Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide

Disclosed is an isolated non-A, non-B hepatitis virus genomic cDNA covering the entire region of the virus gene nucleotide sequence from the 1st to 9416th nucleotides shown in FIG. 2(1) through FIG. 2(16) hereof, wherein the coding region is from the 333rd to 9362nd nucleotides, and the 5'- and 3'- noncoding sequences contain 332 nucleotides and 54 nucleotides, respectively. Part of the cDNA and an antigen polypeptide as an expression product thereof are useful as a diagnostic reagent for non-A, non-B hepatitis. The antigen polypeptide is also useful as an active ingredient for a non-A, non-B hepatitis virus vaccine.

BACKGROUND OF THE INVENTION 
Field of the Invention 
The present invention relates to a non-A, non-B hepatitis virus genomic 
cDNA and a non-A, non-B hepatitis virus genomic cDNA an a non-A, non-B 
hepatitis virus antigen polypeptide. More particularly, the present 
invention is concerned with a non-A, non-B hepatitis virus genomic cDNA 
which is useful for producing a non-A, non-B hepatitis virus antigen 
polypeptide and with a non-A, non-B hepatitis antigen polypeptide which is 
an expression product thereof. The non-A, non-B hepatitis virus genomic 
cDNA of the present invention is also useful for genetically diagnosing 
non-A, non-B hepatitis. Further, the non-A, non-B hepatitis antigen 
polypeptide of the present invention is useful for producing a vaccine for 
non-A, non-B hepatitis, an immunoglobulin, a polyclonal or monoclonal 
antibody, an immunological diagnostic reagent, an agent for screening 
blood for transfusion and an agent for use in affinity chromatography for 
removing non-A, non-B hepatitis virus from blood for transfusion. 
Discussion of Related Art 
Definition of non-A, non-B hepatitis virus 
The viral hepatitis is a liver disease caused by the infection of a 
hepatitis virus. Heretofore, hepatitis A virus, hepatitis B virus and 
hepatitis D (delta) virus have been isolated and identified. The hepatitis 
D virus (delta-hepatitis virus) is a deficient virus which cannot multiply 
by itself and requires for its multiplication the co-presence of hepatitis 
B virus as a helper virus. Therefore, the hepatitis D virus is present 
only in a patient having hepatitis B. In 1974, it was reported that there 
were many patients having hepatitis caused by a factor other than the 
infection with either hepatitis A virus or hepatitis B virus. Such a 
hepatitis was named "non-A, non-B hepatitis", and researches on the non-A, 
non-B hepatitis virus have been made extensively and intensively 
throughout the world. Heretofore, it has been found that a plurality of 
types of non-A, non-B hepatitis viruses exist. Results of the researches 
up to now show that the non-A, non-B hepatitis virus is classified into 
two types according to the infection route, that is, an epidemic hepatitis 
virus, namely an enterically-transmitted non-A, non-B hepatitis virus, 
which is spread through blood by transfusion, etc. Of the non-A, non-B 
hepatitis viruses, only an enterically-transmitted non-A, non-B hepatitis 
virus which spreads over the areas of Africa, India and Southeast Asia has 
been virologically identified, but the blood-transmitted non-A, non-B 
hepatitis virus has not yet been identified. 
Hereinbelow, the blood-transmitted non-A, non-B hepatitis is often referred 
to simply as "NANB hepatitis", and the blood-transmitted non-A, non-B 
hepatitis virus is often referred to simply as "NANBV". Current situation 
of the studies on NANB hepatitis and problems. 
With respect to the epidemiology, clinical examination, diagnosis, 
treatment and prevention of the NANB hepatitis, virological studies have 
been made in the world by the comparison of NANBV with the other hepatitis 
viruses, based on the knowledge of diagnostics, histopathology, 
immunology, molecular biology and the like ["Japan Medical Journal", No. 
3320, pp. 3-10 1987; "Igaku-no Ayumi (Progress of medicine)", 151(13) pp. 
735-923, 1989; "Kan Tan Sui (Liver, Gallbladder, Pancreas)", 21(1) pp. 
5-113, 1990; "Jikken Igaku (Experimental Medicine)", 8(3), pp. 201-233, 
1990 ]. With respect to the NANB hepatitis, the following findings have 
been reported. 
(1) Epidemiology: In Japan, according to the estimation by the Ministry of 
Health and Welfare, about 60% of chronic hepatitis patients (namely about 
720 thousand patients), about 40% of hepatocirrhosis patients (namely 
about 100 thousand patients) and about 40% of liver cancer patients 
(namely about 7 thousand patients) are patients having NANB hepatitis. 
Further, the mortality attributed to the above-described NANB hepatitis 
reaches 16 thousand per year. In U.S.A., the number of post-transfusion 
hepatitis patients reaches 150 to 300 thousand per year and 90% of the 
post-transfusion hepatitis patients are patients having NANB hepatitis. 
Further, it is considered that 1 to 6% of the blood donors are NANBV 
carriers. Further, it is estimated that in the other countries also, the 
incidence of NANB hepatitis and the ratio of the NANBV carrier are equal 
to or higher than those in U.S.A. and Japan. Therefore, prevention, early 
diagnosis and early treatment of the NANB hepatitis are of global 
importance. 
(2) Virology: The NANBV heretofore reported comprises an envelope and 
assumes a viral particle having a spherical shape of about 50 nm in 
diameter. The taxonomic observations suggest that the known NANBV is a 
virus similar to a togavirus or a flavivirus, or a virus of new type 
different from the togavirus or flavivirus. Further, the results of 
pathological observations of the cytoplasm of hepatocytes of a plurality 
of chimpanzees injected with serum of a patient having NANBV hepatitis 
show that the formation of a tubular structure occurs in the cytoplasm of 
a hepatocyte of some of the chimpanzees, but does not occur in the 
cytoplasm of a hepatocyte of the other chimpanzees, and that an 
intranuclear particle is formed in the cytoplasm of a hepatocyte of some 
of the chimpanzees. These results and the results of the epidemiological 
observations, tests on the presence or absence of the chloroform 
sensitivity and immunological diagnosis suggest that a plurality of types 
of NANBV-sexist (see, for example, "Science", Vol. 205, pp. 197-200, 1979, 
"Journal of Infectious Disease", Vol. 148, pp. 254-265, 1983 and 
"Biseibutsu"(Microorganism), Vol. 5, No. 5, pp. 463-475, 1989). The amount 
of the NANBV present in the blood of a patient having NANB hepatitis is 
extremely small as compared to either the amount of a hepatitis A virus 
present in the feces of a patient having hepatitis A or the amount of a 
hepatitis B virus present in the blood of a patient having hepatitis B. 
For example, the amount of hepatitis B virus in the blood of the patient 
is 10.sup.8 to 10.sup.9 per ml in terms of Chimpanzee Infectious dose 
(CID), whereas the amount of NANBV in the blood of the patient is only 
10.sup.4 to 10.sup.5 per ml in terms of CID (Bradley, D. W.: Research 
perspective in post-transfusion non-A, non-B hepatitis, in "Infection, 
Immunity and Blood Transfusion", edited by Dodd, R. Y. & Barker, L. F., 
published by Alan R. Liss, Inc., New York (1985) pp. 81-97). Further it is 
known that except for human, there are no animals except chimpanzee that 
are sensitive to NANBV and that in the cytoplasm of the hepatocyte, a 
typical tubular structure is occasionally formed by NANBV infection. Since 
only chimpanzee can be used as an animal for experiment of the NANBV 
infection, a large number of chimpanzees are required to be used for the 
study of NANBV. However, the chimpanzee is not easily available and 
expensive. Therefore, the study of NANBV, identification of NANBV and 
search for a useful marker for NANBV, is necessarily restricted and delay. 
In order to solve these problems, various attempts have been made for the 
study of NANBV. For example, in an attempt, an NANBV genomic cDNA 
[(referred to as "hepatitis C virus (HCV)"] was cloned from blood plasma 
of chimpanzees suffering from NANB hepatitis (Science, Vol. 244, pp. 
359-362, 1989), and it was confirmed that the antigen (referred to as 
"C-100") obtained by expressing the cDNA exhibited an antigen-antibody 
reaction with the antibody in the blood of an NANB hepatitis patient 
(Science, Vol. 244, pp. 362-364, 1989). Further, in another attempt, a 
chimpanzee was not used and an NANBV genomic cDNA was cloned from the 
blood plasma of NANB hepatitis patients, and it was confirmed that the 
antigen obtained by expressing the cDNA exhibited an antigen-antibody 
reaction with the antibody in the serum of an NANB hepatitis patient 
(Gastroenteriologia Japonica, Vol. 24 pp. 540-544 and pp. 545-548, 1989). 
(3) Clinical observations: Hepatitis is generally classified either into 
epidemic hepatitis and sporadic hepatitis according to the number and 
frequency of the occurrences of hepatitis, or into acute hepatitis, 
fulminant hepatitis, subacute hepatitis, persistent hepatitis and chronic 
hepatitis according to the severeness and stage of the hepatitis patients. 
The latent period of the NANB hepatitis is 2 to 26 weeks. The symptom of 
NANB hepatitis in the early stage is mild as compared to that of hepatitis 
B. For example, a patient having NANB hepatitis only becomes feverish and 
complains of languor. Further, 70% of the patients have anicteric symptom. 
Therefore, the NANB hepatitis is frequently overlooked. However, the NANB 
hepatitis is very dangerous because the NANB hepatitis is likely to become 
chronic and, then, to progress to liver cirrhosis. Illustratively state, 
40 to 50% of the patients having NANB hepatitis whose serum exhibits an 
increased aminotransferase activity develop chronic hepatitis. 10 to 20% 
of the cases of chronic hepatitis suffer from liver cirrhosis. Further, 
0.5 to 1% of blood recipients per year becomes liver cirrhosis patients 
without subjective symptoms. More seriously, the liver cirrhosis may 
further progress to liver cancer or hepatoma. Therefore, for preventing 
biohazard caused by blood transfusion and bleeding, eradication of the 
NANB hepatitis is a matter of global importance from the viewpoint of 
public health. 
(4) Diagnosis: As mentioned above, the NANBV (blood-transmitted type) has 
not yet been identified and a viral marker, such as an NANBV antigen, 
which is useful for the diagnosis of NANB hepatitis has not been known. 
Therefore, diagnosis of NANB hepatitis has been conducted by examining the 
titer of the antibody in serum of a patient, which is specific for each of 
the known pathogenic viruses, such as hepatitis A virus, hepatitis B 
virus, cytomegalovirus, EB virus, varicella virus and herpes simplex 
virus, and diagnosing the patient whose serum is negative with respect to 
the antibody specific for any of the above-mentioned viruses, as having 
NANB hepatitis, or by performing a histopathological examination through a 
biopsy of the liver ("Disease of the Liver and biliary system", 8th 
edition, S. Shenlock, pp. 326-333, 1989, Blackwell Scientific 
Publications). At the same time, another diagnosis method has also been 
used. For example, there have been used a method in which the activity of 
an enzyme in serum, such as GPT [glutamic-pyruvic transaminase, also known 
as "ALT" (alanine aminotransaminase)], GOT [glutamic-oxalo-acetic 
transaminase, also known as "AST" (aspartate aminotransferase)], and 
guanine deaminase (also known as "guanase") is determined ("Kan Tan Sui 
(Liver, Gallbladder, Pancreas)", Vol. 14, pp. 519-522, 1987). With respect 
to the GPT or GOT in serum mentioned above, a standard for the diagnosis 
of NANB hepatitis in which lasting and abnormally high activities of GPT 
and GOT are utilized as a criterion for the diagnosis of NANB hepatitis, 
is employed in Japan ("Journal of Blood Transfusion Society in Japan", 
Vol. 31, No. 4, pp. 316-320, 1985; and "Nippon Rinsho", Vol. 46, p. 
2635-2638, 1988). Regarding the immunological diagnosis, in the present 
situation in which the isolation and identification of NANBV are 
difficult, an antigen-antibody reaction between an antigen obtained by 
expression of NANBV and cDNA clone (which has been isolated using the 
techniques of genetic engineering and the knowledge of immunology) and the 
serum of an NANB hepatitis patient is used as a criterion. Examples of 
known antigens include an expression product of an NANBV cDNA prepared 
from the plasma of an NANB hepatitis patient (European Patent Application 
Publication No. 363025), an expression product of "HCV" cDNA prepared from 
the plasma of a chimpanzee having the symptoms of NANB hepatitis (European 
Patent Application Publication No. 318216 and Japanese Patent Application 
Laid-Open Specification No. 2-500550) an expression product of an NANBV 
cDNA derived from the liver of an NANBV-infected chimpanzee (European 
Patent Application Publication No. 293274, Japanese Patent Publication 
Specification No. 64-2576 and Japanese Patent Application Laid-Open 
Specification No. 1-124387). As a method for determining the 
antigen-antibody reaction, RIA (radioimmunoassay) and EIA (enzyme 
immunoassay) are generally used. However, these expression products are 
different in antigenioity. The antigen which is an expression product of 
HCV cDNA (that is, the C-100 antigen mentioned above) can be some 
criterion or yardstick for the diagnosis of chronic hepatitis caused by 
the HCV infection. However, since the region in which the antigen (C-100) 
exhibits its antigenicity is limited ("Biseibutsu (Microorganism)", Vol. 
5, pp. 453-475, 1989; "Kan Tan Sui (Liver, Gallbladder, Pancreas)", Vol. 
20, pp. 47-51, 1990; and "Igaku-no Ayumi (Progress of Medicine)", Vol. 
151, p. 871, 1989), this antigen is unsatisfactorily from the viewpoint of 
accurate diagnosis of NANB hepatitis and NANBV infection and from the 
viewpoint of accurate determination of the progress of a patient suffering 
from chronic hepatitis and acute hepatitis for treatment thereof. 
Therefore, it has been desired to obtain a reliable method for the 
diagnosis and prognosis of the NANB hepatitis. 
(5) Therapy and Prevention: Recently, the usefulness of .alpha.- and 
.beta.- interferons in the treatment of chronic NANB hepatitis have been 
reported ("Kan Tan Sui (Liver, Gallbladder, Pancreas)" vol. 20, pp. 59-64, 
1990; "Igaku-no Ayumi (Progress of Medicine)", vol. 151, pp. 871-876, 
1990). However, a suitable does of .alpha.- and .beta.- interferons and a 
suitable period for administration thereof have not yet been established. 
On the other hand, for prevention of NANB hepatitis, various vaccines are 
used in which the above-mentioned conventional expression products of 
NANBV cDNAs (European Patent Application Publication No. 363025) or HCV 
cDNAs (European Patent Application Publication No. 318216) are used as an 
antigen. However, as is apparent from the fact that the NANBV itself has 
not yet been isolated and identified before completion of the present 
invention, it has been impossible to specify an antigen useful for NANBV 
vaccines from the above-mentioned expression products each having a 
variety of antigenic determinants (epitopes) and determine the 
effectiveness and safety of such a specific antigen so that the antigen 
can be clinically used. Accordingly, there is no NANBV vaccine which can 
be advantageously put into practical use. 
SUMMARY OF THE INVENTION 
The present invention have made extensive and intensive studies with a view 
toward solving the above-mentioned problems by developing a novel NANBV 
genomic cDNA. As a result, the present invention have surprisingly 
succeeded in cloning an NANBV genomic cDNA, which not only has excellent 
reliability as compared to the known NANBV cDNA but also is larger in 
length than any know NANBV cDNAs and contains the entire region of the 
open reading frame of the NANBV antigen peptide which can reliably exhibit 
an antigen-antibody reaction specific for not only sera from patients 
having chronic NANB hepatitis but also sera from patients having acute 
NANB hepatitis. This success is attributed to a unique technique of the 
present inventors such that in order to obtain an authentic NANBV genome, 
NANBV RNAs are extracted directly from NANBV particles contained in whole 
blood of a patient having NANB hepatitis or a resected liver of a patient 
having NANB hepatitis and liver cancer in combination, without multiplying 
the NANBV in a chimpanzee having unknown factors which are considered to 
have rendered difficult the isolation of NANBV, although the amount of 
NANBV in the blood or resected liver is extremely small, that is, as small 
as about 1/10,000 that of a hepatitis A virus or a hepatitis B virus, but 
with paying minute care in the operating procedure so that the NANBV and 
its genome do not undergo cleavage and/or decomposition by the action of 
body fluids or blood enzymes during the storage of fresh materials for 
NANBV genome. RNAs thus prepared from fresh human materials are then 
converted to double-stranded cDNA by means of a reverse transcriptase to 
obtain a cDNA library. In order to screen an NANBV genome from the cDNA 
library, the cDNAs are individually inserted in lambda gtll phage vectors 
and then expressed on the phage plaques at high concentration, followed by 
screening of NANBV genomic cDNAs by repeatedly conducting enzyme 
immunoassay (EIA) in which both serum from a convalescent patient having 
acute NANB hepatitis and serum from a patient having chronic NANB 
hepatitis are used. Thus, safe production of the NANBV antigen polypeptide 
with high purity on a large scale at low cost without biohazard, has for 
the first time been realized by expressing the cDNA of the present 
invention by recombinant DNA techniques. Based on the above, the present 
invention has been completed. 
Therefore, it is an object of the present invention to provide an NANB 
hepatitis virus genomic cDNA. 
It is another object of the present invention to provide an NANB hepatitis 
virus antigen polypeptide which is useful as an active ingredient for a 
diagnostic reagent and a vaccine for NANB hepatitis. 
It is still another object of the present invention to provide a method for 
producing an NANBV antigen polypeptide. 
It is a further object of the present invention to provide a diagnostic 
reagent for NANB hepatitis. 
It is still a further object of the present invention to provide a vaccine 
for NANB hepatitis. 
The foregoing and other objects, features and advantages of the present 
invention will be appended from the following detailed description, 
appended claims taken in connection with the accompanying drawings.