CA2044535A1 - Non-a, non-b hepatitis virus genome rna, cdna and virus antigen protein - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Non-A, Non-B Hepatitis Virus Genome RNA is disclosed along with cDNA and Virus Antigen Protein.

Description

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Non-A, Non-B l-lepatitis Virus Geno~e RNA, cDNA and 5Virus Antigen Protein Introduction and Bac~ro~lnd This is a continuation~ -part application of patent application serial number 07/451,96~, filed December 19, 1989, which is relied Oll and incorporated herein.
This invention relates to Non-A, Non-B hepatitis virus genome RNA, its cDNA as well as its virus antigen protein.
Viral hepatitis is divided approximately into two categories; enterically transmltted hepatitis and parenterally transmitted (blood-borne) hep~titifi. ~lepatitis A virus in the former and hepatitis B virus in tlle latter have been isolated as causative agen-ts and their virological properties have been elucidated. As the results, diagnosis oE and preventive measures against infection with those viruses have been established and diseases caused by them are held closely under control.
Parenterally transn~itted Non-A, Non-B hepatitis (PT-NANB) i8 said to be about 95% of post-tran3fusion hepatiti~
cases in Japan, however, nothing has been confirmed of its causative virus except for its ex~erimental transmission to chimpanzees as an only susceptive animal other than human beings. Almost all attempts made so far by various J`. ~ 3~ !

researchers have failed in identification of the causative~
agent virus because of the poor reproducibility of the reported results.
Very recently Chiroll's scientists reported hepatitis C virus (HCV) as a causative ~gent of PT-NANB. Its genomic structure is said to resemble that of flavi viruses and an immunoassay using antigenic polypeptide deduced from IICV
genomic sequence is, reportedly, capable of detecting antibodies highly associated with PT-NANB. But, virologically there is no evidence that HCV is the PT-N~NB agent.
~ rima also reported tlle nuc]eotide sequences of cDNA
clones derived from PT-NANB patiellts' plasma aeter Chiron's disclosure of HCV genome. 'rhe nucleot.ide sequences of CHiron's, Arima's and the current inventivn are mutually independent, i.e., there is no homolo~y amvng any pair of them.
Applicants have been strenuously conducting research in Non-A, Non-B hepatitis and have succeeded in isolating cDNA
clone very closely associated with PT-~ANB. There is a clear . ..
association between the nucleotide sequence nf the cDNA clone and PT-NANB; nucleotide-hybridization assay can detect virus and immunoassay using polypeptide deduced from the cDNA can detect antibodies in PT-NANB infected subjects. It has ,, ., --- ?, ~

further been found that they are effective in diagnosis, prevention and therapy of PT-NANB as materials for diagnostic test kits or immunogens.

SummarY oE Inver.tion One feature of the present .invention resides in providing a PT-NANB virus genome RNA having the following base sequence;
5'UGAU A A A A U A A G C C A GGG
UGA U U C U U A A U U IJ U C A GUCU
G A A GUCUUUUUUCCCCUCCC
AGUCCAG U C U C C U C A U U U A C
UAGGG U C A GCAGGGAGA G A G
A G A A G G U C A G C U G U G A A. U GU
U U C C C C U C C C C A G A A U G ~ G G
UGGGCUG G U C C U G A GUUG C A
GCUCGGGGUGGGGG A C GU G A
A C C A GCC3' (hereinafter callecl "N~48~0-RNA");
Another feature of the inventive resides in complementary DNA to PT-NANB virus genome RNA having the following base sequence;
5'GGCTGGTT C A C G T C C C C C
A C C C C G A GCTGC A A C T C A G G
A C C A G C C C A C C C C A T T C T G G

~~ -2~f-~ 3 GGAGGGGAAACAT r c ACAGC
TGACCTTCTCTCTCTCCCTG
CTGACCCTAGTAAATGAGGA
GACTGG A C T G G GP~ G G G G A A A
AAAGACTTCAGACTGAA A A T
T A AGAATCACCCTGGCTTAT
T T T A T C A 3' (hereinafter called "N-43~0-cDNA-T3 "!; and A still further feature of the present invention res:ides in homologous DNA to PT-NANB virus genome RNA having the following base sequence;
5'TG A T A A A A T A A GCCAGGG
TG A T T C T T A A T T T T C A G T C T
G A A GTCTT T T T T C C C C T C C C
A GT C C A GTCTCCT C A T T T AC
TAGGGTCAGCAGGG~G A G A G
AGA A GGTCAGCTGTGAATGT
T T C C C C T C C C C i\GAATGGGG .
TGGGcTGGTcc r GAGT T G C A
GCTCGGGGTGGGGGACGTGA
ACCAG C C 3' (hereinafter called "N-41~0-cDNA-T7"); and Yet another feature of the invent-on resides in a PT-NANB virus antigen protein having the following amino acid residue sequence;

Leu - Val - llis - Val - Pro -~lis - Pro - Glu - Leu - Gln -Leu - Arg - Thr - Ser - Pro -Pro - Hi5 - ser - Gly - Glu -Gly - Lys - ~lis - Ser - C-ln -Leu - Thr - Phe - Se~- - Leu -Ser - Leu - Leu - Thr -- Leu -Val - Asn - Glu - Glu - Thr -Gly - Leu - Gly - Gly - Glu -Lys - Arg - Leu - Gln - Thr -Gl.u - Asn (hereinafter called "N-48~0-P") Bri ~ n of Fi~_res Figure 1 is a photograph and shows detection of antibody against PT-N~NB associated protein antigen (N-4830-P) by Western blotting, Figure 2 i9 a photograph and showq antibody responce of an chimpan2ee infected with PT-NANB
hepatitis along its time-course by antibody detection method used for the data in figure 1.
Fi.gure 3 is a photograph and .~hohs detection of the virus genome RNA by hybridiza~ion method~

` ~ - - 2~`~1153~ ~

Detailed Descri~tiGn of Invention Applicants have refined ar.d produced the nucleic acid and antigen protein as descIibed herein in the following way.
(1) Experimental infection oE chimpanzee~ with PT-NANB. To reproduce PT-NANB hepatitis, chimpanzees were intravenously injected with human serum knowr. to have caused poRt transfusion hepatitis. Since this hepatitis did not sllow antigen-antibody responce specific to hepatitis A or hepati'cis B, i t was concluded to be Non-A, Non--U hepatitjs. Moreover, because of the ultrastructural change3 characteristic to PT-N~.NB !loted in the cytoplasm Or hepatocytes, i t wa~ diagnosed PT-NAN~.

(2) Extraction of nucleic acid from chimpanzee plasma. 6ml of chir.l~anz2e plasma which showed PT-NANB virus titre higller than 107 CIU/ml was layered on top of ~ml of 20% sucrose and centrifuged on tne SW90 ro~or (Beckman) centrifuge at 38, ono rpm for 5.9 hour3 at 9C.
After addition to its pracipitate of SDS and -~ , 3 ? r'~

I'ro-e~ase K 1 2~ ~f lm~/n,l re~pectively and, then, overnight incubation at 37C, nucleic acids extracted from phenol of pll 8.5 was precipitated by ethanol.

(3) cDNA synthesis.
The nucleic a~id obtained under ~2) above was heated at 70~ for 1 minute and had added thereto oligo dT and random hexamer as primers and had further added t.hereto 4 type3 of dNTP
and reverse transcripta3e for reaction to synthesize the primary cDNA chain. Detailed reaction conditions were set according to the protocol provided to the cDNA synthesis kit Amersham (Amersham, England). According to the protocol of the aoove kit, the secondary cDNA
strand was synl.hesized by ribonuc].ease H (RNase ~I) and DNApol.

(4) Phage library o~ cDNA.
The double stranded c~NA obtained under ~3) above was, after treatment with EcoR1 methylase, provided with EcoR1 linker at both ends, ligated with Lamda-gtll DNA at its EcoR1 site, then, packaged in the phage particles and .
infected to Ecoli Y10~0 to obtain a recombinant .

~ 2~l,35 phage library of 2 X 106 PFU. The protocol of the Lamda-gt 11 cloning kit (Amersham, England) 5 was followed in the ehperiment.
(5) Screening of phaye library.
10,000 PFU of the phage library prepared in (~) above was plated onto the LB agar in 90 x 90mm disc and when plague was formed, it was covered with the nitrocellulose membrane impregnated with IPTG for incubation for 2 hours at 370C.
. After incubation, the membrane was removed, and washed with the buffer solution and blocked for 1 hour at the room temperature with the buffer solution containing 40-~ fetal calf serum and 0.05% Tween 20. As the primary antibody, 50 times diluted sera of humall and chimpanzee known to be PT-N~N~ v-.rus careers were used and the membrane w2s immersed i.n each serum for overnigllt incubation at 4C. After washing witll the buffer so;ution contain.ing 0.05~ Tween 20, secondary antibody (peroxidase labeled mixture of antibodie~ against human IgG, IgM
and IgA) was added and incubated for 30 minutes at the room temperature. After washing with '' ' ~ ` 2~ J3j ~

the buffer solution containing 0.05% Tween 20, DAB, Ni, Co alld ~12 ~Z were added for color reactioll .

(6) Preparation of lysogen.
Antigen positive phage prepared under t5) above was isolated and E. Coli Yl089 and infected with it. The lysogen was prepared according to the method described in "Constructing and screening cDNA Libraries in Lamda gT11", Thanh V, I-luynh, et al, DNA Clorirlg, Volume 1, a practical approach edited by D.M. Clover, P99 -7~, IRL Press, Oxford, 19~5.
(7) Purification of B-galactosifase fusion protein.
Lysate was made from the lysogen prepared under (6) above ar,ld was pasqed through affinity column coated with anti-~-galacsitose antibody and column was treated witll g.5M MgClz to elute purified ~-galactosidase fusion protein.
(~) Subcloning and sequencing of cDNA.
Phage DNA of antigen positive plague obtained under (5) above was ~urified and digested with EcoR1 to take out cDNA and subcloned to the EcoR1 site of ~hagescript ~M (S'llRAT~GENE-USA) and sequenced with the Sanger method.
The RNA probe having homologous ~equence to N-~ 0-cDNA-T3 . . . ~ 3 ~

hybridized successfully with the Vil'U5 genome RNA, while the probe having complementary sequence to N-4880-cDNA-Ta did not.
Thus, the genome RNA was concluded to be single stranded and have the same polarity as N-4880-cDN~-T7.
Since the Open Reading Erame (ORF) starting from the third base and terminating at the stop condon at the 159 -161th base of N-48~0-cDNA-T3 forms fusion protein with lac operon of Lamda gtll phage used for expression of the protein, this ORF was translated to deduce the amino acid sequence of N-4880-P.
The present invention is illustrated by the examples described below WhiCIl relate to application of the invention.
(9) Detection of antibody to PT-N~NB viru3 antigen protein (N-g8B0-P).
After treatment for 5 minlltes at 100C in the presence of 1~ SD.S ~nd 1~ 2ME (2-Mercaptoethanol), ~-galactosidase fu3ion protein obtained under ~7) above was subjected to SDS - PAGE (8~) and was tran.~ferred to the nitrocellulose membrane (IYestern blotting).
~fter washing and drying, the membrane was shredded and bloc~ed with 40% ~etal calf serum, and those shereds were applied with antibody samples as the primary an2ibody and incubated overnight at 4C. After washing with the ~ - 2~ 3'.

buffer solution containing the surfactant 0.05%
Tween 20, and immer3ion in biotinylated anti-human IgG or anti-human IgM for 30 minutes incubation at the roon temperature, they were applied with the complex of avidin and biotinylated pero~i~ase, then incubated for 95 minutes at the room temperature. For color reaction, Koni-a Immunostain TM ~Konica, Japan) or DAB, NI,Co, H202 method was used. (Fig. 1) Fig. 1 shows an example of Western blot analysis oE PT-NANB antigen protein (N-9880-P) Eused with B-galactosidase. Primary Antibodies (Test Samples);
a-a':Chimpanzee plasma before Non-A, Non-B hepatitis inEection - (') b-b':Chimpanzee plasma before Non-A, Non-U hepatitis infection - (2) c-c':Chimpanzee plasma before Non-A, Non-B hepatitis infection - ~3) d-d':Chimpanzee plasma with persistent Non-A, Non-B
hepatitis inEection - ~1) e-e':Human plasma with persistent Non-A, Non-B
infection f-f':Chimpanzee plasma with per3istent Non-A, Non-B
hepatitis inEection - ~2) 2 v i 1 . 1 ~, ;
.

g-y':Chimpanzee plasma after recovery from Non-A, Non-B hepatiti~
h-h':Chimpanzee plasma after recovery from Non-A, Non-B hepatitis - (2) i-i':Chimpanzee plasm~ after recovery from Non-A, Non-B hepatitis - (3) X: Anti-B-galactosidase rabbit anti-serum Secondary Antibodies a-i: anti-human IgM
a'-i': anti-human IgG
X: anti-rabbit Ig ~5 shown in the Figur~ 1, hiyllly intensive immuno-stainel bands oE the fusion protein were noted in lanes d and e'. That i3, with persistent Non-A, ~'on-B infection hosts, antibody against N-~3~0-P i9 positive. This ~0 suggests that N-~330-P i5 virus core (gag) protein rather than virus envelope (env) protein.
Second example of the Western blot analysis described in (9) is shown in Fig. 2.

. ~ 3.~-~

Primary Antibodies ~Test Samples) a-t a -t : Same chimpar~zee p7 asma as that used in d-d in Fig. 1 was taken along its time course of infection with PT-N~B hepatitis and was diluted 20 times.
a: Before infection b: 4 weeks after infectior.
c: 5 weeks after infection d: 6.5 weeks after infection e: 7 weeks after inEection f: 8 weeks after .infection g: 9 weeks after infection -h: 10 weeks aEter infection i: 12 weeks after infection j: 14 weeks after infection k: 15.5 weeks after infection l: 21 weeks after infection m: 23 weeks after infection n: 25 weeks after infection o: 26 weeks after infection p: 29 weeks after infection q: 32 weeks after infection r: 40 weeks after infection 20'1~3;~ ~ ~

s: ~2 weeks after infection t: 45 weeks after inEection X: Anti-B galactosidase rabbit-anti-serum Secondary Antibodies (A) Anti-human IgM
(B) Anti-human IgG
X: Anti-rabbit Ig As shown in the Figure, the antibody against Non-A, Non-~
associated antigen protein (N-~880-P) of this invention did not exist in the plasma of the chiMpanzee before its infection witll PT-NANB hepatitis and was found to appear at approximately 7 weeks after infection.
~10) Hybridization assay using the cDNA as probe Strand specific RNA probes were made by transcribing the recombinant phagescript DNA utilizing T~ or T7 promotors which resides at opposite sides of the inserted cDNA.
Fig. 3 shows an example of hybridization using radio isotope labeled probe derived from N-4880-cDNA
obtained in ~10) above. Same plasma as u3ed in the lane d, d' in Fig. 1 was centrifuged in CsCl and fractions with specific gravities 1.22 (1), 1.19 (2), 1.16 (3) dnd 1.13 (4) were obtained.
After dilution of each fraction with the buffer solution, particles of each fraction were 2 ~ 3 ~
..,.

precipitated, its nucleic acid ~as extracted by phenol after digestion with S~S and Protenase K, then, slot blotted on nylon membrane after denaturalization by NaO]I (panel A and B), or formaldehyde (panel C and D). ~s probes for hybridization, RNA pro~e (A and ~) made from Phagescript subclone of N-98~~cDNA by T3 promotor and RNA probe (B and D) made by T7 promotor were used.
As noted in Figure 3, blot 2 of ~he panel C alone showed high signal. This suggests that the v.rus genonle of Non-A, Non-B
hepatitis is single stranded RrlA ar.d is colnplementary to N-48~0-cDNA-Ta, and that it is anti-sense strand.
This further suggests that the particle (virus) including the genome RNA has the specific gravity of 1.19g/cm2 which i9 proven by the infection experiment with chimpanzees.
Of a type of blood-borne Non-A, Non-B hepatitis, applicants have determined chat its causative virus has a specific gravity of l.l~g~cm2 in ceqium chloride density gradient, and that type of genome RNA is single stranded h~ving (-) polarity. In addition, applicants have determined partial nucleotide sequence of the genome RNA, nucleotide sequence of complementary cDNA and antigen protein produced by its translation. RNA, cDNA and protein ~ 3 ~

made available by thi.s invention are useful in the manufacture of PT-NANB hepatitis diaynostic kit, medicine and vaccine.
The present invention also concerns diagnostic test kits for detecting antibodies against PT-NANB in biological samples, including for examp)e blood and serum samples.
Design of the immunoassay is subject to a great deal of variation, and a variety oE these are known in the art For example, the immunoassay may utili~e one viral antigen, for example N-4880-P; alternatively, the immunoassay may use a combination of viral antigens. Protocols may, for example, use solid supports such as pol~vinyl microtiter plates or beads, or may be by immunoprecipitation. The presence of IgG
and/or I~M antibodies to PT-~AN~ antigens such as N-~880-P can be detected using conventional immunoassay~ known in the art.
These include enzyme-lin~ed immunosorbent assay (E~ISA), hemagglutinatioll and radioimmunoassay (RIA) techniques. Most assays involve the use of labeled ~ntibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. These labels have been extensively reported in the patellt and technical literature.
Kits suitable for immunodiagno~is and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including ~ 80-P in suitable . . 2 ~ ~ ~ v ~ .~

containers, along with the remaining reagents and materials required for ~he conduct of the assay, as ~ell as a suitable set of assay instructions.
The present invention also concerns diagnostic test kits for detecting the presence of PT-NANB in biological samples, including for example blood and se~um samples. Use of the disclosed PT-NANB single stranded DNA or RNA is useful as a probe in identification of the viral agent, detection of the virus in diseased individuals, and detection of PT-NANB
RN~. Conventional probe techniques are well known in the art.
The probes can be labeled. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels, fluorescent probes, and chemiluminescent probes.
The probes can be packaged into diagnostic kit~.
Diagnostic kits include the probe DN~ or RNA, which may be labeled; alternatively the probes may be unlabeled and the ingredients Eor labeling may be included in the kit. The kit may also contain other suitably packaged reagents and materials needed Eor the particular protocol, for example, standards, as well as instructions for conducting the test.
The present invention further concerns a prophylactic method oE protecting a patient against becoming infected with PT-NANB. Antibody to PT-NANB antigens can be 2 ~ 3 3 ~ ~

given to Lusceptible persons for a passive immunoprophylaxia and PT-N~NB an~i~ens are used as a vaccine for an active immunoprophylaxis. Antibodies against PT-N~NB antigens can be produced by conventional methods known t:o the art. The active component of the vaccine can be employed with a physiologically acceptable diluent. The v~ccine may be linked to a carrier such as a protein carrier, such as tetnus toxoid, keyhole limpet hemocyanin, or another protein or polypeptide which would elicit a good response in the hcst; non-protein carriers such as polysaccharides, organic polymers, inorganic polymers, lipids, lipid vesicles and liposomes may also be used. The vaccine can be used with or without an adjuvant.
Conventional adjuvants include aluminum hydroxide (alum), murammyl dipeptide, any acceptable oil and water combination.
The amount of the adjuvant which is employed will vary widely depending on the nature of the adjuvant. Such carriers, adjuvants and the like are known in the art.
The vaccine can be adminlstered parenterally, by injection, for example, either subcutaneous]y or intramuscularly. Additional formulationr3 which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. The vaccine can be administered in such amount as wi~l be prophylactically and/or therapeutically effective. The ~uantity to be administered depends on the subject to be treated, capacity of the 20~a3;:~ ~

subject's immune system to syntlle.size antibodies, and the degree of protection desired. The vaccine may be given in a single dose schedule or in a multiple dose schedule. The hepatitis vaccine of the present invention is recommended for all persons at risk of developing non-A, non-B hepatitis and particularly those at especia;ly high risk.
The non-A, non-B hepatitis vaccine of the present invention may contain a peptide, either a synthetic peptide tpeptide produced ~y assembling individual amino acids by chemical means or by expression vectors (D~'A route)) or a peptide derived from natural sources. The vaccine can contain PT-N~NB antigens such as N-9880-P. Any analog of PT-4880-P of the present invention involving amino acid deletion3, amino acid replacements, or amino acid additioilY can ~e utilized, ~o long as the sequences elicit antibcdies recognizing PT-9880-P.
U.S. Patent No. 4,897,080 is incorporated by reference to illustrate techniques and methods that are well known in the art.
Further variations and modiEications of the foregoing will be apparent to those skilled in the act and are intended to be encompassed by the claims appended hereto.

Claims (22)

1. An immunoassay for detecting antibodies directed against non-A, non-B hepatitis antiqens comprising:
(a) incubating a sample suspected of containing anti-non-A, non-B hepatitis antibodies with a non-A, non-B hepatitis antigen under conditions which allow the formation of an antibody-antigen complex; and (b) detecting the antibody-antigen complex containing the probe antigen.

2. An immunoassay according to claim 1 wherein the non-A, non-B hepatitis antigen is a polypeptide.

3. An immunoassay according to claim 2 wherein the polypeptide has the following amino acid sequence:
.

4. A non-A, non-B hepatitis diagnostic test kit for analyzing samples for the presence of antibodies directed against a non-A, non-B hepatitis antigen comprising labeled anti-human IgG and a non-A, non-B hepatitis antigen protein attached to a solid substrate.

5. A non-A, non-B hepatitis diagnostic test kit according to claim 4 wherein said non-A, non-B hepatitis antigen protein has the following amino acid sequence:
.

6. A non-A, non-B hepatitis diagnostic test kit for analyzing samples for the presence of antibodies directed against a non-A, non-B hepatitis antigen comprising labeled anti-human IgM and a non-A, non-B hepatitis antigen protein attached to a solid substrate.

7. A non-A, non-B hepatitis diagnostic test kit according to claim 6 wherein said non-A, non-B hepatitis antigen protein has the following amino acid sequence:
.

8. A method for detecting non-A, non-B hepatitis nucleic acids in a sample comprising:
(a) reacting nucleic acids of the sample with a probe for a non-A, non-B hepatitis polynucleotide under conditions which allow the formation of a polynucleotide duplex between the probe and the non-A, non-B hepatitis nucleic acid from the sample; and (b) detecting a polynucleotide duplex which contains the probe.

9. A method according to claim 8 wherein the probe has the following nucleotide sequence:
.

10. A method according to claim 8 wherein the probe has the following nucleotide sequence:
.

11. A method according to claim 8 wherein the probe has the following nucleotide sequence:
.

12. A non-A, non-B hepatitis diagnostic test kit for analyzing samples for the presence of non-A, non-B hepatitis nucleic acids comprising a probe for a non-A, non-B hepatitis nucleic acid.

13. A non-A, non-B hepatitis diagnostic test kit according to claim 12 wherein said non-A, non-B hepatitis nucleic acid probe has the following nucleotide sequence:
.

14. A non-A, non-B hepatitis diagnostic test kit according to claim 12 wherein said non-A, non-B hepatitis nucleic acid probe has the following nucleotide sequence:
.

15. A non-A, non-B hepatitis diagnostic test kit according to claim 12 wherein said non-A, non-B hepatitis nucleic acid probe has the following nucleotide sequence:
.

16. A vaccine for treatment of non-A, non-B hepatitis infection comprising a non-A, non-B hepatitis antigen and a pharmacologically acceptable carrier wherein the non-A, non-B
hepatitis antigen is present in a pharmacologically effective dose.

17. A vaccine according to claim 16 wherein the non-A, non-B
hepatitis antigen is comprised of an immunogenic polypeptide.

18. A vaccine according to claim 17 wherein the immunogenic polypeptide has the following amino acid sequence:
.

19. A vaccine according to claim 16 wherein the non-A, non-B
hepatitis antigen is present in a dosage of 2-20 micrograms.

20. A vaccine for treatment of non-A, non-B hepatitis infection comprising antibodies directed against a non-A, non-B hepatitis antigen.

21. A vaccine according to claim 20 wherein the antibodies are directed to a non-A, non-B hepatitis protein antigen.

22. A vaccine according to claim 21 wherein the antibodies are directed to a non-A, non-B hepatitis protein antigen which has the following amino acid sequence:
.