Note: Descriptions are shown in the official language in which they were submitted.
W092/0878~PCT/A~'91/~519 ~ ~
: . .
3~ : :
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~
.
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
,. .
~ .
. .
W092/~8785 PCT/AU91/~S19
3 2095539
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.
. :- ; ,............ .. .:,: . . . -
. .
:
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
... . : .- .
~" .
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
. .. ,: : : . , ,, ~
, ~ . ; ... , . . . ., . ,. : :
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
... .
.. . -. .. .:. . . ... . . . . . .. -. ... . :: .. .. :. . .. .. . ..
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
., ~ .
.
.
W092/08~8~ 2 ~ 9 ~ ~ 3 9 PCT/Augl/~sl9
11
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~'.
. . .
~ , . .
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 -~
,
...
... .,. , . ; ... ,. .. ~.. .. . ,.... .. . - . .
W092/0878~ 2 0 9 ~ ~ 3 9 PCT/AU9~/~5,9
13
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.
,:
. ' ' ' ' . " ~ , ': ` ' ' ' ' " , I': '~ ' , "., , ,' ,' ' "
" . . . ~, ... . 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
16
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
',, .
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
19
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
.. ~
.. .. . ..
~, :. . ', . ~ . ' . . , .: ' ':, , , .: ' ' ' " : . ' ' . .
W092/087~ 2 ~ 9 5 5 3 ~ /AUg11~519
22
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 .
~ ,. .
, , . - . : -.
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
24
UUWLE 8
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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