AU620801B2 - Viral antigen, process for its production, and application in diagnosis and therapy (vaccine) - Google Patents

Description

91 tilt: udmI dpiiiiadtI... rctcricu t0 inl paragrapt 01 inis uieciaration were the first application....S.... made in a Convention country in respect of the invention the subject of the application.

Insert place and date of signature.

Signature of declarant(3) (nio sittstation required) Note, Initial all alterations.

Declared at KarJ8ruhe Renate SEELIG/ this 9th dayof Dec. 1988 Han s-Peter L )1Jea n BUR CK H ARD T

CANBERRA,

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l)AVIIS ('OLLISON, MELBOUIRIt!E and 'I -7 4, WELTORGCANISATRtN FOR GEISTIGES ElGENTUM PCT ~Internatiorsales Biiro

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INTERr.qATIONALE ANMELDU G R RFFENTLIGHT NACH DEM VERTRAG OBER DIE INTERNATIONALE ZUSAMM& R T F lIET SP ENW ES(C) (51) Internatiotnale Patentklassifikation 4 at le Ye ffentiitn-snummer: WO088/ 06184 C12Q 1/68, G01N 33/576 Verifffentlichungsdaturai: 25. August 1988 (25.08.88) (21) Internationales Aktenzeichen: PCT/EP88/OO 123 (81),Bestimmungsstaateu: AU, DK, Fl, JP, KR, NO, SU, I us, (22) Internationales Anmeldedatum: 19. Februar 1988 (19.02,88) Yerdffentlicht Mit internationalem Recherchenberich t.

(31) Prioritlitsaktenzei .2en: P 37 05 512.7 P 37 44 242.2 (32) Prioritiitsdaten: 20, Februar 1987 (20.02.87) 24. Dezemnber 1987 (24.12.87) (33) Prior,(titsiand: DE (71)(72) Anmeldei, und Erfinder: SEELIG, Renate (DE/DE]; SEELIG, Hans, Peter [DE/DE]; BURCKHARDT, Jean [CH/DEJ; Kriegsstr. 99, D-7500 Karlsruhe (DE),A.Q 3OT18 (74) Anwa It: DEUFEL, SCHON, HERTEL, LEWALD, OT- -0 .P 13OT 98 AUST RALIAN 14 4SEP 1988 PATENT OFFICE (54)Title: VIRAL ANTIGEN, PROCESS FOR ITS PRODUCTION, AND APPLICATION IN DIAGNOSIS AND THERAPY (VACCINE) (54) Bezelchnung: VIRUSANTIGEN, VERFAHREN ZU SEINER GEWINNUNG UND ANWENDUNG IN DIAG- NOSE UND THERAPIE (IMPFSTOFF) (57) Abstract A DNA of approximately 5 KB, associated with non-A,non-B hepatitis, process for the production of same accord- Ing to known methods, and application of said DNA or of fragments of same in the diagnoi~ of non-A,non-B hepatitis, as well as in the synthesis of proteins for the generation of immunological reagents to dete~t non-A,non-B hepatitis or to produce vaccines, The DNA and fragments of same can be cloned and also be introduced iv~to appropriato vectors, in order to obtain viral expression products.

(57) Zusammenfassung Eine Non-A,Non-B-Hepatitis assozijerte DNA von etwa 5 KB, ein Ver',ahren zur Herstellung derselben nach an sich bekarinten Methoden und die Verwendung dieser DNA oder von Fragmentet, davon zur Diagnose von Non-A,Non-B-H-epatitis sowie zur Synthese von Proteinen zur Erzeugung von lmmunologischt-I, Reagentien for den Nachweis von Non- A,No n. B- Hepatitis (Ader zur Erzeugung von Vaccinen, Die DNA und Fragmen,,e, derselben k~nnen geklont und auch in geeigoete Vektoren eingesetzt werdeii, um Virusexpressi'onsprodukte zu lieferri.

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COMMONWEALTH OF AUSIRALIA PATENTS ACT 1952 CQMWfSPJ= 51Q NAME ADDRESS OF APPLICANT: Renate Seelig Kriegstrasse 99 D-7500 Karlsruhe Federal Republic of Germnany Hans Peter Seelig Kriegsstrasse 99 D-7500 Karlsruhe Fedoral Rqublic of Germany se* ""0.0 *00 0 06 0 Jean Burcklardt Kriegsstrasse 99 D-7500 Karlsruhe Federal Republic of Germany NAME(S) OF INVENTOR(S): Renate SEELIG Hans Peter SEELIG Jean BURCKHARDT ADDRESS FOR SERVICE: DAVIES COUSON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE S.PECIFICAT[ON FOR THE INVENTION ENTITLED: Viral antige,,n,, process for its production, and application in diagnosis and therapy (vacchw-) The following statement is a full description of this invention, including the best methodt of performing it known to me/us:- I 01i I Im"-- VIRUS ANTIGEN, METHOD FOR ITS PREPARATION AND ITS USE FOR DIAGNOSTIC AND THERAPEUTIC PURPOSES (VACCINES) There are numerous extensive descriptions of hepatitis non-A,non-B in the literature. At the same time, the difficulties of isolating a hepatitis non-A, non-B v.rus, and of proper diagnosis of the disease are known.

From feces of patients suffering from hepatitis non-A,non-B we could isolate particles, characterize them with respect to their molecular weight and physical nature, and develop a method for the detection of such particles. We also used the detection of these particles for the diagnosis of the presence of hepatitis non-A,non-B in patients with liver disease.

oe DNA was isolated from the particles prepared from feces, and was cloned by conventional methods. The DNA strands thus obtained were again used for the detection of the presence of DNA homologous or very similar to this DNA in feces, serum, liver tissue, and body fluids of patients with liver 25 disease, where presence of hepatitis non-A,non-B can be diagnosed with sufficient certainity this way.

In summary, the present invention relates to the isolation of hepatitis non-A,non-B associated particles of their DNA and cloning of DNA, furthermore ito the use of those particles as well as of Lhe DNA cloned therefrom for the restriction of liver diseases to the presence of hopatitis non-A,non-B, Further aspects of the present invention deal with the development of broader methods for dotoction of, eventually the development of vaccines against hepatitis non-A,non-B, based on antibodies against these particles, and on synthetic peptides possessing the sequence of the

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Accordingly, the present invention provides a hepatitis non-A, non-B virus DNA selected from the group consisting of a first DNA having the DNA sequence of Fig. 1, a second DNA having deviations up to 5% from the first DNA, a third DNA being a fragment of the first DNA, and a fourth DNA having deviations up to 5% from the third DNA.

A substance was isolated from feces of patients suffering from hepatitis non-A, non-B, which is found with significantly increased frequence in feces of such patients with hepatitis non-A,non-B, but significantly decreased frequence in patients with liver diseases due to other causes, A method for the detection of this substance was developed, wherein polystyrene beads are coated with prediluted serum from patients reconvalescent from hepatitis non-A,non-B.

.The washed beads are then incubated with a 10% fecal suspension from a 15 patient to be analysed, whereby any hepatitis non-A,nonB associated substance present in the feces binds to the antibodies against this substance, adsorbed to the polystyrene beads, thereby being immobilized on them. Binding of hepatitis Snon-A,non-B associated particles can be detected by binding of human IgC, also prepared from serum of patients reconvalescent from hepatitis non-A,non- B, and labelled with radioactive Iodine'2s, Radioactiitiy-labeled immunoglobulin is bound to the hepatitis non-A,non-B associated substance in feces of a patient, if present, and the signal obtained can be used for the detection of the particles.

Using this method to detect the said hepatitis non-A,non-B associated S 25 substance in large populations of patients with liver diseases of various etiologies, it could be shown that it is found with significantly increased frequency in feces of patients in whom hepatitis non-A,non-B could be confirmed (ref. to tab. In patients with other kinds of liver disease, where hepatitis non-A,non-B is not the probable cause, but who resemble in their clinical presentation patients with acute hepatitis non-A,non-B or reconvalescent from hepatitis non-A,non-B the said substance was found only in some rare cases (ref. to tab. 2 and From these findings it becomes evident that presence of this hepatitis S/ t i- 11 .i etiology of any at first unknown, infectious disease of the liver. Furthermore, detection of this substance can be a valuable diagnostic test showing the presence or absence of hepatitis non-A,non-B in a patient.

The hepatitis non-A,non-B associated substance shows a high affinity towards human immunoglobulins as well as fibronectin and its non collagen-binding cleavage products The binding to f (ab) fragments of IgG from healthy probands as well as patients reconvale rent from hepatitis non-A,non-B makes any unspecific binding of the hepatitis non-A,non-B associated substance to the Fc part of the IgG improbable. Its high affinity towards fibronectin and its non-collagen-binding cleavage products is a property shared with other antigenic viral proteins (ref. Seelig et al., 1983). Treatment with organic solvents, such as chloroform or ether, and with heat (70 °C for min.) does not destroy the binding affinity of the hepatitis :pti non-A,non-B associated substance. While digestion with chymotrypsin, trypsin, elastase, and neuraminidase has no influence on the binding affinity of the substance, digestion with papain results in a fast and complete loss of 5 this binding affinity.

After centrifugation at 150 000 x g for 2 h, tne substance aan be detected in the sediment, and is found, after 72 hours centrifugation on a cesium chloride gradient at a densitiy corresponding to 1.3 (1.29 to 1.32) g/ml. Separation of the cesium chloride band at density 1,30 g/ml by electrophoresis on a polyacrylamide gradient gel, and silver staining'of the gel reveals multiple bands corresponding to various molecular weights. Following transfer to nitrocellulose, a Western blot using radioactively labelled IgG and F (ab) fragments from hepatitis non-A,non-B patients and healthy donors shows bands not found in fecal extracts of healthy controls. In total, four bands are visible, namely 1$ hor e tiug to na c si m clrd gain l 4 r 1 two main bands corresponding to a molecular weight of appr.

64 000, and 56 000, resp., and two minor bands with molecular weights of appr. 51 000 and 43 000, resp.. Bands obtained with F (ab) fragments are less strong and show a higher background. Analysis of raw fecal concentrates by SDS-PAGE and blotting revealed, in positive fecal samples, the two main bands with a MW of appr. 60 000.

Further analysis of the hepatitis non-A,non-B associated particles led to the isolation of a DNA and cloning of DNA fragments by conventionl techniques. These cloned DNA fragments from fecal samples of hepatitis non-A,non-B patients could be employed as DNA probes to analyze feces, sera, liver tissue and body fluids, as well as whole blood and plasma derived products from other patients with suspected hepatitis non-A,non-B. Using classical hybridization techniques it could be shown, whether DNA with a sequence of sufficient homology to that of the cloned DNA to give a hybridization signal is present in the unknown fecal samples. Applying this further method to detect hepatitis e non-A,non-B associated DNA sequences, patient samples could be analysed for the presence of hepatitis non-Anon-B associated DNA. Our results obtained so far indicate that the hepatitis non-A, non-B associated substance is a virus 25 particle, and that the DNA isolated from the substance is a S virus DNA, as is evident from both the extraction procedure, and the sequence of the DNA obtained.

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The following examples further illustrate the present invention.

1 Example 1 Isolation of the hepatitis non-A,non-B associated substance from fecal samples of patients Buffer used: Tris-HCl, pH 7.4, 0.05 N; in all steps.

a) Extraction 0.5 g of a fecal sample were suspended in 10 ml of buffer, centrifuged at 8 0000 x g, and the supernatant was collected. The pellet was reextracted twice with 10 ml and 5 ml buffer, resp.. The combined supernatants were subjected to centrifugation at 10 000 x g for 30 min., followed by filtration through a 0.22 micrometer filter (Millipore) to remove bacteria, and PEG 6000 in a final concentration of 0 or 0.4 mol/L was added. After at least 2, but within 12 hours, the precipitate formed was isolated by centrifugation and then redissolved in 10 ml of buffer. This solution was S" 20 extracted with 10 ml of freon, and the two phases were separated by centrifugation. The freon phase was washed once with 5 ml of buffer. The precipitation with PEG and NaCl and0,4mol/L, resp.) was repeated, and the precipitate redissolved in appr. 0.8 ml of buffer.

S" The solution thus obtained was treated with RNase and DNase for 1 h at 37°C, using 0.8 ml PEG precpitate in Tris-HCl buffer, pH 7.4, 0.05 mol/L, 2 000 U/I RNase, and 1 500 U/1 DNase in essentially protease free prieparations (Boehringer) This step could be ommitted, if the preparations were not to be used for the extraction of DNA.

After DNase and RNase digestion the incubation mi tureo is transforred to a ccsium chlorido gradient.

i1 W n i 6 b) Isolation of hepatitis non-A,non-B associated substance by cesium chloride gradient centrifugation Centrifuge tubes were prepared containing 1 ml of a cesium chloride solution with a density of 1.4 g/ml, on top of which 3 ml each of solutions with consecutive densities of 1,3, 1,25, and 1,2 g/ml, resp., were layered. All cesium chloride solutions had been prepared using the buffer formulated as given above. 0.800 ml of fecal extract were layered 1 onto this gradient. Centrifugation time was 65 to 72 h, at a temperature of 100 C and 31 000 rpm. When centrifugation was completed, the lower part of the gradient, at a density of 1,2 to 1,4 g/ml, was collected in fractions of appr, 0.200 ml, while larger volume fractions could be taken in the upper part, density 1.1 to 1.2 g/ml. The density ot each fraction taken was determined by measuring its refraction S*index. All fractions were dialysed extensively against buffer, and 0.050 ml of each fraction were analyzed for the presence of hepatitis non-A,non-B associated substance.

20 Protein content of positive fractions was measured according to the method of Lowry.

Verification and characterization of the substance c) Preparation of a gradient PAGE S. A linear gradient was prepared from two solution with different concentrations of acrylamide using a gradient mixing apparatus. The solution having the higher concentration of acrylamide contained, in addition, 15 sucrose to prevent turbulences when forming the gradient. Otherwise, the solutions had the compositions given by Laemmli (1970) The same was true for the concentrating gel on top of the gradient.

The fractions obtained from the cesium chloride gradient, which had been dialysed and, if necessary, concentrated, 0.005 ml 0.1 9 pyronin were added, and the samples were applied to the gel. Run time was 3.5 h, at 160 to 300 V, to 25 A, and 20 W.

Appr. 5 min. before electrophoresis was completed, another 0.005 ml of pyronin were applied, and the run was terminated when the pyronin had passed the concentrating gel. The gel wa[ stained with silver stain (Wray et al., 1981) or used foi 3 Western blot (Towbin et al., 1979) Blotting was performed over night at 0.5 A, followed by 1 h at 1 A.

d) Treatment of Western blots

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After blotting multiple strips, easily identifiable by their et* 20 pyronin marks, were cut out, and molecular weight markers run in parallel were stained with amido black. Sample strips were treated by incubation in 1 gelatine/PBS for 24 to 72 h with shaking. The IgG fraction from a patient, and the F(ab) fragments, resp., were labelled with "'Iodine (chloramin T) at 0.5 mCi per 0.100 mg of protein, resulting Sin the incorporation of appr. 70 t of the activity. Of this tracer, a volume corresponding to appr. 0.002 mg of protein, was diluted with 20 ml of gelatine/PBS, and the strips were incubated in this mixture for 12 h with shaking. They were Sthen treated for 1 h each by washing in golatine/PBS, three 8O times, PBS-Tween (0.5 t) twice, and once with water. After drying, the strips were transferred to sensitive X-ray film and exposed for 2 to 7 days at -70" C.

Example 2 Method for the detection of hepatitis non-A,non-B associated substance in fecal samples Polystyrene beads (Plasticball Company, Chicago) were incubated for L? t at R.T. with sera of patients reconvalescnet from hepatitis non-A,non-B, diluted 1:200 with carbonate buffer, pH 9.2, 0.01 mol/L. The beads thts coated were washed extensively with PBS (phophate buffered saline). and incubated with fecal samples, applied as 10 (wt./vol.) fecal suspensions, for 2 h at 370 C. Following extensive washing with PBS containing 0.5 t Tween 20, the beads were incubated for 1 h at 37' C with human IgG from sera of patients reconvalescent from hepatitis non-A,non-B, which had been labelled with Itlodine, The beads were then subjected to prolonged washing with destilled water, and bound S 20 radioact 4 'ty was measured in a gamma counter. Fecal samples in which bound radioactivity was threefold higher than the negative control were, by definition, considered positive for the presence of hepatitis non-A,non-B.

25 The data in sach of the tables I to 4 were obtained as described above. Referring to table 1, the substance was found in a large percentage of patients with confirmed hepatitis non-A,non-B. According to table 2, the substance is found only in low percentages, if at all, in groups of patients suffering from a variety of liver diseases of different etiologies, but not related to hepatitis non-A,non-B. Table 3 hhows that the hepatitis non-A,non-B assocldaed substance is found with very low percentages when testing groups of patients with other forms of Infectious hepatitis, A.e. hepatitis A or hepatitis It can bo said that in cases of hepatitis non-A,non-B practically all groups have )ositive findings in 30 or more, while in the groups with hgpatitis A, suspected hepatitis A, hepatitis B,

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I I and suspected hepatitis B these, figures are considerably lower.

The relatively hiqh niuber of positive findings in chronic hepatitis B patients could presumably be due to concurrent infection with hepatitis non-A,non-B virus and hepatitis B virus.

Table 4 gives the results of a study on recipients of blood transfusions. These have to be considered as hic~h risk patients, because of the high iiicidence of hepatitis non-A,non-B infections after blood transfusions. The study was designed as a prospective study. The results clearly show that, depending on the degree of post-transfusion events, patients with no post-transfusion hepatitis excrete in their feces the substance on2ly to a minimal extent. on the other hand, patients with signs of post-transfusion hepatitis, a~nd patients with manifest post-transfusion hepatitis excrete the hepatitis non-'A,non-B associated substance in over 70 b' of the cases studied.

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,vt&~3 L%? Example 3 isolation of DNA from feces,cJloning of DNA sequences# and their incovporactioh into vectors Hepatitis non-A,non-B associated particles were isolatod from feces of patients Wit i hepatitis non-A,non-8, as describec' in Example 1, treated with RVase and ONase, purified over a cesium chlorido gradianto and dialysod oxtofl!ively.

The dialysed fractions of the cesium ohlorido gtd2twere tested for the presonca of the hapatitis non-Alnon-0 associatod substance, as described above 0 Fractions corresponding to a donsItiy of 1.3 cg/inl ware treaited by digosition with 0.050 ing Proteinase K, I 'a SI) and ]CDTA in a final concontration of 10 mnmol/L for 6 h at 371, C. Proteins we remnoved 1 by extracting the reaction mixture with 80 %phenol (wt./vol.) and chloroform. The DNA dissolved in the aqueous phase was precipitated by adding a 2.5 fold volume of ethanol in the presence of 0.3 mol/L sodium acetate and keeping the mixture at -700 C for 60 h, followed by centrifugation. The sediment was washed once with 70 b% ethanol, j dried, and finally redissolved at 0.001 ml per 5 mg of feces extracted in TE buffer, consisting of 10 mnmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA.

0.015 ml of this DNA stock solution were inc,_bAted for 1 h ht in a total volume of 0.050 ml, with 6 U Kienow-Polymerase (DNA polymerase I, large fragjment) ,0.001 ml of a solution containing dATP, dTTP, and dGTP in a con- 15 centration of 1 nuiol/L each, and 0.005 ml aipha-IlP-dCTP, I 000 Ci/mmole, 10 mci/ml, in the incubation buffer for Kienow polymerase according to the manufacturer's (Boehringer, Maninheim) description. Thereafter 0,0G25 ml o.f a 1 mmol/L solution of dCTP were added, and the sample was incubated for another hour at Following inactivation of the enzyme by heating to 680 C for 10 min., the reaction mixture was cooled to 00 C, and then incubated for 16 hi at 160 C with 2 Units of T4-DNA-ligase, 0.001 mg phosphorylated EcoRI linker and 0.006 M! of a 10 mmol/L ATP solution, After adjusting the sodium chloride concentration to 150 tnmol/I,, and addition of 240 U of restriction andonuclease EcoR 1, V. kU/mI, the reaction mixture was incubated for $3 h at 37Q C.

The reaction was stopped by adding 0,005 ml 80 b pher.!51 and 0,001 ml 20 40 S0S. The DNA labelled with radiolotiVity and Provided with linkers was 'freed of salt and Linligatod linkers by passing it over a Sophtiroso 401-CL colurmn, volume 3 ml, length 25 om, and procipitntod with volumes at ethanol for 16 h at -70" C, 'rhe DNA pruoip.tto~ wao~ rocovored by centrifugation for 1O mitn. at 14 000 X q, ond the sediment washed with 0. 150 ml, 70 o thanol, driudo and redissolved In 0,010 ml T1C b'ffoe, 1 11 Incorporation of isolated DNA into lambda-phages, and transfection to bacteria To effect ligation with vector DNA, 0.P02 Mjq DNA of phaqe lambda 1149 were treated by incubation for Ih at 37 C with 2U EcoRI (4 kU/mi) in a buffer according to the manufacturer'Is description in a total volume of 0.006 ml. The enzyme was then inactivated by heating the reaction mixture to 680 C for min.. Ligation was performed at R.T. by adding to this solution 0.003 ml labelled DNA, prepared as described above, 0.001 ml ATP, 5 mnmo2,/L, and I U of T4-DNA ligase, 0.004 nil of the reaction mixture were packed into lambda-phage coats, using a commercially available packaging mix manufactured by Oga-Pack, Vector-Cloning systems. These Packed phages were used to transfect E. coi strain NMl 514. To screen for ir~sertion of DNA, appr. 101 pfu were plated onto screening ~*plates, 22 22 cm in size, (Nunc) and incubated over night 37" C. Phage DNA was then transferred to nitrocellulose f ilters by blotting, and the replica filters thus obtained were hybridized, according to the method described in Maniatis t al., 1982, using 0.001 ml of labelled DA .*9prepared as described above, washed, and visualized by autoradioqraphy for 6 h at -70o C.

Outof7. giving positive hybridizat~ion stgnala, 17 corr* u5,pondinq phage cooiswore pltdat a density of appr, 5 PfU/cmn'. Following plaque, puri fica tion according to I enton and Davis, 197M 16~ remaining positive plajques were used to prepare lysatos, Several of those PhacoQV contained DNA inserts rangincj in lenghth fromn 0,3 to appr, kb, mid giving a positive signial when-t hybridi"-od to the original DNA $tock solutiton, 12 1 Example Characterization and subcloning into the pUC 19 plasmid in E. coli of a 0.45 kb DNA fragment The DNA fragment was cut out from the phage DNA using the appropriate restriction endonucleases (EcoR I) under conditions described above. The fragment thus obtained was separated from the phage DNA by electrophoresis on agarose gel.

and the band corresponding to the DNA insert was cut out and eluted from the gel. thp insert DNA obtained was ligated to the vec'tor plasmid pUJc-19 in the same manner, as described above, and, the resultinq plasmid transfected to the E. coli strain DH 1. Following selh7tion of bacterial colonies which had incorporated the plasmid cirrying the hepatitis non-A, .01 non-B DNA insert, and amplification, the strains were worked up as described in Maniatis et al., "Molecular Cloninig, a Laboratory Manual", 1932.

The same insert was also radioactivity labelled and tested by Southern, blot hybridization to the DNA used as starting material, DNA\ extracted from feces of healthy controls, hepatitis B viral DNA, and plasmid pBR 322 DkNA, 6 Detection of hepatitis n~n-A,non-3 associated DN A in serum 2 to 5 ml of serum were centrifuged at 130 000 x g for 2 h.

The sediment was treated by digestion w~kth 0.200 ml, of a proteinaso K nolution, 0,5 ilg/mI, containing 10 mmol/L DTA, at 37' C fc~ I flour. Then, O.t65 I'll of a saturated oiution of sodiutm Lod Ldo, 2.5 q/iwater at 75.n C, we-,r, r0-osUtinc:T in ;I final Qonontatiori of 12.5 mo.l/L. This Mixture WaS IQated to d00l C tor LO min. and imedintely pised through a rfrctul~( iltte placod in a (lot blot 13 1 ayparatus (Schleicher and Schuell) Following filtration, the n,,*.trocellulose sheet was washed three times with 70 t ethanol, and incubated for 10 min. in 1,30 ml acetic anhydride, containing 0.250 ml triethanolamine at The sheet was then baked for 1 to 2 hours at 800 C under vacuum.

The dry sheet was placed for 3 hours at 65' C in a pre-hybridization solution containing 6 x SSC, I x Denhardt's reagent, 0.5 t SDS, and 0.020 mg heat-denatured herring sperm DNA, according to the method given in Maniatis et al., 1982, cited above. It was then incubated over-night under V, pre-hybri~dization conditions with a DNA probe labelled by nick-translation with P as Oescribed by Naniatis et al., and washed three t~imes in 1) 6 x SSC, 1 x Denhardt's reagent. 0.5% SDS and 0.020 mg herring sperm DNA, 2) 1. x SSC, 0.5% SDS, 1 x Derihardt's reagent, and 3) in 0.1 x 850, 0.05% SDS. After drying the 1415 sheet was placed on X-ray film (Kodak X-Omat) and autoradiographed for 6 hours to 2 days at _-700C In sera with a high content of hepatitis non-A,non-B virus the concentration step, consisting of centrifugation of 2 to 205 ml of serum, can be omiitted, and sera can be subjected directly to proteinase K digestion and applied to nitrocellulose filters, as described by us in Seelig et al., t' V.Klinikarzt 2/1985, p. 86. (ref. to example 7) Exa mle Detection of hepatitis non-A,non-B DNA in serum 0.200 ml of serum -qere incubated with 0.075 mi. of a solution, containing protoinase K (IBoehringer, Mannh~jim) ,4 mg/mi, and 0.025 Ml EDTA, 0.5 mol/L, for 1 hour at 370 C. The incubation mixture was then mixed with 0.100 ml sodium hydroxide, I moi/L, further incubated [orc 10 min. at folliowad by neutralization with 0.250 ml,. of ammonium acetate, 2 mol/L.

0.500 mi this3 mixture, corresponding to 0.101 ml ot A A.t.wink9 14 1 serum, were applied to a nitrocellulose filter using a dot-blot apparatus (Schleicher und Schuell) After neutrali- Zation with another 0.250 ml of ammonium acetate, 2 mau/L, the f ilters were baked under vacuum at 8 00 C f or 4 5 min..

The filters were then pre-hybridized for 3 hours at 65 0 C in 6xSSC, 0.5 %SDS, 1 x Denhardt's reagent, and 0.020 ml heat-denatured (5 min.

at 1000 C) herring sperm DNA. (20 x SSC (standard saline citrate) is a soluition containing sodium chloride, 3 mol/L, and sodium citrate, 1.5 mol/L; SDS is sodium dodecyl sul- 100 x Denhardt's reagent is a solution of Ficoll, 2 polyvinylpyrrolidone, 2 and bovine serumalbumin, 2 Hybzridization was performed over night under pre-hybridisation conditionis usinig the phage clone insert labelled by nick-translation to a specific activity of appr. 1 to 2 x 10' cpm/microgram DNA. The filters were then washed for 20 min. at 65' C once with 1) 6 x SSC, 1 x Denhardt's reagent, 0.5 t SDS, 0.020 mg/ml heat-denatured herring sperm DNA, followed by 2) 1 x SSC, 1I x Denhardt's reagent, 0,5 SDS, and 3) 0.1 x SSC, 0.05 SDS, and bake-dried at 800 C. Autoradiography was done on 20X-ray film using intensifying screens (Kodak) for 6 to 48 hours at -7011 C.

E>xamole 8 *,25 This example describes a simpler variant of the sample preparation for dot-blot: 1 mil of serum was incubated for 30 min. at 37"C viith 0.330 l Of: a solution containing protcainase K, 3 mg/mI, *eTris, 10 mmol/L, pH 7.5, EDTA, 0.5 mmol/L, and. SUS ana 0.125 nil EDTA, 0.5 mol/'L. The solution was then diluted with 8 mil TCA, 2 mol/L, pH 7.0, 3.2 mil ammoniam acetate, 2 Mol/L, 0.020 mil t-1RNA, 10 mag/i, anid 5.3 mil water to a total volume of 16 mil, and tha DNA precipitated with 10 ml) isopropanell at R.T. for 30 min. The DMA was isolated by centrifu- 3, gation for 15 min at 8000 rnm, the pellet was washed with 2 mil of 70 4t ethanol, and redissolved in 0.600 nil Tris, mmol/L, pH 8.4, and EDTA, 1 minol/L. The solution was 7-s- 1 extracted once with phenol, once with chloroform, and then precipitated once more. Aliquots of the DNA precipitate were used for dot-blots or restriction enzyme analysis.

The preparation of particle-associated DNA from fecal samples is done in the same way. Fecal suspensions, however, are first sterilized by filtration and subjected to PEG precipitation.

Example 9 ,If only relatively small amounts of the hepatitis non-A,non-B associated substance are present it has been found useful to amplify the DNA for detection.

The detection of hepatitis non-A,non-B DNA in serum is appropriatly achieved by hybridization to a radioactivitylabelled, cloned hepatitis non-A,non-B-DNA probe following specific enzymatic amplification of the DNA by known tecnni- S'0. ques, as described by Saiki et al., Science 230: 1350 (1985).

0.025 ml of serum and 0.050 mi of a saturated solution of sodium iodide were mixed and incubated first for 2 min. at 370 C, then for 10 min. at 0° C. The incubation mixture was 'then dialyzed for about 20 min. through a polycarbonate 25 membrane Uni Pore, Bio-Rad) against TE-buffer containing Tris, 10 mmol/L, and EDTA, Immol/L. A 0.005 ml sample of the dialysate was drawn to be used for specific enzymatic [S DNA-amplification. Amplification was performed, following the method described by Saiki et al. cited above, using micrograms each of oligo-primers pair A and B, and C and D, resp.. The pairs of oligo-primers were prepared, based on known sequence data, using a DNA synthosizing apparatus (Applied BioSystom 381) After amplification the reaction mixture was separated by oelctrophoresis on a 2 agarose gel, transferred to a nylon membrane (CGenfit) and hybridized to a cloned IINAND-DNA probe label- 4K ,reconvalescent from hepatitis non-A,nonrB the said substance was found only in some rare cases (ref. to tab. 2 and From these findings it becomes evident that presence of this hepatitis

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-16- 1 led with "P according to the method described by Feinberg et Annal. Biochem. 132: 6-13 (1983) Followincj autoradiography, positive samples were identified with the help of standards and length markers run in parallel.

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'A'tV~ Detection' of DNA in certified infected plasma derivat ve: In "Deutsches 3esundheitswesen", Vol. 36, pp. 560-563 (1981) the group of Pr-ofessor RENGER; Charite East Berlin, published a paper on a controlled Hepatitis non-A,non-B epidexny, which was caused by application of infectious -Inti trhesus factor D immunglobulin preparations. The origin of these contaminated preparations couiLd be fully evidenced.

After application of these anti-D-preparation 79 out of 116 immuunised pregnant women became ill. This anti-Dpreparation (batch I, ampules A and C) as well as a batch having lower infectivity (batch II, ampules~ B and D) which bM:d been purifie~i by the same system of columns was made available together with control preparations (Gammavenin, Endobolin, Rhesonativ). These preparations have been used together with buffer controls, serum containing Hepatitis B-virus and positive controls with cloned virus-DNA-Fragments.

The assay was donce by DNA-amplification by primers 237 238 which came from the 0.4 Kb EcoRT-fragment).

Results: I n multiple e:~periments, wqhich have boo~n done under various conditions, a'4tor amplification there was a strong signal in b .ch I (anmpuies A and C) and a weak signal, however, having good amplification yield in batch 11 (ampules B and D) The three lyophilisates Gammavenin, EndoboDln and Rhesonativ used as contr,,ls ga-ve no siqnal., Addition of specific primers of liepatiti B-virus-cJonom to thle diff-orent Gamoglobuljn propa ra tions als~o cjavo no Lndicition of a 3~possible contamination with Hepatitis B-genorn. Thc controls do at the same time were used to ovaluate the efficiency of -17amplification with HBV-specific primers as well as with non-A,non-B-specific primers. The evidence of identity of the DNA in the infected batch with the DNA from the feces of a patient having hepatitis non-A,non-B indicates that a hepatitis non-A,non-B-implicated DNA has been identified.

Furthermore feces of 57 patients of sporadic and posttransfusion hepatitis non-A, non-B have been tested by radioimmunoassay and dot-blot under conditions described above and a significant correlation for both test procedures concerning detectable hepatitis non-A, non-B associated substance (RIA) and detectable DNA (dot-blot, see Example 7) was obtained.

The invention is, further explained by the enclosed figures: Fig. 1 is the sequence ofI the genome Fig. 2 shows the cleavage sites using EcoRI as well as the open reading frames and the reading frames Fig. 3 ard Fig. 4 show the plus- and minus strand of a sequence of about 0.3 kb, that is a partial sequence of the genom O Fig. 5 shows the open reading frames, that is the reading frames lA, 2A and 3A, and Fig. 6 shows the open reading frames for reading frames lB, 2B and 3B.

c SSequencing of the sequences shown in the figures was done according to the method described by Sanger using PUC 8 in M 13 strain host cells an n luescript vector respect vely.

The characterization of the original DNA (Fig. 1) isolated from fecal extracts shows it to be a partially double-stranded circular

DNA.

4 i

A

-u 17A The DNA shows homology to HBV-DN,.A. When hybridcizing either a cloned 0,145 kb fragment or purified material from feces, after labelling with 32P, to HBV'-DNA, both pro'bes gave a positive signal, which, however, was about 1000-fold less strong than when hybridized to themselves. Plasmid -Br, 32Z and phage-lambda D'NA gave no sicjnal.

In pilot tests the purifiod fecal ~a eCould be Offectively labelled using small fragmenQ rEcl plrras wihu need to be denatured first.

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Mr t -18- 1 Fecal samples treated with Klenow polymerase migrated significantly slower in agarose gels than did untreated samples. This indicates that the DNA studied is, as in the case of HBV-DNA, only partially double-stranded, and circular.

The determination of the sequence has confirmed these findings.

Sequence analysis of both DNA-strands was done repeatedly and always resulted in 4,998 base pairs. The sequence is shown from 5' to 3' end. Numbering with 1 starts with the first nucleotide of the 2.5 kb EcoRI-fragment in the DNAstrands which.contains the large open reading frames (see fig. A DNA-fragment, not shown in the figure and having about 10 to 20 base pairs (bp) has been experimentally shown to border upon the 2.5 and 1.5 kb EcoRI-fragments and thus causing ring closure of the DNA shown linearly (see fig. 2) Proof of this fragment was done by an amplification experiment. Purified virus-DNA was incubated with Klenow polyme- 20 rase and both synthetic primers 13 and 17, and a multiple amplification was done on the DNA-sequence between both primers and was identified after electrophoresls by Southernanalysis. As both primers are on both ends of the sequenced DNA, amplification of a fragment is only possible if the DNA is circular. Thus, the virus gencm has a total length of DA am bp. 'his rosulg is additionally 5,010 till a maximum of 5,050 bp. This result is additionally confirmed by independent Southern-analysis of the qenom. i *The restriction enzyme cleavage map has been done. The sequence of, f.i. the EcoR'I-fragmcnzs 1.5/0.45/0.3/0.15 and 2.5 kb.

With rescoct to peculiar sfoatuIs cf cho secquc'nco it is to be mentioned that t long pa[IndOom.ic suquencu (Hai'pin) is situated between bp 2,097 and 2. 149 ai the end of anl opn readinq frame (pos. 4, fig. 2).

NT lit -19- 1 At position 424 and 3,303 there is a "CTG"-box. At bp 3,303 there are also both repeats, having sequence homology with the direct repeats of Hepadnaviridae.

The open reading frames are found only on one strand.

The other strand is closed except for smallish peptides as is also usual with hepatitis B-virus-DNA. While the open DNA strain is able to code without further modification for proteins up to molecular weights over 40,000 in the complementary strain far areas of all three reading frames are closed except for 5 peptides of a very small molecular weight of about 6,000 (see fig. 2 and figs. 5 and 6).

All overlapping clones showed identical results for the same sequence with one exception, where at position 2,331 in one V. case G and in another case A has b-in found. A mistake in sequencing may be excluded.

This confirms that deviations of a few percent, especially 1 to 2 usually have no influence on the function of the sequence, so that functional equivalents may have deviations up to 5 especially up to 2 from the basic structure.

25 The same applies for the proteins coded by such D IAs.

Summarizing the following may be stated: The hepatitis non-A,non-B associated substance is characto- 30 rized by its significantfbinding to immunoglobulins and F (ab) fragments of purified IgGs, by its binding to non-collagen-binding fibronectin cloavago products, and by its infectiosity in human cell lines in culture, which car\ be demonstrated by morphological changes typical for viral infection, and by molecular biological detection of a DNA migrating in gel electrophoresis at an apparent size of 3.2 kb (uncut and under MQKj i-

I

1 native conditions) giving a spec" hybridization signal in these cell lines.

The DNA found in these infected cell lines, as well as in sera of patients with hepatitis non-A,non-B hybridizes to the DNA of appr. 5 kb, isolated from the hepatitis non-A,nonB associated substance, and to the DNA fragmentes cloned from this DNA, ;esp..

The total sequence of appr. 5000 bp according to fig. 1 and a partial sequence of appr. 300 bp according to figs. 3 and 4 in the cloned DNA were compared to other known human DNA sequences, phage-DNA sequences, plasmid-DNA sequences, as well as published virus-DNA sequences. Using these data, they do not correspond to any sequence hitherto described.

.i Having established an open reading frame in the sequence the structures of virus specific proteins, coded for by and S..expressed with the DNA described, can be predicted. Thereby, S 2 hydrophilic and hydrophobic regions within peptides can be identified, and 't is possible to prepare synthetic peptides corresponding to possible antigenic sites within the hydrophilic regions.

The DNA was found, especially genes cont, led in this DNA, may be used for insertion in corresponding expression vectors and the recombinant vectors obtained for transfection of bacteria, such as E. coli, yeast, such as "Saccharomyces cerevisiae, and cell cultures. Thus virus antigens may be produced whereby the diagnosis, particularly of an infected blood bank as well as of an acute, chronical or old infection and production of immune reagents and vaccines are enabled. Antigens produced in cell cultures as well as the corresponding proteins synethesized by this A I..

associated substance is found with very low percentages when testing groups of patients with other forms of infectious hepatitis, i.e. hepatitis A or hepatitis It can be said that in cases of hepatitis non-A,non-B practically all groups have positive findings in 30 or more, while in the groups with hepatitis A, suspected hepatitis A, hepatitis B,

'I

i -21- 1 virus DNA in vivo may be used for making immunological diagnostic agents. The DNA sequence may be used for identifying potential virus proteins and for their synthetic production, that is production of synthetic antigenic peptides. Furthermore the DNA and partial DNA sequences -ay be used for producing synthetic DNA or RNA of DNA- or RNAfragments which may be used as detection or catching probes or primers. Finally, the DNA and the found DNA sequence respectively may be used for making synthetic viruses (total DNA-synthesis) and for insertion of synthetic DNA or DNAfragments in suitable vectors to get virus expression products.

**O

S* *0 S S 0r 3 tested for the presence of the hepatitis non-A.,non-8 as-oCiated substance, as described above. Fractions corresponding( to a densitiy of 1.3 g/ini were treated by digestion with 0.050 mq Proteinase K, 1 SDS and EDTA in a final concentration of 10 inmol/L for 6 h at 37' C. Proteins wern removed

I,

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r' t -22- Table 1 HNANB-associated Substance in Feces of Patients with Sporadic and post-Transfusion hepatitis non-A,non-B Type of hepatitis No. of patients positive ,(amples (a) acute post-triinsfusion HNANB chronical post-transf. I{NANB acute sporadic IINANB chronical sporadic HNANB acute IfNANB in contacts of patients chron. H-NANB in contacts of pat.

healthy persons in qonta,t wit1 HNANB patients 84 44 85 106 4 10 36.7 38 32 26 1,00 W b 9eb~ .9 0*91 9. 99 9 9 9 S L. 059 9 9 99..

*0 99 9 .9 9 p 99 59 9 .9 99 S *s .i 59 -23- Table 2 Excretion of H-NANB associated substance in patients with various diseases affecting the liver Type of Disease No. of No. of patients positives autoimmune hepatitis 16 none primary biliary cirrhosis 18 none M. Wilson 2 none hemochromatosis 2 none M. Meulengracht 3 none absces of the liver I, none liver adenoma 1 none liver hemangioma 1 none idiopathic portal hypertension 1 n in e 'post-hepatitis B I none drug-induced toxic hepatopathy 4 none steato-hepatitis 2 none cholangitis, cholelithisasis 8 none liver metastases 7 2 fatty infiltrates of the liver 51 1 clinical diagnosis of liver disease 7 24 alcohol-induced toxic hepatopathy 14 2 increased sereni gainma-GT without alcoihol anamnesis 13 1 unclear inorease of serumn transarninases 20 2 25 inmononucSleosis 2nn sarcoiclosis I none~ *plasmocytoma I none tuberculosis 2 nano syphilis 2 none streptococcal infection I none holminthosi8 3 nao M. Crohn naI.no 0 30 enteritis 2 n nn pyoionophr'ltis 2 none ulcus ventricull I. nono colon irritablilo nao uncloar fonvor 4 1 222 patieItO halve been tested on different days, approximately three times, and 13 positive findings were obtained.

±v±ic a positive signai when hybridLZCOI to T-n' ui .i.j-L Por stock solution.

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1, -24- Table 3 Hepatitis Non-A,Non-B associated substance in HNANO3ViitUs infections Type of hepatitis No. of patients acute hepatitis A suspected hepatitl., A (not tested for ari)ti-HAV-IgM) acute hepatitis R3 chronic hepatitis B HBs-Ag positive liver cirrhosis No. of positives 5 2 t) 2 (5.1 10 (16 9) 1 2. 9 110 sea.

0*9 9. 9.40 3 positive patients with hepatitis A had acquired the disease in Northern Africa 99 mixture was .ieatod to 930" C for 1.0 min. and iianiediatoly passed k.hrouqh a rnitrocoluose filter placod in a clot blot Table 4 Prospective Study in Recipients of Blood Transfusions Conditions No. of Testing of Fecal Samnples patients negative positive t positives no effects 1514 17.1 increased ganuna-GT 22 19 3 13.6 mnild" hepatitis 5 2 3 manlifest post- 7 2 5 71.4 transfusion hepatitis total 63 48 is 31.3 IA a a 4

I

Claims (9)

1. A hepatitis non-A non-B virus DNA selected from the group consisting of a first DNA having the DNA sequence of Fig. 1, a second DNA having deviations up to 5% from the first DNA, a third DNA being a fragment of the first DNA, and a fourth DNA having deviations up to 5% from the third DNA.

2. The hepatitis non-A, non-B virus DNA according to claim 1, wherein the second DNA deviates up to 2% from the first DNA.

3. The hepatitis non-A, non-B virus DNA according to claimn 1, wherein the third DNA has the sequence of Fig. 3 and Fig. 4 resoectively. S'4. The hepatitis non-A, non-B virus DNA according to claim 1, wherein the fourth DNA deviates up to 2% from the third DNA.

5. A protein corresponding to the amino acid sequence encoded by tb; DNA according to claims 1 to 4.

6. A protein corresponding to the amino acid sequence encoded by the open reading frames of Figures 5 and 6. S 25 7. A process for producing the hepatitis non-A, non-B virus DNA according to claims 1-4 and optionally the protein according to claims 5-6 from feces, tissue, body fluids or virus cultures, wherein the starting material, if desired after filtration to sterility and precipitation M on a 2 b agarose gel, transferred to a nylon membrane (Genofit) and hybridized to a cloned HNAND-DNA probe label- 1 1 t i L 1 I 0 27 with PEG for feces suspension, is incubated with DNase and if desired RNase for digestion, then subjected to a density gradient, especially CsC1 d=1.3 followed by dialysis, digestion with proteinase K, phenol extraction, ethanol precipitation and if desired extraction and again precipitation and isolation and optionally cloning and expression in suitable vectors.

8. Use of the DNA according to claims 1-4 as DNA probe for diagnosis or for expression in suitable vectors.

9. Use of the DNA according to claims 1-4 for synthesis of proteins or peptides for producing immunological reagents for detection of hepatitis non-A, non-B and for production of vaccines. DATED this 26thday of July, 1991 RENATE SEELIG, HANS PETER SEELIG and JEAN BURCKHARDT By their Patent Attorneys DAV'IS COLLISON S. *r M61 i.E Fig. 1. Sequence of hepatitis non-A, non-B virus DNA SEQUENME 4998BP; 996 A; 1130C; 1087 G; ii 9 9 9 99*9 .9 9 9.9 *99* ~t9 9 U 9 999499 9 9 13 4*499* 20 GAA1TIC-GCC ±TMIauIVu 3' CrrAGACGG AGAAGACAAC 80 TCrI'ICG AATAGGCA AGAA.ZAGGTC TIrAAA-CCG 130 140 ATGCrrAA ACAAGAAGCr TACCAATAAT 190 200 T7CAGITA ACIT1OGG AGTC-AAAMAT TcGAAAAGCC 250 -260 G7CGCCCG GTATTTATIAA CAGCQCGkGC CArAAAT-ATI' 310 320 Q;ICi'±-JG TIArACC-TCG CAGAACOSCA AAkAATkGC 370 0 CrACTMLTGG CAACI'ITr CMAA.AACC GTIGAM.TAA 430 440 GGTCG GGATGGAGGT CACCGGAAAC CCTACCTCC 490 500 TATGrCTAC GGCGITACCG *ACTACCACTG CCGCAATGGC 550 560 GACCGTCAG CAGTATGATIT *CTGGCAGG-LC GTCATACTAA 610 620 TPAAGCCT GCIGTTIC *ATTICGCGA CGACrAAGAG 670 680 TATTArIGM TC7CC=GCC ATAATNW-'r AGZGGACTG 730 740 TCA'G~CGC--GCT 7TAACCGCTT ACTCCCc=c; AkTCZGA.A 790 .800 TC1CCIMAC GWCTA ACGGOCA-TG (CAGAGAITAT 850 860 TATCQ:LAcGr ACTGAGA.CGG ATACT-GCA TCACWGC 910 '-920 GACCGACr C.ACTTArM GTCGACXG G=-GATAG 30 40 so ATICTGATr TGcCrCCGGr GC7TCIGG-TC T'rrGGG TAAGACI'ACA ACGG-AGGCCA CGAAGACCAG AAGAACCGCC 90 100 110 120 CGmrCITI1 CICTTIr CkxiCrr= GT IGGGCTGI' GCAAAA GAGACAAAGA GAGGAAACGA AACCGCGACA 150 160 170 180 aAGGCGGT TGACGGATGA CrrrIC"LrGA TIWFICMA CrTICCGCCA ACTGCCACT GAAAAGAACI' AAAGAAGTF 210 220 230 240 TICTGGIGGC GCTCICG~lTCA 'GCcGI'I'G AAGACCACCG CGAGAkGCAGT AACGGCAACA*ACAAAAAG A 270 280 290 300 G-TCGAAAG GATTGGI'IG CA 7GGrAGAC TT~IC CAACAlTT CtACCAACG GTACCATCTG AAACAMAGGC 330 340 350 .360 TITATCGCCA ATG7GKfl1C TATFICIT TIC- i AAAIAGCGGI' TACACCAAG ATA.ACGVkA ACCGAAC 390 400 410 420 TrGGcrr=r Tr-rrGCr'C GClTGICGC TT1 CT ±L AACCAA.ACAG AAAACGAAAG CGAIACAACCG AMACAAGALGA 450 460 470 480 GATAAATAGT GGAAATCCCI' AITATTATCAL ATACITGGG CI'AITATCA CCi AGGGA TAATAATAGT TATIGAACCCA 510 520 530 540 TCI'ATACAGT GCAGTATP.AA GATGCFAGCA CXGCTAT AGATATGTCA CGTCATATrT CTACCATCGT GAACGCTATA 570 580 590 600 ATCkAGGC ATCCGCGCAG GCTGIGCCG ATATGGAC7C TAGAGFICCG TAGGCGCGT CGACAGCGGC TATACCM!AG 630 640 650 660 CI'TGGAGC qTCCI= CCCtCAGGAC CIGCACAGZA G?3MGCCrICG ACG=GGACGA GGGCTCC.-TG GACGTGTC= 690 700 710 720 TCCGCG.AAGG TTATGTiGCCG CA4GQGAzkG AATTACCIT AGGCGCTCC AkTAcACGGC GTIC~±'IC"IY TIAXP3-A 750 760 770 780 ATGWkTG;CCG, CCCAGAT ACTICCGGCIT TGTATGCFMA TACTACTGGC GCGTCGr-rA. 2GGCCG.,A ACATACGaTI' 810 820 830 840, G-TTCACTA~zC TATTrATGGT TGCGGQG ATACCa±T ±x± CAAAGIGATG3 AT1AATACCTIA ACCAAGCCTC TAM2kA%-%

870..' 880 890 900 TC-CACAAGTrC CGGCTATACG TCICAAAGGr ATTICCTATA AC=~GIX.G GCCGATATGC AGACrITCCA Tk;CGa;TAJT 930 940 950 960 ACCIMATCCGC;GGAA TCQCCACLA CTCTCAC.GT TCG%;GGAAT ACCCTCCrA AGG-4TCAMT CC;QGG1CV 4* 4 A 'S Fig. 2 Eco RI-cleavage sites and open reading frames of hepatitis non-A, non-B virus DNA Hairpin .CTG-Box2.. CTG-Box 1 1Kb 2 Kb 3 Kb L DR O 4 Kb' 49918 Eco R, I .215 Kb RI R RIsL 4-l0,1 ,2Kb4'-O4 Kb 1,5 open reading framres reading frames- 3 2 1 i41 I A 2 A 3 A 3 -I 1 2 7~jI~ 6 14 2 .3 I si tuacd b~etaeen bp 2,097 and 2.19 ar- the end of In opcn r-adinq f rame (pos. 4 fig. 2) I I Fia, -i ri 6 9* *r 5'1 TGCAr1CGACGTATCGAACAACTCATArAAGI'TrI C IAGTMT CssLLISKLATFSLLRFICSP* A V R Y Y R S S Q H S V Y G L F-A P L S N Q F V TD I E A R N I Q F T K V Y L L P L V0 71 GAC~r1GC1CAARAGTM ATCCATAC G P C S K V F C F Y P Y R T K N V L C C Y' DL A Q K F S V S I I TEE 0 K3S Y VA NT F 'IT L L K S F L F L S I L K N X K C L L I P S141 TTGr-A=TCATTATAArlG=ACGTCGCIGrrriCICG~rInVZAmr WssYHYFALSS*AAFPRLLQVG GHRIIDILLYRPELLFLACCKWE L V I V S L I F C F I V L SC F,S S L V A S G S 211 GCAGrrTATGA ~rGG AG=CrCAGTVICCG AVL* *VSSVLS SGADSSSRSVL V QFYNECLPFSPLALTQVAQFSE S F I®qS V F R S L L W R L K S L S S L S 281 TCMCCCCCA7WCC- GMMG 301 31 F P II C R SSPIAVE LPPLPL Fig. 3 Plus strand 3' strand) of the partial sequence between nucleotides 3166 and 3467 of hepatitis non-A, non-B virus DNA (see Fig. 1). i i L infection, and by molecular biological detection of a DNA migrating in gel electrophoresis at an apparent size of 3.2 kb (uncut and under j I ii I .9. 9* 99

9999.9 *9 301 C7CAACGGCAATGGGGGAAGACCAGAGAACAGCGACrACIAGCAGCGCCAGCAGAGAACGGA LNGNGGRLELSDYLSQRQRRERK 1 STA®GEDSEN*ATT*VSARGENG 2 Q R Q W G K T Q R T E R L L E S'A P E E R T E 3 231 AGACACICATrhTAAAACITGCCCACrIGCAACAAGCGAGCAAAAGCAGCr-AGGACATAAAGCAA T L I I K L L P L A T S E E K Q L R T I K Q N 1 RHSL*NCSHLQQARKSSSGR* SKI 2 DTHYKTAPTCNKRGKAAQDDKAK 3 161 TATC AATATACGATGACCAAAGTATrAGCAACATAAGACATriv)MT1'AGTATGCATAGAAAC I N D T M T K G I S N I R H F L 3. F S R. N I S®IR*PKVLAT*DIFCSSVWIET 2 Y Q *Y D D Q R Y Q H K T F F V L Q Y G K Q 3 91 AGAAAA=CIAGCAAGGCATrACrAAGGGGAGCAAATAAAcrAGTAAACTGAAT GIGCGGCIT R1KLLSKVITKGSK TLVN*®LRAS 1 E N F A R S L L R G A N P *T E C C E L 2 KTFEQGHY*GEQINLSKLNVASF 3 21 CGATATCArAACGAACTCCA 1 31 I S V T N C RYQ*RTA 2 DISNEL 3 Fig. 4 Minus strand 5' strand) of the partial sequence between nucleotides 3166 and 3467 of hepatitis non-A, non-B virus DNA (see Fig. 1 and 3) _i as well as the corresponding proteins synethesized by this ago, Fig. 5 Open reading frames of hepatitis non-A, non-B virus DNA 3' strand, see Fig. 1 and 2) I INITIATION POSITION i TE. MflTION POSITION1 4 4 157 311 433 1483 1669 1729 2428 2479 2620 2971 2992 3046 3388 3 4 3' 3511 3664 3670 3673 3859 3883 4135 1060 2059 271.9 3133 3520 3697 ***FRAME 1A 1309 1435 I 19 25 2377 2383 487 517 2782 2839 I667 676 3235 '.292 787 796 3619 3661 I1030 1171 3721 3748 1408 1675 I2044 2053 2404 2665 3418 3427 4009 4081 4180 42S2 4393 4468 4642 4'1'20 4897 4945 139 526 721 799 1183 1735 2125 2728 3559 4087 4261 4498 4765 4957 151 556 742 853 1195 1819 ,2278 2788 3571 4093 4279 4531 4828 4972 286 601, 745 883 1276 1825 2287 3004 37/12 4111 4300 4549 4882 4981 481 613 778 898 1339 2035 2365 3022 3868 4150 4354 4636 4888 4987 7 I TOTAL 39 1 T 0T A 84 a lh**F-RA.E 2A 131 539 593 815 1052 1202 I158 266 1205 3539 3833 3875 3986 4040 '1301 1310 4097 4385 1616 1631 1727 1730 1,853 18S6 2060 2258 I2444 2495 12627 2705 12969 2972 I3110 3134 3233 3236 3386 3389 3488 3503 302 3620 3689 3701 878 3818 440 1433 1634 1751 1868 2372 2519 2717 2903 2993 3149 3272 3443 3506 3662 3749 3944 443 1529 1649 1757 1877 2384 2528 2747 2906 3047 3179 3299 3464 3518 3665 37 55 1058 1259 1574 1613 1670 1709 1763 1778 1982 2009 2417 2423 2567 2573 2783 2792 2924 2951 3086 3089 3209 3230 ,1 3323 '3344 1 3476 3485 1 3521 3578 3671 3686 3761 3767 t a.-M*T 3A A* 27 282 483 522 555 693 75 168 189 258 447 540 I741 744 774 930 1194 1275 1 732 1053. 1203 1206 1401 1458 1271476 1599 1656 1770 180.0 29 13'1321 3124 1875 1947 2292 24A03 267 22 07 23 1 38219 23424 It 1865 272 3537 3540 3798 384 3900 .3984 2787 2826 2997 3165 3285 3636 13987 4038 4275 4386 445 4539 3651 3711 3714 .3960 4110 4128 '4686 4992 14179 4254 4263 4299 43Q5 4353 4446 4620 4722 4932 4944 ITOT A L 47 'TO0T AL: 32 Fig. 6 Open reading frames of hepatitis non-A, non-B virus DNA C3'- 5' strand, see Fig. 1 and 2) so. is* *o .o I INITIATION POSITION S TNF*lMATION POSITION FRAME 514 1399 3946 1,7 211 262 292 331 379 430 553 568 583 652 694 700 727 754 766 778 820 871 874 880 991 1111 1180 1186 1255 1270 1303 1363 1369 1396 1432 1495 1678 1699 1735 1768 1789 1792 1822 1987 2014 2146 2173 2233 2272 2356 2425 2431 2479 2536 2566 2581 2626 2647 2812 2920 2923 2977 295b- 3016 3052 3076 3202 329 3235 3247 3274 3280 3355 3367 3424 3505 3523 3544 3565 3649 3652 3700 3718 3730 3760 3769 3778 3805 3808 3922 3928 -3940 4042 4069 4078 4087 4102 4123 4258 4306 4438 4495 46?1 4657 4675 4684 4816n 4816 T TOTAL 3 TOTAL 104 FRAM 2B 1 203 863 896 1115 1139 1172 17 26 59 62 110 164 1685' 1730 2378 2495 2621 2642 245 278 299 347 350 404 3569 3875 4427 4778 0 449 470 530 578 626 719 J 746 749 782 797 800 839 842 1142 1166 1466 1781 1994 l2261 2393 2498 2543 2837. 2945 3323 3530 3881 3968 3977 3983 3986 -034 4049 4052 4082 4157 4184 417 4190 4193 4202 4211 i 4220 4334 4397 4433 4502 4529 4532 4535 4538 4742 4859 4862 I 'T0TA L 16 1 TOTAL 66 r I i I I I ~Xt 3B*** 870 873 1C02 1269 1287' 1362 372 459 522 543 639 723 1542 1677 1600 2172 2646 3114 864 1044 1173 1200 1380 1563 3153 3639 3675 3699 3816 4716 1575 1614 1662 1686 1707 1752 1797 1872' 1896 1923 1953 2007 2019 2097 2139 2160 2166" 2244 2307 2319 2379 2457 2508 2571 2643 2667 2865 3069 3111 3159 3183 3330 3351 3450 3465 3477 3516' 3540 3597 3681 3690 3705 3786 3876 4266 4284 4779 4809 4821 4923 I 'TOTAL 18 TOTAL 62 1 n 4 J Fig.6(cont'd) Proteins encoded by the open reading frames of hepatitis non-A, non-B virus DNA strand, see Fig. 1 and 2) START END MOLECULAR WEIGHT i NO. START EN IOLECUILR WEIGHT FAE IB 514 553 1629.87 1 3946 4042 3550.03 1399 1432 1344.47 2 514 553 1629.87 3946 4042 3550.03 3 1399 1432 1344.47 FRA2 203 245 1707.87 1 3569 3881 11635.17 863 1142 10853.68 2 1172 1466 11262.47 896 1142 9431.19 3 863 1142 10853.68 111,5 1142 1115.33 4 896 1142 9431.19 113S, 1142 149.21 5 2621 2837 7747.*28 1172 1466 '11262-47 6 2642 2837 6866.30 1685 1781 3768.36 7 1685 1781 3768.36 1730 1781 1988.33 8 4778 4859 3218.74 2378 2393 656.78 9 1730 1781 1988.33 2495 2498 149.21 10 203 245 1707.87 2621 2837 7747.28 11 1115 1142 1115.33 2642 2837 6866.30 12 2378 2393 656.78 3569 3881 11635.17 13 3875 3881 °277.38 e 3875 3881 277.38 14 4427 4433 246.32 4427 4433 246.32 15 1139 1142 149.21 4778 4859 3218.74 16 2495 2498 149.21 ****FRAkl 3B*** 87Q 1044 6984.76 1 870 1044 6984.76 873 1044 6853.57 2 873 1044 6853.57 1002 1044 1764.94 3 1269 1380 4059.50 1269 1380 4059.50 4 1287 1380 3436,80 1287 1380 3436.80 5 2172 2244 2682.96 1362 1380 748.87 6 4716 4779 2652.92 1542 1563 879.86 7 1800 1872 2593,86 1677 1686 418.54 8 3816 3876 2182.40 1800 1872 2593.86 9 3114 3159 1826.12 2172 2244 Z682.96 10 1002 1044 1764.94 2646 2667 838.92 11 3639 3681 1629.97 3114 3159 1326.12 12 1542 1563 879,86 3153 3159 248.34 13 2646 2667 838.92 36'9 3681 1629.97 14 1362 1380 748.87 3675 3681 206.26 s 15 1677 1686 418.54 3699 "37-05 236.28 16 3153 3159 248.34 3816 3376 2182.40 17 3699 3705 23(s 28, 4716 4779 2652.92 3675 3681 206.26 1 4INTERNATIONAL SEARCH REPORT PCT/EP88/00123 international Application No 1. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) According to International Patent Classification (IPC) ortto both National Classification and IPC Int.C1 4:C 12 N 15/00;A 61 K 39/29;C 12 Q 1/68;G 01 N 33/576 11, FIELDS SEARCHED Minimum Documentation Searched 7 CiassifIcatlon System IClassification Symbols Int.C1 4 C 12 N;A 61 K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 8 ill, DOCUMENTS CONSIDERED TO BE RELEVANT' Category citation of Document, 11 with Indication, where aprropriate, of the rele vant passages 12 Relevant to Claim Nq, 13 x WOA,84/01107(THE GENERAL HOSPITAL CORP.) 11-10 29 March 1984,.see the whole document x EPAtQ)248965(SEELIG)l4 November 1984,see 7 example 7 A EPA,0066%96(EISAI CO.LTD)08 December 1982 A WQA,82/Q3330(TREPO)14 October 1982 A La Recherche Vol. 14,No. 145, June 1983('PariS FRi A.Zotov."Lles h6patites' pages 874-865 PIA EPIA,0242300(INSTITUT PASTEUR) 21 October 19871 *Spetlal categories Of cIted clocumentsi to 'IT" later doQ~merlt published alter the international filing r~ate "A"f document defining the general stete of the art which Is not Or prority date and not In conflict with the applicat'zs' but considered to be of particuiar relevanice cited' to vrndeistand the principle or theory undoeiylng the a(16 ddcumnt ut pblihed n a afer te Iterntioal lnvrntldn Ming e arle ouetbtpbihd r le h nentoa X" document of particular reievance,. the claimed Invention. fiig d ametwihmyt cannct be considered r~,lor tannot be considered to doumet wichmaytrowdoqubts on priority claim(s) or Involve an Inventive step which Is cited to establihte publictat data Of Another "y oueto atc larrtvancef the claimed Invention citaionor oherspeial easn (s spgifed)cannot be considered to Involve an Inventive step When the document referring to an oral disclosure, Use, enhibition or document Is c ombnd with o or more other such docu* other means ments, such combnation tb ng obvious to a person skilled document published prior to the International. tiing date but In the art, iater than the Priority date claimed 11V document member of the same patent family IV. CERTIFICATION Date of the Actual COmltion of the International 9earch Date of Malting of this International Search Report 29 April1988(29.04.88) 01 Junte 1988(01.06.88) lnterna'onal Searching Authority Signature of Authariged Offcer European, Patent 04 ice Formi PCT11SA1210 (second sheet) (Joflty 186s) I1 l~ I' ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. EP 8800123 SA 20814 This annex lists the patent famiily members relating to the patent documents cited in the above-nientioned international search report. The membters are us contained in the European Patent Office ElDl1 ile on 24/05/88 The European I'uterit Office is in tno i~ay liable for these particulars ishich are merely given for (fhe purpose of iniformation. Patent document cited in scarrh report Publication datI nuemhct-(s) dt Publication date WO-A- 8401107 29-03-84 EP-A- 0119259 26-09-84 CA-A- 1189878 02-07-85 EP-A- 0124896 14-11-84 WO-A- 84043Z6 08-11-84 DE-A- 3316464 08-11-84 AU-A- 2865084 19-11-84 JP-T- 60501241 08-08-85 EP-A- 0066296 08-12-82 JP-A- 57198867 06-12-82 CA-A- 1184846 02-04-85 WO-A- 8203330 14-10-82 FR-A,B 2502154 24-09-82 EP-A,8 0074986 30-03-83 EP-A- 0242300 21-10-87 FR-A- 2597606 Z3-10-837 JP-A- 62249999 30-10-87 1 Por more details ahtot this annex :see (Irilu Journal of the EPuropeant Patent office, No. 12/81 H INTERNATIONALER RECHERCHENBERICHT iternationales Aktenzeichen PCT/ EP 88 /00123 1KLASSIFIKATION DEG ANN1ELDUNGSGErjENSTANDS ibei mehrere,' KIssi#',.aonssymbolen sind, anzugeoenl 6 Nach der Internationalefl Paten tklassifikatiafl iIPC) Oder nach der nationaler. :slassifikation und der IPC Int Ci C 12 N 15/00; A 61 K 39/29; C 12 g 1/68; G 01 N 33/576 11. RECHFRCHIETE SACHGEBIETE Recherchierter Mindestprutstffl 7 Klassifikotionssystem Klassifikatjconssymbole In l 04 T C 12 N; A 61 K Recherchierte nicht zumn Mindestprufstoff gehorendle Veroffentlichungen, sowelt diese unter die recherchier-ten Sachgebiete fallen 8 III. EINSCHLAGIGE VE86FFENTLICHUNGEN 9 An' Kennzeichnung der Verdffentiichungll,sawetc erfarderlich unter Angabe der mailgebiichen Teile 1 2 I Betr, Anspruch Wr. 13 X WO, A, 84/01107 (THE GENTERAL HOSPITAL CORP.) 1-10 29. Md.rz 1984, siehe das ganze Dokument X EP. A, 0124896 (SEELIG) 14. November 1984, 7 siehe Beispiel 7 A EP, A, 0066296 (EISAI CO. LTD) 8. I eember 1982 A WO, A, 82/03330 (TREPO) 14. Oktober 1982 A La Recherche, Band 14, Nr. 145, Juni 1983 (Paris, FR), A. Zotov: "Les h~patites", Seiten 874-865 P,A EP, A, 0242300 (INSTITUT PASTEUR) 21, Oktober 1987 Besandere Kategarien Yon angegebenen Verafientlichungen 1 A" Vera ffentlichu ng, die don aligemelnen Stand der Technik Sp~tere Verdffentlichung, die nach dem internatianalen An- dafinier, aber nicht als besonders bedeutsam anzusehen 1st meldedlaturnm odor dem Prioritdradatum verotfentlicht wardin iteres Dakument, dlas ledoch erst am Odler nach dom Interna. ist und mit der Anmeldung nicht kollidiert, sondern nur zurn tionalen Anmeldedatum verdffenlilcht worden Is Vertndnis des der Erfiridung zugrundellegenden Prinzips L" erfledlhun, ie eegnt 1t,~me Piart~aaOder der lhr 'grundeliwgenden Thiaorle angegetben isi "L"Varfletlihun, de gelget sixs'en rioisaspruch Verdffentllchung van besonderer Bedeutung, die beanspruch- zwelfeihatt erscheinen zu amsen, Oder durch die da, Verof t ridn annctasnuae u ridrshrTtg feniicungdatm lne enere i Reherhenercht go- kelt beruhend betrachtet werden namsten 4dfontllchung belegt warden ioli Oder die aus elne-n enderen besanderen Gnrmnd angegeben 1st Iwie 4usgof~hrf) Verdrfentlichung Yan besanderer Bedeutung: die beanspruch- Verbffontlichung, die sigh allf eama mUndliche Offenbarung, te Erfinciung kann nicht ala auf erfInderischer Tktigkelt be- eine Benuttung, elne Ausstellung Oder andere Maflnahmen ruhend betraghtet viarden, wenn die Verciffentlichung mit bezieht elner Oder mehreren anderen Veraffentlichungen dieser Kate- gonle in Verbinduing gqbracht wird und these Verbindung fur OT" Verdffentlichung, die var dem internationalen Anmeideda. einen Fachmann nahlklieend 1st turn, aber nach dam beanspruchten Pnioritiltsdatum verOf-fent. VecfntchgdeMgIddreinPaetali1t licht warden ist VrfetihndeMtle esle aetail s IV. BESCHEINIGUNG Datum des Abschiusses der Internationalen Recherche Absendedlatum des internatioasen Racherchenberichts 29. April 1988 m .0.8 Int rnati nale Recherchenbehdrcie L rsc rif de s EurookIchs Patentamt E 1.i PUVIYEN Forrnblatl PCT/IsA/210 t814tt 2) W~nLwer 1985) ANHANG ZUNI INTERNATIONALEN RECHERCHENBERIGHT OBER DIE INTERNATIONALE PATENTAN' -ELDUNG NR. EP 8800123 SA 20814 In ieseni Ioln,~n sin, die Mlioieer der I'utinffamilien der irn ohenmainnten incernutionalen Rleehrocnbricht ;unefuihrtCn Viitentdokurncnte unggcgeben. D~ie Anguhen filir die Faiifienrnitglieder entsprchen dcrn Stwid dcr Datei des EFurop~'ischon fPutcntarnts arn 24/05188 IDiese Anguben dienen nur zur Unterrichtung and erfolgcn ohne (;ciwmr. i Reclierchenhericht Diilum der Iitglicd(cr) der Dajtumi der aiigefuhrtes I'utendokurnent Vc riifentliettung I'uicntfurnilie Verofendiehung WO-A- 8401107 29-03-84 EP-A- 0119259 26-09-84 CA-A- 1189878 02-07-85 EP-A- 0124896 14-11-84 WO-A- 8404326 08-11-84 OE-A- 3316464 08-11-84 AU-A- 2865084 19-11-84 JP-T- 60501241 08-08-85 EP-A- 0066296 08-12-82 JP-A- 57198867 06-12-82 CA-A- 1134846 02-04-85 WO-A- 8203330 14-10-82 FR-A,B 2502154 24-09-82 EP-A,B 0074986 30-03-83 EP-A- 0242300 21-10-87 FR-A- 2597606 23-10-87 JP-A- 62249999 30-10-87 Fqr nilhcrc~ Iinlellivitvrt n dieseni AllhUtig 1.siche AnitSblutt des EIuropdischeni IN.!entoints, .Nr.12182