A purified form of a DNA virus which has the following characteristics: molecular weight greater than 2.times.10.sup.6 Daltons; substantial immunoreactivity towards an anti-HBsAg monoclonal antibody obtained from cell line ATCC HB 8058; substantially no immunoreactivity towards an anti-HBsAg monoclonal antibody obtained from cell line ATCC CRL 8018; concentration dependent immunoreactivity towards polyclonal IgG anti-HBsAg antibodies, which increases with increased concentration of said DNA virus; discrete particulate form when observed by immunoelectron microscopy in the presence of IgM antibodies from cell line ATCC HB 8058; the DNA of said virus showing hybridization with DNA from hepatitis B viral DNA; and said DNA virus showing, in chimpanzees, infectivity having the characteristics of non A, non B hepatitis.

1. Field of the Invention 
The present invention deals with the identification, isolation, 
characterization, purification and use of non A, non B hepatitis virus, as 
well as diagnostic methods and vaccine methods therefor. 
2. Brief Description of the Prior Art 
The name non A, non B hepatitis is given to acute and chronic cases of 
viral hepatitis in humans which occur in the absence of infection with any 
known or serologically identifiable virus associated with hepatitis B 
(HBV) or hepatitis A (HAV). The characteristics of non-A, non-B 
(hereinafter "NANB") hepatitis are well described in Dienstag et al, 
Chapter 302 of Harrison's "Principles of Internal Medicine", 9th Ed, 
McGraw-Hill Book Co., 1980) , pp. 1459-1467, and by Robinson, W. S., "The 
Enigma of Non-A, Non-B Hepatitis", The Journal of Infect. Dis., Vol. 145 
No. 3, pp. 387-395 (1982). These two articles are herein incorporated by 
reference, and the following comments are extracted therefrom. 
Sensitive serologic tests for identifying both types A and B hepatitis have 
led to the identification of hepatitis cases with incubation periods and 
modes of transmission consistent with an infectious disease, but without 
serologic evidence of hepatitis A or B infection. 
Transmission of the disease to chimpanzees has clearly established that 
many of the cases are caused by one or more infectious agents. There have 
been intensive efforts in many laboratories throughout the world in the 
past few years to identify and characterize agents that are responsible 
for these infections. 
Clinical diagnosis of NANB hepatitis is made by excluding infection with 
known hepatitis viruses and other known factors that cause hepatitis. The 
infection occurs with high frequency after blood transfusion or parental 
drug abuse, in person to person contact and in other settings that are 
also associated with HBV infections. Endemic and apparently epidemic 
disease has also been observed without obvious overt parental 
transmission. 
Despite these advances and intensive efforts to date, no etiologic agent of 
NANB hepatitis has been unequivocally identified as an antigenic 
ultrastructural or molecular entity. This result suggests that the 
concentration of viral antigen in the serum of patients with NANB 
hepatitis may be much lower than that of HBV antigen in patients with 
hepatitis B, or that appropriate reagents or methods have not been 
heretofore described to identify the virus, its proteins, or its genetic 
material. 
The most important experimental advance in this field to date has been the 
transmission of NANB hepatitis agents to chimpanzees. This provided a 
direct demonstration of a transmissible agent, associated with NANB 
hepatitis, in an animal model of the disease (See, for example, Alter, H. 
J. et al, Lancet 1: 459-463 (1978), Tabor, E. et al, ibid 1: 463-466 
(1978), Hollinger, F. B. et al, Intervirology 10: 60-68 (1978), or Bradley 
D. W. et al, J. Med. Virol. 3: 253-269 (1979), all of which are herein 
incorporated by reference). 
Despite the fact that NANB hepatitis has been transmitted to experimental 
animals, no virus or other infectious agent(s) has been physically 
identified with certainty prior to this invention. Although detection of 
apparently unique antigen/antibodies systems in the sera of patients and 
chimpanzees with NANB hepatitis have been reported, the results have been 
difficult to confirm, and none of these tests has clearly identified sera 
known to contain NANB agents (see for example, Vitvitski, L. et al, Lancet 
22: 1263-1267 (1979), Kabiri, M. et al, Lancet 2: 221-224 (1979), Tabor 
E., J. Med. Vol. 4: 161-169 (1979) and Chircu, L. V. et al, J. Med. Virol. 
6: 147-151 (1980). In addition to antigen, virus-like particulate 
structures have been observed by electron microscopy in serum and liver of 
humans and chimpanzees infected with NANB hepatitis (see for example 
Bradley, D. W., J. Med. Virol. 3: 253-269 (1979) and Bradley, D. W. et al, 
J. Med. Virol. 6: 85-201 (1980)). 
An evaluation of all of these studies has been made by Robinson, supra in 
J. Inf. Dis. Vol. 145, (1982) who stated that "Without more definitive 
evidence concerning these particles and because numerous investigators 
have failed to confirm these findings it is not possible at this time to 
conclude that any HBV-like virus is ever a cause of NANB hepatitis." 
In view of all of the above, it is quite clear that there exists at present 
a great need to identify, isolate and characterize the etiologic agent(s) 
causative of NANB hepatitis. A need also exists for accurate and 
unambiguous identification and detection techniques therefor, which will 
help in the quick and accurate diagnosis of the disease. 
SUMMARY OF THE INVENTION 
It is therefore an object of the invention to provide an accurate and 
specific characterization of the etiologic agent of NANB hepatitis. 
It is another object of the invention to provide for a method of 
identifying and detecting the etiologic agent of NANB hepatitis in 
samples. 
Still another object of the invention is to provide for a method of 
diagnosing NANB hepatitis in animals. 
Yet another object of the invention is to provide a vaccine against NANB 
hepatitis, and a method of immunization which comprises the use of such 
vaccine. 
Still another object of the invention is to provide a method for the 
purification of NANB hepatitis virus. 
These and other objects of the invention as will hereinafter become more 
readily apparent have been attained by providing: 
A purified form of a DNA virus which has the following characteristics: 
(1) molecular weight greater than 2.times.10.sup.6 daltons; 
(2) substantial immunoreactivity towards an anti HBsAg monoclonal antibody 
obtained from cell line ATCC HB 9801. 
(3) substantially no immunoreactivity towards an anti HBsAg monoclonal 
antibody obtained from cell line ATCC CRL 8018; 
(4) concentration dependent binding capacity towards polyclonal IgG 
anti-HBsAg antibodies, which increases with increased concentration of 
said DNA virus; 
(5) discrete particulate form when observed by immunoelectron microscopy in 
the presence of IgM antibodies from cell line ATCC HB 9801. 
(6) a polypeptide profile on sodium dodecyl sulfate polyacrylamide gels, 
when affinity purified with IgM antibody from cell line ATCC HB 9801, 
comprising bands at about 50,000, about 23,000 and about less than 20,000 
molecular weight; 
(7) the DNA of said virus showing partial sequence homology with hepatitis 
B virus DNA by molecular hybridization; and 
(8) said DNA virus showing, in chimpanzees, infectivity having the 
characteristics of non A, non B hepatitis. 
Another object of the invention has been attained by providing a method of 
detecting the presence of non A, non B hepatitis virus in the sample of an 
animal which comprises (A) confirming the presence of said virus in said 
sample, and (B) distinguishing said virus from hepatitis B virus. 
Another object of the invention has been obtained by providing a method of 
purifying NANB virus from an animal sample by immunoaffinity 
chromatography wherein the immunosorbent antibody is a monoclonal antibody 
having substantial imnunoreactivity towards said NANB virus. 
The present invention also provides vaccines and vaccination methods 
utilizing live, attenuated or inactivated forms of the NANB virus.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is based on the discovery of highly specific and 
accurate tests for the identification and characterization of the 
causative agent of non A, non B hepatitis. The inventors have made use of 
a variety of analytical techniques to characterize NANB hepatitis virus 
and distinguish the same from hepatitis virus A (HVA) and hepatitis virus 
B (HBV). These techniques include physical-chemical properties, 
immunological properties, genetic characterization and infectivity 
characterization. 
The discovery of NANB hepatitis virus was made by detecting its presence in 
the blood of persons with the clinical signs of hepatitis but no serologic 
identification by any of the prior art immunoassay techniques using 
polyvalent IgG antibodies. A series of monoclonal antibody screening tests 
were then developed with alternatively positive and negative binding for 
various different monoclonal antibodies, which can readily characterize 
and detect NANB virus and distinguish the same from hepatitis B virus. 
In the discussions that follow, mention is made to a number of antibodies, 
both monoclonal and polyvalent. For clarity purposes and reference, the 
following summarizes the nature, origin and type of these antibodies: 
(1) 5D3: Represents a monoclonal IgM antibody against HBsAg, obtained from 
hybridoma 5D3 on deposit at the ATCC with deposit number HB 9801. 
Reference is made to this antibody in U.S. Pat. No. 4,271,145 to Wands et 
al, as well as in Wands et al, Proc. Nat. Acad. Sci., USA Vol. 78: 
1214-1218, Feb. 1981, both of which are herein incorporated by reference. 
(2) 3D4: Represents a monoclonal IgM antibody having specificity against 
HBsAg (i.e. anti-HBsAg), obtained from cell line 3D4 on deposit at the 
ATCC with deposit number HB-8170. 
(3) 1F8: Represents a monoclonal IgM anti-HBsAg antibody derived from cell 
line 1F8 on deposit at the ATCC have deposit number CRL 8018. This 
antibody is described in the aforementioned Wands et al U.S. Pat. No. 
4,271,145 and Wands et al PNAS Vol. 78, Feb. 1981 paper. 
(4) 5C11: Represents a monclonal IgG.sub.1 anti-HBsAg antibody obtained 
from cell line 5C11 deposited at the ATCC with deposit number HB-8171. 
(5) AUSRIA II: Represents antibodies from a commercially available HBsAg 
test kit (Abbott) containing polyclonal IgG antibodies. 
These and other mentioned antibodies are also described in one or both of 
the following copending U.S. Pat. applications: Ser. No. 188,735, filed 
September 19, 1980 to Wands, Zurawski and Ser. No. 533,161 filed Sept. 19, 
1983, a continuation of Schoemaker, for "Immunoassay Utilizing Monoclonal 
High Affinity IgM Antibodies"; and Ser. No. 372,530, filed Apr. 28, 1982, 
to Wands and Zurawski, by the same title, both of which are also herein 
incorporated by reference. 
Also, a bacterial culture containing a recombinant plasmid with HBV-DNA 
sequences, pAO1 HBV is on deposit with ATCC Number 31873. 
The NANB virus can be isolated from either a human or other animal host, 
e.g., chimpanzee, marmoset, and other suitable hosts for NANB virus, which 
is infected with NANB hepatitis. The presence of the NANB virus has been 
implicated in the prior art by excluding identifiable hepatitis viruses 
(HAV, HBV, Epstein-Barr virus, cytomegalovirus and others) and other 
etiologic factors (for example, hepatotoxic drugs and chemicals). 
Exclusion of other viruses mentioned above can still be used to suggest 
but not establish the presence of the NANB infectious agent in the host. 
However, with the advent by the present invention of highly specific tests 
for NANB virus it is preferred to utilize these, see infra. 
Preferably, affinity chromatography using monoclonal 5D3 IgM anti-HBs can 
be utilized for the purification and subsequent characterization of the 
antigen composition. A suitable material is obtained by coupling 5D3 to 
Sepharose 6B.RTM.. Serum from an appropriate host is placed in contact 
with the monoclonal antibody on the solid phase support and the material 
is incubated for several hours at room temperature. The supports are then 
extensively washed with an appropriate physiological buffer (e.g., 
PBS-phosphate buffer saline) at a physiological pH. Column fractions can 
then be collected with an acidic buffer (for example pH 2-3). The pH of 
each fraction is adjusted to physiological pH, and binding activity is 
determined with the appropriate antibodies. Peak fractions exhibiting the 
highest binding activity can then be pooled to collect the NANB virus. 
Virus can also be isolated from the supernatant of any cell culture (e.g., 
bacteria, yeasts and other eukarotic cells infected with said virus or 
viral DNA) or fermentation broth producing the same. 
The NANB virus can be characterized and identified by at least four 
different characteristics, each of which is described in turn hereinbelow. 
Physical-chemical characteristics. 
NANB virus has a molecular weight of approximately 2.times.10.sup.6 as 
determined by Sepharose 4B.RTM. chromatography. The virus appears as 
distinct particles by immunoelectron microscopy. When the virus is 
isolated from serum by the 5D3-IgM affinity chromatography described 
above, prominent spiculated particles at 220,000.times. are observed, 
suggesting the presence of 5D3 anti-HBs on their surface. When affinity 
purified material is applied to sodium dodecyl sulfate 10% polyacrylamide 
gels and compared to HBsAg virus it is seen that there are similar 
polypeptides in all specimens at a molecular weight of about 50,000 and at 
molecular weights of 22,000-23,000. In addition, however, NANB virus shows 
three additional major protein bands not observed in HBsAg, one has a Mr 
of approximately 80,000 and, of the two others, the first has a molecular 
weight slightly greater than 23,000 and the second has a molecular weight 
less than 20,000. (See FIG. 3). 
Immunological characteristics. 
NANB virus reacts with some monoclonal antibodies having specificity, i.e., 
immunoreactivity, for distinct HBsAg-related epitopes, and not with other 
such anti-HBsAg monoclonal antibodies. For example, NANB will cross react 
at all concentrations with antibody 5D3 or with antibody 3D4 (both of 
which are monoclonal IgM anti-HBsAg antibodies). On the other hand, NANB 
will not cross-react with antibody 1F8 (also a monoclonal IgM having 
specificity against HBsAg) or with monoclonal 5C11 (a IgG.sub.1 antibody). 
This serves to clearly distinguish NANB virus from hepatitis B virus, 
which reacts with these monoclonal antibodies. Immunoreactivity of NANB 
with polyvalent anti-HBs antibodies (commercially available AUSRIA II) is 
concentration dependent. At concentrations of about 1 ng to 100 ng, the 
polyvalent IgG antibodies do not detect or bind the NANB virus. Upon 
concentration of NANB by about 100 fold or larger than these, binding and 
detection by polyvalent IgG can be observed. However, in some instances, 
polyvalent anti-HBsAg antibodies do not detect or bind to NANB hepatitis 
serum even after enrichment by affinity chromatography and 100 fold 
concentration as described above. Preincubation of NANB with 5D3 
anti-HB.sub.s at these higher concentrations blocks the binding by 
conventional polyvalent anti-HBs. 
Genetic Characteristics. 
The DNA sequence of NANB virus is partially homologous related but not 
identical to HBV-DNA. It can thus be detected by hybridization with a 
purified HBV-DNA probe. See, infra. 
Infectivity Characteristics 
NANB virus having the above physico-chemical, morphological, immunologic 
and genetic characteristics is infectious. Infectivity studies of viral 
hepatitis are positive in chimpanzees and in man. The characteristics for 
the infection are different than those normally seen for HBV or HAV. The 
incubation period, as defined from inoculation of infectious material to 
the appearance of virus or viral protein in the blood, is longer than 
previously recognized. Alanine aminotransferase (ALT) elevation precedes 
the appearance of antigenemia by several weeks. Antigenemia may occur in 
the absence of ALT elevations, a phenomenon observed in man. A chronic 
viral carrier state in man and chimpanzees may occur. The period of 
antigenemia and/or viremia appears to persist for weeks to months and 
usually disappears with recovery. Antigenemia is still detectable in the 
resolution phase of illness when ALT levels are normal, a similarity to 
HBV infection in man. Several episodes of antigenemia may occur during the 
course of infection. Pre-existing anti-HBs is not protective in the 
animal, confirming that NANB virus is sufficiently different in antigenic 
composition from HBV. (See FIGS. 6, and 8-10). 
It should be noted, of course, that the aforementioned characteristics are 
but only one possible set. Obviously, as more of these characteristics are 
researched and discovered it may be possible to characterize the virus, 
for example, by additional monoclonal antibodies, or DNA probes including 
one or more that do not cross react at all with hepatitis B. This 
possibility, however, is fully contemplated in the present application 
which, when pertaining to the virus per se, is meant to cover the virus 
itself regardless of any additional or even novel identifying tests. 
The NANB virus characteristics can be used to develop highly sensitive and 
accurate tests for detecting the presence of NANB virus in animal samples, 
such as blood--especially blood to be transfused-, serum, urine, milk, 
tissue samples, feces, and the like. Particularly useful is the detection 
of NANB virus in animal serum, especially human serum, and products 
derived from human blood, such as red blood cells, plasma, platelet 
concentrates, clotting factor concentrates and the like, for the diagnosis 
of NANB hepatitis. Also particularly useful is the detection of NANB virus 
in samples of blood from blood donors, to screen for the possibility of 
transmission of NANB hepatitis infection to recipients. 
The availability of purified NANB virus allows for the development of 
immunoassay methods and systems. Any appropriate antibodies can be used in 
any of the multiple immunoassay procedures currently available to the art 
(see for example, T. Chard "An Introduction to Radioimmunoassay and 
Related Techniques", North-Holland 1978, or Schuurs, A. H. W. M, et al, 
"Enzyme Immunoassay", Clin. Chim. Acta 81: 1-40 (1977), both of which are 
herein incorporated by reference). For example, the presence of the virus 
in a sample can be detected by radioimmunoassay, enzyme immunoassay, or 
latex agglutination immunoassay. The technique utilized can be 
competitive, "sandwich" (forward, reverse or simultaneous), double 
antibody, or enzyme cascade, all of which are well known to those of skill 
in the art. It may be useful for certain techniques to prepare, by art 
known methods, detectably labeled NANB virus such as NANB labeled with a 
radiolabel (I.sup.125, C.sup.14, H.sup.3, P.sup.32, etc.), with an enzyme 
(alkaline phosphatase, peroxidase, etc.) with a fluorescent probe, and the 
like. The antibodies can be either in solution or immobilized, such as for 
example, on the inside of tubes, on polymer or glass beads, on plastic 
strips, and the like. 
Detection can also be carried out by hybridization analysis using a 
detectably labeled probe. The genetic information or code of a specific 
virus comprises a nucleic acid which may be composed of a polymer of 
ribonucleotides (RNA) or deoxyribonucleotides (DNA). It is known that 
nucleotide molecules that are complementary to one another can interact in 
solution by "hydrogen-bonding" to form stable base pairs. Thus, adenine 
recognizes thymidine and guanine recognizes cytosine. When two 
single-stranded, complementary, DNA molecules are present in a solution 
under conditions in which the complementary nucleotides can recognize one 
another, these molecules will interact to form a stable duplex structure. 
This duplex is resistant to attack by certain nucleases which totally 
degrade single-stranded DNA. It is therefore possible to ascertain with 
great precision the extent of duplex formation. This interaction of base 
sequences in polynucleotides reacting in solution is referred to as 
"reannealing" or "molecular hybridization" and can be performed under 
specific and sensitive conditions in which false interactions do not 
occur. 
For substantially stable and recognizable hybrids to be formed, minimum 
complementary sequence lengths of approximately 50-100 nucleotides or more 
often 100-200 nucleotides are required. The ability to form such hybrids 
appears to depend on the experimental conditions of the hybridization 
reaction (ionic strength, polarity, pH and temperature of the 
hybridization solution), the concentration of the complementary nucleic 
acid molecules and the length of time of the incubation. Another variable 
in the reaction is the physical state of the DNA in the test sample, in 
that it can be in solution or fixed to a solid support matrix such as a 
nitrocellulose filter paper. In the latter case, the rate of hybridization 
between the detecting probe and the test sample of DNA affixed to the 
solid support surface is slowed by approximately 30%. The latter method 
is, however, extremely sensitive for detection of hybridizing sequences 
and with a [.sup.32 P] radioactively labeled DNA probe of specific 
activity 2-4 .times.10.sup.8 cpm per .mu.g DNA, as easily obtained by 
workers skilled in the art, a 2-5 mm diameter circular spot on a 
nitrocellulose filter containing 0.1 pgm (10.sup.-13 gm) of specific DNA 
sequence or less can be detected. 
Depending on the various factors mentioned above, the hybridization 
reaction can be performed under very stringent conditions, so that a 
perfect or near perfect match in complementary DNA sequence is required or 
under less stringent conditions in which only a partial match is required. 
As the conditions for stringency of hybridization are relaxed, nucleic 
acid molecules of lesser and lesser sequence homology will form hybrids. 
This, of course, decreases the specificity of the reaction and raises the 
chances of false positive results. Therefore, in the preferred embodiment, 
hybridization conditions of high stringency have been used, so that only 
molecules with sequence regions of approximately 100-200 nucleotides or 
more in common with or nearly identical to HBV-DNA will form stable and 
detectable hybrids on a nitrocellulose filters. This enables the use of 
the hybridization method to identify DNA molecules in any cell, tissue, 
tissue extract, serum, plasma, body fluid, secretum, semen, breast milk, 
vaccine or the like, containing DNA molecules or genetic information 
closely related, nearly identical or identical to NANB-DNA. 
As disclosed in this invention, NANB hepatitis virus(es) contain sequences 
closely related to HBV-DNA and can be detected by hybridization with a 
purified and suitably labeled HBV-DNA probe. DNA or RNA molecules which 
are not closely related to HBV-DNA will not be identified or detected by 
this method. These methods, considerations and conditions as well as many 
variations in hybridization technology as well as means to detect, isolate 
and identify hybrids are well known to those skilled in the art. Details 
concerning the preparation of the recombinant HBV-DNA probe, the labeling 
of the probe, the hybridization conditions are described in Chakrabarty et 
al, Nature, Volume 286, No. 5772, pages 531-533, July 31, 1980; Shouval et 
al, Proceedings National Academy of Science (PNAS) U.S.A., Volume 77, No. 
10, pages 6147-6151, Oct. 1980; Shafritz and Kew, Hepatology, Volume 1, 
No. 1, pages 1-8, Jan.-Feb., 1981; Shafritz, D. A. et al, New Engl. J. 
Med., 305:1067-1073, 1981; and copending U.S. patent application Ser. No. 
249,369, filed Mar. 31, 1981 entitled Diagnostic Test for Hepatitis B 
Virus; now U.S. Pat. No. 4,562,159 all hereby incorporated by reference. 
Regardless of the technique(s) used, the detection of the virus in a sample 
is carried out by an overall two step test, which not only serves to 
confirm its presence but also distinguishes it from HBV, with which it is 
closely related. 
For example, the detection test can comprise a first step of testing for 
immunoreactivity with an antibody such as 5D3 or 3D4, with which NANB 
virus is reactive, followed by a second step of immunoassay with an 
antibody such as 5C11 or 1F8 with which NANB virus is not cross reactive, 
but HBV is. 
Another two step test comprises a first immunoassay step using an antibody 
such as 5C11 or 1F8 (showing no cross reactivity), followed by DNA 
hybridization using an HBV-DNA or an NANB-NA (see infra) detectably 
labeled probe (e.g., .sup.32 P or biotin-labeled probe). 
An alternative test is a two step methodology wherein the first step is an 
immunoassay with 5D3 or 3D4 monoclonal IgM, followed by studying the 
infectivity characteristics in chimpanzees. 
Alternatively, a two step analysis can be used with the first step being an 
immunoassay with 5D3 and in a second step a polyacrylamide gel on sodium 
dodecyl sulfate seeking the differential proteins present in NANB and not 
present HBsAg. 
There are obviously other possibilities, such as procedures utilizing more 
than two steps, for example, screening with 5D3, 3D4, 5C11, 1F8, testing 
for hybridization with a DNA probe, and infectivity characteristics. The 
two step test, (in any desired order) however, is a minimum, in order to 
distinguish over the possibility that the samples may be infected with 
HBV. 
Lack of cross reactivity with polyvalent IgG anti-HBsAg is also indicative 
of the presence of NANB virus and can be added to the battery of the 
aforementioned tests. It is however, not conclusive evidence since 
positive identification such as concentration of antigen is still needed 
to confirm its presence. 
The invention lends itself to the preparation of kits useful in the 
diagnosis of NANB hepatitis. For example, such a kit may comprise a 
carrier being compartmentalized to receive one or more container means 
therein, including a first container containing a monoclonal IgM antibody 
having immunoreactivity towards said NANB virus; and a second container 
containing a monoclonal antibody having immunoreactivity towards HBsAg but 
no immunoreactivity towards the NANB virus. 
The kit may also comprise a third container means containing detectably 
labeled HBV-DNA probe, and/or additional container means containing 
another monoclonal antibody having immunoreactivity towards HBsAg but no 
immunoreactivity towards the NANB virus. 
Detectably labeled HBV-DNA may also be present in the kit in another 
container. 
The use of hybridization techniques initially with purified cloned HBV-DNA 
can be utilized to clone the DNA of NANB hepatitis viruses with partial 
sequence homology to HBV-DNA. This is based on the finding that even under 
very stringent hybridization conditions, the HBV-DNA probe is capable of 
detecting NANB virus in both human and chimpanzee serum. With purification 
of the virus by the monoclonal antibody affinity column described herein, 
the DNA of the virus can be extracted and cloned in bacterial plasmids 
such as pBR 322 or bacteriophages such as bacteriophage .lambda.. 
A series of restriction endonucleases are used to cleave the DNA into 
specific segments with known specific 5' and 3' ends by recognization of 
specific hexanucleotide sequences in double-stranded DNA. These DNA 
fragments can then be introduced into plasmids or bacteriophages treated 
with the same restriction enzymes to produce chimeric recombinant DNA 
molecules. These recombinant DNA molecules are introduced into E. coli, 
amplified and produced in large amounts. Recombinants containing NANB 
virus DNA sequences related to HBV-DNA are identified by molecular 
hybridization using standard screening procedures. A large group of such 
clones can then be used to find additional clones with NANB virus 
sequences only slightly related to HBV-DNA. By this approach, the entire 
molecular structure of NANB hepatitis virus(es) can be reconstructed. With 
this information and these clones, new recombinant DNA clones can then be 
prepared which are unique for NANB hepatitis virus(es). 
The availability of purified isolated NANB virus, substantially free of 
cellular components and other viral or non-viral components, allows for 
the preparation of an NANB vaccine. The vaccine can be prepared according 
to a number of well known methods in the art. Thus, a vaccine can be 
prepared from the whole live virus or from immunologically active but 
non-pathogenic subcomponents thereof, such as capsids and the like, 
obtained by splitting with enzymes or solvents. Chemically attenuated live 
or killed viral vaccines can also be used, for example, by the treatment 
of virus with propio lactone, dilute formalin(i.e., conc. less than 1%), 
ethylene amine, halogenated hydrocarbons, and the like. These agents 
decrease virus pathogenicity while allowing the material to retain 
immunogenicity. 
Another technique for attenuating the virulence of the virus is to develop 
an avirulent or slow growing strain, or a mutant incapable of sustained 
replication in the host. This is generally known in the art as "genetic 
attenuation", and can be done by genetic manipulations or by serial 
passage. For example, the production of live attenuated viruses can be 
carried out by adapting the isolated virus to cultures containing tissue 
cells and attenuation for example by 10-200 passages in such cultures, 
after which said viruses multiply and a vaccine is then prepared. Another 
method of producing live vaccine is to select and culture clones. If the 
infected cells are used for the production of the live vaccine, it is 
advantageous to release the virus from the cells. Techniques for preparing 
vaccines are generally detailed in a publication such as "Newcastle 
Disease Vaccines: Their Production and Use", Allan, W. H., J. E. Lancaster 
and B. Toth; Food and Agricultural Organization, Rome 1978. 
The vaccines, whether live or attenuated, in their many different forms, 
can be prepared in suspension in a manner known per se with a 
pharmacologically acceptable vaccine carrier, such as a bio-acceptable 
oil. It is advantageous to add thereto a stabilizer, particularly if a dry 
preparation is prepared by lyophilization. An adjuvant such as aluminum 
hydroxide may be added. The stabilizing agent can be a carbohydrate such 
as sorbitol, mannitol, starch, dextran or glycose; a protein like albumin 
or casein; a protein-containing agent like bovine serum or skim milk, and 
a buffer such as an alkaline metal phosphate. 1-100 .mu.g of virus can 
normally be present in such composition per unit dosage. 
The vaccine can be administered to animals, especially humans, to prevent 
the same from developing NANB hepatitis. Vaccines (1-100 .mu.g of antigen) 
may be administered intramuscularly followed by 2nd, 3rd and even more 
boosts at 2 two month intervals. It should be noted that vaccines may be 
given subcutaneously or intravenously and the route of administration, 
dosages, and time between primary immunization and secondary boosts will 
depend on the immunogenicity and characteristics of the viral antigens 
employed. 
Having now generally described this invention, the same will become better 
understood by reference to certain specific examples which are included 
herein for purposes of illustration only and are not intended to be 
limiting of the invention unless otherwise specified. 
EXAMPLE 1 
Monoclonal IgM Radioimmunoassay for Hepatitis B Surface Antigen: 
NANB-Binding Activity in Serum that is Unreactive with Polyvalent 
Antibodies 
MATERIALS AND METHODS 
Patients. Patient A was a 26-year-old man with acute hepatitis (AH). At the 
time of study the serum glutamic-oxaloacetic transaminase (SGOT; asparate 
aminotransferase) was 2161 international units (IU)/ml (normal &lt;50), 
bilirubin was 9.2 mg/100 ml (normal &lt;(1.0), and alkaline phosphatase was 
119 IU/liter (normal &lt;45). His disease resolved over 2 months. Patient B 
was a 65-year-old man with chronic active hepatitis (CAH). He developed AH 
2 months after multiple transfusions for gastrointestinal hemorrhage due 
to a duodenal ulcer. Liver biopsy showed a histologic pattern consistent 
with acute viral hepatitis with submassive necrosis. The patient improved, 
with SGOT, bilirubin, and alkaline phosphatase values returning to normal 
over several weeks. However, 2 months later he was again icteric and 
symptomatic; liver biopsy showed CAH with postnecrotic cirrhosis. For the 
last 4 years his disease has remained active, with SGOT values ranging 
between 45 and 221 IU/ml, with mildly increased alkaline phosphate levels. 
Patient C was a 42-year-old woman blood donor. Her physical examination 
and SGOT bilirubin, and alkaline phosphatase were normal. Patient D was a 
58-year-old man with HBsAg-positive CAH proven by liver biopsy. Patient E 
was a 36-year-old man with AH. The SGOT was 650 IU/ml, bilirubin was 2.4 
mg/100 ml, and alkaline phosphatase was 121 IU/liter at the time of study. 
Patient E had no serologic markers for hepatitis A or B [negative for 
HBsAg, antibodies to hepatitis B core antigen (anti-HBc), anti-HBs, and 
IgM antibodies to hepatitis A antigen (anti-HA); tested by Abbott RIAs] 
during the acute phase of his disease. Patient A was positive for anti-HBc 
and anti-HBs but negative for HBsAg and IgM anti-HA. Patient B was also 
negative for HBsAg anti-HBs and IgM anti-HA during AH. However, after the 
development of CAH he became positive for anti-HBc and anti-HBs but not 
HBsAg and remained seropositive for these antibodies for the last 4 years 
in the setting of active liver disease. He was negative for anti-HA IgM. 
Patient C had no serologic markers for hepatitis A or B. Patient D was 
positive only for HBsAg and anti-HBc. 
Patients A, B, C and E were selected for more detailed study because of the 
high binding activity exhibited by their serum in a 5D3--5D3 monoclonal 
sandwich RIA. It should be noted that patient B serum was highly positive 
in the RIA during AH and CAH and he was consistently identified by the 
assay under code. Patient C was of special interest; her blood was 
considered to have transmitted acute hepatitis with no serologic markers 
of hepatitis B or A. Ten units of blood were transfused to the recipient 
and under code her serum was the only one of the eight units available for 
study that was reactive in the monoclonal assay. Patient D was selected as 
a control because his serum was highly reactive for HBsAg with both the 
monoclonal RIA and commerical RIA (AUSRIA II, from Abbott). 
Affinity Purification. Studies were performed to isolate from serum the 
high binding activity detected in the 5D3--5D3 monoclonal RIA. Affinity 
columns of monoclonal 5D3 IgM anti-HBs were prepared by coupling 2-4 mg of 
IgM per ml of cyanogen bromide-activated Sepharose 6B.RTM.. Serum (20-50 
ml) from each patient was placed over the columns and incubated for 
several hours at room temperature; the columns were then extensively 
washed with phosphate-buffered saline (P.sub.i /NaCl) (pH 7.2). 
Subsequently, 1- to 2-ml fractions were collected by elution with glycine 
HCl buffer (pH 2.6). The pH of each fraction was adjusted to 7.4 with 0.1M 
NaOH and the binding activity was determined on the eluates by the 
monoclonal and AUSRIA II RIAs. Peak fractions exhibiting the highest 
binding activity were pooled and concentrated approximately 100-fold by 
the Micro-ProDiCon device (Bio-Molecular Dynamics, Beaverton, Ore.) for 
further studies as outlined below. 
Immunoelectron Microscopy. Serum samples (3-5 ml) and 5D3-affinity-purified 
material from the patients with acute or chronic hepatitis, and serum from 
normal patients and liver disease controls (individuals with halothane 
hepatitis, alcoholic hepatitis, or primary biliary cirrhosis who were 
unreactive in the conventional monoclonal RIA) were incubated for 12 hr at 
4.degree. C. with 100 .mu.g of 5D3 IgM purified by Sepharose 4B 
chromatography. The incubation mixture was centrifuged at 12,000.times.g 
for 1 hr, the supernatant was decanted, and the precipitate was 
resuspended in 30 .mu.l of P.sub.i /NaCl. Drops (5-10 .mu.l) were applied 
to colloidion/carbon-coated specimen grids, negatively stained with 2% 
potassium phosphotungstate (pH 7.2), and examined with a JEOL 100B 
electron microscope. Additional controls consisted of serum and 5D3 
affinity-purified material incubated with 100 .mu.l of serum having an 
anti-HBs titer of 1:500,000 by passive hemagglutination. The latter serum 
was obtained from a multi-transfused hemophiliac. 
Antigenic Characterization. In order to further define the antigenic 
composition of the 5D3 binding material a series of RIAs employing 
monoclonal IgG and IgM anti-HBs antibodies were developed. In brief, 5D3 
IgM anti-HBs was coupled to a solid-phase support, followed by the 
addition of serial dilutions of serum samples or 5D3 IgM affinity-purified 
material and 125I-labeled IC7 and 5C3 (IgG1 and IgG2a monoclonal 
anti-HBs), 2F11, 1F8 and 5D3 (IgM monoclonal anti-HBs). The reaction 
mixture was incubated for 4 hr at 45.degree. C. and then the solid-phase 
support was washed with distilled water. Radioactivity (cpm) bound was 
determined with a Packard gamma counter. The monoclonal antibodies 
employed in the RIAs were shown to recognize different determinants as 
demonstrated by the absence of competitive inhibition in HBsAg binding 
studies [Wands, Jr. et al, Lancet 1:May, 1982 incorporated by reference]. 
The binding activity exhibited by the samples in the monoclonal RIAs was 
also compared to that observed with conventional anti-HBs reagents (AUSRIA 
II). Finally, the 5D3 affinity-purified material was concentrated 
approximately 100-fold as described above and retested with the AUSRIA II 
assay. Under these conditions, the NANB antigen became reactive. 
Analysis of Polypeptides. Binding material (20-25 .mu.l) prepared by 
affinity chromatography from patients was applied to NaDodSO.sub.4 /10% 
polyacrylamide gels (Moriarty et al ibid, 78: 2606 (1981)). Sepharose 4B 
column-purified 5D3 IgM anti-HBs served as control. Therefore, the 
polypeptide profiles on the gels of the affinity-purified material derived 
from patients A, B and C and the HBsAg-positive patient were compared with 
CAH (patient D). 
Molecular Weight Determination. Experiments were performed to determine the 
approximate molecular weight of the 5D3-binding material. Serum samples 
(10-15 ml) from patients B and C were placed over Sepharose 4B columns and 
eluted with P.sub.i /NaCl. The molecular weight markers were blue dextran, 
IgM, IgG and myoglobin. Aliquots of the fractions were tested in the 
5D3--5D3 monoclonal RIA and the binding activity was compared to the 
elution profiles of the molecular weight markers. The fractions exhibiting 
the highest binding activity were pooled, concentrated, and 
immunoprecipitated with 5D3 as noted above and examined by electron 
microscopy. 
RESULTS 
FIG. 1 depicts a typical binding profile of the various fractions eluted 
from the 5D3 IgM anti-HBs affinity columns as measured by the 5D3--5D3 
monoclonal RIA. Binding activity was recovered from serum after elution 
with glycine HCI buffer and, as can be seen in Table 1, the amount of 
radioactivity bound in the peak fractions was higher than that obtained in 
the unfractionated serum. 
TABLE 1 
__________________________________________________________________________ 
Antigenic characterization of 5D3 binding activity by 
polyvalent anti- HBs reagents 
Binding, cpm 
Serum* 5D3 affinity purified.sup.+ 
Concentrate 
Patient 
5D3-5D3 
AUSRIA II.sctn. 
5D3-5D3 
AUSRIA II.sctn. 
5D3-5D3 
AUSRIA II.sctn. 
__________________________________________________________________________ 
A 15,215 
142 34,162 
121 72,510 
8216 (56) 
B 5,610 
128 22,300 
118 66,721 
4432 (42) 
C 8,126 
142 34,136 
102 54,613 
2167 (71) 
D 22,416 
20,618 (11,210) 
46,198 
26,210 (12,617) 
-- -- 
E 34,259 
137 -- 735 -- 5792 (117) 
__________________________________________________________________________ 
*One hundred microliters of serum tested in the simultaneous 5D35D3 
monoclonal or AUSRIA II RIA. Results are positive if the cpm bound are 
greater than 210 or 350, respectively. 
.sup.+ Binding activity isolated from 30-50 ml of serum by affinity 
chromatography. In each RIA, 100 .mu.l was tested. 
Peak binding fractions (see FIG. 1) were pooled (5-7 ml) and 
concentrated to 50 .mu.l by MicroProDiCon. In each RIA, 10 .mu.l was 
tested. 
.sctn.The numbers in parentheses represent the values obtained in AUSRIA 
II after a 12hr preincubation with purified 5D3 IgM monoclonal anti- HBs. 
No binding activity was observed with conventional polyvalent anti-HBs 
reagents. Furthermore, the fractionated serum was devoid of binding 
activity after passage over the columns and elution with P.sub.i / NaCl as 
measured by the monoclonal RIA. (FIG. 1) 
Some of the antigenic characteristics of the 5D3-binding material were 
determined in this Example. (See also below). In one study five monoclonal 
RIAs were employed as shown in FIG. 2. FIG. 2 Right is a semilogarithmic 
plot of the binding profile with serial dilutions of serum tested in RIAs 
using the monoclonal IgM and IgG anti-HBs antibodies (5D3, 2F11, 1F8, 1C7 
and 5C3). All immunoassays showed high reactivity in the patient with 
HBsAg-positive CAH. In contrast, only the 5D3--5D3 RIA identified serum 
from patients B and C as positive, as shown by the absence of significant 
binding activity when the four other monoclonal RIAs were used (FIG. 2 
Left). These findings indicate that the reactivity of these sera in 
5D3--5D3 assay was the result of a specific antigen-antibody interaction 
and not just due to nonspecific binding of serum to murine monoclonal IgG 
and IgM anti-HBs. 
Additional antigenic properties of the 5D3-binding material are also shown 
in Table 1. The degree of binding activity increased in the 5D3--5D3 assay 
as the serum samples from patients A, B and C were affinity purified and 
further concentrated. It is of interest that when all four specimens were 
concentrated approximately 100-fold (by volume) they showed strongly 
positive results with the polyvalent anti-HBs reagents (AUSRIA II). 
However, this binding activity was blocked by preincubation of these 
samples with 5D3 monoclonal anti-HBs. In contrast, only a 50% blockage of 
known HBsAg binding activity was observed in AUSRIA II after preincubation 
of affinity-purified HBsAg from patient D with 5D3 IgM anti-HBs. 
The polypeptide profiles of the affinity-purified material from four 
patients on NaDodSO.sub.4 /polyacrylamide gels were compared, as shown in 
FIG. 3. Some striking similarities in protein bands were observed when 
comparisons were made among patients A, B, C, and HBsAg derived from 
patient D. A major 50,000-dalton protein was found to be common to all 
specimens, although the HBsAg polypeptide migrated slightly ahead of the 
other 50,000-dalton proteins from patients A, B, and C. Two other 
polypeptides in the 22,000- to 23,000-dalton range appeared to be common 
components in all four isolates. More importantly, the polypeptide 
profiles were identical in samples A, B, and C and, although there were 
some similarities to the polypeptides of HBsAg, as a group there were 
distinct differences as well. 
Finally, the molecular weight of the binding material was approximately 
2.times.10.sup.6 in patients B and C as determined by Sepharose 4B.RTM. 
chromatography. 
Discussion 
This Example shows a study which was designed to compare directly the 
properties of the binding material detected only in the monoclonal RIA and 
not in conventional assays (AUSRIA II). If the binding activity measured 
with the monoclonal RIA was identical to HBsAg, it would have been 
expected that the conventional assays should also yield positive results 
in view of the known sensitivity of the monoclonal RIA for HBsAg (100 
.+-.30 pg/ml). The goal of the present study was to assess the 
relationship, if any, of HBsAg to 5D3 affinity-purified material derived 
from patients negative in the serum for HBsAg by conventional RIA with AH 
or CAH, and from a donor whose blood was implicated in transmitting AH to 
a recipient. 
There is no doubt that the monoclonal 5D3 anti-HBs recognized a determinant 
on HBsAg as shown by the present study and previous observations (Wands et 
al, PNAS, 78: 1214-1218 (1981)). HBsAg was isolated from serum by the 5D3 
IgM affinity column. The immunoreactivity of the isolate was confirmed by 
the high binding activity measured both in the 5D3 monoclonal and AUSRIA 
II assays. In addition, when affinity-purified HBsAg was preincubated with 
5D3 IgM anti-HBs and the immunoprecipitate was examined by electon 
microscopy, typical 22- to 25-nm particles were observed. Clumping of the 
particles and their "spiculated" or "fuzzy" appearance is consistent with 
the presence of antibody on the surface. Indeed, this observation provides 
morphologic evidence of the interaction of the 5D3 monoclonal anti-HBs 
with a specific determinant(s) on HBsAg. The polypeptide profile on 
NaDodSO.sub.4 /polyacrylamide gels of the affinity-purified HBsAg isolate 
was consistent with previous reports demonstrating a major 50,000-dalton 
polypeptide and two smaller proteins (23,000 and 27,000 daltons). Finally, 
the immunoreactivity of the HBsAg isolate was further established by high 
binding activity in RIAs using the other four monoclonal IgM and IgG 
anti-HBs antibodies and conventional (commercially available polyvalent 
anti-HBs AUSTRIA II). 
Some similarities were observed between HBsAg and the 5D3 immunoreactive 
material (NANB) isolated from patients A, B, C, and E. First, the binding 
activity recovered from serum by using 5D3-IgM anti-HBs affinity columns 
and the radioactivity bound in the eluate as measured by the monoclonal 
RIA was severalfold higher than that measured in serum. Furthermore, 
concentration of the eluate followed by retesting in the monoclonal RIA 
yielded even higher binding values. Second, immunoprecipitation of the 
affinity-purified material revealed distinct particles by electron 
microscopy. However, no particles were observed in the isolates after the 
addition of high-titer anti-HBs. The appearance and the size of the NANB 
particles was similar but not identical to HBsAg. The density of particles 
on electron microscopic grids was generally less than that observed with 
the 5D3-HBsAg immunoprecipitate. It should be noted be noted that, as with 
the 5D3-HBsAg immunoprecipitate, clumping of particles was observed, which 
presumably represents the presence of 5D3 antibody on their surface. 
Finally, as shown in FIG. 3, NaDodSO.sub.4 /polyacrylamide gel 
electrophoresis revealed three polypeptides in the same molecular weight 
range as previously described for HBsAg. 
Although the 5D3 IgM anti-HBs binding material (NANB) shared certain 
properties with HBsAg, distinct differences were noted. These differences 
were most evident when the antigenic characteristics of the 5D3 
immunoreactive material were examined by using other monoclonal IgG and 
IgM anti-HBs antibodies as well as conventional reagents. Four other 
monoclonal IgM and IgG anti-HBs antibodies were unreactive with 5D3 
binding material when tested in solid-phase RIAs. In contrast, all four 
antibodies were highly reactive with HBsAg in the same RIAs. In this 
regard, it is noteworthy that 5D3 was coupled to the solid-phase support 
and the other .sup.125 I-labeled monoclonal IgM and IgG anti-HBs served as 
the indicator probes. It is likely, therefore, that the 5D3 binding 
material was bound to the solid-phase support but was not detected with 
the RIAs, suggesting that those epitopes were absent or were not available 
in sufficient concentration to be identified by the other monoclonal 
anti-HBs. 
Additional examination of the antigenic characteristics of the 
5D3-affinity-purified material was performed after concentration of the 
eluate (approximately 100-fold by volume). After each concentration all 
these isolates were reactive in AUSRIA II. More importantly, preincubation 
with 5D3 anti-HBs blocked the binding by the conventional anti-HBs. One 
possible interpretation is that the conventionally prepared anti-HBs 
reagents contain small amounts of an antibody like 5D3 IgM anti-HBs or of 
an antibody of the IgG class that competes for the same epitope. However, 
these isolates (Table 1) possessed high binding values with conventional 
anti-HBs in commercial RIAs, which suggests some antigenic crossreactivity 
of the determinants on HBsAg and the NANB 5D3 binding material. In 
contrast, preincubation of 5D3 with HBsAg resulted in an approximately 50% 
reduction in binding activity when retested by AUSRIA II. This result was 
not unexpected, because other unoccupied determinants would be available 
for binding by the commercial polyvalent anti-HBs. These findings 
therefore suggest that 5D3 IgM anti-HBs is directed toward a highly 
represented epitope on HBsAg, but also recognizes an epitope shared with 
the NANB virus. 
It should be emphasized that the patients selected for the present Example 
were part of a much larger group of individuals whose serum gave a 
positive reaction with the 5D3--5D3 monoclonal assay but not with the 
commercial RIA. The patient selection was based primarily on very high 
binding activity of their sera in the monoclonal RIA. Thus, at present it 
is not clear whether isolates obtained from other sera with lower levels 
of binding activity will yield the same properties as described above. 
However, it is evident that the 5D3 IgM material [NANB] may be affinity 
purified from serum derived from patients with acute and chronic 
inflammatory liver diseases and even from an normal an individual free of 
chronic acute hepatitis or acute hepatitis and shows limited 
crossreactivity with HBsAg when analyzed by conventional anti-HBs 
reagents. Furthermore this material is not recognized by four other IgM 
and IgG monoclonal anti-HBs in RIAs, is similar to HBsAg with respect to 
three polypeptides on NaDodSO.sub.4 /polyacrylamide gels, has a molecular 
weight of approximately 2.times.10.sup.6, and appears as distinct 
particles by immunoelectron microscopy. 
EXAMPLE 2 
Demonstration Of Previously Undetected Hepatitis B Viral Related 
Determinants In An Australian Aboriginal Population By Monoclonal Anti-HBs 
Antibody Radioimmunoassays 
Subjects 
Approximately three-quarters of the adults and children of Mornington 
Island, an Aboriginal settlement off the mainland of Queensland in the 
Gulf of Carpentaria were studied The population is very stable and there 
is little interchange with the mainland. The subjects and the Department 
of Health, Queensland, gave permission for blood samples to be taken. 
Peripheral-blood samples were drawn into heparinised tubes, and the plasma 
was separated by centrifugation. 
Production and Characterization of Monoclonal Anti-HBs Antibodies 
The immunization protocols, characteristics and purity of the immunization 
antigen (HBsAg), cell-fusion technique, and growth and cloning of 
hybridomas producing anti-HBs antibodies have been described previously 
(Wands et al, Gastroentrology, 80:225-232). The anti-HBs antibodies have 
been characterized with respect to specificity for determinants on HBsAg, 
ability to agglutinate red blood cells coated with HBsAg (subtypes adw and 
ayw), antibody class and subclass, and affinity for HBsAg-associated 
epitopes. Two IgM and two IgG monoclonal anti-HBs antibodies were selected 
for this study because they recognize all known subtypes of HBsAg and have 
very high affinity constants for HBsAg determinants and also recognizes 
NANB antigenic activity: the monoclonal antibodies 5D3 and 3D4 (IgM), 5C3 
(IgG.sub.2a), and 2C6 (IgG.sub.1) have affinity constants of 
4.times.1O.sup.11, 8.times.1O.sup.10, 4.times.10.sup.10, and 
2.times.10.sup.10 litres/moles per molecule, respectively. 
Monoclonal IgM and IgG Anti-HBs Radioimmunoassays (Test Procedures) 
Previous studies (Shorey, J. et al Hepatology 1:546 (1981) (Abst.)) have 
established that the 5D3 IgM monoclonal anti-HBs antibody recognizes all 
known HBsAg subtypes and, more importantly, has the highest affinity 
constant of the anti-HBs antibodies measured. 5D3 anti-HBs was coupled to 
a solid-phase support, and the other IgM and IgG antibodies were 
radiolabelled with iodine-125 to a specific activity of 4-10 .mu.Ci/.mu.g 
. Before iodination the antibodies were purified from ascites fluid by 
staphylococcal-protein-A affinity chromatography for IgG and 
`Sepharose-4B` chromatography for IgM. For the monoclonal 
radioimmunoassays, approximately 50 ng 5D3-coated beads were incubated 
with 100 .mu.l serum and 100 .mu.l 1 (150,000 cpm) radiolabeled monoclonal 
anti-HBs for 16 h. The solid-phase support was washed three times with 
distilled water, and the radioactivity bound to the bead was measured by a 
Packard gamma well counter. 
All serum samples were evaluated with the 5D3--5D3 "simultaneous sandwich" 
radioimmunoassay in which the antibody on the solid-phase support and the 
radiolabeled indicator antibody are the same. This assay design is the 
most sensitive for detection of an HBsAg-related determinant. Once high 
binding activity was demonstrated in serum, three other monoclonal 
radioimmunoassays were performed in which radiolabeled 3D4, 2C6, or 5C3 
anti-HBs was the indicator probe. It was possible, therefore, to determine 
whether there were additional antigenic determinants in the 
5D3-immunoreactive material which could be detected by the other 
high-affinity monoclonal antibodies. All serum samples from the Aboriginal 
population were also tested for HBsAg, anti-HBs, and hepatitis B core 
antibody (anti-HBc) by commercial radioimmunoassays (`Ausria II`, `Ausab`, 
and `Corab`, respectively; Abbott Laboratories, North Chicago, Ill.). 
Analysis of HBs-Ag-related Determinants 
Competitive-inhibition studies were carried out to determine whether the 
four monoclonal anti-HBs antibodies recognize the same, closely related, 
or separate antigenic determinants on HBsAg. For these investigations 
HBsAg (subtypes adw and ayw) was coated to a solid-phase support and was 
incubated for 16 h with a constant concentration of radiolabeled 5D3 
(150,000 cpm) and various amounts of purified unlabeled 5D3, 3D4, 2C6, or 
5C3 anti-HBs. It would be expected that high concentrations of unlabeled 
5D3 would completely inhibit binding of radiolabeled 5D3 to its 
determinant on HBsAg. If, when another monoclonal anti-HBs such as 2C6 is 
incubated with radiolabeled 5D3, there is no inhibition of 5D3 binding to 
HBsAg, it may be concluded that 5D3 and 2C6 bind to different 
determinants. To provide further evidence in support of this conclusion 
reverse experiments were performed in which, for example, 2C6 was 
radiolabeled and incubated with various concentrations of unlabeled 5D3. 
If there is no inhibition of binding of the labeled antibody in the 
presence of a high concentration of the other, unlabeled antibody, the two 
antibodies must be directed against distinct and separate determinants on 
the hepatitis-B-virus related protein. 
Results 
The IgG anti-HBs antibodies 2C6 and 5C3 had no effect on the binding of 5D3 
to an HBsAg-related determinant (FIG. 4), whereas 3D4, an IgM anti-HBs, 
partially inhibited 5D3 binding. Additional experiments confirmed that 
5C3, 2C6, and 5D3 recognized distinct and separate determinants on HBsAg. 
There was some antigenic cross-reactivity between the 5D3 and 3D4 
epitopes; 3D4 binding was not, however, influenced by the two IgG antiHBs 
HBs antibodies (5C3 and 2C6). The four monoclonal radioimmunoassays used 
in this study detect three separate epitopes and one partially 
cross-reactive epitope on HBsAg. 
Approximately 50% of the study population had been exposed to HBV as shown 
by the presence in serum of HBsAg, anti-HBs and anti-HBc, or both antigen 
and antibodies (Table 2). 
TABLE 2 
__________________________________________________________________________ 
HEPATITIS B VIRUS MARKERS IN MORNINGTON 
ISLAND RESIDENCE 
No. with marker (%) 
Patient Anti- HBs 
Positive by 
group* 
Anti- HBs 
AntiHBc 
anti+ HBc 
Ausria II 
5D3 RIA 
__________________________________________________________________________ 
Adult 
men 
(n = 96) 
23(24.9) 
7(7.3) 
27(28.1) 
5(5.2) 
7(7.3) 
Adult 
women 
(n = 73) 
13(17.8) 
7(9.6) 
15(20.5) 
5(6.8) 
10(13.7) 
Male 
children 
(n = 57) 
5(8.7) 1(1.7) 
15(26.3) 
2(3.5) 
5(8.8) 
Female 
children 
(n = 50) 
4(8.0) 1(2.0) 
6(12.0) 
1(2.0) 
6(12.0) 
Total.sup.+ 
(n = 316) 
51(16.1) 
22(6.98) 
73(23.0) 
14(4.4) 
31(9.8) 
__________________________________________________________________________ 
*Analysis on 276/316 subjects for whom data on age and sex were available 
.sup.+ Total population was 316 subjects. 
14 subjects were positive for HBsAg by the commercial radioimmunoassay 
(AUSRIA II); all were highly positive by the 5D3--5D3 simultaneous 
sandwich monoclonal radiommunoassay, as were 17 other subjects who were 
negative by commercial RIA (AUSRIA II). There was a higher frequency of 
hepatitis B VIRUS markers in male subjects of all ages than in female 
subjects. 
TABLE 3 
__________________________________________________________________________ 
DEMONSTRATION OF HBV-RELATED VIRAL 
DETERMINANTS BY MONOCLONAL-ANTIBODY BINDING 
S/N* measured by 
Sample 
Anti 
Anti Ausria 
5D3- 5D3- 5D3- 5D3- 
No. -HBs 
-HBc II 5D3 5C3 2C6 3D4 
__________________________________________________________________________ 
1 - - 0.6 8.3 4.1 0.7 6.4 
2 - - 0.4 11.0 1.0 0.5 4.3 
3 + + 0.7 4.1 0.5 4.1 0.6 
4 - - 1.1 4.1 8.0 1.2 6.4 
5 + + 0.3 13.0 0.6 0.3 17.0 
6 + - 0.1 4.0 8.0 0.5 1.3 
7 - - 0.9 23.0 2.1 1.1 3.5 
8 + + 1.3 7.1 0.6 0.7 2.8 
9 - + 0.7 5.4 5.4 0.7 2.0 
10 - - 0.9 9.7 0.3 0.9 6.3 
11 + - 0.8 3.1 0.6 0.5 2.1 
12 - - 1.1 11.0 0.9 0.9 1.9 
13 + + 1.3 15.0 0.6 0.6 2.4 
14 - - 0.4 3.9 0.3 0.3 1.6 
15 + - 0.4 11.0 1.4 0.4 4.1 
16 - + 0.8 5.5 0.4 0.5 2.0 
17 + + 1.7 7.0 0.8 0.4 1.7 
Total 
(n = 17) 
8(47%) 
7(41%) 
0(0%) 
17(100%) 
5(29%) 
1(6%) 
12(71%) 
Controls + 
AA 
(n = 14) 
0(0%) 
14(100%) 
14(100%) 
14(100%) 
14(100%) 
14(100%) 
14(100%) 
BD 
(n = 100) 
7(7%) 
5(5%) 
0 1(1%) 
1(1%) 
0 2(2%) 
__________________________________________________________________________ 
*S/N represents signal/noise calculated as mean cmp bound in experimental 
samples divided by the mean cpm of the negative controls. Result 
considered positive if S/N .gtoreq. 2 .multidot. 0. 
No. (%) of subjects positive. 
Australian Aborigines' samples positive by AUSRIA II radioimmunoassay. 
All samples were reactive with monoclonal anti- HBs IgM and IgG 
antibodies. 
BD = blood donors. 
In addition to the 14 HBsAg-positive subjects shown in Table 2, the 
5D3--5D3 monoclonal radioimmunoassay demonstrated significant binding 
activity in 17 other subjects. The results of further monoclonal-antibody 
analysis of these samples are given in Table 3. Several patterns were 
observed. For example, positive results were obtained in sample 1 by the 
5D3--5D3, 5D3--3D4, and 5D3--5C3 radioimmunoassays but not by the 
commercial radioimmunoassay (AUSRIA II) or by the 5D3--2C6 
radioimmunoassay. Samples from other subjects, such 2, 5, and 8 were 
HBsAg-positive by only the 5D3--5D3 and 5D3--3D4 assays; in each of these 
cases 5D3 was coupled to the solid-phase support and the other 
.sup.125.sub.I-labelled monoclonal IgM and IgG anti-HBs served as the 
indicator probe. It is likely that the 5D3-reactive material was bound to 
the solid-phase support but was not detected by some of the 
radioimmunoassays with other monoclonal anti-HBs antibodies because the 
HBsAg viral determinants they recognise were absent or not available in 
sufficient concentration. It is not surprising that there was binding 
activity with 3D4 antibody in 12 of 17 (71%) 5D3-positive samples, since 
competitive-inhibition studies indicated partial antigenic 
cross-reactivity between the 5D3 and 3D4 determinants. The positivity rate 
for all the monclonal radioimmunoassays was negligible in a low-incidence 
blood-donor population (Table 3); this finding provides further evidence 
of the specificity of the monoclonal radioimmunoassays for HBsAg-related 
determinants. 
Discussion 
It has been found that more than 50% of the Aboriginal community on 
Mornington Island had been exposed to HBV. A very high rate of infection 
with HBV would be expected in confined Aboriginal communities such as that 
on Mornington Island because of the amount of close contact within 
household group, the poor socioeconomic conditions, and the very high 
incidence of venereal disease. 
The specificity of the high-affinity IgM and IgG monoclonal antibodies has 
been confirmed by this Example. The antibodies were prepared against 
HBsAg, and each has been demonstrated to react specifically with known 
HBsAg subtypes. Competitive-inhibition experiments indicate that the 
antibodies recognise distinct and separate determinants on HBsAg. All 
serum samples which reacted with conventional polyvalent anti-HBs antisera 
(AUSRIA II) also reacted strongly with the four monoclonal anti-HBs IgG 
and IgM antibodies, and serum samples from a control caucasian population 
known to have a low incidence of HBV exposure, reacted infrequently with 
the monoclonal antibodies. The dilution curves for antibody binding to 
HBsAg in serum are remarkably similar, which indicated that these viral 
determinants are present in high frequency on HBsAg and that they are 
distributed homogenously in the population. 
The 17 subjects whose serum was reactive in the 5D3--5D3 monoclonal 
radioimmunoassay but was negative when tested by polyvalent conventional 
anti-HBs anti-sera are particularly relevant to this invention. The 
5D3--5D3 radioimmunoassay has a sensitivity of 100 pg/ml serum for an 
HBsAg-related determinant, which represents a sensitivity several times 
greater than that of the commercial radioimmunoassay. Therefore, some of 
the positive results may be explained on the basis of the greater 
sensitivity of the assay but other positive results cannot be explained by 
this mechanism. Other monoclonal radioimmunoassays using antibodies which 
recognize different HBsAg determinants have demonstrated enhanced binding 
activity in substantial numbers of the 5D3-positive samples. These 
findings add further support to the concept that the 5D3 binding activity 
is related to the presence of the NANB hepatitis-B-related viral 
determinants in serum. In Example 1 were investigated serum samples that 
exhibited high binding activity in the 5D3--5D3 monoclonal 
radioimmunoassay but were negative by AUSRIA II, and some of the 
properties of the binding material were characterized, supra. The finding 
of antigenic determinants recognized only by high-affinity monoclonal 
antibodies in a high proportion of the Mornington Island population 
without conventional HBV markers, indicates that there are additional 
viruses in this community antigenically related to HBV but not previously 
detected, i.e., NANB virus. This will be proved by subsequent example (See 
below). 
EXAMPLE 3 
Demonstration of NANB viral DNA in Human Serum 
Materials and Methods 
Serum Specimens and RIA's--Serum Specimens and RIA's were those of Examples 
1 and 2. 
Because several individuals who were reactive only in monoclonal RIAs had 
anti-HBs and anti-HBe antibodies in the serum, additional experiments were 
performed to ascertain sentitivity of the monoclonal RIA for an 
HBsAg-related determinant in the HBsAg-anti-HBs immune complexes formed at 
various antigen/antibody ratios. In these investigations, several chronic 
carriers of HBsAg were selected, and serial dilutions were made of their 
serum (with HBsAg-negative serum). Binding activity was measured in each 
specimen by monoclonal RIAs and was compared to that obtained with 
polyvalent anti-HBs antibodies (AUSRIA II).) Dilution of 
HBsAg-positive serum (200 .mu.l) was then incubated with 25 .mu.l of serum 
from a multiply transfused hemophiliac patient (with an anti-HBs titer of 
1-2.2.times.10.sup.6 by passive hemaagglutination) for 12 hr at 20.degree. 
C. After this incubation, the RIAs were performed; monoclonal anti-HBs and 
AUSRIA II for HBsAg and AUSAB for anti-HBs levels. 
HBV DNA Hybridization Studies--For molecular hybridization studies, 10 
.mu.l aliquots of human serum were applied to nitrocellulose filter sheets 
and denatured and fixed to the filter with 0.5M NaOH. The material was 
neutralized, on the filter, with O.5M. Tris-HCL pH 7.4-1.5M NaCl, digested 
with proteinase K (200 .mu.g/ml in 0.3M NaCl/0.03M Na citrate, air dried, 
and baked in vacuo at 80.degree. C. for 2 hr. The bound DNA was 
prehybridized and hybridized with HBV [.sup.32 P] DNA. For these 
experiments, recombinant cloned HBV DNA (.perspectiveto.3,250 base pairs) 
was repurified from plasmid pAOl HBV DNA by digestion of the plasmid with 
restriction endonuclease EcoRI, followed by agarose gel electrophoresis 
and electroelution of the purified HBV DBA band. HBV DNA was labeled with 
[.sup.32 P]dCTP and [.sup.32 P]dATP to a specific activity of 
2-4.times.10.sup.8 cpm/.mu.g of DNA by nick-translation. Hybridization was 
performed in 0.75M NaCl/0.075M Na citrate/0.02% polyvinylpyrrolidone/0.02% 
Ficoll/0.02% bovine serum albumin containing denatured calf thymus DNA 
(150-200 .mu.g/ml) and heat-denatured HBV[.sup.32 p]DNA (1.times.10.sup.6 
cpm/ml) at 65% for 24-36 hr. After hybridization, the unreacted solution 
was discarded, and the nitrocellulose filter was washed, dried, and 
autoradiographed. For control experiments, the test sample was purified 
HBV DNA, DNA extracted from the PLC/PRF/5 cell line, which contains 5 or 6 
copies of HBV DNA per genome equivalent or DNA isolated from serum Dane 
particles. 
Results 
FIG. 5 depicts a serial study on a patient with acute hepatitis B and 
HBsAg-anti-HBs immune complex disease characterized by arthritis, rash and 
arthralgias. In thss figure, the signal/noise ratio for HBsAg (a measure 
of specific binding activity) is higher with monoclonal anti-HBs (IgM) 
than with polyvalent antiHBs (AUSRIA II). More importantly, the monoclonal 
RIA for HBsAg remained positive for .apprxeq.3 wk after the polyvalent 
AUSRIA II RIA had become negative. During this period, anti-HBs was 
present in the serum, suggesting that the monoclonal RIA may detect 
HBsAg-related deteminant in HBsAg-anti-HBs immune complexes formed in 
anti-HBs excess and that such determinants are not detectable by 
polyvalent anti-HBs antisera. 
To further explore this possibility, two additional studies were performed 
in which HBsAg-anti-HBs immune complexes were formed in vitro with serum 
from a chronic HBsAg carrier by the addition of high titer polyvalent anti 
HBs antibodies. When polyvalent anti-HBs was added to serum from an HBsAg 
carrier, the monoclonal RIA (anti-HBs IgM) remained positive up to a 
10-fold greater dilution than did the AUSRIA II RIA. When, in place of IgM 
monoclonal anti-HBs, studies were carried out with IgG monoclonal anti-HBs 
5C3 and 5C11, which recognize distinct and separate determinants on HBsAg, 
similar results were obtained. These findings indicate that monoclonal 
anti-HBs RIAs can recognize specific viral epitopes in the immune 
complexes when HBsAg is no longer detectable by polyvalent anti-HBs 
antibodies. 
To determine whether HBV DNA-related sequences were present in serum 
samples that were positive for HBsAg by RIAs only with monoclonal anti-HBs 
antibodies, sera (10 .mu.l aliquots) were applied as spots to a 
nitrocellulose filter sheet and denatured. The DNA material was fixed, 
hybridized with recombinant-cloned and repurified HBV [.sup.32 P]DNA, 
washed, and autoradiographed. All experiments were preformed under code 
with two investigators independently interpreting the autoradiograms. A 
series of control samples either positive or negative for HBsAg by AUSRIA 
II were correspondingly positive or negative for HBV DNA by hydridization, 
respectively. In several hundred random or unselected specimens from a 
clinical laboratory analyzed, there was no instance in which the HBV DNA 
hydridization test was positive when the AUSRIA II RIA was negative. 
In a select group of specimens that were positive for HBsAg by RIA with 
.sup.125 I-labeled monoclonal anti-HBs IgM (5D3) but were negative by RIA 
with .sup.125 I-labeled polyvalent anti-HBs(AUSRIA II), HBV[.sup.32 P]DNA 
hydridization was performed. Three of seven samples were positive for HBV 
DNA by molecular hybridization. Unlike random specimens from a clinical 
laboratory, some of these specimens which contain HBsAg-related antigenic 
activity as detected by 5D3 anti-HBs RIA (i.e., NANB virus protein) also 
contained HBV-DNA-related sequences (i.e., NANB virus DNA) as detected by 
molecular hybridization with purified HBV-DNA. 
To determine the frequency with which sera negative for HBsAg by AUSRIA II 
but positive for monoclonal anti-HBsAg were positive also for HBV-related 
DNA sequences, 36 selected specimens previously characterized by 
monoclonal RIAs and additional samples were hybridized under code with 
HBV[.sup.32 P]DNA (Table 4). 
TABLE 4 
__________________________________________________________________________ 
Characteristics of patients whose serum was 
reactive by both monoclonal RIAs and HBV DNA Hydridization 
HBV-DNA 
AUSRIA II RIA, 
Monoclonal RIA, Related 
No. 
Diagnosis cpm bound 
cpm bound 
Anti -HBs 
Anti -HBc 
Sequences 
__________________________________________________________________________ 
1 Acute hepatitis 
93 2,641 + - + 
2 Chronic active hepatitis 
141 7,621 - - + 
3 Post-transfusion hepatitis* 
136 2,193 - - + 
4 Blood donor 147 1,862 - - + 
5 Blood donor 141 2,613 - - + 
6 Blood donor 96 1,562 - - + 
7 Blood donor 114 684 - - + 
8 Aus. abor. 119 1,281 - - + 
9 Aus. abor. 88 691 + + + 
10 Aus. abor. 94 663 + + + 
11 Aus. abor. 110 934 + - + 
12 Aus. abor. 113 2,600 + + + 
13 Aus. abor. 138 1,084 + + + 
Controls (100) 
136 .+-. 17 
56 .+-. 9 
__________________________________________________________________________ 
Aus. abor., Australian aborigine. 
*Recipient of blood from patient 4. 
Incriminated in transmitting posttransfusion hepatitis. Serum was not 
available for analysis for recipient of blood from patient 6. 
Table 4 lists the results together with clinical information and data from 
other tests including various RIAs. A total of 13 of 36 samples (36%) of 
specimens from different individuals positive for HBsAg determinants with 
monoclonal anti-HBs but negative with polyvalent anti-HBs were positive 
for HBV DNA sequences by hybridization with recombinant-cloned and 
repurified HBV DNA. Amongst these individual were three patients with 
acute or chronic hepatitis, four blood donors (two of whom have been 
implicated in transmission of hepatitis to recipients of their blood), and 
six Australian aborigines of the isolated population from Mornington 
Island where HBV infection is endemic (See Example 3). 
Discussion 
In the present Example, the monoclonal RIA is able to bind to viral 
epitopes in HBsAg-anti-HBs immune complexes formed in the presence of 
anti-HBs excess. Possible explanations for this phenomenon are: (i) the 
high-affinity monoclonal anti-HBs may compete more effectively for their 
determinant(s) than do naturally occurring anti-HBs or (ii) the antibodies 
may have access to unoccupied determinants in the presence of polyvalent 
anti-HBs excess. Thus, polyvalent anti-HBs may contain only a small amount 
of antibody with immunologic properties of 5D3, 5C3 and 5C11 monoclonal 
antibodies, and, even though immunogenicity is directed against 
HBsAg-related determinants, the region of immunologic reactivity with the 
monoclonal antibodies may extend beyond that present in polyvalent 
antisera. Such a phenomenon could permit detection of HBAg in immune 
complexes by monoclonal RIAs, whereas conventional anti-HBs antibodies 
would demonstrate no binding activity under conditions of anti-HBs excess. 
Although such activity could explain the detection of HBsAg in the presence 
of excess anti-HBs (Table 4 cases 1 and 9-13) additional consideration is 
required concerning the positive binding activity observed in patients 
negative for HBsAg by AUSRIA II RIA who where also anti-HBs negative 
(Table 4 cases 2-8). Some of these results may be explained by the 
increased sensitivity of the monoclonal immunoassays for HBsAg-associated 
determinants as demonstrated by the present and previous examples. In 
addition, HBsAg in some patients may be present in immune complexes 
circulating under conditions of anti-HBs equivalence or excess and, as 
shown here, would be detectable only by monoclonal RIAs. 
In terms of the HBV DNA reacting sequences present in 36% of serum 
specimens positive for HBsAg by monoclonal RIAs but negative by polyvalent 
RIAs(AUSRIA II) the results indicate that DNA sequences related to or 
homologous with HBV DNA are present in these specimens. Aside from these 
selected cases, hybridization with human serum negative for HBsAg by the 
AUSRIA II RIA has not been detected thus far. Therefore, the present 
findings do not represent biologically false-positive results. 
The presence of both immunoreactive material and hybridizable DNA sequences 
provides corroborative evidence for the the presence of the NANB virus in 
a significant proportion of these specimens. Positive results were found 
for both tests under circumstances in which no HBV markers in serum were 
detected by commercially available RIA (cases 2-7, Table 4). It should be 
noted that integrated HBV-DNA has been reported in human hepatocellular 
carcinoma tissue under circumstances in which serum of said patients was 
negative for HBsAg, anti-HBs and anti-HBc by commercial Abbott kits 
(Brechot, C. et al, Hepatology, 1, 499 (abstract 9B), 1981 and Brechot, C. 
et al Hepatology 2, supplement, 27S--34S, 1982 and herein incorporated by 
reference). 
EXAMPLE 4 
Infectivity Studies Of Viral Hepatitis In Chimpanzees: Characterization Of 
NANB Hepatitis B Virus Agents 
RIA's, HBV-DNA hybridization and antibody specificity were as described in 
Examples 1-3, supra. 
Infectivity Studies 
Two chimpanzees were inoculated with one mililiter of serum derived from an 
individual ho had been incriminated in transmitting "non-A, non-B" 
hepatitis through blood transfusions. Another chimpanazee was injected 
with 40 mililiters of a clotting factor concentrate previously shown to 
transmit "non-A, non-B" hepatitis to recipients. The final chimpanzee was 
inoculated with one milliliter from another individual suspected to harbor 
a "non-A, non-B" hepatitis agent. Serial studies were performed and 
immunoreactivity in serum was measured serially by four monoclonal 
anti-HBs, RIAs, the presence of HBV-related DNA sequences by molecular 
hybridization analysis, HBsAg by AUSRIA II RIA, antibodies to hepatitis B 
core antigen (anti-HBc) and anti-HBs (CORAB and AUSAB respectively; Abbott 
Laboratories, North Chicago, Ill.) and on selected samples, IgM antibody 
to hepatitis A virus (HAVAB; Abbott Laboratories). 
Results 
FIG. 6 depicts the observations in a chimpanzee inoculated with the 
clotting factor concentrate. This animal had previously recovered from HBV 
infection and was positive for anti-HBs at the time of inoculation and 
throughout the study period. This chimpanzee was therefor immune to HBV 
infection as currently recognized and defined. The first evidence of liver 
injury was apparent on day 40 with a rise in ALT levels to 70 IU/L (ml &lt;38 
IU/L); ALT elevations persisted for approximately 35 days. Immunoreactive 
antigen appeared briefly in low titer following inoculation of 40 
milliliters of clotting factor concentrate and then disappeared from the 
circulation. On day 64 there was a striking rise in serum IgM anti-HBs 
binding activity from a baseline of 50 CPM to 4010 CPM 
(S/N.perspectiveto.80, nl &lt;2.1). Antigenemia was subsequently present in 
the blood for 56 days although titers fell with resolution of the 
hepatitis. It is noteworthy that ALT levels reached normal values by day 
78 but antigen was still detectable in the blood for an additional 42 
days. Most importantly, the rise in ALT levels preceded the development of 
antigenemia and/or viremia by approximately 30 days and thus gives a more 
accurate description of the incubation period of 64 days (e.g., 24 days 
after the first ALT rise). Correlations were then made between the 
appearance of antigen in the blood and the presence of HBV-related DNA 
hybridizable sequences. As a control, HBV related nucleic acid sequences 
were not detected during the incubation period by molecular hybridization 
analysis. In contrast, there was a striking correlation between the rise 
in antigen titers and the presence of nucleic acid material which 
hybridized to the HBV-DNA probe suggesting that virions were released into 
the circulation, FIG. 7. With respect to other HBV related epitopes, the 
5C3 or 5C11 determinants were not detected in serum by RIAs. This 
observation indicates that NANB virus is antigenically distinct from HBV. 
Finally, a RIA which employs polyvalent anti-HBs antibodies (AUSRIA II) 
was unreactive during the course of infection and anti-HBc and anti-HA 
antibodies were undetectable. 
FIG. 8 demonstrates the clinical and virologic course of a second 
chimpanzee with pre-existing anti-HB inoculated with 1 milliliter of serum 
carrying a "non-A, non-B" agent. In contrast to FIG. 6, there was no rise 
in ALT levels during the observation period. The incubation time was 
judged to be approximately 190 days. The level of antigenemia as reflected 
by the peak binding activity of the IgM monoclonal RIA was, however, 
impressive indeed (S/N.perspectiveto.175). The period of antigenemia was 
prolonged (approximately 65 days), and antigen levels became undetectable 
by day 260. Similar to the first chimpanzee as shown in FIG. 6, 
HBV-related DNA sequences were undetectable during the incubation period 
but were present by HBV-DNA hydridization at the peak of monoclonal IgM 
RIA binding activity. Moreover, other HBV related epitopes were absent as 
determined by the monoclonal RIAs as well as HBsAg (AUSRIA II), anti-HBc 
and anti-HA antibodies. 
FIGS. 9 and 10 illustrate the clinical and virologic course of the final 
two chimpanzees inoculated with 1 milliliter (each) of serum derived from 
another individual who had been incriminated in transmitting "non-A, 
non-B" hepatitis. In FIG. 9, evidence of liver injury as demonstrated by 
ALT elevations was apparent on day 50 and persisted with a relapsing 
pattern for 140 days. Antigenemia appeared on day 92. Antigen titers were, 
however, falling by day 130 and reached undetectable levels on day 180. 
The magnitude of peak binding activity by monoclonal RIA was less 
(S/N.perspectiveto.10) than that observed in previous studies. It should 
be noted that anti-HBs was not present at the time of inoculation or 
during hepatitis infection and recovery. Similar to the chimpanzee shown 
in FIG. 6, the rise in ALT levels precedes the appearance of antigen in 
the blood by approximately 50 days. HBV-related DNA hydridizable sequences 
were not detectable nor were other HBV associated epitopes, HBsAg, 
anti-HBc and anti-HA antibodies. 
FIG. 10 represents the second chimpanzee inoculated with the same serum. In 
contrast to the pattern seen in FIG. 9, ALT elevations were absent. This 
was similar to the pattern observed in FIG. 8. There were three well 
defined spikes of antigenemia with the highest values occuring on day 164. 
HBV-related DNA sequences were not detectable during any of the episodes 
of antigenemia. This chimpanzee was also negative for HBsAg, other HBV 
related epitopes, anti-HBc, anti-HBs (before, during and after infection) 
and anti-HA antibodies. 
In the present Example it is shown that the agent(s) identified by the 
techniques of Examples 1-3 is (are) infectious by infectivity studies of 
viral hepatitis in chimpanzees. 
Thus, it has been possible to reproduce the findings in man (See Example 3, 
Table 4) in an accepted experimental animal model of "non-A, non-B" 
hepatitis. The major observations in the present Example include: (1) 
three different inocula injected into 4 animals were infectious; (2) the 
incubation period, defined as the time from inoculation of infections 
material to the appearance of virus or viral protein in the blood is 
longer than previously recognized; (3) ALT elevations may precede the 
appearance of antigenemia by several weeks; (4) antigenemia may occur in 
the absence of ALT elevations; a phenomenon identical to that observed in 
man; (5) the presence of antigen in the blood as measured by the 
monoclonal IgM anti-HB RIAs correlates well with the appearance of 
HBV-related DNA like sequences by molecular hybridization analysis; (6) 
the period of antigenemia and/or viremia may persist for weeks to months 
and usually disappears with recovery; (7) antigenemia is still detectable 
in the resolution phase of illness when ALT levels are normal, which is 
similar to HBV infection in man; (8) several episodes of antigenemia may 
occur during the course of infection; (9) pre-existing anti-HBs was not 
protective and thus NANB virus is sufficiently different in antigenic 
composition than HBV. In support of this concept is the finding that 
polyvalent anti-HBs antibodies (AUSRIA II) and other monoclonal anti-HBs 
which recognize different HBsAg associated epitopes were unreactive. Taken 
together these and the previous examples provide strong evidence that NANB 
hepatitis agents in many circumstances may be a related but distant or 
distinct variant of hepatitis B virus. 
Having now fully described this invention it will be apparent to those of 
skill in the art that the same can be performed within a wide and 
equivalent range of methods, tests, compositions, procedures and processes 
without affecting the spirit or scope of the invention or of any 
embodiment thereof.