CA2095539A1 - Diagnosis and treatment of multiple sclerosis - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A morbillivirus, multiple sclerosis virus (MSV), is isolated from multiple sclerosis (MS) brain tissue.
Antibodies which recognise or bind to MSV are used in diagnosis or treatment of MS. Serological diagnosis of MS comprises detection of circulating anti-MSV
antibodies. Compositions and methods for producing an immune response against MS are also disclosed.

Description

W092/0878~PCT/A~'91/~519 ~ ~

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DIAGNOSI8 AND ~R2A~MENT O~ MUIT~P~ SCLEROSIS

10This Lnventlon relateg to the dlagno~ls of multlple sclero91s (MS) and ln particular lt rel~tes to a method for dlagnosls of MS by lmmunocytochemlcal ~talnlng of bra$n tl~sue, a~ well a8 to a method for ~erologlcal dlagnosls of MS At prosent there i9 no slngle test lS avallable fo~ the dlagno~l~ of MS, and dlagno~ls is usually ba~ed on the result9 of several technigues lncludlng neurologlcal oxamln~tlon, CAT ~can~, NMR ~ -imaglng and CSF analysls, none of whlch 1~ conclusive on lts own --The present lnventlon also rol~t-s to ~he treatment of MS In thl~ ro-pect, the lnventlon provides antlbodlos to a vlruJ whlc~ has be~n l~ol~t-d from human MS braln tl~uo, and th J- antlbodl-~ may b- used in dlr-ct th~rapy o~ Mg patl-nt~ ~n addltlon, th- present lnv ntlon al~o provlde~ a vaccln for u~e ln produclng an lmmun respon-o to tho vlrus lsolated from MS braln tl~-u~
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There are two maln theorle~ as to tho cause of multlple scloroii One i~ that MS i5 an autoimmune-type dis~ase and 1~ based on the anlmal model of exporlmental allergic enc-phalomyellti- (EAE) ~nd ~hs fact th~t lmmunosuppr-~lon ha~ n b noflcial eff-ct in some MS
3S patlont~ Exten-lve studi~s on EAE ovor the pa~t 50 year~ hsva however f~lled to ~xplain th aetlology of MS
but h~vo h~lpsd to elucldate th~ rols of the i~mune : : ~ . : :

W092/0878~ ~9 ~ r 3 9 PCT/AU91/~519 ~y tem in tho centr~l nervou9 system ~CNS) The second theory is that MS ls a viral dlsease and is based on ~pldemiologlcal ~tudles and the demyellnatlns effect of some vlruse , ln partlcular the morbllllviruses such as measles (MV) and canine dlstemper (CDV) vlruses Many studle~ have ~uggested that multiple sclerosls 18 a vlral dl9ea~e~ and some empha~l~ has been placed on measles2 ~nd canina dlstemper3 vlruses There aro 3everal reports of the lsolation of vlruses from tls~ues of MS patients~ but none of these vlruse has been establlshed as the a-tlolo~lcal agent of thi~ dlsease A
prevlous ~tudy', whlch usod a peroxldase-labelled antibody against a p~ramyxovlrus isolated from the central nervous system of cats, staln d vlru~-like particles and nucleocapsld-like structures in phagocytic cells ln MS
plagues The nuclor~ap-ld-Ilke ~tructureY h~v~
prevlously been referr d to as Hcurvad, llnear proflles"
(C~Ps )6. The antlbody agalnst the fellne vlru~ h~ now been used ln sffinity chromato~raphy to repeatedly lsolate vlru- from the bralns of 8 dlfferent confirmed casos of MS but not from 4 non-MS braln3 Prelimlnary vlrologlcal, lmmunocytoch ~lcal and polyacrylamlde gel electrophor-tlc (PAGE) 8tudle- have lndlc~t-d that thls vlruJ is related to MV and CDV, and thiJ rolatlon~hlp has now b en furthar conflrm d by tho ELISA-typ~ a3say The a~soclation o~ this vlrus wit~ demy-linating lo~lons in th CNS of do~estlc cat~ and MS patlents indicates a po~-lble c~u~al r latlonshlp b~tw~n thq~o anlmal~ and th human diJ-~7 A subcllnical, primary demy llnatlng disea~e has been id-ntlfled ln the CNS o~ appro~lmately 7% of cats examln~d'9 and a parslst-nt, non-perml~slv paramyxov~rus has boen l~ol~t~d from aff-ct~d braln tlssu~
P-roxldas~ b~lled antlbodl~ ral~ d ag~in t the cat vlru~ st~ln vi~u~-llk- p~rtlcl~ ~nd nucloocapsld-like ,. .
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W092/~8785 PCT/AU91/~S19 structure~ (CLP8) withln MS pla~ues5. ~he st~ining reactlon ls blocked by pretreatment of MS bra~n tlssue wlth sera from MS patlents but not by pretreatment with non-MS hum~n ~eraS.
S
Based on thl~ antlgenlc relationshlp, affinity chromatography has now be~n u~ed to lsolate CLPs and vlrus partlcles from human MS braln tlssue. The ~-antlbodies to the cat vlrus, used ln the lmmunocytochemlc~l studle- pr-vlously descrlbed, were absorbed to an afflnlty chrsmatography column contalning one of ~everal matrlces and through thls was passed homogenlsed braln tlssue from MS and non-MS patlents.
CLPs and vlrus p~rtlCles whlch conform to those of morbllllvlruses w~re pre~ent in the eluate of the MS
braln tlssue but not in th~t of tho non-MS ti~sue. The vlrus lsolated from the MS tis~ue has be-n grown ln culture and has been harvested u~lng tho s~me affinlty chromatography technique. It 1~ consldered that the result~ obtaln~d provlde strong evldence that MS is due to perJl~t-nt or l~tent vlral lnfection. Such an aetlology 19 more con~lstent wlth the epldemiology of the disease than that of an autoi~mune-type dise~ss.

The vlru~ 1JO1~ted from MS braln tl~sue, herelna~er r-ferred to as "multiple sclerosl~ vlrus" or ~MSV~ a morbllllvlru~ and i~ closely related to both tha m~a8108 and canlne dlst~mper viru es. The isolated MSV grow~ ~n vltro, for example in CV 1 cell~ and cultures of ollgodendrQcytes, and can be further isolated from cultures of the~e cell~. - -In ona aspect, the present lnventlon provide~ MSV
ln i~olated form, for ex~mple, as a cultura of isolated MSV in CV 1 cells or in primary ollgodendrocyto cultures.
ThQre 1Q al~o provided a method ~or the prep~ration of MSV in ~solated ~orm.

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W092/0878~ PCT/AU91/00519 4 2~g~539 In ~nother a~peet of the pre~ent lnventlon there are provlded antlbodio~ which recognlse or bind to the MSV morbllllviru~, to an antlgen of MSV or to an antlgenlc fr~gment thereof. These antibodles may be elther polyclonal or monoelonal antlbodies. In the case of monoclonal antlbodlQ~, the invention further extends to hybrld cell llnes or hybrldoma~ whlch produce such monoclonal antlbodies.

Tho antibodles of thls aspect of the lnvention may be produeed by eonventlonal teehnlqu-J whlch are well known to person~ ~kllled ln the art using purified MSV
l~olated from MS braln tlQsue or from in v~tro culture as de wrlbed ~bove. ~y way of ex~mple, hybrid cell line or hybridomas produelng monoclonal antlbodle~ may be produeed using th~ w ll known fuslon teGhn~ue f~r~t deserlbed ln 1975 by Kohl~r and M~lJtolnlll2. Polyclon~l antibodles may bo produe~d, for example, in laboratory animal~ sueh as rabblt~, agaln by well known teehnlquesl2 ;
Some monoelonal antlbodle~ produeed by t~e teehnlque~ outlined above have been ~hown to be very ~peelfle for MSV, whilst oth~r monoclonal antlbodie~ show varying de~r~-J of ero~--reaetlvity wlth MV and CDV.
. ' ' ' In onoth-r asp et of tho present inventlon there is provldod a m thod for detoetlon of MSV in a ~ample, --~u~h a- a t~-~uo s~plo, t~ken from a p~tient, which eo~pris-J eont~etlng the s_mplo wlth ~ntlbody whlch 30 reeognl~-s or blnds to MSV or An ~ntlgen of MSV or an ~ -antigenlc fr~g~nt the~eof, _nd det~et~ng blndlng of said ~ntlbody to lndle~t~ th pre~enes of MSV ln the ~mple.

In thl3 _~p et, the polyelonal or monoclonal anti-MSV antlbodies of thl~ inv~ntlon may bG u~ed in lmmunoeytoehamle~l st_lnlng of tls~u~ from MS braln~ to eonfir~ the dlagno~ls of tho di~es~e.
.. ~' ! . ;. ' ' . .: ' W092/08~8~ ~09~ 3 ~ PCT/AU91/~519 In another aspect of the pre~ent lnvention there 19 provlded a method for the serologlcal dlagnosls of MS.
Thls aspect of the lnventlon i3 based on the observatlon that ~era from MS patlents contaln clrculatlng antibodies S to MSV. Using lmmunocytochemlcal tochnlques, lt has been shown that pretreAtment of MS braln tissue wlth sera from MS patlents, especlally from patl~nts who have recently had a relapse, block-~ the lmmunocytochemlcal staining of the braln tlssu- pr-vlou-ly d-~crlbed. Thls blocking demonstrates that the J-ra of MS p~tlent~ contalns antlbodles to the vlrus.

Accordlngly, ln thls aspect of the inventlon, there 18 provlded a method for the dotectlon of antl-MSV
antlbodle~ ln a fluld Qample taX~n from a patlent, whlch comprlses contactlng sald fluld s~mplo with MSV or an antlgen or antlgenlc fragment thereof, and detectlng an*l-MSV antlbodle- bound to s~ld MSV or antlgen or antlgenlc fragment thareof.
The fluld ~ample may b a blood or corebrosplnal fluid s~mple. Preforably, the sample 1~ a blood sample, whlch may bQ whole blood or a d-rlvatlve thereof, for ~xample blood s-rum or blood pla~ma.
The dlagnostlc method of thls asp~ct of the lnv~ntlon may utlllse tho well known prlnclples of enzyme l~munoa~8ay~ or radlo-lmmunoh~say~ to detact the presence of any antl-MSV ~ntlbodle~ from th~ serum sample boumd by ths dotectlng antlg~n. Th~ dotectlng antlgen (MSV or an ~ntlgen or antlgenlc fragm nt thoraof) may, for exAmple, be lmmoblllsad on a olld ~upport, and the pre~ence of bound antl-MSV ~ntlbodl~ can b d~t-ct~d u~lng approprlately lab~lled antl-human immunoglobulin antlbody. Othor alt rnatlvo~ wlll ba wall known to per~ons sklll~d ln the art.

: :: . , - , ~ : , W092/0878~ PCT/AU9l/~5l9 6 209~39 In thl~ aspect of the lnventlon, al~o, there is provlded a dlagno3tic teQt klt for detectlon oS antl-MSV
antlbodles in a fluld sample, whlch 1~ characterlsed in that it include~ MSV or an antlgen or antigenlc f ragment thereof as detecting antlgen, prQferably lmmobllised on a solld support ' :~' '.
In yet anothor a~poct of this lnvention whlch arlses from the lsolatlon and ln vltro culture of MSV, thero ls provlded a vaccln composltlon for stlmulatlng an lmmune re~pon~e agalnst MSV ln a human or animal patlent, whlch comprl~e~ lnactlvat-d or attonuated MSV, or an antigen of MSV or antlgenlc fra~ment thereof, as tho actlve lmmunogen In thl~ aspect of th- lnventlon al~o, there is provlded a method of produclng an lmmune respon~e against MSV ln a human or anlmal patlent, whlch comprlse~
admlnl~tratlon to sald patlent of an effeotlve ~mount of an actlve lmmunogen comprlslng lnactlvated or attenuated MSV, or an antlgen of MSV or antl~onlc fragment thereof -~

If d-~lr~d, th- actlv lmmunogen may be coupled to a carrler mol-cul~ to lmprove lt- lmmunogenlclty Sultable carrl-r molecules may lnclude, for example, haemocyanln~ ~uch a~ keyholo llmp t haemocyanln, bovine 9~ru~ ~lbumln or ovalbum~n In addltlon, the vaccine co~yo d tlon mhy optlonally lnclud- an ad~uvant Known ~dJuvants ~or lncorpor~tlon lnto anlmel and hum~n vacclnos lnclud-, fos example, Freund's complete and lncomplete ad~uv~nts, alum, ~nd the llke Technlque~ are now w ll known for the product~on of lnactiv~t~d or attenuat~d vlru8-J~ and, partl~ularly - 35 ln the c~o o~ MSV whlch 1~ closely relatGd to MV and CDV, attanu~t-d vaccin~s hav~ be~n dev lop~d agalnst both o~ ~h latter viru8-~. Slmllarly, the prep~ration of ... . : .- .
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W092/0878~ 7 2 ~ 9 5 5 ~ 9 PCT/AU91/~519 sub-unlt vlru~ vacclne~ 19 well known, based on the ld~ntlflcation of antlgens or antlgenlc fragments of the vlrus whlch are able to stlmulate an immune response S In the cas~ of the MSV vacclne described herein, the vacclne may bo producod for us~ ln cats, simllar to the canine dl~t~mper vacclne, ~nd/or in humans, partlcularly human lnfants, slmllar to the measles vacclne In yet another aspect of the pre~ent lnventlon thera 19 provlded a method of treatmont of MS in a patient whlch comprises ~dmlnlsterlng to the patlent an effoctlve amount of an antlbody, preferably ~ monoclon~l antlbody, whlch recognlsoJ or blnds to MSV, or an antlgen of MSV or to an antlgenlc fragment ther-of The ~ntlbody u~d ln thl3 ~-pect of the lnvention m~y be comblned wlth a carrler or targetin~ molecule, for exumple c~rrier or targetlng molecule~ ~uch as intercellular ~dheslon molocules which as3i~t the antlbody to penetr~te tho blood braln barrler Furth r ~-ature~ of tho pre~ent lnventlon wlll be 2S app~rent fro0 th- d-t~llod d--crlptlon ln the followlng Example-; -~, ~CWI.~ l; "' " ' ~-ol~tlon o~ M5V ~ro- M8 brn~ tlJ~ue and ln vltro cultlv~tlon th-~-of ln CV 1 c ll~.

Polyclonal antlbodie~ to the p rd~tent, non-perm~slve p~r~myxovlrus l~olated from cat braln tlssue~
w re ralsed ln rabbit~ The lmmunoglobullns wera purl~led from sera of lnoculatod ~nlmal~, extenslvely ad~orbGd ag~ln~t cat llver powder, aceton~ drled Crandell fellne kidn~y (CRFX) ~nd Varo cell~ and concentrated to S

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W092/0878~ 2 0 9 S 5 3 9 PCT/A~I9~

mg of proteln per ml. The prepared antlbody was then ab~orb~d to elther Affi-Gel Proteln A (Blo-Rad) or CN~r-actlvated Sepharose 4B (Phar~acla). One to two grams of frozon, autopsled braln tlssue from 8 dlfferent, S confirmed cases of MS ~nd four non-MS cases were homogenlsed and passed through the afflnlty columns. The colu~n~ were washed wlth large volumos of Trls NaCl buffer prlor to elutlon. Th~ eluat-~ were collected, drops were negatlvely stalned wlth 3% phosphotungstic acld, pH 7.2, prlor to examlnatlon ln an electron mlcroscope. Eluates obtalned from the 12 braln~ were added to cultures of CV 1 cells.

Electron mlcroscopy of the negatlvsly stalned ;~
eluat~ obtalned after passago of MS ~raln tlssue through columns of CNBr-actlvated S~haros~ revealed pleomorphlc, clrcular proflle~ wlth an average dl~mater of approxlmately 300 nm although ~ome were considerably larger. The eluates obtalned fro~ tho Affl-gel Proteln A
columns showed simllar slzed proflle~ but these lacked an ext~rnal membrane and conslsted o~ ~ tl~htly colled, tubular-llke, structure ~pproxlmately 18 nm ln dlameter.
The tubule~ had a thln tran~lucent core and thelr walls had a "herrlng-bono" appearanco con~lstent with a helical structur~. Som fractlon~ contaln d moJtly tubular-like structur0~ whlch were comparable to lsolate~ of the nucl~ocap-id-llk- structures or CLP~. Some eluants contalnlng the vlrus-llke partlcles were pelleted by c-ntrlfugatlon, embedded ln ag~r and flxed and processed for oloctron mlcro~copy. The pGll~tJ contalnod vlrions, up to 300 nm dlameter~ wlth lnternal tubul~r profiles, about 18 nm ln dlameter, s~ctlonod ln varlous planes.
Th~ vlral ~olatlon from MS br~ln tlssuo ha~ been repe~ted mora than 40 ~lme~. - -A~tar 5 p~ssage~, CV 1 calls lnfected with the eluates fro~ th~ MS braln~ show~d some ~ocal cytopathic ... .

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W092/0878~ 2 o 9 5 ~ 3 9 PCT/AU9~/~S,9 effects (CPE) ln the form of small syncytia and sllght cytopl~mlc vacuolatlon Ultrastructur~l examln~tlon of these culture- revealed the presence of cytoplasmlc lnclu~lon~ whlch conslst-d of tubular structures approxlmately 18 nm ln diameter These incluslons were morphologically slmllar to those observed ln the inltial isolation of the feline p~ramyxovirusl Virus particles, simllar to those lsolated from MS br~ins, were present in some cultures and wer~ also lsolated by use of the affinlty chromatography t~chnlque Ultrastructural ex~mlnatlon of the eluates obtalned after pa9sage of non-MS braln tlssue through the columns dld not reveal any viral partlcles Cultures inoculated with the~e eluate~ dld not show any CP~ and nelther cytoplasmlc lncluslon~ nor vlral partlcle~ were seen when exa~lned ln the electron mlcrosco~e Furthermor~, vlral partlcles were not obtalned from these cultures uslng afflnlty chromatography Polyacrylamlde gel electrophoretlc (PAGE) studles have shown that tho pept~de~ of the i~olated virus are comparable wlth tho~e of MV and CDV except for ths absanc- of som membran -splko protelns Thl~ dlfference probably account- for th lack of haemadsorptlon by inf-cted CV 1 cultur~s of erythrocyte~ from chlcken, cow, dog, guln ~-plg, horse, humAn 0, mouse, rabblt, rat and ~h p Th -- obsorvatlons w-r~ conslstont wlth the prop~rtloo o~ th fellne p~ramyxovlrus whlch exlsts in cultur a8 a psrd ~tant non-perml~slvo info~tlon of CRFK
and Vero c~ ' The nucleocap~lds ~nd tho virlon~ l~olated from MS
braln~ show morphologlcal slmll~rltle~ and some 3S lmmunologic~l oros--r-~ctivlty wlth MV and CDV Thls, in ~ddltlon to tho lnltlal PAOE studieQ and the form~tlon of .- . . : ; - , , . - ...... , . ", , - : ~ .. ; . .- .. . ,, . :- - - .

w092~0878~ 2 o 9 ~ 5 3 ~ PCT/A~'9~ 9 , ' .
small syncytla ln tissue culture, suggests that the virus lsolated fro~ MS brain~ may bo morblllivirus ~XAMPL~ 2 Productlon of Polyclon~l Antlbodio~

The nucleocaspld-lik~ structure~ (CLP~) and viral partlcle~ obtained u~lng the ~fflnity chrom~tography procedure as d~scribed in Exumplo 1 abov~ were used ln the production of polyclonal anti-MSV antibodles 1 5 ml of the lsolated MSV (5 ~/ml) w~s thoroughly mlxed with 1 5 ml of complete Freund'~ adJuv~nt and admlnlstered by ~ - -3ubcutaneous ln~ectlon at several -~lte~ to a young adult ~ ~ -rabblt Aftor 10 days, the rabblt was glvon a slmllar in~ectlon contalnlng 1 ml of th vlral lsolate and 1 m}
of lncomplete Freund's ad~uvant Thl~ wa~ repe~ted a furthor 5 tlmo~ at 14 d~y lntervalJ and then the anlmal was bled 8 days after the la~t lnoculation The rabbit was furth~r lnoculated thr-o tlmo- wlth 1 ml of vlrus ev-ry 14 days over 42 day~ follow~d by bleadlng 8 days a~ter the third lnoculatlon Thls was repeatod over a perlod of six months At th tl~ of bl--dlng, th blood waJ allowed to 2S elot and ~-parat- Th a~pl- w~ th n Jpun at 3000 rpm for 20 mlnut-~ ~nd tho cl-an s-rum was a~plrat~d off and ~tor-d at -20 C The lmmu~oglobulln G wa~ pur~fled by addltlon of an equ~l volu~ of satur~ted ammon~um sulph~to ( NH4 ) ~SO~ to thQ ~eru~ to preclpit te the protein 30 ln th~ ~erum The s3mplo wa~ th~n contrlfug~d at 3000 rpm for 30 mlnut~s ~h~ preclplt~t~ wa~ reta~ned ~nd the --~up~rnatant w~ r~treat-d wlth (NH,)2S04 and then recentrlfugea. '' .,.: ' . ' .
The ro~erved preclpltatos w~re ra~u~pend~d and furthor treated wlth an equ~l volu~e of 50/50 ~aturated ~NH~)2S0,/dlstlll2d wat~s~ befor~ the ~olutlon waQ
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W092/08~8~ 2 ~ 9 ~ ~ 3 9 PCT/Augl/~sl9 centrifuyed at 3000 rpm for 30 mlnutes. The preclpltate waQ then di_~olved in 0.01 M phosphate buffer solutlon.

The globulln protelns ln the Qolutlon were then dialysed ag~lnst Qeveral changos of phosphate buffered sallne at 4-C overnlght or for two d~ys. The immunoglobullns were p~--ed through ~ diethylamlnoethyl ~ -cellulose (DEAEC) column of 50 cc capacity and the fractlons collected. Th optlc~l den~ltles of the fractlons were determlned u-lng ~ spectrophotometer and the proteln fr~ctlons pooled. The immunoglobullns were concentrated to about 5 mg/ml of proteln and stored at -20-C.

For lmmunocytoch mlcal studles, the polyclonal antlbody w~ conJug~ted wlth hor9e-radi~h peroxidase accordlng to the technlqyo of Avr~me~ & Ternynck~3. The cross-rQ~ctlvlty betw~n MSV, MV and CDV ba~ been demon~trated uslng an ELISA type a88~y, the results of whlch are shown ln Flgure 1.

~XaMPL~ 3 Productlon of Monoclo~l Antlbodlo~.

Immunl ~tlon Protocol.
Slx to elght w--k old B~lb/C mlce were lmmunlqed wlth an mul~lon con~lstlng o~ 0.1 ml of purifled MSV
(S ~/~1) and O.1 ml of compl-te Fround'q ad~uvant. The mlcJ w re lnJected ln _ever~l sltes ~ubcu~aneously. The boo-ter lmmunl ~tlon, con~lsting o~ 0.1 ml of virus and 0.1 ml of lncompleto Fr~und'~ adJuv~nt, was lnoculated four weeks later subcut~n ously ln 3everal pl~ce~. A
fln~l lnJectlon o four tlm~ th~ orlgln~l dosage of the vlrus ln s~lln~ w~ glven threo to four d~ys prlor to fuslon~'.

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W092/08t8~ PCT/AU91/~519 12 209553~ :
Mvelom~ Cell Llne The my~loma cell llne used was the Balb/C P3-NSl-Ag4-1 cell llne (NSl) The cell-~ were grown in Dulbecco' 9 Modlfied Eagle Medlum (DMEM) supplemented wi th 10% foetal calf serum (FCS) ~nd 0 2 ml of penlclllin/streptomycln (P/S), 120 ~/ml ~nd 25 ~g/ml re~pectlvely Tho myeloma c~lls were incubated in 50 ml flask~ at 37 C ln a 7% C02 humldlfled lncubator, and pa~saged through medium contalnlng 8-azaquanlne (2 ~g/ml) to klll any revertant amlnoptor~n re~lstant cells Myeloma cells wore sub-cultured (~pllt) every 24 hours, three days prlor to fuslon 5pllttlng of the cultureQ involved removlng 50% of the medlum, contalning approxlmately 5 x 10~ cells, and pl~clng lt lnto a new flask 8Oth flaQks wero then reflll~d to the original volume with fresh medl~ ~o that each flask contalned 2 to 3 x 105 cello p r ml Tho myeloma Cell9 wsre nover allowed to grow above a concentratlon of 8 x 105 cells per 20 ml -... .. . . . . .
Per~u~lon of S~le-n to obtaln LYmDhocYtes Immunlsed mlce wero kllled by C02 aQphyxiation, the ~pleen removed aseptlcally nd placed in a ~terlle petri dl~h cont~ln~ng 5 ml of cultur- m dlum Sple-n cellc wor- w~-hed out lnto th p trl dlsh by in~ection of cultur~ m dlum lnto th~ sple n c~psule A cell count was th n porformod Fuslon The protocel used to p~rform fu~lon~ w~s adapted from that describ~d by G~lfr~ *nd Mllstelnls Ten grams of polyothylena glycol (PEG) mol~cular welght 1500 (Sigma Chemlcals), wa~ llquefl~d and dlspsns~d in 0 5 ml allguots lnto pr~-welghed bottles and autoclav~d The bottles wero Shon w lgh~d and the wolght of PEG was calculated Approxlmatoly 10~ mouse ~pleen C811~ ( those -~

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W092/0878~ 2 0 9 ~ ~ 3 9 PCT/AU9~/~5,9 obtalned from 1 spleen) and 10' myeloma cells were mixed and placed ln a sterile 50 ml conlcal-bottom tube. Serum free media was then added to produce a total volume of 50 ml. The mlxture was then centrlfuged at 400 g for flve minutes and because the concentratlon of PEG is crltical for fuslon, all medlum Wa9 removed to prevent dilution.
The 0.5 ml allquot of PEG was dlluted to a flnal concentratlon of 40~ (w/v) wlth serum free medla and kept at approxlmately 40-C. The pellet wa8 brokan by gentle tapplng and 1 ml of PEG ~ddod drop-wl~e over l mlnute.
Thls wa-Q shaken for another 1 to 2 minutes. Then, 1 ml of serum freo medlum (pre~w~rmed to 37-C) w~ added over 1 mlnute. Thls wa~ repeated and th~n performed twlce more wlth the serum belng added over 30 saconds. A
further 7 ml of medlum w~s added over 2 mlnutes, while belng contlnuously shaken. Flnally, 12 to 13 ml of medlum wa3 added and the cells were centrlfuged at 400 g for 5 mlnutes. The Qupern~tant was r~moved by ~uctlon and the pellQt rssusp nded ln 40 ml of HAT selection 20 medla contalnlng 10' feeder cells per ml. EHAT medlum: -50 ml of DMEM supplemented wlth 20~ FCS, 0.4 ml penlclllln/~treptomycln, 0.05 ml Funglzone (TAG0), hypoxanth1ne (136 ~g/ml), ~mlnopterln (0.19 ~g/ml~, thymldine (3.88 ,ug/ml), glutamlne (2 mM) and pyruvate (1 mM).~ The cells were thon dlsponsed ln 100 ,ul allquots lnto 96-w ll culture plato- (Llnbro), and the plateJ were placed ln an lncubator at 37-C.

Po-t-Fusion Cel~ Malntenance.
Cultur- tr4ys were malntalnsd at 37-C ln a 7% C02 humidlfied lncubator. The plates w~re checked da~ly untll Day 5 ~ft~r fuslon when largo-~cale cell death of ~pleen cell~ W~8 expected ~nd ob~-rved. FreJh HAT (50 ml per well) wa8 added. At Day 7 to 14 ~fter fu~lon, medla 35 (100 ml per well) wa~ removad every 2 to 4 days and replaced wlth fre~h HT medla tHT medla 1~ the ~ame as HAT but wlth th~ exclu~lon of amlnopterln]. When the W092/0878~ 2 o ~ ~ ~ 3 9 pcT/A~9l/oosls maJority of hybrld~ reachQd h~lf confluence, 100 ~1 of supernat~nt was ramovQd and screened for the appropriate antlbodles. Posltlve clones were ub-cultured into new plates wlth the addltlon of ~pleen or peritoneal S macrophage feeder cells.

Screenina for Po~ltlve Hybrids.
Posltlv2 hybrldomas were detected u~ing enzyme- - -llnked lmmunosorbQnt a~s~y (ELISA). The Edmonston Strain of the mea~les vlru~ (5 ~g/ml) was dllutad 1 ln 40 ln a ~olutlon contalnlng 15 mM N~CO3 and 33 mM NaHC03 solution (coatlng buffer) at pH 9.6 and 100 ~1 added to each well of ~ 96 well round bottom mlcro-tltre plate (Dlsposable Products Australla). The level of the antlgen used was 15 optlmlsed by tho ELISA chogusr board technlquel'. Plate~ -`
were lncubat~d for 18 hour~ at 4-C and then washed 3 tlmes wlth a 0.5% solutlon of Tween 20 ln phosphate buffered 0.15 M sallns (P~S). Plate~ wera then blocked wlth 10% FCS ln P~S at room temperature for 2 hours, after whlch they were wash~d 3 tlmes wlth P~S-Tween.
Supernatant (100 ul) from ~h3 tlssue culture plateQ was removed aseptlcally, added to tha s0nsltlsed plates and lncubated for 12 hours at 4-C. Well~ were then washed and 100 ~1 of ~ntl-mou~- IgG conJugatod wlth alkallne phosphata~e (Tago, Inc., USA) dllut~d to 1 Ln 1000 was add-d. The pl~t-s wor- lncubated for 2 hours a~ room temp r~tur-, ~nd ~fter washlng, 100 ~1 of the enzyme sub~tr~to 4-nltrophonolphosphate (i-NPP) (Boehrlnger Munnh l~, W.Ger~any), ~t 1 mg/ml ln pH 9.8 10~
dl~thanol~mlne supplemented wlth 0.5 mM MgCl2 (substrate bu~fer) was ~dded. Followlng a 30 mlnute incubation peslod at room tempQratur~, th~ optlc~l denslty was determ~nod on ~ Tltret~k Multl~tre~m mlcrotltra plata read~r u~lng a 405 ~m fllter.

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" . . . ~, ... . 1 , . ', .. .. ~, ' " " . , . ` - ' .. ', ' . ,' ~ ,' , W092/0878~ 2 0 9 ~ ~ 3 9 PCT~AU91~00519 Prellmlnary Scre0nin~ of CDV M,V, and MSV
Separate E~ISA plates wer sensltls~d wlth CDV
vacclne (Websters), Edmonston measles vlru~ and MSV
accordlng to the protocol prev~ously descrlbed Human polyclonal antl-measle~ antlbodies, lot 25754, (Prlncess Margaret Hospltal, West~rn Austr~lia) werQ diluted with PBS to 1 ln 320 The antl-CDV and ant~-MSV antibodles were u~d at a concentr~tlon o~ 1 ln 100, diluted with P~S The enzym- con~ugat-~ used w re ~ntl-rabblt IgG
alkallne phosphata~- (TAGO) for CDV and MSV and anti-human alkaline phosphata~e (TAGO) for th~ measles vlrus After rQcon~titUtiOn, cell coniugat~s w~r~ u~ed at a concentratlon of 1 in 1000 dlluted wlth P~S The ELISAc were carri~d out accordlng to tho protocol descrlbed above and the result~ ~re shown in Flgure 1 Screenlna Monoclonal Antlbodles Slx posltlve hybrldomas w~re also ~creened against CDV, MV ~nd MSV, a~ woll aJ Nowcastle Dlsea~e Vlrus (NDV) (10 ~g/ml) and thro- fellne vlrusss: FQllna panleucopenla, Fellne rhlnotracheltls and Fellne calclvirus tWebstcr~ 3 ln 1 (livlng)~ to check for any non-~peclficity Th- f-llne virus-s woro used after being rocon-tltut-d wlth 1 ml of dlstllled water and subs-quent dllutlon to 1 ln 20 wlth P~S The ELISAs were agaln carslod out accordlng to the protocol pr~viously d~-crlbed ~he re~ults of thls ~creen ar~ shown ln Flgur- 2 ~XA~!LE~ 4 ~au~ocytoch~-lcal 8talnlng Polyclonal antibodl~s ralsad agalnst MSV, as d~scrlbed ln Example 2, ha~ been used ln l~unocytocbemlcal ~tudl~ to ldentlfy MSV protelns wlthln MS braln tls~e, wlthln CVl cells and oligod~ndrocyt~ cultures lnf0cted wlth MSV and w~thin ... . .

W092/0878~ 2 0 9 ~ ~ 3 9 PCT/AU9,/~SI9 CRFX cell~ infected wlth the fellne viral lsolate ~he stalnlng technlque uged was esse~tlally the same a~ that de~crlbed by Cook et. al . ' ln the immunopsroxldase staining of MS braln tlssue uslng polyclon~l ~ntlbodies ag~lnst the fellne derlved agent T~e stalnlng reactlon product obtalned wlth the polyclonal ~ntlbodles to MSV was a~oclated wlth cytoplasmlc lncluQlons mostly withln phagocytlc cells ln MS plaques Ultra~tructural studles h~ve con~l~med th~t the stalned lncluslons were the tubul~r-llke ~tructure8 that h~ve b n called curved-llnear profiles and whlch are in fact the nucleocap~ld o~
the MS vlral isolate These cytopl~mlc lncluslon~ were also stalned wlth horse-radtsh peroxldase-labelled monoclonal antlbodles Cl and El ~see Example 3 and ~lgure 2~ These ln¢luslons w~re not dotQctod ln normal whlte matter ln MS bralns nor ln whlte matter from non-MS
brain~

The lmmunccytochsmlcal stalnlng of Qtoplasmlc lncluslons ln MS plague~ wlth hor~e-radlsh peroxidase-labelled polyclonal antlbodl~s to MSV WA~ blocked when the braln tlsaue was pr--lncub~ted wlth sera from two MS
pationts who wero ~xp rlenclng a relaps- Thls blocklng reactlon wa8 not a- ff-ctlv when arum W~J u~ed from a patl-nt ln a r-ml-~lon pha-- of th dlsoa-e Ther~ was no reductloA ln the lnt n-lty o~ th Jt~lnlng roactlon when th braln tlssue w~8 pre-incub~t~d wlth sera from peopl- who have no medlcal evldonce of MS Furthermore, hor~--radlsh peroxldase-labelled lmmuno~lobulin G (IgG) purlfled ~ro~ the ser~ of two MS patlent~ have been u~ad to staln cytopl~mic inclus~o~s wlthln MS pl~ques The re~ult~ obtalned are comparable to those seen when the l~b~ll~d polyclonal ~ntlbod~-~ to MSV ~ro u~-d Tha~ -re9ult8 lndlcated that MS p~tlent~, e~peclally tho~ ln whlch there i~ dls~ase actlvlty a~ lndlcat~d by cllnlcal ~lgn~, have clrculatlng antlbodl~- to the MSV
, W092/0878~ 2 0 9 ~ 9 PCT/AU91t~19 ~XAMPLE 5 Ollgodendroeyto culturo ~yJt~ for M8 vlru~
Primary cultures of ollgodendrocytes have been grown and lnfected wlth the vlru~ lsol~ted from the braln tlssue of two ca~es of MS. The results show that the vlru8 has an affinity for ollgodendro~lla and does not lnfect astroglla. Furthermore, dlstlnet cytopathlc effects (CPE~ are ob~erved wlthln 6 to 14 days post-lnfectlon. Thls co~p~res with the 6 to 8 weeks that lt takes to ~eQ obvlou~ CPE when the vlru~ 18 grown ln CRFK, Vero or CV 1 eell llnes. Thls culture ~ystom now allows a rapld assessment of the lnfectlvity of the vlru~ and wlll allow a comparlson of straln~ of the vlrus for the deteetlon of attenuated stralns. Th~ afflnlty of the ~ -vlrus for ollgodendroglla provldQ~ further po~slble evidenc that the demyellnatlng proe~s ln MS 1~ due to a vlral lnfoction of the~e cell-.

The ollgodendroeyte culture system permlts an evaluatlon of the effeetlveness of monoclonal antlbodles ralsed agaln~t the vlrus ln lnhlblting the growth of the vlrus. ~-::
one to two day-old Wlstar rat pup~ were euthanased by corvlcal dl~locatlon of CO2 lnhalatlon. ~r~ln tls~ue w~ r mov d ~nd dl--oclated u~lng a modlflcatlon of the tochnlqu- UJ - d by Sarlleve ~t.al.~. Immedlatoly followlng euth~nasl~, ~he pups wora transferred to and lm~rJ-d ln 70~ ethanol in a ba~kar and placod ln a l~min~r flow c~blnat. Bralns wora r~oved aseptically, wa~hod ln Dulbeoco' 8 Mlnlmu~ Ess~ntlal Medlum (DMEM) with 10 Fetal calf ser~ (FCS) and tr~n~f-rrod to a patri-dish cont~lnlng ~ dou~le layor of 8toxill8ed stalnl~s~-~teel mssh (~esh ~iz~ approx 100~). Dlssoclatlon lnto fresh ~edla (DMEM/10~ FCS) waJ p rformod by rep~atedly drawlng and expolling the tls~ue through th~ m sh lnto and from a 20ml Jyrlng~. ~h0 ro~ultant tl98ue su~pen~lon was .. ,. :

W092/0878~ 2 o 9 ~ ~ ~ 9 PCT/AU91/~1s dllutod wlth fre8h m~dla ~nd seeded lnto SOml/25cm Costar tls8ue culture fla9k3 at 1-1 5 bralns per flask with lOml of modta or onto poly-L-ly~lne-co~ted coverslips ln 35 x lOmm petri-dishes at 0 5 br~ins por dlsh with 3ml of DMEM/lO*FCS The cultures wero incub~tod ~t 37 C with 5 C02 Modi~ was changed after the first 4-7 days and then weekly Inoculatlon of Drlmarv culturo~ wlth vlral lsolates from MS br~ln tl~u~
Two lsol~tes were obtalned from br~lns A87/29 and MS-7 uslng the ~fflnlty chromatography tschnlquo prevlously descrlbod Thes- lsolato~ wer- lnocul~ted into 4 day-old prlmary rat gllal culturo~ ~ follows The modl~ w~s r~mov~d from tho culture~ whlch wero washed twlce wlth fresh DMEM The culture~ wsrs thon lnoculated wlth 100~1 of vlru~ susp~nslon ln 1~1 of culturo madlum -~
Flasks wero lncub~ted for 2hr a~ter whlch tho or1glnal medlum wa~ roturned to the cultur- wlthout tho removal of the vlrus suspenslon I~ unocytoch d ~try Fix~tlqn of cultures Cultur-s w r- washed twlc- wlth phosphate buffered salln~ (P~8) to r-mov c-ll d-brl- and flx-d wlth a 5ml solutlon o~ 4~ paraformald~hyd ~nd 1~ glut~raldehyde ln pho-~hat~ buff-r ~or lS mln Tho flxatlve wa8 removed ~nd culture~ w r- washod anothor two tlm~s wlth P~S prlor to b ln~ stored ln fresh P~S untll sta~n~dO
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Immuno~eroxld~so l~b llln~ Qf tlssue ~yl~yro~ -~
Culturo~ wero w~shed twlce wlth PaS ~nd lncubated for 2 mln wlth lce-cold, 70% oth~nol Thl~ wa9 followed by two wash ~ wl~h P~S ov r lS mln ~nd lncubatlon for 30 mln wlth lOd of 0 5% (v/v) H202 ln Trls buffer Flasks w ro wa~had w$th 40ml of Trls buff-r ~nd lncubated for 435 mln wlth 1~1 of prlmary ~ntlbody (olth r HRP lab~lled ~ .. . ..

W092/0878~ 2 o 9 ~ ~ 3 9 PCT,AU9~ 9 antlbody r~ised agalnst 2',3'-cycllc nucleotlde 3'-phosphodiesterase tCNPaQe] or the MS vlral i~olate) at a dllutlon of 1 30 ln Trls buffer or culture~ were left ln Trls buffer alone as controls Followlng incubatlon, S antl~erum was removed by washlng each culture three tlmes over 10 min wlth 40ml of Trls buffer before the additlon of lml of DAe solutlon and lncub~tlon, in the dark, for 15 min After a further three w~she~ in Trl~ buffer, the culture~ were prepared for llght or electron microscopy in a routlne manner Isolatlon of vlru~ ~rtlcle~ from tls~ue culture medla The medlum was collected fro~ culture~ that had been lnfected for at lea~t 11 days, 1 e these culture~
lS had had at least one medium change The pooled medlum wa~ centrlfuged at 35,000 rpm for 3 hr~ ln ~ Beckman ultracentrlfu~e wlth a SW4 rotor The pellet w~s resusp~nded wlth 0 5ml of phosphotungstlc acld and a drop of thls was placod on a Formvar grld for 5 mln prlor to drylng and examlnatlon ln an electron mlcroscope RoJult8 Prlm~rv rat ollaod ndroallal cultur~
Ollgodendrocyte- wlth a round cell antlbody and 1 or 2 pol~r proe~ w-r- ldentl~od aft-r 3 days ln vltro. Although th ~- c-lls n ver rsached confluency, th~y dld ~how prollferatlon above the a~trocytlc bed ~ -lay~ Other oll~od~ndroeytes were comparsble to tho~e d scrlb~d as type I and typo II ollgodendrocytes by Xuhlm~nn-Krleg ~t. al . ~7 . Type I c-lls bad a trl~ngular or ovoid shapa wlth 2 or 3 proc-~e~ and a fla~ cell body Type II cell3 h~d ~ network o~ proc-sses of v~rlous dla~ters and length~ surroundlng the coll body ~hese ollgodendrocytes t~nded to bo above the ~trocytlc bedlayer Astrocytes grew qulckly to form a monolayer wlth cell to cell cont~ct . . .

, , ,' ' ' , - ' . , ` .: ':: ' '. ' ~ . , ~ ' ', ' . ' W092/0878~ 2 o 9 ~ ~ 3 ~ PCT/AU91/~519 Immunocvtochemlcal ldentlflcatlon of ollgodendroc~tes.
The reactlon product to the HRP labelled antlserum to CNP~se, ~n enzyme 9peelflc to oligodondrocytes, wa~
located ~n the c~lls d-serlbed above as ollgodendrocyte~.
There waR no stalnlng reactlon with the underlyln~ layer of astrocytes. The reactlon product to CNP~se wa~
d~trlbuted throughout the cytopla~m of the ollgodendrocytos and ltY locatlon wa~ the same lrrespectlv- of tho age o~ th- culture. Thero WA9, however, a sllght deereas- in th- lnten~lty of the stalnlng ln the longer-term culture~. -.
Controls.
There w~s vory llttle, lf any, DA8 re~ctlon 15 product ln control flask~. ~
- ~ .
El~e~p mleroseoDv.
The above re-ult~ were conflrmod by eleetron mlcroseopy. Tho ~ntlbody to CNPa8e gave a stalnlng reactlon whlch showed that thl~ enzyme wa3 ln cells that had the ultrastructusal ch~racteri~tlc3 of ollgodendroglla ~nd was located throughou~ tha cytopl~sm. ~;
Cells wlth fe~tur-~ ty~lcal of a~trocyte~ dld not show posltlve stalnlng.
Inoeulatlon o~ cultur-~ with tho MS vlral i~olate.
6 d~y- ~o~t-lnoculatlon (10 days ln vlt~o).
Som~ of th~ ollgod-ndroeyte~ appeared to be enl~rg d ~nd con~ls~ed of ~ eentral rlng of eondensed cytoplasm ~ound which wa~ a large m~mbranous ~h~t-llke strueture. Som- of theso cell~ con~alned 2 to 3 nucle1.
Thes~ cell~ w~r- wlthln th~ conflu nt l~yer of a~trocytes rather than on top and th r W~9 a retractlon of a~troeyte~ around tho oll~od ndroeyt-s.

W092~0878~ 2 ~ ~ ~ 5 3 ~ PCT/AU9l/~5,9 9 to 14 days post-lnfectlon with the MS vlral lsolate In contrast to the 6 day post-lnfected cultures, these l~ter cultures showed very obvlous CPE There were several large multlnucleated gllal cells withln each culture Some of these cells contalned up to 30 nuclei, arranged as a rlng around the periphery of the cytoplasm These cells were posltlve for the labelled CNPase and contained cytoplasmic lncluslons whlch were po~lt~ve to the l~belled antlbody to the MS vlrus The bedlayer of astrocytQs h~d receded ~om~ conslder~bl- dlstance from the perlphery of these cells su~g~tlng that the multlnucleated cells were produclng soluble sub~t~nces whlch may be cytoklnes Examlnatlon of the pell~t ob~ained by ultra-centrlfugatlon of the pooled medla fro~ the cultures lnfected wlth the MS vlru~ show-d that vlral partlcles were present Thls obsorvatlon lndlcates that vlru~ ls belng produced from the lnfected c0113 and that thls is a mor- convenlent and rapld m thod of obtalnlng l~rge quantitles of the vlru~ Althou~h vlrus has b~en obtalned from lnfected CRFK, Vero ~nd CV 1 colls, the productlon of thls vlru~ ha~ not been observed untll 50 days or mor post-lnfectlon It ~hould be noted that cultured oll~odondroglla do not appear to be ~u~coptlble to th- Edmon-on ~traln of mea-le8 vlru~ Culture~ -lnf cted wlth thls vlrus dld not show any CPE

~XaMPL~ 6 D-t-ctlnn of antl-M8 antlbodl-~ ln -ru~ or cerebro~p~nnl fluld Example 4 provld~s lmmunocytochsmlcal evldence that there are clrculatlng antlbodlo~ to MSV ln the serum of MS p~tlcnts PurlflQd MSV, an antlg-n or antlgenlc fr~gment derl~ed from purl~led MSV, or a synthetlc .. ~
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W092/087~ 2 ~ 9 5 5 3 ~ /AUg11~519 polypeptlde havlng a seguQnce ~ubstantlally homologous (at least 75% homologous, preferably at least 90%
homologous) with an antlgen or antigenlc fragment of MSV
18 used as the detectlng antlgen in an enzyme-llnked S immuno~orbent assay (E~ISA) Uslng known techniques, the detectlng antlg~n 19 lmmobillQed by belng coated onto a solld support or carrier such as tha surface of the wells of a mlcrotltre plate, u~lng coatlng buffer After a washlng step, the s~rum or cerebrosplnal fluld sample ls added to the well~ Followlng lncubatlon, the wells are further wa~hed, and an approprlate detectlng antlbody havlng an enzyme conJug~ted th-reto (such aR a goat antl-human IgG-horseradl_h peroxlda-- conJugate) 1~ added Enzyme actlvlty bound to the ~elld ~upport or carrler 18 detected uQlng an approprlate enzy~ substrate (such a-Q
4-chloronaphthol or dlamlnobenzldlne ln the case of hors-radlsh peroxldas-) to lndlcate the pr~sQnce of antl-MS antlbodleJ ln the sample EXAMPL~ 7 D-v-lop ent of ~ vaccln- to pr-v~nt MS.

The g-nu~ ~orbllllvlru~ lnclud-~ m a-les and canlne dlstsmp~r a~ w-ll as oth~r vlruse~ that havo not b~n d~toct-d ln Au~tralla sueh as rinderpest vlru~, - p~t- d-- p tlt~ rumlnant3, bovln- enc~phalitl~ and phocln- dlst-mpar vlrus ThQse viruseQ ~how a close antlg nlclty and there ls conslder~ble lmmuno-cro~s reactlvlty b tw~en some of th vlral protelns On the ba~ls of lmmunologlcal, morphologlc~l and vlrologleAl studle~, the MS virus 18 anothe~ membsr of thl~ genu~ and has ~imllar prop~rtles to measles and canlne dlstemper vlru3es .

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W092/~78~ 2 o 9 5 ~ 3 ~ PCT/AU9l/~s~g ~ lve vlrus vacclnes ~ra con~ldered to be better immunogens th~n lnactlve or kllled vlrnl vacclne~.
Effectlve measles and canlne dlstemper vacclnes are based on the use of a llve vlru~ and the production of these vacclnes 1 well developed. The evldence that MSV is related to mea~le~ and canlne dlstemper vlruses, based on the lmmuno-cros~ reactlvlty de~crlbed ln Example 3, lndlcate~ that ~mllar technique~ can be used in the development of a vacclne using MSV.
10 ';
The ollgodendrocyte culture sy~t~m (soe Example 5) provld0s a me~ns by whlch thq lnfectlvity of 1 olateq or attenuated straln~ of MSV are assayed. Tls~u~ culture lnfoctlve doses (TCID) of the lsolates or 3traln~ of the vlrus aro evaluat~d and compar~d to ldentlfy an attenuat6d str~ln of the vlrus. The TC~D of MS vlrus that h~s been grown in Vero and CVl c~118 for several year~, or vlru~ th~t has been passaged through laboratory anlmals, are comparod wlth the TCID of 1301ate~ obtaining dlrectly from MS brain tlssue to detormine whether or not there is attenuatlon of th virus in the form of docreased infectivity of the ollgodendroglial cell~ in culture.

Th- ollgod-ndrogli~l culture syst m al~o permit~
an evaluatlon of the effOEctlven J~ of polyclonal and monoclon~l ~ntlbodl-~ ral~ed agalnst MSV on the vlrus in cultur-. Th eff~ctlv ness sf Antibodl-~ ral~d against att~nu~ted form- of the viru~ 18 evaluated a~ well aq the qu stion of whothor or not the att~nuated for~ produce a qufflciant antibody re~pon~e to act a~ an effective immuns~n.
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WO 92/0878~2 o 9 5 5 ~ 9 PCr/AU91/OOS19 D-velop lt of ~ tre-t~t for M8. .

Thls lnventlon provides forms of therapy of MS
S The flrst of the~ is the uso of an inoculum of a monoclonal antlbody, such as C6 (Flgure 2), which shows di tinct speclficlty to MSV Th~ lnoculatlon of varying quantltle~ of thls antlbody wlll help to boost the patlent's lmmune response to the vlru~ It 1Q well recognlsed that not all MS loJlons show an lmmune re~ponse ln the form of a lymphocytlc lnflltrate of the tlssuel~ and there 19 a lack of antlbody produclng plasma cells dosplte the presence of vlral prot-ln~ in these lo~lons However, ~ yurlflod, monoclonal antlbody m~y not ponotrat- the blood-braln b~rrlor to provlde an effectlve actlon ~galnst the vlru- v~n though thls b~rrlor 1~ conJld red to be damag-d du- to oedema~ The antlbody ¢an then b~ con~ug~ted wlth a carrler molecule, such as a cellul~r adhoslon mol~culo or an ollgodendrocyte speclflc moleculo, whlch c~n target the antlbody Tre~tment of MS may al80 tak~ the form of an attenuated vaccln ~ de-crlb d ~bov or a sub-unlt vaccln- ba-od on an antlg n or antlg nlc fr~gment of MSV
(or a ~ynth tlc polyp ptlde ~ub-t~ntl~lly hologou~ with ~n ~ntlg n or untigenlc fragmont of MSV), for exs~ple, th~ lnoculat~on of ~ speclflc antlgenlc fragm nt of the ~-vlru~ such aJ the epltop~ to whlch tho monoclon~l ~ntlbody C6 (Flgur~ 2) was produced Th~ advantage of thls 15 th~t the immunogon wlll ~ti~ul~t~ th lmmune Sy8tQm to produce B-lymphocyt-- whlch wlll produce large amounts of ~peclfic ~ntlbodl-J ~g~lnst MSV The stlmul~tlon of tho lm~una sy~t~ in thls m~nner will also 3S increA~o the production of T-help~r lymphocyte~ which wlll a~sist $n th~ l~mune response ln th~ le.810n8.

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Claims (33)

CLAIMS:

1. The morbillivirus, multiple sclerosis virus (MSV), in isolated form

2. A culture of isolated MSV in CV 1 cells.

3. A culture of isolated MSV in primary

4. A method for the preparation of MSV in isolated form, which comprises the step of contacting MSV-containing tissue with an immobilised antibody raised against feline paramyxovirus isolated from the central nervous system of cats, and recovering isolated MSV bound by said immobilised antibody.

5. A method for the preparation of MSV in isolated form, which comprises the step of contacting MSV-containing tissue with an immobilised antibody raised against MSV isolated from multiple sclerosis (MS) brain tissue, and recovering isolated MSV bound by said immobilised antibody.

6. A method of claim 4 or claim 5, wherein said MSV-containing tissue is MS brain tissue.

7. A method of claim 4 or claim 5, wherein after recovery said isolated MSV is cultured in CV 1 cells or primary oligodendrocyte cultures.

8. An antibody which recognises or binds to MSV, to an antigen of MSV, or to an antigenic fragment thereof.

9. An antibody of claim 8, which is a polyclonal antibody.

10. An antibody of claim 8, which is a monoclonal antibody

11. A diagnostic reagent which comprises a labelled antibody of any one of claims 8 to 10

12. A reagent of claim 11, wherein said labelled antibody comprises an enzyme-antibody conjugate.

13. A reagent of claim 12, wherein said conjugate comprises a horse-radish peroxidase-antibody conjugate.

14. A hybrid cell line or hybridoma which produces a monoclonal antibody which recognises or binds to MSV, to an antigen of MSV, or to an antigenic fragment thereof

15. A method for the detection of MSV in a sample taken from a patient, which comprises contacting said sample with an antibody of any one of claims 8 to 10 or a diagnostic reagent of any one of claims 11 to 13, and detecting binding of said antibody or diagnostic reagent to indicate the presence of MSV in said sample.

16. A method of claim 15 wherein said sample is a brain or other tissue sample, and said detecting step comprises detecting binding of said antibody or diagnostic reagent to said sample to indicate the presence of MSV in said sample.

17. A method of claim 16, wherein said detecting step comprises immunocytochemical staining of said sample.

18. 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, an antigen or antigenic fragment thereof, or a synthetic polypeptide substantially homologous with an antigen or antigenic fragment of MSV, and detecting anti-MSV antibodies bound to said MSV or antigen or antigenic fragment thereof

19. A method of claim 18, wherein the detecting antigen comprises MSV or an antigen or antigenic fragment thereof immobilised on a solid support

20. A method of claim 18 or claim 19, wherein bound anti-MSV antibodies are detected using labelled anti-human immunoglobulin antibody

21. A method of any one of claims 18 to 20, wherein said fluid sample comprises a blood sample

22. A method of any one of claims 18 to 20, wherein said fluid sample comprises a fluid sample.

23. A test kit for the detection of anti-MSV
antibodies in a fluid sample taken from a patient, characterised in that it includes MSV, an antigen or antigenic fragment thereof, or a synthetic polypeptide substantially homologous with an antigen or antigenic fragment of MSV, as detecting antigen.

24. A test kit of claim 23, wherein the detecting antigen comprises MSV or an antigen or antigenic fragment thereof immobilised on a solid support.

25. A composition for stimulating an immune response against MSV in a human or animal patient, which comprises inactivated or attenuated MSV, an antigen of MSV or antigenic fragment thereof, or a synthetic polypeptide substantially homologous with an antigen or antigenic fragment of MSV, as the active immunogen.

26. A composition of claim 25, wherein said active immunogen is coupled to a carrier molecule.

27. A composition of claim 26, wherein said carrier molecule is selected from keyhole limpet haemocyanin or another haemocyanin, bovine serum albumin or ovalbumin.

28. A composition of any one of claims 25 to 27, further comprising an adjuvant.

29. A composition of claim 28, wherein said adjuvant is selected from Freund's complete or incomplete adjuvants, or alum.

30. A method for producing an immune response against MSV in a human or animal patient, which comprises administration to said patient of an effective amount of a vaccine composition of any one of claims 25 to 29.

31. A method of treatment of MS in a patient, which comprises administering to said patient an effective amount of an antibody which recognises or binds to MSV, to an antigen of MSV, or to an antigenic fragment thereof.

32. A method of claim 31, wherein said antibody is a monoclonal antibody.

33. A method of claim 30 or claim 31, wherein said antibody is combined with a carrier or targeting molecule.