CA2121030A1 - Type c-like human retrovirus linked to multiple sclerosis (ms) - Google Patents

Abstract

The present invention relates to a type C-like human retrovirus.
In nested PCR analysis using high stringency PCR conditions and primer pairs and probes used for HIV-I or HTLV-I detection, no genomic sequences are detectable; in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 or p24 or the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind. When performed on purified retrovirus-containing material, reverse transcriptase activity is detectable. A cell culture which additionally is infected with a herpes group virus which enhances the production of the retrovirus has been deposited. Within the scope of the present invention is diagnostic agents comprising genomic fragments of the retrovirus which can be used as nucleotide probes in PCR
and diagnostic agents comprising retroviral antigens capable of binding antibodies specific for the retrovirus which can be used for the diagnosis of multiple sclerosis as well as a method for obtaining a protective immunity against multiple sclerosis comprising administering a vaccine against Epstein-Barr virus.

Description

TYPE C-LIKE HUMAN RETROYIRUS LINKED TO MULTIPLE SCLEROSIS ~MS) Multiple s~lerosis Multiple sclerosis (NS) is a neurological disease of the central nervous system in which smaller or larger ulcera-tions and/or scars can be found in the central nervoussystem. In these ulcerations and scars, nerve conduction is more or less - 50m times totally - damaged, because of d~emyelination o~ the nerve sheaths. Clinically, multiple sclerosis is highly variable and the rate of progression varies to a high degree from patient to patient (McFarlin and~McFarland, 1982).

~; The disease is associated with clinical symptoms such as sensory, visual and motor~dysfunction because of lesions in the nervvus system as mentioned above caused by the ~reak-15~ down of the myelin sheaths. The lesions can be ranging from 1 mm~to~several~;centimeters. Cl;inical diagnusis of the dis~ease can be~made by electrophysiologic evaluation, magnetic~resonanoe~(MR)~and cerebral~spinal fluid examina-tion (~CSF) ~McFarl~in~and McFarland,~1982). So far, no ZO~speaific~diagnostic test is available and the diagnosis is based~ on~clinical~and~pathologlcal criteria. The diagnosis may be~based~on c~linical symptoms a~ mentioned above~ that is~sensor~y~, V~isual~ and~motor~dysfunction. Futhermore, the diagnose may~be~based on~pathology. The documentation of 25~1esions~that have occured~on more~than one occasion and at more~than one~si~e~ and which~are~not explained by other ~, ~ mechanisms is consideredjas a definitive diagnosis (McFar-lin and McFar~land,~1982~

The~ early sta~e of the disease is manlfested by re~issions with~`complete or ;nearly complete~return of normal neurolo-gical functions,~whereas ~in the later stages of MS, neuro-logi~c dysfunctions~progress with only partly or no return of normal neurological functlons~. In the acute forms of MS
no reniissions with~complete return to normal neurological : :: : : :

~, :

W093/07259 ~ 0 3 ~ PCT/DK92/002g9

2 ,.
functions occurs and the process progresses rapidly over a few weeks or months.

In a mild form of MS also seen, only a few neurological dysfunctions followed by complete recovery is observed and the patients may remain relatively asymptomatic for years thereafter.

Finally, autopsy has indicated that subclinical forms of the disease may exist in which demyelination occurs without resulting in symptoms as those described above.

The possible invol~emant of retroviru~ and Epstein-Baxr virus i~ multiple ~clero~is ; Tropical spastic paraparesis (TSP) is a chronic progressive ~; myelopathy associated with HTLV-I infection. The disease : affects females more often than males and has many simila-rities to multiple sclerosis (M~) (Poser, l989). On the hasis of epidemiological obser~ations, it has been sug-: gested that MS is;associated with an environmental agent(Martyn, 1991). Simi1arity between MS and TSP has led to the hypothesis that a retrovirus could be involved in MS.

:20 In 1985 antibodies a~ainst human retroviruses were claimed ~ , ~
o occur more often in:MS patients than in controls (Ko-prowski et al.,~1985),~ but~ it has~not been possible to : con~irm thesé;findings:(Hauser et al., 1986). In l98g the : same group ala~imed tha;t sequences~of HTLV-I could be found :; 25 more often in MS patients than in controls (Reddy et al., 1989), but these results have not been co~firmed either (Prayoonwiwat et al., l99l) 7 : : The conc:lusion from a symposium held in Copenhagen in 1988 : on Retrovirus~in MultiPle Sclerosis and Related Diseases : 30~ was that mu1tiple:sc1erosis m1ght be associated with retro-virus and more research:should be undertaken to explore this possibil~ity (Haahr, 1992).

3 2121030 Isolation of a leptomeningeal cell line containing retro-virus from a patient with MS (Perron et al., 1989) has been reported in 1989j and in 1991 the same group has reported retroviral particles associated with positive reverse tran-scriptase activity in mononuclear cells from patients with MS (Perron et al., 1991).

~he present inventors have analyzed long-term cultures of cerebrospinal fluid cells and long-term peripheral blood mononuclear cell cultures from MS patients, patients with other neurological~diseases and hea~thy controls for growth ;; characteristics, cell morphology, reverse transcriptase and 2',5'-oligoadenylate synthetase activit~ies. None of these parameters differed between the groups (Hollsberg et al., 1989j.
, In another study ~Sommerlund et al., 1991), multinucleated giant~macrophagés~in long-term~culture~s derived from MS
patients and healthy contro1s~were compared. No differences could~be found~ Electron~microscopy (~ M) was performed on these~ cultures~,~but~no signs of retrovirus like particles 2~0 were observed~

Previously, retrovirus-1ike;~partio~les~have been foun~ in blood~ lymphocyte~cu~ltures~established~from a patient with mul~tiple~s¢lerosis~(Haahr~et al~ 1991). The cells were initially~st ~ lated with phytoba~emagglutinin (PHA) and ;; 25~cultured~with~medlum containing interleukin-2 (IL-2), and the established cell line was~an IL-2-d~pendent T-cell ~line.~These cells~can oniy~bè kept in culture for 1-3 months. Because;of;~this,~only;marginal amounts of these retrovirus-like~;partlcles~could be~produced, and further characterlzation~of~ this~putative;virus could not be per-for~ed.~

: , :

W093/07259 21~10 3 0 4 PCT/DK92/00299 EBV

Transactivation of retrovirus by EBV (Ohtsuki et al., lg90) ~ and other viruses of the herpes virus group (Skolnik et :~ al., 1988) is a well known phenomenon.
: ;~
: 5 For several years it has been known that~depending on techniques and antigens used, 99-100% of patients with MS
have antibodies against EBV, ln contrast to controls where only ~6-95% have antibodies against this virus (Sumaya et al., 1980; Bray et al., 198~3; Larsen et al., 1985; Sumaya et al., 1985).~ These studies have been confirmed by the present inventors, but it is still unknown whether EBV
infection ~is a prerequisite for development of MS or whet-her the 100% EBV seroposltivity is a:consequence of the MS
disease. : : : :

In a ~ase-control study of MS~patients, a strong positive correlation with a~previous story of infectious mononucleo-sis;~was~ found,~ dicating older~age~at 2xposure to EBV
(Operskals~i et al~.~, 1989).~In:another study, patients with inf~éGtious~mononuGl~eosis ~were~matched:with an MS registry, 20~ and~a~higher risk~for MS:to::occur:~subsequently to infec-tious~:mononucleosis:was~f~ound (Lindberg et al., 1991) If~MS~is~:cau~ed~by~an~agent~similar~to~HTLV-I, one could imagine~that~this~agènt~had:the same epidemiology as HTLV-I. Therefore~ the~intrafamilial epidemiology of MS was . ,. j - , , .,; ~ ~ .
compared with the~known~intrafamilial epidemiology of in-fections with~HTLV-I::~(Haahr~et~al., 1991~). The study did not~reveal any:~;common intrafamilial pattern of MS and HTLV-I: infections~.~.It~was concluded that if~MS is associa-3:0~ ted:with a~:~spe~i:fic~"MS v~irus"~ it is hardly one with: thesame epidemiology~a:s~HTLV~ ,:perhaps:because NS could~be a multifactorial~di~sease~only~developing if various factors : : coincide in the same~person~

W093/07259 PCT/DK92/~299 21210~0 The cause of MS is thus unknown ev~n though a wide number of possibilities including different types of retroviruses have been proposed as the cause~of MS. However, no definite proof of such an association between a retrovirus and MS
has been presented until now.

Bri~f qescription of the invention Recently, a B-lymphoblastoid cell line (LCL) of polyclonal origin was isolated~from a 30-year-old male patient with a chronic progressive myelopathy, clinically resembling multiple sclerosis as described in Example 1. The LCL
expresses Epstein-Barr virus (EBV) encoded proteins and on EM the LCL was shown to produce both EBV particles and retrovirus-like particles spontaneously. The LCL was nega-tive for human~retrovirus (HIV-1 and~HTL~-I) sequences by a 15~ polymerase chain reaction ~PCR) as~ay using high stringency PCR conditions as;described in Example 9. Furthermore, as described in Example 4, the patient was seronegative to these~retroviruses as well as HTLV-II and HIV-2. It is con-templated that~the LCL~is double-lnfected with EBV and a - 20 hi~therto uncharacterized human retrovirus. The EM pictures was~examined~by~Dr~.~Hans~Gelderblom,~who~ confirmed the observation.~The~spontaneous production~of EBV is re~ark-able~because;thi~s~usually takes place only in cell lines induced~by exogenaus~factors like halagenated pyrimidines 25~ or~infection by a seoond virus~. Thus,~human retroviruses have~been found~to induce EBV in cell lines (Lai et al., 1989)-, The cell ~line~MS-1533j is now~established and growing continuausly~in~the inventors~' laboratory, producing 0.5-1 bill~ion;cells~per~week.;Production of virus has been fol-, : ~ .
lowed~by transmissi~on~EM. Production o~ retrovirus-like particles takes place~for~a~period of at least 5-6 months after~establishln~g the;cultures~fram~frozen ampoules. Pro-duction of intact~EBV~part1cles only takes place initially, :: : ~: : : : :
.
:~
.
: - :

later on defect EBV is produced. Both viruses have been purified by a two-fold sucrose gradient ultracentrifugation as described in Example 5A. Verification of this purifica-tion is performed by negative staining ~M as described in Example 8.

Im~unoelectron microscopy gives the possibility of cap-turing virus particles so that they are seen in greater numbers than what would be seen by normal negative staining procedures where the virus solution is spread directly onto the grids. Furthermore, a more "clean" picture is seen since cell organelles or other ~mall cell parts are washed away. In the material shown in Figure 2b, the antibody was a mouse monoclonal antibody to HTLV-1 gp46 which had been shown to react with MS 1533 in Western blotting assays. The viral solution was cell supernatant double purified by sucrose gradient centrifugation.

Initially it was not possible to obtain a positive reverse transcriptase assay. However, in the purified material, the reverse transcriptase assay is clearly positive in the gradients where EM shows retrovirus-like particles as , described in Example 6. Electrophoresis on material from these gradients~has ~been performed and in material from the gradient where retrovirus is seen, one antigen has been found, this antigen reac~s with an antibody raised against an HTL~-I antigén;~(see Example 7 for details). The fact that polyclonal~HTLV-I~ antibodies detected only one of sevaral antigens clearly indicates that it is not a ques-tion of HTLV-I.
:
The synopsis o~ these~results has led to the tantalizing hypothesis that the e~lological;agent for development of MS
and M5-like diseases is a hitherto uncharacteriæed retro-: ~:
virus, but development of neurologic disease is related toor even dependent on~a delayed EBV infection. This dual in-fectlon hypothesls has been analyzed and was found to be in :.
:

.. 7 2121030 accordance with the most consistent epidemiological charac-teristics of MS (Martyn, 1991).

The poto~tial role o~ ~ytokinss as pathoge~ic factors Even if retrovirus and EBV would be found to be the etiolo-gical agents in MS, it would most probably still be neces-sary to explain the pathogenic process in ~S as an immuno-logical reaction. Here cytokines may play a~ essential ro~e.

A spontaneous higher production of IL-6 has been found in mononuclear cells from MS patients compared with cells from controls (Sommerlund et al., 1:991). A spontaneous produc-tion of IL-6, ~-IFN and TNF has been found in the cell line MS 1533 (see Example 3).

IL-6 is a cytokine which influences both T and B lympho-cytes, playing a special role for antibody production. This could explain the polyclonal B-cell activation in MS pa-tients and the high amount of antibody in cerebrospinal fluid and serum. TNF has been found toxic to oligodendro-cytes and it can cause demyelination (Selmaj et al., 1988).
Evidence of intrathecal synthesis of TNF in MS has been found, and the level of TNF in cerebrospinal fluid c~rre-: : lates with the ~everity and progr~ssion of the disease (Sharief et al., 1992).

Retro~irus :

Reverse transcripta~e :~ Reverse transcriptase:(RT), a ~rucial marker of retrovirus, :
has ~een found~in:cells~from MS patients by one group ~;`: (Perron et al., 1989~; Perron et al., 1991). The present ::~ inventors have been searching for RT activity for an ex-tended period without significant results, since only a ~; ~ little more than borderline actlvity could be detected.

With the large number of cells available from the cell line MS 1533, it has been possible to purify and concentrate supernatants to a degree where RT activity is clearly seen.
A further characterization of the reverse transcriptase and of the optimum conditions for its detection is currently being performed.

Antigen char~cterisation MS 1533 has been characterized antigenetically, using seve-;

ral polyclonal as well as monoclonaI antibodies raised ~ 10 against the various HTLV-I antigens. The antibodies were ; ~ from several different s~urces.

Using Western Blotting techniques on purified ~iral par-ticles obtained by double-gradient centrifuged super-natants, it has been found that the retrovirus is not HTLV-I; this is~described~in detail in Example 7. Two of the most prominent HTLV-I antigens~could not be identified.
On the other hand,~ a po~sitive signal has been identified as described in detail~;in Example 7~using a monoclonal anti-b~dy against~a~specific antigen, which signal cannot be caused~by~backg~ound or unspecific~binding. This distinct protein ~shows~the~expected siz~e in~mo~leculàr weight, indi-cat;ing~the~presence~ Oe a~retroviral related protein. -8eguenoe~n~ly-~5~

The fact~that~one~of~the antigens has been positi~ely iden-tified facilit~ates~the further characterization of the retro~irus at tHe nucleic acid level. Oligonucleotide primersj their~sequence either~being~ deduced from the antigen or bàsed;~on~known~retrovir~al~"consensus" sequences, is uti~lized for~PCR~analysis of~cDNA~synthesized from RNA
3~0 isolated from~the~LCL~as~described~in Example 9. The syn-thesized DNA~fragments~may~be~used bath as probes for the relevant retrovir;al~seguences ~in the LCL genome and as templates for a~;prellmlnary sequence analysis.

:: , ~ . :

g The oligonucleotides as well as the characterized PCR pro-ducts can be used as probes for the assessment of viral ex-pression levels, either by quantitakive PCR analysis or by various blotting techniques. The concomitant use of oligo-nucleotides specific for either EBV or the retrovirus willhelp elucidating the double infection and may be used as a diagnostic tool.

Antibody studies , ~

The purification and~concentration of antigens as describ~d in Example 5 make it possible to use Western Blotting tech-niques for antibody studies and to develop an ELISA for both the viruses involved in MS, thus greatly facilitating the processing ~of larger numbers of patient sera.
,, ~ ; Establisbing DeW~ Gell lines ~ ~
~: , 15 ~ Mononuclear peripheral~blood cells are~continuously being grown, a~nd new cell cultures~are being es~ablished from ; both patients with~chronic~neurological diseases and heal-thy controls in order to look for~retrovirus-like particles in the cells~by EM. The cells are grown without activating 20~ factors but with~and~without Ih-2~. In this way, it is pos-sible~to establish~both;T-cell lines~and B-lymphoblastoid cell ;lines.

` EBV

~;~ In order to further~elucidate the association of EBV with MS,~it is deslrabIe to~determine the~occurrence of IgA
antibodies in serum~;from NS~patlents~and controls. In patient~s with nasopharyngeal carcinoma, a disease which is suggested to~be associated with~EBV,;~high titers of IgA
hàve been found in~serum~ Henle et~al., 1976).

~: -2121020 10 ,,~
If possible, CSF and blood from patients with MS will be examined as well as controls for cells secreting antibodies against EBV, thereby examining whether EBV antibodies are produced within the CNS in MS. As mentioned above, oligo-nucleotides specific for EBV may also be used in order toelucidate the infection in cells with EBV, for example in plaques.

To clarify previous studies ~Operskalski et al., 1989;
Lindberg et al., 1991~ showing a higher risk for MS to occur subsequent to infectious mononucIeosis, information has been obtained about 4000 persons who showed a positive Paul-Bunnell reaction 20-25 years ago, confirming an on-going infectious mononucleosis. These 4000 persons have been searched in the Danish MS ~egistry in which informa-tion about more than 95% of all Danish MS cases are : :, :
registered. At the same time a control group of 14000persons suspected~for~having infectlous mononucleosis but showing a negative Paul-Bunnell reaction, indicating that ,. ~ , they did not have~an~acute infection with EBV at the time of examination~was searched in the Danish MS Registry.

In~the group~of ~4000 persons with a positive Paul-Bunnell (PB)~reaction, 11~persons~later developed multiple sclero-sis, whereas only ~14~ out of~the 14000 persons with a-nega-tive Paul-Bunne~ reaction later developed MS. None of the 4000 persons~with a;confirmed~infectious mononucleosis had signs of~MS~befare~this infection. This is in contrast to 9 per~sons from~the~control group, who had a diagnosed MS
befnre beeing tested with a PB reaction. The study showed a 2.75 times highër~risk of developing MS subsequent to ::: : ` :
30~ infectlous mononucleosls. ;~

Cytokines ~ ~

Studies on the production of IL-6 in MS patients and con-trols n~vivo and~ln vltro as described in Example 3 are continued The production of cytokines in LCL (MS 1533) is further evaluated, and new cell lines established is analyzed for these cytokines using quantitative PCR analysis~
. ~
Det~iled de~cription of the invention According to the present inventlon, a new retro~irus has now been found which is associated wi.th MS, and which, ;~ therefore, opens up a wide range of possibilities for reliable diagnosis of MS.;Also, the production of the retrovirus has been found to co-exist with production of '10 Epstein-Barr virus for which réason it is justified to assume that the~retroviros~is~activated by Epstein-Barr virus; this gives rise to the provision of a strategy for the development of a vaccine treatment for MS, such as will appear from the~following.~

15~ The retrovirus~of the invention is found in cells from patients sufferi;ng from MS,~including early stages,of MS.
Thus, onè cell~culture producing the~retrovirus is a lym-phoblastoid~B~cell~cu;lturè from~a~patient with a chrsnic progrèssive neurological~disease with a marked spasticity ' 20 of~;the legs. The~cell culture~wàs established from blood mononuclear ce~l~ls~without stimulation~with PHA and without ' interleukin-2 in'the~medium. The first weeks, the culture ' appeared~as a~aul~ture of~;adherent~macrophages with~few non-adherènt cells~in~the~medium.~Aftcr several weeks, small 2~5~` clones-~of~;larger~ lymphocytes~appeared in the cuIture. In the beginning,~thèse~sma;ll clones~ were adhering to the macrophages. Thls~cell~culture~is described in greater detai~l ~in~Example~ and~in the~following.

The~retrovirus~,appe~ars~ in transml~ssion~electron micro-scopy performed as~described~in Examples 2 or 8, to be a ;' type'~C-like retrovirus~,~;and;a number of tests as repor*ed in~the followlng~i~have~confirmed non-identity between the ` re~rovirus and~a number of known retroviruses.

2121~3Q

Thus, i~ one aspect, the invention relates to a cell culture comprising cells which are infected with a type C-like retro-virus which is present in human patients who have symptoms indicating an early stage of multiple sclerosis, the retro-virus being a retrovirus which can exist in the form of aspherical particIe structure with a diameter of 80-120 nm containing a core-like condensation:and without visible projections on its outer membrane when studied in transmis-sion electron microscopy at a magnification of 50,000 times`, the retrovirus showing the following negative tests:
' .~ ' ;

: A) in nested PCR analysis on the cell culture using high : stringency PCR conditions, the:following primer pairs ~nd probes used for HIV-I detection:

LSTl (SEQ ID NO~ /LST2 (SEQ ID NO:2), SK38 (SEQ ID
NO:3)/SK39 :(SEQ ID NO:4), LST3 (SEQ ID N0:5)/LST4 ~SEQ ID
NO:~), SK68;;(SEQ~ID NO:7)~/SK6:9 tSEQ ID NO:8), LST5 (SEQ
NO:9)/hST6~(SEQ:ID NO:10)~, SK29 (SEQ I~ NO:ll)/SK30 (SEQ ID NO:12)~, SKl9 (SEQ ID~NO:13;), SK70 (SEQ ID NO:14) and SK31 (SEQ:ID NO:~15) ~

20~ : and:the following primers~used for:HTLV-I detection:

HT~V-I/026~ (SEQ~ID ~NO:16)~ and~HTLV-I/029 (SEQ ID NO:17) : no~genomic~sequenc~s~are detectàble, : B) in immunofluorescence analysis, antibodies directed agai~nst HT~V~ antlgens~:pl9~:and~p24 do not bind to the - ~
25:: cell cul:tùre, ? ~: in immuno;fluorescence~analysis,~antibodies directed ayainst~the ~re~troviruses MuLV, S;S~-l (p28), FeLV or RD114 do~ not~bind to~the~cell~;culture:.

SU~315TITUlI E SHF;~

In a further aspect, the inven~ion relates to a cell culture comprising cells which are infected with a type C-like human retrovirus, the retrovirus being a retrovirus which can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studied in transmission electron microscopy at a magnification of 50,000 times, the retrovirus showing t~e following negative tests:

A) in nested PCR analysis on the cell culture using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:

LSTl (SEQ ID N0:1)/LST2 (SEQ ID N0:2), SX38 (SEQ ID
N0:3)/SK39 (SEQ ID No:a)l LST3 (SEQ ID NO:5)/~ST4 (SEQ ID
: N0:6), SK68 (SEQ ID N0:7)/SK69 (~EQ ID N0:8), LST5 (SEQ
ID NO:9)/LST6 (SEQ ID N0:10), SK29 (SEQ ID N0:11)/SK30 (SEQ ID NO:12), SKlg (SEQ ID N0:13), SK70 (SEQ ID NO:14) and SK31 (SEQ ID N0:15) and in PCR using the following primers used ~or HTLV- I
detection:

-20 HTLv r/02~6 ~SEQ ID N0:16)-and HTLV=I~9 ~SEQ-ID NO~

no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens pl9 and p24 do not bind to the cell culture~

C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV l (p28~, FeLV or RD114 do not bind to the cell culture, and the retrovirus showing the folIowing positive tests:
.

2121031~

A) in Western blotting performed on retro~irus- containing material purified by sucrose gradient purification or Triton X-114 temperature induced phase separation or purified by adsorption to antibody-conjugated microbeads, binding by the antibodies anti-rat HTLV-I gp46/gp21: 30g, le, 5a, 69b; and anti-mouse HTLV-I gp46/gp21: 46 to the ~irus envelope protein is detectable, B) in reverse transcriptase assay perfor~ed on retrovirus-containing material doubble purified on sucrose gradi-ents, revers transcriptase activity is detectable, c) in PCR analysis using low stringency PCR conditions and using the following primer pairs 477 (SEQ ID N0:18)/478 (SEQ ID N0:19), 189a ~SEQ ID
N0:20)/1899 (SEQ ID N0:21), 1900 (SEQ ID N0:22)/1901 (SEQ
ID N0:23), 1956 (SEQ ID N0:24)/1957 (SEQ ID N0:2S) and 2345 (SEQ ID N0:26)/2346 (SEQ ID N0:27), PCR products are detectable.

The above tests, which:are desribed in detail in ~he exam-.... ples, conf.irm that the retrovirus is not identical with known retroviruses.
: .
The appearance of the retrovirus particles in transmission electron microscopy appears from Fig. lc and ld. Using nega-~i tive staining electron microscopy the spherical particles have substantially the appearance as shown ~.n figures 2a an~
: 25 2b.

The retrovirus o~ the invention is identifled as a type C-like retrovirus in accordance with the description given in Dalgleish et al. 1990,: as the retrovirus possesses various morphologic features characteristic to retroviruses o~ type C. No visible projections from the outer cell membrane is ob-. served, only an ill defined central core structure is present .

-*

14~
and no identifiable intracytoplasmic precursor forms are observeid, all of these features being key features in the identification of type C viruses. Furthermore, -: , ....
`

. _ --: ~ :
`` :

:

W093/07259 PCT/~K92/00299 - 15 21210~
the size of the retrovirus being 80-120 nm is in accordance with the morphological features of a type C virus.
:, .
The cell culture is preferably one which is capable of actively producing the retrovirus, so that the retrovirus can be produced using the cell culture and then isolated, concentrated and purified, and so that antigens and nucleo-tide sequences characteristic to the retrovirus can be obtai~ed from the culture. Thereby, the cell culture ac-cording to the invention constitutes an important source of diagnostic materials and starting materials for diagnos-tic materials, such as wiIl appear from the following. It has been found that the culture of the invention actively produces the retrovirus when the cell culture is additio-nally containing a herpes group virus, in particular Ep-stein-Barr virus. It~is believed that the expression/pro-duct of the herpes~group virus, such as Epstein-Barr virus enhances the production of the retrovirus; it is well known that such an intèraction between different types of viruses can occur. Another~herpes group virus which is contemplated to~be particu~arly interesting for~enhancing the production of~the retrovirus~ lS human~;herpes virus-6 (Schonnebeck et al., l991).

The~cel~l culture~may;be a~mammalian cell culture, in parti-cular~a simian~or human cell culture.

;-25 Examples;of cells~useful or~;contemplated to be useful for establishing the~cell~culture;~are lymphoblastoid cells or ~;~ - myelomonoc~tary~cells, in particular lymphoblastoid B
cells. An interest~ing cell culture is one in which the cells are~capable of growi~ng without adherence to surfaces.

The cell culture~accordlng to tXe invention may be estab-lished on the basis of cells~from body fluidsj such as blood, or from tissu~e~samples, from patients suffering from : MS as established :by clinical or definitive diagnosis or f~rom MS-llke diseasés~such as chronic progessive myelo-WO g3/(~7259 PCr/DK92/00299 ~121030 16 pathies, said patients being infected with the retrovirus,and in particular such patients whose serum does not con-tain antibodies against other retroviruses. The cell cul-ture may be established using conventional techniques, such as described in Example l, and the presence of the retro-virus in the cell cultures is confirmed by transmission electron microscopy and/or negative staining electron microscopy and the various tests as described herein, in particular the tests described above and PCR or hybridi-zation tests as described below.

A cell culture of the invention, named MS1533, has beendeposited with ECACC, European Collection of Animal Cell Cultures, Porton Down, Sa~lisbury, Wilts, SP4 OJG, UK, on

4 October, 1991 and was given the provisional deposit num-ber V 91100401. Because of a low number of cells in theinitial deposit, cultivation of cells was made at the ECACC. A new deposit of MS1533 was made on 8 Apxil, 1992 ; and was given the provisional deposit number V 92040805-It will be understood that~whenever the provisional deposit number V 91100401 is referred to in the present specifica-tion and claims, the culture~is~derived from the same source as the cu~lture given the provisional deposit number V 92040805 and that the~latter number can therefore be substituted for the~former number~ .
25~ The cell culture~of the~invention can also be defined as a cell culture;inf:ected with the retrovirus with which said ; deposited cell cultures is infected or with a retrovirus which is identical therewlth~except.for genetic variations which are commonly found in~retroviruses and which do not ; 30 change the abové-defined properties of the retrovirus. It is well known that~all~living organlsms are subject to genetic variation~ànd~mutation in most cases without losing neither its identifylng characterlstica nor its charac-teristic functions,~and~hence, it is evident that not only 35~ the specific retrovirus~of the deposited cell culture, but also~a range;;of~funct~i~o~nally and morphologically identical retroviruses with sl~ight genetic~variation will be useful ~ 17 2121 03 0 as diagnostic material and starting material for producing diagnostic materials such as disclosed herein, for which reason the invention, when characterized on the basis of the deposited cell culture, also comprises cell cultures infected with such variations of the retrovirus, provided that the retrovirus passes the above-mentioned tests.

Another way of characterizing the range of cell cultures according to the invention is by reference to the fact that they contain genomic fragments which can also be found by PCR in blood samples from diagnosed multiple sclerosis patients, but not in blood samples from healthy persons.

~y the term "healthy person" is meant a per~on who is not infected with the retrovirus of the invention.

A further way of characterizing the range of cell cultures ~accordlng to the invention is by reference to the fact that they contain antigens capable of~binding antibodies which are~present in sera from diagnosed~multiple sclerosis patients, such antigens being antigens which do not bind antibodies presènt~in sera from~healthy persons.

; 20~The i;nvention~also~relates to~the~purified retrovirus in the~form of whole~retrovirus or fragments thereof. Such puri~f~ied retrovirus~or~fragments;~ther~of may be obtained by known~methods,~e.g.~by rupturing the cells of a cell cul-ture~as~defined~ab~ve~and~concentrating/purifying the retrovirus or fragments thereof, e.g.~by affinity chromato-~raphy, such as; antibody affinity chromatography using immobilized antibod;ies~;specific to the retrovirus. The produGtion~of such~antibodies is described in the follow-ing. Alternatively,; the retrovirus~containing material can 30 ~be purified~by the~methods descrlbed in Example 5 ~sucrose ; grad;ient purifi~ation,; Triton X-114 temperature induced phase separation or~purification~by adsorption to antibody-; conjugated microbeads). The purified retrovirus or frag-ments thereof are~useful,~e.g. as starting material for sequencing purposes, as antigenic~immunogenic material for the production of further antibodies, and as diagnostic agents or starting materials for diagnostic agents, e.g.
diagnostic agents as discussed below.

The invention also relates to an antigen or epitope derived from, produced by, or induced by the retrovirus with which the above-identified c~ll culture is infected, or derived from the above-mentioned purified retrovirus, the antigen showing the following negative tests:

B) in immuno~luorescence analysis, antibodies directed against HTLV-I antigens pl9 and p24 do not bind to the antigen or epitope, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the antigen or epitope, :
D) in immuncassays, antibodies directed against HIV-I, and HIV-II do nok bind to the antigen or epitope, the antigen or epitope being obtainable by subjecting cell fragments a:nd/or medium from a cell culture as dissussed above or purified:retrovixus as described above to ge-l electrophoresis, applying~serum from a diagnosed multiple sclerosi~ patient~to:the resulting gel and visualizing bound~antibody:~by means~of labelled anti-human antibody, comparing the visualized gel bands with a similar prepara-tion made~ using sera from a number of healthy persons, identifying the bands which are antibody-bound in the pre-paration using the serum from a~diagnosed patient and which are not bound in the prepara~ions using sera from the heal-thy persons:, is~ol:ating such bands containing the antigen or epitope from corresponding gel electrophoresis, and option-ally extracting the antigen or epitope from the bands and purifying the antigen or epitope. It will be understood that once an ldentification as described above has been :: :

performed, that is, once the bands in the gel characteristic of the antigen or epitope have been identified, further productions of the antigen or epitope can be performed simply by securing identlty with the bands first found by the use of 'the sequences of test criteria described abo~e, e.g. by ~ repeating the gel electrophor~esis under exactly the same ; ~ conditions and isolating the antigen or epitope from the bands having the position identified to be the position of the characteristic antigens or~epitopes.

In a further aspect~, the antigens or epitopes of the inven-tion further show the following positive test:

A) in Western blot~ting;performed on retrovirus- containing material purified by su~rose~gradient purification or Triton X-114 temperature induced phase separation or '15~ ~ purified by adsorption to antibody-conjugated micr'obeads, ; binding by~the antib~odies;anti-ra~ HT~V-I gp46/gp2~1: 30g, 'le, 5a, 69bj~ and anti-mouse~HTLV-I gp46/gp21: 46 to the vlrus envelope protein~is det~e~ctable.

Th~e~antigens or~epitopes of~the inventlo~, characteristic to 20~ the cell~culture~andlor~;the~retrovirus,~are very ~aluable diagnostic agents;for~thei~d~agnosis of MS i~ any i~fected stage~, ~such as~ac~ive stage or early stage or prestage or ; ' subclinic s~tage~or latent infection, all of which are de~ined -;f~below,~ which~infected~stage;~antibodies against the antigen are~prodùced in'the~patîent.~Thereby, a specific and easy diagnostic test~for~MS~ i9 made avaialable. Likewise, anti-idiotypic~antibodies~ as~déf~ined below may~be ~aluable diag- - -nostic~agent~s~for~ the~diagnosis of MS in any of the below def~ined~infective~stages~in~pat~ienti 3~0~ In connection with the;~diagnostic and other aspects of the present invent~i~on~,;the~fo1lowing explanations/definitions of the various s~tages of MS are relevant:

:: ~ ..

W093/07259 2121 ~ PCT/DK92/00299 With the term "prestagel' is meant a stage of infection with the retrovirus in a patient, in which the clinical and/or pathological symptoms have not yet developed but in which the immune system has been presented to the retrovirus and in which the retrovirus is activated ~nd will give rise to MS over an unknown period of time.

With the term "early stages of infection" is meant a stage of infection with the retrovirus in a patient, in which the retrovirus is activated and has given rise to clinical symptoms comprising sensory andtor visual and/or motor dysfunction and/or pathological symptoms comprising lesions but where remission to complete or nearly complete return of normal neurological functions occurs. In this stage, lesions of vaxiable dimensions have been observed.

With the term "dlagno~ed multiple sclerosis" is meant a stage of infection with the retrovirus in a patient, in which the clinical~symptoms comprising sensory, visual and/or motor dysfunction and pathological symptoms compris-ing lesions of the nervous system have been diagnosed, by a definitive diagnosis.

With the term "subclinic infection" is meant an infection with the retrov~irus in a patient;, in which the retrovirus gives rlse to~only~pathological signs such as demyelina-tion shown under autopsy,~but where no clinical symptoms 25 ~characteristic~to MS~can be observed or was observed.

With the term "latent infection'i is meant an infection in a patient with theiretrovirus, in which the retrovirus will not give~rise~to any cl~inlcal and/or pathological symptoms indicatlng~MS ~or an early stage~thereof unless activated, possib:le;by an inPection with a herpes group virus such as ~ Epstein-Barr virus.

;~ ~ The invention also relates ~o a;method of diagnosing multi-~ ple sclerosis or a prestage thereof, a latent infection or : :
:~:

W093/07259 PCT/DK92/~299 .. 21 21 21 0~ 0 a subclinical infection with the retrovirus of the culture ~ according to the invention expressing the antibody as : defined above, comprising contacting a sample of body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent comprising an antigen or epitope of the invention or an anti-idiotypic antibody:as defined below, and determin-: ing the presence of any ant~ibody from the sample bound to the antigen, epitope~,~or anti-idiotypic antibody.

;10 The diagnostic test may be~performed using a diagnostic agent which compris~es~an~:antigen or epitope of the inven-' tion or an anti~-idiotypic antibody as described below, bound to a carr~ier~or~support~as~described below. Any an-tibodies from:the sample binding to the antigen, epitope, or anti-idiotypic:~antibody~may be~:detected using a secon-dary antibody~capable~:of binding to the first bound anti-:body~and providedi~with-~a:label;~as:~described below.

The~: ubstance:~used;as~:~labe~l~may~:~be~selected from;any sub-stance~:which~i's~in~itself detectable or which may be react-2~0~ ed~with~another.~substance~to~produGe;:a' detectable product.Thus,.~the label~may~be:selécted~ from:radioactive isotopes, ènzymes,~chromophorè~s~ fluor:escent~:~or~chemiluminescent bstances, and~complex-ng~agene~

Exampl;es~of~ enz ~es~useful~as:~labels are ~-galactosidase, 2S~ .:urease,~ gluc ~ ~'oxida~se,~carbonic~;anhydrase, peroxidases e~ g.~horseradish~peroxidase~ 'p~hosphatases (e.g.:alkaline or acid phosphatase)~ glucose-6-phosphate dehydrogenase and ribonuclease.~

EnZymes~are~not~in~:themse~lves~detectable, but must be 3~ combined with::~a~substrate to:~catalyze a reaction the end product of~which~ s~:detectab`le~.:Thus, a substrate may be added~to~the reaction~mixture~résulting in a coloured, fluorescent or~:chemil:uminescent product or in a colour :change~or 1n~a;~ch:ânge ln~the intensity of the colour, 2121030 22 ~-~
fluorescence or chemiluminescence. Examples of substrates which are useful in the present method as substrates for the enzymes mentioned above are H202, p nitrophenylphos-phate, lactose, urea, ~-D-glucose, C02, RNA, starch, or malate. The substrate may be combined with, e.g. a chromo-phore which is either a donor or acceptor.

Fluorescent substances which may be used as labels for the detection of the components as used according to the of invention may be 4-methylumbelliferyl-phosphate, 4-methyl-umbelliferyl-D-galactopyranoside, and 3-(p-hydroxyphenyl) propionic acid. These substances may be detected by means of a fluorescence spectrophotometer. Chemiluminescent ~ substances which may be peroxidase/eosin/EDTA, isoluminol/-; EDTA/H202 and à substrate therefor.
Chromophores may be o-phenylenediamine or similar com-pounds. These substances may be detected by means of a spectrophotometer~

Radioactive isotopes~may b~ any detectable and in a labora-2Q tory~acceptable~isotope, e.g.~125I 131I 3H 32P 35S
4C. The radioactivity may~be measured in a ~-counter or a scintillation counter or by radioautography followed by dens~tometry~

Complexing~agents~may~be;~Protein~A, Protein G (which forms 25~a~complex~with~immunoglQbulins),~biotin ~which forms a complex~with~avidin~i~;and streptavidin), and lectin (which forms a complex with~carbohydrate determinants, e.g. recep-tors). In this aase, the complex is not in itself directly ; detectable,~necessltating labelling of the substance with which the complexing~agent~forms a complex. The marking may be~performed with~any~of~the labelling substances described above.

~s~indlcated above, this type of diagnostic agent normally comprlses the ant~igen or epitope or the anti-idiotypic : ~ : : : :

~ .

antibody bound to a carrier or support so that appropriate washing or other treatments as appropriate may be performed wi~hout risk of appreciable loss of the bound antigen, epitope, or anti idiotypic antibody. The carrier or support is normally solid, and the antigen, epitope or anti-idioty-pic antibody is bound to the carrier or support by any suitable mode of binding, such as hydrogen bonding, hydro-phobic bonding, van der Waals' forces, covalent bonding, etc.

In an embodiment of the invention, the antigen, epitope or anti-idiotypic antibody of the invention may be indirectly coupled to a solid support via a bridging compound or "linker'i. The linker, which is designed to link the solid support and the antigen, the epitope may be hydrazide, Protein A, glutaraldehydé, carbodiimide, or lysine.

The solid support employed is e.g. a polymer or it may be a matrix coated wlth~a polymer. The matrix may be of any sui-table solid material, e.g. glass, paper or plastic. The polymer may be a plastic, cellulose such as specially trea-20 ted paper, nitroc~ellulose paper or cyanogenbromide-acti~a-ted paper, silicone; silica, or a polysaccharide such as agarose or dextran. Examples of suitable plastics are latex, a polystyrene,~polyvinylchloride, polyurethane;
polyacrylamide, polyvinylacetate and any suitable copolymer ;25 thereof~. Examples~o~f silicone polymers include siloxane.

The solid support~may~be~in the form of a tray, a plate such as a mitrotiter~plate, e.g. a thin layer or, preferab-ly, strip, film, threads~, solid particles such as beads, including Proteln A-coated bacterla, or paper.

The invention also relate~to a monoclonal or polyclonal antibody which binds to an antigen or epitope as defined above and also to an anti-idiotypic antibody as described below. A most useful type of~antibody is a monoclonal antibody; however, also a polyclonal antibody may be of W093/07259 212 ~ 0 3 24 PCT/DK92/00299 great importance provided it shows a sufficient selectivi-ty, which may be obtained, e.g., by means of known absorp-tion methods.

The term "antibody" refers to a substance which is produced by ~ mammal or more precisely a cell of mammalian origin belonging to the immune system as a response to exposure to the polypeptides of the invention.

The variant domain of an antibody is composed of variable and constant sequences. The variant part of the domain is called the idiotype of the antibody. This part of the antibody is responsible for the interaction with the anti-gen, the antigen binding.

The idlotypic structure is antigenic and can thus give rise to specific antibodies directed against the idiotypic structure. Production of such anti-idiotypic antibody has been done in mice. The antibodies raised against the idio type, the anti-idiotypic antibodies, may mimic the struc-ture of the original antigen and there~ore may function as the original antigen to raise antibodies reactive with the original antigen. This approach may be ~dv~ntageous as it circumvents the problem associated with the characteriza-tion and synthes;is~of the important immunogenic parts-of the antigen in question. Thi is most important in the case of conformational epitopes,~which might otherwise be dif-~icult to;identify. The present inventisn therefore also :: ~
relates to an anti-idiotypic antibody which is directed against the site of an antibody which binds the antigen or the epitope according to the invention.

The antibodies of the present invention may be produced by a method which comprises administering in an immunogenic form at least a part of the antigen or epitope of the invention or an anti-idiotypic antibody as defined above to obtain cells producing antibodies reactive with said poly~
peptide and isolating the antibody containing material ~:

W093/07259 PCT/DK92/002g9 from the organism or the cells. The methods of producing antibodies of the invention will be explained further below.

In a further aspect, the invention relates to a diagnostic agent which comprises an antibody as defined above, prefe~
rably a monoclonal antibody. The diagnostic agent may comprise the antibody coupled to a carrier or support.
Alternatively, the diagnostic agent may be in the form of a test kit comprising in a container an antibody as defined above. The diagnostic agent may be used in the diagnosis of ; MS or a prestage thereof, a latent infection or a subclini-cal inection with the retrovirus of the culture according to the invention expressing the antigen or epitope of the invention, comprising contacting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis pat;ient with a diagnostic agent compris-` ing an antibody as defined above, and determining the presence of any antigen or epitope from the sample binding ~; ~ to the antibody.

The diagnostic agent may be one which is suited for use inan~agglutination~assay in which solid particles to which the antibody is coupled agglutinate in the presence of an antigen or~an~eplt~pe of~the invention in the sample~sub-jected to testing.~In this type of testing, no labelling of antibody is~necessary. For mos~ uses it i~, however, preferred that~the antibody;is bound to a carrier or sup-port, using,~e.g~., t he techniques described above in con-nection with dia~nostic agents based on an an~igen, epi-tope, or anti-idiotypic antibody, and tha~ the binding of antigen or epitope from the sample to the diagnostic agent ; lS detected using a secondary~an~ibody which is capable of binding to the thus bound~antigen or epitope, the second antibody being provided with~a label for the detection of bound secondary antibody. The~substance use~ as label may be selected ~rom~any substance which is in itself detec-table or whlch may be reacted with another substance to produce a detectable product. Thus, the label may be selec-ted from radioactive isotopes, enzymes, chromophores, fluorescent or chemiluminescent substances, and complexing agents, all of which are described in greater detail above.

The antibody of the invention may be used in an assay for the identification and/or quantification of at least a form and/or a part of the antigen or epitope of the invention present in a sample. The identification and/or quantifica-tion performed by the use according to the present inven-tion may be any identification and/or quantification in-volving the antigen or epitope of the invention. The iden-tification and/or quantification may be performed for both a scientific, a clinical and an industrial purpose. As will be further described below, it is especially important in clinical routine to identify or quantify antigens or epi-topes of the invention.

~: :
Furthermore, the antigen or epitope or anti-idiotypic antibody may be~used in an assay for the identification and/or quantification of antlbodies reactive with the 20~ antigen or epitope of the invention and being present in a sample, e.g. as defined above. This assay may be carried out by use~of a method comprising~contacting the sample with the antigen or~epit~ope of~the invention and detecting the~presence of~;bound~a~ntibody resuIting from said contact-ing and correlating~the result with a reference value.

~ In one preferred embodiment~of the invention it is prefer-; red that the antibody used in the method of the invention . , , is a monoclonal antibody as this generally provides a high-er precision~and accuracy of~the;~assay, at the same time possibly requiring less time to~perform. Furthermore, a mixture of two or more different monoclonal antibodies may ; be employed as~this may~increase the detection limit and sensiti~ity~of the test. The mon;oclonal antibody may be obtained by the method described below. Antibodies possess-~ ~ .

W093/07259 PCT/DK92tO0299 .. 27 21 21 03 D
ing high avidity such as poloclonal antibodies may be selected for catching techniques.

The antibody used in the present method is preferably in substantially pure form (purified according to suitab1e

5 techniques or by the methods of the invention, see below) in order to improve the precision and/or accuracy of the assays of the invention.

Another field of the invention is a method for producing an antibody which binds to the antigen or the epitope of the invention, which comprises immunizing an animal with the antigen or epi~ope or an anti-idiotypic antibody or an antigen or epitope produced by cultivating cells harbouring a p1asmid which contains and is capable of expressing a nucleotide sequence as described below which codes for a polypeptide which has the properties of the antigen or epitope as described above, or by synthetically producing a poIypeptide having an amino acid sequence derived from the nucleotide sequence of:the retrovirus of the invention, whereby cells producing an antibody specific for the anti-gen is obtained~and the antibody is isolated from theanimal or the ¢ells~. :

The antibody is preferably a monospecific antibody. The monospecifi:c antibo~y~may:be prepared by injecting a sui-table:animal with~a~substantlally pure preparation of the po1~ypeptide~of the~invention followed b~l one or more boos-ter injections:at~:suitable intervals ~e.g. one or two weeks to a month) up o four or~five months before the first bleeding. The established immunization schedule is continu-ed, and the animals are bled about one week after each :: , ~ ::: 30 booster immunization, and antibody is iso1ated from the ~ ~:
~; ~ serum in a suitable manner (cf. e.g. Harboe and Ingi1d, 1973~.

For purposes not requiring a high assay specificity, the antibody may be a polyclonal antibody. Polyclonal anti-.

W093/072~9 PCT/DK92/00299 21210~ 28 ..
bodies may be obtained, e.g. as described in Harboe andIngild, see above. More specifically, when polyclonal antibodies are to be obtained, the compound comprising an antigen or epitope of the invention or an anti-idiotype antibody as described above is prepared and preferably after addition of a suitable adjuvant, such as Freund's incomplete or complete adjuvant, injected into an animal.
The animals are bled regularly, for instance at weekly intervals, and the blood obtained is separated into an antibody containing serum fraction, and optionally said fraction is subjected to further conventional procedures ~: for antibody purification, and/or procedures involving use of purified compounds comprising an antigen or epitope of the invention or idio-typic antibody as described above.

In another preferred embodiment, monoclonal antibodies are obtained. The monocIonal antibody may be raised against or . ~ directed substantlally against an antigen or epitope of the : : invention as described above or an anti-idiotypic antibody ~:: as described above. The monoclonal antibody may be produced by conventional techniques (e.g. as described by Kohler and Milstein, 1975~, e.g.~ by use of a:h~bridoma cell line, or by clones or subclones thereof or by cells carrying genetic information from the hybridoma cell line coding for said monoclonal antibody. The monoclonal antibody may be pro-: 25 duced by fus:ing cells producing th~e monoc~nal antibody ` with cells of a suitable cell line, and selecting and clo-ning he resulti:ng~hybridoma cells producing said monoclo-nal antibody.:Alternatively, the:monoclonal antibody may be produced by immortalizing an unfused cell line producing said monoclonal antibody, subsequently growing the cells in : a suitable medium to produce said antibody, and harvesting :: the monoclonal~ antibody f`rom the growth medium.
~ ::: ` :
:: ~ The immunized animal used for the preparation of antibodies ~ , :: : of the invention is preferably selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, ~: horse and guinea pigs. The cells producing the antibodies ':

of the invention may be spleen cells, lymph cells or peri-pheric lymphocytes.

When hybridoma cells are used in the production of anti-bodies of the invention, these may be grown in vitro or in a body cavity of an animal. The antibody-producing cell is injected into an animal such as a mouse resulting in the formation of an ascites tumour which releases high con-centrations of the anti~ody in the ascites of the animal.
Although the animals will also produce normal antibodies, these will only amount to a minor percentage of the mono-clonal antibodies which may be purified from ascites by standard purification procedures such as centrifugation, filtration, precipitation, chromatography or a combination thereof.

An example of a suitable manner in which the monoclonal : : antîbody may be produced is as a result of fusing spleen ; cells from immunized mice (such as Balb/c mice) with mye-loma cells using aonventional techniques (e.g. as described by Dalchau et:al. 1980).~The fusions obtained are screened by aonventional techniques~such as binding assays employing compounds comprising antigen or epitope o~ the invention or an anti-idiotypic antibody as::described above isolated by ; the above-described:methods.

: The invention also re~lates to a nucleic acid having a nucleotide~ sequence which is cha~acteristic to the above-identified~ retrovirus. In other words, the nu~leotide ~; sequence ls one which is distinct from sequences from known retroviruses, and at the same time is indicative of the presence of th~e above-identified:new retrovirus. Such : 30 nucleic acids are nucleotide sequences which, when used as a probe on samples of~body fluid such as blood samples or tissue samples from`a number of healthy persons and blood samples or tissue~samples from diagnosed multiple sclero-~; ~ sis patienes, respectively, :detects~nucleotide sequences : ~ ' .

.

W093/07259 PCT/DK92/0029g 2 1 2 1 0 3 ~ 3~ b5 from diagnosed multiple sclerosis patients, which can notbe detected in samples from the healthy persons.

In other words, the nucleotide sequences serve as a speci~
fic marker of the retroviru~ of the invention. The nucleo-tide sequences can be obtained by isolating nucleotide sequences derived from the cell cult~re described above or the purified retrovirus or fragments thereof as described above by the use of retrovirus-specific nucleotide primers recognizing specific regions of the nucleotide sequences from the above-described retrovirus. The virus~specific nucleotide primers ~which also constitute an aspect of the invention) can be developed using on the one hand a cell culture according to the invention and on the other hand primers which are not specific to the present retrovirus, but which contain nucleotide sequences which will recognize retrovirus generally, such ~s known conserved re~ions of nucleotide sequences from various retroviruses, using the following strategy: The non-specific, but generally retro-virus-recognizing primers can be used to obtain nuclsotide sequences derived ~rom the cell culture according to the invention or ~he purified r~etrovirus according to the inven ion by PCR. In the next stage, the nucleotide sequen-ces thus obtained can be sequenced to identify regions thereof which are distinct from~sequences from known retro-` 25 viruses, and these distinct sequences can then be tested byusing them as primers on samples from a number of healthy persons and ~amples from diagnosed MS patients, respective-ly, and~selecting the sequences which give rise to the ~ttainment of oligonucleotide.sequences from diagnosed MS
patients, but do; not give rise to attainment of oligo nucleotide se~uences from the healthy persons. Evidently, the sequencing could in prlnciple be excluded, but this would make more testing on samples necessary.

In a specific embodiment, the invention relates to a nu-cleic acid having a nucleotide sequence ~S) obtainable by using a retrovirus-related nucleotide primer recognizing , W O 93/07259 PC~r/DK92/00299 31 21 21 0 ~ Q

tide sequences derived from the cell culture according to the invention or, alternatively, the purified retrovirus according to the invention by PCR, optionally sequencing the nucleic acids obtained to identify sequences which are distinct from sequences from known retroviruses, testing the nucleic acids obtained by PCR or the se~uences iden-tified by sequencing by using them as primers on blood samples fr~m a number of healthy persons and blood samples from diagnosed multiple sclerosis patients, respectively, and selecting, as: the nucleotide sequence (S), the nucleic : ~ acids or sequences which give rise to the attainment of nucleotide sequences from diagnosed multiple sclerosis : ~ patients in PCR, but:do not give rise to attainment of .lS nucleotide sequences from healthy persons, or using such :~ nucleic acids or sequences as primers for an additional PCR
obtainment:of nucleic acids from the cell culture according :to the invention:,~performing the testing defined above using such second~generation:nucleic acids or sequences identified therein as primers and~select~ng, as the nucleo-:tide sequence (5)~,:the~nuc~leic~acids or sequences which gi~ve rise to the;attainme:nt of nucleotide sequences from diagnosed multiple sclerosis patients in PCR, but do not ; give rise:to atta~inmen~t: of~nucleotide sequences from heal-25:; thy~persons. ~

By the term "nucleotide:sequences" is meant any nucleotidesequences of various~le~ngth:,;~pref:erably an oligonucleotide sequence. The;~:nucleotide~sequence may be either a~RNA
nucleotide sequence or a:DNA nucleotide sequence.

In a specific embodiment,~the invention relates to a diag-nostic:agent comprislng~a nucleotide probe which is capable of~ detect1ng a~nucle~otide~sequence according to the inven-:` The lnvention also rel:ates to a nucleotide prob~ which is capable of detecting a nucleotide sequence as defined ~::

W093/072~9 2~ 3 0 PCT/DK92/00299 above. Both the nucleotide sequence and the nucleotideprobe are valuable ~or use in dia~nostic agents, such as PCR kits, for diagnosing ~S.

Whereever used, the te~m l'probe" describes any nucleotide sequence, preferably an oligonucleotide sequence, which may be used to obtain nucleotlde sequences complementary to the probe, e.g. nucleotide sequences suitable for use as pri-mers in PCR or for use as probes in hybridization techni-ques.

The substance used to label the probe may be selected from any substance which is in itself detectable or which may be reacted with another substance to produce a detectable product. Thus, the label may b selected from radioactive isotopes, enzymes, chromophores, fluorescent or chemilumin-escent substances, and complexing agents.

Examples of enzymes useful as labels are ~-galactosidase, urease, glucose oxidase, carbonic anhydrase, peroxidases e.g. horseradish peroxidase), phosphatases (e.g. alkaline or acid phosphatase~, glucose-6-phosphate dehydrogenase and ribonuclQase. ~ ~

Radioactive isotopes~may be any detectable and in a labora-tory~acceptable isotope, e~.g. 125I 131I 3H 32P 35S or 4C~. The radioactiv~ty may~be measured in a ~counter or a scintillation~counter or by radioautography followed by densitometr~y.~

, ~ , Examples of detection systems based on enzymes are the DIG-system (digoxygenin;~ Boehringer) and the Tropix-system (Stratagene). Complexing agents~used in the detection may be biotin (which forms a;complex w~th avidin and strep-tavidin~.~In this case, the complex is not in itself di-rectly detectable, necessltating labelling of the substance with which the complexing agent forms a complex. The mark-.
' . ~

ing may be performed with any of t~el ~Q~Qng substancesdescribed above.

As indicated above, this type of diagnostic agent normally comprises the labelled probe, which reacts with or detects the presence of complementary sequences in a sample of a body fluid, such as a blood sample, or a tissue sample.
Either probe or sample may be bound to a carrier or sup-port, so that appropriate treatments may be performed without risk of appreciable loss of the bound material. The carrier or support is normally solid, and the probe or ~; ~ sample is bound to the carrier or ~support by any suitable method of binding, such as hydrogen bonding, van der Waal's forces or covalent binding.
' :
In an embodiment of the invention, the labelled probe or sample may be indirectly coupled to a solid support via a bridging compound or a linker. The linker, designed to link the solid~support and the labelled probe or sample, may be streptavidin. ~

The solid support;~employed is e.g.;a polymer or it may be a 20~ matrix~coated~with polymer. The matrix may be of any suita-b}e solid material,~e~.g.~glass~, paper~or plastic. The polymer may be~a nylon or nitrocellulose. Examples of suitable~plasti~s~are~polystyrene ~or polyvinylchloride~ The solid support may be;in~the;for~m of a tube, a thin layer or ;strlp, threads,~sclid partlcles such as beads or paper.

Thus, the invention also relates to a method of diagnosing ;~ multipl~e sclerosis~,~an early;stage or a pre-stage thereof, a latent~infection or~a~subclinlcal infection with the retro~irus of~the~culture~defined~above, the method com prising subjecting~a~sampl~e of a body fluid, such as a blood sample, or a t;lssue~sample~from a suspected multiple sclerosis patient~to~a PCR analysis in which the sample is contacted with~a~dlagnostlc~agent comprising the nucleotide probe or the nucleotide sequence defined above, and any W~93/07259 PCT/DK92/00299 2121030 34 ,~
detected nucleotide sequence is allowed to be amplified, followed by detection of any amplified target nucleotide sequence. The PC~ analysis is a well-established technique described, e.g., in Sambrook, 1990.

Alternative diagnostic methods can be be hybridization as described in s~andard textbooks in the field. These can be performed e.g. as described in EP 326 395 which describes a method of detecting and identifying a human retrovirus in a human blood or tissue sample comprising (a) amplifying a portion of the human retrovirus env or consensus RNA pre-sent in the sample.

In a further aspect, the invention relates to a method of diagnosing multiple sclerosis, an early stage or a pre-stage thereof, a latent infection or a subclinical infec-: : 15 tion with the retrovirus of the culture according to the : invention comprising subjecting a sample of a body fluid, : :: such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient to a PCR analysis in which the s:ample is contacted with a diagnostic agent according to the invention~allowing any nucleotide sequence to be ampli-` fied followed by detection of any amplified target nucleot-ide sequence. ~ : ~

In another aspect, the invention relates to a method of in vitro diagnosing mul:tiple sclerosis, an early stage or a 2:5 pre-stage thereof, a latent infection or a subclinical infection~with the retrovlrus of the culture according to the invention, optionally combined with a method for detec-tion of an infection with another virus such as a herpes : group virus, comprising contacting a sample of a body fluid, such as a blood sample, or a tissue sample from a : suspected multiple sclerosis patient with a diagnostic agent according to the invention comprising a nucleic acid : accordlng to the invention and determining the presence of any identical or homologous nucleotide sequences in the sample.

. .

212103~
In another aspect, the invention relates to a method of in vitro diagnosing multiple sclerosis, an early stage or a prestage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to the invention comprising contacting a sample of body fluid, such as a blood sample, or a tissue sample from a suspected : multiple sclerosis patient with a diagnostic agent accor-ding to the invention and determining the presence of bound antibody from the sample.
:: :
1~ In yet another aspect, the invention relates to a method of in vitro diagnosing multiple sclerosis or a prestage there-~:: of, a latent infection or a~subclinical infection with the retrovirus of the culture according to the invention com-prising contacting a sample of a body fluid, such as a 15~ blood sample, or a tissue sample from a suspected multiple .: ~
sclerosis patient with a diagnostic agent according to the invention and:~determining the presence of baund antigen from the sample. ~ ~

An~important embodiment~ of~the~ invention relates to a 2~0~ method~for~producing an:antigen or~epitope characteristic to~the retrovlrus,~ whlch comprises~;cultivating cells har-bouring a plasmid~whlch contains and is capable of express-ing a nucleotide~sequence which codes ~or a polypeptide which;has~the~pr~opertles~of;the antigen or epitope of the 2:5~ invention.~ ~n~:alternative:method for producing an antigen or epitope characteristic to~the~ retroviru~, comprises `~: synthesizing a peptide having an~amino acid sequence deri-ved from the nucle~tide sequence a~ccording to the inven-The term "targ~t:nucleo~ide~sequence" describes any nucleo-tide sequence~ as~def~lned above, preferably an oligonucleo-: tide sequence:, wh~ich contains a nucleotide sequence comple-~mentary to~the probe`used in the various assays such as PCR or hybridizat~ion.

:

W093/07259 PCr/DK92/00299 In a further aspect, the invention relates to a method for obtaining a protective immunity in an animal, including a human beiny, against multiple sclerosis caused by the retrovirus of the culture according to the invention, comprising administering, to the animal, an immunogenically effective amount of a vaccine against a herpes group virus such as Epstein-Barr virus, thereby preventing the previ-ously mentioned interaction between a herpes group virus such as Epstein-Barr virus and the retrovirus of the inven-tion which interaction is assumed to result in the activa-tion of the retrovirus.
;

The vaccine should be made so as to allow an optimal stimu-lation of the relevant parts of the immune system, i.e to present the immunogenic agent for a period of time and in a form being optim~l with respect to the recognization, the :~ uptake or any other interaction or processing necessary for the s~imulation.

The term "vaccinel' is to be understood to comprise any preparation con~aining an immunologically effective part of : 20 a herpes group virus, eOg. an antigen or epitope of the : herpes group virus suited for administration to living organisms for the prevention of MS by providing an animal, such as a human, with a~protectiv ~ immunity against a ~: herpes group virus such as an Epst~ln-Barr virus.
: ~ 25 The invention also relates~ to a me~hod for obtaining a protective immunity in an animal, including a human being, against multiple sclerosis caused by the retrovirus of the invention, comprising administering, to the animal, an : immunogenicall~ effective amount of a vaccine comprising ~ 3Q any preparation containing an lmmunologically effective : part of a cell culture~infected:with the retrovirus of the invention, or purified retrovirus or fragments thereof all : of which are defined above, e.g. an antigen or epitope of the invention, or comprising an anti-idiotypic antibody of .

W093/0725~ PCT/DK92/00299 the invention suited for a~minis~r~Q ~Q living organlsms f~r the prevention of MS so as to prove the animal, such as a human, with a protective immunity against the retrovirus according to the invention.

The vaccines described above may be used seperately or the two vaccines may be used in combination.

The term "immunization" is understood to comprise the process of evoking a specific immunologic response with the expectation that this will result in humoral, and/or secre-tory, and/or cell-mediated immunity to a herpes group virus such as Epstein-Barr virus or to the retrovirus of the in~ention, i.e. immunity is to be understood to comprise the ability of the individual to resist or overcome infec-tion or to overcome infection "more easily" compared to in-: 15 di~iduals who have not been immunized or to tolerate theinfection without being clinically affected or to block transmission. Thus, in~one aspect the immunization accord-ing to the present invention is a process of increasing resistance to infection with a herpes group virus such as : 20 Epstein-Barr virus, thereby preventing such a herpes group virus from activating any retrovirus according to the in-vention present in th~e: same i ndividual. An overall aspect : in the preparat~ion of:~the vaccines of the inven~ion is the physi~logical~acceptability of the componen~s and of the :~ 25 total composition of the vaccine. The final formulation of ~: the vaccine should:be;a mixture~of substances supporting and enhancing~the immune response induced by the specific immunogenic component. In another aspect, the immunization according to the present invention is a process of increas-: 30 ing resistance to infection with a retrovirus of the inven-tion.~

~: By using the above-mentioned vaccine against a herpes group virus such as a Epstein-Barr virus and vaccine against the ~: retrovirus of the invention i:n combination, the effect of the immunization may be enhanced.
.

.38 21~1031) In a further aspect, the invention relates to a method as explained above, in~which the vaccine is a live or dead Epstein-Barr virus administered to the mammal at a stage in ~ the development of the~:mamma~l in:~which the disease caused :~ 5 by Epstein-Barr virus~has a mild clinical cause and cannot induce active:production~of the retrovirus of the invention to result in multiple~;sclerosis, an:early stage or a pre-stage thereof. The~vaccine may:be administered to a mammal, preferably a human,:and~the~stage in which the administra-lO .~tion is~performed is the pre-puberty stage. The mammal, : such as:a huma:n,~:to~which the:~vaccine is administered may be~mammal which~has~been shown t~o carry~the retrovirus of the :culture as-de~fined above~

The vaccine may be:~an~attenuated Epstein-Barr virus or an S~: immunogenic antigen~:~ch~aracteristic;to Epste~in-Barr virus and eliciting:~the~formatio~n of:~antibodies against Epstein-Barr virus. : : ~

The-~.administration of~ the~vaccinè~against the retrovirus of the~i ntion;t ~a~m m al,~:such~as~a~hum 2~b~ ~formed;~at~àny~s ~ge~in~the~;development of the mammal.
Preferably,~ the~vaccine~against the~retrovirus from the .cùlture:~:~of~:the~ ~ ntion~may~be~administered to a~mam~al which~has~been shown~to~carry~the~retrovirus:of the culture as;defined~ above~or~.which:; s~-be ~ h ~ n to carry~a her es 25~.group:virus:such~:as:.~an-~Epstein-Barr virus.

W093/07259 PCT/DKs2/002g9 ,- ....
39 2121031~
Lege~a to figures Figure la.

An Epstein-Barr virus particle with a diameter of 150 nm is seen outside the cell membrane of a B-cell (EBV), i.e. a core surrounded by an icosahedral capsid measuring maximal-ly lOOnm in diameter and an outer irregularly shaped en-velop~ with projections.

Figure lb.

~ Eppstein-Barr viral capsids are seen in the nucleus : 10 (EBV.VC). The central nucleic acid:of the virion is sur-rounded by an icosahedral capsid (Crawford and Edwards, 1990) -Figures lc and 1~

Retrovirus-like particles located just outsi~e the cell ~ 15 memb~ane measuring 90-100 nm:in diameter (RVLP). The par-: ;: ticl s have a core-like condensation and the envelope does : not contain visible projec~ions. Note ~he variable size of :the~particles~and the diffuse spherical cores`as those illustrated in;a~textbook~on human retroviruses (Dal~leish 20~ and~Weiss, 19~0). :~

~ ~ ~ : Figure Za.~

.~ Viral particles together with a mixture of cell organelles : and other small "cell-parts" are seen. The material is a ::
pelleted fraction: o f ;a~sucrose gradient centrifuged as described in Example~:5.~After resuspension of the pellet, the~material is spread on grids~and stained as described in Example~8. . ;~ : ;
. :: , ~ ::: : : : : :
: .

:' :

; , .

W093/07259 ~ ~ i Q 3 ~ PCT/DK92/00~99 Figure 2b~ .

Viral particles captured by monoclonal anti~odies to gp46 are seen. The material is prepared as described above in Figure 2a. Small cellular fragments have been washed away as described in Example 8. Finally, the material is stai-ned.

:
, ' : `: ` : :

:

.

::::

.. ~
41 2~2103Q

E~A~P~E 1 Cultivatio~ of the lymphobl~toid cell culture The lymphoblastoid B-cell line was established as follows.
Heparinized blood was diluted in phosphate-buffered saline PBS (pH 7.4, ~0 i.u./ml heparin), before separating the mononuclear cells by Ficol Isopaque density gradient ren-trifugation. The mononuclear cells were harvested and wash-ed twice in cold PBS. Finally the mononuclear cells were seeded at a density of 20x106/5 ml and grown in RPMI 1640 (Seromed) supplemented with 200 i.u./ml penicillin (Leo), 0.2 mg/ml streptomycin (Rosco), 0.29 mg/ml glutamine (Sig-ma), 0.01 M hepes buffer (Bioproduct), 5% human heat inac-tivated sera in Falcon Primaria bottles. During the first 7 weeks of cultivation, the serum percentage was 5%. After appearances of clones in the culture, the medium contained ; 10% of human serum.

: In the lag period before:appearance of the clones in the : cell culture, the morphology of the cells was studied. The non-adherent cells became fewer and small. The adherent : 20 cells which were initially small and round became increas-~:~:: in~Iy larger wi~h ~ormation of:multinucleated giant cells.
After:6 weeks:~of culture, few large:~multinucleated gi~nt cells with 12 to 17 nuclei were present and small clones of B-celIs were attached~to these cells. By the 7th week of culture, ~he B-cell clones were more numerous and were also - :
seen in the culture without attachment to adherent cells.
After 7 weeks of culture, the B-cell clones were suhcul-tured in medium containing different percentages of human serum. I~t wa~ found to be esse~ntial for survival of the culture that the~cell density was initially not below 0.5x106 cells/mI, and only half~of the medium was changed when subcultured.~Furthermore, it~was found that medium containing 10% of human serum supported the growth of the ~; cell l:ine ~etter ln long-term culture than a similar medium containing fetal calf serum. The cells were subcultured , -' .

.

three times a week with a split ratio of 1:3 or 1:4 with a cell density of 0.5x105 cells/ml.

The cells were capable of growing in clumps or singly with-out adherence to the surface o~ the container in which the cultivation took place. The population doubling time was initially 17 hours but rose to 26 hours after 5 months of culture. The maximum cell density obtainable was around 2X106 cells/ml and a poor cell proliferation was observed ~at cell densities below 0.35x106 cells/ml.

: 10 ~XANPLE 2 Examination of the lymphobla~toid cell culture using tran~-miY~ion electron micro copy Naterials a~d methoas :
~:~ Fixation :~ 15 2X106 cells were flxed:with cold ~.5% glutaraldehyde buf-fered at pH 7.2 with 0.1 M sodium cacodylate for at least l ;~ hour. The cel~ls were;then pelleted by centrifug~tion at ::~ : 1500 rpm for:5 minutes. The pellet was washed 3 times in cacodylate buff:e~,:pH~7.2 and then postfixed for 1 hour in 1% osmium~tetroxide buffered~at pH 7.2 with veronal acetate buffer and:then wàshed:3:times in veronal acetate buffer.
~:~ Between these washing steps, pelleting was carried out at 1500 rpm for 5 minutes.

.
:~ Block staiDi~g -2X106 cells were~washed 3 times with maleate buffer, pH
5.2; and stained:~for~l hour in 0.05 M maleate buffer + 0.5%
~: uranyl acetate~ pH:6Ø They were then washed 3 times with : maleate buffer, pH 5.2. Between these washing steps, pel-: leting was carried out at 1500 rpm for 5 minutes.

W093/07259 PCTtDK92/00299 43 21~

Dehydration a~d embedding 2X106 cells were dehydrated in increasing concentrations of alcohol (70%, 90~, 96% absolute alcohol), followed by 3 changes in propanol. Be~ween these dehydration steps, pel-leting was carried out at 1500 rpm for 5 minutes.

The ceIls were then treated for 1 hour in 75% propanol +
25% TAAB812 resin mixture (TAAB Laboratories), for 1 hour in 50~ propanol ~ 50% TAAB812 resin mixture, and for 1 hour in 25% propanol ~ 75% TAAB812 resin mixture, followed by 24 ~; ~ 10 hours in fresh resin mixture.

Between each of these treatments, pelleting was carried out at 2000 rpm for 10-15 minutes.

; After blocking, the resin was polymerized for 48 hours at 60~C.

~eationing and stiaining Sections for electron microscopy of light grey or grey in colour (about 40 nm) were cut on an~LKB Ultratome with a diamond kni~fe~. They were then stained with uranyl acetate por 10 minutes, followèd by lead citrate for 2 minutes.
.
0 Ele~tron microsGopy The sections were examined in a Jeol 100 B electron micro-j , , scope at an accelerating vo1tage of 60 KV.

Particles with herpes group virus morphology were observed both inside and outside some of~the cells as shown in Figure la. Immature via1 capsids were seen in the nucleus (Figure lb). Viral capsids without core were also seen.
Another type of particles, that is, the retro~irus accord-ing to the invention, was seen along the outer cell mem-~:

2~2103 44 ~
brane in approximately 1-2% of the cells~ The particles were spherical structures with a diameter of 90-lO0 nm, containing a core-like condensation as seen in Figures lc and ld. The outer membrane did not contain visible projec-S tions. The structures were indistinguishable from viruspar~icles and had a close resemblance to known retrovirus.
Due to the morphology of these particles, the retrovirus was characterized as being a type C-like retrovirus in accordance with the description given in Dalgleish et al., 10 l9gO.

EXAMP~E 3 Characterization of the lymphoblastoid cell culture : The cell culture was examined for production of various products, and the presence of leukocyte differentiation antigens was analyzed.

: The presence of interleukin-6 (IL-6) was measured by a bio-logical assay using :the IL-6 sensitive Bg cell-line as de-scri~ed in Rozenberg et al., 1991. Interferon assay was performed as descri~ed in Haahr et al., 1976, and neutra lization was made w~ith a polyclonal antibody to interferon-(Boehringer Mannheim).

Tumour necrosls factor activity~was measured in a cytotoxi-city assay in~L-929;cells. In brief, monolayers of cells : ~
seeded the day before (2x104 cellslwell) were overlayed with two-fold dilutions of the supernatants to be tested, : ~
obtained from the superna~ant of ~ell culture of the cell :~ line 1533, and incubated:at 38.5C with actinomycin D
ltml; Calbiochem, Behr~ing Diagnostics, La Jolla) for 18 hours. The plates were fixed in 1% formaldehyde and stained : 30 with crystal violet (1 mg/mI) for 20 minutes, washed and read for light absorbance at 600 nm in an ImmunoReader (InterMed NJ-2000).~TNF titers were assessed as the dilu-212103~
tion resulting in 50% cytotoxicity and compared with stan-dard TNF titrated on each plate. The cytotoxic substance was identified as TNF-~ by neutraliæation by the specific antiserum to human TNF-~ (rabbit anti-human TNF-~ polyclo-nal antibody 80 ~1/ml (80-800 neutralizing units), Genzyme) and was not neutralized by specific antiserum to human TNF-~ (rabbit anti-human TNF-~ 80 neutralizing units/ml, : Walther Fies Gent).

The cell culture was shown to have an autocrine production ~: 10 of interleukin-6 (2 units/ml) and to produce interferon-~
;;~: spontaneously (2.74 unitslml) . Tumour~necrosis factor-~
;~ : (ioo units/ml) was also produced by the cell culture spon-: , taneously.

The identification of the presence of leukocyte differen-tiation antigens was~performed by ~he~use of the immunohis-tochemical~ method as described in the following.

Cytocentrifuge~specimens were a:ir-dried and fixed in ace-tone~for 10 minutes at room~:temperature whereafter the spe-c:imens were stained with each:of:the monoclonal antibodies 2~0~ listed in~Table~1~below;using~the alkaline phosphatase ant:i-alkaline phosph~ataae~(APAAP) or a three-stage immuno-peroxidase method as:described~in Pallesen et al., 1~91.
Phenotyp~c characteriæation~of the~:lymphoblastoid cells was performed~with~a~wide:panel:of~ monoclonal antibodies to 2:5: leukocyte~:dif~ferentiation~antigens in accordanc with prin-ciples described~;in:Palleseni::1988~.

The results obtained appear from Table ~ below. The cell population did:nQt contain cells labelled with T-lympho-cyte- or~ma~rophage-speci:fic monoclonal antibodies.

, : :
~: :
.:

: : :

WO 93/07259 ~ 12 1 0 3 0 4 6 PCI`/I)K92/00299 TAsLE 1 Expre ision of leukocyte differentiation antigens of the LCL MS 1533 5 B-cell antigensa) Reactivityb) CDl9 (DAX0-CD19) 2+
CD20 (L26) 2+
CD21 (OKB7) 3+
CD22 (Tol5~ 1+
CD23 (MHM6) 3~
CD77 (424/4A11) ~ -/1+
Polytypic immunoglobulin 3+
T-cell antigens (various) CD4 (DAK0-T4) Monocyte and granulocyte antigens (various) CD15 (DAK0-Ml) Cell adhesion molecules CD54 (LB-2) 2+
CD58 (TS2/9) 3+
CDlla (F110.22) 2+
~cti~ation-associat~d antigens CD30 lBer-H2) 3+
CD2~ (TU69) -/1+
HLA antigens ~: c 2 5 HLA- I ( DAKO-HhA-ABC ) 3 HLA I I ( Tu3 5 ) 3 ~
: Proliferation-associated antigens (Ki-67) 95%

`
: ~ a) Monoclonal antibody designation is indicated in brack-e~s.
b) Score: - = negative, + - positive, -/~ = <50% cells positive, 1+, 2+, 3+ = weak, medium and strong stain-; ing, respectively.:

EX~PLE 4 :
.T~ ting flor variou~ retroviru~-rel~ted a~tigans in the cell culture ineated with the retroYiru~ ~

~ The lymphoblastoid cell culture was examined by an immuno-:~ fluorescence analysis for the expression of the HT~V-I
antigens pl9 and p24 by the use of the following monoclonal antibodies directed against HTLV-I pl9 and p24: 12G4, MAS
197b, 6G9 and MAS l99b (Sera-Lab).

WO 93/0725g PCI`/DK92/11)0299 ,,~ 47 2121030 As appears from Table 2 in Example 9 below, no binding of monoclonal antibodies directed against the HTLV-~ antigens plg and p24 was observed, which means that the lymphoblas-toid cell culture did not express these antigens.

To characterize other possible antigens present in the lymphoblastoid cell culture infected with the retrovirus, the culture was examined for animal virus antigens from murine-leukemia virus (MuLV), simian sarcoma ~irus-l (SS~ p28), feline leukemia virus (FeLV) and the endoge-nous cat virus (RDI14) using immunofluorescence analysis.The polyclonal antibodies used were provided by Bjorn Nexo, The Fibiger Laboratory, Copenhagen, Denmark (see Kaltoft et al., 1988).

As appears from Table 2, no binding of antibodies was observed, which means that the lymphoblastoid cell culture :: infected with the retrovirus did not express antigens characteristic of the abovs-mentioned animal viruses.

Finally, the antibodies present in the serum from the pa-tient from which~the lymphoblastoid cell culture was deri ved were examined in order to further support the above - ~ ~
results showing tha~ the retrovirus:from the lymphoblastoid cell culture was~ n~ot an HIV-I, HIV-II or HTLV-I virus: By use o~ ELI5A, the ~erum from:the patient was examined for : IgG antibodies to:HIV-I and HIV-II, and HTLV antibodies were determined~by an immunofluorescence method at the State Serum Instltute~,~Copenhagen. No positive result was obtained showing that no antibodies capable of binding to HIV-I, HIV-II or ~TLV-I viruses were produced as a re~ult f infection with the retrovlrus from the lymphoblastoid culture.:Furthermore~,~ as there is no serological difference between HTLV-I:and~HTLV-II with respect to the antibodies produced in an infection, the retrovirus from the cell culture was not an HTLV-II virus.

ExAMpLE 5 2 1 2 103 Purific~tion of ~iral particles o~ antige~s from the lym~
phobl~stoid call culture , Several approaches were used in the purification of viral particles or antigens The principles of this type of purification are outlined in Poiesz et al , 1980 ~: A. ~u¢rose gradisnts 1x108 cells were sonicated and harvested for 10 minutes by centrifugation at 2000 rpm in a sorvall GLC-2 centrifuge, and the pellet of~the~supernata~nt was layered on the fo -lowing gradient after~the supernatant had been spun for 3 hours at 19,000 rpm~in a VTi 55 rotor (30,000 x g) at 4C, and the~pellet~had been resuspended~in l-2 ml of TNE (50 mM
Tris-HCl pH 7 5, ~100~mM NaCl, ~l mM EDTA) 15~5% sucrose ~ 12 5 q of sucrose In;25 ol of 2xTNE (100 mM
Tris-HCl pH~7 5, 20!0 mM~NaCl, 2 mM EDTA) and 2~5~ml of~distilled water;
4~5% sucrose ~2~2;~5;~g~of~sucrose in~25~ml of 2xTNE ~100 mM
Tris-HCl pH 7 5,~200~mM~NaCll 2 mM EDTA) and 2;0 ~ 25 ml of~d~lsti~lled~wa~ter The gradient~mixer~was~rinsed~and~the~gradient was made up in~9/16x3~1/2~(14~x~89~mm) tubes~

The gradient was spun for 16 hours~at 38,000 rpm in an SW41 rotor at 4C,~and 6 fractions were ta~en and diluted three 25~ times~with~TNE-~(5~0~mM~Tris-HCl~pH~7~5~, ~100 mM NaCl, 1 mM
EDTA) ~entri~fugation was then ~performed for 3 hours at 38~,000 rpm in an~SW41~rotor (4~5,0~00~x;g) at 4C, and the retrovirus~fraction~;~was then contained in ~the pellet :. : :: :

No bands were visible in the first gradients which were divided into 6 fractions. Each fraction was pelleted and 6 new gxadients were run separately.

B. Purification by ~dsorption to ~ntibody-conjugated micro-bead~
~ .
The labelling of microspheres with rabbit anti-mouse/anti-rat IgG was performed~as follsws:
:: , l g of beads (Tessek, Aarhus; = 3 x lO9 beads/g) was washed in lO ml of Buffer~A (700 mM (NH4)2SO4; lO0 mM sodium bo-rate, pH 9). The beads were spun down and the supernatantwas removed. 2-5~ ml of IgG (5-lO mg/ml IgG) in Buffer A was added, and the mixture was shaken overnight at room tempe-rature. The beads~were aga~in spun down and the supernatant was removed. The m1xture was washed several times in double 15 ~distilled wat~r;~and~OD280 was~read in;a photometer (ap-proximately O~if~washed~efficientlyj.~ The mixture was then suspended in l;ml~of~Buffer~B (lOO mM ~sodium borate; lO0 mM
;ethanol amine,~ pH 9)~and incubated overnight at room tempe-rature. The beads were~spun down and washed in double 20~distilled~water until pH was~about 6-7~. The~mixture was washe`d~and~`resuspended 1n~PBS. ;~

The antibodi~es~wére con3ugated~to the~;labelled microspheres (about~3xlO9~;spheres/g were~use~ in~the following manner:
incubation~was~Garried~out~with l~m1 of antibodies:~anti-5~ rat~HTLU~ 30g~ le, 5a,~ 69b;~or anti-mouse HTLV-I 46 (T.
Schultz,~Chester~Beat~ty Lab.,; UKj (+~, or the following antibodies raisèd~against other ~TLV-I antigens: anti-mouse HTLV-I pl9~(~asc~ites~ or ant1-mouse~HTL~I p~4 ~K~. Kaltoft, Bartholin,~Aarhus~University~ 3~ 3000 x diluted in PBS
for 1 hour at room~temperature. The~beads were spun down and washed~several~times~in PBS/Twee~n.

; The~ incubation~w1th;~sample~ was performed by adding 104 W093/07259 ~121 0 ~ O PCT/DK92/00299 spheres to 500 ~l of sample supernatant. The mixture was then incubated at 37C while shaking.

Two methods wexe employed to remove bound particles and : IgG. Either the beads were incubated for 5 minutes at room temperature with lO0 mM glycine, pH 2.5, followed by neu-tralization by addition of l/lO volume of sample of l M
Tris-HCl, pH 9. Alternatively, the beads were incubated with 4 M MgCl2 fox 5 minutes at room temperature, followed by lOOx dilution with PBS, pH 7.4.

C. Purific~tion~of membr~ne-~ssociated proteins by Triton X-114 temperature induced~phase epar~tion 5 x lo8 cells were harvested and washed twic~ in PBS, pH
7.4 containing 1 mg/ml BSA.

$he cells were resuspended and lysed in 4 ml of lO0 mM
Tris-HCl, 1% Triton X-114j lO mM EDTA, 1 mM PMSF (phenyl-methylsulfonyl fluoridej, pH 8.1 at 0C for lO minutes~

Thè lysate was clarified by centr~ifugation at 50Q0 x g for lO minutes at 4C and was transferred to Falcon tubes and : incubated for::lO minutes at 37C to induce the phase sepa-2~0 ration.

:~ : :: : : :
The detergent phase at the bottom of the tube was collected : after centrifugation~:~for lO minutes at 20C and 1800 x g.
An equa1 volume~of 100 mM~Tr1s-HC1, pH 8, was added and the procedure was repeated.
' 1 ' ' ~ ' ' : ~ ~ 25 The detergent:phase ~`~100-600 ~:l) was suspended in 2 ml of 100 mM Tris-HC1, pH 8,~w1th 0.5%~CHAPS (3-[(3-cholamidopro-: pyl)dimethylammonio] l-propanesulfonate; Boehringer) to ~` avoid temperature induced phase s~eparation during subse-quent handling.

::

.
' .

W093~07259 PCT/DK92/00299 51 2121~30 For Western blots, an equal volum~ of sample and sample buffer was used, but the final SDS concentration should not exceed 0.1%.

The viral particles or antigens prepared by these methods were used to further characterize the virus as exemplified in the following.

EXAMPL~ 6 Reverse txa~¢ripta~a assays : The supernatants obtained from cell line 1533 were concen-trated and purified by ultracentrifugation on sucrose gra-dientsj followed by pelIeting of the various fractions as described in Example 5 above.
~: : : : :
:20 ~l of the test material thus obtained was placed in a Widal glass (3 m~ and to~this the following mixture was added~

5 ~l of Tris 0.5 M/DTT (dithiothreitol) 0.04 M (pH 8.2) : : 5 ~l of NaCl O.l M
5 ~l of MgC12~0.3~M~
23~1 of double distilled~water:::
; 2:0 lO~l of NP-40~2~(Boehringer) 2 ~.1 of poly rC~-::oligo dGl~_l8 (Pharmacia Prod.No. ~7-: 7944):~
10 ~1 of Decxy{:8-3H} guanosine~triphosphate (Amersham TRK-.350~

: : 2s~ The test tubes were;incubated~at:37~C for 90 minutes. The reaction was stopped by adding 100 ~1 of a cold solution of distilled~water containing 12.5~ of water sa~urated with sodium phosphate,~ 12.:5% of water:saturated with sodium pyrophosphate, ~and ~20~ of trichloroacetic acid (TCA). After : 30 30 minutes on ice, the tubes were emptied onto a Millipore W093/072~9 PCT/DK92/~299 membrane filter (cat.no. GVWP02500), and the membrane fil-ter was washed 4 times with 5 ml of 5% TCA u~der depres-~: sion of 2 atm. The membrane filters were dried, placed in counting tubes, covered with 5 ml of scintillation fluid (Lumasafe-Packard), and counted in a beta-counter (Packard) for 5 minutes per test tube. Results were measured in counts per minute tCPM). The various samples were run in duplicate. The principles of this assay are described in ~: Poiesz et al., 1980.

Results During the initial phases of the growth of MS 1533, the supernatants were tested either unconcentrated or concen-:
trated to a level of concentration lower than what can beobtained by centrifugation on sucrose gradients by a method essentially as outlined above. Tests run on the former material only showed borderline reverse transcriptase activity. In the purified material, a reverse transcriptase activity 3 fold~background was found.

:
Te~ting purified retrovirus:for various retroviru3-rel~ted antig~ns ; Retroviral:particles~or:antigens purified by methods A, B
: and C as described in;Example 5~above were subjected to .
SDS-PAGE. The 10% SDS-PA:gels were subsequently used either ~' 25 for direct visualization of the proteins in the samples by ~ ~ silver staining or;for;Western:~Blotting. In either case, :;~ 5 ~1 of prestained Mr:ma~rker (~Sigma) was loaded in one lane of the gel~

W093/07259 2 ~ ~1 o ~/D~9~/00299 ~ilver staining Gels to be silver stained were run on ~he Pharmacia PHAST
System. The samples were prepared before loading by mixing 10 ~1 of sample with 2.5 ~l of sample buffer (100 mM Tris-HCl, pH 6.8, 10% glycerol, 1~% SDS and 5% ~PB) and 1.25 ~1of 100 mM DTT. The samples were boiled for 3 minutes, and 1.4 ~l of iodoacetamide was added. The gels were run at 50 V overnight.

After electrophoresis, the gels were silver stained by incubating the gels consecutively in the following:

1 hour at room temperature in 150 ml of 96% ethanol;
20 ml of 100% HAc; 12:0 ml of distilled water, -~ 1 hour at room temperature in 15 ml of 96% ethanol; 21 ml of 100% HAc; 264 ml of distilled water, :
: ~ 15 1 hour at room temperature in 120 ml of glutardialdehyde;
: 180 ml of distilled water; washing in 300 ml of d~stilled : ` :water 3 x 10 minute~:, 1 x l:hour, 1 x 10 seconds, 30 minutes at room temperature in 97,5 ~l of 100 mM DTT;
30:0 ml of distilled water; washing in 300 ml of distilled : :: :20 :water 2 x 5 minutes, : 30 minute~ at:room~temperature::in 300 ml of distilled water ~; with 0,3 g~AgN03, l~minute, followed by 3 minutes in 150 ~l of 37~ form lin in 300 ml of 3% Na2C03, 10 minutes:~at room:temperature in 200 ml of distilled water with 4,85 g of~citr~ic acid; washing in 300 ml of distilled water:2 x 30 minutes; washlng in 50 ml of 96% ethanol;

6,8 ml of 87% glycer~ol; 14~3 ml of distilled water, and drying the gel on~a paper towel.
:, '`

.

Wester~ Blotting The samples were prepared before loading by mixing 30 ~il of sample with 7.5 ~l of sample buffer ~100 mM Tris-HCl pH
6.8, 10% glycerol, 12% SDS and 5% BPB) and 3~7 ~l of 100 mM
DTT. The samples were boiled for 3 minutes, and 4,4 ~l of iodoacetamide was added. The gels were run at 50 V over-nig~t -After electrophoresis, the gels were blotted to Immob.ilon (Millipore~ membrans in a Kem-En-Tec wet blotter. The gels were equilibrated in transfer buffer for 15 minutes, while the membranes were equilibrated in methanol, water and transfer buffer (25 mM Tris-HCl, pH 8.3; 192 mM glycine;
20% ethanol; 0.1% SDS).

The gels and membranes were made up as sandwiches with Whatman filter paper and blott~d 500 Vh (overnight at 25 V). The sandwich was dismantled and the membrane washed in double distilled water. The membrane was then blocked in TBS/0.1% Tween ~or 15 minutes at room temperature and could e stored as such~in~TBSITween at 4C for a week.

The membranes~were scre~ned with the following antibodies, all raised against~HTLV-I envelope antigens: anti-rat HT~V-I: 30g, le, 5a,~69b; and anti-mouse HT~V-I 46 (T.
Schultz, Chester Beatty Lab., UK)~(~), and the following antibodies raised~aga`inst other;HTLV-I antigens: anti-mouse ;~ 25 HTL~-I pl9 (~ascités)~ and anti-mouse HTLV-I p24 (K. Kaltoft, ~ Bartholin, Aarhus University) (-~.
, The s~creening procedure was as follows: Antibodies were 3000 diluted in PBS ~ith 0.05`% Tween, incubated ove~night at room temp~erature on a shaker and washed 3 x 5 minutes in TBS with 0.05%~ Tween and~350 mM NaCl. The membranes were then incubated for 1 hour with 1:3000 diluted rabbit anti-mouse or anti-ra~ antibodies and washed 3 x 5 minutes in TBS with 0.05% Tween and 350 mM NaCl. The membranes were : :

~ :
::

W093/07259 PCT/DK92/~299 then incubated for 1 hour with 1:5000 diluted AP~conjugated goat anti-rabbit antibodies and washed 3 x 5 minutes in TBS
with 0.05% Tween and 350 mM NaCl. The membranes were then washed for 5 minutes at room temperature in 100 mM ethanol amine, pH 9, and the substrate (1/10 vol NBT (4-nitroblue tetrazolium chloride; Boehringer); l/100 vol of BCIP ~5-bromo-4-chloro-3-indolyl phosphate; Boehringer); 1/500 vol of 2 M MgC12) in 100 mM ethanol amine, pH 9, was added. The membranes were washed in water after the colour reaction.

The fact that some, but not all, of the antibodies gave a positive reaction (+) indicates similarity to, but not ~ identity with HTLV-I.

:~ :

Electron microscopy - negative staining A small drop of ~irus containing solutian from the double ;~ purified fractions as~described in Example 5A was placed on ; carbon coated~G400 grids (Gilder) which wera initially coated with parlodion 2% in amyl~acetate, and a drop of negative stain ~(1%~phosphotunqstic acid (PTA) adjusted to 20 pH 6.5 with lN~KOH) was~added. After 20 seconds, exc~ss moisture was ~emoved with the torn edge of a fiIter paper.
The grids were then air-dried and examined in an electron microscope (Jeol 100 B, Jeol,;Japan). The result of one of the above-described~experiments~is~illustrated in fi~ure 2a.
;

~; Immunoelectron microscopy The grids~were floated (carbon slde down) on a 15 ~l drop of a solution of protein A ~O.Ol mglml in PBS buffer) for : ~ :
10 minutes, and~excess moisture was removed with the torn 30; edge of a f1lter paper. The grids were washed by transferr-; ing them through 3 drops of PBS buffer, 1-2 minutes on top ~,:

2 1 2 1 0 3 0 56 " h, of each drop. The grids were floated on 15 ~l of antiserum (mouse monoclonal antibody to HTLV-I gp46 diluted 1:100 in PBS) for 10 minutes. Excess moisture was removed with ~he torn edge of a filter paper, and the grids were then washed in buffer as described. The grids were then floated on lS ~l of virus-containing solution from the double purified fractions as described in Example 5A and were washed in buffer as described. Then a drop of negative stain (potas-sium phosphotungstate (PTA)) was placed on the grids, and excess moisture was removed with the torn edge of a filter ::: paper. Excess stain was removed after 20 seconds, and the ~; grids were air-dried and examined in an electron microsco-pe (Jeol 100 B, Jeol, Japan). The result of one of the : above-described experiments is~illustrated in figure 2b.

Testing the culture containing~the retrovirus for ~ucleo-~, ~
tide sequences from HIV-I and HT~V-~

Various PCR methods were employed to identify and partly : characterize the; nucleic acids~of retroviral origin in : 20 MS 1533~. PCR :strategies were performed as described in i Innis et al.~,~l990 (PCR Protocols, A guide to methods and applications, Academic~Press, Inc.j. In order to avoid endogenous retroviral~sequences~,~cytoplasmatic RNA was ;purified,~subjected to;reverse transcription and subjected to PCR analysis. lst~strand cDNA~was: used as a template in all PCR éxperiments described herein. Cytoplasmatic RNA was purlfied and ampllfied~as ~ollovs: .

Approximately 120 x ~106 cells were~harvested for 5 minutes at lOOO~rpm in a c~entrlfuge and were then rinsed twice in : 30 PBS. The cells:were resuspended:in 8:00 ~l of PBS in an Eppendorf tube and harvested ~for 10 seconds in an Eppendorf centrifuge after which the supernatant was discarded. The cells were lysed in :500 ~l of lysis buffer (140 mM NaCl, :~ , ,,, , , , . . , _ ... , , _ _ . _ , .. .. ..... . ... . ... . . .. .

1.5 mM MgC12, lo mM Tris-HCl pH 8~6, 0.5~ 43P and were placed on ice for 5 minutes. The mixture was pelleted cold ~or 5 minute~ at 10,000 x g in the Eppendorf centrifuge.

To the supernatant was added sodium acetate, pH 7.0, to 150 mM and SDS to 1%, after which 5 ~1 of 20 mg/ml protei-nase K (Merck) was added and the suspension was incubated at 37C for 30 minutes. Phenol/chloroform extraction was performed twice; chloroform extraction was performed once.
The aqueous phase was adjusted to 300 mM sodium acetate, pH

-7, and 2.5 volumes of 96% ethanol was added. The RNA was harvested cold for 30 minutes, washed with 80% ethanol and , resuspended in distilled water.

; Detection of radioactively labelled PCR products was accom-plished by end label;ling the~ oligonucleotide primers as follows:

The following~mixture~was;prepared:

O pmol/~l~oligonucleotide;~(prepared synthetically), 2 pl 0f 10~x T4PNK buffer~commercially available buffer from Boehringer~Mannheim)~, ~5~ of~32P-ATP (from NEN, New ~England~Nuclear)~ 11.4-~1 of~distilled water, and 8 units of~T4-po~lynucleotide~kinase. ~ -The~mixture~ was~incubated~for 45~minutes at 37~C and for 10 ;; minutes~at 68C.~To~the mixture was~added 40 ~1 of distil-led~water,~ 240~ of~7~M~NH4Ac~and~750 ~1 of g6% ethanol, and the tube wa~s~placed on an ice bath for 30 minutes.

The;mixture was harve~sted~for 20~minutes at l2,000 rpm 5,000 x g)~in~an~Eppendorf~aentrifuge at 4C and then washed with 80%;~ethanol.~;The~mixture was resuspended in 100 ~1 of~TE~ 10~mM Tris-HCl~ mM~EDTA, pH 8).

~First strand cDNA~synthesis and PCR were performed with , ~ equipment and~kits from Perkin Elmer Cetus (USA). Reverse f 212103~

transcription was performed in a bu~fer with S mM MgCl2, lx PCR, ~ mM of each of the dNTP~s, 1 ~/~1 RNase inhibitor, 2,5 ~/~1 reverse transcriptase, 2.5 ~M random hexamers and 1 ~g of RNA. The mix was incubated at 42~C for 15 minu~es, at 99~C for 5 minutes, and at 5~C for 5 minutes. Amplification was performed by adding MgCl2 to 2 mM, lx PCR, 2.5 ~ Taq DNA
polymerase and double distilled water to 100 ~l. In low stringency reactions, the annealing temperature was between 37C and 48~C, in high stringency reactions it was 60C.
These criteria generally apply, where ~low stringency PCR
conditions" and "high stringency PCT conditions", respec-tively, are referred to in the present specification and claims.

The lymphoblastoid cell culture infected with the retrovirus was analyzed for the presence of nucleotide sequences speci-: . fic to the retrovirus HIV-I using high stringency nes~ed PCR
as described in Teglbj2rg et al., 1992. The following primer pairs and probes were used: .
:
~: LST1/LST2, SK3~8/SK39, LST3/LST4, SK68/SK69, hST5/LST6, SK29/-:~: 20 SK30, SK70, gK19 and SK31.
:
- The~DNA sequences of the primers and probes were as fo~lows:

~ ~ Primers:
*
: LSTl: 5' ATC A~G CAG CCA TGC AAA TG ~SEQ ID N0:1) LST2: 5' CTA ~AT AGT CTC TAA AGG GT (SEQ ID N0:2) SK38: 5' ATA AT~ CAC CTA TCC CAG TAG GAG AAA T (SEQ ID
NO:3) :~ SK39: ~ 5' TTT GGT CCT TGT CTT ATG T~C AGA ATG C (SEO ID
N~: 4 ) :- I.ST3: 5~' AGG AGG AGA TAT GAG GGA CAA TTG (SEQ ID NO: S ) ` 30 1ST4: 5' &GA GCT GTT GAT CCT TTA GGT ATC ~ SEQ ID NO:6) SK6 8: 5 ' AGC AGC AGG AAG CAC TAT GG ( SEQ ID NO: 7 ) SK69: 5' CCA GAC TGT GAG TTG CAA CAG (SEQ ID NO:8) LST5: 5' G¢C ~GG GAG CTC TCT GGC TA (SEQ ID N0:9) ~ST6: 5' CGG GCG C~A CTG CTA GAG A (SEQ ID ~O:10) ~T~T~r~ T

' o ` - - 2 1 2 1 0 3 ~

SK29: 5' ACT AGG GA~ CCC ACT GCT (SEQ ID NO:ll) SK30: 5' GGT CTG AGG GAT CTC TA (SEQ TD NO:12) Probes:
SKl9: 5' ATC CTG GGA TTA AAT AAA ATA GTA AG~ ATG TAT AGC
CCT AC (SEQ ID NO:13) SK70: 5' ACG GTA CAG GCC AGA CAA TTA TTG TCT GGT ATA GT
(SEQ ID N0:14) SK31: 5' ACC AGA GTC ACA CAA CAC ACG GGC ACA CAC TAC T (SEQ
ID NO~15) The lymphoblastoid cell culture was also analyzed for the presence of nucleotide sequences specific to the retrovirus HTLV-I using PCR. The followiny primers were used:

HTLV-I/026 and HTLV-I/029 .
HThV-I/026: 5' ~AG GCA GAT GAC AAT G~C CAY G~R CC (SEQ ID
N0:16) HT~V-I/029: 5' NAG CCA CCT NCT GAA CTG TC (SEQ ID NO:17) : ~ , As appears from Tab~e 2 beLow,; no:nucleotide~sequences speci-fic for HIV-I or HTLV-I were detected usi~g high stringency : ~ PCR condltions.:

Lo~_stri~gsncy a~neallng:

:
Using low stringency PCR conditions and a pa~el of primer pairs derived either from HTLV-I or from known retroviral consensus sequences,~PCR~products were obtained (indicated as positive:result in Table 2). The result~ of the PCR experi-me~ts are illustrated in Table 2. Th~_~ximer sequences were based on known:sequences from other retroviruses. The sequen-ces were based on~various~ principles for reverse translation.
The following primers were syn~hesized and used:

::

S~B~T~ T

6~
477/478, 1898/1899, l~00/1901, 1956/1957 and 2345/2346. Their sequences were as follows:

477: 5' AAT TGA AGC GAG CTG ATT GGT TAG TTT (SEQ ID NO:18) 47a: 5' A~T TAA ACT AAC C~A TCA GCT CGC TTC (SEQ ID NO:l9) 1898: 5' CTC GTC GAC AAC ATG TCA TCA ATG TA (SEQ ID NO:20) 1899: 5' CTC GAA TTC GAT GCA TAT TTC ACA AT~ CC (SEQ ID
. NO:~l) 1900: 5' CTC GAA TTC TAA AAG ATG CCT:TCT T (SEQ ID NO:22), 1901: 5' CTC GTC~GAC ATC GTC CAC ATA~GA (SEQ ID NO:23) 1956: 5' GAG:GCA GAT GAC AAT GA CAY GAR CC (SEQ ID NO:24) :~ . 1957: 5' NAG CCA CCT NCT GAA CTG TC (SEQ ID NO:25) 2345~: 5' TGG AAT GTI CTI CCI CAI GG (SEQ ID NO.26) 2346: 5' GCT AGG AGA ATI TCI TCC ~TI TA (SEQ ID NO:27) The following~oligonucleotides for~PCR are contemplated to be 15~ usefùl in~the~further analysis of the nucleotide sequenc~ of -th~ retrovirus~

1. 5' TGC AAG GCC CTG CAG GAG CAG TGC TGC (SEQ ID NO 2 a ) 2.~ ~5' T~G GGC CTG~AAC:TGG~G~C:CTG GGC CTG TCC CAG TGG
SEQ ID NO~:29) 3 :5~' CCA CTG GGA CAG GCC;CAG GTC CCA GTT C~ GCC CCA
(SEQ ID NO:30)~

: 4. 5' CGG TAC CCC CAC TAC:TCC (SEQ ID NO:31) hC~ r 5. 5' GGA GTA GTG G&G GT~ CCG (ShQ ID NO:32) 6. 5' AAC ACA GAG CCC TCC CAG CTG CCC (SEQ ID MO:33) 7. 5' CCT GTG CCC ACC CTG GGC TCC CGG T (SEQ ID NO:34)

8. 5' ACC GGG AGC CCA GGG TGG GCA CAG G (SEQ ID NO:35) . ' :
: 5 9. 5' ATC CCC TGG AAG TCC AAG CTG CTG ACC (SEQ ID NO:36) . .
.10. 5' CTG GTG CAG CTG ACC CTG CAG TCC (SEQ ID NO:37) 11. 5' AAC TGG ACC CAC TGC TTT GAC CCC CAG ATC CAG (SEQ, ID NO:38) 12. 5' CAC TGG CAT GTG CTG TAC TCC CCA ~T (SEQ ID NO:39) ~ , ~ ~ 10 13 . 5 ' ATT GGG GAG TAC M C ACA TGC CAG TG ~SEQ ID NO:40) :

.

- _ _ :: ~ : : :
: :~ : : :

~:` :

::: : ::
:

61~

~ntibody, PCR and Immunofluorescence analysis _ Antibody against the following retroviruses:
HIV-I negative HXV-II negative HTLV-I some negative, some positive High s~ringency PCR analysis for the following retroviruses:
HIV-I ~ negative HTLV-I negatlve Low stringency PCR analysis for the following retroviruses:
: HTLV-I positive various retroviral consensus positive Immunofluorescence analysis for HTL~-I antigens:
: pl9 negatlve ; p24 negative Immunofluorescence analysis for animal virus.antigens:
MuL~ ~ negative SSV-l, p28 negative FeLV ~ negatlve RD114 ~ ; negative ., ~ . .
~: ` : ~:
, .. .

:
, :
::

~;U~TITU~ ~

W O 93/07259 PC~r/DK92/00299 Eau~MpLE 1 ~ 1 2 1 0 3 ~unalysis of the lyn~phoblastoid cell culture for Epstein-Barr ~iru~ encod~a protei~s.

Immunohistochemistry was performed as described in Pallesen et al., 1991 in order to analyze the lymphoblastoid cell culture for ex~ression of Epstein-Barr virus proteins. The expression of the latent infection protein EBNA 1 was de-termined by a complement fixation assay as described in Reedman & Klein, 1973. The expression of the latent infec-tion proteins EBNA 2 and LMP was determined using the mono-; clonal antibodies PE2 and CS.1-4 which were obtained from L.S. Young and M. Rowe (Birmingham, UK). To determine the expression of immediate-early proteins BZLF1, the monoclo-nal antibody BZ~ also obtained from L.S. Young and M. ~owe (Birmingham, UK) was used.~FînalIy, the determination of the expression of productive cycle proteins, EA, VCA and MA, was performed by~the use of the monoclonal antibodies 37G11, 2F2 and~1~ID7; obtained from Prof. H. Wolf (Munich, Germany)~ A detailed~cha~acterization of these antibodies has recently been~published (Pallesen et al., 1991; Palle-~ ~ , sen et al., lg913.~

Expression~of Epstein-Barr~irus encoded proteins in the LCL MS 1533 ntigena)% positiveb) , Latent infection proteins: EBNA 1 100%
EBNA 2 (PE2) 100%
~ LMP-I (CS.1-4) 91%
Immediate-earIy~protein~ BZLF1 (BZl) 9%
ProductIve cycIe~proteins: EA-D (37G1l)3~
VCA (2F2)0.1%
MA~ (llD7)0.1%

a) Monoclonal ~ntibody indicated in brackets.
`~ b) ~Percentage of cells labelled.

: ::::: : :
~`: : :

~ 6~ 21210~0 The results obtained appear from Table 3 above and from the following. The latent infection protein EBNA 2 was loca-lised to the nuclei, but occasionally, cells show~d addi-tional fine granular cytoplasmic labelling. The latent infection protein LMP was localised to the cytoplasm and cell membrane. The immediate-early proteins BZLFl were localised to the nucleus but a weaker, diffuse cytoplasmic staining was also ~een in many cells, either alone or in combination with nuclear labelllng. Productive cycle pro-teins in the form~of EA were seen as an intense nuclear and a weaker cytoplasmic reaction, whereas VCA was mainly nuclear and~with a~granular appearance. MA was localised to the cytoplasm.
:
The~a~ove results showed that the lymphoblastoid cell culture expressed latent infection and partly productive cycle proteins~, ind~icating that~the cell line was trans-formed~by Epstein-Barr virus with an increased frequency of cells entering~ the lytic cycle. During the first month of cultivation,~ to 5% of the~cells were positive for VCA, ZO but after ~5 months~this~percentage was reduced to 0.1%.

, . :

:`~ `
:
: :
~ '

9 21210 3 ~ 64 PCI/DK92/00299 REFERE~NCES

- Bray, P.~., L.C. Bloomer, V.C. Salmon, M.H. Bagley, P.D.
Larsen: I'Epstein-Barr virus infection and antibody syn-thesis in patients with multiple sclerosis", Arch. N u~
rol. 40, 406-4~8, 1983.

- Crawford, D.H., J.M.B. Edwards: "Epstein-Barr virus".
In: A.J. Zuckermann, J~E. Banatvala and J.R. Patison (eds.), "Principles and Practice of Clinical Virology", 2nd edition, John Wiley & Sons, Chichester, 103-128, 199~.

- Dalchau R., J. Kirkley, J~W. Fabre: "Monoclonal antibody to a human leukocyte-specific membrane glycoprotein pro-bably homologous to the leukocyte-common (L-C) antigen : of the ratl', Eur. J. Immunol. 10, 737-744, 1980.

- Dalgleishj A.G., R.A. Weiss: "Human Retroviruses". In:
A.J. Zuckermann, J.E. Banatvala and J.R. Patison (eds.~, "Principles and Practice of Clinical Virology", 2nd edition, John Wiley & Sons, Chichester, S73-602, l9gO.

Gerber, P.: "Actlvation of Epstein-Barr virus by 5 bromo-: 20 deoxyuridine in virus-free human cells", Proc. .Natl.
Acad. Sci.~USA 69, 83~-85, 1972.

Haahr, S~ L~Rasmussen, T.C. Merigan: I'Lymphocyte Trans-formation and Interferon~Production in Human Mononuclear Cell Microcultures for Assay of Cellular Immunity to Herpes Simplex Virus", Inféct. Immun. 14, 47-54, 1976.

- Haahr, S., M. Sommerlundj A. M0ller-Larsen, ~. Nielsen, ~;: N.J. Hansen:~"Just another dubious virus in cells from a patient with multiple sclerosis?", Lancet 337, 863, 1991.

- Haahr, S. Second International Symposium on Retrovirus in Multlple Sclerosis and Related Diseases. Report and ab-.

212103~
stracts from a symposium arranged by Sven Haahr on behalf of the Danish Multiple Sclerosis Society in ~openhagen, August, 1991. J. of NeuroimmunoloqY 38, 163-174, 1992.

- Harboe and Ingild: "Immunization, Isolation of Immunoglo-bulins, Estimation of Antibody Titre", Scand. J. Immun. 2 (Suppl. 1), 161-164, 1973.

- Hauser, S.~., C. Aubert, J.S. Burks, C. Kerr, O. Lyon-Caen, G. deThe, M. Brahic: "Analysis of human T-lympho-tropic virus sequences in multiple sclerosis tissue", Nature 322, 176-177, lg86.

- Henle, G~, W. Henle: "Epstein-Barr virus-specific IgA
serum antibodies as an outstanding feature of nasopharyn-geal carcinoma", Int. J. Cancer 17, 1-7, 1976.

- Hollsberg P., A. M0ller-Larsen~ F. Skou Pedersen, J. Jus-tesen, H.J. Hansen, S. Haahr: "Search for a retrovirus in long-term cultured cerebrospinal fluid cells and peri-pheral blood mononuclear cells from patients with multi-ple sc1erosis", Acta Neuroloqica Scand. _80, 603-609, 1989.
:: :
- Innis et al., PC~ Protocols, A guide to method~ and applications, Academic Press, Inc., ~990.

Kaltoft, K., S. Bisballe, H.F. Rasmussen, K. Thestrup Pedersenl W.H. Boehncke, H. Vol~er, W. Sterry: "C-type particles are inducible in SeAx, a continuous T-cell line from a patient with Sézary's syndrome", rch. Dermatol.
Res. 280; 264-267, 1988.

- Koprowski, H., E. DeFreitas, M.~ Harper, M. Sandberg Woll-heim, C.W. Saxinger,~M.B. Feinberg, F. Wong-Staal, R.C~
Gallo: "Multiple sclerosis and:T-cell lymphotropic retro-viruses", Nature 318~, 154-160, 1985.

W093/072S9 2121 ~ 3 0 66 PCT/DK92/00299 - Kohler and Milstein: "Continuous cultures of fused cells secreting antibody of predefined specificity" r Nature 256~ 495-4g7, 1975.

- Lai, ~.K., X. Li, D.J. Volsky: "Induction of Epstein-Barr ~irus in B-lymphoblastoid cells by human immunodeficiency virus type 1", Int. J. Cancer 43, 1104~ , 1989.
.
- Larsen, P.D., L.C. Bloomer, P.F. Bray: "Epstein-Barr virus nuclear antigen and viral capsid antigen antibody titers in multiple sclerosis", Neurol. 35, 435-438, 1985.

~; ; 10 ~ Lindberg, C., O. Andersen, A. Vahlne, M. Dal~on, B. Re-marker: "Epidemiological investigation of the association beteween infectious mononucleosis and multiple sclero-: ~ SlS~, Neuroepidemioloqy~l-o~ 62-65, 1991.

Martyn, C.: "The; pidemiology of multiple sclerosis". In:
W.B.~ Matthews (ed.~,~ McAl~ine's Multi~le Sclerosis, Churchill Livingstone, Edinburgh, 3-40, 1991.

McFarlln, D.E. and~H.F. McFarland: "Multiple Sclerosis", The New~Enqland Journal o~ Medicine 307, nr. 19, 1183-1188 and~1246-1250, 1982.~ ~

2~0 - Montagnier,~L.,~ J. Gruest, S. Chamaret, C. Dauguet, ~.
Axler, D~; Guetard,~ M.T.~ Nugeyre, F. Barxé-Sinoussi, J.C.
Chermann, J.B;.~; Brunet,~ D. ~Klatzmann, J.C. Gluckm~n:
Adaptation of ~ phadenopathy~Associated Virus (LAV) to Replication in EBV-Transformed B Lymphoblastoid Cell . I ~ ! : . : , Lines"~, Sciencè 2_S, 63-66, 1984.

Ohtsuki,~ Y., S. Yano,~ I. Kubonishi, K. Hayashi, H. So~
nobe, J.;Iwata,~M.~ Fur~ihata,~ T. Hlkita, H. Wakiguchi, T.
Kurashige, I.~ Miyosh~ "Ribosome-lamella complexes ob-served in~an~Epstein-Barr virus-producing B-cell line, infected with human~ T-lymphotropic virus type I", J.
Pathol. 161, 191-194, 1g90. ~
:

~ :

- Operskalski, E.A., B.R. Visscher, R.M. Malmgren, R. De tels: "A case-control study of multiple sclerosis", Neuroloay 39, ~5-829, 1989.

- Pallesen, G., S.J. Hamilton-Dutoit, M. Rowe, I. Lisse, E.
Ralfkiaer, K. Sandvej, L.S. Young: Expression of Epstein-Barr ~irus replicative proteins in AIDS-related non Hodgkin's lymphoma cells", J. Pathol. 165, 289-299, 1991.

- Pallesen, G.: "Immunophenotypic markers for characteriz-ing malignant lymphoma, malignant histiocytosis and tumors derived from accessory cells", Cancer Rev. _8, 1-65, 198~.

- Pallesen, G., S.J. Hamilton-Dutoit, M. Rswe, L.S. Young:
Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin's disease", Lancet 337, 320-322, : ~5 19~1.

- Perron, H., C. Geny, A. Laurent, C. Mouriquand, C. Pel-lat, J. Perret, J. Seiynerin: "Leptomeningeal cell line from multiple sclerosis with: reverse transcriptase ac-tivity and viral particles", Res Virol. 140, 551-561, 19~

Perron, H., B~ :Lalande, B. Gratacap, A. Laurent, CO ~ou-~ riquand,~ J. Pellat, J. Perret, J.M. Seigneurin: "Issla-: tion of retrovirus from patients with multiple sclero-sis", Lancet i, 862-863, 1931.
~ 2S - Poiesz, B.J., F.~W. Ruscetti, A.F~ Gazdar, P.A. Bunn, J.D.
Minna, R.C. Gallo: "Detection and isslation sf type C
: retrovlrus particles from fresh and cultured lymphocytes ~: of a patient with cutaneous T-cell lymphoma", Proc. Natl.
Acad._Sci. U5A 77, 7415-7419, 1980.

.

21210~ 0 68 ,,~
- Poser, C.M.: ~Multiple Sclerosis and HTLV-I". In: Roman, G.C., J.-C. Vernant and M. Osame (eds), Neuroloa~ and Neurobioloay_51, HTLV-l and the Nervous system. Alan R.
Liss, New York, 373-384, 1989.

- Prayoonwiwat, N., L.R. Pease, M. Rodriguez: "Human T-cell lymphotropic virus type I sequences detected by nested polymerase chain reactions are not associated with multi-ple sclerosis", MaYo _Clinic Proceedinqs 66, 665-680, 1991 .

- Reddy, E.P., M. Sandberg-Wollheim, R.V. Mettus, P.E. Ray, E. DeFritas, H~. Koprowski, Science 243, 529-533, 1989.

- Reedman, B.M. and G. Klein: Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing ; antigen in producer and non-producer lymphoblastoid cell lines", Int. J. Cancer 11, 499-520, 1973.

- Rozenberg, F., S. Lefe~vre, C. Lubetzki, P. Lebon, O.
Lyon-Caen, M.~ Brahic,~J-F. Bureau: "Analysis of Retro-viral Sequences in th Spinal Form of Multiple Sclero-sis", Ann. Neurol. 29, 333-336, 1991.

~20 - Sambrook, J., Molecular Clonina, A Laboratorv Manual., 2nd Edition, Co~d Spring~Harbor~Laboratory Press, 1990.

: :
Schonnebeck, M~., C.R.F. Krueger, M. Braun, M. Fischer, B.
` ~ Koch, D.V. Ablashi, N. B;alachandran: "Hu~an Herpesvirus-6 Infection May Predispose Cells to Superinfection by Other Vi~uses", in vivo 5, 255-264, 1991.
: :
Selmaj, K.W., C.S~. Ralne: "Tumor necrosis factor mediates myelin and :oligodendrocyte damage in vitro", Ann. Neurol.
23, 339-346, 19~8.

- Sharief, M., R. Hentges: "Association between tumor ne-crosi~s factor-alpha and disease progression in patients W093/072~9 PCT/DK92/00299 with mul~iple sclerosis", New Enql. J. of Med. 325~ 467-472, 1992.

- Skolnik, P.R., B.R. Kosleff, M.S. Hirsch: "Bidirectional interactions between human immunodeficiency virus type 1 5and cytomegalovirus", J. Infect. Dis. 157, 508-514, 1988.

- Sommerlund, M., A.:M0ller-Larsen~ H.J. Hansen, S. Haahr:
Multinucleated giant macrophages in long-term cultures derived from mul~iple sclerosis patients", Immu~ol.
In~ect. Dis., June 1992.

- Sumaya, C.V., L~W.; Myers, G.W. Ellison: "Epstein-Barr virus antibodies in multiple sclerosis", Arch. Neurol.
37, 94-9~, 19~0.

:~ - Sumaya, C.V., L.W:. Myers, G.W.~ElIison, Y. Ench: 'IIn-creased prevalence and titer of Epstein-Barr virus anti-: 15bodies in patients with:multiple sclerosis", Ann. Neurol.
7, 371-377, ~985.

- TegIbjærg,~ L.L.S., C.:Nielsen, J-E:.S. Hansen: "Sensitive non-radioactive detection:of:HIV-1: use of nested primers for the amplificat1on~of HIV DNA", Molecular and Cellular 20: Probes 6, 175-180~,~ 1992;. ~

::

~ ~ .
: ., ~ :
:~ :: :

Claims (50)

72

1. A cell culture comprising cells which are infected with a type C-like retrovirus which is present in human patients who have symptoms indicating an early stage of multiple sclero-sis, the retrovirus being a retrovirus which can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visib-le projections on its outer membrane when studied in trans-mission electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests:
A) in nexted PCR analysis on the cell culture using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:

LST1 (SEQ ID NO:1)/LST2 (SEQ ID NO:2), SK38 (SEQ ID
NO:3)/SK39 (SEQ ID NO:4), LST3 (SEQ ID NO:5)/LST4 (SEQ ID
NO:6), SK68 (SEQ ID NO:7)/SK69 (SEQ ID NO:8), LST5 (SEQ
ID NO:9/LST6 (SEQ ID NO:10), SK29 (SEQ ID NO:11)/SK30 (SEQ ID NO:12), SK19 (SEQ ID NO:13), SK70 (SEQ ID NO:14) and SK31 (SEQ ID NO:15) and the following primers used for HTLV-I detection:
HTLV-I/026 (SEQ ID NO:16) and HTLV-I/029 (SEQ ID NO:17) no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the cell culture, C) in immunofluorescence analysis antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the cell culture.

2. A cell culture comprising cells which are infected with a type C-like human retrovirus, the retrovirus being a retro-virus which can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studied in transmission electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests:
A) in nested PCR analysis on the cell culture using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:
LST1 (SEQ ID NO:1)/LST2 (SEQ ID NO:2), SK38 (SEQ ID
NO:3)/SK39 (SEQ ID NO:4), LST3 (SEQ ID NO:5)/LST4 (SEQ ID
NO:6), SK68 (SEQ ID NO:7)/SK69 (SEQ ID NO:8), LST5 (SEQ
ID NO:9)/LST6 (SEQ ID NO:10), SK29 (SEQ ID NO:11)/SK30 (SEQ ID NO:12), SK19 (SEQ ID NO:13), SK70 (SEQ ID NO:14) and SK31 (SEQ ID NO:15) and the following primers used for HTLV-I detection:
HTLV-I/026 (SEQ ID NO:16) and HTLV-I/029 (SEQ ID NO:17) no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the cell culture, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the cell culture, and the retrovirus showing the following positive tests:
A) in Western blotting performed on retrovirus-containing material purified by sucrose gradient purification or Triton X-114 temperature induced phase separation or purified by adsorption to antibody-conjugated microbeads, binding by the antibodies anti-rat HTLV-I gp46/gp21: 30g, 1e, 5a, 69b; and anti-mouse HTLV-I gp46/gp21: 46 to the virus envelope protein is detectable, B) in reverse transcriptase assay performed on retrovirus-containing material double purified on sucrose gradients, revers transcriptase activity is detectable, c) in PCR analysis using low stringency PCR conditions and using the following primer pairs , PCR products are detectable.

3. A cell culture according to claim 1 or 2, in which the spherical particles have substantially the appearance as shown in Fig. 1c and 1d in transmission electron microscopy.

4. A cell culture according to claim 3, in which the spheri-cal particles have substantially the appearance as shown in figures 2a and 2b using negative staining electron microsco-py.

5. A cell culture according to any of claims 1-4, which is capable of producing the retrovirus.

6. A cell culture according to claim 5 which additionally is infected with a herpes group virus which enhances the produc-tion of the retrovirus.

7. A cell culture comprising cells which are infected with a type C-like retrovirus which can exist in the form of a spherical particle structure with a diameter of 80-120 mm containing a core-like condensation and without visible projections on its outer membrane when studies in transmis-sion electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests:
A) in nested PCR analysis on the cell culture using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:
and the following primers used for HTLV-I detection:
no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the cell culture, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p26), FeLV or RD114 do not bind to the cell culture, the retrovirus being the retrovirus with which cells of the cell culture MS1533 - provisioral deposit no.
V 91082701 are infected or a retrovirus which is identical therewith except for genetic variations which are commonly found in retroviruses and which do not change the above-defined properties of the retrovirus.

8. A cell culture comprising cells which are infected with a type C-like human retrovirus, the retrovirus being a retro-virus which can exist in the form of a spherical particle structure with a diameter of 80-120 mm containing a core-like condensation and without visible projections on its outer membrane when studies in transmission electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests:
A) in nested PCR analysis on the cell culture using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:
and the following primers used for HTLV-I detection:
no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the cell culture, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the cell culture, and the retrovirus showing the following positive tests:
A) in Western blotting performed on retrovirus-containing material purified by sucrose gradient purification or Triton X-114 temperature induced phase separation or purified by adsorption to antibody-conjugated microbeads, binding by the antibodies anti-rat HTLV-I gp46/gp21; 30g, 1e, 5a, 69b; and anti-mouse HTLV-I gp46/gp21: 46 to the virus envelope protein is detectable, B) in reverse transcriptase assay performed on retrovirus-containing material double purified on sucrose gradients, revers transcriptase activity is detectable.
c) in PCR analysis using low stringency PCR conditions and using the following primer pairs , PCR products are detectable, the retrovirus being the retrovirus with which cells of the cell culture MS1533 - provisional deposit no.
V 91082701 and V 92040805 are infected or a retrovirus which is identical therewith except for genetic variations which are commonly found in retroviruses and which do not change the above-defined properties of the retrovirus.

9. A cell culture according to any of claims 7 or 8, which contains genomic fragments which can also be found by PCR in blood samples from diagnosed multiple sclerosis patients and which are not present in sera from persons who are not infec-ted with the type C-like retrovirus.

10. A cell culture according to any of claims 7-9, which contains antigens capable of binding antibodies which are present in sera from diagnosed multiple sclerosis patients and which are not present in sera from persons who are not infected with the type C-like retrovirus.

11. A cell culture according to any of claims 1-10, which is infected with a retrovirus identical to the retrovirus with which cells of the cell culture MS1533 provisional deposit no. V 91100401 are infected.

12. A cell culture according to any of claims 1-11, which is infected with a retrovirus identical to the retrovirus with which cells of the cell culture MS1533 - provisional deposit no. V 92040805 are infected.

13. A purified retrovirus in the form of whole retrovirus or fragments thereof which is a type C-like retrovirus which is present in human patients who have symptoms indicating an early stage of multiple sclerosis, the retrovirus being a retrovirus which, in a cell culture, can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studies in transmis-sion electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests:
A) in nested PCR analysis on the retrovirus or the fragments using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:
and the following primers used for HTLV-I detection:
no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the retrovirus or the fragments, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the retrovirus or the fragments.

14. A purified retrovirus in the form of whole retrovirus or fragments thereof which is a type C-like human retrovirus, the retrovirus being a retrovirus which, in a cell culture, can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studied in transmission electron microscopy at a magnifica-tion of 50,000 times, the retrovirus showing the following negative tests:
A) in nested PCR analysis on the retrovirus or the fragments using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:
and the following primers used for HTLV-I detection:
no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the retrovirus or the fragments, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the retrovirus or the fragments, and the retrovirus showing the following positive tests:
A) in Western blotting performed on retrovirus- containing material purified by sucrose gradient purification or Triton X-114 temperature induced phase separation or purified by adsorption to antibody-conjugated microbeads, binding by the antibodies anti-rat HTLV-I gp46/gp21: 30g, 1e, 5a, 69b; and anti-mouse HTLV-I gp46/gp21: 46 to the virus envelope protein is detectable, B) in reverse transcriptase assay performed on retrovirus-containing material double purified on sucrose gradients, revers transcriptase activity is detectable, c) in PCR analysis using low stringency PCR conditions and using the following primer pairs , PCR products are detectable.

15. A purified retrovirus in the form of whole virus or fragments thereof, the retrovirus being a type C-like retro-virus which, in a cell culture, can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studies in transmis-sion electron microscopy at a magnification of 50,000 times, the retrovirus showing the following negative tests:

A) in nested PCR analysis on the retrovirus or the fragments using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:
and the following primers used for HTLV-I detection:
no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the retrovirus or the fragments, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the retrovirus or the fragments.
the retrovirus being the retrovirus with which cells of the cell culture MS1533 - provisional no. V 91082701 are infected or a retrovirus which is identical therewith except for gene-tic variations which are commonly found in retroviruses and which do not change the above-defined properties of the retrovirus.

16. A purified retrovirus in the form of whole virus or fragments thereof, the retrovirus being a human type C-like retrovirus which, in a cell culture, can exist in the form of a spherical particle structure with a diameter of 80-120 nm containing a core-like condensation and without visible projections on its outer membrane when studies in transmis-sion electron microscopy as a magnification of 50,000 times, the retrovirus showing the following negative tests:
A) in nested PCR analysis on the retrovirus or the fragments using high stringency PCR conditions, the following primer pairs and probes used for HIV-I detection:
and the following primers used for HTLV-I detection:
no genomic sequences are detectable, B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the retrovirus or the fragments, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the retrovirus or the fragments and the retrovirus showing the following positive tests:
A) in Western blotting performed on retrovirus-containing material purified by sucrose gradient purification or Triton X-114 temperature induced-phase separation or purified by adsorption to antibody-conjugated microbeads, binding by the antibodies anti-rat HTLV-I gp46/gp21: 30g, 1e, 5a, 69b; and anti-mouse HTLV-I gp46/gp21: 46 to the virus envelope protein is detectable, B) in reverse transcriptase assay performed on retrovirus-containing material double purified on sucrose gradients, revers transcriptase activity is detectable, c) in PCR analysis using low stringency PCR conditions and using the following primer pairs and PCR products are detectable, the retrovirus being the retrovirus with which cells of the cell culture MS1533 - provisional no. V 91082701 and provi-sional no. V 92040805 are infected or a retrovirus which is identical therewith except for genetic variations which are commonly found in retroviruses and which do not change the above-defined properties of the retrovirus.

17. A retrovirus according to any of claims 15 or 16, which contains genomic fragments which can also be found by PCR in blood samples from diagnosed multiple sclerosis patients and which are not present in sera from persons who are not infec-ted with the type C-like retrovirus.

18. A retrovirus according to any of claims 15-17, which contains antigens capable of binding antibodies which are present in sera from diagnosed multiple sclerosis patients and which are not present in sera from persons who are not infected with the type C-like ratrovirus.

19. An antigen or epitope derived from, produced by, or induced by the retrovirus with which the cell culture accor-ding to any of claims 1-13 is infected or derived from the purified retrovirus according to any of claims 14-18, the antigen showing the following negative tests:

B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p19 and p24 do not bind to the antigen or epitope, C) in immunofluorescence analysis, antibodies directed against the retrovirus MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the antigen or epitope, D) im immunoassays, antibodies directed against HIV-I, and HIV-II do not bind to the antigen or epitope, the antigen or epitope being obtainable by subjecting cell fragments and/or medium from a cell culture according to any of claims 1-13 or purified retrovirus according to any of claims 14-18 to gel electrophoresis, applying serum from a diagnosed multiple sclerosis patient to the resulting gel and visualizing bound antibody by means of labelled anti-human antibody, comparing the visualized gel bands with a similar preparation made using sera from a number of persons who are not infected with the type-C retrovirus, identifying the bands which are antibody-bound in the preparation using the serum from a diagnosed patient and which are not bound in the preparations using sera from the persons who are not infected with the type-C retrovirus, and isolating such bands contain-ing the antigen or epitope from corresponding gel electro-phoresis, and optionally extracting the antigen or epitope from the bands and purifying the antigen or epitope.

20. An antigen or epitope derived from, produced by, or in-duced by the retrovirus with which the cell culture according to any of claims 1-13 is infected or derived from the purifi-ed retrovirus according to any of claims 14-18, the antigen showing the following negative tests:
B) in immunofluorescence analysis, antibodies directed against HTLV-I antigens p15 and p24 do not bind to the antigen or epitope, C) in immunofluorescence analysis, antibodies directed against the retroviruses MuLV, SSV-1 (p28), FeLV or RD114 do not bind to the antigen or epitope, D) in immunoassays, antibodies directed against HIV-I or HIV-II do not bind to the antigen or epitope, and the antigen showing the following positive test:
A) in Western blotting performed on retrovirus-containing material purified by sucrose gradient purification or Triton X-114 temperature induced phase separation or purified by adsorption to antibody-conjugated microbeads, binding by the antibodies anti-rat HTLV-I gp46/gp21: 30g, 1e, 5a, 69b; and anti-mouse HTLV-I gp46/gp21: 46 to the virus envelope protein is detectable, the antigen or epitope being obtainable by subjecting cell fragments and/or medium from a cell culture according to any of claims 1-13 or purified retrovirus according to any of claims 14-18 to gel electrophoresis, applying serum from a diagnosed multiple sclerosis patient to the resulting gel and visualizing bound antibody by means of labelled anti-human antibody, comparing the visualized gel bands with a similar preparation made using sera from a number of persons who are not infected with the type-C retrovirus, identifying the bands which are antibody-bound in the preparation using the serum from a diagnosed patient and which are not bounds in the preparations using sera from the persons who are not infected with the type-C retrovirus, and isolating such bands contain-ing the antigen or epitope from corresponding gel electro-phoresis, and optionally extracting the antigen or epitope from the bands and purifying the antigen or epitope.

21. A diagnostic agent which comprises an antigen or epitope according to claim 19 or 20.

22. A diagnostic agent according to claim 21, wherein the antigen or epitope is provided with a label.

23. A diagnostic agent according to claim 22, wherein the label is selected from enzymes, fluorescent substances, radioactive isotopes and ligands such as biotin.

24. A diagnostic agent according to claim 23, wherein the antigen or epitope is coupled to a solid support directly or via a spacer.

25. An antibody which binds to an antigen or epitope accord-ing to claim 19 or 20.

26. An antibody according to claim 25, which is a monoclonal antibody.

27. An antibody according to claim 25, which is a polyclonal antibody.

28. An anti-idiotypic antibody which is directed against the site of an antibody according to claim 25 which is reactive with the epitope or the antigen according to claim 19 or 20.

29. A diagnostic agent which comprises an antibody according to any of claims 25-27 or an anti-iditypic antibody accord-ing to claim 28.

30. A diagnostic agent according to claim 25, wherein the antibody is provided with a label.

31. A diagnostic agent according to claim 30, wherein the label is selected from enzymes, fluorescent substances, radioactive isotopes and ligands such as biotin.

32. A diagnostic agent according to claim 31, wherein the antibody is coupled to a solid support directly or via a spacer.

33. A nucleic acid having a nucleotide sequence which is distinct from sequences from known retroviruses and which when used as a probe on samples of body fluid, such as blood samples, or tissue samples from a number of persons who are not infected with the type-C retrovirus and samples of body fluid, such as blood samples, or tissue samples from diagno-sed multiple sclerosis patients, respectively, detects nu-cleotide sequences from diagnosed multiple sclerosis pati-ents, which cannot be detected in samples from the persons who are not infected with the type-C retrovirus, the nucleo-tide sequence being obtainable by isolating nucleic acids from the cell culture according to any of claims 1-13 or the purified retrovirus according to any of claims 14-18 and determining the sequence.

34. A nucleic acid according to claim 33 being obtainable by isolating nucleic acids from the cell culture according to any of claims 1-13 or the purified retrovirus according to any of claims 14-18 by the use of virus-specific nucleotide primers recognizing specific regions of the nucleotide se-quences from the retrovirus.

35. A nucleic acid having a nucleotide sequence (S) obtain-able by using a retrovirus-related nucleotide primer recogni-zing conserved regions of known retroviruses to obtain nu-cleotide sequences derived from the cell culture according to any of claims 1-13 or the purified retrovirus according to any of claims 14-18 by PCR, optionally sequencing the nucleic acids obtained to identify sequences which are distinct from sequences from known retroviruses, testing the nucleic acids obtained by PCR or the sequences identified by sequencing by using them as primers on blood samples from a number of per-sons who are not infected with the type-C retrovirus and blood samples from diagnosed multiple sclerosis patients, respectively, and selecting, as the nucleotide sequence (S), the nucleic acids or sequences which give rise to the attain-ment of nucleotide sequences from diagnosed multiple sclero-sis patients in PCR, but do not give rise to attainment of nucleotide sequences from persons who are not infected with the type-C retrovirus, or using such nucleic acids or sequen-ces as primers for an additional PCR obtainment of nucleic acids from the cell culture according to any of claims 1-21, performing the testing defined above using such second gene-ration nucleic acids or sequences identified therein as primers and selecting, as the nucleotide sequence (S), the nucleic acids or sequences which give rise to the attainment of nucleotide sequences from diagnosed multiple sclerosis patients in PCR, but do not give rise to attainment of nu-cleotide sequences from persons who are not infected with the type-C retrovirus.

36. A diagnostic agent comprising a nucleotide probe which is capable of detecting a nucleotide sequence according to any of claims 33-35.

37. A method of diagnosing multiple sclerosis, an early stage or a pre-stage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to any of claims 1-13, comprising subjecting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient to a PCR analysis in which the sample is contacted with a diagnostic agent accord-ing to claim 36 allowing any nucleotide sequence to be ampli-fied followed by detection of any amplified target nucleotide sequence.

38. A method of in vitro diagnosing multiple sclerosis, an early stage or a pre-stage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to any of claims 1-13, optionally combined with a method for detection of an infection with another virus such as a herpes group virus, comprising contacting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent according claim 36 comprising a nucleic acid according to any of claims 33-35 and determining the presence of any identical or homologous nucleotide sequences in the sample.

39. A method of in vitro diagnosing multiple sclerosis, an early stage or a prestage thereof, a latent infection or a subclinical infection with the retrovirus of the culture according to any of claims 1-13, comprising contacting a sample of body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent according to any of claims 29-32, and deter-mining the presence of bound antibody from the sample.

40. A method of in vitro diagnosing multiple sclerosis or a prestage thereof, a latent infection or a subclinical infec-tion with the retrovirus of the culture according to any of claims 1-13, comprising contacting a sample of a body fluid, such as a blood sample, or a tissue sample from a suspected multiple sclerosis patient with a diagnostic agent according to any of claims 21-24 and determining the presence of bound antigen from the sample.

41. A method for producing an antigen or epitope characte-ristic to the retrovirus, which comprises cultivating cells harbouring a plasmid which contains and is capable of expres-sing a nucleotide sequence according to any of claims 33-35 which codes for a polypeptide which has the properties of the antigen or epitope of claim 19 or 20.

42. A method for producing an antigen or epitope characte-ristic to the retrovirus, which comprises synthesizing a peptide having an amino acid sequence derived from the nu-cleotide sequence of any of claims 33-35.

43. A method for producing a antibody according to claim 25, which comprises immunizing an animal with an antigen or epitope according to claim 19 or 20 or an anti-idiotypic antibody according to claim 28 or an antigen or epitope produced by the method according to claim 41 or 42 to obtain cells producing an antibody specific for the antigen, and isolating the antibody from the animal or the cells.

44. A method according to claim 43 for producing a monoclonal antibody according to claim 26, which comprises immunizing an animal with an antigen or epitope according to claim 19 or 20 or an anti-idiotypic antibody according to claim 28 or an antigen or epitope produced by the method according to claim 41 or 42 to obtain cells producing an antibody specific for the antigen, fusing the cells with cells of cell line capable of rendering the fused cells immoral, and selecting and cloning the resulting hybridoma cells producing the monoclonal antibody, or immortalizing an unfused cell line producing the monoclonal antibody, followed by growing the cells in a medium to produce the monoclonal antibody, and harvesting the monoclonal antibody from the growth medium.

45. A method for obtaining a protective immunity in an ani-mal, including a human being, against multiple sclerosis caused by the retrovirus of the culture according to any of claims 1-13, comprising administering, to the animal, an immunogenically effective amount of a vaccine against Ep-stein-Barr virus.

46. A method according to claim 45, in which the vaccine is live or Epstein-Barr virus administered to the mammal at a stage in the development of the mammal in which the disease caused by Epstein-Barr virus has a mild clinical cause and cannot induce active production of the retrovirus of the culture according to any of claims 1-13 to result in multiple sclerosis, an early stage or a pre-stage thereof.

47. A method according to claim 46 in which the mammal is a human, and the stage in which the administration is performed is the pre-puberty stage.

48. A method according to claim 47, in which the vaccine is attenuated Epstein-Barr virus or an immunogenic antigen cha-racteristic to Epstein-Barr virus and eliciting the formation of antibodies against Epstein-Barr virus.

49. A method according to any of claims 45-48, in which the mammal to which the vaccine is administered is a mammal which has been shown to carry the retrovirus of the culture accord-ing to any of claims 1-13 by any of the methods according to any of claims 37-40.

50. A method for obtaining a protective immunity in an ani-mal, including a human being, against multiple sclerosis caused by the retrovirus of the culture according to any of claims 1-13, comprising administering, to the animal, an immunogenically effective part of a cell culture according to any of claims 1-12, a purified retrovirus according to any of claims 13-18 or a fragment thereof, an antigen according to any of claims 19 or 20 or an anti-idiotypic antibody accord-ing to claim 28.