WO2006039739A1

WO2006039739A1 - Hepatitis b viral (hbv) mutants detection and application

Info

Publication number: WO2006039739A1
Application number: PCT/AU2005/001538
Authority: WO
Inventors: Angeline Ingrid Bartholomeusz; Stephen Alister Locarnini; Anna Ayres; Sharon Ruth Lewin; Joseph John Sasadeusz
Original assignee: Melbourne Health; Bayside Health
Priority date: 2004-10-13
Filing date: 2005-10-06
Publication date: 2006-04-20

Abstract

The present invention relates generally to viral variants exhibiting reduced sensitivity to agents and in particular one or more nucleoside or nucleotide analogs and/or non-nucleoside or non-nucleotide agents. More particularly, the present invention is directed to hepatitis B (HBV) variants RTh234Y and preS1 191V which correlate with a higher risk of an adverse disease outcome and exhibiting complete or partial resistance to abacavir (ABC) and lamividuine (LMV). The present invention further contemplates assays for detecting such viral variants which assays are useful in diagnosing a higher risk of an adverse disease outcome, in monitoring antiviral therapeutic regimes and in developing new or modified nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents. The present invention also contemplates the use of viral variants to screen for agents capable of inhibiting infection, replication and/or release of the virus. Such agents are useful in treating hepatitis.

Description

VIRAL MUTANTS DETECTION AND APPLICATIONS

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to viral variants exhibiting reduced sensitivity to agents and in particular one or more nucleoside or nucleotide analogs and/or non- nucleoside or non-nucleotide agents. More particularly, the present invention is directed to hepatitis B virus (HBV) variants or quasi species which correlate with a higher risk of an adverse disease outcome and exhibiting complete or partial resistance to one or more nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents. The present invention further contemplates assays for detecting such viral variants which assays are useful in diagnosing a higher risk of an adverse disease outcome, in monitoring antiviral therapeutic regimes and in developing new or modified nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents. The present invention also contemplates the use of the viral variants to screen for agents capable of inhibiting infection, replication and/or release of the virus. Such agents are useful in treating hepatitis.

DESCRIPTION OF THE PRIOR ART

Bibliographic details of the publications referred to in this specification are also collected at the end of the description.

Specific mutations in an amino acid sequence are represented herein as "Xaa.ιήKaa.₂ ' where Xaεn is the original amino acid residue before mutation, n is the residue number and Xaa₂ is the mutant amino acid. The abbreviation "Xaa" may be the three letter or single letter (i.e. "X") code. The amino acid residues for Hepatitis B virus DNA polymerase are numbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD) being residue number 204. Specific mutations in a nucleic acid sequence are represented herein as "N₁IiN₂" where N₁ is the original nucleotide before mutation, n is the nucleotide number and N₂ is the mutant nucleotide.

Nucleoside analog antiviral therapy has been successfully introduced for the treatment of chronic hepatitis B virus (HBV) infection. Lamivudine (LMV) and Adefovir dipivoxil (ADV) are now registered for use in chronic HBV patients, whilst other compounds are in various stages of clinical development including Entecavir (ETV) and Famciclovir (FCV). Whilst these agents are highly effective in inhibiting HBV DNA synthesis, there is the potential for resistant mutants of HBV to emerge during long term antiviral chemotherapy. In patients on prolonged LMV therapy, key resistance mutations are selected in the reverse transcriptase (rt) domain within the polymerase at rtM204I/V +/- rtL180M. The nomenclature used for the polymerase mutations is in accordance with that proposed by Stuyver and colleagues [Stuyver et al, Hepatology, 53:751-757, 2001].

A number of the antiviral agents including LMV and Adefovir dipivoxil ADV were developed for HIV and were found to have HBV activity as well. Abacavir ([lS,4R]-4-[2- amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-l -methanol; ABC) is another such agent that has recently been shown to have anti-HBV activity [Walters et al., Antimicrob Agents Chemother. 47:1936-42, 2003.]. This agent is being used to treat HIV patients [Staszewski et al, AIDS. /2:197-202, 1998.].

HIV and HBV are both blood borne viruses and have similar potential routes of transmission. Approximately 10% of HIV infected individuals are co-infected with HBV. The improved prognosis of HIV-infection that has occurred since the introduction of highly active antiretroviral therapy (HAART) has resulted in renewed emphasis being placed on co-morbidities associated with HIV-infection, and chronic viral hepatitis in particular. Hepatitis B virus (HBV) infection is an important infection in HIV-I infected individuals because of the influence of HIV-I co-infection on the natural history of HBV infection. Antiviral therapies with activity against both viruses have enabled targeted therapy in co-infected individuals. However, antiviral resistance is an important issue for both HIV and HBV.

In work leading up to the present invention, the inventors identified the selection of HBV variants a patient co-infected with HBV and HIV, that was treated with both Lamivudine and Abacavir.. In accordance with the present invention, the inventors have identified variants of HBV, selected following LMV and ABC treatment, with mutations in the HBV

DNA polymerase gene which reduce the sensitivity of HBV to this nucleoside analogue.

The identification of these HBV variants is important for the development of assays to monitor Abacavir and/or LMV resistance and/or resistance to other nucleoside analogue therapeutic regimes and to screen for agents which are useful as alternative therapeutic agents

There is a need, therefore, to monitor for newly emerging variants of HBV that exhibit reduced sensitivity to nucleoside or nucleotide analogs. The early detection of such variants enables appropriate therapeutic protocols to be rapidly developed and/or therapeutic protocols altered. There is also a need to monitor HBV strains isolated from subjects exposed to nucleoside or nucleotide analogs to ascertain whether these strains exhibit resistance to nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents other than the ones to which the patient was exposed.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l [SEQ ID NO: 1], <400>2 [SEQ ID NO: 2], etc.

HBV variants exhibiting reduced sensitivity to nucleoside or nucleotide analogs and/or non-nucleoside or non-nucleotide agents are identified from patients on antiviral chemotherapy. These mutations are detected in the context of the genetic framework of HBV. Furthermore, such variants may also arise in patients co-infected with HBV and human immunodeficiency virus (HIV) or in HBV mono-infected individuals.

Thus, the identification of HBV variants with these mutational patterns is important inter alia for the development of assays to detect HBV variants associated with a higher risk of developing an adverse disease outcome including severe fulminant hepatitis and/or death, for the development of assays to screen for agents which are useful in treating and/or preventing infections by those variants and/or other HBV isolates and for the development of alternative therapeutic regimes for managing HBV infections. They are also important in high risk groups such as co-infected patients such as in HBV and HIV co-infected subjects.

In accordance with the present invention, HBV resistant variants were identified in a patient (patient A) with chronic hepatitis B and HIV treated with both LMV and ABC. In combination therapy, accordance with the present invention, resistant variants of HBV were identified, following LMV and ABC treatment, with mutations in the HBV DNA polymerase gene which reduce the sensitivity of HBV to these nucleoside or nucleotide analogs and potentially others such as ADV and/or TDF and/or FTC. Corresponding mutations in the surface antigen also occur. Strains of HBV may also be selected which exhibit resistance to nucleoside or nucleotide analogs other than LMV or ABC. The identification of these HBV variants is important for the development of assays to monitor LMV and/or ABC resistance and/or resistance to other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide analogs and to screen for agents which are useful as alternative therapeutic agents.

The detection of such HBV variants is particularly important in the management of therapeutic protocols including the selection of appropriate agents for treating HBV infection. The method of this aspect of the present invention is predicated in part on monitoring the development in a subject of an increased HBV load in the presence of a nucleoside or nucleotide analog. The clinician is then able to modify an existing treatment protocol or select an appropriate treatment protocol accordingly.

The present invention further provides an isolated HBV variant comprising a nucleotide mutation at rtH234Y in DNA polymerase reverse transcriptase (rt) region wherein said variant exhibits decreased sensitivity to one or more of (i) ABC and/or LMV (ii) ADV; (iii) TDF; (iv) ETV, (v) FTC C; or a combination of (i), (ii) (iii) (iv) and/or (v).

Yet another aspect of the invention is an isolated HBV variant comprising nucleotide mutation in a gene encoding or corresponding to the DNA polymerase reverse transcriptase (rt) region said mutation resulting in an rtH234Y change wherein said variant exhibits decreased sensitivity to one or more of ABC, LMV, ADV, TDF₅, ETV or FTC or combinations thereof such as (i) ABC and/or LMV, (ii) ADV and/or (iii) TDF and/or ETV and (iv)_TDF and/or FTC. Decreased sensitivity to ABC and/or LMV is particularly preferred. Corresponding changes may also occur to a surface antigen of HBV. In addition, HBV variants resistant to other nucleoside analogs may also be selected even if these are not resistant to ABC or LMV or TDF or ETV or FTC. The present invention provides, therefore, an isolated HBV variant comprising a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to the DNA polymerase and which exhibits decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs or or non-nucleoside or non-nucleotide agents. In one embodiment, the DNA polymerase exhibits reduced sensitivity to ABC alone or in addition to LMV. The variant HBV comprises a mutation in an overlapping open reading frame in its genome in a region defined by one or more of domains F and A through E of HBV DNA polymerase.

The present invention further contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to ABC and/or LMV or optionally other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents by isolating DNA or corresponding mRNA from the HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and A through E or a region proximal thereto of the DNA polymerase and associated with resistance or decreased sensitivity to ABC and/or LMV; or optionally in addition one or more of ADV, TDF and/or ETV and/or FTC. The presence of such a mutation is an indication of the likelihood of resistance to said ABC and/or LMV. In one embodiment, the HBV variant exhibits reduced sensitivity to ABC, or both ABC and LMV.

The present invention also provides a composition comprising a variant HBV resistant to ABC and/or LMV and optionally other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents or a derivative form thereof or its chemical equivalent and one or more pharmaceutically acceptable carriers and/or diluents. Yet another aspect of the present invention provides a use of the aforementioned composition or a variant HBV comprising a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to the DNA polymerase and a decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs such as one or more of ADV, TDF and/or ETV and/or FTC or a non-nucleoside or non-nucleotide agent in the manufacture of a medicament for the treatment and/or prophylaxis of HBV virus infection.

The present invention also contemplates a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or non-nucleoside or non- nucleotide agent by isolating DNA or corresponding mRNA from the HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase wherein the presence of the following mutations in the polymerase at rtH234Y is indicative of a variant which exhibits a decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs such as ADV₅ TDF and/or ETV and/or FTC or or non-nucleoside or non-nucleotide agents.

The present invention is predicated in part on the identification and isolation of variants of HBV that have decreased or reduced sensitivity to one or more nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents, relative to wild-type HBV. Thus, the identification of HBV variants with these mutational patterns is important inter alia for the development of assays to detect HBV variants and assays to screen for agents which are useful in treating and/or preventing infections by those variants and/or other HBV isolates and for the development of alternative therapeutic regimes for managing HBV infections.

Accordingly, the present invention is also directed to an isolated HBV variant with resistance to one or more nucleoside or nucleotide analogs or non-nucleoside or non- nucleotide agents.

Reference to nucleoside analogs including ABC, LMV, ADV, TDF and/or ETV and/or FTC.

Yet another aspect of the present invention provides an isolated HBV variant with a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase wherein said variant exhibits decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents.

Another aspect of the present invention contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV by generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in a plasmid vector and then transfecting said cells with said construct, contacting the cells, before, during and/or after transfection, with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agents; and the subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral- component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent. In a preferred embodiment, the plasmid vector in a baculovirus vector and the method comprises generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in or fused to an amount of a baculovirus genome effective to infect cells and then infecting said cells with said construct, contacting the cells, before, during and/or after infection, with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

In an alternative embodiment, the method comprises generating a continuous cell line comprising an infectious copy of the genome of the HBV in a replication competent effective amount such that said infectious HBV genome is stably integrated into said continuous cell line such as but not limited to 2.2.15 or AD, contacting the cells with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus- like particles if resistant to the agent and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

In an alternative embodiment, the present invention also contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV polymerase in an in vitro polymerase assay. The HBV polymerase activity can be examined using established assays (Gaillard et ah, Antimicrob Agents Chemother. 46(4): 1005-1013, 2002; Xiong et ah, Hepatology. 28(6): 1669-73, 1998). The HBV polymerase may be a wild-type or reference HBV polymerase or mutant HBV polymerase.

In connection with these methods, the plasmid vector may include genes encoding part or all of other viral vectors such as baculovirus vectors or adenovirus vectors (see Ren and Nassal, J. Virol. 75(3): 1104-1116, 2001).

The abbreviations defined in Table 1 are used in the subject specification

TABLE 1

Abbreviations

A summary of sequence identifiers used throughout the subject specification is provided in Table 2.

TABLE 2 Summary of sequence identifiers

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagrammatic representation showing the partially double stranded DNA HBV genome showing the overlapping open reading frames encoding surface (S), core (C)₅ polymerase (P) and X gene.

Figure 2 is a diagrammatic representation of the chemical structure of ABC.

Figure 3 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene from Patient A during LMV/ ABC combination therapy.

Figure 4 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene from Patient A during LMV/ ABC combination therapy.

Figure 5 is a representation showing the deduced amino acid sequence of the envelope gene from Patient A during LMV/ ABC combination therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the identification and isolation of nucleoside or nucleotide analog resistant variants of HBV following treatment of patients with ABC or LMV or ABC and LMV and optionally other nucleoside or nucleotide analogs including ADV, TDF, ETV or FTC or non-nucleoside or non-nucleotide agents. In particular, ABV, or ABC and LMV treated patients give rise to variants of HBV exhibiting decreased or reduced sensitivity to ABC and/or LMV. Reference herein to "decreased" or "reduced" in relation to sensitivity to ABC and/or LMV includes and encompasses a complete or substantial resistance to the nucleoside or nucleotide analogs or non-nucleoside or non- nucleotide agents as well as partial resistance and includes a replication rate or replication efficiency (yield phenotype) which is more than a wild-type in the presence of a nucleoside or nucleotide analog. In one aspect, this is conveniently measured by an increase in viral load to a level similar or greater than pre-treatment levels.

Accordingly, one aspect of the present invention is directed to an isolated HBV variant wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase and wherein said variant exhibits decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents.

Preferably, the decreased sensitivity is in respect of ABC, or both ABC and LMV, and optionally in addition one or more of ADV, TDF and/or ETV and/or FTC.

In addition to a mutation in the gene encoding DNA polymerase, due to the overlapping nature of the HBV genome (Figure 1), a corresponding mutation may also occur in the genes encoding the HBV envelope proteins (including PreSl, PreS2 or HBsAg). The present invention extends, therefore, to an HBV variant exhibiting decreased sensitivity to ABC and/or LMV wherein the variant is selected for following ABC and/or LMV combination or sequential treatment. The term "sequential" in this respect means ABC followed by LMV or LMV followed by ABC or multiple sequential administrations of each of ABC and LMV, or LMV and ABC.

A viral variant may, therefore, carry mutation only in the DNA polymerase or both in the DNA polymerase and the HBV envelope genes. The term "mutation" is to be read in its broadest context and includes multiple mutations.

The present invention extends to a mutation and any domain of the HBV DNA polymerase and in particular regions F and A through E provided the mutation leads to decreased sensitivity to ABC alone or in combination with LMV and optionally one or more of ADV, TDF and/or ETV and/or FTC.

Preferably, the mutation results in an altered amino acid sequence in any one or more of domains F and A through E or regions proximal thereto of the HBV DNA polymerase.

Another aspect of the present invention provides an HBV variant comprising a mutation in an overlapping open reading frame in its genome wherein said mutation is in a region defined by one or more of domains F and A through E of HBV DNA polymerase and wherein said variant exhibits decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs including ADV, TDF and/or ETV and/or FTC or non-nucleoside or non-nucleotide agents.

It should be noted that exposure of a subject to ABC or ABC and LMV may result in other variants, still sensitive to ABC or ABC and LMV but now resistant to other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents.

The term "combination therapy" means that both ABC and LMV are co-administered in the same composition or simultaneously in separate compositions. The term "sequential therapy" means that the two agents are administered within seconds, minutes, hours, days or weeks of each other and in either order. Sequential therapy also encompasses completing a therapeutic course with one or other of ABC or LMV and then completing a second therapeutic course with the other of ABC or LMV.

Preferably, the variants are in isolated form such that they have undergone at least one purification step away from naturally occurring body fluid. Alternatively, the variants may be maintained in isolated body fluid or may be in DNA form. The present invention also contemplates infectious molecular clones comprising the genome or parts thereof from a variant HBV. Furthermore, the present invention provides isolated components from the variant HBVs.

Preferred mutations in the HBV DNA polymerase include variants selected from patients with HBV recurrence following ABC and/or LMV treatment. Preferably, the treatment involves ABC or both ABC and/or LMV in combination or sequential therapy. Nucleoside or nucleotide analog treatment or treatment by a non-nucleoside or non-nucleotide agent may occur following treatment of patients diagnosed with hepatitis in patients with HBV alone or co-infected with HIV. Following selection of variants, viral loads are obtainable at levels greater than pre-treatment levels.

Preferred mutations in the HBV DNA polymerase include rtH234Y mutation that is indicative of a variant wherein said variant exhibits a decreased sensitivity to ABC and/or

LMV and optionally other nucleoside or nucleotide analogs, including ADV, TDF and/ior

ETV and/or FTC or a non-nucleoside or non-nucleotide agents. It should be noted that the nomenclature system for amino acid positions is based on the methionine residues in the

YMDD motif being designated codon rtM204. This numbering system is different to that in Australian Patent No. 734831 where the methionine residue in the YMDD motif within the polymerase gene is designated codon 550. The term "SPACER" means a region that has been designated between two functional regions: Terminal protein and reverse transcriptase. It provides the correct folding for the functional regions and no other specific function has been designated for this region. Corresponding mutations may also occur in envelope genes such as in one or more of PreSl, PreS2 and HBsAg. Particular mutations are as follows: PreSl 19 IV or rtH234Y or combinations thereof or an equivalent one or more other mutation is indicative of a variant wherein said variant exhibits a decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents.

The identification of the variants of the present invention permits the generation of a range of assays to detect such variants. The detection of such variants may be important in identifying resistant variants to determine the appropriate form of chemotherapy.

Still another aspect of the present invention contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to ABC and/or LMV or optionally other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and A through E or a region proximal thereto of said DNA polymerase and associated with resistance or decreased sensitivity to ABC and/or LMV wherein the presence of such a mutation is an indication of the likelihood of resistance to said ABC and/or LMV.

Preferably, the assay detects one or more of the following mutations in the rt region: rtH234Y wherein the mutation is indicative of a variant wherein said variant exhibits a decreased sensitivity to ABC and/or LMV and optionally other nucleoside or nucleotide analogs, including ADV, TDF and/or ETV and/or FTC or non-nucleoside or non- nucleotide agents

Accordingly, another aspect of the present invention contemplates a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or a non-nucleoside or non-nucleotide agent, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase wherein the presence of the following mutation in the rt region at rtH234Y is indicative of a variant wherein said variant exhibits a decreased sensitivity to ABC and/or LMV and optionally other nucleoside analogs, including ADV, TDF and/or ETV and/or FTC.

The detection of HBV or its components in cells, cell lysates, cultured supernatant fluid and bodily fluid may be by any convenient means including any nucleic acid-based detection means, for example, by nucleic acid hybridization techniques or via one or more polymerase chain reactions (PCRs). The term "bodily fluid" includes any fluid derived from the blood, lymph, tissue or organ systems including serum, whole blood, biopsy and biopsy fluid, organ explants and organ suspension such as liver suspensions. The invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others. Other forms of detection include Northern blots, Southern blots, PCR sequencing, antibody procedures such as ELISA, Western blot and immunohistochemistry. A particularly useful assay includes the reagents and components required for immobilized oligonucleotide- or oligopeptide-mediated detection systems.

One particularly useful nucleic acid detection system is the reverse hybridization technique. In this technique, DNA from an HBV sample is amplified using a biotin or other ligand-labeled primer to generate a labeled amplificon. Oligonucleotides immobilized to a solid support such as a nitrocellulose film are then used to capture amplified DNA by hybridization. Specific nucleic acid fragments are identified via biotin or the ligand. Generally, the labeled primer is specific for a particular nucleotide variation to be detected. Amplification occurs only if the variation to be detected is present. There are many forms of the reverse hybridization assay and all are encompassed by the present invention.

Detecting HBV replication in cell culture is particularly useful. Another aspect of the present invention contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV by:

generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in a plasmid vector and then transfecting said cells with said construct;

contacting the cells, before, during and/or after transfection, with the agent to be tested;

culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agents; and

then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

In a preferred embodiment, the plasmid vector may include genes encoding part or all of oher viral vectors such as baculovirus or adenovirus (Ren and Nassal, 2001, supra) and the method comprises:

generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in or fused to an amount of a baculovirus genome or adenovirus genome effective to infect cells and then infecting said cells with said construct;

contacting the cells, before, during and/or after infection, with the agent to be tested; culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and

In an alternative embodiment, the method comprises:

generating a continuous cell line comprising an infectious copy of the genome of the HBV in a replication competent effective amount such that said infectious HBV genome is stably integrated into said continuous cell line such as but not limited to 2.2.15 or AD;

contacting the cells with the agent to be tested;

culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to the agent; and

As indicated above, variants may also be detected with reference to the HBV envelope genes including PreSl. Preferred mutations in this regard include PreSl I91V. A "non-nucleoside" or "non-nucleotide" agent includes an antibody or other immunoglobulin molecule or chemical or proteinaceous molecule, naturally occurring or synthesized.

The detection of amino acid variants of DNA polymerase is conveniently accomplished by amino acid sequencing. The polymorphisms shown represent the variations shown in various databases for active pathogenic HBV strains. Where an HBV variant comprises an amino acid different to what is represented, then such an isolate is considered a putative HBV variant having an altered DNA polymerase activity.

The present invention further contemplates agents which inhibit ABC and/or LMV resistant HBV variants. Such agents will be particularly useful if long term treatment by ABC and/or LMV and/or optionally other nucleoside or nucleotide analogs or non- nucleoside or non-nucleotide agents is contemplated by the clinician. The agents may be DNA or RNA or proteinaceous or non-proteinaceous chemical molecules. Natural product screening such as from plants, coral and microorganisms is also contemplated as a useful potential source of masking agents. The agents may be in isolated form or in the form of a pharmaceutical composition and may be administered sequentially or simultaneously with the nucleoside or nucleotide analog or a non-nucleoside or non-nucleotide agent.

Accordingly, another aspect of the present invention contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV, exhibiting resistance or decreased sensitivity to ABC and/or LMV, said method comprising:

generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in a plasmid vector and then transfecting said cells with said construct;

contacting said cells, before, during and/or after transfection, with the agent to be tested; culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and

subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.

Still another aspect of the present invention provides a method for detecting an agent which exhibits inhibitory activity to an HBV, exhibiting resistance or decreased sensitivity to ABC and/or LMV, said method comprising:

generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in or fused to an amount of a baculovirus genome effective to infect cells and then infecting said cells with said construct;

contacting said cells, before, during and/or after infection, with the agent to be tested;

culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and

subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral- component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.

Still another aspect of the present invention provides a method for detecting an agent which exhibits inhibitory activity to an HBV₅ exhibiting resistance or decreased sensitivity to ABC and/or LMV, said method comprising: generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in or fused to an amount of a baculovirus genome effective to infect cells and then infecting said cells with said construct;

Preferably, the HBV genome is stably integrated into the cells' genome.

Whilst the baculovirus vector is a particularly useful in the practice of the present invention, the subject invention extends to a range of other vectors such as but not limited to adenoviral vectors.

The present invention further extends to cell lines carrying genetic constructs comprising all or a portion of an HBV genome or a gene or part of a gene therefrom.

A similar method may also be used to detect HBV variants resistant to other nucleoside analogs whether or not the variants themselves are resistant to ABC or ABC and LMV.

The present invention also provides for the use of the subject HBV variants to screen for anti-viral agents. These anti-viral agents inhibit the virus. The term "inhibit" includes antagonizing or otherwise preventing infection, replication, assembly and/or release or any intermediate step. Preferred anti-viral agents include nucleoside or nucleotide analogs, however, the present invention extends to non-nucleoside or non-nucleotide molecules.

In addition, rational drug design is also contemplated to identify or generate chemical molecules which either mimic a nucleoside or which interact with a particular nucleotide sequence or a particular nucleotide. Combinatorial chemistry and two hybrid screening are some of a number of techniques which can be employed to identify potential therapeutic or diagnostic agents.

In one example, the crystal structure of polymerase is used to rationally design small chemical molecules likely to interact with key regions of the molecule required for function. Such agents may be useful as inhibitors of polymerase activity.

Several models of the HBV polymerase have been prepared due to the similarity with reverse transcriptase from HIV (Das et al, J. Virol. 75(10): 4771-4779, 2001;

Bartholomeusz et al, Intervirology 40(5-6): 337-342 1997; Allen et al, Hepatology 27(6):

1670-1677, 1998). The models of the HBV polymerase can be used for the rational drug design of new agents effective against HBV encoding the resistant mutations as well as wild type virus. The rational drug that is designed may be based on a modification of an existing antiviral agent such as the agent used in the selection of the HBV encoding the mutations associated with resistance. Viruses or clones expressing HBV genomic material encoding the mutations may also be used to screen for new antiviral agents.

The above methods are particularly useful in identifying an inhibitor of a ABC- and/or LMV -resistant HBV. The present invention extends, therefore, to compositions of the inhibitors. The inhibitors may also be in the form of antibodies or genetic molecules such as ribozymes, antisense molecules and/or sense molecules for co-suppression or the induction of RNAi. Reference to RNAi includes reference to siRNA.

The term "composition" includes a "pharmaceutical composition" as well as a vaccine composition. The inhibitor is referred to below as an "active ingredient" or "active compound" and may be selected from the list of inhibitors given above.

The composition may include a defective HBV variant or an agent identified through natural product screening or rational drug design (including combinatorial chemistry).

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of encoding an aspartyl protease inhibitor. The vector may, for example, be a viral vector.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the active ingredient and optionally other active ingredients as required, followed by filtered sterilization or other appropriate means of sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and the freeze-drying technique which yield a powder of active ingredient plus any additionally desired ingredient.

When the active ingredient is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, b varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 200 mg of active compound. Alternative dosage amounts include from about 1 μg to about 1000 mg and from about 10 μg to about 500 mg. These dosages may be per individual or per kg body weight. Administration may be per hour, day, week, month or year.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavouring. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non¬ toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

An aspect of the present invention provides a composition comprising a variant HBV resistant to ABC and/or LMV and optionally other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents and one or more pharmaceutically acceptable carriers and/or diluents.

Insofar as ribozyme, antisense or co-suppression (RNAi) repression is concerned, this is conveniently aimed at post-transcription gene silencing. DNA or RNA may be administered or a complex comprising RNAi or a chemical analog thereof specific for HBV niRNA may be employed.

All such molecules may be incorporated into pharmaceutical compositions.

The present invention is further directed to the use of defective HBV variants in the manufacture of therapeutic vaccines to vaccinate individuals against infection by HBV strains having a particular nucleotide sequence or encoding a particular polymerase proteins.

In one embodiment, for example, an HBV variant may be identified having a particular mutation in its polymerase conferring resistance or decreased sensitivity to a nucleoside or nucleotide analog or a non-nucleoside or non-nucleotide agent. This variant may then be mutated to render it defective, i.e. attenuated or unable to cause infection. Such a defective, nucleoside or nucleotide analog-resistant virus may then be used as a therapeutic vaccine against virulent viruses having the same mutation in its polymerase.

The subject invention extends to kits for assays for variant HBV resistant to ABC and/or LMV. Such kits may, for example, contain the reagents from PCR or other nucleic acid hybridization technology. A particularly useful assay includes the reagents and components required for immobilized oligonucleotide- or oligopeptide-mediated detection systems.

Suitably, the antiviral agent is another nucleoside analogue effective in treating HBV such as but not limited to ABC and/or LMV or optionally ADV, TDF and/or ETV and/or FTC or combinations thereof.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1 Overlapping genome of HBV

The overlapping genome of HBV is represented in Figure 1. The gene encoding DNA polymerase (P), overlaps the viral envelope genes, Pre-Sl and Pre-S2, and partially overlaps the X and core (C) genes. The HBV envelope comprises small, middle and large HBV surface antigens. The large protein component is referred to as the HBV surface antigen (HBsAg) and is enclosed by the S gene sequence. The Pre-Sl and Pre-S2 gene sequences encode the other envelope components.

EXAMPLE 2

Patient

Patient A is a 55 year old male with HBV and HIV co-infection. He had a CD4 of 40 and an HIV viral load of 97,300 c/ml and ALT 36. The patient was on antiretroviral therapy Azidothymidine (AZT) and Nevirapine. The AZT was ceased after 1 month due to anaemia. The patient was subsequently treated with ABC and LMV.

While on combination retroviral therapy including ABC and LMV the patient's HBV DNA became 5pg/ml. In a subsequent analysis his CD4 was 197 (8%), HIV viral load<50, The patients liver function test remained at ALT 45. While on this combination treatment the Patient selected unique mutations in the HBV polymerase gene and HBV PreSl gene (refer to Example 5).

The patient subsequently was treated with TDF and responded.

EXAMPLE 3 Detection of Viral Markers

Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), anti-HBe and hepatitis B core antigen (HBcAg) specific IgG and IgM were measured using commercially available immunoassays (Abbott Laboratories, North Chicago, IL, USA). Hepatitis B viral DNA levels were measured using a capture hybridisation assay according to the manufacturer's directions (Digene Hybrid Capture II, Digene Diagnostics Inc., Beltsville, MD). The manufacturer's stated cut-off for detecting HBV viremia in clinical specimens was 0.7 x 10⁶ copies/ml or 2.5 pg/ml, [Hendricks DA, et ah, Am J CHn Pathol 104: 537-46, 1995].

EXAMPLE 4 Sequencing of HBV DNA

HBV DNA was extracted from lOOμl of serum collected at 6 different time points (Figure 2) as described previously by Aye et ah, J Hepatol. 26: 1148-53, 1997. Oligonucleotides were synthesized by Geneworks, Adelaide, Australia. Amplification of the HBV polymerase gene has been described by Aye et ah, 1997, supra.

The specific amplified products were purified using PCR purification columns from MO BIO Laboratories Iiic (La Jolla, CA) and directly sequenced using Big Dye terminator

Cycle sequencing Ready Reaction Kit (Perkin Elmer, Cetus Norwalk, CT). The PCR primers were used as sequencing primers, OSl 5'- GCC TCA TTT TGT GGG TCA CCA

TA -3' (nt 1408-1430) [SEQ ID NO: 1], TTA3 5'-AAA TTC GCA GTC CCC AAA -

3'(nt2128-2145) [SEQ ID NO: 2], JM 5'- TTG GGG TGG AGC CCT CAG GCT - 3'(ntl 676-1696) [SEQ ID NO: 3], TTA4 5' -GAA AAT TGG TAA CAG CGG -3'(nt

2615-2632) [SEQ ID NO: 4], OS2 5' TCT CTG ACA TAC TTT CCA AT 3' (nt 2798-

2817) [SEQ ID NO: 5], to sequence the internal regions of the PCR products.

EXAMPLE 5 Analysis of HBV DNA

Patient A: The ABC resistant mutations at rtH234Y were detected by sequencing after (Table 3) A unique mutation was also selected at PreSl 19 IV. These unique changes were compared to reference sequences from each of the seven genotypes A-G as well as a consensus sequence from pretreatment samples to determine unique changes. TABLE 3

Summary of HBV mutations inpatient A treated with TDF and LMV

Nomenclature according to Stuyver et al, 2001, supra

EXAMPLE 6 ABC

Abacavir ([lS,4R]-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-l- methanol; ABC) is a bio-oral drug that has activity against both HIV And HBV. ABC (Ziagen, formerly 1592 from Glaxo Wellcome) was approved by the FDA for the treatment of HIV in 1999. The structure of ABC is shown in Figure 2.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

Alien ed/., Hepatology 27(6): 1670-1677, 1998

Aye et al., J. Hepatol. 2(5:1148-53, 1997

Das et al, J. Virol. 75(10): 4771-4779, 2001

Gaillard et al, Antimicrob Agents Chemother. 46(4): 1005-1013, 2002

Hendricks et al, Am. J. Clin. Pathol. 104:437-46, 1995

Ren andNassal, J. Virol. 75(3): 1104-1116, 2001

Staszewski et al, AIDS. 72:197-202, 1998

Stuyver et al, Hepatology 53:751-57, 2001

Walters et al, Antimicrob Agents Chemother. 47:1936-42, 2003

Xiong et al, Hepatology. 28(6): 1669-73, 1998

Claims

1. An isolated Hepatitis B virus (HBV) variant wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase and wherein said variant exhibits decreased sensitivity to one or more nucleotide analogs selected from the list consisting of ABC; ABC and LMV; and another nucleoside or nucleotide analog selected from ADV, TDF and/or ETV and/or FTC or a non-nucleoside or non-nucleotide agent.

2. The isolated HBV variant of Claim 1 wherein the decreased sensitivity is in respect of ABC.

3. The isolated HBV variant of Claim 1 wherein the decreased sensitivity is in respect of ABC and LMV.

4. The isolated HBV variant of Claim 1 or 2 or 3 wherein the decreased sensitivity is in respect of ADV.

5. The isolated HBV variant of Claim 1 or 2 or 3 wherein the decreased sensitivity is in respect of TDF.

6. The isolated HBV variant of Claim 1 or 2 or 3 wherein the decreased sensitivity is in respect to FTC.

7. The isolated HBV variant of Claim 1 or 2 or 3 wherein the decreased sensitivity is in respect of ETV.

8. The isolated HBV variant of Claim 1 wherein said variant comprises a rtH234Y mutation in the DNA polymerase.

9. The isolated HBV variant of Claim 1 or 7 wherein said variant comprises a pre Sl 19 IV mutation in the envelope antigen.

10. A method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside or nucleotide analog selected from ABC; and ABC and LMV or optionally or alternatively other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and A through E or a region proximal thereto of said DNA polymerase and associated with resistance or decreases sensitivity to one or more of ABC or ABC with LMV, wherein the presence of such a mutation is an indication of the likelihood of resistance to said one or more of ABC, LMV, ADV, TDF and/or ETV and/or FTC.

11. The method of Claim 10 wherein the mutation screened for is rtH234Y or an equivalent mutation.

12. The method of Claim 10 wherein the mutation screened for is Pre Sl I91V or an equivalent mutation.

13. A method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or non-nucleoside or non-nucleotide agent, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase wherein the presence of a mutation in a region selected from the F to G domain, between F and A domains, the A domain, between the A and B domains, the B domain, between the B and C domains, to C domain, between the C and D domains, the D domain, between the D and E domain and the E domain or combinations thereof or an equivalent one or more other mutation is indicative of a variant wherein said variant exhibits a decreased sensitivity to one or more of ABC, LMV, ADV, TDF and/or ETV and/or FTC optionally or alternatively other nucleoside or nucleotide analogs or non-nucleoside or non-nucleotide agents.

14. The method of Claim 13 wherein the mutation screened for is rtH234Y or an equivalent mutation.

15. The method of Claim 13 or 14 wherein the mutation screened for is Pre Sl 19 IV or an equivalent mutation.

16. A method for detecting an agent which exhibits inhibitory activity to an HBV which exhibits resistance or decreased sensitivity to one or more of ABC, LMV, ADV, TDF and/or ETV and/or FTC said method comprising:

17. The method of Claim 16 wherein the HBV variant comprises a mutation in rtH234Y.

18. The method of Claim 16 or 17 wherein the HBV variant comprises a mutation in Pre Sl I91V.

19. A computer product for assessing the likely usefulness of a viral variant or biological sample comprising same for determining an appropriate therapeutic protocol in a subject, said product comprising:

(1) code that receives as input code for at least two features associated with said viral agents or biological sample comprising same, wherein said features are selected from:

(a) the ability to exhibit resistance for reduced sensitivity to a particular compound or immunological agent;

(b) an altered DNA polymerase from wild-type HBV;

(c) an altered surface antigen from wild-type HBV; or

(d) morbidity or recovery potential of a patient;

(2) code that adds said input code to provide a sum corresponding to a value for said viral variants or biological samples; and

(3) a computer readable medium that stores the codes.

20. The computer product or composition of Claim 19 wherein the input code is resistant to one or more ABC, LMV, ADV, TDF and/or ETV and/or FTC.

21. The computer product or composition of Claim 19 or 20 wherein the input code is a rtH234Y mutation.

22. The computer product or composition of Claim 19 or 20 or 21 wherein the input code is Pre Sl 19 IV mutation.

23. Use of an HBV variant resistant to ABC or ABC and LMV in the manufacture of a medicament for the treatment or prophylaxis of HBV infection.

24. Use of an HBV variant resistant to ADV, TDF, ETV or FTC and selected following exposure to ABC or ABC and LMV in the manufacture of a medicament from the treatment or prophylaxis of HBV infection.

25. Use of Claim 23 or 24 wherein the HBV variant comprises a rtH234Y mutation.

26. Use of Claim 23 or 24 or 25 wherein the HBV variant comprises a Pre Sl I91V.

27. A kit for an assay for variant HBV resistant to ABC or ABC and LMV and/or one or more of ADV₅ TDF, ETV or FTC, said kit comprising genetic agents capable of detecting an altered DNA polymerase gene on the HBV variant.