1 7

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- 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 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 sequences identical, partially or in total, or equivalent to the sequence defined in the sequences 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.

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, the determination of disease stage, selection of medical treatment and the determination of the prognosis of the individual patient as outlined above with respect to diagnostic 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- 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 invention, there is provided the use of such particles as a diagnostic agent.

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. 1 8

The invention is further illustrated in the following, non-limiting examples.

EXAMPLE 1

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- 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 expression of Epstein-Barr virus (EBV) proteins and occasionally mature EBV particles (Sommerlund et al., 1 993; Munch et al., 1 995).

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., 1 997) . The B-lymphoblastoid cell lines were estab- 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- pies were obtained by drawing blood into Venoject VT1 00H tubes containing 1 50 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 1 0⁶ cells/5 ml and cultivated in RPMI 1 640 (Seromed) supplemented with 200 i.u./ml penicillin (Leo), 0.2 mg/ml streptomycin (Rosco), 290 mg/ml glutamine (Sigma),

10 mM HEPES (Bioproduct) and 1 0% heat inactivated human serum (serum pool obtained 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 1 9

than two months) and are continuously growing. The established cell lines were supplemented 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 by repeated analyses (standard Hoechst stain) .

Table 1

MS B-lymphoblastoid cell lines

Patient Age, Diagnosis Duration,

No. Sex years years

MS1 533 M 30 Chronic progressive 7 myelopathy

MS 1 844 F 40 Benign MS 1 1

MS1 845 M 33 Relapsing-Remitting MS 2

MS1 851 F 24 Primary progressive MS 2

Expression of RGH variants in retroviral particles in B-lymphoblastoid cell lines established from MS patients

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 template and subsequent sequencing of the obtained amplicons. Retroviral particles were purified from cell line supernatants by ultracentrifugation in self-forming Optiprep gradients. 20

1 .3-2 litres of suspension cultures were centrifuged at 4°C 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°C for 2 hours at 45,000 x g (Beckmann ultracentrifuge). Optiprep is an iodinated, nonionic 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 μm filter (Acrodisc 32, Gelman Sciences) to remove any cellular de- bris, adjusted to 20% Optiprep and centrifuged in 1 1 .2 ml Optiseal (Beckmann) tubes in a Beckmann NVTi rotor at 364,000 x g for 3.5 hours. 400-500 μl fractions ( 1 2 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 1 00 μl of beads/sample (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°C. Before use, each RNA sample was treated in a 20 μl final volume with amplification grade DNAse (Life Technologies) according to the manufacturer's instructions. After cooling on ice, the buffer was exchanged with 1 0 μl of DEPC-treated ddH₂0, and 2 μl aliquots were 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 fragments with homology to RGH were identified (Hirose et al., 1993) in the retroviral par- tides produced by the cell lines indicated in Table 1 (MS1 533, MS 1 844, MS1 845 and MS1 851 ).

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 reaction. All relevant controls were included. First strand cDNA synthesis was primed with random hexamers, in conjunction with the bead-oligo-dT complexed RNA template. 21

Primer sets for the PCR amplification of gag and env were as follows:

5'-CTTTTATTACCCAATCTGCTCCCGAYAT-3' (SEQ ID NO:3)

5'-TTAGTGGTGGACAGTCTCTTTTCCARTG-3' (SEQ ID N0:4) (45°C), (Medstrand et al., 1 992)

5'-ATTTTATTACCCAATCTGCTCCAAACAT-3' (SEQ ID N0:5)

5'-AGGTGAGTTGAACAGTCTGATTTTTA-3' (SEQ ID N0:6) (50°C)

5'-CGTTTACATATCACTCCCTTCCTAGTCTCTGT-3' (SEQ ID NO:7)

5'-GCATTAACCTTGACTATGTCTTTAGCTCCAG-3' (SEQ ID NO:8) (50°C).

5'-GATCCTCCCCACTGGGTTCACCATT-3' (SEQ ID NO:9)

5'-GGAAGTATTGGAGGGTGCCCTGCC-3' (SEQ ID NO: 10) (60°C) .

All reactions were run in a Perkin Elmer DNA Thermocycler. Conditions for first strand synthesis: 5 minutes at room temperature, 45 minutes at 42°C, and 5 minutes at 95°C, followed by cooling to 4°C. PCR: one cycle for 2 minutes at 95°C was followed by 45/50 cycles with annealing temperatures as indicated above: 1 minute at 94°C, 2 minutes annealing, 3 minutes at 72°C with 1 0 seconds extension. This was followed by a final extension for 7 minutes at 72°C. 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).

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. 22

EXAMPLE 2

Expression of RGH variants in retroviral particles in peripheral blood from MS patients

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 nucleic 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 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. RNA extracted from particles isolated from plasma was assayed by RT-PCR with subsequent 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 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 respective clinics into VT-1 00SCPDA 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 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 samples were obtained from the citrate blood samples (40 ml of blood -» appr. 1 0 ml of plasma). After the initial separation by centrifugation at room temperature for 1 5 min. at 500 x g, the plasma was aspirated and subjected to another centrifugation at 4°C for 30 minutes at 1 000 x g to pellet any remaining cells. The cell-free plasma was aspirated and samples layered on 50% Optiprep, ultracentrifuged and filtered as de- 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 .

If not used directly, the RNA-coupled beads were stored in 80% EtOH at -80°C. Before use, each RNA sample was treated in a 20 μl final volume with amplification grade DNAse (Life Technologies) according to the manufacturer's instructions. After cooling on ice, the buffer was exchanged with 1 0 μl of DEPC-treated ddH₂0, and 2 μl aliquots were used for each cDNA synthesis.

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 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 samples from 24 of 33 MS patients. The sequences were absent in all of 29 cell-free 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.

A large subgroup of MS cases is thus associated with the production in the blood of retroviral particles containing RGH sequence variants.

24

Table 2

All MS patients had clinically definite MS, the MS type and disease activity is indicated. Chronic progressive MS with no stationary phase, and exacerbations in relapsing/remitting MS were defined as active MS. RR: relapsing-remitting, SP: secondary 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

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

M 68 Hairy cell leukemia

F 42 Prurigo nodularis Hide

2 F 35-61 Colitis ulcerosa

2 M 31-32

F 52 SLE

F 55 Arteritis temporalis

F 53 Rheumatoid armritis

M 47-61

F 63 Thyreotoxicosis

M 55

M 47 Sarcoidosis

M 50 Collagenosis

F 58 Poly yositis

F 76 Myelomatosis

M 58 Leukemia/lymphoma F 72 Polymyalgia rheumatoides 25

EXAMPLE 3

Characterization of expressed RGH sequence variants

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 clinical samples, i.e. the paniculate 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- mic RNA from retroviral particles.

RGH belongs to the RTVL-H/HERV-H (priming of transcription with tRNA^His) 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), and to ERV-9. A genomic RTVL-H clone which potentially encodes functional proteins has been described (Wilkinson et al., 1 991 ) .

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- compasses a partial pol and an env 3'LTR region and thus lacks gag, whereas the published RGH-2 clone is 871 5 bp long and includes the complete coding potential: 5'LTR gag-pol-env 3'LTR. They are present in about 1 00 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.

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 potential of being transcribed to an RNA sequence variant.

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. 26

Table 3

SEQ ID Nθ: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 Nθ: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 Nθ:13 CCCGACATTA AATAAAGCTC CAAAAATTAG ATTCCAGCCC GGAAACCCCA CAACAGGACT

TAACCTCACC TTCAAGGTGT ACAATAATAG AGAGGAGTCT CAATTCTTGC CTCTGCTGTG

AGAGAAACCC CAGCCACATC TCCAGCACAC AAGAACTTCA AAATGCCT

SEQ ID Nθ:14 CCAAACATTA AATAAAACTC CAAAAATTAA ATTCCGGCCC TCAAACCCCA CAACAAGGCT

TAATTAACCT CACCTTCAAG GTGTTCAATA ACAGAGTAGA GGCAACCAAG TAGCAATGTA

TTTCTAAGTT GCAATTCCTT ACCTCCACTG TAAGACAAAC CCCAGCCACA TCTCCAGCAC

ACAAGAACTC CAAATGCCCG

SEQ ID Nθ: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

27

EXAMPLE 4

The present invention also relates to diagnosis and prognosis by immunological means 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 antigens for diagnostic and purification purposes.

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 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.

Having defined peptides with an amino acid sequence derived from the known sequences of RGH, e.g. the gag and env regions, RGH subsequences or sequence variants, the peptide comprising an epitope can be chemically synthesized, e.g. by the conventional Merrifield synthesis technique.

Peptides or proteins may be produced in commercially available prokaryotic or eukary- otic expression systems. Typical prokaryotic expression systems are based on E. coli 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 prokaryotic expression system (GST Gene Fusion System, Pharmacia Biotech) is described in the following: Based on the sequence of RGH, RGH subsequence or sequence 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 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 fragment/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 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- 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 affinity chromatography and using methods based on antibodies e.g. as outlined in Harris and Angal, 1 990 and Harlow and Lane, 1 988.

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

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 characterized as follows:

a) Serologic reactivity characterized by a standard peptide ELISA: Sera can be ob- tained by standard procedures and the peptide may be coupled to a carrier as outlined in Harlow and Lane, 1 988, or used for passive coating of microtiter plates. Free binding 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.

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. 29

All serum samples were obtained from blood samples as described in Example 2, except for the type of Venoject tubes, which in this case was VT-100UX Venoject without additions. After the initial separation by centrifugation at room temperature for 1 5 min. at 500 x g, the serum was aspirated, aliquoted into 1 ml portions and stored at - 70°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- nologies, Denmark) at a concentration of 0.1 μg or 1 μg/100 μl/well in coating buffer (1 5 mM Na₂C0₃ , 35 mM NaHC0₃, 0.2% NaN₃, pH 9.6). The plates were incubated in a wet chamber at room temperature for 48 hrs, washed 3 times with 200 μl/well TBS- Tween (20 mM Tris-HCI, 1 37 mM NaCI, 0.05 % Tween 20) and incubated with 1 00 μl serum/well for 24 hrs at 4°C. The sera were diluted at 1 :20, 1 :25, 1 :250 and 1 :500 in TBS-Tween. The sera were aspirated and the plates were washed three times in TBS-Tween before adding 1 00 μl bionylated rabbit anti-human IgG (Dako E0482)/well diluted 1 : 1 000 in TBS-Tween. The plates were washed 3 times with TBS-Tween and 100 μl/well of Eu-labelled streptavidin ( 1 244-363; LKB, Wallac, Turku, Finland) was added at 1 : 1000 dilution in TBS-Tween supplemented with 25 μM EDTA. 200 μl/well of 1 0 μM diethylene-triaminepentaacetic acid (LKB, Wallac), 0.1 % Triton X-100, 1 5 μM 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).

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: 1 7 GLDLLTAEKGGLCIFLNE

SEQ ID NO: 18 STHLAATSRAPGTLAQG

SEQ ID NO: 1 9 QPGLVYDNIKKLKERDQKL

SEQ ID NO:20 VLQAKVDSLAAVVLQNCR 30

SEQ ID N0:21 YKKPSPVHGSFGSNPET

SEQ ID N0:22 FITQSAAPNIKIKLQKLNSGP

SEQ ID NO:23 LQNRCGLDLLTAEKGGLCI

SEQ ID N0:24 DNIKKLKERDQKLANQAS

Table 5 summarizes the results for the peptides shown in Table 4.

Table 5. Testing of peptide antigens shown in Table 4

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 diseases; 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; 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 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 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 protocols, 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- ers, 1 990) 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 increase in RGH transcripts or by an increase in serologic reactivity towards RGH variants.

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.

Characterization of RGH-associated antigens in samples from patients

RGH-derived antigens can be purified, identified, and characterized from patient samples, e.g. by methods as outlined above. Specifically, the antigens can be identified by using methods based on antibodies, e.g. RGH-specific antibodies, e.g. derived as outlined below. Ultimately, the purified RGH-derived antigens can be characterized by protein sequencing, e.g. as outlined in Harris and Angal, 1 990.

RGH-specific antibodies

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 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, 1 980. Antibody reactivity may be characterized as outlined above.

33

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Claims

35CLAIMS

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 in a sample derived from a patient suspected of suffering from multiple sclerosis, the presence 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- 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- pie 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 disease including multiple sclerosis, the presence of SEQ ID NO:1 or a subsequence or variant thereof.

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:1 of at least 80%.

5. A method according to any of claims 1-3 wherein the detection is accomplished by using 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%.

6. A method according to any of claims 1-3 which further comprises the detection of the presence in a retroviral particle of 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 Virus (HTLV), a sequence derived from a Multiple Sclerosis Retrovirus (MSRV) and a sequence derived from Human Endogenous Retrovirus-K (HERV-K). 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.

8. A diagnostic agent for the diagnosis, subgrouping or monitoring of a demyelinating disease including multiple sclerosis by the detection of the presence, in a sample material derived 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 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 disease including multiple sclerosis by detection of the presence, in a sample material derived 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 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%.

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 encapsidated virion-like particle of 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 Virus (HTLV), a sequence derived from a Multiple Sclerosis Retrovirus (MSRV) and a sequence derived from Human Endogenous Retrovirus-K (HERV-K).

12. A method of diagnosing, subgrouping or monitoring demyelinating disease including multiple sclerosis, the method comprising detecting the presence, in a sample material derived 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 variant of said sequence. 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 sequence derived from Human Endogenous Retrovirus-K (HERV-K).

14. A diagnostic agent for the diagnosis, subgrouping or monitoring of demyelinating disease 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 peptide encoded by SEQ ID NO:1 or by a subsequence or variant of said sequence.

1 5 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 Immunodeficiency 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 Retrovirus-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 disease 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 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.

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 epitope of a Human T-cell Leukemia Virus (HTLV), an epitope of a Multiple Sclerosis Retrovirus (MSRV) and an epitope of a Human Endogenous Retrovirus-K (HERV-K).

20. A method of isolating or purifying, from a sample material, a retroviral particle or an encapsidated 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.

21. A method according to any of claims 1-7 comprising that, prior to the detection, in a retroviral 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.

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- tein or a peptide encoded by SEQ ID NO:1 or by a subsequence or variant thereof, a retroviral particle or an encapsidated virion-like particle comprising said said sequence, subsequence 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 23.

25. Use of an isolated and/or purified RGH virus particle or encapsidated virion-like particle, or a fragment hereof, as a diagnostic agent. 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- cle, or a fragment hereof, in the manufacturing of a medicament for the treatment of a demyelinating disease including multiple sclerosis.