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WO1993007259A1 - Retrovirus humain analogue au type c lie a la sclerose en plaques - Google Patents

Retrovirus humain analogue au type c lie a la sclerose en plaques Download PDF

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Publication number
WO1993007259A1
WO1993007259A1 PCT/DK1992/000299 DK9200299W WO9307259A1 WO 1993007259 A1 WO1993007259 A1 WO 1993007259A1 DK 9200299 W DK9200299 W DK 9200299W WO 9307259 A1 WO9307259 A1 WO 9307259A1
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WO
WIPO (PCT)
Prior art keywords
retrovirus
htlv
cell culture
antigen
antibody
Prior art date
Application number
PCT/DK1992/000299
Other languages
English (en)
Inventor
Mette Sommerlund
Sven Haahr
Anné MØLLER-LARSEN
Arne Willy Jensen
Tove Christensen
Original Assignee
Scleroseforeningen (The Danish Ms-Society)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scleroseforeningen (The Danish Ms-Society) filed Critical Scleroseforeningen (The Danish Ms-Society)
Priority to AU27709/92A priority Critical patent/AU664049B2/en
Priority to EP92921610A priority patent/EP0609305A1/fr
Publication of WO1993007259A1 publication Critical patent/WO1993007259A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Definitions

  • MS Multiple sclerosis
  • the disease is associated with clinical symptoms such as sensory, visual and motor dysfunction because of lesions in the nervous system as mentioned above caused by the break-down of the myelin sheaths.
  • the lesions can be ranging from 1 mm to several centimeters.
  • Clinical diagnosis of the disease can be made by electrophysiologic evaluation, magnetic resonance (MR) and cerebral spinal fluid examination (CSF) (McFarlin and McFarland, 1982). So far, no specific diagnostic test is available and the diagnosis is based on clinical and pathological criteria.
  • the diagnosis may be based on clinical symptoms as mentioned above, that is sensory, visual and motor dysfunction. Futhermore, the diagnose may be based on pathology.
  • the documentation of lesions that have occured on more than one occasion and at more than one site, and which are not explained by other mechanisms is considered as a definitive diagnosis (McFarlin and McFarland, 1982).
  • TSP Tropical spastic paraparesis
  • MS multiple sclerosis
  • Similarity between MS and TSP has led to the hypothesis that a retrovirus could be involved in MS.
  • antibodies against human retroviruses were claimed to occur more often in MS patients than in controls (Koprowski et al., 1985), but it has not been possible to confirm these findings (Hauser et al., 1986).
  • sequences of HTLV-I could be found more often in MS patients than in controls (Reddy et al., 1989), but these results have not been confirmed either (Prayoonwiwat et al., 1991).
  • the present inventors have analyzed long-term cultures of cerebrospinal fluid cells and long-term peripheral blood mononuclear cell cultures from MS patients, patients with other neurological diseases and healthy controls for growth characteristics, cell morphology, reverse transcriptase and 2',5'-oligoadenylate synthetase activities. None of these parameters differed between the groups (Höllsberg et al., 1989). In another study (Sommerlund et al., 1991), multinucleated giant macrophages in long-term cultures derived from MS patients and healthy controls were compared. No differences could be found. Electron microscopy (EM) was performed on these cultures, but no signs of retrovirus-like particles were observed.
  • EM Electron microscopy
  • retrovirus-like particles have been found in blood lymphocyte cultures established from a patient with multiple sclerosis (Haahr et al., 1991). The cells were initially stimulated with phytohaemagglutinin (PHA) and cultured with medium containing interleukin-2 (IL-2), and the established cell line was an IL-2-dependent T-cell line. These cells can only be kept in culture for 1-3 months. Because of this, only marginal amounts of these retrovirus-like particles could be produced, and further characterization of this putative virus could not be performed.
  • PHA phytohaemagglutinin
  • IL-2 interleukin-2
  • MS is caused by an agent similar to HTLV-I, one could imagine that this agent had the same epidemiology as
  • LCL B-lymphoblastoid cell line
  • EBV Epstein-Barr virus
  • PCR polymerase chain reaction
  • the LCL is double-infected with EBV and a hitherto uncharacterized human retrovirus.
  • the EM pictures was examined by Dr. Hans Gelderblom, who confirmed the observation.
  • the spontaneous production of EBV is remarkable because this usually takes place only in cell lines induced by exogenous factors like halogenated pyrimidines or infection by a second virus.
  • human retroviruses have been found to induce EBV in cell lines (Lai et al., 1989) .
  • the cell line (MS-1533) is now established and growing continuously in the inventors' laboratory, producing 0.5-1 billion cells per week. Production of virus has been followed by transmission EM. Production of retrovirus-like particles takes place for a period of at least 5-6 months after establishing the cultures from frozen ampoules. Production of intact EBV particles only takes place initially, later on defect EBV is produced. Both viruses have been purified by a two-fold sucrose gradient ultracentrifugation as described in Example 5A. Verification of this purification is performed by negative staining EM as described in Example 8.
  • Immunoelectron microscopy gives the possibility of capturing virus particles so that they are seen in greater numbers than what would be seen by normal negative staining procedures where the virus solution is spread directly onto the grids. Furthermore, a more "clean" picture is seen since cell organelles or other small cell parts are washed away.
  • the antibody was a mouse monoclonal antibody to HTLV-l gp46 which had been shown to react with MS 1533 in Western blotting assays.
  • the viral solution was cell supernatant double purified by sucrose gradient centrifugation.
  • the reverse transcriptase assay is clearly positive in the gradients where EM shows retrovirus-like particles as described in Example 6. Electrophoresis on material from these gradients has been performed and in material from the gradient where retrovirus is seen, one antigen has been found; this antigen reacts with an antibody raised against an HTLV-I antigen (see Example 7 for details). The fact that polyclonal HTLV-I antibodies detected only one of several antigens clearly indicates that it is not a question of HTLV-I.
  • cytokines may play an essential role.
  • IL-6 is a cytokine which influences both T and B lympho- cytes, playing a special role for antibody production. This could explain the polyclonal B-cell activation in MS patients and the high amount of antibody in cerebrospinal fluid and serum. TNF has been found toxic to oligodendrocytes and it can cause demyelination (Selmaj et al., 1988). Evidence of intrathecal synthesis of TNF in MS has been found, and the level of TNF in cerebrospinal fluid correlates with the severity and progression of the disease (Sharief et al., 1992).
  • RT Reverse transcriptase
  • MS 1533 has been characterized antigenetically, using several polyclonal as well as monoclonal antibodies raised against the various HTLV-I antigens.
  • the antibodies were from several different sources.
  • HTLV-I HTLV-I
  • HTLV-I HTLV-I
  • Two of the most prominent HTLV-I antigens could not be identified.
  • a positive signal has been identified as described in detail in Example 7 using a monoclonal antibody against a specific antigen, which signal cannot be caused by background or unspecific binding.
  • This distinct protein shows the expected size in molecular weight, indicating the presence of a retroviral related protein.
  • Oligonucleotide primers their sequence either being deduced from the antigen or based on known retroviral "consensus" sequences, is utilized for PCR analysis of cDNA synthesized from RNA isolated from the LCL as described in Example 9.
  • the synthesized DNA fragments may be used both as probes for the relevant retroviral sequences in the LCL genome and as templates for a preliminary sequence analysis.
  • the oligonucleotides as well as the characterized PCR products can be used as probes for the assessment of viral expression levels, either by quantitative PCR analysis or by various blotting techniques.
  • the concomitant use of oligonucleotides specific for either EBV or the retrovirus will help elucidating the double infection and may be used as a diagnostic tool.
  • Example 5 The purification and concentration of antigens as described in Example 5 make it possible to use Western Blotting techniques for antibody studies and to develop an ELISA for both the viruses involved in MS, thus greatly facilitating the processing of larger numbers of patient sera.
  • Mononuclear peripheral blood cells are continuously being grown, and new cell cultures are being established from both patients with chronic neurological diseases and healthy controls in order to look for retrovirus-like particles in the cells by EM.
  • the cells are grown without activating factors but with and without IL-2. In this way, it is possible to establish both T-cell lines and B-lymphoblastoid cell lines.
  • oligonucleotides specific for EBV may also be used in order to elucidate the infection in cells with EBV, for example in plaques.
  • a new retrovirus has now been found which is associated with MS, and which, therefore, opens up a wide range of possibilities for reliable diagnosis of MS. Also, the production of the retrovirus has been found to co-exist with production of Epstein-Barr virus for which reason it is justified to assume that the retrovirus is activated by Epstein-Barr virus; this gives rise to the provision of a strategy for the development of a vaccine treatment for MS, such as will appear from the following.
  • the retrovirus of the invention is found in cells from patients suffering from MS, including early stages of MS. Thus, one cell culture producing the retrovirus is a lymphoblastoid B cell culture from a patient with a chronic progressive neurological disease with a marked spasticity of the legs.
  • the cell culture was established from blood mononuclear cells without stimulation with PHA and without interleukin-2 in the medium. The first weeks, the culture appeared as a culture of adherent macrophages with few non-adherent cells in the medium. After several weeks, small clones of larger lymphocytes appeared in the culture. In the beginning, these small clones were adhering to the macrophages. This cell culture is described in greater detail in Example 1 and in the following.
  • the retrovirus appears, in transmission electron microscopy performed as described in Examples 2 or 8, to be a type C-like retrovirus, and a number of tests as reported in the following have confirmed non-identity between the retrovirus and a number of known retroviruses.
  • the invention relates to a cell culture comprising cells which are infected with a type C- like retrovirus which is present in human patients who have symptoms indicating an early stage of multiple sclerosis, the retrovirus being a retrovirus which can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studied in transmission electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests:
  • HTLV-I/026 and HTLV-I/029 no genomic sequences are detectable
  • RD114 do not bind to the cell culture.
  • the invention relates to a cell culture comprising cells which are infected with a type C-like human retrovirus, the retrovirus being a retrovirus which can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studied in transmission electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests: a) in which the retrovirus shows the following negative tests:
  • HTLV-I/026 and HTLV-I/029 no genomic sequences are detectable
  • the appearance of the retrovirus particles in transmission electron microscopy appears from Fig. 1c and 1d.
  • the spherical particles have substantially the appearance as shown in figures 2a and 2b.
  • the retrovirus of the invention is identified as a type C-like retrovirus in accordance with the description given in Dalgleish et al. 1990, as the retrovirus possesses various morphologic features characteristic to retroviruses of type C. No visible projections from the outer cell membrane is observed, only an ill defined central core structure is present and no identifiable intracytoplasmic precursor forms are observed, all of these features being key features in the identification of type C viruses. Furthermore, the size of the retrovirus being 80-120 nm is in accordance with the morphological features of a type C virus.
  • the cell culture is preferably one which is capable of actively producing the retrovirus, so that the retrovirus can be produced using the cell culture and then isolated, concentrated and purified, and so that antigens and nucleotide sequences characteristic to the retrovirus can be obtained from the culture.
  • the cell culture according to the invention constitutes an important source of diagnostic materials and starting materials for diagnostic materials, such as will appear from the following. It has been found that the culture of the invention actively produces the retrovirus when the cell culture is additionally containing a herpes group virus, in particular Epstein-Barr virus. It is believed that the expression/product of the herpes group virus, such as Epstein-Barr virus enhances the production of the retrovirus; it is well known that such an interaction between different types of viruses can occur.
  • Another herpes group virus which is contemplated to be particularly interesting for enhancing the production of the retrovirus is human herpes virus-6 (Schonnebeck et al., 1991).
  • the cell culture may be a mammalian cell culture, in particular a simian or human cell culture.
  • cells useful or contemplated to be useful for establishing the cell culture are lymphoblastoid cells or myelomonocytary cells, in particular lymphoblastoid B cells.
  • An interesting cell culture is one in which the cells are capable of growing without adherence to surfaces.
  • the cell culture according to the invention may be established on the basis of cells from body fluids, such as blood, or from tissue samples, from patients suffering from MS as established by clinical or definitive diagnosis or from MS-like diseases such as chronic progessive myelo pathies, said patients being infected with the retrovirus, and in particular such patients whose serum does not contain antibodies against other retroviruses.
  • the cell culture may be established using conventional techniques, such as described in Example 1, and the presence of the retrovirus in the cell cultures is confirmed by transmission electron microscopy and/or negative staining electron microscopy and the various tests as described herein, in particular the tests described above and PCR or hybridization tests as described below.
  • a cell culture of the invention has been deposited with ECACC, European Collection of Animal Cell Cultures, Porton Down, Salisbury, Wilts, SP4 OJG, UK, on 4 October, 1991 and was given the provisional deposit number V 91100401. Because of a low number of cells in the initial deposit, cultivation of cells was made at the
  • the cell culture of the invention can also be defined as a cell culture infected with the retrovirus with which said deposited cell cultures is infected or with a retrovirus which is identical therewith except for genetic variations which are commonly found in retroviruses and which do not change the above-defined properties of the retrovirus.
  • Another way of characterizing the range of cell cultures according to the invention is by reference to the fact that they contain genomic fragments which can also be found by PCR in blood samples from diagnosed multiple sclerosis patients, but not in blood samples from healthy persons.
  • the term “healthy person” is meant a person who is not infected with the retrovirus of the invention.
  • a further way of characterizing the range of cell cultures according to the invention is by reference to the fact that they contain antigens capable of binding antibodies which are present in sera from diagnosed multiple sclerosis patients, such antigens being antigens which do not bind antibodies present in sera from healthy persons.
  • the invention also relates to the purified retrovirus in the form of whole retrovirus or fragments thereof.
  • Such purified retrovirus or fragments thereof may be obtained by known methods, e.g. by rupturing the cells of a cell culture as defined above and concentrating/purifying the retrovirus or fragments thereof, e.g. by affinity chromatography, such as antibody affinity chromatography using immobilized antibodies specific to the retrovirus. The production of such antibodies is described in the following.
  • the retrovirus containing material can be purified by the methods described in Example 5 (sucrose gradient purification, Triton X-114 temperature induced phase separation or purification by adsorption to antibody-conjugated microbeads).
  • the purified retrovirus or fragments thereof are useful, e.g. as starting material for sequencing purposes, as antigenic/immunogenic material for the production of further antibodies, and as diagnostic agents or starting materials for diagnostic agents, e.g. diagnostic agents as discussed below.
  • the invention also relates to an antigen or epitope derived from, produced by, or induced by the retrovirus with which the above-identified cell culture is infected, or derived from the above-mentioned purified retrovirus, the antigen showing the following negative tests: B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the antigen or epitope,
  • antibodies directed against HIV-I, and HIV-II do not bind to the antigen or epitope, the antigen or epitope being obtainable by subjecting cell fragments and/or medium from a cell culture as discussed above or purified retrovirus as described above to gel electrophoresis, applying serum from a diagnosed multiple sclerosis patient to the resulting gel and visualizing bound antibody by means of labelled anti-human antibody, comparing the visualized gel bands with a similar preparation made using sera from a number of healthy persons, identifying the bands which are antibody-bound in the preparation using the serum from a diagnosed patient and which are not bound in the preparations using sera from the healthy persons, isolating such bands containing the antigen or epitope from corresponding gel electrophoresis, and optionally extracting the antigen or epitope from the bands and purifying the antigen or epitope.
  • the antigens or epitopes of the invention further show the following positive test:
  • the antigens or epitopes of the invention characteristic to the cell culture and/or the retrovirus, are very valuable diagnostic agents for the diagnosis of MS in any infected stage, such as active stage or early stage or prestage or subclinic stage or latent infection, all of which are defined below, in which infected stage antibodies against the antigen are produced in the patient. Thereby, a specific and easy diagnostic test for MS is made avaialable. Likewise, anti-idiotypic antibodies as defined below may be valuable diagnostic agents for the diagnosis of MS in any of the below defined infective stages in patients.
  • stage is meant a stage of infection with the retrovirus in a patient, in which the clinical and/or pathological symptoms have not yet developed but in which the immune system has been presented to the retrovirus and in which the retrovirus is activated and will give rise to MS over an unknown period of time.
  • the term “early stages of infection” is meant a stage of infection with the retrovirus in a patient, in which the retrovirus is activated and has given rise to clinical symptoms comprising sensory and/or visual and/or motor dysfunction and/or pathological symptoms comprising lesions but where remission to complete or nearly complete return of normal neurological functions occurs. In this stage, lesions of variable dimensions have been observed.
  • diagnosisd multiple sclerosis is meant a stage of infection with the retrovirus in a patient, in which the clinical symptoms comprising sensory, visual and/or motor dysfunction and pathological symptoms comprising lesions of the nervous system have been diagnosed, by a definitive diagnosis.
  • subclinic infection an infection with the retrovirus in a patient, in which the retrovirus gives rise to only pathological signs such as demyelination shown under autopsy, but where no clinical symptoms characteristic to MS can be observed or was observed.
  • lymphosens an infection in a patient with the retrovirus, in which the retrovirus will not give rise to any clinical and/or pathological symptoms indicating MS or an early stage thereof unless activated, possible by an infection with a herpes group virus such as Epstein-Barr virus.
  • a herpes group virus such as Epstein-Barr virus.
  • the invention also relates to a method of diagnosing multiple sclerosis or a prestage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to the invention expressing the antibody as defined above, comprising contacting a sample of body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent comprising an antigen or epitope of the invention or an anti-idiotypic antibody as defined below, and determining the presence of any antibody from the sample bound to the antigen, epitope, or anti-idiotypic antibody.
  • the diagnostic test may be performed using a diagnostic agent which comprises an antigen or epitope of the invention or an anti-idiotypic antibody as described below, bound to a carrier or support as described below. Any antibodies from the sample binding to the antigen, epitope, or anti-idiotypic antibody may be detected using a secondary antibody capable 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.
  • the label may be selected from radioactive isotopes, enzymes, chromophores, fluorescent or chemiluminescent substances, and complexing agents.
  • enzymes useful as labels are ⁇ -galactosidase, urease, glucose oxidase, carbonic anhydrase, peroxidases (e.g. horseradish peroxidase), phosphatases (e.g. alkaline or acid phosphatase), glucose-6-phosphate dehydrogenase and ribonuclease.
  • peroxidases e.g. horseradish peroxidase
  • phosphatases e.g. alkaline or acid phosphatase
  • glucose-6-phosphate dehydrogenase 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.
  • 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.
  • substrates which are useful in the present method as substrates for the enzymes mentioned above are H 2 O 2 , p-nitrophenylphosphate, lactose, urea, ⁇ -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 components as used according to the of invention may be 4-methylumbelliferyl-phosphate, 4-methyl- umbelliferyl-D-galactopyranoside, and 3-(p-hydroxyphenyl) propionic acid. These substances may be detected by means of a fluorescence spectrophotometer. Chemiluminescent substances which may be peroxidase/eosin/EDTA, isoluminol/- EDTA/H 2 O 2 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 labora- tory acceptable isotope, e.g. 125 I, 131 I, 3 H, 3 2 p , 35 S or 1 4 C.
  • the radioactivity may be measured in a ⁇ -counter or a scintillation counter or by radioautography followed by densitometry.
  • Complexing agents may be Protein A, Protein G (which forms a complex with immunoglobulins), biotin (which forms a complex with avidin and streptavidin), and lectin (which forms a complex with carbohydrate determinants, e.g. receptors).
  • 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.
  • this type of diagnostic agent normally comprises the antigen or epitope or the anti-idiotypic antibody 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 antigen, epitope, or anti-idiotypic antibody.
  • the carrier or support is normally solid, and the antigen, epitope or anti-idiotypic antibody 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.
  • the antigen, epitope or anti-idiotypic antibody of the invention may be indirectly coupled to a solid support via a bridging compound or
  • linker which is designed to link the solid support and the antigen, 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.
  • suitable plastics are latex, a polystyrene, polyvinylchloride, polyurethane, polyacrylamide, polyvinylacetate and any suitable copolymer thereof.
  • 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.
  • the invention also relate to a monoclonal or polyclonal antibody which binds to an antigen or epitope as defined above and also to an anti-idiotypic antibody as described below.
  • a most useful type of antibody is a monoclonal antibody; however, also a polyclonal antibody may be of great importance provided it shows a sufficient selectivity, which may be obtained, e.g., by means of known absorption methods.
  • antibody refers to a substance which is produced by a mammal or more precisely a cell of mammalian origin belonging to the immune system as a response to exposure to the polypeptides of the invention.
  • the variant domain of an antibody is composed of variable and constant sequences.
  • the variant part of the domain is called the idiotype of the antibody. This part of the antibody is responsible for the interaction with the antigen, the antigen binding.
  • the idiotypic structure is antigenic and can thus give rise to specific antibodies directed against the idiotypic structure. Production of such anti-idiotypic antibody has been done in mice.
  • the antibodies raised against the idiotype, the anti-idiotypic antibodies may mimic the structure of the original antigen and therefore may function as the original antigen to raise antibodies reactive with the original antigen.
  • This approach may be advantageous as it circumvents the problem associated with the characterization and synthesis of the important immunogenic parts of the antigen in question. This is most important in the case of conformational epitopes, which might otherwise be difficult to identify.
  • the present invention therefore also relates to an anti-idiotypic antibody which is directed against the site of an antibody which binds the antigen or the epitope according to the invention.
  • the antibodies of the present invention may be produced by a method which comprises administering in an immunogenic form at least a part of the antigen or epitope of the invention or an anti-idiotypic antibody as defined above to obtain cells producing antibodies reactive with said polypeptide and isolating the antibody containing material from the organism or the cells.
  • the methods of producing antibodies of the invention will be explained further below.
  • 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.
  • the diagnostic agent may be in the form of a test kit comprising in a container an antibody as defined above.
  • the diagnostic agent may be used in the diagnosis of MS or a prestage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to the invention expressing the antigen or epitope of the invention, comprising contacting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent comprising an antibody as defined above, and determining the presence of any antigen or epitope from the sample binding to the antibody.
  • a body fluid such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient
  • 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 an antigen or an epitope of the invention in the 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 an antigen, epitope, or anti-idiotypic antibody, and that the binding of antigen or epitope from the sample to the diagnostic agent is detected using a secondary antibody which is capable of binding to the thus bound antigen 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 detectable or which may be reacted with another substance to "produce a detectable product.
  • the label may be selected from radioactive isotopes, enzymes, chromophores, fluorescent or chemiluminescent substances, and complexing agents, all of which are described in greater detail above.
  • the antibody of the invention may be used in an assay for the identification and/or quantification of at least a form and/or a part of the antigen or epitope of the invention present in a sample.
  • the identification and/or quantification performed by the use according to the present invention may be any identification and/or quantification involving the antigen or epitope of the invention.
  • the identification and/or quantification may be performed for both a scientific, a clinical and an industrial purpose. As will be further described below, it is especially important in clinical routine to identify or quantify antigens or epitopes of the invention.
  • the antigen or epitope or anti-idiotypic antibody may be used in an assay for the identification and/or quantification of antibodies reactive with the antigen or epitope of the invention and being present in a sample, e.g. as defined above.
  • This assay may be carried out by use of a method comprising contacting the sample with the antigen or epitope of the invention and detecting the presence of bound antibody resulting from said contacting and correlating the result with a reference value.
  • the antibody used in the method of the invention 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 possess ing high avidity such as poloclonal antibodies may be selected for catching techniques.
  • the antibody used in the present method is preferably in substantially pure form (purified according to suitable techniques or by the methods of the invention, see below) in order to improve the precision and/or accuracy of the assays of the invention.
  • Another field of the invention is a method for producing an antibody which binds to the antigen or the epitope of the invention, which comprises immunizing an animal with the antigen or epitope or an anti-idiotypic antibody or an antigen or epitope produced by cultivating cells harbouring a plasmid which contains and is capable of expressing a nucleotide sequence as described below which codes for a polypeptide which has the properties of the antigen or epitope as described above, or by synthetically producing a polypeptide having an amino acid sequence derived from the nucleotide sequence of the retrovirus of the invention, whereby cells producing an antibody specific for the antigen is obtained and the antibody is isolated from the animal or the cells.
  • the antibody is preferably a monospecific antibody.
  • the monospecific antibody may be prepared by injecting a suitable animal with a substantially pure preparation of the polypeptide of the invention followed by one or more booster injections at suitable intervals (e.g. one or two weeks to a month) up to four or five months before the first bleeding.
  • suitable intervals e.g. one or two weeks to a month
  • the established immunization schedule is continued, and the animals are bled about one week after each booster immunization, and antibody is isolated from the serum in a suitable manner (cf. e.g. Harboe and Ingild, 1973).
  • the antibody may be a polyclonal antibody.
  • Polyclonal anti- bodies may be obtained, e.g. as described in Harboe and Ingild, see above. More specifically, when polyclonal antibodies are to be obtained, the compound comprising an antigen or epitope of the invention or an anti-idiotype antibody as described above is prepared and preferably after addition of a suitable adjuvant, such as Freund's incomplete or complete adjuvant, injected into an animal.
  • a suitable adjuvant such as Freund's incomplete or complete adjuvant
  • the animals are bled regularly, for instance at weekly intervals, and the blood obtained is separated into an antibody containing serum fraction, and optionally said fraction is subjected to further conventional procedures for antibody purification, and/or procedures involving use of purified compounds comprising an antigen or epitope of the invention or idio-typic antibody as described above.
  • monoclonal antibodies are obtained.
  • the monoclonal antibody may be raised against or directed substantially against an antigen or epitope of the invention as described above or an anti-idiotypic antibody as described above.
  • the monoclonal antibody may be produced by conventional techniques (e.g. as described by Kohler and Milstein, 1975), e.g.
  • the monoclonal antibody may be produced by fusing cells producing the monoclonal antibody with cells of a suitable cell line, and selecting and cloning the resulting hybridoma cells producing said monoclonal antibody.
  • the monoclonal antibody may be produced by immortalizing an unfused cell line producing said monoclonal antibody, subsequently growing the cells in a suitable medium to produce said antibody, and harvesting the monoclonal antibody from the growth medium.
  • the immunized animal used for the preparation of antibodies of the invention is preferably selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse and guinea pigs.
  • the cells producing the antibodies of the invention may be spleen cells, lymph cells or peri- pheric lymphocytes.
  • hybridoma cells When hybridoma cells are used in the production of antibodies of the invention, these may be grown in vitro or in a body cavity of an animal.
  • the antibody-producing cell is injected into an animal such as a mouse resulting in the formation of an ascites tumour which releases high concentrations of the antibody in the ascites of the animal.
  • the animals will also produce normal antibodies, these will only amount to a minor percentage of the monoclonal antibodies which may be purified from ascites by standard purification procedures such as centrifugation, filtration, precipitation, chromatography or a combination thereof.
  • An example of a suitable manner in which the monoclonal antibody may be produced is as a result of fusing spleen cells from immunized mice (such as Balb/c mice) with myeloma cells using conventional techniques (e.g. as described by Dalchau et al. 1980).
  • the fusions obtained are screened by conventional techniques such as binding assays employing compounds comprising antigen or epitope of the invention or an anti-idiotypic antibody as described above isolated by the above-described methods.
  • the invention also relates to a nucleic acid having a nucleotide sequence which is characteristic to the above-identified retrovirus.
  • the nucleotide sequence is one which is distinct from sequences from known retroviruses, and at the same time is indicative of the presence of the above-identified new retrovirus.
  • Such nucleic acids are nucleotide sequences which, when used as a probe on samples of body fluid such as blood samples or tissue samples from a number of healthy persons and blood samples or tissue samples from diagnosed multiple sclerosis patients, respectively, detects nucleotide sequences from diagnosed multiple sclerosis patients, which can not be detected in samples from the healthy persons.
  • nucleotide sequences serve as a specific marker of the retrovirus of the invention.
  • the nucleo- tide sequences can be obtained by isolating nucleotide sequences derived from the cell culture described above or the purified retrovirus or fragments thereof as described above by the use of retrovirus-specific nucleotide primers recognizing specific regions of the nucleotide sequences from the above-described retrovirus.
  • the virus-specific nucleotide primers (which also constitute an aspect of the invention) can be developed using on the one hand a cell culture according to the invention and on the other hand primers which are not specific to the present retrovirus, but which contain nucleotide sequences which will recognize retrovirus generally, such as known conserved regions of nucleotide sequences from various retroviruses, using the following strategy:
  • the non-specific, but generally retrovirus-recognizing primers can be used to obtain nucleotide sequences derived from the cell culture according to the invention or the purified retrovirus according to the invention by PCR.
  • the nucleotide sequences thus obtained can be sequenced to identify regions thereof which are distinct from sequences from known retroviruses, and these distinct sequences can then be tested by using them as primers on samples from a number of healthy persons and samples from diagnosed MS patients, respectively, and selecting the sequences which give rise to the attainment of oligonucleotide sequences from diagnosed MS patients, but do not give rise to attainment of oligonucleotide sequences from the healthy persons.
  • the sequencing could in principle be excluded, but this would make more testing on samples necessary.
  • the invention relates to a nucleic acid having a nucleotide sequence (S) obtainable by using a retrovirus-related nucleotide primer recognizing conserved regions of known retroviruses to obtain nucleotide sequences derived from the cell culture according to the invention or, alternatively, the purified retrovirus according to the invention by PCR, optionally sequencing the nucleic acids obtained to identify sequences which are distinct from sequences from known retroviruses, testing the nucleic acids obtained by PCR or the sequences identified by sequencing by using them as primers on blood samples from a number of healthy persons and blood samples from diagnosed multiple sclerosis patients, respectively, and selecting, as the nucleotide sequence (S), the nucleic acids or sequences which give rise to the attainment of nucleotide sequences from diagnosed multiple sclerosis patients in PCR, but do not give rise to attainment of nucleotide sequences from healthy persons, or using such nucleic acids or sequences as primers for an additional PCR obtainment of nucleotide sequence
  • nucleotide sequences any nucleotide sequences of various length, preferably an oligonucleotide sequence.
  • the nucleotide sequence may be either a RNA nucleotide sequence or a DNA nucleotide sequence.
  • the invention relates to a diagnostic agent comprising a nucleotide probe which is capable of detecting a nucleotide sequence according to the invention.
  • the invention also relates to a nucleotide probe which is capable of detecting a nucleotide sequence as defined above. Both the nucleotide sequence and the nucleotide probe are valuable for use in diagnostic agents, such as PCR kits, for diagnosing MS.
  • probe describes any nucleotide sequence, preferably an oligonucleotide sequence, which may be used to obtain nucleotide sequences complementary to the probe, e.g. nucleotide sequences suitable for use as primers in PCR or for use as probes in hybridization techniques.
  • the substance used to label the probe may be selected from any substance which is in itself detectable or which may be reacted with another substance to produce a detectable product.
  • the label may be selected from radioactive isotopes, enzymes, chromophores, fluorescent or chemiluminescent substances, and complexing agents.
  • enzymes useful as labels are ⁇ -galactosidase, urease, glucose oxidase, carbonic anhydrase, peroxidases (e.g. horseradish peroxidase), phosphatases (e.g. alkaline or acid phosphatase), glucose-6-phosphate dehydrogenase and ribonuclease.
  • peroxidases e.g. horseradish peroxidase
  • phosphatases e.g. alkaline or acid phosphatase
  • glucose-6-phosphate dehydrogenase ribonuclease.
  • Radioactive isotopes may be any detectable and in a laboratory acceptable isotope, e.g. 125 I, 131 I, 3 H, 3 2 p , 35 S or 1 4 C.
  • the radioactivity may be measured in a ⁇ -counter or a scintillation counter or by radioautography followed by densitometry.
  • detection systems based on enzymes are the DIG-system (digoxygenin; Boehringer) and the Tropix-system
  • Complexing agents used in the detection may be biotin (which forms a complex with avidin and streptavidin). 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 mark ing may be performed with any of the labelling substances described above.
  • this type of diagnostic agent normally comprises the labelled probe, which reacts with or detects the presence of complementary sequences in a sample of a body fluid, such as a blood sample, or a tissue sample.
  • a sample of a body fluid such as a blood sample, or a tissue sample.
  • Either probe or sample may be bound to a carrier or support, so that appropriate treatments may be performed without risk of appreciable loss of the bound material.
  • the carrier or support is normally solid, and the probe or sample is bound to the carrier or support by any suitable method of binding, such as hydrogen bonding, van der Waal's forces or covalent binding.
  • the labelled probe or sample may be indirectly coupled to a solid support via a bridging compound or a linker.
  • the linker designed to link the solid support and the labelled probe or sample, may be streptavidin.
  • the solid support employed is e.g. a polymer or it may be a matrix coated with polymer.
  • the matrix may be of any suitable solid material, e.g. glass, paper or plastic.
  • the polymer may be a nylon or nitrocellulose. Examples of suitable plastics are polystyrene or polyvinylchloride.
  • the solid support may be in the form of a tube, a thin layer or strip, threads, solid particles such as beads or paper.
  • the invention also relates to a method of diagnosing multiple sclerosis, an early stage or a pre-stage thereof, a latent infection or a subclinical infection with the retrovirus of the culture defined above, the method comprising subjecting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient to a PCR analysis in which the sample is contacted with a diagnostic agent comprising the nucleotide probe or the nucleotide sequence defined above, and any detected nucleotide sequence is allowed to be amplified, followed by detection of any amplified target nucleotide sequence.
  • the PCR analysis is a well-established technique described, e.g., in Sambrook, 1990.
  • the invention relates to a method of diagnosing multiple sclerosis, an early stage or a prestage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to the invention comprising subjecting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient to a PCR analysis in which the sample is contacted with a diagnostic agent according to the invention allowing any nucleotide sequence to be amplified followed by detection of any amplified target nucleotide sequence.
  • a body fluid such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient
  • a diagnostic agent according to the invention allowing any nucleotide sequence to be amplified followed by detection of any amplified target nucleotide sequence.
  • the invention relates to a method of in vitro diagnosing multiple sclerosis, an early stage or a pre-stage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to the invention, optionally combined with a method for detection of an infection with another virus such as a herpes group virus, comprising contacting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent according to the invention comprising a nucleic acid according to the invention and determining the presence of any identical or homologous nucleotide sequences in the sample.
  • a body fluid such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient
  • a diagnostic agent according to the invention comprising a nucleic acid according to the invention and determining the presence of any identical or homologous nucleotide sequences in the sample.
  • the invention in another aspect, relates to a method of in vitro diagnosing multiple sclerosis, an early stage or a prestage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to the invention comprising contacting a sample of body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent according to the invention and determining the presence of bound antibody from the sample.
  • the invention relates to a method of in vitro diagnosing multiple sclerosis or a prestage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to the invention comprising contacting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent according to the invention and determining the presence of bound antigen from the sample.
  • a body fluid such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient
  • a diagnostic agent according to the invention
  • An important embodiment of the invention relates to a method for producing an antigen or epitope characteristic to the retrovirus, which comprises cultivating cells harbouring a plasmid which contains and is capable of expressing a nucleotide sequence which codes for a polypeptide which has the properties of the antigen or epitope of the invention.
  • An alternative method for producing an antigen or epitope characteristic to the retrovirus comprises synthesizing a peptide having an amino acid sequence derived from the nucleotide sequence according to the invention.
  • target nucleotide sequence describes any nucleotide sequence as defined above, preferably an oligonucleotide sequence, which contains a nucleotide sequence complementary to the probe used in the various assays such as PCR or hybridization.
  • the invention relates to a method for obtaining a protective immunity in an animal, including a human being, against multiple sclerosis caused by the retrovirus of the culture according to the invention, comprising administering, to the animal, an immunogenically effective amount of a vaccine against a herpes group virus such as Epstein-Barr virus, thereby preventing the previously mentioned interaction between a herpes group virus such as Epstein-Barr virus and the retrovirus of the invention which interaction is assumed to result in the activation of the retrovirus.
  • a herpes group virus such as Epstein-Barr virus
  • the vaccine should be made so as to allow an optimal stimulation of the relevant parts of the immune system, i.e to present the immunogenic agent for a period of time and in a form being optimal with respect to the recognization, the uptake or any other interaction or processing necessary for the stimulation.
  • vaccine is to be understood to comprise any preparation containing an immunologically effective part of a herpes group virus, e.g. an antigen or epitope of the herpes group virus suited for administration to living organisms for the prevention of MS by providing an animal, such as a human, with a protective immunity against a herpes group virus such as an Epstein-Barr virus.
  • the invention also relates to a method for obtaining a protective immunity in an animal, including a human being, against multiple sclerosis caused by the retrovirus of the invention, comprising administering, to the animal, an immunogenically effective amount of a vaccine comprising any preparation containing an immunologically effective part of a cell culture infected with the retrovirus of the invention, or purified retrovirus or fragments thereof all of which are defined above, e.g. an antigen or epitope of the invention, or comprising an anti-idiotypic antibody of the invention suited for administration to living organisms for the prevention of MS so as to prove the animal, such as a human, with a protective immunity against the retrovirus according to the invention.
  • the vaccines described above may be used seperately or the two vaccines may be used in combination.
  • immunological is understood to comprise the process of evoking a specific immunologic response with the expectation that this will result in humoral, and/or secre- tory, and/or cell-mediated immunity to a herpes group virus such as Epstein-Barr virus or to the retrovirus of the invention, i.e. immunity is to be understood to comprise the ability of the individual to resist or overcome infection or to overcome infection "more easily” compared to individuals who have not been immunized or to tolerate the infection without being clinically affected or to block transmission.
  • the immunization according to the present invention is a process of increasing resistance to infection with a herpes group virus such as Epstein-Barr virus, thereby preventing such a herpes group virus from activating any retrovirus according to the invention present in the same individual.
  • An overall aspect in the preparation of the vaccines of the invention is the physiological acceptability of the components and of the total composition of the vaccine.
  • the final formulation of the vaccine should be a mixture of substances supporting and enhancing the immune response induced by the specific immunogenic component.
  • the immunization according to the present invention is a process of increasing resistance to infection with a retrovirus of the invention.
  • the invention relates to a method as explained above, in which the vaccine is a live or dead Epstein-Barr virus administered to the mammal at a stage in the development of the mammal in which the disease caused by Epstein-Barr virus has a mild clinical cause and cannot induce active production of the retrovirus of the invention to result in multiple sclerosis, an early stage or a prestage thereof.
  • the vaccine may be administered to a mammal, preferably a human, and the stage in which the administration is performed is the pre-puberty stage.
  • the mammal, such as a human, to which the vaccine is administered may be mammal which has been shown to carry the retrovirus of the culture as defined above.
  • the vaccine may be an attenuated Epstein-Barr virus or an immunogenic antigen characteristic to Epstein-Barr virus and eliciting the formation of antibodies against Epstein-Barr virus.
  • the administration of the vaccine against the retrovirus of the invention to a mammal, such as a human, may be per-formed at any stage in the development of the mammal.
  • the vaccine against the retrovirus from the culture of the invention may be administered to a mammal which has been shown to carry the retrovirus of the culture as defined above or which has been shown to carry a herpes group virus such as an Epstein-Barr virus.
  • a herpes group virus such as an Epstein-Barr virus.
  • Epstein-Barr virus particle with a diameter of 150 nm is seen outside the cell membrane of a B-cell (EBV), i.e. a core surrounded by an icosahedral capsid measuring maximally 100nm in diameter and an outer irregularly shaped envelope with projections.
  • EBV B-cell
  • EBV.VC The central nucleic acid of the virion is surrounded by an icosahedral capsid (Crawford and Edwards, 1990).
  • Retrovirus-like particles located just outside the cell membrane measuring 90-100 nm in diameter (RVLP).
  • the particles have a core-like condensation and the envelope does not contain visible projections. Note the variable size of the particles and the diffuse spherical cores as those illustrated in a textbook on human retroviruses (Dalgleish and Weiss, 1990).
  • Viral particles together with a mixture of cell organelles and other small "cell-parts" are seen.
  • the material is a pelleted fraction of a sucrose gradient centrifuged as described in Example 5. After resuspension of the pellet, the material is spread on grids and stained as described in Example 8. Figure 2b.
  • Viral particles captured by monoclonal antibodies to gp46 are seen.
  • the material is prepared as described above in Figure 2a. Small cellular fragments have been washed away as described in Example 8. Finally, the material is stained.
  • the lymphoblastoid B-cell line was established as follows. Heparinized blood was diluted in phosphate-buffered saline PBS (pH 7.4, 20 i.u./ml heparin), before separating the mononuclear cells by Ficol-Isopaque density gradient centrifugation. The mononuclear cells were harvested and washed twice in cold PBS.
  • PBS phosphate-buffered saline
  • the mononuclear cells were seeded at a density of 20 ⁇ 10 6 /5 ml and grown in RPMI 1640 (Seromed) supplemented with 200 i.u./ml penicillin (Leo), 0.2 mg/ml streptomycin (Rosco), 0.29 mg/ml glutamine (Sigma), 0.01 M hepes buffer (Bioproduct), 5% human heat inactivated sera in Falcon Primaria bottles. During the first 7 weeks of cultivation, the serum percentage was 5%. After appearances of clones in the culture, the medium contained 10% of human serum.
  • the cells were capable of growing in clumps or singly without adherence to the surface of the container in which the cultivation took place.
  • the population doubling time was initially 17 hours but rose to 26 hours after 5 months of culture.
  • the maximum cell density obtainable was around 2 ⁇ 10 6 cells/ml and a poor cell proliferation was observed at cell densities below 0.35 ⁇ 10 6 cells/ml.
  • Fixation 2 ⁇ 10 6 cells were fixed with cold 2.5% glutaraldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate for at least 1 hour. The cells were then pelleted by centrifugation at 1500 rpm for 5 minutes. The pellet was washed 3 times in cacodylate buffer, pH 7.2 and then postfixed for 1 hour in 1% osmium tetroxide buffered at pH 7.2 with veronal acetate buffer and then washed 3 times in veronal acetate buffer. Between these washing steps, pelleting was carried out at 1500 rpm for 5 minutes.
  • the cells were then treated for 1 hour in 75% propanol + 25% TAAB812 resin mixture (TAAB Laboratories), for 1 hour in 50% propanol + 50% TAAB812 resin mixture, and for 1 hour in 25% propanol + 75% TAAB812 resin mixture, followed by 24 hours in fresh resin mixture.
  • TAAB Laboratories 75% propanol + 25% TAAB812 resin mixture
  • 50% propanol + 50% TAAB812 resin mixture for 1 hour in 25% propanol + 75% TAAB812 resin mixture, followed by 24 hours in fresh resin mixture.
  • pelleting was carried out at 2000 rpm for 10-15 minutes.
  • Sections for electron microscopy of light grey or grey in colour (about 40 nm) were cut on an LKB Ultratome with a diamond knife. They were then stained with uranyl acetate for 10 minutes, followed by lead citrate for 2 minutes. Electron microscopy
  • the sections were examined in a Jeol 100 B electron microscope at an accelerating voltage of 60 KV.
  • FIG 1a Immature vial capsids were seen in the nucleus ( Figure 1b). Viral capsids without core were also seen.
  • the retrovirus according to the invention was seen along the outer cell mem brane in approximately 1-2% of the cells.
  • the particles were spherical structures with a diameter of 90-100 nm, containing a core-like condensation as seen in Figures 1c and 1d.
  • the outer membrane did not contain visible projections.
  • the structures were indistinguishable from virus particles and had a close resemblance to known retrovirus. Due to the morphology of these particles, the retrovirus was characterized as being a type C-like retrovirus in accordance with the description given in Dalgleish et al., 1990.
  • the cell culture was examined for production of various products, and the presence of leukocyte differentiation antigens was analyzed.
  • interleukin-6 (IL-6) was measured by a biological assay using the IL-6 sensitive B 9 cell-line as described in Rozenberg et al., 1991. Interferon assay was performed as described in Haahr et al., 1976, and neutralization was made with a polyclonal antibody to interferon- ⁇ -(Boehringer Mannheim).
  • Tumour necrosis factor activity was measured in a cytotoxicity assay in L-929 cells.
  • monolayers of cells seeded the day before (2 ⁇ 10 4 cells/well) were overlayed with two-fold dilutions of the supernatants to be tested, obtained from the supernatant of cell culture of the cell line 1533, and incubated at 38.5°C with actinomycin D
  • TNF titers were assessed as the dilu tion resulting in 50% cytotoxicity and compared with standard TNF titrated on each plate.
  • the cytotoxic substance was identified as TNF- ⁇ by neutralization by the specific antiserum to human TNF- ⁇ (rabbit anti-human TNF- ⁇ polyclonal antibody 80 ⁇ l/ml (80-800 neutralizing units), Genzyme) and was not neutralized by specific antiserum to human TNF- ⁇ (rabbit anti-human TNF- ⁇ 80 neutralizing units/ml, Walther Fies Gent).
  • the cell culture was shown to have an autocrine production of interleukin-6 (2 units/ml) and to produce interferon- ⁇ spontaneously (2.74 units/ml). Tumour necrosis factor- ⁇ (100 units/ml) was also produced by the cell culture spontaneously.
  • the identification of the presence of leukocyte differen- tiation antigens was performed by the use of the immunohistochemical method as described in the following.
  • CD15 (DAKO-M1) -
  • the lymphoblastoid cell culture was examined by an immunofluorescence analysis for the expression of the HTLV-I antigens p19 and p24 by the use of the following monoclonal antibodies directed against HTLV-I p19 and p24: 12G4, MAS 197b, 6G9 and MAS 199b (Sera-Laa). As appears from Table 2 in Example 9 below, no binding of monoclonal antibodies directed against the HTLV-I antigens p19 and p24 was observed, which means that the lymphoblastoid cell culture did not express these antigens. To characterize other possible antigens present in the lymphoblastoid cell culture infected with the retrovirus, the culture was examined for animal virus antigens from murine-leukemia virus (MuLV), simian sarcoma virus-1
  • the antibodies present in the serum from the patient from which the lymphoblastoid cell culture was derived were examined in order to further support the above results showing that the retrovirus from the lymphoblastoid cell culture was not an HIV-I, HIV-II or HTLV-I virus.
  • the serum from the patient was examined for IgG antibodies to HIV-I and HIV-II, and HTLV antibodies were determined by an immunofluorescence method at the
  • sucrose 22.5 g of sucrose in 25 ml of 2xTNE (100 mM
  • the gradient mixer was rinsed and the gradient was made up in 9/16 ⁇ 3 1/2 (14 ⁇ 89 mm) tubes.
  • the gradient was spun for 16 hours at 38,000 rpm in an SW41 rotor at 4°C, and 6 fractions were taken and diluted three times with TNE (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA). Centrifugation was then performed for 3 hours at 38,000 rpm in an SW41 rotor (45,000 ⁇ g) at 4°C, and the retrovirus fraction was then contained in the pellet. No bands were visible in the first gradients which were divided into 6 fractions. Each fraction was pelleted and 6 new gradients were run separately.
  • microspheres with rabbit anti-mouse/anti-rat IgG were labelling of microspheres with rabbit anti-mouse/anti-rat IgG as follows:
  • Buffer B 100 mM sodium borate; 100 mM ethanol amine, pH 9
  • the beads were spun down and washed in double distilled water until pH was about 6-7.
  • the mixture was washed and resuspended in PBS.
  • the antibodies were conjugated to the labelled microspheres (about 3 ⁇ 10 9 spheres/g were used) in the following manner: incubation was carried out with 1 ml of antibodies: anti-rat HTLV-I: 30g, 1e, 5a, 69b; or anti-mouse HTLV-I 46 (T. Schultz, Chester Beatty Lab., UK) (+), or the following antibodies raised against other HTLV-I antigens: anti-mouse HTLV-I p19 (ascites) or anti-mouse HTLV-I p24 (K. Kaltoft, Bartholin, Aarhus University) (-) 1:3000 ⁇ diluted in PBS for 1 hour at room temperature. The beads were spun down and washed several times in PBS/Tween.
  • the incubation with sample was performed by adding 10 4 spheres to 500 ⁇ l of sample supernatant. The mixture was then incubated at 37°C while shaking.
  • the cells were resuspended and lysed in 4 ml of 100 mM Tris-HCl, 1% Triton X-114, 10 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride), pH 8.1 at 0°C for 10 minutes.
  • the lysate was clarified by centrifugation at 5000 x g for 10 minutes at 4°C and was transferred to Falcon tubes and incubated for 10 minutes at 37° C to induce the phase separation.
  • the detergent phase at the bottom of the tube was collected after centrifugation for 10 minutes at 20° C and 1800 ⁇ g. An equal volume of 100 mM Tris-HCl, pH 8, was added and the procedure was repeated. The detergent phase (100-600 ⁇ l) was suspended in 2 ml of 100 mM Tris-HCl, pH 8, with 0.5% CHAPS (3-[(3-cholamidopro-pyl)dimethylammonio] 1-propanesulfonate; Boehringer) to avoid temperature-induced phase separation during subsequent handling. For Western blots, an equal volume of sample and sample buffer was used, but the final SDS concentration should not exceed 0.1%.
  • the viral particles or antigens prepared by these methods were used to further characterize the virus as exemplified in the following.
  • the supernatants obtained from cell line 1533 were concen- trated and purified by ultracentrifugation on sucrose gradients, followed by pelleting of the various fractions as described in Example 5 above.
  • test tubes were incubated at 37° C for 90 minutes. The reaction was stopped by adding 100 ⁇ l of a cold solution of distilled water containing 12.5% of water saturated with sodium phosphate, 12.5% of water saturated with sodium pyrophosphate, and 20% of trichloroacetic acid (TCA). After 30 minutes on ice, the tubes were emptied onto a Millipore membrane filter (cat.no. GVWP02500), and the membrane filter was washed 4 times with 5 ml of 5% TCA under depression of 2 atm.
  • TCA trichloroacetic acid
  • Retroviral particles or antigens purified by methods A, B and C as described in Example 5 above were subjected to SDS-PAGE.
  • the 10% SDS-PA gels were subsequently used either for direct visualization of the proteins in the samples by silver staining or for Western Blotting. In either case, 5 ⁇ l of prestained M r marker (Sigma) was loaded in one lane of the gel.
  • the gels were silver stained by incubating the gels consecutively in the following:
  • sample buffer 100 mM Tris-HCl pH 6.8, 10% glycerol, 12% SDS and 5% BPB
  • sample buffer 100 mM Tris-HCl pH 6.8, 10% glycerol, 12% SDS and 5% BPB
  • the gels were blotted to Immobilon (Millipore) membrans in a Kem-En-Tec wet blotter.
  • the gels were equilibrated in transfer buffer for 15 minutes, while the membranes were equilibrated in methanol, water and transfer buffer (25 mM Tris-HCl, pH 8.3; 192 mM glycine; 20% ethanol; 0.1% SDS).
  • the gels and membranes were made up as sandwiches with Whatman filter paper and blotted 500 Vh (overnight at
  • the sandwich was dismantled and the membrane washed in double distilled water. The membrane was then blocked in TBS/0.1% Tween for 15 minutes at room temperature and could be stored as such in TBS/Tween at 4°C for a week.
  • the membranes were screened with the following antibodies, all raised against HTLV-I envelope antigens: anti-rat
  • HTLV-I 30g, le, 5a, 69b; and anti-mouse HTLV-I 46 (T.
  • the screening procedure was as follows: Antibodies were 1:3000 diluted in PBS with 0.05% Tween, incubated overnight at room temperature on a shaker and washed 3 x 5 minutes in TBS with 0.05% Tween and 350 mM NaCl. The membranes were then incubated for 1 hour with 1:3000 diluted rabbit anti-mouse or anti-rat antibodies and washed 3 x 5 minutes in TBS with 0.05% Tween and 350 mM NaCl. The membranes were then incubated for 1 hour with 1:5000 diluted AP-conjugated goat anti-rabbit antibodies and washed 3 x 5 minutes in TBS with 0.05% Tween and 350 mM NaCl.
  • the membranes were then washed for 5 minutes at room temperature in 100 mM ethanol amine, pH 9, and the substrate (1/10 vol NBT (4-nitroblue tetrazolium chloride; Boehringer); 1/100 vol of BCIP (5-bromo-4-chloro-3-indolyl phosphate; Boehringer); 1/500 vol of 2 M MgCl 2 ) in 100 mM ethanol amine, pH 9, was added.
  • the membranes were washed in water after the colour reaction. The fact that some, but not all, of the antibodies gave a positive reaction (+) indicates similarity to, but not identity with HTLV-I.
  • Electron microscopy - negative staining A small drop of virus containing solution from the double purified fractions as described in Example 5A was placed on carbon coated G400 grids (Gilder) which were initially coated with parlodion 2% in amyl acetate, and a drop of negative stain (1% phosphotungstic acid (PTA) adjusted to pH 6.5 with 1N KOH) was added. After 20 seconds, excess moisture was removed with the torn edge of a filter paper. The grids were then air-dried and examined in an electron microscope (Jeol 100 B, Jeol, Japan). The result of one of the above-described experiments is illustrated in figure 2a.
  • PTA phosphotungstic acid
  • the grids were floated (carbon side down) on a 15 ⁇ l drop of a solution of protein A (0.01 mg/ml in PBS buffer) for 10 minutes, and excess moisture was removed with the torn edge of a filter paper.
  • the grids were washed by transferring them through 3 drops of PBS buffer, 1-2 minutes on top of each drop.
  • the grids were floated on 15 ⁇ l of antiserum (mouse monoclonal antibody to HTLV-I gp46 diluted 1:100 in PBS) for 10 minutes. Excess moisture was removed with the torn edge of a filter paper, and the grids were then washed in buffer as described. The grids were then floated on
  • cytoplasmatic RNA was purified, subjected to reverse transcription and subjected to PCR analysis. 1st strand cDNA was used as a template in all PCR experiments described herein. Cytoplasmatic RNA was purified and amplified as follows:
  • Eppendorf tube and harvested for 10 seconds in an Eppendorf centrifuge after which the supernatant was discarded.
  • the cells were lysed in 500 ⁇ l of lysis buffer (140 mM NaCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl pH 8.6, 0.5% NP-40) and were placed on ice for 5 minutes. The mixture was pelleted cold for 5 minutes at 10,000 ⁇ g in the Eppendorf centrifuge.
  • lysis buffer 140 mM NaCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl pH 8.6, 0.5% NP-40
  • T4PNK buffer commercially available buffer from Boehringer Mannheim
  • 5 ⁇ l of ⁇ 32 P-ATP from NEN, New England Nuclear
  • 11.4 ⁇ l of distilled water 11.4 ⁇ l of distilled water
  • 8 units (1 ⁇ l) of T4-polynucleotide kinase 2 ⁇ l of 10 ⁇ T4PNK buffer (commercially available buffer from Boehringer Mannheim)
  • 5 ⁇ l of ⁇ 32 P-ATP from NEN, New England Nuclear
  • distilled water 11.4 ⁇ l
  • 8 units (1 ⁇ l) of T4-polynucleotide kinase 8 units (1 ⁇ l) of T4-polynucleotide kinase
  • the mixture was incubated for 45 minutes at 37° C and for 10 minutes at 68° C.
  • To the mixture was added 40 ⁇ l of distilled water, 240 ⁇ l of 7 M NH 4 Ac and 750 ⁇ l of 96% ethanol, and the tube was placed on an ice bath for 30 minutes.
  • the mixture was harvested for 20 minutes at 12,000 rpm (15,000 ⁇ g) in an Eppendorf centrifuge at 4°C and then washed with 80% ethanol.
  • the mixture was resuspended in 100 ⁇ l of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8).
  • First strand cDNA synthesis and PCR were performed with equipment and kits from Perkin Elmer Cetus (USA).
  • Reverse transcription was performed in a buffer with 5 mM MgCl 2 , 1 ⁇ PCR, 1 mM of each of the dNTP's, 1 ⁇ / ⁇ l RNase inhibitor, 2,5 ⁇ / ⁇ l reverse transcriptase, 2.5 ⁇ M random hexamers and 1 ⁇ g of RNA.
  • the mix was incubated at 42 °C for 15 minutes, at 99° C for 5 minutes, and at 5°C for 5 minutes.
  • Amplification was performed by adding MgCl 2 to 2 mM, 1 ⁇ PCR, 2.5 ⁇ Taq DNA polymerase and double distilled water to 100 ⁇ l.
  • the annealing temperature was between 37°C and 48° C, in high stringency reactions it was 60°C.
  • the lymphoblastoid cell culture infected with the retro- virus was analyzed for the presence of nucleotide sequences specific to the retrovirus HIV-I using high stringency nested PCR as described in Teglbjaerg et al., 1992.
  • the following primer pairs and probes were used:
  • LST1 5' ATC AAG CAG CCA TGC AAA TG
  • LST3 5' AGG AGG AGA TAT GAG GGA CAA TTG
  • LST4 5' GGA GCT GTT GAT CCT TTA GGT ATC
  • LST5 5' GCC TGG GAG CTC TCT GGC TA
  • LST6 5' CGG GCG CCA CTG CTA GAG A
  • the lymphoblastoid cell culture was also analyzed for the presence of nucleotide sequences specific to the retrovirus HTLV-I using PCR.
  • the following primers were used: HTLV-I/026 and HTLV-I/029
  • HTLV-I/026 5' GAG GCA GAT GAC AAT GAC CAY GAR CC
  • HTLV-I/029 5' NAG CCA CCT NCT GAA CTG TC
  • PCR products were obtained (indicated as positive results in Table 2).
  • the results of the PCR experiments are illustrated in Table 2.
  • the primer sequences were based on known sequences from other retroviruses.
  • the sequences were based on various principles for reverse translation. The following primers were synthesized and used:
  • BZLF1 BZ1
  • the latent infection protein EBNA 2 was localised to the nuclei, but occasionally, cells showed additional fine granular cytoplasmic labelling.
  • the latent infection protein LMP was localised to the cytoplasm and cell membrane.
  • the immediate-early proteins BZLF1 were localised to the nucleus but a weaker, diffuse cytoplasmic staining was also seen in many cells, either alone or in combination with nuclear labelling.
  • Productive cycle proteins in the form of EA were seen as an intense nuclear and a weaker cytoplasmic reaction, whereas VCA was mainly nuclear and with a granular appearance.
  • MA was localised to the cytoplasm.
  • lymphoblastoid cell culture expressed latent infection and partly productive cycle proteins, indicating that the cell line was transformed by Epstein-Barr virus with an increased frequency of cells entering the lytic cycle.
  • Epstein-Barr virus with an increased frequency of cells entering the lytic cycle.
  • EBV Epstein-Barr virus

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Abstract

La présente invention se rapporte à un rétrovirus humain analogue au type C. Dans une analyse par imbrications de la polymérisation en chaîne (PCR) effectuée dans des conditions très strictes et avec des paires d'amorces et des sondes utilisées dans la détection du VIH-1 ou du HTLV-1, aucune séquence génomique ne peut être détectée; dans une analyse par immunofluorescence, des anticorps dirigés contre des antigènes p19 ou p24 du HTLV-I ou contre les rétrovirus MuLV, SSV-1 (p28), FeLV ou RD114 ne se lient pas. Lorsque l'analyse est effectuée sur une structure purifiée contenant un rétrovirus, une activité transcriptase inverse peut être détectée. Une culture cellulaire qui est en outre contaminée par un virus du groupe de l'herpès stimulant la production du rétrovirus a été déposée. La présente invention se rapporte également à des agents de diagnostic comprenant des fragments génomiques du rétrovirus qui peuvent être utilisés comme sondes nucléotidiques dans la polymérisation en chaîne, ainsi que des agents de diagnostic comprenant des antigènes rétroviraux capables de lier des anticorps spécifiques du rétrovirus que l'on peut utiliser dans le diagnostic de la sclérose en plaques. Un procédé d'obtention d'une immunité protectrice contre la sclérose en plaques consistant à administrer un vaccin contre le virus Epstein-Barr est également décrit.
PCT/DK1992/000299 1991-10-11 1992-10-12 Retrovirus humain analogue au type c lie a la sclerose en plaques WO1993007259A1 (fr)

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