NZ528739A

NZ528739A - A mutant human hepatitis B viral strain and uses thereof

Info

Publication number: NZ528739A
Application number: NZ52873998A
Authority: NZ
Inventors: Chong Jin Oon; Gek Keow Lim; Yi Zhao; Wei Ning Chen
Original assignee: Government Of The Republic Of
Priority date: 1998-06-19
Filing date: 1998-06-19
Publication date: 2005-07-29

Abstract

An isolated strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-133 Oon Strain (Methionine to Threonine). Also, an isolated nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than methionine. Such nucleic acids are useful for determining whether a patient is infected with the said strain, wherein diagnosis involves testing a nucleic acid sample for the presence of a nucleic acid encoding a protein as discussed above. (62) Divided out of 508890

Description

Patents Form No. 5 Divisional Application From NZ 508890 OurRef: JC220192 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION A MUTANT HUMAN HEPATITIS B VIRAL STRAIN AND USES THEREOF We, GOVERNMENT OF THE REPUBLIC OF SINGAPORE, a body corporate organised under the laws of Singapore of Ministry of Health, College of Medicine Building, 18 College Road, Singapore 169854, hereby declare the invention, for which We pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: PT053818348 (Followed by Page 1a) 100253016 1 WO 99/66048 PCT/SG98/00046 la A MUTANT HUMAN HEPATITIS B VIRAL STRAIN AND USES THEREOF Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

This specification is a divisional specification filed out of NZ 508890.

BACKGROUND OF THE INVENTION The present invention concerns the human hepatitis B virus genome, isolated from hepatocellular carcinoma (HCC) , and with a mutation at amino acid residue 13 3 (Methionine to Threonine) within the major surface antigen, its nucleotide sequence, the deduced amino acid sequence of the four major proteins, antigen, antibody, detection systems, development of effective vaccines-, and antiviral agents.

HCC is one of the most common human liver cancers, particularly in Asia where 7 0% of the worldwide new cases are found every year. It usually arises in cirrhotic livers as a complication of chronic liver disease. The clinical manifestations of HCC patients are unspecific with signs and symptoms only appearing in the later stages of the cancer.

One of the major causes of chronic liver diseases is hepatitis B viral infection. First discovered in 1963 as a human virus that is transmitted parenterally, chronic hepatitis B viral infection has been most commonly implicated in serological undefined pathogenesis of HCC. Despite the fact that hepatitis B virus does not display features of a complete viral oncogene, its involvement in the development of HCC can be attributed to various aspects of its interaction with host hepatocyte cells. These include the promiscuous transcriptional activity of the smallest viral protein, X, which enhances the expression level of many cellular target genes including proto-oncogenes. On the other hand, integrated viral DNA in the host chromosomes is regularly found in HCC patients. There is also evidence for an active WO 99/66048 PCT/SG98/00046 2 role in the development of "HCC by the major surface antigen. This protein has served as the main detection marker for carriers of hepatitis B virus. The most antigenic epitope is a highly conserved region spanning 23 amino acid residues and 5 located from amino acid position 124 to 147 of the major surface antigen. This small region designated as the group specific determinant "a" is found in all subtypes and isolates of hepatitis B viral genomes. Its antigenic properties seem due to its proposed double loop structure, to which the 10 vaccine-induced neutralizing antibody binds.

Our epidemiological data indicate that the wild type major surface antigen has been found in most HCC patients. "Furthermore, observation indicates that several variants of 15 the major surface antigen from HCC patients may be involved in the pathogenesis of HCC. Direct sequencing analysis indicated that 24 out of 63 HCC patients (around 38%) carry various mutations in the "a" epitope of the major surface antigen. When both the wild type and. variant cases are combined, the 20 proportion of the variant virus carrying a mutation at amino acid residue 133, located in the first loop of the "a" epitope of the major surface antigen (Methionine to Threonine) , is as high as 12.7% in 63 HCC patients from the Southeast Asia region, and present in 5 local HCC cases. However, the same 25 mutation is found in only 2% of hepatitis B virus carriers in a random population (more than 10 0 cases). The significance of this variant at amino acid residue 13 3 is further strengthened by the fact that the proportion of variant virus at amino acid residue 145 (Glycine to Arginine) , Better known 30 as a vaccine-induced mutant and located in the second loop of the "a" epitope, remains constant at 8% in hepatitis B virus carriers in the random population sample.

Although this variant hepatitis B viral strain, carrying a 35 mutation at amino acid residue 13 3 (Methionine to Threonine) of the major surface antigen in HCC patients, may arise differently from those induced following vaccination (i.e. with a mutation at amino acid residue 145 of the major surface 3 antigen) , this strain shares similar characteristics in that both are stable and cases of vertical transmission of these strains have been reported, despite effective hepatitis B virus prophylaxis and hepatitis B immunoglobulin (HBIG) being used.

The emergence of the variant human hepatitis B virus, carrying mutations in the "a" epitope of the major surface antigen, in HCC is of concern. The high proportion of the mutant virus with a substitution at amino acid residue 13 3 of the major surface antigen is of particular interest as it may point to a close association with the pathogenesis of HCC. This correlation would therefore require the urgent development of specific detection systems as well as effective prophylactic and therapeutic vaccines and antiviral agents. Determination of the nucleotide sequence of this mutant virus constitutes the first step towards these aims and will certainly be helpful for developing the above-mentioned diagnostic and treatment schemes. 4 SUMMARY OF THE INVENTION This invention provides an isolated strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-' S' -5 133 Oon Strain (Methionine to Threonine) which constituent viral genome is deposited under Accession Nos. P97121501, P97121502 and P97121503 with the European Collection of Cell Culture on I5ch December 1997.

This invention further provides an isolated nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine rather than a methionine.

This invention further provides a method of producing the polypeptide and a method of obtaining the polypeptide in purified form so as to recover a purified polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than a methionine.

This invention further provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with 30 sequences of only the mutant viral strain of hepatitis B virus.

This invention further provides a method of obtaining antibodies to the polypeptide and to the antibodies produced.

This invention also provides use of the above-identified nucleic acid, polypeptide, peptide or antibody for determining whether a subject is infected with the above-identified viral strain.

This invention also provides use of the above-identified nucleic acid, polypeptide, peptide or antibody for determining whether a subject has a predisposition for hepatocellular carcinoma.

This invention also provides a vaccine for preventing and treating hepatocellular carcinoma, as well as, a vaccine for treating or preventing infection by the above-identified mutant strain of hepatitis B virus.

This invention also provides a method for identifying a chemical compound which is capable of treating or preventing hepatocellular carcinoma and compositions containing such compounds.

This invention also provides a method for identifying a chemical compound which is capable of treating or preventing infection by the above-identified mutant strain and compositions containing such compounds.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 Structure of the four open reading frames of human hepatitis B viral genome isolated from HCC (with a Methionine to Threonine mutation at amino acid residue 13 3 of the major surface antigen, as labeled by an asterisk) . The major viral proteins: DNA polymerase, large/middle/major surface antigen, precore, core and transactivating X are denoted as P, PreS l/PreS2/S, PreC, C and X respectively.

Figure 2 Figure 4 .

Figure 6 3:> Figure 7 Strategy of cloning and sequence determination of the same hepatitis B viral genome.

B virus, isolated from HCC and carrying a mutation at amino acid residue 133 (Methionine to Threonine) of the major surface antigen (SEQ I.D. No. 1) . The mutation is shown at nucleic acids numbered 551-553 .

De iced amino acid sequence of the DNA poiyn ;ase from the nucleotide sequence of Figure .(SEQ. I.D. No. 2) .

Deduced < . acid sequence of the large surface antigen n the nucleotide sequence of Figure 3 (SI D. No. 3) . The mutated amino acid residue (J^Jro T) is numbered 3 07 Deduced amino acid sequS protein from the nucleotide 3 (SEQ. I.D. No. 4). of the core of Figure Deduced amino acid sequence the trans-activating X protein from the nucl^^tide -o -> fS^IQ-- T FI TvTo c; 1 T Figure 9.

Figure 10 Figure 11.

Figure 12 75 Figure 13 initiation site of the coding region of DNA polymerase, at position 2307 of the viral genome and matches the coding strand (sense oligonucleotide) (SEQ.I.D.No.6).

Oligonucleotide sequence corresponding to ;ition 25 0 of the viral nucleotide sequence matches the complementary strand (anti-je oligonucleotide) (SEQ.I.D.No.7).

Oligoniic tide sequence corresponding to position ) of the viral nucleotide sequence and ma1 ls the coding strand (sense oligonucleotide) (SEQ.I.D.No.8).

Oligonucleotide sequence corresponding to the stop codon of tHk coding region of DNA polymerase, at posiMon 1623 of the viral genome and matches th^complementary strand (anti-sense oligonucleotide) (SEQ.I.D.No.9).

Oligonucleotide sequence corresponding to position 142 0 of the viral genome and matches the coding strand (sense oligonucleotide) (SEQ.I.D.No.10).

Oligonucleotide sequence correspondiUcr to position 2340 of the viral genome'and mat\ies the complementary strand (anti-serf! 8 DETAILED DESCRIPTION OF THE INVENTION Throughout this application, references to specific nucleotides are to nucleotides present on the coding strand of the nucleic acid. The following standard abbreviations are used throughout the specification to indicate specific nucleotides: C=cytosine A=adenosine T=thymidine G=guanosine The present invention provides the nucleotide sequence of human hepatitis B virus genome isolated from hepatocellular carcinoma (HCC), which carries a mutation at amino acid residue 13 3 (Methionine to Threonine) of the major surface antigen, consisting of 3215 nucleotides (Figure 3) coding for 4 overlapping viral proteins shown in Figure's 4-7.

The invention provides the amino acid sequences of the four major viral proteins, including the DNA polymerase, large/middle/major surface antigen, core and trans-activating X. These proteins can be produced using recombinant technology, and used in developing polyclonal or monoclonal antibodies.

The present invention also provides a human hepatitis B virus diagnostic system, specific for the mutation at amino acid residue 133 (Methionine to Threonine) of the major surface antigen, using the nucleotide or protein sequences, or antibodies described above.

This invention provides an isolated strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-13 3 Oon•Strain (Methionine to Threonine) which constituent viral genome is deposited under Accession Nos. P97121501, P97121502 and P97121503 .

This invention also provides an isolated nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than a methionine. In a specific embodiment, the polypeptide is being encoded by nucleotides 155 through 835 of the nucleic acid sequence designated SEQ. I.D. No. 1, comprising nucleotides "ACG" in position 551-553, instead of "ATG".

The isolated nucleic acid can be DNA or RNA, specifically cDNA or genomic DNA.

In another embodiment, the polypeptide has an amino acid sequence substantially identical to amino acid residues 174 through 400 of the amino acid sequence designated SEQ. I.D. No. 3.

This invention also provides an isolated nucleic acid which encodes el peptide/' wheirein the peptide is encoded by a nucleic acid molecule comprising nucleotides 527 through 595 of SEQ. I.D. No. 1.

This invention also provides an isolated nucleic acid which encodes a peptide, wherein the peptide has an amino acid sequence comprising amino acid residues 298 through 320 of the amino acid sequence designated SEQ. I.D. No. 3.

This invention further provides a vector- comprising an isolated nucleic acid encoding a peptide, wherein the peptide is encoded by a nucleic acid molecule comprising nucleotides 52 7 through 595 of SEQ. I.D. No. 1. ■ Specifically the vectors above comprise viral DNA.

This invention also provides a host vector system for the production of a polypeptide or peptide and comprises the 10 above-identified vectors in a suitable host.

Also in this invention, is a method of producing a polypeptide which comprises growing in the host vector systems described above, under suitable conditions permitting production of the 15 polypeptide or the peptide and recovering the polypeptide or peptide.

This Invention also provides a method of obtaining a polypeptide or peptide in purified form which comprises: 20 (a) introducing any of the above-describes vectors into a suitable host cell; (b) culturing the resulting host cell so as to produce the polypeptide; (c) recovering the polypeptide produced into step (b); and (d) purifying the polypeptide or peptide so recovered.

This invention also provides a purified polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an "amino acid sequence which differs from the amino acid sequence of a ma-jor surface 30 antigen of a wild type hepatitis 3 virus in that the amino acid at position number 133 of such-polypeptide is a threonine rather than a methionine. Specifically, one can obtain the purified polypeptide or peptide using the above methods.

This invention provides a purified peptide, wherein the peptide has' an amino acid sequence comprising amino acid residues 298 through 320 of the amino acid sequence designated SEQ. I.D. No. 3. wo 99/66048 11 This invention also provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides within a nucleic acid which encodes a polypeptide which is a mutant major surface antigen of a 5 strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than a methionine, without hybridizing to 10 any sequence of nucleotides within a - nucleic acid which encodes the major surface antigen of a wildtype hepatitis B virus. In an embodiment, the oligonucleotide comprises nucleotides 527 through 595 of SEQ. I.D. No. 1.

This invention also provides a composition capable of ^— stimulating or enhancing antibody production for the polypeptide.

This invention also provides a method of obtaining antibodies 20 to a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is 25 a threonine, rather than a methionine, and not to the major surface antigen of a wild type hepatitis B virus, comprising: (a) obtaining the polypeptide in a purified form; (b) immunizing an organism capable of producing antibodies against the purified polypeptide; (c) collecting the produced 30 antibodies; (d) combining the produced antibodies and the purified polypeptide under conditions to form a complex; and (e) determining which produced antibodies form a complex with the purified polypeptide so as to obtain antibodies to the polypeptide. In a specific embodiment, the polypeptide is 35 being encoded by nucleotides 155 through 83 5 of the nucleic acid sequence designated SEQ. I.D. No. 1. In another embodiment, the polypeptide has an amino acid sequence substantially identical to amino acid residues 174 through 400 12 of the amino acid sequence designated SEQ. I.D. No. 3.

One can obtain these antibodies from organism such as a rabbit or a mouse.

This invention also provides a method of obtaining antibodies to a peptide, wherein the peptide has an amino acid sequence comprising amino acid residues 298 through 32 0 of the amino acid sequence designated SEQ. I.D. No. 3, comprising: (a) 10 obtaining the peptide in a purified form; (b) immunizing an organism capable of producing antibodies against the purified peptide; (c) collecting the produced antibodies; (d) combining the produced antibodies and the purified peptide under conditions to form a complex; and (e) determining which 15 produced antibodies form a complex with the purified peptide so as to obtain antibodies to the peptide.

This invention also provides for the antibodies obtained using the methods described above, specifically when the antibodies 20 are monoclonal antibodies.

This invention also provides for antibodies capable of detecting a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide 25 having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine, and incapable of detecting the raa-j or surface 30 antigen of a wild type hepatitis B virus.

This invention further provides for antibodies capable of detecting a peptide, wherein the peptide has an amino acid sequence comprising amino acid residues 298 through 320 of the 35 amino acid sequence designated SEQ. I.D. No. 3.

This invention provides use * of a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a ;WO 99/66048 ;PCT/SG98/00046 ;13 ;strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino, acid at position, number 133 of such polypeptide is a threonine, rather than a methionine for determining whether a subject is infected with a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S' -13 3 Oon Strain (Methionine to Threonine), wherein such determination comprises: (a) obtaining an appropriate nucleic acid sample from the subject; and (b) determining whether the nucleic acid sample from step (a) is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine. ;In one embodiment, the nucleic acid sample in step (a) comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine, and wherein the determining of step (b) , which comprises: (i) contacting the mRNA with the oligonucleotide under conditions permitting binding of the mRNA to the oligonucleotide so as to form a complex; (ii) isolating the complex so formed; and (iii) identifying the mRNA in the isolated complex so as to thereby determine whether the mRNA is, or is derived from, a nucleic acid which encodes the polypeptide. ;In another embodiment, the nucleic acid sample in step (a) comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a ;WO 99/66048 ;PCT/SG98/00046 ;14 ;strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is 5 a threonine, rather than a methionine, and wherein the determining of step (b) comprises: (i) translating the mRNA under suitable conditions to obtain an amino acid sequence; and (ii) comparing the amino acid sequence of step (i) with the amino acid sequence encoded by the above-described 10 isolated nucleic acid so as to thereby determine whether the nucleic acid sample is, or is derived from, a nucleic acid which encodes the polypeptide. ;Further, one can determine step (b) by: (i) amplifying the 15 nucleic acid present in the sample of step (a); and {ii} detecting the presence of polypeptide in the resulting amplified nucleic acid. ;This invention also provides use of antibodies that recognize 20 a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is 25 a threonine, rather than a methionine for determining whether a subject is infected with a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S' -13 3 Oon Strain (Methionine to Threonine), wherein such determination comprises: (a) obtaining an appropriate sampl-e from the 30 subject; and (b) determining whether the sample from step (a) is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major 35 surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine by contacting the sample under appropriate conditions to bind to the antibodies ;WO 99/66048 ;PGT/SG98/00046 ;15 ;recognizing the polypeptide so as to determine whether a subject is infected. ;In the above-identified uses, the isolated nucleic acid, oligonucleotide or antibody may be labeled with a detectable marker. Examples of detectable markers include radioactive isotopes, fluorophors, and enzymes. ;In embodiments, the sample may comprise blood, tissue or sera. ;This invention provides use of a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine for determining whether a subject has a predisposition for hepatocellular carcinoma, wherein such determination comprises: (a) obtaining an appropriate nucleic acid sample from the subject; and (b) determining whether the nucleic acid sample from step (a) is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine. - ;In one embodiment, the nucleic acid sample in step (a) comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, and wherein the determining of step (b) , which comprises: (i) contacting the ;WO 99/66048 ;PCT/SG98/00046 ;16 ;mRNA with the oligonucleotide under conditions permitting binding of the mRNA to the oligonucleotide so as to form a complex; (ii) isolating the complex so formed; and (iii) identifying the mRNA in the isolated complex so as to thereby determine whether the mRNA is, or is derived from, a nucleic acid which encodes the polypeptide. ;In another embodiment, the nucleic acid sample in step (a) comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine, and wherein the determining of step (b) comprises: (i) translating the mRNA under suitable conditions to obtain an amino acid sequence; and (ii) comparing the amino acid sequence of step (i) with the amino acid sequence of the isolated nucleic acid which encodes a polypeptide, wherein the polypeptide has an amino acid sequence substantially identical to amino acids 174 through 4 00 of the amino acid sequence designated SEQ. I.D. No. 3, so as to thereby determine whether the nucleic acid sample is, or is derived from, a nucleic acid which encodes the polypeptide. ;In another embodiment, the determining of step (b) comprises: (i) amplifying the nucleic acid present in the sample of step (a); and (ii) detecting the presence of polypeptide in the resulting amplified nucleic acid. ;This invention further provides use of antibodies that recognize a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a ;WO 99/66048 ;PCT/SG98/00046 ;17 ;methionine for determining whether a subject has a predisposition for hepatocellular carcinoma, wherein such determination comprises: (a) obtaining an appropriate sample from the subject; and (b) determining whether the sample from step (a) is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine by contacting the sample under appropriate conditions to bind to the antibodies that recognize the polypeptide so as to determine whether a subject is-infected. ;In the above-described uses, the isolated nucleic acid, oligonucleotide or antibody may be labeled with a detectable marker. Examples of detectable markers include radioactive isotopes, fluorophors, and enzymes. ;In embodiments, the sample may comprise blood, tissue or sera. ;This invention provides a method for identifying a chemical compound for the manufacture of a medicament which is capable of treating infection by a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S' -13 3 Oon Strain {Methionine to Threonine) which comprises: (a) contacting a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such-polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, with the chemical compound under conditions permitting binding between the polypeptide and the chemical compound; (b) detecting specific binding of the chemical compound to the polypeptide;"and (c) determining whether the chemical compound inhibits the polypeptide so as to identify ;WO 99/66048 ;PCT/SG98/00046 ;18 ;a chemical compound which is capable of treating infection by the viral strain. ;This invention also provides a method' for identifying a chemical compound for the manufacture of a medicament which is capable of preventing infection by a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-13 3 Oon Strain (Methionine to Threonine), which comprises: (a) contacting a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine, with the chemical compound under conditions permitting binding between the polypeptide and the chemical compound; (b) detecting specific binding of the chemical compound to the polypeptide; and (c) determining whether the chemical compound inhibits the polypeptide so as to identify a chemical compound which is capable of preventing infection by the VI IT 3.1 st2rsiii- ;This invention also provides a method for identifying a chemical compound for the manufacture of a medicament which is capable of treating hepatocellular carcinoma which comprises: (a) contacting a polypeptide which is a mutant major, surface antigen of a strain of hepatitis B virus,, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of 5. wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, with the chemical compound under conditions permitting binding between the polypeptide and the chemical compound; (b) detecting specific binding of the chemical compound to the polypeptide; and' (c) determining whether the chemical compound inhibits the polypeptide so as to identify a chemical compound which is capable of treating infection by the viral strain. ;WO 99/66048 ;PCT/SG98/00046 ;19 ;This invention also provides a method for identifying a chemical compound for the manufacture of a medicament which is capable of preventing hepatocellular carcinoma, which comprises:(a) contacting a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, with the chemical compound under conditions permitting binding between the polypeptide and the chemical compound; (b) detecting specific binding of the chemical compound to the polypeptide; and (c) determining whether the chemical compound inhibits the polypeptide so as to identify a chemical compound which is capable of preventing infection by the viral strain. ;This invention also provides a composition comprising the chemical compound identified by the above-described methods in an amount effective to treat or prevent infection by the strain and a pharmaceutically effective carrier. ;This invention provides a composition comprising a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 13 3 of such polypeptide is a threonine, rather than a methionine, or derivative thereof, the amounts of such polypeptide being effective to stimulate or enhance antibody production in a subject, and a pharmaceutically acceptable carrier. ;The actual effective amount will be based upon the size of the polypeptide, the biodegradability of the polypeptide, the bioactivity of the polypeptide and the bioavailability of the polypeptide. If the polypeptide does not degrade quickly, is bioavailable and highly active, a smaller amount will be ;WO 99/66048 ;PCT/SG98/00046 ;20 ;required to be effective. The effective amount will be known to one of skill in the art; it will also be dependent upon the form of the polypeptide, the size of the polypeptide and the bioactivity of the polypeptide. Use of an adjuvant for 5 example, would lower the required amount of the polypeptide. One of skill in the art could routinely perform empirical activity tests to determine the bioactivity in bioassays and thus determine the effective amount. ;10 Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0. 1M and preferably Q.05M phosphate buffer or 0.8%- saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. 15 Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. 20 Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils . Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other 25 additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. ;This invention also provides a composition comprising a 30 peptide, wherein the peptide has an amino acid sequence comprising amino acid residues 298 through 320 of the amino acid sequence designated SEQ. I.D. No. 3. or derivative thereof, the amounts of such peptide being effective to stimulate or enhance antibody production in a subject, and a 35 pharmaceutically acceptable. ;This invention also provides compositions comprising the chemical compound identified by the above-described methods in ;WO 99/66048 ;PCT/SG98/00046 ;21 ;an amount effective to treat or prevent hepatocellular carcinoma and a pharmaceutically effective carrier. ;This invention also provides a composition comprising the chemical compound identified by the above-described methods in an amount effective to treat or prevent infection by a strain of hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-133 Oon Strain (Methionine to Threonine) and a pharmaceutically effective carrier. ;This invention further provides use of the above-identified compositions for treating a subject infected with a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-133 Oon Strain (Methionine to Threonine). ;This invention also provides use of the above-identified compositions for preventing infection by a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen's' -133 Oon Strain (Methionine to Threonine) in a subject. ;This invention also provides use of the above-described compositions for treating or preventing hepatocellular carcinoma. ;This invention also provides a method of screening bodily fluids from a subject for a strain of hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-' S'-133 Oon Strain (Methionine to Threonine) which comprises: (a) obtaining an appropriate sample of bodily fluid from the subject; (b) determining the presence of a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wild type hepatitis B virus in that the amino acid ■ at position number 133 of such polypeptide is a threonine, rather than a methionine in the sample of step (a) so as to screen the sample' for the strain. Specifically, wherein the bodily fluid comprises blood, sera, or a nucleic ;WO 99/66048 ;PCT/SG98/00046 ;22 ;acid sample of blood or sera. ;This invention further provides a method of treating a subject infected with this viral strain. ;This invention also provides a method of screening tissues and bodily fluids for this viral strain. ;This invention provides a hepatitis vaccine, comprising a mutant form of the surface antigen of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the wild type amino acid sequence of the major surface antigen of hepatitis B in that the amino acid at position number 13 3 of such polypeptide is a threonine rather than a methionine. ;This invention also provides the above-described vaccine, further comprising an adjuvant. ;This invention is illustrated in the Experimental Details section which follows. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter. ;WO 99/66048 ;PCT/SG98/00046 ;23 ;Experimental Details ;In the method described below, the human hepatitis B virus carrying the mutation at amino acid residue 13 3 (Methionine to 5 Threonine) of the major surface antigen was isolated, and its nucleotide sequence was determined. ;Serum sample (5194) was obtained from a S3-year old Chinese female patient of surface antigen carrier. She was confirmed 10 as a HCC patient by subsequent biopsy. The hepatitis B virus from her serum carried a mutation at amino acid.residue 133 (Methionine to Threonine) in the major surface antigen, as shown by previous sequencing analysis of the wa" epitope. Viral DNA was extracted prior to the determination of its 15 sequence in the present invention. ;As described in the examples below, the genome of this hepatitis B mutant virus from HCC and carrying a mutation at the amino acid residue 13 3 of the major surface antigen, 20 consists of 3215 nucleotides which is identical to that of the wild type virus of the same subtype (a.dr) . Open reading frames (ORF) coding for the major viral proteins are found at corresponding positions when compared to the wild type virus. Position 1 in the mutant hepatitis B virus genome is defined 25 according to.that in the wild type virus. ;The structure of the different ORFs in the mutant human virus genome are reported here and summarized in Fig. 1. The locations are indicated as follows: ;30 - DNA polymerase gene starts at position 2307 and ends at position 1623, therefore consisting of 2532 nucleotides and coding for 843 amino acid residues; ;- Large surface antigen gene starts at position 2848 and ends at position 835, therefore consisting of 1203 nucleotides and 35 coding for 400 amino acid residues. This large surface antigen overlaps the middle surface antigen which starts at position 3205 and the major surface antigen which starts at position 155. Both the middle (consisting of 281 amino acids ;WO 99/66048 ;PCT/SG98/00046 ;24 ;residues) and the major (consisting of 226 amino acid residues) surface antigens end at the same position as the large surface antigen; ;- Core gene starts at position 1814 and ends at position 2452, 5 therefore consisting of 63 9 nucleotides and coding for 212 ;amino acid residues; and ;- Trans-activating X gene starts at position 1374 and ends., at position 183 8, therefore consisting of 465 nucleotides and coding for 154 amino acid residues. ;iO ;Furthermore, sequence analysis has established this mutant hepatitis B virus belongs to the adr subtype as indicated by a lysine residue and an arginine residue at positions 122 and 16 0 respectively in the major surface antigen. Consistent 15 with previous analysis of the na" epitope by direct sequencing, the mutation (from Methionine to Threonine) is found at amino acid residue 133 of the major surface antigen. ;Compared with the wild type hepatitis B virus deposited in the 20 Genbank database (accession number D16665), the identity of this hepatitis B viral strain is at 90.3% for the nucleotide sequence. The identity' of different viral proteins of the present mutant hepatitis B virus as compared with its counterpart, the wild type virus, is 95.8%,97.5%, 95.1% and 25 94.8% for DNA polymerase (PIR - . Protein Identification Resources accession number S43491) , large surface antigen (PIR accession number JQ2107), core (PIR accession number S43490) and trans-activating X (PIR accession number S35529) proteins respectively. Conversely, numerous amino acid substitutions 30 are present in each of the viral proteins as compared to their wild type counterparts, these include: 5 mutations in DNA polymerase, 5 in large surface antigen (including the Methionine to Threonine change for the initiation codon of the major surface antigen) , 5 in core and 4 in trans-activating X 35 protein. ;The human hepatitis B virus" genome in the present invention, isolated from HCC and carrying mutation at amino acid residue ;WO 99/66048 ;PCT/SG98/00046 ;25 ;133 (Methionine to Threonine) of the major surface antigen, can be used as material to design oligonucleotides specific to the mutant virus genome. These oligonucleotides can be used as material for highly specific diagnostic agents that detect virus derived from HCC carrying a mutation at amino acid residue 133 of the major surface antigen. ;The human hepatitis B virus genome in the present invention, with a mutation at amino acid residue 133 (Methionine to Threonine) of the major surface antigen, can be used as material to produce proteins of the invention by expressing a vector that carries the relevant coding region, and which can replicate in a host cell such as Escherichia coli by standard DNA recombinant technology. ;Proteins of the present invention are useful as material for highly specific diagnostic agents capable of detecting hepatitis B virus from HCC, carrying a mutation at amino acid residue 13 3 (Methionine to Threonine) of the major surface antigen. Using known methods, these same proteins can be used to produce polyclonal and monoclonal antibodies. ;Polyclonal and monoclonal antibodies can be used as material for diagnostic agents to detect with high specificity antigens of hepatitis B virus, from HCC, with a mutation at amino acid residue 13 3 (Methionine, to Threonine) of the major surface antigen. ;A detection system using each protein of the present invention or proteins with partial replacement of amino acids, and a detection system using monoclonal or polyclonal antibodies to such proteins are useful as highly specific diagnostic agents capable of detecting of human hepatitis B virus from HCC to detect this virus from patients who are carriers of hepatitis ;* B surface antigen who may then be at risk of developing HCC. The proteins, or antibodies of such proteins can be used as a material for development of prophylactic and therapeutic vaccines against this viral strain. 26 It is well known that one or more nucleotides in a DNA sequence can be substituted by other nucleotides to produce the same protein. The present invention also concerns such nucleotide changes which code for proteins reported in this invention. It is equally well known that one or more amino acids in a protein sequence can be replaced by other analogous amino acids, as defined by their hydrophilic properties or charges, to produce an analog of the amino acid sequence. Any analogs of the proteins of the present invention involving amino acid replacement, deletions, isosteres (modified amino acids that bear close structural and spatial similarity to protein amino acids) , or additions, can be utilized, provided that the resulting sequences elicit antibodies recognizing hepatitis B virus from HCC with a mutation at amino acid mutation 133 (Methionine to Threonine) of the major surface antigen.

EXAMPLES The nucleotide sequence and the deduced amino acid sequence of human hepatitis B virus, isolated from HCC and carrying a mutation at amino acid residue 133' (Methionine to Threonine) of the major surface antigen, were determined in the following manp s 2r ^ 1. Isolation of Viral DNA The viral DNA was isolated from a serum sample (5194) obtained from a 63-year old Chinese female patient of surface antigen carrier. She was confirmed as an HCC patient by biopsy. The hepatitis B virus from her serum carried a mutation at amino acid residue 133 (Methionine to Threonine) in the major surface antigen, as shown by our previous sequencing analysis o^ the "a" epitope.

The isolation method used was: 200 /ii of the serum sample was added with 400 ^ 1 of the lysis buffer (Tris chloride 10 mM, pH7.4, EDTA 1 mM, and sodium 27 dodecyl sulfate 2%) and 25 fil of proteinase K (20 mg/ml) , 0 incubated at 65 C for 3 hours. Viral DNA was then extracted by phenol/chloroform and precipitated by ethanol. 2. Amplification of Viral DNA by Polymerase Chain Reaction (PCR) The virus genome in the present invention was amplified by polymerase chain reaction (PCR) using 3 sets of overlapping oligonucleotides, which were designed according to the wild type hepatitis B virus. Various restriction enzyme sites were included to facilitate the cloning of the PCR products. The position of these oligonucleotides is shown in Fig. 2 and indicated as follows: - Flag 1 (ATAAGCTTATGCCCCTATCTTATCAACACTTCCGGA) (SEQ. I.D. No. 6) starts at the initiation site of the coding region of DNA polymerase, at position 23 07 of the viral nucleotide sequence and matches the coding strand-(sense oligonucleotide). An additional HindiII restriction enzyme site is underlined; - Xba3 (GAGTCTAGACTCTGCGGTATTGTGA) (SEQ. I.D. No. 7) starts at the internal restriction enzyme site Xbal, at position 25 0 of the viral nucleotide sequence and: matches the complementary strand (anti-sense oligonucleotide). An additional Xbal restriction enzyme site is underlined; - Xba5 (GAGTCTAGACTCGTGGTGGACTTCT) (SEQ. I.D. No. 8) starts at the internal Xbal site, at the same location as that of Xba3 oligonucleotide but matches the coding strand (sense oligonucleotide). An additional Xbal restriction enzyme site is underlined; - Common 3 (TGAGAATTCTCACGGTGGTCTCCATGCGACGT) (SEQ. I.D. No. 9) starts at the stop codon of the DNA polymerase, at position 1623 of the viral nucleotide sequence and matches the complementary strand (anti-sense oligonucleotide). An additional EcoRI restriction enzyme site is underlined; - Vll (TTTGTTTACGTCCCGT) (SEQ. I.D. No. 10) starts near the initiation site of the X gene, at position 1420 of the viral nucleotide sequence and matches the coding strand (sense oligonucleotide); 28 - Hindi IIADW3 (CTAAGCTTAGTTTCCGGAA.GTGTTGAT) (SEQ. I.D. No . 11) starts close to the initiation site of the DNA polymerase, at position 2340 and matches the complementary strand (anti-sense oligonucleotide). An additional Hind III restriction enzyme site is underlined.

Using viral DNA as template, PCR was then carried out on a DNA Thermal Cycler (Perkin-Elmer. Cetus) for 35 cycles using Pfu polymerase (Stratagene, U.S.A.), each cycle consisting of 1.5 minutes at a denaturing temperature of 94° C, 2 minutes at an o annealing temperature of 53 C and 4 minutes at an extension temperature of 72° C. The following combinations of oligonucleotides were used: Flagl/Xba3, Xba5/Common3 and Vll/HindIIIADW3, and generated amplification products of 1.2 kb, 1.4 kb and 1.1 kb respectively. 3. Cloning of the Amplified Viral DNA Fragments The amplified viral DNA fragment from Flagl/Xba3 (1.2 kb) was subjected to restriction enzyme digestion by Hindlll and Xbal, prior to cloning in a BlueScript plasmid pre-treated by the same restriction enzymes. Similar digestion with Xbal and EcoRI was applied to PCR product from Xba5/Common3 (1.4 kb) , prior to cloning in a BlueScript plasmid pre-treated by Xbal and EcoRI. On the other hand, the DNA fragment amplified with Vll and HindIIIADW3 (1.1 kb) was directly cloned into a ZeroBlunt plasmid, developed by InvitroGen (U.S.A.) for cloning blunt-end DNA fragments. 4. Determination of Nucleotide Sequence Nucleotide sequence of the human hepatitis B virus isolated from HCC and reported in the present invention was determined on plasmid DNA template by chain-terminating inhibitors, using the Sequenase DNA Sequencing Kit (United States Biochemical Corp.). To facilitate the sequencing procedure, various internal oligonucleotides were designed (from VI to V16) according to the wild type hepatitis B virus, and their 29 positions are indicated in Figure 2.

From the analysis described above, the full-length nucleotide sequence of the hepatitis B virus, isolated from HCC and 5 carrying a mutation at amino acid residue 133 (Methionine to Threonine) of the major surface antigen, was determined as shown in Figure 3. The deduced amino acid sequences coding for the major viral proteins are shown in Figures 4-7: hepatitis B viral DNA polymerase (Figure 4) , the large surface 10 antigen, including the middle and major surface antigen (Figure 5) , the core protein (Figure 6) and the trans-activating X protein (Figure 7).

Alignment of the virus sequence in the present invention with 15 other hepatitis B viral sequences, available in the Genbank database, will point to specific sequence differences which in turn can be used to design DNA probes. A detection system using polymerase chain reaction (PCR) can then be developed. Such PCR reactions will involve combinations of 20 oligonucleotides specific to human hepatitis B virus in the present invention, with a mutation at the amino acid residue 13 3 (Methionine to Threonine) of the major surface antigen, thereby allowing highly specific detection of these mutant viral DNA. Briefly, viral DNA can be extracted as described 25 in this invention. PCR. reaction can be performed using specific oligonucleotides using the similar cycling conditions described above. Results can then be analyzed after resolving PCR products on a 1% agarose gel.

According to known immunological procedures, it is possible to determine epitopes from protein sequences as those in Figures 4-7. Determination of these epitopes specific to human hepatitis B virus, from HCC and carrying a mutation at amino acid residue 133 (Methionine to Threonine) of the major 35 surface antigen, will allow synthesis of peptides using known genetic engineering methods , synthesis of proteins, production of antibodies specific to" these epitopes, development of specific diagnostic reagents, development of prophylactic and therapeutic vaccines, and antiviral agents.

A detection system for antibody against hepatitis B virus isolated from HCC and carrying a mutation at amino acid 5 residue 133 (Methionine to Threonine) of the major surface antigen can be'developed using polyvinyl microtiter plates and the sandwich method. Briefly, 5 0 fil of 5 p.g/ml concentration of a peptide from the hepatitis B virus in the present invention, isolated from HCC and carrying a mutation at amino 10 acid residue 133 (Methionine to Threonine) of the major surface antigen, can be dispensed in each well of the microtiter plates and incubated overnight at room temperature for consolidation. A similar procedure can be applied to a protein purified from host cells such as Escherichia coli. 15 The microplate wells can be washed five times with physiological saline solution containing 0.05% Tween 20. For overcoating, 100 fil of NaCl buffer containing 3 0% (v/v) of calf serum and 0.05% Tween 20 (CS buffer) can be dispensed in each well and discarded after incubation for 3 0 minutes at 20 room temperature.

To determine antibodies specific for the mutant (Methionine to Threonine at amino acid residue 133 of the major surface antigen) hepatitis B virus in serum, the primary reaction can 25 be carried out such that 5 0 p.1 of the CS buffer and 10 y1 of a serum sample can be dispensed in each microplate well and incubated on a microplate vibrator for one hour at room temperature. After completion of the primary reaction, microplate wells are washed five times as described above.

In the secondary reaction, 1 ng of horseradish peroxidase labeled anti-human IgG mouse monoclonal antibodies dissolved in 5 0 /zl of calf serum can. be dispensed in each microplate well and incubated on a microplate vibrator for one hour at 35 room temperature. Upon completion,- wells can be washed five times in the same way. After addition of hydrogen peroxide (as substrate) and 50 fil of "O-phenylenediamine solution (as color developer) in each well, and after incubation for 3 0 31 minutes at room temperature, 5 0 fil of 4M sulphuric acid solution can be dispensed in each well to stop further color development and for reading absorbance at 490nm.

The present invention makes possible detection of the mutant human hepatitis B virus, in particular those carrying a mutation at amino acid residue 133 (Methionine to Threonine) of the major surface antigen. The present invention also provides detection systems capable of a highly specific and sensitive detection at an early stage of infection or development of HCC when the HCC may be treated and cured.

In addition, these features allow accurate diagnosis of patients at an early stage in the HCC and also help to inhibit with higher efficiency mutant hepatitis B virus.

Proteins and their antibodies under the present invention can be utilized for development of prophylactic and therapeutic vaccines, as well as, immunological pharmaceuticals. Sequence information on structural genes of these mutant viruses will be helpful for developing detection systems of the relevant protein antigens and antibodies.

Antigen-antibody complexes can be detected by known methods. Specific monoclonal and polyclonal antibodies can be raised by immunizing animals such as mice and rabbits with peptides or proteins specific to mutant hepatitis B viruses from HCC. Inhibitory antiviral agents can be designed and targeted against these proteins and molecules in cell culture or in vivo.

The present invention is based on studies on human hepatitis virus genome isolated from HCC and carrying a mutation at amino acid residue 133 (Methionine to Threonine) of the major surface antigen. The invention makes possible highly specific detection of these mutant hepatitis B virus from HCC, and provides material such as protein, polyclonal and monoclonal antibodies for development of such detection system. 32 REFERENCES 1. Oon, C-J. , "Viral hepatitis B in Singapore.- epidemiology, prevention and control-monitoring the hepatitis B programme and management of carriers" J. Roval Collecre Physic. London (1997), in press. 2. Ogata, N. , et al. "Infectivity and pathogenicity in chimpanzees of a surface gene mutant of hepatitis B virus that emerged in a vaccinated infant" J. Infec. Disease (1997) 175: 511-523. 3. Oon, C-J., "Issues associated with HBV mutant strains" J. Royal College Physic. London (1997), in press. 4. Oon, C-J., et al. "Hepatitis B surface antigen (HBsAg) mutants - their significance" Annals Acad. Med. Singapore (1997), in press. 5. Tsai, J.F., et al. "Additive effect modification of hepatitis B surface antigen and e antigen on the development of hepatocellular carcinoma" Brit. J. Cancer (1996) 73: 1498-1502. 6. Oon, C-J., "Molecular epidemiology of hepatitis B 'a' variants and mutants: significance in immune population" JAMA (1996) 12: pp.5-6. 7. Goh, K-T, "Hepatitis B immunization in Singapore" Lancet 0 (1996) 348: 1385-1385. 8. Oon, C-J., et al. , "Natural history of hepatitis B surface antigen mutants in children" Lancet. . (1996) 34 8: 1524-1525. 9. Harrison, T.J., "Genetic variation in hepatitis B virus" Eur. J. Gastroenter. & "Hepatol. (19 9 6) 8 : pp.306-311. 33 . Oon, C-J., et al. , "Molecular epidemiology of hepatitis B virus vaccine variants in Singapore" Vaccine (1995) 13 : 699-702. 11. Oon, C-J., et al. "Molecular epidemiology of hepatitis B variants and mutants - significance and transmissibility" Proc.3nd Internatl. Svmp. Viral Hepatitis & HCC. Singapore (1995) pp.39-43. 12. Carman, W. , et al. , "Viral genetic variation: hepatitis B virus as a clinical example" Lancet (1993) 341: 349-353 . 13". Harrison, T. J. , "Variants of hepatitis B virus" Vox Sang (1992) 63: 161-167. 14. Carman, W.F., et al. , "Vaccine-induced escape mutant of hepatitis B virus" Lancet (1990) 336: 325-329. 34 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) TITLE OF INVENTION: A MUTANT HUMAN HEPATITIS B VIRAL STRAIN AND USES THEREOF (ii) NUMBER OP SEQUENCES: 11 (2) INFORMATION FOR SEQ ID NO: 1: (Figure 3) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3215 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CTCCACAACA TTCCACCAAG CTCTGCTAGA TCCCAGGGTG AGGGGCCTAT ATTTTCCTGC 60 TGGTGGCTCC AGTTC CGGAA CAGTAAACCC TGTTCCGACT ACTGCCTCTC CCATATCGTC 120 AATCTTCTCG AGGACTGGGG ACCCTGCACC GAACATGGAG AACACAACAT CAGGATTCCT 180 AGGACCCCTG CTCGTGTTAC AGGCGGGGTT f TTCTCGTTG ACAAGAATCC TCACAATACC 240 GCAGAGTCTA GACTCTGGTG GACTTCTCTC AATTTTCTAG GGGGAGCACC CACGTGTTCC 300 TGGCCAAAAT TCGCAGTCCC CAACCTCCAA TCACTCAC CA ACCTCTTGTC CTCCAATTTG 360 TCCTGGCTAT CGCTGGATGT GTCTGCGGCG TTTTAT CATA TTCCTCTTCA TCCTGCTGCT 420 ATGCCTCATC TTCTTGTTGG TTCTTCTGGA CTACCAAGGT ATGTTGCCCG TTTGTCCTCT 480 ACTTCCAGGA ACATCAACCA C CAGCACGGG GCCATGCAAG ACCTGCACGA CTCCTGCTCA 540 AGGAAACTCT ACGTTTCCCT CTTGTTGCTG TACAAAACCT TCGGACGGAA ACTGCACTTG 600 TATTCCCATC CCA.TCATCCT GGGCTTTCGC AAGATTCCTA TGGGAGTGGG CCTCAGTCCG 660 TTTCTCCTGG CTCAGTTTAC TAGTGCCATT TGTTCAGTGG TTCGTAGGGC TTTCCCCCAC 720 TGTTTGGCTT TCAGTTATAT GGATGATGTG GTATTGGGGG CGAAGTCTGT ACAACATCTT 780 GAGTCCCTTT TTACCTCTAT TACCAATTTT CTTTTGTCTT TGGGTATACA TTTAAACCCT 840 AATAAAACCA AACGTTGGGG CTACTCCCTT AACTTCATGG GATATGTAAT TGGAAGTTGG 900 GGTACTTTAC CGCAGGAACA TATTGTACTA AAACTCAAGC AATGTTTTCG AAAACTGCCT 960 GTAAATAGAC CTATTGATTG GAAAGTATGT CAAAGAATTG TGGGTCTTTT GGGCTTTGCT 1020 GCCCCTTTTA CACAATGTGG CTATCCTGCC TTGATGCCTT TATATGCATG TATACAATCT 1080 AAGCAGGCTT TCACTTTCTC GCCAACTTAC AAGGCCTTTC TGTGTAAACA ATATCTGAAC 114 0 CTTTACCCCG TTGCCCGGCA ACGGTCCGGT CTCTGCCAAG TGTTTGCTGA CGCAACCCCC 1200 ACTGGATGGG GCTTGGCCAT AGGCCATCAG CGCATGGCTG GAACCTTTCT GGCTCCTCTG 1260 CCGATCCATA CTGCGGAACT CCTAGCAGCT TGTTTTGCTC GCAGCCGGTC TGGAGCAAAA 1320 CTTATCGGAA CCGACAACTC TGTTGTCCTC TCTCGGAAAT ACACCTCCTT TCCATGGCTG 1380 CTAGGGTGTG CTGCCAACTG GATCCTGCGC GGGACGTCCT TTGTCTACGT CCCGTCGGCG 1440 CTGAATCCCG CGGACGACCC GTCTCGGGGC CGTTTGGGGC TCTACCGTCC CCTTCTTCAT 1500 CTGCCGTTCC GGCCGACCAC GGGGCGCACC TCTCTTTACG CGGTCTCCCC GTATGTGCCT 1560 TCTCATCTGC CGGACCGTGT GCACTTCGCT TCACCTCTGC ACGTCGCATG GAGACCACCG 162 0 TGAACGCACG CCAGGTCTTG CCCAAGGTCT TATATAAGAG GACTCTTGGA CTCTCAGCAA 168 0 TGTCAACGAC CGACCTTGAG GCATACTTCA AAGACTGTGT GTTTAAAGAC TGGGAGGAGT 1740 TGGGGGAGGA GATTAGGTTA AAGATTTATG TACTAGGAGG CTGTAGGCAT AAATTGGTCT 1800 GTTCACCAGC ACCATGCAAC TTTTTCTCCT CTGCCTAATC ATCTCATGTT CATGTCCTAC 1860 TGTTCAAGCC TCCAAGCTGT GCCTTGGGTG GCTTTGGGAC ATGGACATTG ACCCGTATAA 1920 AGAATTTGGA GCATCTGCTG AGTTACTCTC TTTTTTGCCT TCTGACTTCT TTCCGTCTAT 1980 TCGAGATCTC CTCGACACCG CCTCTGCTCT GTATCGGGAG GCCTTAGAGT CTCCGGAACA 2040 TTGTTCGCCT CACCATACAG CACTCAGGCA AGCTATTTTG TGTTGGGGTG AGTTGATGAA 2100 TCTGGCCACC TGGGTGGGAA GTAATTTGGA AGATCCAGCA TCCAGGGAAT TAGTAGTCAG 216 0 CTATGTCAAC GTTAATATGG GCCTAAAACT CAGACAAATA TTGTGGTTTC ACATTTCCTG 2220 TCTTACTTTT GGAAGAGAAA CTGTTCTTGA GTACTTGGTA TCTTTTGGAG TGTGGATTCG 2280 CACTCCTACC GCTTACAGAC CACCAAATGC CCCTATCTTA TCAACACTTC CGGAAACTAC 2340 TGTTGTTAGA■CGACGAGGCA GGTCCCCTAG AAGAAGAACT CCCTCGCCTC GCAGACGAAG 2400 GTCTCAATCG CCGCGTCGCA GAAGATCTCA ATCTCGGGAA TCTCAACGTT AGTATTCCTT 2460 GGACTCATAA GGTGGGAAAC TTTACTGGGC TTTATTCTTC TACTGTACCT GTCTTTAATC 2520 CCGAGTGGCA AATTCCTTCC TTTCCTCACA TTCATTTACA AGAGGACATT ATTAATAGAT 2580 GTCAACAATA TGTGGGCCCT CTTACAGTTA ATGAAAAAAG AAGATTAAAA TTAATTATGC 264 0 CTGCTAGGTT TTATCCTAAC CTTACTAAAT ATTTGCCCTT AGACAAAGGC ATTAAACCGT 2700 ATTATCCTGA ACATGCAGTT AATCATTACT TCAAAACTAG GCATTATTTA CATACTCTGT 2760 36 GGAAGGCTGG CATTCTATAT AAGAGAGAAA CTACACGCAG CGCCTCATTT TGTGGGTCAC 282 0 CATATTCTTG GGAACAAGAG CTACAGCATG GGAGGTTGGT CTTCCAAACC TCGAGAAGGC 288 0 ATGGGGAGCA ATCTTGCTGT TCCCAATCCT CTGGGATTCT TTCCCGATCA CCAGTTGGAC 294 0 CCTGCGTTCG GAGCCAACTC AAACAATCCA GATTGGGACT TCAACCCCAA CAAGGATCAC 3000 TGGCCAGAGG CAAATCAGGT AGGAGTGGGA GCATTCGGGC CAGGGTTCAC CCCACCACAC 3060 GGCGGTCTTT TGGGGGGGAG CCCTCAGGCT CAGGGCATAT TGACAACAGT GCCAGCAGCA 3120 CCTCCTCCTG CCTCCACCAA TCGGCAGTCA GGAAGACAGC CTACTCCCAT CTCTCCACCT 318 0 CTAAGAGACA GTCATCCTCA GGCCACGCAG TGGAA 3215 (2) INFORMATION FOR SEQ ID NO: 2: (Figure 4) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 843 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO-.2: Met Pro Leu Ser Tyr Gin His Phe Arg Lys Leu Leu Leu Leu Asp Asp 15 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly 2 0 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val 35 40 45 Ser He Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gin He Pro Ser Phe Pro 65 70 75 80 His lie His Leu Gin Glu Asp lie lie Asn Arg Cys Gin Gin Tyr Val 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu lie Met Pro 100 105 110" Ala Arg Phe Tyr Pro Asn Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gly 115 120 125 lie Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Lys Thr 130 135 140 37 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly lie Leu Tyr Lys Arg 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 165 170 175 Gin Glu Leu Gin His Gly Arg Leu Val Phe Gin Thr Ser Thr Arg His 180 185 190 Gly Asp Glu Ser Cys Cys Ser Gin Ser Ser Gly lie Leu Ser Arg Ser 195 200 205 Pro Val Gly Pro Cys Val Arg Ser Gin Leu Lys Gin Ser Arg Leu Gly 210 215 220 Leu Gin Pro Gin Gin Gly Ser Leu Ala Arg Gly Lys Ser Gly Arg Ser 225 230 235 240 Gly Ser lie Arg Ala Arg Val His Pro Thr Thr Arg Arg Ser Phe Gly 245 250 255 Gly Glu Pro Ser Gly Ser Gly His lie Asp Asn Ser Ala Ser Ser Thr 260 265 270 Ser Ser Cys Leu His Gin Ser Ala Val Arg Lys Thr Ala Tyr Ser His 275 280 285 Leu Ser Thr Ser Lys Arg Gin Ser Ser Ser Gly His Ala Val Glu Leu 290 295 300 His Asn lie Pro Pro Ser Ser Ala Arg Ser Gin Gly Glu Gly Pro lie 305 310 315 320 Phe Ser Cys Trp Trp Leu Gin Phe Arg Asn Ser Lys Pro Cys Ser Asp 325 330 335 Tyr Cys Leu Ser His lie Val Asn Leu Leu Glu Asp Trp Gly Pro Cys 340 345 ' 350 Thr Glu His Gly Glu His Asn lie Arg lie Pro Arg Thr Pro Ala Arg 355 360 .365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala 370 375 380 Glu Ser Arg Leu Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro 385 390 3 95 400 Thr Cys Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gin Ser Leu Thr 405 410 ' "415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala 420 425 430 Ala Phe Tyr His lie Pro Leu His Pro Ala Ala Met Pro His Leu Leu 435 440 445 38 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Thr 450 455 460 Ser Arg Asn lie Asn His Gin His Gly Ala .Met Gin Asp Leu His Asp 465 470 475 480 Ser Cys Ser Arg Lys Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Thr 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro lie lie Leu Gly Phe 500 505 510 Arg Lys lie Pro Met Gly Val Gly .Leu Ser Pro Phe Leu Leu Ala Gin 515 520 525 Phe Thr Ser Ala He Cys Ser Val Val Arg Arg Ala Phe Pro His Cys 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Val 545 550 555 560 Gin His Leu Glu Ser Leu Phe Thr Ser He Thr Asn Phe Leu Leu Ser 565 570 575 Leu Gly lie His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Ser 580 585 590 Leu Asn Phe Met Gly Tyr Val lie Gly Ser Trp Gly Thr Leu Pro Gin 595 600 605 Glu His lie Val Leu Lys Leu Lys Gin Cys Phe Arg Lys Leu Pro Val 610 ' 615 " 620 Asn Arg Pro He Asp Trp Lys Val Cys Gin Arg lie Val Gly Leu Leu 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gin Cys Gly Tyr Pro Ala Leu Met Pro 645 650 655 Leu Tyr Ala Cys lie Gin Ser Lys Gin Ala Phe Thr Phe Ser Pro Thr 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gin Tyr Leu Asn Leu Tyr Pro Val Ala 675 680 685 Arg Gin Arg Ser Gly Leu Cys Gin Val Phe Ala Asp Ala Thr Pro Thr 690 695 700 Gly Trp Gly Leu Ala lie Gly His Gin Arg Met Ala Gly Thr Phe Leu 705 710 715 " 720 Ala Pro Leu Pro lie His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu lie Gly Thr Asp Asn Ser Val Val 740 745 750 39 Leu Ser Arg Lys Tyr Thr Ser Phe 755 760 Asn Trp lie Leu Arg Gly Thr Ser 770 775 Asn Pro Ala Asp Asp Pro Ser Arg 785 790 Leu Leu His Leu Pro Phe Arg Pro 805 Ala Val Ser Pro Tyr Val Pro Ser 820 Ala Ser Pro Leu His Val Ala Trp 835 840 Pro Trp Leu Leu Gly Cys Ala Ala 765 Phe Val Tyr Val Pro Ser Ala Leu 780 Gly Arg Leu Gly Leu Tyr Arg Pro 795 800 Thr Thr Gly Arg Thr Ser Leu Tyr 810 815 His Leu Pro Asp Arg Val His Phe 825 830 Arg Pro Pro ) INFORMATION FOR SEQ ID NO:3: (Figure 5) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 400 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Gly Gly Trp Ser Ser Lys Pro Arg Gin Gly Met Gly Thr Asn Leu 1 5 10 15 Ala Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gin Leu Asp Pro 20 25 30 Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Asn 35 40 45 Lys Asp His Trp Pro Glu Ala Asn Gin Val Gly Val Gly Ala Phe Gly 50 55 60 Pro Gly Phe Thr Pro Pro His Gly Gly Leu Leu Gly Gly Ser Pro Gin 65 70 75 80 Ala Gin Gly lie Leu Thr Thr Val Pro Ala Ala Pro Pro Pro Ala Ser 85 90 95 Thr Asn Arg Gin Ser Gly Arg Gin Pro Thr Pro lie Ser Pro Pro Leu 100 105 110 Arg Asp Ser His Pro Gin Ala Thr Gin Trp Asn Ser Thr Thr Phe His 115 120 125 Gin Ala Leu Leu Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly 130 135 140 40 Gly Ser Ser Ser Gly Thr Val Asn Pro Val Pro Thr Thr Ala Ser Pro 145 150 155 160 lie Ser Ser lie Phe Ser Arg Thr Gly Asp Pro Ala Pro Asn Met Glu 165 170 175 Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gin Ala Gly 180 185 190 Phe Phe Ser Leu Thr Arg lie Leu Thr lie Pro Gin Ser Leu Asp Ser 195 200 205 Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys Pro Gly 210 215 220 Gin Asn Ser Gin Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro 225 ' 230 235 240 Pro lie Cys Pro Gly Tyr Arg Trp Asn Cys Leu Arg Arg Phe lie lie 245 250 255 Phe Leu Phe lie Leu Leu Leu Cys Leu lie Phe Leu Leu Val Leu Leu 260 265 270 Asp Tyr Gin Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly Thr Ser 275 280 285 Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gin Gly 290 295 300 Asn Ser Thr Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn 305 310 " 315 320 Cys Thr Cys lie Pro lie Pro Ser Ser Trp Ala Phe Ala Arg Phe Leu 325 330 335 Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro 340 345 350 Phe Val Gin Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Val 355 360 365 He Trp Met Met Trp Tyr Trp Gly Arg Ser Leu Tyr Asn He Leu Ser 370 375 380 Pro Phe Leu Pro Leu Leu Pro lie Phe Phe Cys Leu Trp Val Tyr He 385 390 395 400 INFORMATION FOR SEQ ID NO: 4 : (Figure 6) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 212 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 41 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 : Met Gin Leu Phe Leu Leu Cys Leu lie lie Ser Cys Ser Cys Pro Thr 15 10 15 Val Gin Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp Met Asp lie 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Ser Ala Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser He Arg Asp Leu Leu Asp Thr Ala Ser 50 55 SO Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gin Ala lie Leu Cys Trp Gly Glu Leu Met Asn 85 90 95 Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu 100 105 110 Lsu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Leu Arg Gin 115 120 125 lie Leu Trp Phe His lie Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 13 0 13 5 14 0 Leu Glu Tyr Leu Val Ser Phe Gly Val Trr> lie Arg Thr Pro Thr Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro lie Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg Arg Ser Gin Ser Arg 195 200 205 Glu Ser Gin Arg 210 INFORMATION FOR SEQ ID NO: 5: (Figure 7) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 154 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 42 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Met Ala Ala Arg Val Cys Cys Gin Leu Asp .Pro Ala Arg Asp Val Leu 15 10 15 Cys Leu Arg Pro Val Gly Ala Glu Ser Arg Gly Arg Pro Val Ser Gly 20 25 30 Pro Phe Gly Ala Leu Pro Ser Pro Ser Ser Ser Ala Val Pro Ala Asp 35 40 45 His Gly Ala His Leu Ser Leu Arg Gly Leu Pro Val Cys Ala Phe Ser 50 55 60 Ser Ala Gly Pro Cys Ala Leu Arg Phe Thr Ser Ala Arg Arg Met Glu 65 70 75 80 Thr Thr Val Asn Ala Arg Gin Val Leu Pro Lys Val Leu Tyr Lys Arg 85 90 95 Thr Leu Gly Leu Ser Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe 100 105 110 Lys Asp Cys Val Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu lie Arg 115 120 125 Leu Lys lie Tyr Val Leu Gly Gly Cys Arg His Lys Leu Val Cys Ser 130 135 140 Pro Ala Pro Cys Asn phe Phe Ser Ser Ala 145 150 (2) INFORMATION FOR SEQ ID N0:6: (Figure 8) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH; 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATAAGCTTAT GCCCCTATCT TATCAACACT TCCGGA 36 (2) INFORMATION FOR SEQ ID NO:7: (Figure 9) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 43 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GAGTCTAGAC TCTGCGGTAT TGTGA (2) INFORMATION FOR SEQ ID NO:8: (Figure 10) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAGTCTAGAC TCGTGGTGGA CTTCT (2) INFORMATION FOR SEQ ID NO:9: (Figure 11) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TGAGAATTCT CACGGTGGTC TCCATGCGAC GT (2) INFORMATION FOR SEQ ID NO:10: (Figure 12) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TTTGTTTACG TCCCGT 44 (2) INFORMATION FOR SEQ ID NO:11: (Figure 13) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ATAAGCTTAT GCCCCTATCT TATCAACACT TCCGGA 36

Claims

-45- What we claim is:

1. Use of a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, selected from the groups comprising SEQ ID Nos: 1, 2 or 3, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine; for determining whether a subject is infected with a strain of hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-133 Oon Strain (Methionine to Threonine), wherein such determination comprises; determining whether a nucleic acid sample from the subject is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine.

2. The use of claim 1, wherein the nucleic acid sample in step (a) comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, selected from the group comprising SEQ ID Nos: 1, 2 or 3, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, and wherein the determining step comprises: (i) contacting the mRNA with an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides within a nucleic acid which encodes a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than a methionine, without hybridizing to any sequence of nucleotides within a nucleic acid which encodes the major surface antigen of a wild type hepatitis "intellectual property ofice of n.z -6 APR 2005 RECEIVED -46- B virus, under conditions permitting binding of the mRNA to the oligonucleotide so as to form a complex; (ii) isolating the complex so formed; and (iii) identifying the mRNA in the isolated complex so as to thereby determine whether the mRNA is, or is derived from, a nucleic acid which encodes the polypeptide.

3. The use of claim 2, wherein the nucleic acid sample comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, and wherein the determining step comprises: (i) translating the mRNA under suitable conditions to obtain an amino acid sequence; and (ii) comparing the amino acid sequence of step (i) with the amino acid sequence of the isolated nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than a methionine, and wherein the polypeptide has an amino acid sequence is at least more than 95% identical to amino acid residues 174 through 400 of the amino acid sequence designated SEQ. I.D. No. 3, so as to thereby determine whether the nucleic acid sample is, or is derived from, a nucleic acid which encodes the polypeptide.

4. The use of claim 1, wherein the determining step comprises: (i) amplifying the nucleic acid present in the sample; and (ii) detecting the presence of polypeptide in the resulting amplified nucleic acid. intellectual property office of n.z. -6 APR 2005 -47-

5. Use of an antibody that recognises a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, selected from the group comprising SEQ ID Nos: 1, 2 or 3, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine; for determining whether a subject is infected with a strain of hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-l33 Oon Strain (Methionine to Threonine), wherein such determination comprises; determining whether a sample from the subject is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine by contacting the sample under appropriate conditions to bind to: (i) antibodies capable of detecting a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, and incapable of detecting a major surface antigen of a wildtype hepatitis B virus; or (ii) antibodies capable of detecting a polypeptide, wherein the peptide has an amino acid sequence comprising amino acid residues 298 through 320 of the amino acid sequence designated SEQ. I.D. No. 3; so as to determine whether a subject is infected.

6. The use of claim 1, 2 or 5, wherein the isolated nucleic acid, oligonucleotide or antibody is labeled with a detectable marker.

7. The use of claim 6, wherein the detectable marker is a radioactive isotope, a fluorophor or an enzyme. intellectuaTproperty ofice of n.z - E APR 2005 -48-

8. The use of claim 1, wherein the sample comprises blood, tissue or sera.

9. Use of a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, selected from the group comprising SEQ ID Nos: 1, 2 or 3, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine; for determining whether a subject has a predisposition for hepatocellular carcinoma, which comprises; determining whether a nucleic acid sample from the subject is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine.

10. The use of claim 9, wherein the nucleic acid sample comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, selected from the group comprising SEQ ID Nos: 1, 2 or 3, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, and wherein the determining step, comprises: (i) contacting the mRNA with an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides within a nucleic acid which encodes a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than a methionine, without hybridizing to any sequence of nucleotides within a nucleic acid which encodes the major surface antigen of a wild type hepatitis llntellectup^property ofice of n.z. -6 APR 2005 -49- B virus, under conditions permitting binding of the mRNA to the oligonucleotide so as to form a complex; (ii) isolating the complex so formed; and (iii) identifying the mRNA in the isolated complex so as to thereby determine whether the mRNA is, or is derived from, a nucleic acid which encodes the polypeptide.

11. The use of claim 9, wherein the nucleic acid sample comprises mRNA corresponding to the transcript of DNA encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, and wherein the determining step comprises: (i) translating the mRNA under suitable conditions to obtain an amino acid sequence; and (ii) comparing the amino acid sequence of step (i) with the amino acid sequence of the isolated nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine rather than a methionine, and wherein the polypeptide has an amino acid sequence substantially identical to amino acid residues 174 through 400 of the amino acid sequence designated SEQ. I.D. No. 3, so as to thereby determine whether the nucleic acid sample is, or is derived from, a nucleic acid which encodes the polypeptide.

12. The use of claim 9, wherein the determining step comprises: (i) amplifying the nucleic acid present in the sample; and (ii) detecting the presence of polypeptide in the resulting amplified nucleic acid. WElectuaT property" ofice of m.z -6 APR 2005 -50-

13. Use of an antibody that recognises a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, selected from the group comprising SEQ ID Nos: 1, 2 or 3, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine; for determining whether the subject has a predisposition for hepatocellular carcinoma, wherein such determination comprises; determining whether a sample from the subject is, or is derived from, a nucleic acid encoding a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than any methionine by contacting the sample under appropriate conditions to bind to: (i) antibodies capable of detecting a polypeptide which is a mutant major surface antigen of a strain of hepatitis B virus, such polypeptide having an amino acid sequence which differs from the amino acid sequence of a major surface antigen of a wildtype hepatitis B virus in that the amino acid at position number 133 of such polypeptide is a threonine, rather than a methionine, and incapable of detecting a major surface antigen of a wildtype hepatitis B virus; or (ii) antibodies capable of detecting a polypeptide, wherein the peptide has an amino acid sequence comprising amino acid residues 298 through 320 of the amino acid sequence designated SEQ. I.D. No. 3; so as to determine whether a subject has a predisposition for hepatocellular carcinoma.

14. The use of claim 10, 11 or 13, wherein the isolated nucleic acid, oligonucleotide or antibody is labeled with a detectable marker. intellectual property ofice OF IM.Z - 6 APR 2005 RECEIVED -51-

15. The use of claim 14, wherein the detectable marker is a radioactive isotope, a fluorophor or an enzyme.

16. The use of claim 9, wherein the sample comprises blood, tissue or sera.

17. The use according to any one of claims 1, 2, 5, 9, or 13, substantially as herein described with reference to any one of the Examples and/or Sequence Listing thereof.

18. The use according to any one of claims 1 to 16, substantially as herein described with reference to any one of the Examples and/or the Sequence Listing thereof. GOVERNMENT OF THE REPUBLIC OF SINGAPORE By Its Attorneys BALDWINS