This invention provides an isolated strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'Ś-145 Singapore Strain (Glycine to Arginine) which constituent viral genome is deposited under Accession Nos. P97121504, P97121505 and P97121506 with the European Collection of Cell Culture on 15th Dec. 1997. 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 wild type hepatitis B virus in that the amino acid at position number 145 of such polypeptide is an arginine rather than a glycine, and the purified peptide. This invention further provides an isolated nucleic acid which encodes a peptide, wherein the peptide is encoded by a nucleic acid molecule comprising nucleotides 527 through 595 of SEQ. ID. No. 1, and the purified peptide. This invention also provides various methods using the disclosed isolated nucleic acids and polypeptides. This invention also provides various uses of the viral strain and its proteins.

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 a hepatitis B virus genome, which carries a vaccine-induced mutation at amino acid residue 145 (Glycine to Arginine) of the major surface antigen, consisting of 3215 nucleotides (SEQ ID NO: 1) coding for 4 overlapping viral proteins.

The invention provides amino acid sequences of the four major viral proteins, these include 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 hepatitis B virus diagnostic system, specific for the vaccine-induced mutation at amino acid residue 145 (Glycine to Arginine) of the major surface antigen, using nucleotide or protein sequences or antibodies described herein.

The present invention provides an isolated strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-145 Singapore Strain (Glycine to Arginine) which constituent viral genome is deposited under Accession Nos. P97121504, P97121505 and P97121506.

The 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 the major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 145 of such polypeptide is an arginine rather than a glycine. 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, specifically, comprising nucleotides "AGA" in position 587-589, instead of "GGA." This nucleic acid can be DNA or RNA, specifically, cDNA or genomic DNA.

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

This invention further provides an isolated nucleic acid which encodes a peptide, wherein 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 also provides a vector comprising 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 145 of such polypeptide is an arginine rather than a glycine and operatively linked to a promoter of RNA transcription.

Further, this invention provides a vector comprising an isolated nucleic acid encoding a peptide, wherein the peptide is encoded by a nucleic acid molecule comprising nucleotides 527 through 595 of SEQ. I.D. No. 1.

In both of the above-identified vectors, the vector may comprise viral DNA.

This invention also provides a host vector system for the production of a polypeptide which comprises the above-described vectors in a suitable host.

This invention also provides a method of producing a polypeptide or a peptide which comprises growing the host vector systems described above, under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

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

This invention further 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 major surface antigen of a wild type hepatitis B virus in that the amino acid at position number 145 of such polypeptide is an arginine rather than a glycine. One means of obtaining the polypeptide is by the above-described method.

This invention also 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. One means of obtaining this peptide is by the above-describe method.

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 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 145 of such polypeptide is an arginine rather than a glycine, without hybridizing to any sequence of nucleotides within a nucleic acid which encodes the major surface antigen of a wild type hepatitis B virus. Specifically the oligonucleotide comprises nucleotides 527 through 595 of SEQ. I.D. No. 1.

This invention also provides a method of obtaining antibodies 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 145 of such polypeptide is an arginine, rather than a glycine, 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 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. Specifically, the polypeptide is being encoded by nucleotides 155 through 835 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 of the amino acid sequence designated SEQ. I.D. No. 3.

One could perform the above-described well-known method in rabbits or mice.

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 320 of the amino acid sequence designated SEQ. I.D. No. 3, comprising: (a) 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 produced antibodies form a complex with the purified peptide so as to obtain antibodies to the peptide.

One could perform the above-described well-known method in rabbits or mice.

This invention also provides the antibodies obtained from the above-described methods, specifically the monoclonal antibodies. Further, the invention provides 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 wild type hepatitis B virus in that the amino acid at position number 145 of such polypeptide is an arginine, rather than a glycine, and incapable of detecting the major surface antigen of a wild type hepatitis B virus, as well as, antibodies capable of detecting 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 also provides use of 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 145 of such polypeptide is an arginine, rather than a glycine for determining whether a subject is infected with a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-145 Singapore Strain (Glycine to Arginine), 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 145 of such polypeptide is an arginine, rather than a glycine.

A means of determination is where 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 145 of such polypeptide is an arginine, rather than a glycine, and wherein the determining of step b) comprises: (i) contacting the mRNA with the above-described 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.

Another example is where 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 145 of such polypeptide is an arginine, rather than a glycine, 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) which the amino acid sequence of an isolated nucleic acid which encodes a polypeptide, 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 cucleic acid which encodes the polypeptide. A further example is where 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 provides the use of an antibody that recognizes a polypeptide which is a mutant major surface antigen of a train of hepatitis B virus for determining whether the 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 145 of such polypeptide is an arginine, rather than a glycine, by contacting the sample under appropriate conditions to bind to the antibodies of claim 35 so as to determine whether the subject has a predisposition for hepatocellular carcinoma.

The invention also provides use of antibodies capable of detecting a polypeptide which is a mutant major surface antigen of a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-145 Singapore Strain (Glycine to Arginine) for determining whether a subject is infected with a strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-145 Singapore Strain (Glycine to Arginine), 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 145 of such polypeptide is an arginine, rather than a glycine by contacting the sample under appropriate conditions to bind to the antibodies so as to determine whether a subject is infected. Furthermore, the antibody may also be capable detecting a peptide, wherein the peptide has an amino acid sequence comprising amino acid residues 298-320 of the amino acid sequence designated SEQ. I.D. No. 3.

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 a specific embodiment, the sample includes, but is not limited to, blood, tissue or sera.

This invention also 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'-145 Singapore Strain (Glycine to Arginine) 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 145 of such polypeptide is an arginine, rather than a glycine, 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.

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'-145 Singapore Strain (Glycine to Arginine), 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 145 of such polypeptide is an arginine, rather than a glycine, 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 further 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 posit on number 145 of such polypeptide is an arginine, rather than a glycine, 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 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 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.

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 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. 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. 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 additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

This invention further provides a composition comprising 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 or derivative thereof, the amounts of such peptide being effective to stimulate or enhance antibody production in a subject, and a pharmaceutically acceptable carrier.

This invention further provides compositions 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'-145 Singapore Strain (Glycine to Arginine) and a pharmaceutically effective carrier.

This invention provides the use of the above-described compositions as medicaments for treating and/or preventing hepatocellular carcinoma.

This invention also 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'-145 Singapore Strain (Glycine to Arginine).

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'-145 Singapore Strain (Glycine to Arginine) in a subject which comprises administering an effective amount.

This invention further provides a method of screening tissues and bodily fluids from a subject for a strain of hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-145 Singapore Strain (Glycine to Arginine) 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 145 of such polypeptide is an arginine, rather than a glycine in the sample of step (a) so as to screen the sample for the strain. In embodiments of this method, the bodily fluid comprises blood, sera, or a nucleic acid sample of blood or sera.

This invention provides a method for determining whether a subject has a predisposition for hepatocellular carcinoma, which 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 145 of such polypeptide is an arginine, rather than a glycine, thereby determining whether the subject has a predisposition for hepatocellular carcinoma.

This invention also provides the above-described method, wherein the nucleic acid sample in step (a) comprises mRNA 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 145 of such polypeptide is an arginine, rather than a glycine, and wherein the determining of step (b) comprises: (i) contacting the mRNA with the above-described oligonucleotides 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.

This invention further provides the above-described method, wherein the nucleic acid sample in step (a) comprises mRNA 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 145 of such polypeptide is an arginine, rather than a glycine, 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 isolated nucleic acid described above so as to determine whether the nucleic acid sample is, or is derived from, a nucleic acid which encodes the polypeptide.

This invention also provides the above-described method, wherein 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 the above-described method for determining whether a subject has a predisposition for hepatocellular carcinoma, which 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 145 of such polypeptide is an arginine, rather than a glycine, by contacting the sample under appropriate conditions to bind to the above-described antibodies so as to determine whether the subject has a predisposition for hepatocellular carcinoma.

This invention provides the above-described methods, wherein the oligonucleotide or antibody is labeled with a detectable marker.

This invention also provides the above-described methods, wherein the detectable marker is a radioactive isotope, a fluorophor or an enzyme.

This invention also provides the above-described methods, wherein the sample comprises blood, tissue or sera.

This invention further 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 a wild type hepatitis B virus in that the amino acid at position number 145 of such polypeptide is an arginine, rather than a glycine, 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 binds to the polypeptide so as to identify a chemical compound which is capable of treating hepatocellular carcinoma.

This invention 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 145 of such polypeptide is an arginine, rather than a glycine, 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 binds to the polypeptide so as to identify a chemical compound which is capable of preventing hepatocellular carcinoma.

Additionally, this invention provides a composition comprising the chemical compound identified by the above-described methods in an amount effective to treat 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 prevent hepatocellular carcinoma and a pharmaceutically effective carrier.

This invention further provides a method treating a subject with hepatocellular carcinoma which comprises administering an effective amount of the above-described compositions.

This invention further provides a method preventing hepatocellular carcinoma in a subject which comprises administering an effective amount of the above-described compositions.

This invention also 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 amino acid sequence of the major surface antigen of hepatitis B in that the amino acid at position number 145 of such polypeptide is an arginine rather than a glycine.

This invention also provides the above-described vaccine and an adjuvant.
 

Claim 1 of 10 Claims

1. An isolated strain of Hepatitis B virus designated Human Hepatitis B Virus Surface Antigen-'S'-145 Singapore Strain (Glycine to Arginine) and deposited under any one of Accession Nos. P97121504, P97121505, or P97121506 with the European Collection of Cell Culture on 15^th Dec. 1997.

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