JP3266612B2 - Diagnosis and treatment of multiple sclerosis - Google Patents

Description

The present invention relates to the diagnosis of multiple sclerosis (MS), and more particularly to a method for diagnosing MS by immunocytochemical staining of brain tissue and a method for serodiagnosis of MS. Currently there is no single test available for the diagnosis of MS, and the diagnosis is usually made based on the results of various techniques including neurological examination, CAT scan, NMR imaging and CSF analysis, Neither of these is decisive in itself.

The invention also relates to the treatment of MS. In this regard, the present invention provides antibodies against viruses isolated from human MS brain tissue, which can be used directly in the treatment of MS patients. Further, the present invention provides a vaccine for use in generating an immune response against a virus isolated from MS brain tissue.

There are two main theories regarding the causes of multiple sclerosis. One is that MS is an autoimmune-type dis
Based on animal models of experimental allergic encephalomyelitis (EAE), it is also the fact that immunosuppression has a beneficial effect on some MS patients. But EA over the last 50 years
Numerous studies of E failed to explain the etiology of MS, but did help elucidate the role of the immune system in the central nervous system (CNS). The second theory is that MS is a viral disease and epidemiological studies and some viruses, especially measles virus (MV)
And the demyelinating effect of morbilliviruses such as canine distemper virus (CDV).

Studies show that multiple sclerosis is a viral disease
¹ Some highlight measles virus ² and canine distemper virus ³ . MS patients of various viruses isolated from tissue have been reported not established ⁴ as causative agents of both of these diseases of these viruses. A previous study ⁵ using peroxidase-labeled antibodies to paramyxovirus isolated from the central nervous system of cats stained virus-like particles and nucleocapsid-like structures in phagocytes of MS plaques. The nucleocapsid-like structure was previously described as “curved, linear profile (cu
rved, linear profiles) ”(CLPs) ⁶ .
Currently, antibodies to feline viruses are used in affinity chromatography to detect eight different established cases.
The virus was repeatedly isolated from MS brains, but could not be isolated from four non-MS brains. Preliminary virological, immunocytochemical and polyacrylamide gel electrophoresis (PAGE) studies show that the virus is associated with MV and CDV, and that this relationship is now confirmed by an ELISA-type assay I have. Association of this virus with demyelinating disorders in the CNS of domesticated cats and MS patients indicates a possible causal relationship between these animal and human diseases ⁷ .

Asymptomatic primary demyelinating di
sease) was confirmed in about 7% of the CNS of cats tested ^{8, 9,}
Persistent, non-permissive paramyxovirus was isolated from infected brain tissue ¹⁰ . Peroxidase-labeled antibodies raised against feline virus stain virus-like particles and nucleocapsid-like structures (CLPs) within MS plaques ⁵ . Staining reaction is not inhibited by pre-treatment with a non-MS-human serum is inhibited by pretreating the MS brain tissue with sera from MS patients ^5.

Based on this antigenic relationship, CLPs and virus particles are currently being isolated from human MS brain tissue using affinity chromatography. Antibodies to feline virus were used in the immunocytochemical studies described previously, and were absorbed on affinity chromatography columns containing one of a variety of matrices, through which homogenized brain tissue from MS and non-MS patients was I passed. CLPs and virus particles similar to those of morbillivirus were present in the eluate of MS brain tissue but not in non-MS tissue. Viruses isolated from MS tissues were propagated in culture and collected using similar affinity chromatography methods. The result obtained is
It is believed that MS provides strong evidence that it is due to persistent or latent virus infection. Such etiology is more consistent with the epidemiology of this disease than the epidemiology of autoimmune diseases.

Viruses isolated from MS brain tissue, hereinafter referred to as "multiple sclerosis virus" or "MSV", are morbilliviruses and are closely related to both measles and canine distemper virus. The isolated MSV is used in vitro, for example, CV1.
Cells and oligodendrocytes can be grown in culture and further isolated from cultures of these cells.

In one aspect, the invention relates to isolated forms of MSV, such as isolated MV in CV1 cells or primary oligodendrocyte cultures.
Provide a culture of SV. Also provided is a method of preparing MSV in isolated form.

In another aspect of the present invention, there is provided an antibody that recognizes or binds to MSV morbillivirus, an antigen of MSV or an antigenic fragment thereof. These antibodies may be either polyclonal or monoclonal antibodies. In the case of monoclonal antibodies, the invention further extends to hybrid cell lines or hybridomas producing such monoclonal antibodies.

Antibodies of this aspect of the invention can be produced using MS brain tissue or purified MSV isolated from in vitro cultures as described above by conventional methods well known to those skilled in the art. By way of example, hybrid cell lines or hybridoma-producing monoclonal antibodies can be produced using the known fusion methods ^11,12 first described by Kohler and Milstein in 1975. Polyclonal antibodies, for example, in experimental animals such as rabbits, can be further produced by known methods ^12.

Some monoclonal antibodies produced by the methods outlined above have been shown to be very specific for MSV, while other monoclonal antibodies have varying degrees of cross-reactivity with MV and CDV. Is shown.

In another aspect of the present invention, there is provided a method for detecting MSV in a sample, e.g., a tissue sample, obtained from a patient, the method comprising the steps of: And detecting the binding of the antibody to indicate the presence of MSV in the sample.

In this regard, the polyclonal or monoclonal antibodies of the present invention can be used for immunocytochemical staining of MS brain-derived tissue to ensure disease diagnosis.

Another aspect of the present invention provides a method for serodiagnosis of MS. This aspect of the invention is based on the observation that serum from MS patients contains circulating antibodies to MSV. Using immunocytochemical methods,
Pretreatment of MS brain tissue with sera from MS patients, especially recently relapsed patients, has been shown to inhibit the previously described immunocytochemical staining of brain tissue. This inhibition indicates that the sera of MS1 individuals contain antibodies to this virus.

Thus, in this aspect of the invention, there is provided a method of detecting anti-MSV antibodies in a fluid sample taken from a patient, the method comprising contacting MSV or an antigen or antigenic fragment thereof with said fluid sample. And detecting an anti-MSV antibody bound to the MSV or the antigen or an antigenic fragment thereof.

The fluid sample may be a blood or cerebrospinal fluid sample. Preferably,
The sample is a blood sample and may be whole blood or a derivative thereof, such as serum or serum.

The diagnostic methods of this aspect of the invention can detect the presence of any anti-MSV antibody from a bound serum sample by detecting the antigen using an enzyme immunoassay or radioimmunoassay of known principles. Detection antigen (MSV or antigen or antigenic fragment thereof)
Is, for example, an anti-MSV bound and immobilized on a solid support
The presence of the antibody can be detected using an appropriately labeled anti-human immunoglobulin antibody. Other methods are well known to those skilled in the art.

Also provided in this aspect of the invention is a diagnostic test kit for detecting an anti-MSV antibody in a fluid sample, comprising MSV or an antigen or antigenic fragment thereof, preferably immobilized on a solid support, as a detection antigen. It is characterized by including.

In yet another aspect of the present invention resulting from MSV isolates and in vitro cultures, there is provided a vaccine composition for stimulating an immune response to MSV in a human or animal patient, which is an active immunogen, It includes activated or attenuated MSV, or an antigen of MSV or an antigenic fragment thereof.

Also provided in this aspect of the invention is a method of generating an immune response to MSV in a human or animal patient, which comprises inactivating or attenuating MSV, or MV, in said patient.
Administering an effective amount of an active immunogen comprising the SV antigen or antigenic fragment thereof.

If desired, the active immunogen can be conjugated to a carrier molecule to improve its immunogenicity. Suitable carrier molecules may include, for example, hemocyanin, such as keyhole limpet hemocyanin, bovine serum albumin or ovalbumin. In addition, the vaccine composition may optionally include an adjuvant. Known adjuvants for binding to animal and human vaccines include, for example, Freund's complete and incomplete adjuvants, alum, and the like.

There are currently well-known methods for producing inactivated or attenuated viruses, and attenuated vaccines have been developed for both viruses, especially in the case of MSV, which is closely related to MV and CDV . Similarly, subunit virus vaccines are well known and are based on the identification of viral antigens or antigenic fragments that can stimulate an immune response.

In the case of the MSV vaccine described herein, the vaccine can be produced for use in cats, as can the dog distemper vaccine, and / or in humans, especially human infants, the measles vaccine.

In yet another aspect of the invention, a method of treating MS patients comprising administering to the patient an effective amount of an antibody, preferably a monoclonal antibody, that recognizes or binds MSV or an antigen or antigenic fragment thereof. I will provide a.

The antibodies used in this aspect of the invention can be conjugated to a carrier or targeting molecule, such as an intracellular adhesion molecule that assists the antibody and crosses the brain-blood barrier.

Further features of the present invention will become apparent from the detailed description of the following embodiments.

Example 1 Isolation of MSV from MS brain tissue and its in vitro culture on CV1 cells.

A polyclonal antibody ¹⁰ against persistent, non-permissive paramyxovirus isolated from cat brain tissue was raised in rabbits. Immunoglobulin was purified from the sera of the inoculated animals, and cat liver powder, acetone-dried Crandel (Cr
andell) was adsorbed extensively to cat kidney (CRFK) and Vero cells and further concentrated to 5 mg protein / ml. Next, the prepared antibody was subjected to Affi-Gel Protein A (Bio-Rad) or C
Absorbed on NBr activated Sepharose 4B (Pharmacia). 8 different, MS confirmed and 4 non-MS
1-2 g of frozen, dissected brain tissue from the case of MS was homogenized and passed through an affinity column. The column was washed with a large volume of Tris NaCl buffer before elution. Collect the eluate,
Aliquots were negatively stained with 3% phosphotungstic acid, pH 7.2 before being examined by electron microscopy. Eluates from 12 brains were added to cultures of CV1 cells.

Electron microscopy of the negatively stained eluate obtained after passing MS brain tissue through a CNBr-activated Sepharose column showed a polymorphic, spherical profile with an average diameter of about 300 nm but somewhat larger diameters . The eluates from the Affi-Gel Protein A column showed similar sized profiles but consisted of a tightly coiled, tubular, approximately 18 nm diameter structure lacking the outer membrane. The tubing has a thin translucent nucleus whose wall conforms to a helical structure, "herrbon (herrbon).
ing-bone) "appearance. Some fractions mainly contained nucleocapsid-like structures or tubular structures comparable to isolate ⁶ of CLPs. Some eluate containing virus-like particles was pelleted by centrifugation, embedded in agar, fixed and processed for electron microscopy. 300nm
Pellets containing virions, having a tubular profile inside, about 18 nm in diameter, up to the diameter of, were cut in various planes. Virus isolation from MS brain tissue was repeated more than 40 times.

After 5 passages, CV1 cells infected with eluate from MS brains showed some intensive cytopathic effect (CP) in the form of small syncytium and slight cytoplasmic vacuolation.
E) indicated. Microstructural examination of these cultures was approximately 18n in diameter
The presence of cytoplasmic inclusions consisting of m tubular structures was revealed. These inclusions were morphologically similar to those observed in early isolates of feline paramyxovirus ¹⁰ . Virus particles, similar to those isolated from MS brains, were present in some cultures and were further isolated by using affinity chromatography.

Ultrastructural examination of the eluate obtained after passing non-MS brain tissue through the column did not reveal any virus particles. Cultures inoculated with these eluates showed no CPE and did not show any cytoplasmic content or virus particles when examined by electron microscopy. In addition, virus particles were not obtained from these cultures using affinity chromatography.

Polyacrylamide gel electrophoresis (PAGE) studies showed MV and CDV except that the isolated viral peptide lacked some membrane-spike protein.
It was shown to be comparable to that of. This difference is probably due to chicken, cow, dog, guinea pig: horse, human O (huma
n O), which causes erythrocytes from mice, rabbits, rats and sheep to not adhere to infected CV1 cultures. These observations were consistent with the characteristics of feline paramyxovirus present in culture as a persistent non-permissive infection of CRFK and Vero cells ¹⁰ .

The nucleocapsids and virions isolated from MS brain show morphological similarities and show some immunological cross-reactivity with MV and CDV. This indicates that in addition to early PAGE studies and the formation of small syncytia in tissue culture, the virus isolated from MS brain may be a morbillivirus.

Example 2 Production of Polyclonal Antibodies Nucleocapsid-like structures (CLPs) and virus particles obtained using affinity chromatography as described in Example 1 above were used for the production of polyclonal anti-MSV antibodies. 1.5 ml of isolated MSV (5 μg / ml) was mixed well with 1.5 ml of Freund's complete adjuvant and administered to young adult rabbits by subcutaneous injection at various sites. After 10 days, the rabbits received 1 ml of virus isolate and 1
A similar injection was made containing incomplete Freund's adjuvant. This was repeated five more times at 14 day intervals and the animals were bled for 8 days after the last inoculation. Rabbits were further inoculated three times with 1 ml of virus every 14 days for 42 days, and then bled for 8 days after the third inoculation. This was repeated over a period of six months. Simultaneously with the blood collection, the blood was coagulated and separated. The samples were then spun at 3,000 rpm for 20 minutes to aspirate pure serum and stored at -20 ° C. Immunoglobulin G was purified by adding an equal amount of saturated ammonium sulfate (NH ₄ ) ₂ SO ₄ to the serum to precipitate proteins in the serum. The sample was then centrifuged at 3,000 rpm for 30 minutes. The precipitate was retained and the supernatant was re-treated with (NH ₄ ) ₂ SO ₄ and centrifuged again.

The stored precipitate was resuspended and treated with an equal volume of 50/50 saturated ammonium sulfate / distilled water before centrifuging the solution at 3,000 rpm for 30 minutes. The precipitate was then dissolved in a 0.01 M phosphate buffer solution.

The globulin proteins in the solution were then dialyzed overnight at 4 ° C. or 2 days against variously changed phosphate buffer solutions. The immunoglobulin was passed through a 50 cc capacity diethylaminoethylcellulose (DEAEC) column to collect fractions. The optical density of the fractions was determined using a spectrophotometer and the protein fractions were pooled. The immunoglobulin was concentrated to about 5 mg / ml protein and stored at -20 ° C.

For immunocytochemical studies, polyclonal antibodies were combined with Avrameas and Ternyn.
ck) was coupled to horseradish peroxidase by method ¹³ . Cross-reactivity between MSV, MV and CDV was demonstrated using an ELISA-type assay and the results are shown in FIG.

Example 3 Production of Monoclonal Antibodies Immunization Method Balb / C mice 6-8 weeks old were purified with 0.1 ml of purified MSV (5 μg /
(ml) and 0.1 ml of Freund's complete adjuvant. Mice were injected subcutaneously at various sites. Four weeks later, booster immunized injections consisting of 0.1 ml of virus and 0.1 ml of Freund's incomplete adjuvant were inoculated subcutaneously at various locations. 4 times the last injection of the initial dose of physiological saline virus was given 3-4 days prior to fusion ^14.

Myeloma cell line As the myeloma cell line, a Balb / CP3-NS1-Ag4-1 cell line (NS1) was used. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 120 μg / ml and 25 μg / ml, respectively, of 0.2 ml penicillin / streptomycin (P / S). Myeloma cells in a 50 ml flask in a 7% CO ₂ humidified incubator at 37 ° C
And then add 8-azaguanine (8
-Azaquanine) (2 μg / ml) and subcultured through a medium containing any revertant aminoptera.
n) Kill resistant cells.

Myeloma cells were subcultured (split) every 24 hours for 3 days before fusion. Splitting the culture involved removing 50% of the medium, containing approximately 5 × 10 ⁵ cells, and placing it in a new flask. Then refilled 2-3 each flask per 1ml with × until the first volume with fresh medium to both flasks 10 ⁵
Cells were included. Myeloma cells were never grown above 8 × 10 ⁵ cells per ml.

Perfusion of the spleen to obtain lymphocytes The immunized mice were killed by asphyxiation with CO ₂ and the spleen was aseptically removed and placed in a sterile Petri dish containing 5 ml of medium. Spleen cells were flushed into petri dishes by injecting the medium into the spleen capsule. Cell counting was then performed.

Fusion The method used to perform the fusion was Galfr
e) and those described by Milstein^FifteenBreak
It was good. 10 g polyethylene glycol with a molecular weight of 1,500
(PEG) (Sigma Chemical Co.) was melted and pre-weighed
Autoclay in 0.5 ml aliquots in bottles
Processed. Then weigh the bottle and weigh the PEG.
Calculated. About 10⁸Mouse spleen cells (obtained from one spleen)
And 10⁷50m sterile mixed myeloma cells
l conical-bottom tube
Was placed. Then add medium without serum and add
The volume was 50 ml. The mixture is then centrifuged at 400g for 5 minutes
Because all PEG concentrations are absolute for fusion,
Was removed to prevent dilution. 0.5 ml aliquot of PEG serum
Diluted to a final concentration of 40% (W / V) in a medium without
Maintained. Tap the pellet lightly for 1 minute
PEG was dropped and broken. Shake it for another 1-2 minutes
did. Then, 1 ml of medium without serum (pre-
(Warmed) was added over 1 minute. Repeat this
More than once, then serum was added for 30 seconds.
With continuous shaking, add another 7 ml of medium for 2 minutes.
Just added. Finally, add 12-13 ml of medium and add cells
Was centrifuged at 400 g for 5 minutes. Aspirate the supernatant and remove the pellet.
10 per ml^Five40 ml HAT selection medium containing individual feeder cells
Resuspended in the ground. [HAT medium: 50 ml containing 20% FCS
DMEM, 0.4 ml penicillin / streptomycin, 0.0
5ml Fungizone "Fungizone "[TAGO],
Hypoxanthine (136 μg / ml), aminopterin (0.19
μg / ml), thymidine (3.88 μg / ml), glutamine (2m
M) and pyruvate (1 mM)]. The cells are then 96 wells
100 µl of the culture plate [Linbro]
Distribute in aliquots and place plate in 37 ° C incubator
did.

Maintaining cells after fusion Culture trays were maintained at 37 ° C. in a 7% CO ₂ humidified incubator. Plates were checked daily up to 5 days after fusion, where massive spleen cell death is expected and observed. Fresh HAT (5 per well
0 ml) was added. On the 7th to 14th days after the fusion, the medium (100 ml per well) was removed every 2 to 4 days and replaced with a fresh HT medium (HT medium was the same as HAT medium except aminopterin was removed). Many hybrids are semi-confluent (helf conflue)
Once reached, 100 μl of supernatant was removed and selected for appropriate antibodies. Positive clones were subcultured to new plates by adding spleen or peritoneal macrophage feeders.

Screening for positive hybrids Positive hybrids are screened for enzyme-linked immunosorbents
Detection was performed using an assay (ELISA). Measles virus
Edmonston Strain (5μg / ml)
15 mM Na_TwoCO_ThreeAnd 33 mM NaHCO_ThreeSolution (coating loose
Solution) and dilute 40-fold in a pH 9.6 solution containing
Well round bottom microtiter plate [Disposer
Bull Products Australia (Disposable Products)
Australia)]. Review of the antibodies used
Bell is ELISA checker board techniqu
e)¹⁴Optimized. Place plate at 4 ° C for 18 hours
Incubate and then 0.15M phosphate buffer solution (PBS)
Was washed three times with a 0.5% Tween 20 solution. Then 2
Incubate with 10% FCS in PBS at room temperature for
The plate was washed three times with PBS-Tween 20. Tissue culture
Aseptically remove the supernatant (100 μl) from the plate and
Plate at 4 ° C for 12 hours.
Incubated. Then wash the wells and 100μl
Alkaline phosphatase-conjugated anti-mouse I diluted 1,000 times
gG (Tago, USA) was added. Plate at room temperature
Incubate for 2 hours, wash and 100 μl enzyme
Substrate 4-nitrophenol phosphate
nolphosphate) (4-NPP) (Paeringer, Germany)
Mannheim) 0.5 mM MgCl_Two(Substrate buffer) was added
In 10% diethanolamine with pH 9.8
At 1 mg / ml. Incubate for 30 minutes at room temperature
After that, using a filter with an optical density of 405 μm,
retek »Multistream Microtiter Plate
Measured with a reader.

Prescreening of CDV, MV and MSV Separate ELISA plates were sensitized with CDV vaccine (Websters), Edmonston measles virus and MSV by the method described previously. Human polyclonal anti-measles antibody, lot 25754, (Princess Margaret Hospital, Western Australia) was diluted 320-fold with PBS.
Anti-CDV and anti-MSV antibodies were used after being diluted 100 times with PBS. Enzyme conjugates used were anti-rabbit IgG alkaline phosphatase (TAGO) against CDV and MSV and anti-human alkaline phosphatase (TAGO) against measles virus. After reconstitution, the cell conjugate was diluted to 1,000-fold with PBS and used. ELISA was performed according to the method described above, and the results are shown in FIG.

Screening of monoclonal antibodies Six types of positive hybridomas were identified as CDV, MV and MSV,
And Newcastle disease virus (NDV) (10 μg / m
l) and three cat viruses; feline panleukopenia, feline rhinotracheitis and feline calicivirus (calciv
irus) [Screened against Webster 3 as 1 (survival)] to confirm any non-specificity. The feline virus was used after reconstitution with 1 ml of distilled water followed by a 20-fold dilution in PBS. ELISA was performed again by the method described previously. Figure 2 shows the results of this screening.
Shown in

Example 4 Immunocytochemical Staining Polyclonal antibodies raised against MSV as described in Example 2 were used for immunocytochemical studies in MS brain tissue, CV1 cells and oligodendrocytes infected with MSV. CRFK infected in glial cell culture and with feline virus isolates
The MSV protein in the cells was confirmed. The staining method used was immunoperoxidase staining of MS brain tissue using a polyclonal antibody against a cat-induced pathogen.
dase staining) was essentially identical to that described by Cook et al. The staining reaction product obtained using a polyclonal antibody against MSV was largely associated with the cytoplasmic content of phagocytes in MS plaques. Microstructural studies confirmed that the stained inclusions were tubular structures, which are actually nucleocapsids of MS virus isolates, referred to as curved linear profiles. These cytoplasmic contents were stained with horseradish peroxidase-labeled monoclonal antibodies C1 and E1 (Example 3).
And FIG. 2). These inclusions were not detected in normal white matter in MS brains or white matter from non-MS brains.

Immunocytochemical staining of cytoplasmic content in MS plaques using a horseradish peroxidase-labeled polyclonal antibody to MSV was inhibited upon pre-incubation of brain tissue with serum from two relapsed MS patients. This inhibitory response was not effective when serum from patients in remission of the disease was used. Preincubation of brain tissue with sera from individuals without medical evidence of MS did not reduce the intensity of the staining reaction. further,
Cytoplasmic content in MS plaques was stained using horseradish peroxidase-labeled immunoglobulin G (IgG) purified from the sera of two MS patients. The results obtained are comparable to those found when using a labeled polyclonal antibody against MSV. These results indicated that MS patients, especially those who exhibited disease activity as indicated by clinical signs, had circulating antibodies to MSV.

Example 5 Oligodendrocyte Culture System for MS Virus Primary cultures of oligodendrocytes were grown and infected with virus isolated from brain tissue of MS in two cases. The results showed that the virus had affinity for oligodendrocytes and did not infect astrocytes. In addition, a distinct cytopathic effect (CPE) is observed within 6-14 days after infection. This is comparable to the 6-8 weeks required to find a clear CPE when growing the virus on CRFK, Vero or CV1 cell lines. At present, this culture system allows a quick assessment of the infectivity of the virus and allows comparison of virus strains for detection of attenuated strains. The affinity of the virus for oligodendrocytes provides other possible evidence that demyelination in MS is due to viral infection of these cells.

The oligodendrocyte culture system allows the evaluation of the efficacy of monoclonal antibodies raised against the virus in inhibiting the growth of the virus.

Wistar rat pups 1-2 days old
p) was euthanized by cervical dislocation with CO ₂ inhalation. Method used by Sarlieve et al. To remove brain tissue
^A modified version of ¹⁶ was used to separate. Immediately after euthanasia,
The pups were transferred to a beaker, immersed in 70% ethanol and placed in a laminar flow cabinet. Dulbecco's minimal medium (DMEM) with aseptically removed brain and 10% fetal calf serum (FCS)
And transferred to a Petri dish containing a double layer of sterilized stainless steel mesh (mesh size about 100 μm). Separation into fresh medium (DMEM / 10% FCS) was performed by repeatedly sucking and discharging the tissue through a mesh into a 20 ml syringe. Dilute the resulting tissue suspension with fresh medium to 50 ml
/ 25cm Costar tissue culture flask
1 to 1.5 brains per flask with 10 ml of medium or 0.5 × brains per dish with 3 ml of DMEM / 10% on a poly-L-lysine coated cover glass in a 35 × 10 mm Petri dish. Inoculated with FCS. 37 cultures
Incubate at 5 ° C. with 5% CO ₂ . Medium was changed after the first 4-7 days and then weekly.

Inoculation of primary cultures with isolates from MS brain tissue Two isolates were obtained from brain A87 / 29 and MS-7 using affinity chromatography. These isolates were inoculated into 4-day-old primary rat glial cultures as follows. The medium was removed from the culture washed twice with fresh DMEM. One ml of the culture medium was then inoculated with 100 μl of the virus suspension. The flask was incubated for 2 hours, after which the initial medium was returned to the culture without removing the virus suspension.

Immunocytochemistry Culture Fixation The culture was washed twice with phosphate buffered saline (PBS) to remove cell debris and a 5 ml solution containing 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer was used. And fixed for 15 minutes. After washing twice more with PBS, the fixative was removed and the culture was stored in fresh PBS until staining.

Immunoperoxidase labeling of tissue cultures Cultures were washed twice with PBS and incubated with ice-cold, 70% ethanol for 2 minutes. It is then washed twice with PBS for 15 minutes and washed with 10 ml of 0.5% (v / v) H
Incubated with ₂ O ₂ Tris buffer for 30 minutes. The flask was washed with 40 ml of Tris buffer and 1 ml of primary antibody (either 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase [CNPase] or HRP-labeled antibody raised against MS virus isolate). In Tris buffer
Incubate with the 1:30 dilution for 453 minutes or as a control, leave the culture in Tris buffer alone. After incubation, antiserum was removed by adding 1 ml of DAB solution and washing each culture 3 times with 40 ml of Tris buffer for 10 minutes before incubation for 15 minutes in the dark. After washing three more times in Tris buffer, cultures were prepared for light or electron microscopy in a conventional manner.

Isolation of virus particles from tissue culture medium Cultures that had been infected with the medium for at least 11 days, ie, those cultures that had undergone at least one medium change, were collected. The pooled medium was centrifuged at 35,000 rpm for 3 hours in a Beckman ultracentrifuge equipped with a SW4 rotor. The pellet was resuspended in 0.5 ml of phosphotungstic acid and the
Drops were placed on a Formvar grid for 5 minutes, dried and tested for electron microscopy.

Results Primary rat oligodendrocyte cultures Oligodendrocytes with round cell antibodies and one or two polar processes were identified in vitro after 3 days. Although these cells never become confluent, they show growth on the astrocyte bed layer. Other oligodendrocytes were comparable to those described by Kuhlmann-Krieg et al. As type I and type II oligodendrocytes ¹⁷ . Type I cells have a triangular or oval shape with two or three types of projections and a flat cell body. Type II cells have a network of projections of various diameters and lengths surrounding the cell body. These oligodendrocytes tended to spread on the astrocyte bed layer. Astrocytes grew rapidly and formed a monolayer in which cells contacted each other.

Immunocytochemical identification of oligodendrocytes HR for CNPase, an enzyme specific to oligodendrocytes
The reaction product for P-labeled antiserum was located in the cells described above, such as oligodendrocytes. No staining reaction occurred with the underlying astrocyte layer. The product of the reaction to CNPase was located in the cytoplasm of oligodendrocytes and its location was similar regardless of the age of the culture. However, longer incubations resulted in a slight decrease in staining intensity.

Control Even if there were DAB reaction products in the control flask,
Very few.

Electron microscope The results were confirmed by an electron microscope. Antibodies to CNPase elicited a staining reaction indicating that this enzyme was present in cells with ultrastructural properties of oligodendrocytes and located in the cytoplasm. Cells with typical astrocyte characteristics did not show positive staining.

Inoculation of culture with MS virus isolate 6 days after inoculation (10 days in vitro).

Some oligodendrocytes were thought to have a large membrane-like memblanous sheet-like structure consisting of a central ring of swollen and concentrated cytoplasm. Some of these cells contained a few nuclei. These cells were present in the astrocytes but not in the apical confluent layer, and the astrocytes around the oligodendrocytes contracted.

9-14 days post-infection with MS virus isolates Compared to cultures 6 days post-infection, these latter cultures showed very distinct CPE. Various large multinucleated glial cells were present in each culture. Some of these cells
It contained up to 30 nuclei and was arranged as a ring around the perimeter of the cytoplasm. These cells contained cytoplasmic material that was positive for labeled CNPase and positive for labeled antibody to MS virus. The astrocyte bed layer has receded a considerable distance from the periphery of these cells, indicating that the multinucleated cells are producing soluble substances that may be cytokines.

Examination of the pooled ultracentrifuged pellets of media from cultures infected with MS virus indicated the presence of virus particles. This observation indicated that virus was produced from infected cells and that this was a more convenient and rapid way to obtain large amounts of virus. Virus is infected CRF
Although obtained from K, Vero and CV1 cells, production of this virus was not observed until more than 50 days after infection. It should be noted that cultured oligodendrocytes were not considered susceptible to the measles virus Edmonson strain. Cultures infected with this virus did not show any CPE.

Example 6 Detection of Anti-MS Antibodies in Serum or Cerebrospinal Fluid Example 4 provides immunocytochemical evidence that circulating antibodies to MSV are present in the sera of MS patients. Purification
Enzyme-linked immunosorbents of MSV, an antigen or antigenic fragment derived from purified MSV, or a synthetic polypeptide having at least the same sequence as an antigen or antigenic fragment of MSV (at least 75% identical, preferably at least 90% identical) Used as detection antigen in assay (ELISA). Using known methods, the detection antigen was immobilized by coating it with a coating buffer on a solid support or carrier, such as the surface of a well in a microtiter plate. After washing, a serum or cerebrospinal fluid sample was added to the wells. After incubation, the wells are further washed and an appropriate detection antibody having a conjugated enzyme (eg, goat anti-IgG-horseradish peroxidase conjugate) is added. The activity of the enzyme bound to the solid support or carrier is determined by the presence of a suitable enzyme substrate, such as 4-chloronaphthol (4 in the case of horseradish peroxidase).
-Chloronaphtol) or diaminobenzidine] to indicate the presence of anti-MS antibodies in the sample.

Example 7 Development of Vaccine to Prevent MS Morbillivirus and other viruses not found in Australia such as measles and canine distemper and rinderpest virus, small ruminant plague, bovine encephalitis and seal distemper virus Is included. These viruses show similar antigenicity and show significant immune cross-reactivity among some viral proteins. Based on immunological, morphological and virological studies, MS viruses are members of this genus and have properties similar to measles and canine distemper viruses.

Live virus vaccines are considered more preferred immunogens than inactivated or killed virus vaccines. Effective measles and canine distemper vaccines are based on the use of live viruses and the production of these vaccines develops successfully. Evidence that MSV is associated with measles and canine distemper virus indicates that similar methods can be used to develop vaccines with MSV based on the immune cross-reactivity described in Example 3.

The oligodendrocyte culture system (see Example 5) provides a means to assay the infectivity of an isolate or attenuated strain of MSV. The tissue culture infective dose (TCID) of the virus isolate or strain is evaluated and compared to identify attenuated strains of the virus. Vero and
Infectivity of oligodendrocytes in culture by comparing the TCID of MS virus grown for several years in CV1 cells, or of the virus passaged through laboratory animals, with the TCID of isolates obtained directly from MS brain tissue It was determined whether there was an attenuation of the virus in a reduced form.

The oligodendrocyte tissue culture system can also evaluate the efficacy of polyclonal and monoclonal antibodies raised against MSV in virus in culture. The effectiveness of the antibodies raised against the attenuated form of the virus as well as the question of whether the attenuated form produces a sufficient antibody response to function as an effective immunogen is evaluated.

Example 8 Development of Treatment for MS The present invention provides a form of treatment for MS. The first of these is the use of an inoculum of a monoclonal antibody such as C6 (FIG. 2), which shows a distinct specificity for MSV. Inoculation of this antibody in varying amounts helps to increase the patient's immune response to the virus. It is well known that not all MS disorders show an immune response in the form of tissue lymphocytic infiltrates, but the loss of antibody-producing plasma cells occurs despite the presence of viral proteins in these lesions ¹⁸ . However, purified, monoclonal antibodies it may never provide effective action against the virus even when the blood brain barrier to street this barrier is considered damaged for edema ^18. The antibody can then bind to a carrier molecule, such as a cell adhesion molecule or oligodendrocyte-specific molecule, which can target the antibody.

In addition, treatment of MS can be performed using an attenuated vaccine as described above or MS.
V forms of subunit vaccines based on antigens or antigenic fragments of V (or synthetic polypeptides that are almost identical to antigens or antigenic fragments of MSV), eg, specificity of a virus such as an epitope producing monoclonal antibody C6 (FIG. 2) Inoculation of a specific antigenic fragment may be performed. The advantage is that the immunogen stimulates the immune system
To produce B lymphocytes that produce large amounts of specific antibodies against Stimulation of the immune system in this way also increases the production of T helper lymphocytes that assist the immune response in the disorder.

References: 1. Liebowitz, S. in “Multiple Sclerosis” (eds. Hallpik
e, JF, Adams, CWMand Tourtellotte, WW) (Chapma
n & Hall, London, 1983). 2. Norrby, E. Prog. Med. Virol. 24 , 1-39 (1978). 3.Cook, SD, Dowling, PCand Russell, WCJNeurol, S
ci. 41 , 61-70 (1979). 4.ter Meulen, V. and Stephenson, JRin “Multiple Scl
erosis "(eds. Hallpike, JF, Adams, CWMand Tourtel
lotte, WW) (Chapman & Hall, London, 1983). 5.Cook, RD, Flower, RLPand Dutton, Neuropath.Appl
d. Neurobiol. 12 , 63-79 (1986). 6. Prineas, J. Human Pathol. 6 , 531-554 (1975). 7. Cook, RDMed. Hypoth. 7 , 147-154, (1981). 8.Cook, RDNeuropath.Appld.Neurobiol. 5 , 395-404,
(1979). 9.Cook, RDand Wilcox, GENeuropath.Appld.Neurobio
l. 11 , 361-367, (1985). 10. Wilcox, GEet. Al. Vet. Microbiol. 9 , 355-366, (19
84). 11.Kohler, G. and Milstein, C. Nature, 256 , 495-497 (19
75). 12.Goding, JW, “Monoclonal Antibodies: Principles
and Practice "(Academic Press, 1986) 13. Avrameus, S. and Ternynck, T. Immunocytochemistry,
8 , 1175-1179 (1971). 14.Campbell, AM “Monoclonal Antibody Technology”
(Elsevier Publishers, Netherlands, 1984). 15.Galfre, G. and Milstein, C. Methods in Enzymology, 7
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Claims (24)

(57) [Claims] 1. An isolated form of multiple sclerosis virus (MS), which is a morbillivirus associated with multiple sclerosis (MS).
V). 2. Culture of isolated MSV in CV1 cells. 3. A culture of isolated MSV in primary oligodendrocytes. 4. An isolated antibody produced by contacting an immobilized antibody raised against a feline paramyxovirus isolated from the central nervous system of a cat with an MSV-containing tissue and further binding with the immobilized antibody.
M in isolated form, comprising the step of recovering MSV.
How to prepare SV. 5. MS isolated from multiple sclerosis (MS) brain tissue
Contacting the immobilized antibody generated against V with MSV-containing tissue,
A method for preparing an isolated form of MSV, further comprising the step of collecting the isolated MSV bound by the immobilized antibody. 6. The method according to claim 4, wherein the MSV-containing tissue is MS brain tissue. 7. The method according to claim 4, wherein after the recovery, the isolated MSV is cultured in CV1 cells or primary oligodendrocytes. 8. An antibody that recognizes or binds to MSV, an antigen of MSV, or an antigenic fragment thereof. 9. The antibody according to claim 8, wherein said antibody is a polyclonal antibody. 10. The antibody according to claim 8, wherein said antibody is a monoclonal antibody. A diagnostic reagent comprising the labeled antibody according to any one of claims 8 to 10. 12. The reagent according to claim 11, wherein said labeled antibody contains an enzyme-antibody conjugate. 13. The reagent of claim 12, wherein said conjugate comprises a horseradish peroxidase antibody conjugate. 14. A hybrid cell line or hybridoma which produces a monoclonal antibody that recognizes or binds to MSV, an antigen of MSV, or an antigenic fragment thereof. 15. The method according to claim 8, wherein the sample collected from the patient is
10. The antibody or claim 11 to any one of claims 10 to
14. A method for detecting MSV in a sample, comprising bringing the sample into contact with the diagnostic reagent according to any one of 13 and detecting the binding of the antibody or the diagnostic reagent to indicate the presence of MSV in the sample. 16. The method of claim 16, wherein the sample is a brain or other tissue sample, and wherein the detecting step detects the binding of the antibody or diagnostic reagent to the sample to indicate the presence of MSV in the sample. The method of claim 15. 17. The method of claim 16, wherein said detecting step comprises immunocytochemical staining of said sample. 18. A fluid sample collected from a patient is contacted with MSV, an antigen or antigenic fragment thereof or a synthetic polypeptide almost identical to an antigen or antigenic fragment of MSV, and further contacted with said MSV or antigen or antigenic fragment thereof. A method for detecting an anti-MSV antibody in the sample, comprising detecting the bound anti-MSV antibody. 19. The method according to claim 18, wherein the detection antigen comprises MSV immobilized on a solid support or an antigen or antigenic fragment thereof. 20. The method according to claim 1, wherein the bound anti-MSV antibody is detected using a labeled anti-human immunoglobulin antibody.
18. The method according to 18 or 19. 21. The method according to claim 18, wherein said fluid sample comprises a blood sample. 22. The method according to claim 18, wherein said fluid sample comprises a cerebrospinal fluid sample. 23. A fluid sample collected from a patient, characterized in that the test kit contains MSV, its antigen or antigenic fragment, or a synthetic polypeptide almost identical to the antigen or antigenic fragment of MSV as a detection antigen. Test kit for detecting anti-MSV antibodies. 24. The test kit according to claim 23, wherein the detection antigen comprises MSV immobilized on a solid support or an antigen or an antigenic fragment thereof.