NO177310B - HBxAg antigenic polypeptide, antibody that reacts with the polypeptide, diagnostic assay system to determine the presence of HBxAg in a body sample, and use of the polypeptide for in vitro diagnosis - Google Patents

Description

Technical area

The present invention relates to antigenic synthetic polypeptide that corresponds to an antigenic determinant in HBxAg, antibody that reacts immunologically with the polypeptide, diagnostic analysis system for determining the presence of HBxAg in a body sample, as well as use of the polypeptide for in vitro diagnosis.

Background technology

A. Cloning and vectors

The introduction of exogenous DNA into eukaryotic cells has become one of the most powerful tools for the molecular biologist. This method requires efficient delivery of DNA to the nucleus of the recipient cell and subsequent identification of cells expressing the foreign DNA.

Engineered vectors, such as plasmids or bacteriophages (phages), or other DNA sequence capable of replicating in a host cell, can be used to engineer cells that act as factories that produce large amounts of specific viral proteins. Recombinant plasmids will be used here as exemplary vectors, also called cloning vehicles. See US Patent No. 4,378,397.

Plasmids are extrachromosomal genetic elements found in many different bacterial species. They are usually double-stranded, closed, circular DNA molecules. The most widely used plasmid is pBR 322, a vector whose nucleotide sequence and endonuclease cleavage sites are well known.

Nucleic acid production using plasmid or phage vectors has become very simple. The plasmid or phage DNA is cleaved with a restriction endonuclease and ligated in vitro to a foreign DNA of choice. The resulting recombinant plasmid or recombinant phage is then introduced into a cell, such as E. coli, and the resulting cell is induced to produce many copies of the engineered vector. Once a sufficient amount of DNA has been produced using the cloning vector, the produced foreign DNA is removed and placed into a second vector to produce or transcribe the protein or polypeptide encoded by the foreign gene.

Depending on the DNA (intact gene, cDNA or bacterial gene), it may be necessary to provide eukaryotic transcription and translation signals to direct expression in the recipient cell. These signals can be provided by mixing foreign DNA in vitro with an expression vector.

Expression vectors contain sequences of DNA that

is required for the transcription of cloned genes and the translation of their messenger RNAs (mRNAs) into proteins. Typically, such required sequences or control elements are: (1) a promoter that signals the start point of transcription, (2) a terminator that signals the stop point of transcription, (3) an operator that regulates the promoter, (4) a ribosome binding site for the first binding of the cell's protein synthesis machinery, and (5) start and stop codons that signal the beginning and end of protein synthesis.

To be useful, an expression vector should have several additional properties. It should be relatively small and contain a strong promoter. The expression vector should carry one . or more selectable markers to enable identification of transformants. It should also contain a recognition site for one or more restriction enzymes in regions of the vector that are not essential for expression.

The construction of expression vectors is therefore a complicated and somewhat unpredictable event. The only true test of the efficiency of an expression vector is to measure the frequency with which synthesis of the correct mRNA is initiated. However, quantification of mRNA is laborious, and it

is often difficult to obtain accurate measurements. Other, more practically applicable ways of detecting transformation have therefore been developed.

One such way has been to monitor the synthesis of foreign proteins in transformed cells with enzymatic analyses. Several marker genes have been developed to indicate that transformation has occurred.

Another way that has been used to monitor transformation involves the use of immunological reagents. If the level of expressed protein is sufficiently high, then cytoplasmic or surface immunofluorescence with an antibody conjugated to a fluorescent dye, such as fluorescein or rhodamine, can be used to detect vector-specific protein expression products.

More commonly, transformed cells are cultured in the presence of radioactivity after immunoprecipitation. In this method, Staphylococcus aureus protein A selection of immune complexes has been used (Kessler, (1975), J. Immunol., 115: 1617-1624), and the method of "Western blotting"

(Renart et al., (1979), Proe. Nati. Acad. Sei. USA, 76: 3116-3120) to detect transformation-specific markers.

Analysis of gene expression using simian virus 40 (SV40) vectors is clearly the most explored eukaryotic transformation technique at the biological and immunochemical levels. Genetic and biochemical information regarding the organization of the SV40 genome has been provided or confirmed using the nucleotide sequence of the viral genome. Review by Tooze (1980), Molecular Biology of Tumor Viruses, 2nd ed., Part 2, Cold Spring Harbor Laboratory, Cold Spring Harboir, New York. The design of various SV40 vector molecules has been based on the precise mapping of genetic signals and the use of restriction endonucleases for the isolation of specific fragments from the SV40 genome.

SV40 was first developed as a eukaryotic transfer vector using a lytic system. Mulligan et al.,

(1979), Nature (London), 277: 108-114. Subsequently, transforming (non-lytic) vectors were constructed with isolated segments of the SV40 genome. Review by Elder et al., (1981), Annu. Fox. Genet., 15: 295-340. Hamer et al. were the first to propose that SV40 could be used to clone genes for which no probe was available. They proposed that double-stranded cDNA copies from a heterogeneous mRNA population could be "shot" into an SV40 vector, and that viruses carrying the desired sequence could be identified by using a radioactive or fluorescent antibody. Hamer et al. first reported the construction of SV40 recombinant expression vector containing an expression marker in 1979. Hamer et al., (1979), Cell, 17: 725-735. Their SV40 vector contained the viral DNA sequences from the BamHI endonuclease restriction site at 0.14 map units clockwise to the Haell restriction site at 0.82 map units. In addition to the entire early gene A and the origin of viral DNA replication, the vector contained the viral promoter, the leader, an intermediate sequence of the virus, the 5<1> part of the genome of the virus and the 3'-end sequences for the late 195 mRNA of the virus. It did not contain 1660 base pairs (bp) of the sequences in the late region encoding the viral protein UPI, 2 and 3. Priers et al., (1978), Nature, 273: 113-120 and Reddy et al., (1978 ), Science, 200: 494-502. Coding sequences from the rabbit beta-globin gene were ligated into the above-mentioned vector as an expression marker. To determine whether rabbit beta-globin was synthesized in monkey cells infected with their recombinant vector, Hamer et al., supra, used a radioimmunoassay capable of detecting as little as 1.0 nanograms of globin. Although Hamer et al. were able to show positive signs of beta-globin expression, they expressed several reservations regarding the applicability of the SV40/beta-globin recombinant system. As globin is only slightly soluble, firstly considerable losses may have been incurred during the preparation of samples for measurement. The determination of the amount of globin in the infected cells can thus be incorrect by as much as a factor of 10. Second, the assay cannot distinguish between authentic globin and other immunologically related products, such as "read-through" protein or polypeptide fragments. A factor that Hamer et al. not tackled, is the high degree of homology between all eukaryotic globins. This homology makes it difficult to distinguish vector-induced globin expression from globin endogenous to the host cell system.

B. Hepatitis B virus peptides and

anti-polypeptide antibodies

Hepatitis viruses are distinctly different infectious agents. They are grouped together solely because of the "target" organ that they attack, the liver. Although a number of viruses attack the liver as part of systemic infections, the term "hepatitis virus" usually refers to type A (HAV), type B (HBV) and the non-A, non-B infectious agents. Of the three virus types, HBV is by far the most explored at the biological, immunochemical and clinical levels.

HBV is classified as a DNA virus and differs

in many respects from all other families of DNA viruses. HBV is composed of an outer envelope (firmer than a membrane or envelope) consisting of protein, lipid and carbohydrate, and which carries a unique antigen complex; i.e. the hepatitis B surface antigen (HBsAg). It also contains an inner nucleocapsid with an antigenic specificity that is different from the specificity of the surface antigen, i.e. the hepatitis B core antigen (HBcAg).

Within the technique, one is also aware of soluble antigen, HBeAg. This antigen is believed to consist of HBcAg polypeptides that are not assembled into HBV cores, and consequently has a unique antigenic specificity in the unassembled state.

In typical self-limiting acute HBV infections, the following serological markers appear successively in the serum of an infected host: HBsAg, HBeAg, anti-HBC, anti-HBE and anti-HBS. The appearance of anti-HBE signals the final loss of detectable HBsAg. This applies in all cases of self-limiting acute HBV infection. After HBS has disappeared there is a delay of 5 weeks to several months before the appearance of anti-HBS.

During chronic HBV infection, HBsAg and anti-HBC are present. The host's serum can show either HBeAg or anti-HBE serological markers, i.e. the patient can be either HBeAg or anti-HBE positive.

Sensitive and specific radioimmunological assays and enzyme immunoassays for several of the HBV markers are widely used. These highly sensitive serological tests have provided a basis for monitoring the emergence of virus and immune response markers during HBV infection.

In the last few years, many studies have indicated that each serological marker denotes specific viral events of host response during HBV replication. The profile of serological markers at different stages during the clinical course of disease can thus provide useful diagnostic and prognostic information.

The connection between hepatitis B virus and human hepatocellular carcinoma (HCC), liver cancer, has been thoroughly investigated and seroepidemiological and histopathological findings strongly suggest that HBV is directly or indirectly involved in the etiology of liver cancer. A number of hepatoma cell lines have been derived from human HCC, and detection and HBV-specific DNA integrated into the genome of two such cell lines, PLC/PRF/5 and EPH3B, have been reported. In these cell lines, however, only hepatitis B and the surface antigen have been expressed in tissue culture as a virus-specific gene product. Other markers for HBV, such as hepatitis B core antigen, hepatitis BE antigen and a DNA polymerase, have not been detected.

Some DNA tumor viruses in animals can produce transformation through the action of viral genes that regulate the replication and integration of the viral genome, and cells transformed with such viruses can carry a T-(tumor) or neo-(new) antigen expressed by of the transforming genes. Recent evidence for an antigen analogous to T antigen has been obtained in human hepatoma cells containing integrated HBV genes, using the anti-complement immunofluorescence staining technique. This antigen has been termed HBV-associated core antigen (HBNA). Wen et al., (1983) Infect. Immun., 39: 1361-1367.

HBNA was detected in sera from several HBsAg-positive HCC patients, and expression of the antigen was demonstrated both in cell culture and tumor tissue. In addition, anti-HBNA antibodies were found in the sera of some HBsAg-positive patients with HCC. HBNA may account for the previously unrecognized expression of the HBV gene.

In 1979, Galibert et al., Nature, 281: 646-650, reported the nucleotide sequence of the HBV genome, a circular DNA of approx. 3200 bases that are partly double-stranded and partly single-stranded, a feature unique among viruses. The genome's long strand, or L strand, (completely circular) was found to contain four reading frames large enough to be responsible for viral proteins. These areas were called S, C, P

and X, and is shown schematically in Figure 1. Areas S and C have been found to contain the genes for HBsAg and HBeAg respectively. Region P is thought to encode a protein similar in size and amino acid residue content to a DNA polymerase. Region X postulated by Wen et al., supra, to be one of the likely sources of the gene encoding HBNA.

The preliminary indication of function for the genes in the P and X regions alone demonstrates the gap that still exists in understanding the genetic organization and molecular biology of HBV. One reason for this gap has been the absence of an in vitro system for propagation of the virus.

Until recently, the only source of HBV was the serum of human patients. The failed attempts to grow the virus in cell culture are the result of its very narrow host cell range.

One approach to the problem of producing HBV DNA and its gene products has been to use recombinant DNA techniques. This technique enables large-scale production of nucleic acid sequences that code for a specific viral protein.

Tiollåis et al., (1981), Science, 213: 406-411 reported transformation of E. coli with pBR322 containing the gene encoding HBsAg and reported production of significant amounts of this isolated gene. These researchers also wanted to investigate the HBsAg gene's location in the HBV genome, and the factors that influenced its expression into protein. For this, they constructed expression vectors containing the HBsAg gene, for use in both bacterial and mammalian cells.

One of the expression vectors constructed by Tiollais et al., supra, achieved biosynthesis of protein in E. coli containing HBsAg antigenic determinants. It was constructed by inserting part of the gene encoding HBsAg into the plac5-lUV5 bacteriophages to preserve the reading frame of native HBV.

To examine HBV gene expression in mammalian cells, Tiollais et al., supra, constructed a series of HBsAg expression plasmids by inserting transfection elements at various locations throughout the HBV genome. The transfection elements allowed the entire HBV genome to be integrated in several different orientations into the genome of mouse cells transformed with the vector. By creating vectors in this way, these researchers attempted to use HBV's naturally occurring genetic control elements.

Only three of the six expression vectors reported by Tiollais et al., supra, gave expression of the entire HBsAg protein. Its production was demonstrated by testing for its presence in tissue culture fluids using sheep anti-HBsAg antiserum. The other viral markers known at the time of the investigation (ie HBeAg, HBeAg and DNA polymerase) were not detected in the transformed cells.

At the time of the above-mentioned study by Tiollais et al. both the possible existence of region X and the possibility that it may code for an HBV protein were known. That is that it was known that the HBV genome had an ability to code for more proteins than those previously associated with the virus.

This problem has been called genotyping by phenotype. It is, among other things, this problem that Wen et al., supra, tackled when they postulated that area X contains the gene that codes for HBNA.

Before the above-mentioned investigations by Tiollais et al. and Wen et al., Sutcliffe et al., (1980), Nature, 287: 801-805, among others, demonstrated a general technique for solving this problem. They synthesized a polypeptide chemically from the protein predicted by the nucleotide sequence of a viral gene with a protein product that was unknown. Antibodies were raised against the polypeptide and reacted against all the proteins made by cells infected with the virus. By using antibody to parts of the predicted protein, Sutcliffe et al. a previously unknown and unrecognized viral protein product.

To date, the application of this or any other technique to unambiguously identify protein products from HBV genome region X has not been reported. This may be because although the general concept of the preparation of synthetic antigens and their use to induce antibodies of predetermined specificity has been described, there remains a large area of this technique that continues to elude predictability.

There are several reasons for this. First, protein amino acid residue sequences derived from a genetic sequence are of a hypothetical nature unless the nucleotide sequence reading frame is definitely established, due to the redundancy of the genetic code.

Second, a synthetic antigen does not necessarily induce antibodies that react immunologically with the intact protein in its natural environment. Third, a host's natural antibodies to an immunogen rarely react immunologically with a polypeptide that corresponds to a short linear portion of the immunogen's amino acid sequence.

Brief summary of the invention

The present invention is based on two aspects. One aspect is a recombinant DNA comprising an expression vector linked to the gene encoding HBxAg. In a particularly preferred recombinant DNA, the DNA expression vector comprises a first DNA sequence comprising the simian virus 40 viral DNA sequence from the BamHI endonuclease restriction site at base position 2468 clockwise to the Haell endonuclease restriction site at base position 767 according to Figure 2, and the gene encoding HBxAg is provided by means of a second gene DNA sequence comprising the hepatitis B virus DNA sequence from the Haell endonuclease restriction site at base position 1437 clockwise to the BamHI endonuclease restriction site at base position 28 according to Figure 1. The first and second DNA sequences are operably linked to promote expression of HBxAg. Another aspect on which this invention is based is constituted by a recombinant DNA comprising a gene which codes for HBxAg or for a polypeptide which forms a substantial part of HBxAg. A particularly preferred recombinant DNA contains the gene encoding HBxAg, and has the base sequence shown in Figure 1 covering base positions 1437 clockwise to 28, or a substantial part thereof.

A plasmid which consists of at least one recombinant DNA comprising a gene which codes for HBxAg or for a polypeptide which forms a substantial part thereof, is used in connection with this invention. The plasmid may comprise the base sequence shown in Figure 1 covering base positions 1437 clockwise to 28, or a substantial part thereof. In a particularly preferred embodiment, the plasmid comprises a first DNA sequence comprising simian virus 40 (SV40) viral DNA sequence from the BamHI endonuclease restriction site at base position 2468 clockwise to the EcoRI endonuclease restriction site at base position 1717 according to Figure 2; a second DNA sequence comprising the DNA sequence of plasmid pBR322 from the BamHI endonuclease site at base position 375 clockwise to the EcoRI endonuclease restriction site at base position 0 according to Figure 3; and a third DNA sequence comprising the hepatitis B virus DNA sequence from the BamHI endonuclease restriction site at base position 1400 clockwise to the BamHI endonuclease restriction site at base position 28 according to Figure 1. In this plasmid, the first and second DNA sequences are operative bound in their respective EcoRI endonuclease restriction sites; the second and third DNA sequences are operably bound in their respective BamHI endonuclease restriction sites; and the first and third DNA sequences are operatively ligated into their respective BamHI endonuclease restriction sites of Figure 4.

A method for producing HBxAg or a substantial polypeptide part of HBxAg constitutes another aspect on which the invention is based. A host containing an above-mentioned expression system is cultured in a culture medium until HBxAg or a substantial polypeptide portion thereof is produced and accumulated in the host or the culture medium. The accumulated HBxAg or the essential polypeptide part thereof is then recovered.

One aspect of the invention relates to an antigenic synthetic polypeptide. This synthetic polypeptide corresponds in sequence to the sequence of an antigenic determinant in HBxAg, and comprises an amino acid residue sequence selected from the group of polypeptide sequences represented by the formula written from left to right and in the direction from amino end to carboxyl end: Gln-Leu-Asp-Pro- Ala-Arg-Asp-Val-Leu-Cys-Leu-Arg-Pro-Val-Gly,

Ser-Ala-Val-Pro-Thr-Asp-His-Gly-Ala-His-Leu-Ser-Leu-Arg-Gly-Leu-Pro-Val-Cys and

Met-Glu-Thr-Thr-Val-Asn-Ala-His-Gln-Ile-Leu-Pro-Lys-Val-Leu-His-Lys-Arg-Thr-Leu-Gly.

Antibodies comprising an antibody binding site capable of reacting immunologically with one of the previously mentioned antigenic polypeptides are also included. The particularly preferred polypeptides with which these antibodies react immunologically are illustrated by means of the polypeptides described above. These antibodies also react immunologically with HBxAg or with a substantial polypeptide part thereof.

A diagnostic analysis system for determining the presence of a detectable amount of HBxAg in a body sample to be analyzed is also included. This system comprises at least one container containing a reagent comprising the antibodies described above. This system can further comprise a second reagent in a second container, the second reagent being the antigenic synthetic polypeptide with which the antibodies react immunologically. A marker to signal the immunoreaction between the antibodies and HBxAg can form a further part of the diagnostic analysis system.

A use of an antigenic synthetic polypeptide for in vitro diagnosis of a detectable amount of HBxAg in a body sample constitutes a further aspect of the invention. Here, proteins from a body sample to be analyzed for the presence of a detectable amount of HBxAg are mixed with the above-mentioned antibodies in the presence of a marker for signaling an immunoreaction between the receptors and HBxAg. The mixture is kept for a period of time sufficient for the marker to signal that an immunoreaction has occurred. The presence of this signal is then ascertained.

Brief description of the drawings

the figures which form part of the description of the invention:

Figure 1 is a schematic representation showing the physical structure and genetic organization of the HBV/adw genome from Tiollais et al., (1981), Science, 213: 406-411 as modified by Ono et al., (1983) , N.A.R., 11: 1747-1757. The 5' end of the long (L) strand is reported to base pair with the 5<1> end of the short (S) strand. Certain restriction sites indicated by arrows adjacent to the circle arcs and the abbreviation for the restriction endonuclease correspond to the physical map of the HBV/adw genome shown by analysis by Ono et al., supra. The broad arrows surrounding the genome correspond to the four large, open regions of the L strand. These four possible coding regions are designated S, P, X and C. The number of amino acid residues in parentheses (aa) after each of the four designated regions corresponds to the length of the hypothetical polypeptide coded for by each region. The two regions corresponding to the defined genes S and C are indicated by means of dotted regions within the broad arrows. The single EcoRI endonuclease cleavage site is used as the starting point for the map. Figure 2 schematically illustrates an SV40 DNA restriction map prepared from the primary sequence published by Reddy et al., (1978), Science, 200: 494-501, as modified by Van Heuverswyn and Fiers (1979), Eur. J. Biochem. 100: 51-60. Figure 3 schematically illustrates a plasmid pBR322 re-string ion map prepared from primary sequence data according to Sutcliffe (1979), Cold Spring Harbor Symposium on Quantitative Biology 43, 77. Figure 4 schematically illustrates the procedure described in the Materials and Methods section below, used for insertion of DNA fragments in vectors of this invention made from AM6 (wavy lines) pBR322 (solid lines) and SV40 (dotted lines) to produce the cloning vector SVAM191 and then the expression vector SVHBV-3 which expresses a substantial part of HBxAg. Relative positions

IO

for restriction endonuclease sites for BamHI, EcoRI and Haell are shown by closed circles, open circles and closed triangles, respectively. The origin (ori) and early and late promoters in the SV40 genome are also indicated.

Figure 5 shows a linear portion of the SVHBV-3 recombinant expression vector containing the genes for the SV40 sen promoter, the amino-terminal 99 amino acid residues (99aa), and an essential polypeptide portion (132aa; 133 of 154 amino acid residues) of the HBxAg gene sequence encoding the 132 carboxyl-terminal amino acid residues. mRNA for the VP2 HBxAg fusion protein expressed using the vector is also shown. Figure 6 illustrates the translated amino acid residue sequence shown from left to right and in the direction from amino end to carboxyl end in the gene that codes for HBxAg. The major part of HBxAg expressed by SVHBV-3 is illustrated by the arrow at amino acid residue position 23 (Ala), which is the amino terminal residue of the expressed HBxAg polypeptide. The relative positions of synthetic polypeptides designated 8, 42 and 79 according to the invention, as well as 99, 100 and 142, in the HBxAg sequence are illustrated by means of the three marked, underlined amino acid residue sequences. The amino acid residue sequence of synthetic polypeptide 99 therefore corresponds to positions 100 - 115 from the amino-terminal Met residue shown in the figure, the sequence of synthetic polypeptide 100 corresponds to positions 115 - 131 from the amino-terminal residue in the figure, and the sequence of synthetic polypeptide 142 corresponds in sequence to positions 144 - 154 from the amino-terminal Met residue in the figure; i.e. the 11 residues at the carboxyl end. Figure 7 is a photograph of an autoradiogram showing the activity of anti-X antisera with two human hepatoma cell lysates. Two human hepatoma cell lines, PLC/PRF/5 and HepG2, were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum to confluence (day 6 of a 1:5 aliquot from a confluent culture). Supernatants were removed, the flasks containing the monolayers were placed on ice for 5 min before the cells were collected by scraping, and the collected cells were pelleted by centrifugation. Cell pellets were washed with phosphate buffered saline (PBS), snap frozen at -70°C and lyophilized overnight. The resulting cell powders were dissolved in PBS and brought to a final concentration of 2 milligrams per milliliters (mg/ml) in sample buffer. The samples were boiled for 5 minutes and cell debris was removed by centrifugation in a Beckman microcentrifuge for 30 minutes. Fifty micrograms of cell lysate was incubated for 15 minutes in RIPA buffer (PBS containing 1% "Nonidet P-40"

(polyoxyethylene-(9)octylphenyl ether), 0.05% sodium deoxycholate and 0.1% SDS), and radioactively labeled with 3 microCurie 125 I by means of the chloramine T-reaction ti.l McConahey et al.,

(1966), Int. Arch. Allergy, 29: 185-189. 1.5 x IO<6> counts per minute (cpm) for each of the radiolabeled hepatoma lysates was reacted with 10 microliters of anti-X polypeptide for 60 minutes in RIPA. The antigen-antibody complexes (immunoreaction products) were precipitated with formalin-fixed Staphylococcus aureus. The pellets were washed once with RIPA and twice with 500 millimolar (mM) LiCl, 100 mM Tris (pH = 8.5) and were analyzed for radioactivity. All pellets were dissolved in 40 microliters of sample buffer (0.0625M Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.01% bromophenol blue), boiled for 3 minutes, centrifuged to remove cell debris and subjected to SDS-PAGE as follows.

The radiolabeled cell lysates were loaded onto denaturing sodium dodecyl sulfate-polyacrylamide gels (12.5%)

(SDS-PAGE), and subjected to electrophoresis according to the methods of Laemmli, (1970), Nature, 297: 689-685. The proteins were electrophoretically transferred to the nitrocellulose sheet as described by Towbin et al., (1979), Proe. Nati. Acad. Pollock. USA, 76: 4350-4354. The nitrocellulose stains were stained

in amido black before incubation at 4°C overnight in BLOTTO (Johnson et al. (1983), J. Exp. Med., 159: 1751-1756) to reduce non-specific binding. Nitrocellulose strips were incubated for 3 hours at room temperature with a 1:350 dilution of anti-polypeptide antibodies in a final volume of 10 ml BLOTTO. The strips were washed in BLOTTO before a 1-hour incubation with 125 -I-labeled S aureus protein A (10 6 cpm per

10 ml). Where competition with polypeptide is indicated below, the anti-polypeptide antisera were incubated with 100 µg

polypeptide for 60 minutes before the addition of the radiolabeled antigen. The strips were washed as above, rinsed with water, dried and autoradiographed. Lanes 1 and 5 were reacted with anti-polypept-id 99 antibodies (anti-99), lane 2 was reacted with anti-99 preincubated with polypeptide 99, lane 3 was reacted with anti-99 preincubated with an unrelated polypeptide, and lane 4 was reacted with a preimmune serum control. Lysates from HepG2 cells, lanes 1-4, did not react with anti-polypeptide 99. The numbers in the left margin indicate relative protein migration positions. Arrows in the right margin indicate the migration positions of proteins with molecular weights of 28,000 and 25,000 daltons. Figure 8 is a photograph showing the reactivity of anti-X antiserum with chimpanzee (C; lanes 3 and 4) and human (H; lanes 1 and 2) liver tissue. Liver sections from chimpanzees and humans were quickly frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. Cell powders were solubilized in sample buffer and were subjected to gel electrophoresis and stained as described for Figure 7. Nitrocellulose strips were developed with a 1:50 dilution of antiserum as described in Figure 7, with two exceptions: 1) 1% solution of bovine serum albumin (BSA ) was used instead of BLOTTO in all incubations and washes, and 2) the antigen-bound antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG. The antigen-antibody immunoreaction products were visualized by dipping the strips in the following developing solution. The developing solution was prepared from 50 ml of TBS buffer at 37°C to which 250 microliters of absolute ethanol containing 8 mg of 4-chloro-1-naphthol and 330 microliters of 30% hydrogen peroxide were added. Antiserum against polypeptide 99 (anti-99) was used in the two panels on the left, blocked and unblocked, while antiserum against polypeptide 142 (anti-142) was used in the two panels on the right, blocked and unblocked. The numbers on the left side of the figure show the gel migration positions for the proteins with molecular weights of 66, 45, 31, 21 and 14 kilodaltons respectively. Figure 9 is a photograph of a Southern blot analysis of restriction enzyme digested liver DNAs from human and chimpanzee liver tissue that exhibited X protein, using hepatitis B virus (HBV) DNA as the binding probe. Total DNA was extracted from the cell powders made from the samples described in Figure 8. 10 micrograms of DNA was loaded into each well of a 1% agarose gel. Restriction enzyme dissolution buffers were according to the manufacturer. All dissolutions were performed overnight at 37°C with 50 units of enzyme (5 x DNA concentration in micrograms). Lanes 1-3 show chimpanzee A-243 liver DNA digested with BamHI, EcoRI or uncleaved, respectively; lanes 4-6 show chimpanzee 343 liver DNA digested with BamHI, EcoRI or uncleaved, respectively; lanes 7-10 show human HBcAg-positive liver DNA cut with HindIII, BamHI, respectively

EcoRI or no resolution; and lanes 11 - 14 show human HBsAg-negative liver DNA resolved with HindIII, BamHI, EcoRI or unresolved, respectively.

Figure 10 is a photograph of an autoradiogram showing the reactivity of anti-X polypeptide antisera with an X-directed gene product. Confluent monolayers of BSC-1 cells (2x10<7 >cells) were infected with the recombinant SVHBV-3 standard virus (a mixture of tsA^g and SVHBV-3 virions) or wild-type SV40 (Moriarty et al. ( 1981), Proe. Nat. Acad. Sei.

USA, 78: 2606-2610). The cultures were incubated in serum-free DMEM at 40°C for 48-72 hours when approximately 50% cytopathic effect had occurred. Supernatants were removed and the flasks containing the monolayers were placed on ice for 5 min before the cells were harvested by scraping. The collected cells were centrifuged and the resulting cell pellets were washed with PBS, snap frozen at -70°C and lyophilized overnight.

The resulting cell powders were dissolved in PBS and brought to a final concentration of 2 mg/ml in sample buffer. The samples were boiled for 5 minutes and cell debris was removed by centrifugation in a Beckman microcentrifuge for 30 minutes. 50 to 100 micrograms of cell lysate was loaded onto denaturing polyacrylamide gels (12.5%) and subjected to electrophoresis as described previously. The proteins were electrophoretically transferred to nitrocellulose sheets as described previously. The nitrocellulose blots were stained in amido black before incubation at 4°C overnight in BLOTTO to reduce non-specific binding. Nitrocellulose strips were incubated for 3 hours at room temperature with a 1:350 dilution of anti-peptide antibodies in a final volume of 10 ml BLOTTO. The strips were washed in BLOTTO before a 1-hour incubation with 1<25>I-labeled S. auereus protein (10<6> cpm per 10 ml). The strips were washed as above, rinsed with water, dried and autoradiographed. In cases where blocking with polypeptide was performed, the antisera were preincubated with 100 micrograms of polypeptide solution overnight at 4°C or 2 hours at 37°C before incubation with the nitrocellulose strips. Molecular weights were based on standards with low molecular weights, and are shown in the left margin of the figure. Lanes 1 and 3 were reacted with anti-polypeptide 99 and anti-polypeptide 142 antibodies, respectively. Lanes 2 and 4 were reacted with the same antibodies pre-incubated with polypeptide 99 or with polypeptide 142, respectively. Lanes 5 and 6 show the reaction between the anti-polypeptide 99 and anti-polypeptide 142 antibodies, respectively, and control cell lysates.

The present invention has several benefits and advantages. One of these benefits is that, for the first time, the presence of HBxAg in a cell has been demonstrated.

Another advantage is that the invention provides an analysis system for detecting the presence of HBxAg in a chronic hepatitis B-infected host animal.

Yet another good thing is that the application of the invention has made it possible to detect HBxAg in cells extracted from human hepatomas, whereby the presumed connection between hepatitis B virus and this form of cancer has been strengthened.

One of the advantages of the invention is that it provides a substantial polypeptide part of HBxAg which can be used as an antigen in a diagnostic analysis with regard to the presence of antibodies against HBxAg (anti-X antibodies) which are present in the serum of humans.

Yet another advantage of the invention is the production of a synthetic polypeptide with an amino acid residue sequence that corresponds to the sequence of an antigenic determinant in HBxAg, which can be used to produce antibodies that react immunologically with HBxAg, as well as with polypeptides that have an antigenic determinant in common with HBxAg .

Even further benefits and advantages of the present invention will become clear to those skilled in the art from the detailed description and requirements that follow.

Detailed description of the invention

Definitions

Transfection: is the acquisition of new genetic markers by incorporating added DNA into eukaryotic cells.

Transformation; is the acquisition of new genetic markers by incorporation of added DNA into prokaryotic cells.

Cloning vector; is any plasmid or virus into which a foreign DNA can be inserted to be cloned.

Plasmid: is an autonomous self-replicating extrachromosomal circular DNA.

Open reading frame: is a DNA sequence that is (potentially) translatable into protein.

Helper virus: is a virus that provides functions absent in a defective virus, enabling the latter to complete the infection cycle during a mixed infection.

Gene (Cistron): is the segment of DNA involved in the production of a polypeptide chain; it includes areas before and after the coding region (leader and follower), as well as intermediate sequences (introns) between individual coding segments (exons).

Expression: the process by which a structural gene undergoes to produce a polypeptide. It is a combination of transcription and translation.

Clone: describes a large number of identical cells or molecules with a single stem cell or a single stem molecule.

Base pair (bp): is a pair ratio between adenine (A) and thymine (T); or between cytosine (C) and guanine (G) in a DNA double helix.

Expression vector: is any plasmid or virus into which a foreign DNA can be inserted or expressed.

Downstream: identifies sequences that follow

in the direction of expression; e.g. is the polypeptide coding re-

19

rule for a gene downstream from the initiation codon.

A. General review

Region X of the hepatitis B virus HBV genome is of interest, among other things, because recent evidence indicated the possibility that it is expressed, and traditional HBV serology had not found its protein product or antibodies to such a product. Recently developed recombinant DNA and synthetic polypeptide techniques are used here to overcome some of the errors encountered with traditional methods with respect to expression of HBxAg, and to illustrate the expression of HBxAg in cells from cell lines derived from human hepatoma and in liver cells from chimpanzees and humans with a chronic HBV infection.

In this respect, the present invention relates to synthetic polypeptides that correspond to antigenic determinants in HBxAg, and antibodies raised against these synthetic polypeptides that bind to the polypeptides as well as to HBxAg, or to a significant part thereof, as well as analysis systems with regard to the presence of HBxAg.

The cloning vectors mentioned here are used, among other things, to produce usable amounts of an HBxAg-containing gene. This gene can in turn be used, among other things, to produce quantities of HBxAg and polypeptide fragments thereof which can be used in the examination of the hepatitis B disease itself.

The new synthetic polypeptides according to the invention can, among other things, be used as antigens in the production of antibodies which react immunologically with HBxAg and significant polypeptide parts thereof, as well as react immunologically with the synthetic polypeptides themselves. The antibodies produced in this way can then be used to analyze the presence of HBxAg or a significant polypeptide part thereof in the cells of an infected animal host or in tissue culture.

The expression vector can be used to transfect cells and induce expression of a polypeptide comprising a substantial part of HBxAg. The expressed polypeptide can then be used as an antigen in an assay for determining the presence of antibodies against HBxAg in a body sample.

The expression vector and the antibodies used can also be used as a marker for transfected cells that contain the expression vector, and furthermore comprise a further ligated gene whose presence can be relatively difficult to confirm. The expression vector designated SVHBV-3 described below can thus be opened and ligated to yet another, foreign gene that codes for a protein that can be relatively difficult to analyze with regard to. After new ring formation, infection of appropriate cells with the new vector thus formed, and plating into single-cell colonies, the cells that incorporated the new vector can be identified by their expression of the portion of HBxAg that the vector encodes fused to the protein encoded by the foreign gene, by using the antibodies according to the invention. Such a marker can be particularly useful when the vector is desired to be expressed in eukaryotic cells; ie, when drug resistance markers present in bacterial cell vectors, such as pBR322, are not present.

1. Cloning vectors

The recombinant DNA that is used and that contains the HBxAg gene can be produced, e.g. by cloning part of the HBV DNA. To obtain HBV genomic material, Dane particle DNA containing a single-stranded portion is converted into a complete double-stranded DNA using the virus's endogenous DNA polymerase.

The ring-shaped, double-stranded HBV thus obtained contains two BamHI endonuclease restriction sites. Cleavage with BamHI usually yields three straight-chain products classified by size. The largest is a fragment of 3200 base pairs (bp) that constitutes the entire HBV genome cleaved in only one BamHI site. Fragments containing 1850 bp and 1350 bp are produced when HBV is cleaved in both BamHI sites.

Analysis of the HBV nucleotide sequence according to Galibert et al. (1979), Nature, 281: 646-650 discloses that the 1850 bp HBV-BamHI fragment comprises the gene encoding HBxAg. However, all three fragments have BamHI complementary ends and can therefore be inserted into the BamHI site of a cloning plasmid. Plasmid pBR322 has a single BamHI restriction site located within its tetracycline resistance-conferring gene, as can be seen by examination of Figure 3. Digestion of pBR322 DNA with BamHI yields a single linear fragment with ends complementary to each of the three BamHI - the HBV DNA fragments.

Ligation of each of the three BamHI HBV DNA fragments into pBR322 at its BamHI site with T4 DNA ligase to reconstitute and circularize the plasmids resulted in three unique recombinant plasmid DNAs. The recombinant cloning plasmids are circular DNAs, and are designated: AM7, containing pBR322 DNA with the 1350 bp BamHI HBV DNA fragment inserted into pBR322's BamHI site; AM6, containing pBR322 DNA with the entire HBV genome inserted into pBR322's BamHI site; and AMI, containing pBR322 DNA with the 1850 bp BamHI HBV DNA fragment including the HBxAg gene inserted into pBR322's BamHI site.

When the BamHI-HBV-DNA fragments are ligated into the pBR322-BamHI site with T4-DNA ligase, the resulting recombinant plasmids no longer have the ability to confer tetracycline resistance. Ampicillin-resistant and tetracycline-sensitive transformants were thereby selected from among colonies of Escherichia coli (E. coli) strain HB101 transformed with the above-mentioned ligation products.

Three drug-selected strains of E. coli HB101 transformed with recombinant plasmids AMI, AM6 and AM7 are designated ECAM1, ECAM6 and ECAM7, respectively. The above-mentioned plasmids were produced in relatively large quantities by growing the above-mentioned transformants in a suitable medium, such as LB nutrient fluid, as described below.

From the above-mentioned plasmids AML and AM6, a gene fragment containing all or part of the HBxAg gene base sequence can be removed by using appropriate restriction enzymes. E.g. a DNA fragment with 1850 bp included in the gene encoding HBxAg was isolated from the reaction products when the plasmids AMI and AM6 were digested with the restriction enzyme BamHI. By cultivating the recombinant plasmid-transformed E. coli HB101 strains ECAM6 or ECAM1, a relatively large amount of the DNA sequence encoding HBxAg can be obtained.

From the HBxAg gene for which the DNA base sequence and location on the HBV genome have been determined, it is clear that there are no introns. This means the gene can be transcribed as it is, leading to phenotypic expression as a messenger RNA in animal cells as well as in bacterial cells.

2. Expression vectors

Introduction of the HBxAg gene together with a suitable expression system into an animal cell, e.g. an African green monkey kidney cell, using the method of Ganem et al. (1976), J. Mol. Biol., 101: 57-83 can lead to HBxAg synthesis in the cell. Furthermore, cultivation of these monkey kidney cells containing the HBxAg gene together with an appropriate expression vector enables inexpensive mass production of HbxAg.

Advantageously, a recombinant DNA capable of expressing the HBxAg gene was constructed by inserting, without frameshifting, the complete HBxAg gene, or a fragment thereof, into simian virus 40 (SV40) downstream of the promoter last in SV40's area. A vector for expression using the promoter last in the SV40 region was produced in large quantities in the following manner.

SV40 DNA and pBR322 DNA were subjected to BamHI and EcoRI double restriction enzyme digestion. The larger SV40 and pBR322 fragments were ligated using T4 DNA ligase. The ligation product was subjected to BamHI digestion to form a linear DNA with BamHI cohesive ends. Ligation of this SV40/pBR322 DNA with the 1850 bp BamHI-HBV DNA resulted in a circular recombinant plasmid designated SVAM-191.

SVAM191 recombinant DNA contains an intact E. coli ampicillin resistance gene. E. coli HB101 transformed with SVAM-191 is ampicillin resistant and tetracycline sensitive, and can thus be selected for on the basis of drug sensitivity.

E. coli HB101 transformed with SVAM191 is called EC-SVAM191, and can be grown in a suitable medium, such as LB nutrient fluid containing ampicillin, and a relatively large amount of the plasmid can thereby be obtained.

3. Cell culture

The transformation of a host cell with the thus obtained plasmids AMI, AM6 and SVAM191 with recombinant DNA can be carried out using known methods (Cohen et al., Proe. Nati. Acad. Sei. USA, 69: 2110-2114 (1972)) or a modification thereof. The host cells include, among others, such microorganisms as Escerichia coli (E. coli), Bacillus subtilis and yeast and are preferably a strain of Escerichia coli, such as the strains designated 294 (ATCC 31446), W3110 (ATCC 27325) or RR1

(ATCC 31447). E. coli strain HB101 (ATCC 33694) is a particularly preferred host cell line.

Isolation of a cell strain carrying the HBxAg gene-containing new recombinant plasmid DNA can be carried out by known methods, including e.g. the following technique.

Dane particle DNA that is only partially double-stranded

can be radioactively labeled by refilling the single-stranded portion with <3>H>-containing dNTPs using the reaction with endogenous DNA polymerase. Robinson (1975), Am. J. Med. Pollock.,

270: 151-159. Using the labeled product as an applicant, a positive clone can then be picked out among the already obtained drug-selected transformants by means of the known "Southern blot" hybridization method (Southern

(1975), J. Mol. Biol., 98: 503), or by means of the known colony hybridization method (Grunstein et al. (1975), Proe. Nati. Acad. Sei. USA, 72: 3961-3965).

Host cells transformed and isolated in this way are cultured in a known medium. The medium can e.g. be LB nutrient solution, Penassay nutrient solution or M9 medium containing glucose

and casamino acids (Miller, Experiments in Molecular Genetics, 431-433 (Cold Spring Harbor Laboratory, New York, (1972)).

Cultivation is generally carried out at 15 - 43°C, preferably 28 - 40°C, for 2 - 24 hours, preferably for 4 - 16 hours, if necessary with aeration and/or stirring.

After cultivation, the bacterial cells are collected, the cells are suspended in a buffer and lysed, e.g. by means of lysozyme, freezing/thawing or ultrasound treatment. The gene encoding HBxAg can be isolated using a procedure generally known for DNA purification from the centrifugation supernatant obtained after cell lysis.

To prepare a vector for expression of HBxAg in eukaryotic cells, a portion of SVAM191 DNA, including all DNA of pBR322 origin, was removed. This was done by subjecting SVAM191 to Haell restriction enzyme solution. When the largest of the Haell SVAMl91 lysates were cyclized, a vector capable of expressing HBxAg in animal cells, designated SVHBV-3, was formed.

A mammalian cell host can be transfected with SVHBV-3

using known methods, whereby an expression system is obtained. E.g. SVHBV-3, when cleaved with the restriction enzyme HindIII, can be introduced into COS-7 cells (ATCC CRL 1651). (See Gluzman (1981), Cell, 23: 175-182 and Siddiqui

(1983), Mol. and Cell Biology, 3: 143-146). SVHBV-3 can also be inserted into bovine papillomavirus and thereby used to transfect murine cell lines.

Preferably, an SVHBV-3 together with SV40tsA23g helper virus is used to transfect the kidney cell line BSC-1 (ATCC CCL 26) from African green monkeys. After being transfected in this manner, BSC-1 cells produced a polypeptide containing HBxAg antigenic determinants.

It should be understood that the nucleotide sequence or gene fragment inserted into the selected restriction site in the cloning or expression aid may comprise nucleotides which are not part of the relevant gene for the desired protein, or may comprise only a fragment of this gene. Due to the known redundancy in the genetic codes, the inserted gene thus does not have to be identical to the HBxAg gene described here, but only needs to code for HBxAg. In addition, a DNA sequence encoding HBxAg may comprise additional bases at either one or both of the 3' or 5' ends of the sequence encoding HBxAg, as long as the reading frame is unchanged. Put another way, the present invention, regardless of which DNA sequence is inserted, only requires that the transformed or transfected host produce a DNA which codes for a polypeptide comprising a substantial part of the HBxAg protein.

4. Polypeptides, antigens and antibodies

As described below, the expression of HBxAg can be detected using antibodies induced by means of synthetic polypeptides with amino acid residue sequence corresponding to the amino acid residue sequence of an antigenic determinant in HBxAg. The polypeptides according to the invention contain respectively 15, 19 and 20 amino acid residues.

The amino acid residue sequence of the synthetic polypeptides designated 8, 42 and 79 according to the invention, as well as 99 and 100 and 142 shown below and in Figure 6, was determined from the HBV nucleotide sequence as published by Galibert et al., supra.

Polypeptide 8: Gln-Leu-Asp-Pro-Ala-Arg-Asp-Val-Leu-Cys-Leu-Arg-Pro-Val-Gly,

polypeptide 42: Ser-Ala-Val-Pro-Thr-Asp-His-Gly-Ala-His-Leu-Ser-Leu-Arg-Gly-Leu-Pro-Val-Cys,

polypeptide 79: Met-Glu-Thr-Thr-Val-Asn-Ala-His-Gln-Ile-Leu-Pro-Lys-Val-Leu-His-Lys-Arg-Thr-Leu-Gly,

polypeptide 99: Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp-Cys,

polypeptide 100: Cys-Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val, and

polypeptide 142: Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala,

where each of the amino acid residue sequences is shown in the direction from left to right, and in the direction from amino end to carboxyl end.

a. X protein in transfected cells

Rabbits immunized with polypeptides 99, 100, or 142 linked to a keyhole lymph-hemocyanin carrier (KLH) as a conjugate produced antibodies (anti-99, anti-100, and anti-142, respectively) that reacted immunologically with the HBxAg polypeptide expressed in BSC -l cells transfected with SVHBV-3, thereby demonstrating for the first time the expression of an HBxAg polypeptide in these or in cells at all.These anti-polypeptide antibodies did not react immunologically with any polypeptide found in untransfected BSC-l- cells, i.e. cells infected with wild-type (wt) SV40.

In addition, the immunoreactivity of the anti-polypeptide antibodies expressing the HBxAg polypeptide was inhibited or blocked by preincubation of the antibodies with their complementary (corresponding) polypeptide; ie, the polypeptide immunogen against which they were raised. This blocking indicates that the antibodies specifically recognized an antigenic determinant in the polypeptide predicted to be HBxAg, and that the synthetic polypeptides corresponded in amino acid residue sequence to antigenic determinants in the predicted HBxAg protein. These blocking results were obtained using the Western blot technique (Materials and Methods) followed by localization of bound

125

antibodies with an I-labeled Staphylococcus aureus protein A followed by autoradiographic development of the sample.

These results are shown in Figure 10 for antisera against polypeptides 99 and 142. Field 1 in Figure 10 shows the immunoreaction between anti-99 and the SVHBV-3-infected cell lysate, while field 2 shows blocking of the immunoreaction by pre-incubation of the antiserum with polypeptide 99. Likewise, anti-142 reacted immunologically with a cell lysate protein in lane 3, and this immunoreaction was blocked by preincubation of this antiserum with polypeptide 142. Lanes 5 and 6 show that no immunoreaction took place with lysates from control

cells.

Polypeptide expression product from SVHBV-3 transfected cells as it was specifically identified by antisera against polypeptides 99 and 142, as shown in Figure 10, had a molecular weight of approx. 24,000 daltons. As discussed below, a polypeptide with a molecular weight of approx. 28,000 daltons have been identified in lysates from the human hepatoma-derived cell line PLC/PRF/5. It is believed that the molecular weight difference between the polypeptides (proteins) expressed by the two cell lines originates from the fact that the polypeptides expressed result from the fusion of the X protein with other proteins or polypeptides.

Thus, as already noted, SVHBV-3 lacks part of the complete X-coding genome. Figure 6 shows that the part of the X gene included in SVHBV-3 excludes codons for the first 22 amino acid residues in the putative X protein which includes the methionine initiation codon ATG.

It was therefore predicted that the polypeptide expressed by cells transfected with SVHBV-3 would be a fusion product with the SV40 structural protein sequences VP2 present in the vector. The predicted size of the fusion protein (polypeptide) was predicted to be 24,500 daltons. The discovery of an expressed polypeptide with a molecular weight of approx. 24,000 daltons thus corresponds to this prediction. The 28,000 dalton fusion protein (polypeptide) is discussed below.

b. X protein expressed in hepatoma cells

Expression of HBxAg is also believed to be a function of certain HBV disease states. E.g. recognized antibodies against polypeptide 99 (anti-99) specifically a 28,000 dalton protein expressed in the human hepatoma-derived cell line PLC/PRF/5 (ATCC CRL 8024). Normal rabbit serum did not recognize this protein and anti-99 recognition was blocked by preincubation with polypeptide 99. This cell line is known to contain integrated HBV DNA. Chakrabarty et al. (1980), Nature, 286: 531-533, Edman et al. (1980), Nature, 286: 535-538 and Marion et al. (1980), Virol, 33: 795-806. HBsAg has been detected in this hepatoma cell line, (McNab et al. (1976), Cancer, 34: 509-515 and Aden et al. (1979), Nature, 282: 615-616), while all standard assays for HBeAg and HBeAg have been negative. The cell line designated HepG2 (ATCC CCL 23) is also a human hepatoma cell line, but does not contain integrated HBV DNA sequences. Aden et al., supra.

These results were obtained by radiolabeling the PLC/PRF/5 cell proteins followed by repeated immunoprecipitations using anti-99 and Staphylococcus aureus protein A, and then electrophoresis of the recovered precipitate. The 28,000 dalton protein was then identified by autoradiography. This method is also discussed in the section on materials and methods.

Results from a similar study using lysates from both PLC/PRF/5 and HepG2 cells are shown in Figure 7. Figure 7 shows that an X-specific protein of 28,000 daltons was detected in PLC/PRF/5 cells ( field 5). Immunoreactivity was lost when antibody was preincubated with polypeptide 99 (lane 6), but not with an unrelated polypeptide (lane 7). The preimmunoserum control did not react immunologically (lane 8). No X-specific reactivity was detected in the control HepG2 lysates (lanes 1 - 4). These results establish that an X gene product is expressed in a human hepatoma cell line containing integrated HBV DNA sequences.

c. X protein expression in liver cells

In addition, four human and chimpanzee liver samples, either with or without prior HBV infection, were examined in a Western blot analysis for the presence of an X-related protein using anti-X polypeptide antibodies. The results of this investigation are shown in figure 8. One human sample (H) was from a chronic HBV-infected hepatoma liver and the other from an acute-phase infection (H). An uninfected chimpanzee (C) and one chronically infected with HBV (C) were both used to obtain liver cell lysates which were reacted with anti-X polypeptide antibodies.

Anti-99 antibodies reacted against both a 45,000 and a 28,000 (arrow in left margin, p28 in right margin) dalton protein. Both reactivities were blocked when these antibodies were preincubated with polypeptide 99. When anti-142 antibodies were reacted against these same cell lysates, specific bands were detected at 40,000 (p40), 28,000 (p28) and 17,000 (pl7). The reactivities against these proteins are considered specific because they are removed when anti-142 antibodies are preincubated with polypeptide 142.

The control sample, an uninfected chimpanzee liver (field 3 in all panels), showed approx. 45,000 dalton protein (p45) with anti-99 antibodies, and both p40 and pl7 with anti-142 antibodies. Protein p28 was expressed only in HBV-infected tissues (lane 5). Immunoreactivity was lost when antibody was preincubated with polypeptide 99 (lane 6), but not with an unrelated polypeptide (lane 7). The pre-immunoserum control did not react immunologically (lane 8). No X-specific reactivity was detected in the control HepG2 lysates (lanes 1 - 4). These results establish that an X gene product is expressed in a human hepatoma cell line containing integrated HBV DNA sequences.

The presence of a 45,000 dalton protein was also observed in a relatively small amount in the lysates from PLC/PRF/5 cells shown in Figure 7. The immune reaction between anti-99 antibodies and the 45,000 dalton protein was also blocked by preincubation of the antibodies with the immunizing polypeptide 99. This is shown in field 6 of Figure 7.

Normal human liver cell extracts (HBV seronegative) were found to express only the 45,000 dalton protein in the Western blot analysis using anti-99. In addition, control assays performed using commercially available antibodies raised against HBs, HBc and HBe did not identify any of the proteins localized by the antibodies of the invention. This evidence again suggests that the 45,000 dalton protein has antigenic determinants homologous to HBxAg, but that the 28,000 dalton protein recognized by anti-99 is specific for an HBV disease state and is distinct from HbsAg, HBeAg, and HBeAg.

d. X gene in liver cells

The expression of p28 in PLC/PRGF/5 cells occurred from HBV DNA integrated into host DNA. It was of interest to determine whether this was the only way in which X protein could be expressed.

The chimpanzee and human liver DNAs that expressed X protein were therefore probed with HBV DNA (Figure 9). Total DNA was isolated from the liver tissues shown in Figure 8, and was analyzed for the presence of HBV DNA sequences. DNA from the chimpanzee containing the 28,000 dalton protein revealed homologous bands when digested with BamHI, EcoRI or when unresolved (Figure 9, lanes 1, 2 and 3, respectively). BamHI cleaves circular HBV DNA into two fragments (1850 and 1350 base pairs) (bp), with the band at 5.8 kilobases (Kb) (lane 1) representing an integrated sequence larger than the genome size. As no band was seen below the high molecular weight DNA band (lane 3), it is concluded that only integrated sequences of HBV are present in this DNA.

In addition, DNA from an HBcAg-positive human liver was prepared and tested for HBV DNA. Restriction enzyme digestion with HindIII, BamHI, EcoRI or unresolved DNA (Figure 9, lanes 7-10, respectively) revealed homologous sequences that were either equal to or smaller than the genome size, eliminating any indication that these human DNA samples contain integrated sequences . Chimpanzee and human liver tissues lacking the 28,000 dalton protein were also negative for HBV DNA sequences (Figure 9, lanes 4 - 6 and 11 - 14, respectively). The data shown in Figure 9 confirm that X protein is expressed in HBV-infected tissues regardless of the state of the HBV genome, therefore eliminating a prerequisite for HBV DNA integration for X expression.

e. Summary of results

The results described above demonstrate the presence of a previously unidentified virus-encoded protein in HBV-infected human and chimpanzee liver tissue. This 28,000 dalton protein (p28) is coded for by the HBV genome regardless of whether it exists free in an extrachromosomal form or is found integrated in cell DNA.

The 28,000 dalton protein (p28) is not the product of the X region alone, but contains additional HBV sequences. The predicted size of X protein, based on the sequence reported by Galibert et al., supra, is approx. 17,000 daltons (figure 6). However, the molecular weight of the X protein detected by anti-X peptide antibodies in PLC/PRF/5 cells, as well as in chimpanzee- and human-HBV-infected tissues is 28,000 daltons. For-

the shell in size is due to a fusion of the X gene with cell sequences as the lack of integrated HBV DNA in human tissue expressing p28 (Figure 9, lanes 7-10) indicates that the X protein (p28) is translated alone from the HBV genome.

The increased size of the X protein can be explained if the protein is either an HBsAg-X or an X-HBcAg fusion. The probability of such a fusion is suggested by the proximity of these HBV genes to each other on the genome.

An attempt to determine the existence of such a fusion at the RNA level was not successful, due to the fact that the transcripts obtained in the PLC/PRF/5 cells, which were found to contain X sequences, also exhibited either surface or core sequences as well as X (data not shown). Similar results were reported by Gough (1983), J. Mol. Biol., 165: 683-699. Evidence for an X-core fusion comes from the DNA sequence of

another member of the hepadnavirus family, duck hepatitis B virus (DHBV), in which both genes are translated as a single protein (Mandart et al. (1984), J. Virol., 49: 782-792) and from work by Feitelson et eel. (1982), J. Virol., 43: 687-696.

The fact that the X region of HBV is expressed has been established. However, the function of this gene product remains unknown. A protein molecule that has been proposed to be the translation product of the X region is found in association with the HBV genome, or more precisely is covalently bound to

The 5'-"nick"-L strand, Gerlich et al. (1980), Cell, 21: 801-809. The idea that X protein constitutes a DNA binding protein was eliminated as DNAse treatment of PLC/PRF/5 cells or HBV-infected tissues did not remove or alter p28 activity (data not shown).

Currently, little is known about the biology of HBV infection, or the connection between HBV infection and the subsequent attack of hepatocellular carcinoma. For this purpose, any additional marker for HBV infection or for the presence of HBV sequences is of great importance. Human sera are currently being examined for the existence of anti-X antibodies and the X protein.

The expression "corresponding to" in its various grammatical forms is used here and in the claims in connection with polypeptide sequences in the sense of the described polypeptide sequence plus or minus up to three amino acid residues in either one of or in both the amino and carboxyl ends, and containing only conservative substitutions in specific amino acid residues along the polypeptide sequence.

The term "conservative substitution", as used above, is intended to denote that an amino acid residue has been replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of a hydrophobic residue, such as Ile, Val, Leu or Met, with another,

or the substitution of one polar residue with another, such as between Arg and Lys, between Glu and Asp or between Gin and Asn, and the like.

In some cases, the replacement of an ionic residue with an oppositely charged ionic residue, such as Asp with Lys, has been called conservative in the art because these ionic groups are believed to provide only solubility assistance. As the exchanges discussed here are on relatively short synthetic polypeptide antigens compared to an entire protein, however, generally exchange of an ion residue with another ion residue of opposite charge is considered here to be a "radical exchange", which also applies to exchange between nonionic and ionic residues, and bulky residues, such as Phe, Tyr, or Trp, and less bulky residues, such as Gly, Ile, and Val.

The terms "non-ionic" and "ionic" residues are used here in their usual meanings to denote those amino acid residues which normally either carry no charge or normally carry a charge at physiological pH values. Examples of non-ionic residues include Thr and Gin, while examples of ionic residues include Arg and Asp.

The word "antigen" has historically been used to denote an entity that is bound by an antibody, and also to denote the entity that induces the production of the antibody. More recent usage limits the meaning of antigen to the entity that is bound by an antibody, while the word "immunogen" is used for the entity that induces antibody production.

In some cases, the antigen and the immunogen are the same entity, as when a synthetic polypeptide is used to induce the production of antibodies that bind to the polypeptide. However, the same polypeptides (99, 100 or 142) also induce antibodies that bind to an entire protein, such as HBxAg, in which case the polypeptide is both immunogenic and antigenic, whereas HBxAg is an antigen. When an entity discussed herein is both immunogenic and antigenic, it will generally be called an antigen.

5. Analysis systems and methods

The above results indicate that SVHBV-3 can be used as an expression vector. It provides expression control elements for foreign DNA sequences inserted into the reading frame downstream of the HBxAg gene.

The results also indicate that synthetic polypeptides 99, 100 and 142, and antipeptide antibodies against these can be used as part of an analysis system and an analysis method for detecting the presence of HBxAg in a body sample to be analyzed, such as hepatoma cells or liver cells from an animal host that suspected of having HBxAg, to demonstrate successful transfection with SVHBV-3, and to monitor expression when SVHBV-3 is used as an expression vector.

Such a system comprises at least one first reagent containing antibodies comprising an antibody binding site,

essentially whole antibodies, or the well-known Fab and F(ab' )2 antibody parts, and are capable of reacting immunologically with a synthetic polypeptide according to the invention; i.e. elicited against the synthetic polypeptides according to the invention. Markers, such as a fluorescent dye such as fluorescein or rhodamine, a radioactive element such as iodine-125 or carbon-14, or an enzyme-linked antibody raised against the receptors of the first reagent (eg, peroxidase-linked goat anti-rabbit IgG), to signal immunoreaction between the receptors of the first reagent and the expression-specific HBxAg, is also provided. The labels can be pre-bound or free of the first reagent. Typical applications of such an HBxAg assay reagent system include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, and immunofluorescence (IF) assays.

Diagnostic analyzes for determining the presence of HBxAg in a body sample using the polypeptides according to the invention and their anti-polypeptide antibodies have already been extensively described. In a commercial embodiment of such a diagnostic analysis, a diagnostic analysis system is concerned.

Such a system comprises providing at least one container with an anti-polypeptide antibody, such as anti-8, -42, -79, -99 or -142, as the first reagent. A second container with an amount of the polypeptide as anti-polypeptide antibody was developed against, e.g. polypeptide 8, 42, 79, 99 or 142, may also be provided to provide a second reagent. Additional containers of the previously described markers, one or more buffers, and the like as are well known, may also be provided to the diagnostic analysis system.

A method of assaying for the presence of a detectable amount of HBxAg has also been generally described above. Briefly, this method comprises mixing proteins from a body sample to be analyzed, such as hepatoma or liver cells, with antibodies comprising an antibody binding site capable of reacting immunologically with a synthetic polypeptide according to the invention and with HBxAg, such as anti -8, -42, -79, -99 or -142, or a Fab or F(ab') portion thereof, in the presence of an indicator, such as <125>I-labeled Staphylococcus aureus ( S. aureus ) protein or an enzyme marker, such as horseradish peroxidase, bound to one of the above-mentioned antibodies for signaling an immunoreaction between HBxAg and the antibodies. The mixture is maintained for a period of time sufficient for the marker to signal that an immunoreaction has occurred, and the presence of the signal is confirmed, e.g. using an autoradiogram. The body sample or the proteins from it are preferably adsorbed or attached in another way (bonded) to a solid matrix, such as a nitrocellulose sheet or a glass slide, before it is mixed with the antibodies.

When the marker is a separate molecule, such as radioactively labeled S. aureus protein A, the bound body sample and the antibodies are first mixed in the absence of the marker, and the mixture is kept for a period of time sufficient for the formation of a bound immunoreaction product. The bound immunoreaction product is then rinsed, the separate molecule marker is then mixed with the bound immunoreaction product and this mixture is kept for a period of time sufficient for a bound second reaction product to form. The bound second reaction product is then washed away, and the presence of a signal from the marker is determined.

When the marker is part of the antibodies according to the invention, these labeled antibodies are mixed with the bound body sample to be analyzed, and the mixture is kept for a period of time sufficient for an immunoreaction product to form. The bound immunoreaction product is then washed away and the presence of a signal from the marker is determined.

The previously mentioned second reagent, e.g. polypeptides 8, 42, 79, 99 or 142 can be used as a control reagent in an immunoreaction blocking assay to confirm that the specific, and not the non-specific, immunobinding has occurred. Such blocking investigations are illustrated in figures 7, 8 and 10.

To use SVHBV-3 as an expression vector, foreign DNA is inserted downstream of the HBxAg gene, preferably

in its unique BamHI restriction site, and the resulting new vector is used to transfect cells. Expression of HBxAg in a transfected cell is presumptive evidence of expression of the foreign DNA inserted into SVHBV-3. Expression of HBxAg detected using the above HBxAg expression assay system indicates the expression of foreign DNA inserted into SVHBV-3.

Examination of cell colonies produced after the attempt at transfection using the above-mentioned analysis system can thus be used to select those colonies where transfection was successful. Cells from colonies resulting from the attempt to introduce the vector into eukaryotic cells are harvested, lysed and the cellular proteins extracted, e.g. The extracted cellular proteins are then separated on an SDS-polyacrylamide gel by electrophoresis, and the separated proteins are transferred onto nitrocellulose. Contacting the transferred proteins with an excess of antibody and marker to the previously described assay system, such as <125>I-labeled antibodies against synthetic polypeptide 99 or polypeptide 142, followed by washing to remove the non-immunoreacted antibodies (receptors ), can then be used to produce an autoradiograph indicating the presence of the expressed HBxAg portion fused to the polypeptide encoded by the foreign gene.

The previously described analysis methods and systems are particularly applicable for identifying the X protein or a significant part thereof which may be present in a body sample, in particular a tissue sample, such as in cells from a liver biopsy. The diagnostic assay and method described below is particularly useful for determining the presence of antibodies against X proteins that may be present in a body sample, such as whole blood, serum or plasma. A Western blot analysis will be used as an example of such a diagnostic procedure. A commercial embodiment in which the method is used, however, is an ELISA (enzyme-linked immunosorbent assay) diagnostic system.

Here, an expression product from SVHBV-3 vector-transfected cells, such as the protein (polypeptide) of approximately 24,000 daltons expressed in BSC-1 cells discussed in connection with Figure 10, is used as the antigen, although such a polypeptide as polypeptide 99 or polypeptide 142 , can also be used as an antigen. Thus, the HBxAg molecule itself, used in a substantially purified form as obtained by affinity chromatography as discussed below, can a substantial part of the HBxAg molecule, such as that expressed by SVHBV-3-transfected BSC-1 cells (the polypeptide of 24,000 daltons), or a polypeptide that corresponds in amino acid residue sequence to an antigenic determinant in HBxAg, is used as the antigen.

The antigen is preferably bound on (adsorbed to) 38 or otherwise attached to a solid matrix, such as the cross-linked dextran available under the trade name "Sephadex", agarose, glass beads, polyvinyl chloride, polystyrene, cross-linked acrylamide, nitrocellulose or the wells of a microtiter plate , whereby a solid carrier is formed.

The antigen, preferably bound to a solid matrix as part of a solid carrier, is mixed with a liquid body sample that is analyzed, whereby a mixture of solid and liquid phase is formed. The mixture is maintained for a period of time sufficient for anti-X protein (anti-HBxAg) antibodies (HBxAg) present in the body sample to react immunologically with the antigen. The presence of this immunoreaction is then determined e.g. with an indicator that signals the presence of anti-X protein antibodies in the analyzed body sample.

During a survey, e.g. SVHBV-3 vector-transfected BSC-1 cell lysates were prepared, separated electrophoretically, and transferred and bound to nitrocellulose sheets as a solid matrix, in a manner substantially similar to that discussed in connection with Figure 10, thereby forming a solid phase. Sera diluted 1:50 from six patients with different hepatitis diseases were then separately mixed with nitrocellulose-bound transfected BSC-1 cell proteins, thereby forming solid and liquid phase mixtures. These mixtures were kept for a period of time (2 hours) sufficient for anti-antibodies present in the sera to react immunologically with the bound antigen.

After washing to separate the solid and liquid phases, the solid phases obtained from the above steps were separately mixed with liquid solutions containing 1:200 dilutions of goat anti-human or goat anti-rabbit antibodies conjugated to horseradish peroxidase as a marker, whereby the other mixtures of solid and liquid phase were formed. The separate, second mixtures of solid and liquid phase were kept for a period of time (1 hour) sufficient for the labeled antibodies to react with human anti-X antibodies that had reacted immunologically with the matrix-bound antigen. Rabbit anti-polypeptide antibodies were used as controls for the goat anti-rabbit antibodies. The resulting mixture of solid and liquid phases was again separated and rinsed. The resulting solid phases were then mixed using solutions containing 4-chloro-1-naphthol as described for Figure 8.

The results of this investigation indicate that serum from a patient diagnosed as having a hepatocellular carcinoma contained antibodies that bound to the polypeptide expressed by the SVHBV-3 vector in the transfected BSC-1 cells. These antibodies are thus anti-X antibodies and their presence establishes that the X gene product is an authentic antigen at one stage or another of HBV infection.

When the polypeptide expression product of approximately 24,000 daltons used in the Western blot assay described above is used as the antigen in an ELISA, as discussed below, or in other similar assays, this polypeptide is preferably purified and isolated prior to such use. The purification and isolation can be achieved using well-known techniques.

E.g. can anti-8, anti-42, anti-79, anti-99 or anti-142 antibodies, or the antibody-binding sites thereon, be prepared as described here and bound to a solid matrix, such as the agarose and the cross-linked agarose derivatives sold under the trademarks "Sepharose 6B", "CL6B", "4B" and "CL4B", or those sold under the trademarks "Bio-Gel A-0.5", "A-1.5M" and "A- 50M". The previously described cross-linked dextran sold under the trade name "Sephadex" and the polyacrylamide beads sold under the trade names "Bio-Gel P-2", "P-30", "P-100" and "P-300" are also usable fixed matrices. Use of a matrix consisting of an agarose derivative is discussed below as an illustration.

The agarose matrix is typically activated for binding using cyanogen bromide. The activated matrix is then washed and bound to the desired antibody molecules without drying the activated matrix. The matrix-bound antibody is then washed and is ready for use. Unreacted reactive groups on the matrix can, if desired, be reacted with an amine, such as ethanolamine, or Tris, although these reactive groups degrade quickly.

The affinity sorbent can be used in its loose state, such as in a beaker or flask, or it can be enclosed in a column. Before use, it is preferred that the affinity sorbent is washed in the buffer or other aqueous medium used for the purification of cell lysates containing the polypeptide of approximately 24,000 daltons, in order to eliminate non-specifically bound proteins or the antibodies that were bound unstable to the carrier.

An aqueous mixture containing the SVHBV-3 vector-transfected cell lysate is provided and then mixed

with the affinity sorbent. This mixture forms a reversible, bound complex of antibody and antigen (receptor and ligand) between the bound antibody (the receptor) and the approximately 24,000 dalton polypeptide (the antigen).

The bound complex of receptor and ligand is then separated from the remainder of the aqueous mixture which has not formed a complex, whereby the polypeptide antigen is thereby obtained in purified form bound to the affinity sorbent. When the mixture takes place in a beaker or flask, this separation can be done by filtration and washing. When the sorbent is in a column, the separation can take place by elution of the aqueous medium which did not form a complex, again preferably followed by a washing step.

When the purified polypeptide antigen is desired free of the affinity sorbent, it can usually be obtained by several different methods. In any of these methods, the reversibly bound receptor/ligand complex is dissociated into its components of matrix-bound receptor and polypeptide antigen ligand, followed by separation of the polypeptide antigen ligand from the bound receptor, thereby obtaining a solution of the purified polypeptide antigen ligand free of the affinity sorbent. The solution can be used as such, or it can be concentrated or dried before use if desired. In some cases, it may be desirable to desalt the solution before use, which is known.

The dissociation of the reversible complex can be effected in several ways. A 0.2 molar glycine hydrochloride solution with a pH value of approx. 2.5 is usually used. Alternatively, the bound polypeptide antigen ligand can be outcompeted from the bound receptor by mixing the reversible complex with an excess of the immunogenic polypeptide used to elicit the antibodies, e.g. polypeptide 99 where anti-99 antibodies are bound to the matrix. With such competition, possible denaturation of the polypeptide antigen is avoided. Separation of the dissociated polypeptide antigen from the affinity sorbent can be achieved as above.

The preparation of affinity sorbents and their use are generally known. However, such materials and applications which include the antibody and antigen molecules according to the invention have not been available until now. A more detailed description of affinity sorbents, their methods of production and application where the antigen is bound to the matrix, can be found in Antibody as a Tool, Marchalonis and Warr, editors, John Wiley & Sons, New York, pp. 64- 67 and 76-96 (1982).

In an exemplary ELISA where the above-mentioned method is used, a solid carrier is used consisting of a previously described antigen according to the invention adsorbed on, or attached in some other way to, a solid matrix consisting of the walls of a twelve- or ninety-six-well microtiter plate made of polystyrene or polyvinyl chloride which constitutes the solid carrier. Non-specific binding sites on the microtiter well walls are then typically blocked with a protein, such as bovine serum albumin (BSA). Any unbound antigen and BSA are removed from the microtiter well, e.g. when rinsing.

A body sample aliquot, such as human serum, blood or

-plasma, is mixed with the antigen-bound solid carrier described above, whereby a mixture containing solid and liquid phases is obtained. The mixture of solid and liquid phase is maintained for a period of time sufficient for anti-X protein antibodies in the body sample to react immunologically with the polypeptide antigen, thereby forming a polypeptide-containing immunoreaction product, e.g. about. 30 minutes to approx. 2 hours. The solid and liquid phases are then generally separated.

A liquid solution of a second, labeled, marker-containing antibody, antibody binding site or S. aureus protein A that reacts with the first-mentioned antibody is then mixed with the solid phase, whereby a second mixture of solid and liquid phase is formed; i.e. a labeling reaction mixture. An example of a second antibody is a peroxidase-labeled goat anti-human Ig antibody where the first-mentioned antibodies are from a human body sample. In addition, useful enzyme markers include alkaline phosphatase, beta-D-galactosidase and glucose oxidase.

The mixture formed from the solid phase (antigen/antibody immunoreaction product bound to the solid matrix) and the second, labeled antibody solution is maintained (incubated) for a period of time (e.g. approx. 1 hour) which is sufficient to form a reaction product between the bound, first-mentioned antibody and the indicator, such as a second immunoreaction between the two antibodies. The solid and liquid phases are then separated.

The second antibody described above may also be specific for reacting immunologically with only one of the classes of immunoglobulin (eg, IgG, IgM, IgE, IgA, or IgD). Such antibodies make it possible to identify the immunoglobulin class of anti-X protein antibody present in the body sample. In addition, the second antibody or antibody binding site may be specific for, and react immunologically with, only one of the two types of immunoglobulin light chains (eg, kappa or lambda). These antibodies make it possible to identify the isotype of the immunoglobulin molecule present in the body sample, and are well known.

A solution containing a substrate for the enzyme marker, such as hydrogen peroxide for peroxidase, and a color-forming dye precursor, such as o-phenylenediamine or 4-chloro-1-naphthol, or p-nitrophenyl phosphate for alkaline phosphatase, is then mixed with the solid phase. The optical density at a preselected wavelength (e.g. 490 or 405 nanometers respectively) can then be determined after a sufficient period of time has elapsed (e.g. 60 minutes) and compared with the optical density of a control for to determine whether anti-X protein antibodies were present in the body sample.

The diagnostic analysis system according to the invention can be used in kit form which comprises a solid carrier consisting of a solid matrix, such as a twelve-well microtiter strip of polystyrene, and a previously described antigen according to the invention absorbed (bound) or attached in some other way to it solid matrix, so that a solid carrier is formed. This system preferably also includes separately packaged anti-human Ig antibodies with an attached label, such as peroxidase-labeled goat anti-human Ig antibodies, and may also include a substrate for the attached label, such as hydrogen peroxide and a color-forming dye precursor , such as o-phenylenediamine, in further separate packages. Hydrogen peroxide is usually not included in the kit due to its relative instability, and is usually supplied by the end user. Although a container of hydrogen peroxide can also be a component of the diagnostic system. Buffer salts that can be used in an analysis where this system is used can also be included in one or more separate packages in dry or liquid form. A preferred polypeptide according to the invention may also be included in a separate package for use in studies with competitive binding as a control. An analysis with regard to the presence of anti-X protein antibodies in a body sample, such as serum, can be carried out with this diagnostic system, using the previously described method.

6. Coupling of polypeptides to protein carriers

The synthetic polypeptides used herein were linked to keyhole limpet hemocyanin (KLH) using the following well-known method. The KLH carrier was first activated with m-maleimidobenzoyl-N-hydroxysuccinimide ester, and was then linked to the polypeptide through a cysteine residue present in the polypeptide amino terminus (polypeptide 100), the carboxyl terminus (polypeptide 99) or by using the central cysteine in polypeptide 142, using a Michael addition reaction as described in Liu et al. (1979), Biochem., 80: 690.

A polypeptide according to the invention can also be linked to a carrier through various means, and can be linked to carriers other than KLH. E.g. a polypeptide can be linked to a tetanus toxoid carrier through free amino groups, using a 0.04% glutaraldehyde solution which is well known. See e.g. Klipstein et al. (1983), J. Infect. Haze, 147:318.

Cysteine residues present in the amino or carboxyl ends, or in the middle part of the synthetic polypeptide, have been found to be particularly useful for the formation of conjugates via disulfide bonds, and Michael addition reaction products, but other methods well known in the art for the preparation of conjugates, can also be used. Exemplary methods for further binding (linking) include the use of dialdehydes, such as glutaraldehyde (discussed above) and the like, or the use of carbodiimide techniques, such as by the use of a water-soluble carbodiimide, e.g. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, whereby amide bonds are formed between the carrier and the polypeptide.

Useful carriers are well known in the art and are generally proteins themselves. Examples of such carriers are keyhole limpet hemocyanin (KLH), edestin, thyroglobulin, such albumins as bovine serum albumin or human serum albumin (BSA or HSA, respectively), red blood cells such as e.g. sheep erythrocytes (SRBC) with tetanus toxoid, cholera toxoid and polyamino acids such as e.g. poly(D-glycine:D.glutamic acid) and the like.

As is also well known in the art, it is often advantageous to bind the synthetic polypeptide to its carrier by means of an intermediate linking group. As indicated earlier, glutaraldehyde is one such linking group.

When cysteine is used, however, the intermediate linking group is preferably an m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), as also discussed earlier. MBS is usually first added to the support by means of an ester-amide exchange reaction. Then the above-mentioned Michael reaction can be followed or the MBS addition can be followed by a Michael addition of a blocked mercapto group, such as thiolactic acid (CH^COSH) over the maleimido double bond. After cleavage of the blocking acyl group, a disulfide bond is formed between the linking group mercaptan from which the blocking group has been removed and the mercaptan in the added cysteine residue in the synthetic polypeptide.

The choice of carrier is more dependent on the final intended use of the antigen than on the determinant part of the antigen, and is based on criteria that are not particularly involved in the present invention. If e.g. an inoculum is to be used in animals, as for the production of anti-polypeptide antibodies to be used to analyze for the presence of HBxAg, a carrier should be selected which does not generate an adverse reaction in the particular animal.

7. Immunization procedures

The inocula used for immunizations contain an effective amount of polypeptide, as a polymer of the individual polypeptides bound together through oxidized cysteine residues, or as a conjugate of the polypeptide bound to a carrier. The effective amount of polypeptide per inoculation depends, among other things, on the species of animal being inoculated, the body weight of the animal and the chosen inoculation schedule, which is well known. Inocula are usually prepared from the dried, solid polypeptide conjugate by suspending the polypeptide conjugate in water, saline or adjuvant.

These inocula usually contain polypeptide concentrations of approx. 20 micrograms to approx. 500 milligrams per inoculation. The indicated amounts of polypeptide refer to the weight of polypeptide without the weight of a carrier, when a carrier was used.

The inocula also contain a physiologically tolerable (acceptable) diluent, such as e.g. water, phosphate-buffered saline or saline, and further usually comprises an adjuvant as part of the diluent. Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum are materials well known in the art and are commercially available from several sources.

Standard solutions of inoculum are prepared with CFA, IFA or alum in the following way: An amount of the conjugate with synthetic polypeptide that is sufficient to obtain the desired amount of polypeptide per inoculation, dissolve in phosphate-buffered saline (PBS) at a pH value of 7.2. Equal volumes of CFA, IFA or alum are then mixed with the polypeptide solution, whereby an inoculum containing polypeptide, water and adjuvant is obtained, where the ratio between water and oil is approx. 1:1. The mixture is then homogenized, whereby the standard solution of inoculum is obtained.

Rabbits used here to raise anti-polypeptide antibodies were injected subcutaneously with an inoculum comprising 200 micrograms of a polypeptide conjugate (polypeptide plus carrier) emulsified in complete Freund's adjuvant (CFA), 200 micrograms of polypeptide conjugate, incomplete in Freund's adjuvant (IFA ), and 200 micrograms of polypeptide conjugate with 4 milligrams of alum, injected intraperitoneally on days 0, 14 and 21 of the immunization schedule, respectively. Each inoculation (immunization) consisted of four injections of the inoculum. Mice can be immunized in a similar way using approx. one tenth of the above-mentioned dose per injection.

Blood samples are usually taken from the animals 4 and 15 weeks after the first injection. Control pre-immune serum was obtained from each animal by blood sampling just before the first immunization.

Standard solutions of control inoculum can also be prepared with keyhole limpet hemocyanin (KLH), KLH in IFA (incomplete Freund's adjuvant), KLH-alum absorbed, KLH-alum absorbed-pertussis, edestin, thyroglobulin, tetanus toxoid, tetanus toxoid in IFA, cholera toxoid and cholera toxoid in IFA.

After injection or other introduction of the antigen or inoculum into the host animal, the immune system of the animal responds by producing large amounts of antibody against the antigen. As the specific antigenic determinant of the processed antigen, i.e. the antigen formed by the synthetic polypeptide attached to the carrier corresponds to the determinant of the relevant natural antigen, the host animal produces antibodies not only against the antigen of synthetic polypeptide, but also against the protein or polypeptide which the antigen of synthetic polypeptide corresponds to, i.e. against HBxAg.

8. Deposits

The materials listed below were deposited at the American Type Culture Collection, March 8, 1984, and have the deposit numbers indicated for each. All designations for cell lines, vectors and the like that include the letters "ATCC" followed by numbers and/or letters and numbers refer to materials deposited at the above-mentioned American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20862.

In addition to the above-mentioned materials deposited at ATCC by the inventors, materials produced by others and used here have also been deposited at ATCC. A list of these materials is given below:

B. Materials and methods

1. HBV DNA isolation and production

The region of XHBV DNA was isolated from Dane particles, subtype adw, from the plasma of an HBsAg-positive human donor (National Institute of Health No. 737139) following the procedure of Robinson (Am. J. Med. Sei., ( 1975) 270: 151-159). Briefly, 1.0 milliliters of plasma was diluted to 3.0 ml with TN buffer containing 0.001 mol (M) Tris-HCl (pH 7.4) and 0.5 M HCl. The diluted plasma was centrifuged at 10,000 x g for 10 min to remove large debris, and then loaded onto 2.5 milliliters (ml) of 30% (w/v) sucrose containing TN, 0.01 M EDTA (0.1 % 2-mercaptoethanol and 1 milligram/milliliter (mg/ml) bovine serum albumin (BSA) that had previously been centrifuged at 10,000 x g for 10 minutes to remove precipitate (BSA). After centrifugation for 4 hours at 50,000 rpm, 4°C in a Spinco SW-65 centrifuge, the supernatant was carefully removed and the pellet was resuspended in 50 microliters of TN buffer containing 1% "Nonidet P40" (polyoxyethylene (9)octylphenyl ether) and 0.1% 2-mercaptoethanol.

The single-stranded portion of the HBV DNA isolated above was made double-stranded by adding 25 microliters of mixture E (0.2 M Tris-HCl (pH 7.4), 0.08 M MgCl2, 0.24

NH4Cl, 1.0 millimoles (mM) each of dATP, dTTP, dGTP and dCTP)

to 50 microliters of resuspended HBV DNA, followed by incubation at 37°C for 3 hours. Robinson (1975), Am. J. Med. Sei., 270: 151-159.

Radiolabeled HBV DNA to be used as a genome probe was made using the above replenishment procedure by replacing dCTP and dGTP with 0.25 microliters of 3HdCTP and 3HdGTP, respectively (both 21 curie per mM).

2. HBxAg cloning

A portion of the HBV genome containing the DNA sequence encoding HBxAg was cloned using the plasmid pBR322 introduced into E. coli. The double-stranded, unlabeled HBV DNA isolated above was digested with the restriction endonuclease BamHI (50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl, 1 mM dithiothreitol). The reaction mixture was electrophoresed in a 0.6% agarose horizontal plate gel at 100 amps for 2 hours (0.04 Tris-acetate, 0.002 M EDTA).

Staining with 1% ethidium bromide revealed three HBV DNA fragments, indicating the presence of two BamHI restriction sites in the genome. A 3200 base pair (bp) fragment comprised the entire HBV genome in linear form as a result of being cut in only one of the BamHI sites. The 1850 and 1350 bp fragments constituted the genome cut into two fragments as a result of complete BamHI digestion.

Copies of the three BamHI HBV DNA restriction fragments isolated above were obtained by cloning into plasmid pBR322

(ATCC 37017) (Sutcliffe (1978), Proe. Nati. Acad. Sei., USA, 75: 3737-3791). To insert the HBV DNA BamHI fragments into pBR322, the plasmid was linearized by digestion with BamHI (50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol) formed ends that were complementary to the ends of the HBV fragments. The three HBV DNA fragments and the pBR322 fragment were mixed and subjected to DNA ligation with T4 DNA ligase (66 mM Tris-HCl (pH 7.6), 66 mM

<MqC1>2' 10 mM dithiothreitol and 0.4 mM ATP). The ligation reaction product obtained in the above reaction is a circular DNA derived from the linearized pBR322 and HBV fragments joined together at their complementary ends.

The circular recombinant plasmids were introduced into

E. coli strain HB101 (ATCC 33694, provided by Dr. Dean Hunter at the National Cancer Institute, NIH, Bethesda, MD) following the procedure of Cohen et al. (1972), Proe. Nati. Acad. Sei., USA, 69: 2110-2114. Briefly, a culture of E. coli HB101 was incubated in a solution of 0.03 M CaCl3 at 0 o C for 20 minutes. About. 5 x 10 9 bacterial cells were added to 0.3 milliliters of the same solution to which was added 100 nanograms (ng) of recombinant plasmids. This transformation reaction mixture was incubated at 0°C for 60 minutes.

The plasmid pBR322 contains genes conferring ampicillin and tetracycline resistance. Drug-sensitive bacterial cells transformed with this plasmid exhibit ampicillin and tetracycline resistance. As pBR322 DNA has only one BamHI cleavage site which is on the tetracycline resistance gene, insertion of the HBV fragments into the BamHI site disrupts this gene. Drug-sensitive E. coli HB101 transformed with a plasmid derived from pBR322 with an HBV DNA fragment inserted into the BamHI site (cloning vector) therefore exhibits ampicillin resistance and tetracycline sensitivity.

Transformed E. coli, i.e. those containing the plasmid gene conferring ampicillin resistance, were selected by plating the transformation reaction mixture on a medium of LB nutrient agar (a culture medium containing per liter 10

g bactoagar, 10 g bactotryptone, 5 g bacto yeast extract and 5 g NaCl) containing 50 micrograms of ampicillin per milliliters. The plated E. coli was incubated at 37°C for 2 days.

From the colonies that showed ampicillin resistance after transformation using the above-mentioned method, tetracycline-sensitive colonies were selected by plating the colonies on an LB nutrient medium as indicated above, containing 50 micrograms of tetracycline per milliliters instead of ampicillin. These plated colonies were also incubated at 37°C for 2 days. Strains of E. coli HB101 containing pBR322 with HBV DNA fragments inserted into the BamHI cleavage site of the tetracycline resistance gene were thereby obtained.

3. Plasmid DNA extraction

The cells of E. coli HB101 containing the recombinant DNA isolated as indicated above were grown in the medium of LB nutrient fluid containing 20 micrograms/milliliter ampicillin, with the addition of chloramphenicol to a concentration of 170 micrograms/milliliter in the logarithmic growth phase. Cultivation was continued for several hours to increase the number of copies of plasmid DNA.

The cells were then lysed by mixing 5 milliliters of a solution of 25% sucrose in 50 mM Tris-HCl (pH 8.0) with 500 milliliters of the cells. After incubation for 10 minutes at 0°C, 1 milliliter of a 1% lysozyme in 0.25 M Tris-HCl (pH 7.5) solution was added. After incubation for 10 minutes at 0°C, 2 milliliters of 0.25 M EDTA (pH 8.0) was gently added. After incubation for 10 minutes at 0°C, 8 milliliters of a 1% solution of "Triton X-100" were added

(polyoxyethylene (9) octylphenyl ether) in 0.15 M Tris-HCl (pH 8.0), 0.2 M EDTA (pH 8.0) and re-incubated for 10 minutes at 0°C.

The resulting lysate was centrifuged at 30,000 rpm in a Spinco SW-65 centrifuge to remove denatured protein and cell debris. DNA was precipitated from the clarified lysate by mixing one-tenth volume of 2.5 sodium acetate and 2.5 volume of 99% ethanol, and incubating at -20°C for 30 minutes. The DNA precipitate was pelleted by centrifugation at 10,000 x g for 30 min.

Deproteinization was performed twice with 1 milliliter of phenol saturated with 0.01 M Tris-HCl (pH 7.6), 0.1 M NaCl, and 0.0012 M EDTA. Landers et al. (1977), J. Virol., 23: 368-376. The aqueous layer from the above procedure was taken out and DNA precipitated from this by adding a tenth volume of 2 M sodium acetate and 2.5 volume parts of 99% ethanol, and incubating at -20°C for 20 minutes. Centrifugation for 10 minutes at 10,000 x g yielded a pellet of complete double-stranded DNA comprising the gene encoding HBxAg.

4. Plasmid DNA analysis

The presence of HBV DNA in the clones was confirmed using the technique of Southern blotting and hybridization. Southern (1975), Mol. Biol., 98: 503. 10 milligrams of plasmid DNA from each clone isolated as above was resolved with BamHI (50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM dithiothreitol). The reaction mixture was electrophoresed in a 0.6% agarose horizontal plate gel at 100 amps for 2 hours (0.04 M Tris-acetate, 0.002 M EDTA).

DNA in the gel was denatured by soaking the gel in several volumes with 1.5 M NaCl and 0.5 M NaOH for 1 hour at room temperature with constant stirring or shaking. In some cases, DNA in the gel was hydrolyzed by acid wash before alkali denaturation by soaking the gel twice for 15 min in 0.25 M HCl at room temperature. After denaturation, the gel was neutralized by soaking in several volumes of a solution of IM Tris-HCl (pH 8.0) and 1.5 M NaCl for 1 hour at room temperature with constant shaking.

DNA in the gel was transferred onto nitrocellulose essentially as described in Maniatis et al. (1982), J. Molecular Cloning, Cold Spring Harbor. Briefly, nitrocellulose was placed on top of the gel, and several layers of Whatman 3MM paper were placed over the nitrocellulose. This "sandwich" was then placed gel side down on a Whatman 3MM wick with the ends submerged in a buffer containing 0.9 M NaCl and 0.09 M sodium citrate. The capillary movement of the buffer thereby transferred the DNA from the gel to the nitrocellulose. DNA was attached to the nitrocellulose by heating at 80°C for 2 hours under vacuum.

Plasmid DNA on the nitrocellulose prepared above (Southern filters) was tested for the presence of HBV DNA fragments using the method of Maniatis et al., supra.

Briefly, Southern filters were prehybridized by soaking in prehybridization fluid, 0.9 M NaCl, 0.09 M sodium citrate, 0.5% SDS (sodium dodecyl sulfate), 100 mg/ml denatured salmon sperm DNA, and 5X Denhardf's solution (containing 1 g Ficoll, 1 g polyvinylpyrrolidone and 1 g BSA fraction V per litre) for 4 hours at 68°C.

Hybridization was performed in a heat-sealable plastic bag with just enough hybridization solution to keep the Southern filter wet (50 microliters/cm 2 filter). Hybridization solution contained 0.9 M NaCl, 0.09 M sodium citrate, 0.01 M EDTA, 5X Denhardf's solution, 0.5% SDS, 100 micrograms/ml denatured salmon sperm DNA and 5 x 10<7>cpm of the radioactively labeled HBV DNA prepared above. The Southern filter was incubated in the hybridization solution for 12 hours at 68°C.

After washing the filter for 2 hours (0.3 M NaCl, 0.03

M sodium citrate), X-ray film (XRP-1) was exposed to the filter, whereby an autoradiographic image was obtained.

Three recombinant plasmids were isolated using the above procedure and designated AM6, AM7 and AMI. AM6 contained pBR322 DNA and the entire HBV genome. AM7 contained pBR322 DNA and a 1350 bp HBV DNA fragment. AMI contained pBR322 DNA and an 1850 bp HBV DNA fragment including the gene encoding HBxAg.

5. Construction of a replication plasmid containing

an SV40/HBxAg expression vector DNA

Defective SV40 virus obtained from Dean Hamer, Supra, was subjected to BamHI and EcoRI digestion in a buffer containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , and 1 mM dithiothreitol. Plasmid pBR322 DNA was subjected to the double resolution described earlier to form ends complementary to SV40 DNA cleaved in this manner. The cleavage products from each of these solutions were separated and purified by cesium chloride density gradient centrifugation. Tanaka et al. (1975), J. Bact., 121:354-362. The 4492 bp SV40 fragment and 3987 bp pBR322 fragment thus isolated were subjected to DNA ligation with T4 DNA ligase in a buffer containing 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl 2 , 10 mM dithiothreitol and 0.4 mM ATP, thereby generating pBRSV (Figure 4).

The ligation reaction product obtained with the above reaction is a circular DNA derived from the linearized pBR-322 3987 bp and SV40 4492 bp fragments ligated at their complementary EcoRI and BamHI ends. This circular plasmid was then linearized by cleavage at the BamHI restriction site formed during ligation, creating BamHI cohesive ends at each end.

An 1850 bp HBV DNA fragment comprising the gene encoding HBxAg was isolated by subjecting plasmid AM6 isolated above to resolution with BamHI in a buffer containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.5) , 10 mM MgCl2 and 1 mM dithiothreitol. The reaction mixture was electrophoresed at 100 amps for 2 hours using 0.8% agarose, and the 1850 bp band was excised and melted at 68°C for 15 minutes. A 1/10 volume of 3 M sodium acetate was added and the resulting solution was subjected to the deproteinization procedure described previously.

The HBxAg gene-containing HBV DNA fragment thus isolated had BamHI complementary ends. This DNA fragment was then ligated to the pBRSV prepared above using T4 DNA ligase in a buffer containing 66 mM Tris-HCl (pH 7.6), mM MgCl 2 , 10 mM dithiothreitol and 0.4 mM ATP.

The product of the above ligation reaction was a circular plasmid DNA designated SVAM191. It contained, in clockwise order, a 4492 bp SV40 DNA fragment from the BamHI site at base position 2468 to the EcoRI site at base position 1717, a 3987 bp pBR322 DNA fragment from the EcoRI site at base position 0 to BamHI the site at base position 375, and an 1850 bp HBV DNA fragment from the BamHI site at base position 1400 to the BamHI site at base position 28. Plasmid SVAM191 is shown schematically in figure 4.

SVAM191 was introduced into E. coli HB101 using the transformation procedure described above. As these transformed E. coli were also ampicillin resistant and tetracycline sensitive, they were subjected to the same isolation procedure described previously for the E. coli cloning vector. The procedures for preparation, isolation, purification and analysis described earlier were used to obtain milligram quantities of substantially pure SVAM191 DNA.

6. Construction of SV40/HBxAg expression vector

A vector capable of inducing the production of HBxAg in eukaryotic cells was made by removing part of the SVAM191 DNA. This was performed by digesting SVAM191 DNA with Haell endonuclease in a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 1 mM dithothreitol, to cleave SVAM191 in the Haell sites at base position 767 in SV40 -DNA region and at base position 1437 in the HBV DNA region.

The two DNA fragments obtained from the above reaction were separated using the electrophoretic agarose gel method described earlier. The thus isolated large fragment containing only SV40 and HBV DNA was made circular by subjecting its Haell complementary ends to the T4 DNA ligation procedure described previously.

The circular DNA expression vector generated above was designated SVHBV-3 (Figures 4 and 5). It contains expression control elements from SV40 and a gene that codes for HBxAg, from HBV. 7. Transfection of eukaryotic host cells with SVHBV-3 The BSC-1 African green monkey kidney cell line (ATCC CCL 26, obtained from Dr. G.B. Thornton, Johnson & Johnson Bio-technology Center. Inc., La Jolla, Calif.), a host that allows SV40, was selected for transfection with SVHBV-3. Confluent monolayer of approx. 10 7 cells were obtained by cultivation at 37°C in Eagle's minimal essential medium (EMEM) supplemented with 10% fetal calf serum, 100 units penicillin/ml, 100 micrograms/milliliter streptomycin and 3 mM L-glutamine. The monolayers were infected with vector SVHBV-3 DNA in the presence of DEAE-dextran as described by Ganem et al. (1976), J. Mol. Biol., 101: 57-83.

A flask was infected with 1.6 micrograms of the recombinant SVHBV-3 together with 0.05 micrograms of SV40-tsA239~<D>NA

which helps. Controls received equal amounts of the vector helper DNAs alone. The cultures were incubated at 40°C for 12 days and then lightened by freeze/thaw, and stored at -70°C.

Cellular proteins were extracted from the frozen/thawed cell powders obtained above by first adding 2 milligrams of cell powder to 1 milliliter of protein extraction buffer (2% SDS, 10% glycerol, 0.08 M Tris-HCl (pH 6.8),

2 mM phenylmethylsulfonyl fluoride, 0.1 M dithiothreitol,

0.001 percent bromophenol blue). The solution was then boiled in

5 minutes, centrifuged at 15,000 rpm for 10 minutes and the supernatants collected from this.

An amount of supernatant sufficient to yield 100 micrograms of sample protein was then subjected to SDS-polyacrylamide gel electrophoresis in the Western blot method described below.

8. Polypeptide syntheses

The polypeptides according to the invention were synthesized chemically using the solid phase method as described in Merrifield et al. (1963), J. Am. Ghem. Soc, 85: 2149; Merrifield et al. (1970), to Rev. Biochem., 39: 841-866 and Houghten et al.

(1980), Int. J. Peptide Prot.. Res., 16: 311-320. The relatively short polypeptides used here correspond to antigenic determinants in HBxAg. Figure 6 shows the 154 amino acid residue long sequence of HBxAg. The amino acid residue sequences of the synthetic polypeptides described here (99, 100 and 142) are also shown in Figure 6. The composition of both synthetic polypeptides was confirmed by amino acid analysis.

In general, an immunogenic or synthetic polypeptide is made by the steps of providing a large number of suitably protected amino acids corresponding to the amino acid residues of an antigenic determinant region of HBxAg, and synthesizing these amino acids into a polypeptide having an amino acid residue sequence corresponding to the polypeptide amino acid residue sequence of this antigenic determinant. The synthetic polypeptide produced can be used to prepare an inoculum, usually by binding it to a carrier, thereby forming a conjugate, and then dispersing an effective amount of the conjugate in a physiologically tolerable diluent.

The polypeptides are preferably synthesized according to the solid phase methods referred to above, using a cysteine resin. See Merrifield, et al., J. Am. Chem. Soc, supra. Using this method, the alpha-amino group of each added amino acid is usually protected by a tertiary butoxycarbonyl group (t-BOC) before the amino acid is added to the growing polypeptide chain. The t-BOC group is then removed before adding the next amino acid to the growing polypeptide chain. The side chains of some amino acids are protected as follows: Arg-tosyl; Ser-, Thr-, Glu- and Asp-O-benzyl; Tyr-O-bromobenzyloxycarbamyl; Trp-N-formyl; S-methoxybenzyl for cysteine; 2-chlorobenzoxycarbonyl for lysine and dinitrophenyl for histidine. When asparagine is used, an equal molar amount of N-hydroxy-benzotriazole is added together with the protected amino acid and dimethylformamide (DMF) is used as the coupling solvent. The N-formyl group on the Trp residues is removed after cleavage of the polypeptide from the resin carrier by treatment with 1.0 molar ammonium bicarbonate at a polypeptide concentration of 1.0 milligrams/milliliter for 16 hours at room temperature. Yamashiro et al. (1973), J. Org. Chem., 38: 2594-2597. The coupling efficiency in each step can be monitored with ninhydrin or picric acid, and is preferably greater than 99% in all cases. See Gisin (1972), Anal. Chem. Acta., 58: 248-249 and Kaiser (1980), Anal. Biochem., 34: 595-598.

After production of a desired polypeptide, a portion of the resulting protected polypeptide (approx. 1 gram) is treated with 2 milliliters of anisole, and anhydrous hydrogen fluoride, approx.

20 milliliters, is condensed in the reaction vessel at dry ice temperature. The resulting mixture is stirred at approx. 4°C

for about. 1 hour to deprotect and remove the polypeptide from the resin. After evaporating the hydrogen fluoride at a temperature of 4°C with a stream, the residue is extracted with anhydrous diethyl ether three times to remove the anisole, and the residue is dried under vacuum.

The vacuum-dried material is extracted with 5% aqueous acetic acid (3 times 50 milliliters) to separate the free polypeptide from the resin. The extract-containing solution is lyophilized, whereby a monomeric non-oxidized polypeptide is obtained.

As a generalized procedure for each polypeptide, briefly react 4 milligrams of KHL in 0.25 milliliters of 10 millimolar sodium phosphate buffer (pH 7.2) with 0.7 milligrams of MBS dissolved in DMF, and the resulting mixture is stirred for 30 minutes at room temperature. The MBS solution is added dropwise to ensure that the local concentration of DMF is not too high, as KLH is insoluble at DMF concentrations of approx. 30% or higher. The reaction product, KLH-MB, is passed through a chromatography column prepared with "Sephadex G-25" equilibrated with 50 millimolar sodium phosphate buffer (pH 6.0) to remove free MBS. KLH recovery from peak fractions in the column eluate, monitored at 280 nanometers, is typically about 80%.

The thus prepared KLH-MB is then reacted with 5 milligrams of polypeptide dissolved in 1 milliliter of buffer. The pH value of the resulting reaction mixture is adjusted to 7 - 7.5, and the reaction mixture is stirred at room temperature for 3 hours, whereby a conjugate of polypeptide and carrier is obtained.

9. "Western blotting"

The anti-polypeptide antibodies (anti-99, anti-100 and anti-142) were examined using the Western blot technique to confirm their predicted specificity for HBxAg, and to confirm the expression of the major polypeptide portion of HBxAg in transfected cells. The cellular proteins, including HBxAg, were separated by 12.5% SDS-polyacrylamide electrophoresis. See Laemmli (1979), Nature, 277: 680-685 and Towbin et al. (1979), Proe. Nati. Acad. Pollock. USA, 76: 4350-4354.

Proteins were electrophoretically transferred to nitrocellulose as described by Towbin et al., supra, using an electroblot apparatus with a buffer consisting of 25 mM Tris base, 192 mM glycine, 20% methanol, and 0.1% SDS (pH 8 ,3). After the transfer, the nitrocellulose was blocked in BLOTTO (Bovine Lacto Transfer Technique Optimizer, Johnson et al.

(1983), J. Exp. Med., 159: 1751-1756; 5% (w/v) non-fat dry milk, 0.01% "Anti-foam A" and 0.001% merthiolate in PBS at pH 7.2), to reduce non-specific binding. The blots were reacted with 100 microliters of antipeptide antibody in 10 milliliters of BLOTTO for 3 hours, and then washed three times for 1 hour with 50 ml of fresh BLOTTO.

Anti-polypeptide antibodies bound to vector-specific protein were detected by reacting the blots with 20 microliters of 125 I-labeled protein A from Staphylococcus aureus in 10 milliliters of BLOTTO for 1 hour. The spots were then washed in 50 milliliters of fresh BLOTTO for 15 minutes 4 times, and then under a continuous stream of water for 20 minutes.

125

10. I-markxng of

hepatoma cell extracts

Monolayers of the human hepatoma-derived cell line PLC/PRF/5 known to contain integrated sequences of HBV (Alexander et al. (1976), African Med. J., 50: 21-24; ATCC CRL 8024) were lyophilized. The cell powder was dissolved in phosphate-buffered saline (PBS), whereby a protein concentration of 1 milligram/milliliter was obtained. After centrifugation at 10,000 x g to remove cell debris, 50 μl of the above solution was mixed with 75 μl RIPA (0.15 M NaCl, 10 mM sodium phosphate (pH 7.5), 1% "Nonidet P-40", 0.5 % sodium deoxycholate, 0.1% SDS) and 20 microliters of 0.2 M sodium phosphate (pH 7.5). In an investigation, the solubilized cell protein was labeled with 5 millicuries of 125

In using the chloramine-T reaction. In the study shown in Figure 7, the cell lysates were labeled with 3 microCurie

125

of I using the same reaction.

The above reaction mixture was then passed through the Sephadex G-25 column washed with PBS. The obtained radioactively labeled peak fraction (9 x 10^ cpm/ml) was pre-incubated with . normal rabbit serum to remove non-specific binding. Briefly, 20 microliters of a top fraction was mixed with 1.0 ml RIPA, 20 microliters of "Trasylol"

(a trademark for a protein) and 100 microliters of NRS. After incubation for 1 hour at 0°C, 500 microliters of formalin-fixed cells of Staphylococcus aureus (Staph A) were incubated at 0°C for 30 minutes. The precipitate of Staph A was removed by centrifugation at 10,000 x g for 10 minutes and the supernatant was recovered.

11. Immunoprecipitations (IP)

Rabbit anti-polypeptide antiserum against polypeptide 99 (anti-99) was reacted with the cell proteins obtained above which were labeled with radioactive iodine. Briefly, 10 microliters of anti-99 was mixed with 2 x 10 cpm labeled extract and incubated for 1 hour at 0°C. 40 microliters of Staph A was then added and incubated for 30 minutes at 0°C. The precipitates of Staph A were removed by centrifugation at 10,000 x g for 10 minutes, and the pellet was recovered. The pellet was resuspended in 1 milliliter of RIPA and centrifuged as above. The resulting pellet was resuspended in 1.0 milliliters of LiCl solution (100 mM Tris-HCl, 500 mM LiCl), and then centrifuged again. The LiCl solution treatment was repeated and the pellet recovered.

The pellet obtained above was resuspended in 50 microliters of sample buffer and boiled for 3 minutes. The mixture was centrifuged at 10,000 x g for 1 minute and the supernatant recovered and electrophoresed on 12.5% SDS-polyacrylamide gel. The above gels were exposed to XRP-1 X-ray film, whereby an autoradiography was obtained.

12. Preparation and analysis of chimpanzee and human liver extracts

Liver samples from two chronically infected HBV chimpanzees and one human, and from normal chimpanzee and human livers (HBV-seronegative) were snap-frozen in liquid nitrogen and ground to a powder. The powders were mixed in sample buffer (0.0625 M Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue) and boiled for 5 minutes. The mixture was centrifuged at 10,000 x g for 30 min to remove cell debris. The samples were then subjected to "Western blot" analysis as described earlier using 75 micrograms of protein per gel field.

13. Identification of anti-HBxAg anti-

substances in human sera

20 micrograms per fields of SVHBV-3-transfected BSC-1 cell extracts were subjected to SDS-PAGE on 12% acrylamide gels, and the separated proteins were electrophoretically transferred to nitrocellulose sheets as described previously. The resulting nitrocellulose sheets were mixed with 1:50 dilutions of serum from six people who had been diagnosed as having HBV-related infections, including a symptomatic carrier and a patient with a hepatocellular carcinoma. Control sheets were mixed with rabbit anti-polypeptide antibodies according to the invention.

The nitrocellulose-protein-serum mixtures were stored

for 2 hours at room temperature. The sheets were then rinsed and mixed with a 1:200 dilution of goat anti-human or anti-rabbit antibodies linked to horseradish peroxidase, as appropriate. This mixture was kept for a period of 1 hour to allow bound anti-X antibodies to react with the correct anti-antibodies. The sheets were washed and then developed with 4-chloro-1-naphthol, as described previously. The serum from the hepatocellular carcinoma patient showed strong immunoreactivity with the approximately 24,000 dalton polypeptide expressed by the SVHBV-3-transfected cells.

14. Cell lines and tissue samples

PLC/PRF/5 and HepG2 cells were provided by Dr. D. Milich Department of Basic & Clinical Research, Scripps Clinic and Research Foundation (Scripps), La Jolla, CA. Chimpanzee liver tissue samples were provided by Dr. R. Purcell and P. Kaplan at the National Institute of Allergy and Infectious Diseases, Bethesda MD and Ortho Diagnostics, Inc., Raritan NJ, respectively. Human liver tissue samples were provided by Drs F. Chisari, J. Dienstag and A. Yu, Scripps, Department of Medicine, Harvard University, Boston, MA and Department of Pediatrics, University of California-San-Diego, La Jolla CA, respectively .

Examples

1. Production and application of diagnostic systems

for the detection of anti-HBxAg antibodies (HBxAb) Polypeptides 8, 42, 79, 99, 100 and 142 (here also referred to as p8, p42, p79, p99, pl00 and pl42) were synthesized by symmetrical anhydride chemistry on a Model 430A Applied Biosystems solid-phase peptide synthesizer according to the method of Hagenmeier et al., Hoppe-Seyler's Z. Physiol. Chem., 353: 1973-1976 (1972).

Each polypeptide was dissolved in deionized water

(pH 6) at a concentration of 1 milligram per milliliters (mg/ml). 100 microliters (µl) of 10 millimole (mM) sodium borate buffer, pH 9, containing 1 microgram (µg) of the polypeptides p8, p42, p79, p99, p100 or p142 were mixed into the wells of 96-well EIA microtiter plates. The plates were

then stored for approx. 16 hours at 37°C to allow the buffers to evaporate and the polypeptides to adsorb on (attach to) the walls of the wells. 300 microliters of T-wash (Tris-buffered saline: 50 mM Tris base, 150 mM NaCl, pH 7.6) containing 10% normal horse serum, 5% normal goat serum, 5% fetal calf serum and 0.05% "Tween 20", was then mixed in each well to block excess protein binding sites.

The wells were kept for 2 hours at 37°C, the blocking solution was removed by shaking and the wells were dried by keeping them for 1 hour at 37°C, thereby forming a diagnostic system according to the present invention, i.e. an HBxAg- related polypeptide-containing solid support (polypeptide-coated well).

The polypeptide-containing solid carriers were used to perform an ELISA assay for the presence and amount of HBxAb in several different human sera. 100 microliters of each serum diluted 1:50 in T-wash was mixed into a polypeptide-coated well. The resulting solid/liquid-phase immunoreaction mixture was kept at 37°C for 2 hours to allow formation of polypeptide-containing immunoreaction products. The wells were then rinsed 4 times with PBS containing 0.05% "Tween 20", followed by a final rinse in deionized water (pH 6), whereby any polypeptide-containing (solid phase) immunoreaction products were separated from unreacted human antibodies.

Three hundred µl of a horseradish peroxidase-labeled goat anti-human IgG diluted 1:5000 in T-wash was then added to each well. The resulting label reaction mixtures (the other solid/liquid phase mixtures) were then kept in 1

hour at 37°C to enable formation of polypeptide-containing (solid phase-bound) labeled immunoreaction product. The wells

was then washed as described previously to remove unreacted labeled antibody.

One hundred µl of o-phenylenediamine (OPD) was then added

in each well, whereby a development reaction mixture was formed. After holding the development reaction mixture for 30 minutes at approx. 20°C, 50 ul of 4N H2SO4 was added to each well to stop the development reaction, and the resulting

titer solutions were analyzed for absorbance at 490 nanometers (nm) using a microtiter plate reader.

2. ELISA assays to detect

the presence of HBxAb

A total of 130 serum samples were analyzed for the presence of HBxAb using the ELISA systems and methods described in Example 1. The samples were grouped according to the diagnosis of the patient at the time of collection. All serum samples from patients chronically infected with HBV (HBsAg positive) were from Tokyo, Japan. The panel of normal serum was obtained from the General Clinical Research Center (GCRC) Scripps Clinic, La Jolla, CA. Serum samples from acute phase hepatitis B were from both Tokyo and La Jolla. The serum samples were classified as acute hepatitis B [AH(B)], asymptomatic carrier (ASC), chronic hepatitis (CH), hepatocellular carcinoma (HCC), or normal.

Table 1 summarizes the results obtained with serum samples analyzed using each of the X polypeptides. A positive score was recorded if the sample was reactive with either one or more of the polypeptides.

^ Diagnosis of patient at time of sample collection for investigation: AH(B) = acute hepatitis B, ASC = asymptomatic carrier, CH = chronic hepatitis, HCC = hepatocellular carcinoma, Normal = no detectable time-

lower exposure to HBV (HbsAb-negative).

<2> Percentage of each category of sera that gave a positive result against at least one polypeptide.

As can be seen from Table 1, no HBxAb was detected in the 21 acute phase serum samples analyzed. Samples from the asymptomatic group and the group with chronic carriers contained approximately the same percentage of positive samples (26.9 and 30.7%, respectively). A significant number of samples (85.7%) from the hepatocellular carcinoma (HCC) group were found to contain HBxAb. The large number of positive samples in the HCC group was in agreement with previous investigations where eight out of eleven serum samples from patients with HCC were reactive with peptides 100-115 and 144-154 (Moriarty et al., Science, 227: 429-433 (1985 )). All samples that were positive for HBxAb were also HBsAg positive. HBeAg/HBeAb status had no influence on the predictability of HBxAb detection; approximately two thirds of the positive samples were HBeAb positive.

The absorbance values (A40,g) for positive samples are shown in Table 2 to illustrate the polypeptides to which the antibody response of each serum sample was directed.

Diagnosis of the patient at the time of sample collection:

ASC = asymptomatic carrier, CH = chronic hepatitis B,

HCC = hepatocellular carcinoma.

<2> Serum accession number.

<3> Polypeptide used as phase antigen in ELISA

^ Absorbance value obtained in ELISA measured at 49 0 nm.

Table 2 shows that the specificity of HBxAb in the ASC group was between amino acid residues 79 - 131 of the HBx protein, with a "hot spot" around the 7 9 - 99 region, i.e. polypeptide 79. Interestingly, serum samples from individuals who had signs on liver damage (CH and HCC groups), HBxAb with a specificity that covered the entire protein, i.e. that the majority of these samples were reactive with all six peptides. The significance of this finding is unclear, but suggests that HBxAg may exist in more than one conformation in the various disease states.

3. Analysis of serial samples of sera from

acute hepatitis B with regard to HBxAb

The lack of detectable HBxAb in a particular group of serum samples from patients with acute HBV infections [AH(B) in Table 1] prompted an investigation to determine whether the 26 analyzed samples from this group were taken at a time point early in the infection to detect HBxAb, or whether the appearance of antibody in reality reflects a chronic infection with HBV. Serial serum samples were obtained from four individuals acutely infected with HBV. The times for the samples began at the onset of symptoms and continued until HBsAb became apparent. The serial samples were examined for the presence of HBxAb by means of ELISA assays using the polypeptides 79, 99 and 100 as described in Example 1.

All four panels were negative with regard to HBxAb

at all stages of the acute infection. Although no significant HBxAb levels were detected, the classical markers of an acute HBV infection were observed, i.e. early detection of HBsAg, sera conversion to HBsAb within six months, with final viral shedding determined by the detection of HBeAb. Thus, although it is not known at this time whether the X protein is expressed during the replication stage of the virus, the above data suggest that the appearance of HBxAb is more closely associated with chronic HBV infection.

In these studies, HBsAg and HBsAb levels were determined by passive hemagglutination assays according to the method of Vyas et al., Science, 170: 332-333 (1970). HBeAg and HBeAb levels were determined using a commercially available kit. PreSAg levels were determined using an ELISA assay where a PreS2-specific monoclonal antibody was attached to microtiter plate wells (100 nanograms per well). The solid-phase bound monoclonal antibody was mixed and reacted with the serum samples to form an immunoreaction product which was detected using a horseradish peroxidase labeled anti-HBs monoclonal antibody using the conditions described in Example 1.

The above mentioned is intended to be illustrative of the present invention.

Claims (6)

1. Antigenic synthetic polypeptide, characterized in that it corresponds to an antigenic determinant in HBxAg, and which comprises an amino acid residue sequence selected from the group of polypeptides with the formulas, written from left to right and in the direction from the amino end to the carboxyl end, 2. Antibody, characterized in that it comprises an antibody f binding site which is able to react immunologically with an antigenic synthetic polypeptide according to claim 1. 3. Diagnostic analysis system for determining the presence of HBxAg in a body sample, characterized in that it comprises at least one container with a first reagent, where the first reagent comprises antibodies comprising an antibody binding site capable of reacting immunologically with HBxAg, and with an antigenic synthetic polypeptide corresponding to an antigenic determinant in HBxAg, and comprising an amino acid residue sequence selected from the group of polypeptides of the formulas, written from left to right and in the direction from amino terminus to carboxyl terminus, 4. Diagnostic analysis system according to claim 3, characterized in that it further comprises a second reagent in a second container, where the second reagent is the antigenic synthetic polypeptide according to claim 1 with which the antibody reacts immunologically. 5. Diagnostic analysis system according to claim 4, characterized in that it further comprises a marker for signaling the immunological reaction between the antibodies and HBxAG. 6. Use of an antigenic synthetic polypeptide according to claim 1 for in vitro diagnosis.