EP0557388A1 - Diagnose und behandlung von multipler sclerose - Google Patents

Diagnose und behandlung von multipler sclerose

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Publication number
EP0557388A1
EP0557388A1 EP91920470A EP91920470A EP0557388A1 EP 0557388 A1 EP0557388 A1 EP 0557388A1 EP 91920470 A EP91920470 A EP 91920470A EP 91920470 A EP91920470 A EP 91920470A EP 0557388 A1 EP0557388 A1 EP 0557388A1
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EP
European Patent Office
Prior art keywords
msv
antibody
antigen
antigenic fragment
isolated
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English (en)
French (fr)
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EP0557388A4 (en
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Robert David Cook
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UNIVERSITY Co Pty Ltd
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UNIVERSITY Co Pty Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18421—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to the diagnosis of multiple sclerosis (MS) and in particular it relates to a method for diagnosis of MS by immunocytochemical staining of brain tissue, as well as to a method for serological diagnosis of MS.
  • MS multiple sclerosis
  • diagnosis is usually based on the results of several techniques including neurological examination, CAT scans, NMR imaging and CSF analysis, none of which is conclusive on its own.
  • the present invention also relates to the treatment of MS.
  • the invention provides antibodies to a virus which has been isolated from human MS brain tissue, and these antibodies may be used in direct therapy of MS patients.
  • the present invention also provides a vaccine for use in producing an immune response to the virus isolated from MS brain tissue.
  • MS is an autoimmun -type disease and is based on the animal model of experimental allergic encephalomyelitis (EAE) and the fact that immunosuppression has a beneficial effect in some MS patients.
  • EAE allergic encephalomyelitis
  • CNS central nervous system
  • MS is a viral disease and is based on epidemiological studies and the demyelinating effect of some viruses, in particular the morbilliviruses such as measles (MV) and canine distemper (CDV) viruses.
  • MV measles
  • CDV canine distemper
  • CLPs curved, linear profiles
  • the antibody against the feline virus has now been used in affinity chromatography to repeatedly isolate virus from the brains of 8 different confirmed cases of MS but not from 4 non-MS brains.
  • Preliminary virological, immunocytochemical and polyacrylamide gel electrophoretic (PAGE) studies have indicated that this virus is related to MV and CDV, and this relationship has now been further confirmed by the ELISA-type assay.
  • the association of this virus with demyelinating lesions in the CNS of domestic cats and MS patients indicates a possible causal relationship between these animals and the human disease 7 .
  • a subclinical, primary demyelinating disease has been identified in the CNS of approximately 7% of cats examined 8-9 and a persistent, non-permissive paramyxovirus has been isolated from affected brain tissue 10 .
  • Peroxidase-labelled antibodies raised against the cat virus stain virus-like particles and nucleocapsid-like structures (CLPs) within MS plaques 5 .
  • the staining reaction is blocked by pretreatment of MS brain tissue with sera from MS patients but not by pretreatment with non-MS human sera 5 .
  • affinity chromatography has now been used to isolate CLPs and virus particles from human MS brain tissue.
  • the antibodies to the cat virus used in the immunocytochemical studies previously described, were absorbed to an affinity chromatography column containing one of several matrices and through this was passed homogenised brain tissue from MS and non-MS patients.
  • CLPs and virus particles which conform to those of morbilliviruses were present in the eluate of the MS brain tissue but not in that of the non-MS tissue.
  • the virus isolated from the MS tissue has been grown in culture and has been harvested using the same affinity chromatography technique. It is considered that the results obtained provide strong evidence that MS is due to persistent or latent viral infection. Such an aetiology is more consistent with the epidemiology of the disease than that of an autoimmune-type disease.
  • MSV multiple sclerosis virus
  • the present invention provides MSV in isolated form, for example, as a culture of isolated MSV in CV 1 cells or in primary oligodendrocyte cultures. There is also provided a method for the preparation of MSV in isolated form.
  • the antibodies of this aspect of the invention may be produced by conventional techniques which are well known to persons skilled in the art using purified MSV isolated from MS brain tissue or from in vitro culture as described above.
  • hybrid cell lines or hybridomas producing monoclonal antibodies may be produced using the well known fusion technique first described in 1975 by Kohler and Milstein 11,12 .
  • Polyclonal antibodies may be produced, for example, in laboratory animals such as rabbits, again by well known techniques 12 -
  • a method for detection of MSV in a sample such as a tissue sample, taken from a patient, which comprises contacting the sample with antibody which recognises or binds to MSV or an antigen of MSV or an antigenic fragment thereof, and detecting binding of said antibody to indicate the presence of MSV in the sample.
  • the polyclonal or monoclonal anti- MSV antibodies of this invention may be used in immunocytochemical staining of tissue from MS brains to confirm the diagnosis of the disease.
  • a method for the serological diagnosis of MS is based on the observation that sera from MS patients contain circulating antibodies to MSV.
  • a method for the detection of anti-MSV antibodies in a fluid sample taken from a patient which comprises contacting said fluid sample with MSV or an antigen or antigenic fragment thereof, and detecting anti-MSV antibodies bound to said MSV or antigen or antigenic fragment thereof.
  • the fluid sample may be a blood or cerebrospinal fluid sample.
  • the sample is a blood sample, which may be whole blood or a derivative thereof, for example blood serum or blood plasma.
  • the diagnostic method of this aspect of the invention may utilise the well known principles of enzyme immunoassays or radio-immunoassays to detect the presence of any anti-MSV antibodies from the serum sample bound by the detecting antigen.
  • the detecting antigen (MSV or an antigen or antigenic fragment thereof) may, for example, be immobilised on a solid support, and the presence of bound anti-MSV antibodies can be detected using appropriately labelled anti-human immunoglobulin antibody.
  • Other alternatives will be well known to persons skilled in the art.
  • a diagnostic test kit for detection of anti-MSV antibodies in a fluid sample which is characterised in that it includes MSV or an antigen or antigenic fragment thereof as detecting antigen, preferably immobilised on a solid support.
  • a vaccine composition for stimulating an immune response against MSV in a human or animal patient which comprises inactivated or attenuated MSV, or an antigen of MSV or antigenic fragment thereof, as the active immunogen.
  • a method of producing an immune response against MSV in a human or animal patient which comprises administration to said patient of an effective amount of an active immunogen comprising inactivated or attenuated MSV, or an antigen of MSV or antigenic fragment thereof.
  • the active immunogen may be coupled to a carrier molecule to improve its immunogenicity.
  • suitable carrier molecules may include, for example, haemocyanins such as keyhole limpet haemocyanin, bovine serum albumin or ovalbumin.
  • the vaccine composition may optionally include an adjuvant.
  • adjuvants for incorporation into animal and human vaccines include, for example, Freund's complete and incomplete adjuvants, alum, and the like.
  • the vaccine may be produced for use in cats, similar to the canine distemper vaccine, and/or in humans, particularly human infants, similar to the measles vaccine.
  • a method of treatment of MS in a patient which comprises administering to the patient an effective amount of an antibody, preferably a monoclonal antibody, which recognises or binds to MSV, or an antigen of MSV or to an antigenic fragment thereof.
  • an antibody preferably a monoclonal antibody, which recognises or binds to MSV, or an antigen of MSV or to an antigenic fragment thereof.
  • the antibody used in this aspect of the invention may be combined with a carrier or targeting molecule, for example carrier or targeting molecules such as intercellular adhesion molecules which assist the antibody to penetrate the blood brain barrier.
  • a carrier or targeting molecule for example carrier or targeting molecules such as intercellular adhesion molecules which assist the antibody to penetrate the blood brain barrier.
  • EXAMPLE 1 Isolation of MSV from MS brain tissue and in vitro cultivation thereof in CV 1 cells.
  • Polyclonal antibodies to the persistent, non- permissive paramyxovirus isolated from cat brain tissue 10 were raised in rabbits.
  • the immunoglobulins were purified from sera of inoculated animals, extensively adsorbed against cat liver powder, acetone dried Crandell feline kidney (CRFK) and Vero cells and concentrated to 5 ⁇ mg of protein per ml.
  • the prepared antibody was then absorbed to either Affi-Gel Protein A (Bio-Rad) or CNBr- activated Sepharose 4B (Pharmacia).
  • Affi-Gel Protein A Bio-Rad
  • CNBr- activated Sepharose 4B Pharmacia
  • the tubules had a thin translucent core and their walls had a "herring-bone” appearance consistent with a helical structure.
  • Some eluants containing the virus-like particles were pelleted by centrifugation, embedded in agar and fixed and processed for electron microscopy.
  • the pellets contained virions, up to 300 nm diameter, with internal tubular profiles, about 18 nm in diameter, sectioned in various planes.
  • the viral isolation from MS brain tissue has been repeated more than 40 times.
  • CV 1 cells infected with the eluates from the MS brains showed some focal cytopathic effects (CPE) in the form of small syncytia and slight cytoplasmic vacuolation.
  • CPE focal cytopathic effects
  • Ultrastructural examination of these cultures revealed the presence of cytoplasmic inclusions which consisted of tubular structures approximately 18 nm in diameter. These inclusions were morphologically similar to those observed in the initial isolation of the feline paramyxovirus 10 .
  • Virus particles, similar to those isolated from MS brains were present in some cultures and were also isolated by use of the affinity chromatography technique.
  • the nucleocapsids and the virions isolated from MS brains show morphological similarities and some im unological cross-reactivity with MV and CDV. This, in addition to the initial PAGE studies and the formation of small syncytia in tissue culture, suggests that the virus isolated from MS brains may be morbiUivirus.
  • the nucleocaspid-like structures (CLPs) and viral particles obtained using the affinity chromatography procedure as described in Example 1 above were used in the production of polyclonal anti-MSV antibodies.
  • 1.5 ml of the isolated MSV (5 ⁇ g/ml) was thoroughly mixed with 1.5 ml of complete Freund's adjuvant and administered by subcutaneous injection at several sites to a young adult rabbit. After 10 days, the rabbit was given a similar injection containing 1 ml of the viral isolate and 1 ml of incomplete Freund's adjuvant. This was repeated a further 5 times at 14 day intervals and then the animal was bled 8 days after the last inoculation. The rabbit was further inoculated three times with 1 ml of virus every 14 days over 42 days followed by bleeding 8 days after the third inoculation. This was repeated over a period of six months.
  • the blood was allowed to clot and separate.
  • the sample was then spun at 3000 rpm for 20 minutes and the clean serum was aspirated off and stored at -20°C.
  • the immunoglobulin G was purified by addition of an equal volume of saturated ammonium sulphate (NH 4 ) 2 S0 4 to the serum to precipitate the protein in the serum.
  • the sample was then centrifuged at 3000 rpm for 30 minutes. The precipitate was retained and the supernatant was retreated with (NH 4 ) 2 S0 4 and then recentrifuged.
  • the reserved precipitates were resuspended and further treated with an equal volume of 50/50 saturated (NH 4 ) 2 S0 4 /distilled water, before the solution was centrifuged at 3000 rpm for 30 minutes. The precipitate was then dissolved in 0.01 M phosphate buffer solution.
  • the globulin proteins in the solution were then dialysed against several changes of phosphate buffered saline at 4°C overnight or for two days.
  • the immunoglobulins were passed through a diethylaminoethyl cellulose (DEAEC) column of 50 cc capacity and the fractions collected.
  • the optical densities of the fractions were determined using a spectrophotometer and the protein fractions pooled.
  • the immunoglobulins were concentrated to about 5 mg/ml of protein and stored at -20°C.
  • mice Six to eight week old Balb/C mice were immunised with an emulsion consisting of 0.1 ml of purified MSV (5 ⁇ g/ml) and 0.1 ml of complete Freund's adjuvant. The mice were injected in several sites subcutaneously. The booster immunisation, consisting of 0.1 ml of virus and 0.1 ml of incomplete Freund's adjuvant, was inoculated four weeks later subcutaneously in several places. A final injection of four times the original dosage of the virus in saline was given three to four days prior to fusion 14 . Myeloma Cell Line.
  • the myeloma cell line used was the Balb/C P3-NS1- Ag4-1 cell line (NS1).
  • the cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal calf serum (FCS) and 0.2 ml of penicillin/streptomycin (P/S), 120 ⁇ g/ml and 25 ⁇ g/ml respectively.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS foetal calf serum
  • P/S penicillin/streptomycin
  • the myeloma cells were incubated in 50 ml flasks at 37°C in a 7% C0 2 humidified incubator, and passaged through medium containing 8-azaquanine (2 ⁇ g/ml) to kill any revertant aminopteran resistant cells.
  • Myeloma cells were sub-cultured (split) every 24 hours, three days prior to fusion. Splitting of the cultures involved removing 50% of the medium, containing approximately 5 x 10 s cells, and placing it into a new flask. Both flasks were then refilled to the original volume with fresh media so that each flask contained 2 to 3 x 10 5 cells per ml. The myeloma cells were never allowed to grow above a concentration of 8 x 10 5 cells per ml.
  • mice were killed by C0 2 asphyxiation, the spleen removed aseptically and placed in a sterile petri dish containing 5 ml of culture medium. Spleen cells were washed out into the petri dish by injection of culture medium into the spleen capsule. A cell count was then performed.
  • the protocol used to perform fusions was adapted from that described by Galfre and Milstein 15 .
  • Approximately 10 8 mouse spleen cells (those obtained from 1 spleen) and 10 7 myeloma cells were mixed and placed in a sterile 50 ml conical-bottom tube. Serum free media was then added to produce a total volume of 50 ml.
  • the mixture was then centrifuged at 400 g for five minutes and because the concentration of ' PEG is critical for fusion, all medium was removed to prevent dilution.
  • the 0.5 ml aliquot of PEG was diluted to a final concentration of 40% (w/v) with serum free media and kept at approximately 40°C.
  • the pellet was broken by gentle tapping and 1 ml of PEG added drop-wise over 1 minute. This was shaken for another 1 to 2 minutes. Then, 1 ml of serum free medium (pre-warmed to 37°C) was added over 1 minute. This was repeated and then performed twice more with the serum being added over 30 seconds. A further 7 ml of medium was added over 2 minutes, while being continuously shaken.
  • HAT medium 50 ml of DMEM supplemented with 20% FCS, 0.4 ml penicillin/streptomycin, 0.05 ml Fungizone® (TAGO), hypoxanthine (136 ⁇ g/ml), aminopterin (0.19 ⁇ g/ml), • thymidine (3.88 ⁇ g/ml), glutamine (2 mM) and pyruvate (1 mM).
  • the cells were then dispensed in 100 ⁇ l aliquots into 96-well culture plates (Linbro), and the plates were placed in an incubator at 37°C.
  • Post-Fusion Cell Maintenance Culture trays were maintained at 37°C in a 7% C0 2 humidified incubator. The plates were checked daily until Day 5 after fusion when large-scale cell death of spleen cells was expected and observed. Fresh HAT (50 ml per well) was added. At Day 7 to 14 after fusion, media (100 ml per well) was removed every 2 to 4 days and replaced with fresh HT media [HT media is the same as HAT but with the exclusion of aminopterin] . When the majority of hybrids reached half confluence, 100 ⁇ l of supernatant was removed and screened for the appropriate antibodies. Positive clones were sub-cultured into new plates with the addition of spleen or peritoneal macrophage feeder cells.
  • ELISA enzyme- linked immunosorbent assay
  • the Edmonston Strain of the measles virus (5 ⁇ g/ml) was diluted 1 in 40 in a solution containing 15 mM Na 2 C0 3 and 33 mM NaHC0 3 solution (coating buffer) at pH 9.6 and 100 ⁇ l added to each well of a 96 well round bottom micro-titre plate (Disposable Products Australia).
  • the level of the antigen used was optimised by the ELISA chequer board technique 14 . Plates were incubated for 18 hours at 4°C and then washed 3 times with a 0.5% solution of Tween 20 in phosphate buffered 0.15 M saline (PBS). Plates were then blocked with 10% FCS in PBS at room temperature for 2 hours, after which they were washed 3 times with PBS-Tween.
  • Polyclonal antibodies raised against MSV has been used in immunocytochemical studies to identify MSV proteins within MS brain tissue, within CV1 cells and oligodendrocyte cultures infected with MSV and within CRFK cells infected with the feline viral isolate.
  • the staining technique used was essentially the same as that described by Cook et. al . 5 in the immunoperoxidase staining of MS brain tissue using polyclonal antibodies against the feline derived agent.
  • the staining reaction product obtained with the polyclonal antibodies to MSV was associated with cytoplasmic inclusions mostly within phagocytic cells in MS plaques.
  • oligodendrocytes Primary cultures of oligodendrocytes have been grown and infected with the virus isolated from the brain tissue of two cases of MS. The results show that the virus has an affinity for oligodendroglia and does not infect astroglia. Furthermore, distinct cytopathic effects (CPE) are observed within 6 to 14 days post- infection. This compares with the 6 to 8 weeks that it takes to see obvious CPE when the virus is grown in CRFK, Vero or CV 1 cell lines. This culture system now allows a rapid assessment of the infectivity of the virus and will allow a comparison of strains of the virus for the detection of attenuated strains. The affinity of the virus for oligodendroglia provides further possible evidence that the demyelinating process in MS is due to a viral infection of these cells.
  • the oligodendrocyte culture system permits an evaluation of the effectiveness of monoclonal antibodies raised against the virus in inhibiting the growth of the virus.
  • Dissociation into fresh media was performed by repeatedly drawing and expelling the tissue through the mesh into and from a 20ml syringe.
  • the resultant tissue suspension was diluted with fresh media and seeded into 50ml/25cm Costar tissue culture flasks at 1-1.5 brains per flask with 10ml of media or onto poly-L-lysine- ⁇ oated coverslips in 35 x 10mm petri-dishes at 0.5 brains per dish with 3ml of DMEM/10%FCS.
  • the cultures were incubated at 37°C with 5% C0 2 . Media was changed after the first 4-7 days and then weekly.
  • Cultures were washed twice with phosphate buffered saline (PBS) to remove cell debris and fixed with a 5ml solution of 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer for 15 min. The fixative was removed and cultures were washed another two times with PBS prior to being stored in fresh PBS until stained.
  • PBS phosphate buffered saline
  • the medium was collected from cultures that had been infected for at least 11 days, i.e. these cultures had had at least one medium change.
  • the pooled medium was centrifuged at 35,000 rpm for 3 hrs in a Beckman ultracentrifuge with a SW4 rotor.
  • the pellet was resuspended with 0.5ml of phosphotungstic acid and a drop of this was placed on a Formvar grid for 5 min prior to drying and examination in an electron microscope.
  • Oligodendrocytes with a round cell antibody and 1 or 2 polar processes were identified after 3 days in vitro . Although these cells never reached confluency, they did show proliferation above the astrocytic bed layer. Other oligodendrocytes were comparable to those described as type I and type II oligodendrocytes by Kuhlmann-Krieg et.al. 17 .
  • Type I cells had a triangular or ovoid shape with 2 or 3 processes and a flat cell body.
  • Type II cells had a network of processes of various diameters and lengths surrounding the cell body. These oligodendrocytes tended to be above the astrocytic bedlayer. Astrocytes grew quickly to form a monolayer with cell to cell contact. Immunocytochemical identification of oligodendrocytes.
  • the reaction product to the HRP labelled antiserum to CNPase an enzyme specific to oligodendrocytes, was located in the cells described above as oligodendrocytes. There was no staining reaction with the underlying layer of astrocytes. The reaction product to CNPase was distributed throughout the cytoplasm of the oligodendrocytes and its location was the same irrespective of the age of the culture. There was, however, a slight decrease in the intensity of the staining in the longer-term cultures.
  • oligodendrocytes appeared to be enlarged and consisted of a central ring of condensed cytoplasm around which was a large membranous sheet-like structure. Some of these cells contained 2 to 3 nuclei.
  • Example 4 provides immunocytochemical evidence that there are circulating antibodies to MSV in the serum of MS patients.
  • Purified MSV, an antigen or antigenic fragment derived from purified MSV, or a synthetic polypeptide having a sequence substantially homologous (at least 75% homologous, preferably at least 90% homologous) with an antigen or antigenic fragment of MSV is used as the detecting antigen in an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the detecting antigen is immobilised by being coated onto a solid support or carrier such as the surface of the wells of a microtitre plate, using coating buffer. After a washing step, the serum or cerebrospinal fluid sample is added to the wells.
  • an appropriate detecting antibody having an enzyme conjugated thereto such as a goat anti- human IgG-horseradish peroxidase conjugate
  • an appropriate enzyme substrate such as 4-chloronaphthol or diaminobenzidine in the case of horseradish peroxidase
  • the genus MorbiUivirus includes measles and canine distemper as well as other viruses that have not been detected in Australia such as rinderpest virus, peste desient ruminants, bovine encephalitis and phocine distemper virus. These viruses show a close antigenicity and there is considerable immuno-cross reactivity between some of the viral proteins.
  • the MS virus is another member of this genus and has similar properties to measles and canine distemper viruses.
  • Live virus vaccines are considered to be better immunogens than inactive or killed viral vaccines.
  • Effective measles and canine distemper vaccines are based on the use of a live virus and the production of these vaccines is well developed.
  • the oligodendrocyte culture system provides a means by which the infectivity of isolates or attenuated strains of MSV are assayed.
  • Tissue culture infective doses (TCID) of the isolates or strains of the virus are evaluated and compared to identify an attenuated strain of the virus.
  • the TCID of MS virus that has been grown in Vero and CV1 cells for several years, or virus that has been passaged through laboratory animals, are compared with the TCID of isolates obtaining directly from MS brain tissue to determine whether or not there is attenuation of the virus in the form of decreased infectivity of the oligodendroglial cells in culture.
  • the oligodendroglial culture system also permits an evaluation of the effectiveness of polyclonal and monoclonal antibodies raised against MSV on the virus in culture.
  • the effectiveness of antibodies raised against attenuated forms of the virus is evaluated as well as the question of whether or not the attenuated forms produce a sufficient antibody response to act as an effective immunogen.
  • EXAMPLE 8 Development of a treatment for MS.
  • This invention provides forms of therapy of MS.
  • the first of these is the use of an inoculum of a monoclonal antibody, such as C6 ( Figure 2), which shows distinct specificity to MSV.
  • the inoculation of varying quantities of this antibody will help to boost the patient's immune response to the virus. It is well recognised that not all MS lesions show an immune response in the form of a lymphocytic infiltrate of the tissue 18 and there is a lack of antibody producing plasma cells despite the presence of viral proteins in these lesions.
  • a purified, monoclonal antibody may not penetrate the blood-brain barrier to provide an effective action against the virus even though this barrier is considered to be damaged due to oedema 18 .
  • the antibody can then be conjugated with a carrier molecule, such as a cellular adhesion molecule or an oligodendrocyte specific molecule, which can target the antibody.
  • Treatment of MS may also take the form of an attenuated vaccine as described above or a sub-unit vaccine based on an antigen or antigenic fragment of MSV (or a synthetic polypeptide substantially homologous with an antigen or antigenic fragment of MSV), for example, the inoculation of a specific antigenic fragment of the virus such as the epitope to which the monoclonal antibody C6 ( Figure 2) was produced.
  • the advantage of this is that the immunogen will stimulate the immune system to produce B-lymphocytes which will produce large amounts of specific antibodies against MSV. The stimulation of the immune system in this manner will also increase the production of T-helper lymphocytes which will assist in the immune response in the lesions.

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EP19910920470 1990-11-14 1991-11-14 Diagnosis and treatment of multiple sclerosis Withdrawn EP0557388A4 (en)

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US7575929B2 (en) 2002-10-02 2009-08-18 Dmi Life Sciences, Inc. Diagnosis of multiple sclerosis with diketopiperazines
US7732403B2 (en) 2003-05-15 2010-06-08 Dmi Biosciences, Inc. Treatment of T-cell mediated diseases
US9623072B2 (en) 2011-10-10 2017-04-18 Ampio Pharmaceuticals, Inc. Treatment of degenerative joint disease
US9808454B2 (en) 2013-03-15 2017-11-07 Ampio Pharmaceuticals, Inc. Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same
US9925300B2 (en) 2011-10-10 2018-03-27 Ampio Pharmaceuticals, Inc. Implantable medical devices with increased immune tolerance, and methods for making and implanting
US9956217B2 (en) 2014-08-18 2018-05-01 Ampio Pharmaceuticals, Inc. Treatment of joint conditions
US10039760B2 (en) 2000-08-04 2018-08-07 Ampio Pharmaceuticals, Inc. Method of using diketopiperazines and composition containing them
US10881710B2 (en) 2011-10-28 2021-01-05 Ampio Pharmaceuticals, Inc. Treatment of rhinitis
US11389512B2 (en) 2015-06-22 2022-07-19 Ampio Pharmaceuticals, Inc. Use of low molecular weight fractions of human serum albumin in treating diseases

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DK173091D0 (da) * 1991-10-11 1991-10-11 Schleroseforeningen The Danish Biologisk materiale
GB9310657D0 (en) * 1993-05-24 1993-07-07 Univ London Retrovirus
WO1999053103A1 (en) * 1998-04-08 1999-10-21 Ms Research A/S Diagnosis of multiple sclerosis and other demyelinating diseases
US8217047B2 (en) 2008-05-27 2012-07-10 Dmi Acquisition Corp. Therapeutic methods and compounds
CA2810844C (en) 2010-09-07 2017-03-21 Dmi Acquisition Corp. Diketopiperazine compositions for the treatment of metabolic syndrome and related conditions

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NZ218050A (en) * 1985-11-13 1989-05-29 Wistar Inst Test for the presence of htlv-1v
EP0326395A2 (de) * 1988-01-29 1989-08-02 City Of Hope Verfahren zum Nachweis und zur Identifizierung von bestimmten viralen Sequenzien

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BIOLOGICAL ABSTRACTS, xol. 82, no. 2 1986, Philadelphia, PA, US; abstract no. 15669, R.D. COOK ET AL. 'LIGHT AND ELECTRON MICROSCOPICAL STUDIES OF THE IMMUNOPEROXIDASE STAINING OF MULTIPLE SCLEROSIS PLAQUES USING ANTISERA TO A FELINE-DERIVED AGENT AND GALACTOCEREBROSIDE.' page AB-669 ; & NEUROPATHOL. APPL. NEUROBIOL. vol. 12, no. 1 , 1986 pages 63 - 80 *
See also references of WO9208785A1 *

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US10039760B2 (en) 2000-08-04 2018-08-07 Ampio Pharmaceuticals, Inc. Method of using diketopiperazines and composition containing them
AU2003279761B2 (en) * 2002-10-02 2009-11-12 Luoxis Diagnostics, Inc. Diagnosis and monitoring of diseases
US7575929B2 (en) 2002-10-02 2009-08-18 Dmi Life Sciences, Inc. Diagnosis of multiple sclerosis with diketopiperazines
US7732403B2 (en) 2003-05-15 2010-06-08 Dmi Biosciences, Inc. Treatment of T-cell mediated diseases
US11369598B2 (en) 2003-05-15 2022-06-28 Ampio Pharmaceuticals, Inc. Treatment of T-cell mediated diseases
US9707227B2 (en) 2003-05-15 2017-07-18 Ampio Pharmaceuticals, Inc. Treatment of T-cell mediated diseases
US9730924B2 (en) 2003-05-15 2017-08-15 Ampio Pharmaceuticals, Inc. Treatment of T-cell mediated diseases
US10828296B2 (en) 2003-05-15 2020-11-10 Ampio Pharmaceuticals, Inc. Treatment of T-cell mediated diseases
US10251930B2 (en) 2011-10-10 2019-04-09 Ampio Pharmaceuticals, Inc. Treatment of degenerative joint disease
US9925300B2 (en) 2011-10-10 2018-03-27 Ampio Pharmaceuticals, Inc. Implantable medical devices with increased immune tolerance, and methods for making and implanting
US10471178B2 (en) 2011-10-10 2019-11-12 Ampio Pharmaceuticals, Inc. Implantable medical devices with increased immune tolerance, and methods for making and implanting
US10842847B2 (en) 2011-10-10 2020-11-24 Ampio Pharmaceuticals, Inc. Treatment of degenerative joint disease
US11058798B2 (en) 2011-10-10 2021-07-13 Ampio Pharmaceuticals, Inc. Implantable medical devices with increased immune tolerance, and methods for making and implanting
US9623072B2 (en) 2011-10-10 2017-04-18 Ampio Pharmaceuticals, Inc. Treatment of degenerative joint disease
US10881710B2 (en) 2011-10-28 2021-01-05 Ampio Pharmaceuticals, Inc. Treatment of rhinitis
US9808454B2 (en) 2013-03-15 2017-11-07 Ampio Pharmaceuticals, Inc. Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same
US11026940B2 (en) 2013-03-15 2021-06-08 Ampio Pharmaceuticals, Inc. Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same
US9956217B2 (en) 2014-08-18 2018-05-01 Ampio Pharmaceuticals, Inc. Treatment of joint conditions
US10342793B2 (en) 2014-08-18 2019-07-09 Ampio Pharmaceuticals, Inc. Treatment of joint conditions
US11090301B2 (en) 2014-08-18 2021-08-17 Ampio Pharmaceuticals, Inc. Treatment of joint conditions
US11389512B2 (en) 2015-06-22 2022-07-19 Ampio Pharmaceuticals, Inc. Use of low molecular weight fractions of human serum albumin in treating diseases

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WO1992008785A1 (en) 1992-05-29
AU8955591A (en) 1992-06-11
CA2095539A1 (en) 1992-05-15
JPH06502764A (ja) 1994-03-31
AU646522B2 (en) 1994-02-24
EP0557388A4 (en) 1994-07-06
JP3266612B2 (ja) 2002-03-18

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