NON-A, NON-B HEPATITIS VIRUS GENOME RNA, CDNA AND VIRUS ANTIGEN PROTEIN

ARN GENOMIQUE, ADNC ET ANTIGENE PROTEIQUE DU VIRUS DE L'HEPATITE NON A - NON B

English Abstract

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

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
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:
<IMG> .

- 2 -
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:
<IMG> .
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.

- 3 -
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:
<IMG>.
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.

- 4 -
9. A method according to claim 8 wherein the probe has the
following nucleotide sequence:
<IMG> .
10. A method according to claim 8 wherein the probe has the
following nucleotide sequence:
<IMG> .

- 5 -
11. A method according to claim 8 wherein the probe has the
following nucleotide sequence:
<IMG>.
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:
<IMG>

- 6 -
<IMG>.
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:
<IMG>.
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:
<IMG>

- 7 -
<IMG> .
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:
<IMG> .

- 8 -
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:
<IMG> .

Description

',L 3 3 S
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.