AU3141501A - An assay for detecting variant hapatitis b viruses (hbvs) which exhibit altered sensitivity to agents - Google Patents

Description

WO 01/57244 PCT/AUO1/00098 An assay for detecting variant Hepatitis B viruses (HBVs) which exhibit altered sensitivity to agents FIELD OF THE INVENTION 5 The present invention relates generally to an assay for detecting variant Hepatitis B viruses (HBVs) which exhibit altered sensitivity to agents. The variant HBVs are delivered to cells using a baculovirus vector. The same agents generally have a particular effect or absence of effect on a reference HBV. The altered sensitivity is in relation to the effects of the agent on one or more stages of infection, replication, assembly or release of virus or virus 10 like particles including any intermediary steps during the processes of viral infection, replication, assembly and/or release. The identification of variant HBVs with altered sensitivities to anti-HBV agents provides a means of monitoring cross resistance, or the development of new therapeutics effective against variant HBVs with altered sensitivities to other anti-HBV agents, as well as monitoring therapeutic protocols which may then 15 need to be modified to ensure the appropriate anti-HBV agent is administered or that the appropriate therapeutic protocol is instituted. The present invention further provides variant HBVs detected by the assay of the present invention and to components thereof as well as recombinant, chemical analogue, homologue and derivative forms of such components. 20 BACKGROUND OF THE INVENTION Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common 25 general knowledge in Australia or any other country. Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. 30 Specific mutations in amino acid sequence are represented herein as "XaainXaa 2 " where Xaa 1 is the original amino acid residue before mutation, n is the residue number and Xaa 2 WO 01/57244 PCT/AUO1/00098 -2 is the mutant amino acid. The abbreviation "Xaa" may be the three letter or single letter amino acid code. A mutation in single letter code is represented, for example, by XinX 2 where X 1 and X 2 are the same as Xaal and Xaa 2 , respectively. The amino acid residues for Hepatitis B virus DNA polymerase are numbered with the residue methionine in the motif 5 "Tyr Met Asp Asp (YMDD)," being residue number 550. The reference HBV is considered herein to comprise a composite or consensus nucleotide or amino acid sequence from HBV genotypes A through G (1, 2). 10 The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating advances in the medical and allied health fields. This is particularly the case with the generation of therapeutic compositions and recombinant vaccines. Recombinant technology is providing the genetic bases for screening or identifying useful components for therapeutic compositions. 15 Hepatitis B virus (HBV) can cause debilitating disease conditions ranging from subclinical infection to chronic active hepatitis and can lead to acute liver failure or fulminant hepatitis. 20 Most patients will suffer an acute hepatitis during which time the virus is eliminated. In flminant hepatitis, patients have acute liver failure and this frequently leads to patient death. About 5% of patients in North America and Europe fail to eliminate the virus, whereas in West Africa, up to 15% of infected patients fail to clear HBV (3). Persistent HBV infection predisposes the host to chronic liver disease and hepatocellular carcinoma 25 (4). The HBV genome comprises a series of overlapping genes in a circular, partially double stranded DNA molecule (5) [see also Figure 1]. These genes encode for four overlapping open reading frames. For example, the gene encoding the DNA polymerase overlaps the 30 viral envelope genes (Pre-Sl, Pre-S2 and S) and partially overlaps the X and core genes. The protein component of the small HBV surface protein is generally referred to as the WO 01/57244 PCT/AUO1/00098 -3 HBV surface antigen (HBsAg) and is encoded by the S gene sequence. The Pre-S1 and Pre-S2 gene sequences encode the other envelope components (6). The core open reading frame encodes for both the hepatitis B core protein (HBcAg) and HBeAg, which starts from a precore initiation codon. HBV variants can have single or multiple mutations in one 5 or more of the overlapping genes. The HBV DNA polymerase is a reverse transcriptase (i.e. an RNA dependent DNA polymerase) and also has DNA dependent DNA polymerase as well as primase and RNase H activity. Nucleoside analogues have been used to inhibit HBV DNA replication. 10 However, mutations have arisen in the gene encoding the HBV DNA polymerase resulting in the development of HBV variants resistant to the nucleoside analogues. Resistance may occur to a single nucleoside analogue or cross-resistance may also occur to an entire family of nucleoside analogues. Furthermore, when the mutation occurs in a region overlapping with the gene encoding HBsAg, alterations may occur to the HBsAg itself leading to the 15 development of vaccine escape mutants. Some precore variants of HBV result in hepatitis B e antigen (HBeAg)-negative hepatitis B. Seven to 30% of patients with chronic HBV infection worldwide are HBeAg-negative and are positive for HBV DNA by hybridisation using commercial tests. One such variant 20 is unable to synthesize HBeAg. A single base substitution (G-to-A) at nucleotide 1896

(A

189 6 ; numbering from the unique EcoRI site) gives rise to a translational stop codon in the second last codon (codon 28) of the precore gene. Other precore and basal core promoter (BCP) mutations are listed in Table 1. Since the core gene itself is not affected, synthesis of the core protein proceeds normally enabling production of virions. Precore 25 A 189 6 mutations occur in both anti-HBe-positive patients with mild disease and those with high level viraemia and severe chronic hepatitis, suggesting that there is not a direct causal association with chronic progressive disease. However, infection with precore mutant virus has been associated with fulminant hepatitis and in the transplantation setting, graft failure (15). 30 WO 01/57244 PCT/AUO1/00098 -4 The HBsAg comprises an antigenic region referred to as the "a" determinant (7). The "a" determinant is complex, conformational and dependent upon disulphide bonding among highly conserved cysteine residues. Genetic variation leading to changes in the "a" determinant has been implicated in mutants of HBV which escape the immunological 5 response generated to conventional vaccines (8-12). One particularly common mutation is a glycine (G) to arginine (R) substitution at amino acid position 145 (G145R) of HBsAg. This mutation affects the "a" epitope region. The increasing reliance on chemical and immunological intervention in treating or 10 preventing HBV infection is resulting in greater selective pressure for the emergence of variants of HBV which are resistant to the interventionist therapy. Due to the overlapping genomic structure of HBV, HBV variants, may be directly or indirectly selected for by the use of chemical agents or vaccines. 15 It is important to be able to detect variant HBVs so that appropriate steps can be taken to modify a therapeutic protocol. This is also particularly important in the development of new therapeutic agents to be effective against known resistant variants of HBV and also when cross resistance develops within a family of chemically related anti-viral agents. 20 HBV baculovirus mediated HBV replication is a transient system and does not require integration of the HBV viral genome. This system was recently described by Delaney et al. (13, 14). The HBV baculovirus system has a number of advantages over standard transient transfection systems and cell lines expressing HBV. 25 In the study of HBV replication and the development of therapeutic agents directed against HBV, some cell lines have been developed which are capable of expressing HBV DNA. However, these cell lines were developed using HBV DNA sequences under the control of heterologous promoters or heterologous regulatory sequences which are unlikely to mimic the situation in a naturally infected cell. 30 WO 01/57244 PCT/AUO1/00098 -5 Furthermore, cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Hepadnavirus genomes are maintained in the nucleus of infected cells in vivo as a pool of episomal, covalently closed circular (CCC) DNA molecules. Although the integration of HBV DNA in human liver has been reported, it is not an obligatory part of 5 the HBV lifecycle and integration is not required for HBV replication. In addition, when integrated HBV DNA is found, it is frequently rearranged and is often transcriptionally silent. Because HBV expressing cell lines contain stably integrated HBV DNA, viral gene expression and replication is continuous; therefore, it is not possible to experimentally control the time or conditions under which these processes are initiated. Stable HBV 10 expressing cell lines contain fixed numbers of integrated typically head-to tail orientated HBV genomes and, as such, HBV gene expression and replication levels cannot be regulated and are restricted to the number of integrated copies which each cell line contains. Consequently, it is not possible to study the effects of increasing or decreasing the copy number of integrated HBV genomes without transfecting the cell line and/or 15 selecting new cell lines. HBV baculovirus infection, even at high multiplicities, is not toxic to cells such as HepG2 or Huh-7. HBV expression can be enhanced or prolonged in a population of HBV baculovirus infected cells simply by superinfection of the cultures. 20 One major difference between baculovirus-mediated gene transfer of HBV to HepG2 cells and stably transfected cell lines is the ability to synchronously initiate the replication process. In a stably transfected HBV cell line, such as a derivative of the HepG2 cell line referred to as "2.2.15", each cell contains virus at all phases of the replication cycle. In 25 contrast, HBV baculovirus infection can be used to synchronously start HBV replication in, for example, HepG2 cells because these cells contain no viral products before infection. In HBV baculovirus infected HepG2 cells, it is possible to follow the time course for secretion of both HBsAg and HBeAg with time after infection using the appropriate recombinant HBV baculovirus. 30 WO 01/57244 PCT/AUO1/00098 -6 There is a need, therefore, to develop a baculovirus system to screen for specific HBV variants having altered sensitivities to particular agents.

WO 01/57244 PCT/AUO1/00098 -7 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 5 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, i.e. <400>1, <400>2, etc. A sequence listing is provided after the claims. 10 The present invention relates generally to- an assay for detecting a variant HBV which exhibits altered sensitivity to agents. In particular, the assay comprises the use of a baculovirus system to screen for sensitivities of HBV variants to particular agents. The agents contemplated herein include chemical agents such as nucleotide and nucleoside 15 analogues and non-nucleoside analogues, immunological agents such as antibodies and cytokines as well as other therapeutic molecules. These agents generally but not exclusively have a known effect against a reference HBV such as inhibiting or reducing HBV infection, replication and/or assembly or release of virus or virus-like particles. The assay generally comprises detecting a variant HBV which exhibits an altered sensitivity to 20 an agent by first generating a genetic construct comprising a replication competent amount of the genome from the HBV variant contained in or fused to an amount of baculovirus genome capable of infecting cells. Before, during or after the cells are infected, an agent to be tested is brought into contact with the cells. There is an optional further step where the cells are again infected with the same construct or a genetic construct comprising the 25 genome of an HBV wild-type or other HBV variant. The cells are then cultured for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences and/or assembly and/or release virus or virus-like particles if resistant to the agent. The cells, cell lysates or culture supernatant fluid are then subjected to viral- or viral component-detection means to determine whether or not the HBV variant has replicated, 30 expressed genetic material and/or assembly and/or has been released in the presence of an WO 01/57244 PCT/AUO1/00098 agent. The presence or absence of the detectable components provides an indication of resistance or sensitivity to the agent.

WO 01/57244 PCT/AUO1/00098 -9 BRIEF DESCRIPTION OF THE FIGURES Figure 1A is a diagrammatic representation showing overlapping genome of HBV. 5 Figure 1B is a diagrammatic representation showing examples of the major mutations detected in the precore, envelope and polymerase genes. Figure 2 is a representation of the amino acid consensus sequence from HBV DNA polymerase proteins encompassing regions which are conserved in the RNA polymerase 10 protein. These regions are shown as domains A-E and are underlined. In the consensus sequence the M in the YMDD motif is designated as amino acid number 550. The amino acids which are subject to mutation during 3TC and/or FCV treatment are shown in bold. An asterisk (*) indicates greater than three amino acid possibilities at this position of the consensus sequence. The HBsAg major hydrophilic region containing the neutralisation 15 domain is indicated by a double line and the polymerase mutations which alter the HBsAg are indicated in italics. Figure 3 is a representation of the nucleotide sequence from various strains of HBV encoding the surface antigen. The amino acid sequence of the surface antigen beginning at 20 amino acid 108 is shown above the nucleotide sequence. Figure 4A is a diagrammatical representation of pBBHVB1.28 carrying the HBV 1.28 genome. 25 Figure 4B is a diagrammatical representation of pBBHVBl.5 carrying the HBV 1.5 genome. Figure 5A is the representation of the nucleotide sequence of HBV 1.28 genome. 30 Figure 5B is the representation of the nucleotide sequence of HBV 1.5 genome.

WO 01/57244 PCT/AUO1/00098 -10 Figures 6A and 6B are graphical representations of antiviral testing performed with wild type HBV baculovirus using A, 3TC; B, PMEA. Figure 6C is a photographic representation of a Southern blot showing the effect of PCV 5 on wild-type HBV baculovirus. Figures 7A to 7C are graphical representations of antiviral testing performed with L526M HBV baculovirus using A, 3TC; B, PMEA; C, PCV. 10 Figures 8A to 8C are graphical representations of antiviral testing performed with L526M/M550V HBV baculovirus using A, 3TC; B, PMEA; C, PCV. Figures 9A to 9C are graphical representations of antiviral testing performed with M5501 HBV baculovirus using A, 3TC; B, PMEA; C, PCV. 15 Figure 10 is a graphical representation showing the competition of radio-labelled [a 2

P]

dCTP by cold dCTP using the endogenous polymerase assay with particles prepared from baculovirus infected cells. 20 Figures 11A to iC are photographic representations showing Southern blot of the intracellular and extracellular HBV DNA production from HepG2 cells transduced with wildtype (WT) and precore (G1896A) recombinant HBV-baculovirus exposed to increasing concentrations of (A) adefovir, (B) lamivudine and (C) penciclovir. IC, intracellular; EC, extracellular; RC, relaxed circular HBV DNA; DS, linear double 25 stranded HBV DNA; SS, single-stranded HBV DNA. Figure 12 is a photographic representation showing Southern blot of intracellular and extracellular HBV DNA production from HepG2 cells transduced with various recombinant HBV-baculovirus M5501, precore/M550I, L526M/M550V and 30 precore/L526M/M550V.

WO 01/57244 PCT/AUO1/00098 -11 Figures 13A to 13F are photographic representations showing Southern blot of intracellular and extracellular HBV DNA production from HepG2 cells transduced with recombinant HBV-baculovirus [M550I and precore/M5 501 (Figures 13A, 13C, 13E) L526M/M550V and precore/L526M/M550V (Figures 13B, 13D, 13F)] exposed to various 5 concentrations of adefovir, or lamivudine, or penciclovir. The extracellular virus production from cells transduced with L526M/M550V was too low to be measured. IC, intracellular; EC, extracellular; RC, relaxed circular HBV DNA; DS, linear double stranded HBV DNA; SS, single-stranded HBV DNA.

WO 01/57244 PCT/AUO1/00098 - 12 The following abbreviations are used in the subject specification: Abbreviations Definitions 3TC, LAM (-)-b-L-2',3'-dideoxy-3'-thiacytidine PMEA 9-(2-phosphonylmethoxyethyl)adenine PCV 9-[4-hydroxy-3-(hydroxymethyl)but- 1-yl]guanine YMDD Amino acid motif from HBV DNA polymerase; Try Met Asp Asp; the Met is residue 550 HBV Hepatitis B virus Xaa Any amino acid Pre-S1, Pre-S2, S Viral envelope genes; S encodes HBsAg HBsAg HBV surface antigen CCC DNA Covalently closed circular DNA moi Multiplicity of infection pfu plaque forming units DNA Deoxyribonucleic acid HBeAg hepatitis B e antigen RNA Ribonucleic acid PCR Polymerase Chain Reaction ELISA Enzyme Linked Immunosorbent Assay p.i. Post infected P Polymerase gene IC intracellular EC extracellular RC Relaxed circular HBV DNA DS Linear double stranded HBV DNA SS Single-stranded HBV DNA LDH lactate dehydrogenase FCV Famciclovir WO 01/57244 PCT/AUO1/00098 - 13 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides an assay using variant HBV baculovirus to screen for the sensitivities of HBV variants to particular agents. These agents generally but not 5 exclusively have a known effect against a reference HBV such as inhibiting or reducing HBV infection, replication and/or assembly and/or release of virus or virus-like particles. The present invention provides an assay to screen for the sensitivities of HBV variants to particular agents. These agents generally but not exclusively have a known effect against a reference HBV such as inhibiting or reducing HBV infection, replication and/or assembly 10 and/or release of virus or virus-like particles. The present invention is predicated in part on the identification of HBV variants which have altered sensitivity to agents which would, under standard conditions, have a particular effect or absence of effect on a reference HBV. 15 Generally, although not exclusively, the agent inhibits or reduces HBV infection, replication and/or assembly and/or release of virus or virus-like particles. The assay determines whether the agent has the same effect on a particular variant HBV. The assay is also useful in determining the extent of cross resistance within a group of chemically or 20 functionally related anti-viral agents. In one embodiment, the variant HBV is an escape mutant. The present invention extends, however, to the case where an agent has little effect on a reference HBV but is effective against the variant HBV. For the present purposes, a "reference" HBV is conveniently regarded as a "wild-type" HBV. 25 The term "variant" is used in its broadest context and includes mutants, derivatives, modified and altered forms of an HBV relative to a reference HBV. A variant generally contains a single or multiple nucleotide substitution, addition and/or deletion or truncation mutation in the viral genome and a corresponding single or multiple amino acid substitution, addition and/or deletion or truncation in a viral peptide, polypeptide or 30 protein.

WO 01/57244 PCT/AUO1/00098 -14 Accordingly, one aspect of the present invention contemplates a method for detecting a variant HBV which exhibits an altered sensitivity to an agent, said method comprising: generating a genetic construct comprising a replication competent amount 5 of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable 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; 10 optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant 15 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 variant virus has 20 replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent. Preferably, the altered sensitivity includes an effect on viral infection, replication and/or assembly and/or release of virus or virus-like particles or an effect on intermediary steps 25 during the processes of infection, replication, assembly and/or release. In a particularly preferred embodiment, the identification of whether the HBV variant is resistant to an agent is determined. Resistance to an agent includes resistance to two or more chemically or functionally related agents as may occur during the development of cross resistance. 30 Accordingly, another aspect of the present invention provides a method for detecting a variant HBV which is capable of infecting, replicating, assembly and/or release in the WO 01/57244 PCT/AUO1/00098 - 15 presence of an agent which inhibits or reduces infection, replication, assembly and/or release of a reference HBV said method comprising: generating a genetic construct comprising a replication competent amount 5 of said variant HBV genome contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting cells with said construct; contacting said cells, before and/or during and/or after infection, with the agent to be tested; 10 optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant 15 HBV to replicate, express genetic material and/or assemble and/or release virus or virus like particles if resistant to said agent; and subjecting the cells or cell lysates to viral- or viral component-detection means to determine whether or not the variant virus has replicated, expressed genetic 20 material or assembled and/or been released in the presence of said agent. The optional step referred to above encompasses testing of the effects of co-infection by the same or other HBVs. 25 Accordingly, another aspect of the present invention provides a method for detecting a variant HBV which is capable of infecting, replicating, assembly and/or release in the presence of an agent which inhibits or reduces infection, replication, assembly and/or release of a reference HBV said method comprising:- WO 01/57244 PCT/AUO1/00098 -16 generating a genetic construct comprising a replication competent amount of said variant HBV genome contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting cells with said construct; 5 contacting said cells, before and/or during, and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant at one 10 or more times after the initial infection; culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic material and/or assemble and/or release virus or virus like particles if resistant to said agent; and 15 subjecting the cells or cell lysates to viral- or viral component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled and/or been released in the presence of said agent. 20 The optional step referred to above encompasses testing of the effects of superinfection by the same or other HBVs. The detection of HBV or its components in cells, cell lysates and culture supernatant fluid may be by any convenient means. For example, total HBV DNA or RNA may be 25 determined, replicative intermediates may be detected or HBV-specific products or gene transcripts may be determined. Suitable assay means includes PCR, nucleic acid hybridization protocols such as northern blots and Southern blots and antibody procedures such as ELISA and Western blot may be employed. 30 An example of an HBV variant of the present invention is a variant obtained following selective pressure in the clinical setting. One form of selective pressure is chemical WO 01/57244 PCT/AUO1/00098 -17 pressure (e.g. via nucleoside analogues) directed to the HBV DNA polymerase which selects for a mutation in the gene encoding HBV DNA polymerase. Due to the overlapping nature of the HIBV genome, a corresponding mutation may also occur in the gene encoding HBsAg (see Figure IB). Accordingly, a mutation in one or more nucleotides encoding 5 HBV DNA polymerase may have an effect on the nucleotide sequence encoding HBsAg. A viral variant may, in accordance with the present invention, carry a mutation only in the DNA polymerase or the surface antigen or may carry a mutation in both genes. The term "mutation" is to be read in its broadest context and includes a silent mutation not 10 substantially affecting the normal function of the DNA polymerase or surface antigen or may be an active mutation having the effect of selection of nucleoside analogue resistance or a vaccine escape mutant phenotype. Where multiple mutations occur in accordance with the present invention or where multiple phenotypes result from a single mutation, at least one mutation must be active or the virus must exhibit at least one altered phenotype such 15 as nucleoside analogue resistance or reduced immunological interactivity of anti-HBs to the surface antigen of a reference HBV. The present invention extends to assaying any HBV mutant carrying a single or multiple substitution, addition and/or deletion or truncation in the amino acid sequence of the 20 catalytic region of the HBV DNA polymerase as compared to the amino acid sequence set forth in Formula I which is considered herein to define a reference HBV: FORMULA I 25 S Z1 L S W L S L D V S A A F Y H Z2 P L H P A A M P H L L Z3 G S S G L Z4 R Y V A R L S S Z 5 S Z 6

Z

7 X N Z 8 Q Z 9 Zio X X X Z 1 L H Z 12

Z

13 C S R Z 14 L Y V S L Z 15 L L Y Z 16 T Z 17 G Z 1 8 K L H L Z 19

Z

2 H P I Z 2 1 L G F R K Z 22 P M G Z 2 3 G L S P F L L A Q F T S A I Z 24

Z

25

Z

26

Z

27

Z

2 8 R A F Z 29 H C Z 3 0 Z 3 1 F Z 32 Y M* D D Z 33 V L G A Z 34

Z

3 5

Z

36

Z

37 H Z 3 8 E Z 39 L Z 4 0

Z

41

Z

42

Z

43

Z

44

Z

45

Z

46 L L Z 4 7

Z

4 8 G I H L N P Z 49 K T K R W G 30 YSLNFMGYZ 5 0

IG

WO 01/57244 PCT/AUO1/00098 - 18 wherein: X is any amino acid; 5 Z 1 is N or D;

Z

2 is I or P;

Z

3 is I or V;

Z

4 is S or D;

Z

5 is T or N; 10 Z 6 is R or N;

Z

7 is N or I;

Z

8 is N or Y or H;

Z

9 is H or Y; Zio is G or R; 15 Zul is D or N;

Z

12 is D or N;

Z

13 is S or Y;

Z

14 is N or Q;

Z

15 is L or M; 20 Z 16 is K or Q;

Z

17 is Y or F;

Z

18 is R or W;

Z

1 9 is Y or L;

Z

20 is S or A; 25 Z 21 is I or V;

Z

22 is I or L;

Z

23 is V or G;

Z

24 is C or L;

Z

25 is A or S; 30 Z 26 is V or M;

Z

27 is V or T; WO 01/57244 PCT/AUO1/00098 - 19 Z 2 8 is R or C;

Z

2 9 is F or P;

Z

3 0 is L or V;

Z

3 1 is A or V; 5 Z 32 is S or A;

Z

33 is V or L or M;

Z

34 is K or R;

Z

35 is S or T;

Z

36 is V or G; 10 Z 3 7 is Q or E;

Z

3 9 is L or S or R;

Z

39 is S or F;

Z

4 0 is F or Y;

Z

4 1 is T or A; 15 Z 42 is A or S;

Z

43 is V or I;

Z

44 is T or C;

Z

4 5 is N or S;

Z

46 is F or V; 20 Z 47 is S or D;

Z

48 is L or V;

Z

4 9 is N or Q;

Z

50 is V or I; and M* is amino acid 550. 25 Accordingly, another aspect of the present invention provides an assay for detecting an HBV variant having an altered sensitivity to an agent, said variant HBV comprising a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the amino acid sequence set forth in Formula I and wherein the variant HBV is selected 30 for by a nucleoside analogue of the HBV DNA polymerase said method comprising:- WO 01/57244 PCT/AUO1/00098 -20 generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and infecting said cells with said construct; 5 contacting said cells, before and/or during, and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; 10 culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus like particles if resistant to said agent; and 15 subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled and/or been released in the presence of said agent. 20 In a related embodiment the present invention is directed to an assay for an HBV variant, in which the HBV variant exhibits reduced sensitivity to an agent which otherwise inhibits or reduces infection, replication or assembly and/or release by a reference HBV, said variant HBV comprising a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the amino acid sequence set forth in Formula I, said 25 method comprising: generating a genetic construct comprising a replication competent amount of said variant HBV genome contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting cells with the construct; 30 WO 01/57244 PCT/AUO1/00098 -21 contacting said cells, before, and/or during, and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a 5 genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant HBV, express genetic material and/or assemble and/or release virus or virus-like particles if resistant to said agent; and 10 subjecting the cells or cell lysates to viral- or viral component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled and/or been released in the presence of said agent. 15 Another HBV variant contemplated by the present invention is in the precore or the basal core promoter sequences. Precore mutations contemplated by the present invention include those listed in Table 1 and include. Particular precore gene and basal core promoter (BCP) mutations include A1814T, C1856T, G1896A, G1897A, G1898A, G1899A, G1896A/ G1899A, A1762T/ G1764A, T1753C, G1757A and C1653T (where the numbering is from 20 the unique EcoRl site in HBV). Accordingly, another aspect of the present invention provides an assay for an HBV variant with an altered sensitivity to an agent, said variant HBV comprising a nucleotide sequence containing a single or multiple nucleotide substitution, addition and/or deletion to the 25 precore nucleotide sequence, said method comprising: generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and infecting said cells with said construct; 30 WO 01/57244 PCT/AUO1/00098 -22 contacting said cells, before and/or during, and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a 5 genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences or assemble if resistant to said agent; an d 10 subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled in the presence of said agent. Preferably, the HBV variant according to this aspect of the present invention has a precore 15 gene or a BCP mutation as listed in Table 1. In a particularly preferred embodiment, the precore or a BCP mutation is selected from A1814T, C1856T, G1896A, G1897A, G1898A, G1899A, G1896A/G1899A, A1762T/ G1764A, T1753C, G1757A and C1653T (where the numbering is from the unique EcoRl site in HBV). 20 In a related embodiment, the present invention contemplates an assay for an HBV variant which exhibits reduced sensitivity to an agent which otherwise inhibits or reduces infection, replication or assembly and/or release by a reference HBV, said variant HBV comprising a nucleotide sequence comprising a single or multiple nucleotide substitution, addition and/or deletion to the precore nucleotide sequence, said method comprising: 25 generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and infecting said cells with said construct; 30 contacting said cells, before, and/or during, and/or after infection, with the agent to be tested; WO 01/57244 PCT/AUO1/00098 - 23 optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; 5 culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences or assemble 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 variant virus has 10 replicated, expressed genetic material or assembled in the presence of said agent. In another embodiment, the HBV variant comprises a precore mutation together with a mutation in the DNA polymerase or the corresponding mutation in the hepatitis B surface antigen (HBsAg). Preferred mutations according to this embodiment result from resistance 15 to lamivudine (LAM) such as but are limited to a precore mutation together with L526M+M550V [M1951] or a precore mutation together with M550I [W196L, W196S or W196STOP]. The mutations given in parenthesis are the corresponding HBsAg mutations following a mutation in the HBV DNA polymerase gene. Preferred precore gene and BCP mutations are A1814T, C1856T, G1896A, G1897A, G1898A, G1899A, G1896A/G1899A, 20 A1762T/G1764A, T1753C, G1757A, G1653T (Table 1). Accordingly, another aspect of the present invention provides an assay for an HBV variant with an altered sensitivity to LAM or another agent, said variant HBV comprising a nucleotide sequence comprising a single or multiple nucleotide substitution, addition 25 and/or deletion to the precore nucleotide sequence, and the DNA polymerase gene and optionally the overlapping HBsAg nucleotide sequence said method comprising: generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus 30 genome capable to infect cells and infecting said cells with said construct; WO 01/57244 PCT/AUO1/00098 -24 contacting said cells, before and/or during, and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a 5 genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences or assemble if resistant to said agent; and 10 subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled in the presence of said agent. In a related embodiment, the present invention contemplates an assay for an HBV variant 15 which exhibits reduced sensitivity to LAM or another agent and which otherwise inhibits or reduces infection, replication or assembly and/or release by a reference HBV, said variant HBV comprising a nucleotide sequence encoding a single or multiple nucleotide substitution, addition and/or deletion to the precore nucleotide sequence, and in the DNA polymerase gene and optionally the HBsAg nucleotide sequence said method comprising: 20 generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and infecting said cells with said construct; 25 contacting said cells, before, and/or during, and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; 30 WO 01/57244 PCT/AUO1/00098 -25 culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences or assemble if resistant to said agent; and subjecting the cells, cell lysates or culture supernatant fluid to viral- or 5 viral-component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled in the presence of said agent. Preferably, the HBV variants according to these aspects of the present invention has a precore gene and BCP mutation as listed in Table 1. In a particularly preferred 10 embodiment, the precore or a BCP mutation is selected from A1814T, C1856T, G1896A, G1897A, G1898A, G1899A, G1896A/ G1899A, A1762T/ G1764A, T1753C, G1757A and C1653T (where the numbering is from the unique EcoRl site in HBV). Preferably, the mutation of the DNA polymerase gene [and HBsAg] is L526M+M550V [M1951] or M5501 [W196L, W196S or W196STOP]. 15 Another example of a variant to be tested in accordance with the present invention is one with an altered immunological profile. Such a variant would substantially not be affected by a neutralizing immune response directed to a conventional HBV vaccine such as a vaccine comprising a reference HBV or a surface component thereof. Similarly, the variant 20 HBV may substantially not be affected at the infection, replication, assembly or release level or another level during the life cycle of the HBV by an agent which is capable of inhibiting or reducing infection, replication, assembly or release of a reference HBV. The expression "substantially not affected" includes reduced susceptibility to the immune response generated by a vaccine or reduce susceptibility to chemical agents such as 25 nucleoside analogues which target HBV genes such as the DNA polymerase. Due to the overlapping nature of reading frames for DNA polymerase and certain viral surface components, an altered surface component may have a corresponding alteration in the DNA polymerase. 30 A preferred surface component of the HBV of the present invention is HBsAg. It is proposed in accordance with this aspect of the present invention that the HBsAg of the WO 01/57244 PCT/AUO1/00098 - 26 HBV variants exhibit an altered immune profile relative to an HBsAg from a reference HBV. For the purposes of the present invention, a reference HBV conveniently comprises an HBsAg with an amino acid sequence substantially as set forth by Norder et al. (1) and Stuyver et al. (2) which encompasses all known genotypes of HBV (currently A through 5 G). The assay of the present invention can be used to screen for the effect of the altered HBsAg on the sensitivity of a variant HBV to a particular agent. The present invention extends, therefore, to any single or multiple amino acid substitution, addition and/or deletion or truncation in the amino acid sequence of HBsAg relative to the 10 amino acid sequence set forth in Formula II below as defined by a single or multiple amino acid substitution, addition and/or deletion to the catalytic region of the HBV DNA polymerase set forth above in Formula I. The amino acid sequence of an HBsAg and which is considered to define a reference HBV 15 is set forth below in Formula II: FORMULA I1 M X 1

X

2

X

3

X

4 S G X 5 L X 6 P L X 7

VLQAX

8

X

9 F X 1 o L T X 11 I X 12

X

13 I P X 14 S L 20 X 1 5 S W W T S L N F L G X 1 6

X

1 7

X

1 s X 1 9 C X 2 o GX 21 N X 22 Q S X 23

X

24 S X 25 H X 26 P

X

27

X

28 C P PX 2 9 C X3GYR W MC LX 3 1 RFIIF LX 32 ILLLC LIF LL V LLD

X

33 Q GMLX 34 V C P L X 35 P X 3 6

X

37

X

3 8 T T S X 39

X

4 0 X 41 C X 42 T C X 43

X

44

X

45

X

4 6 Q GX 47 S X 4 s X 49 P X 5 o X 51 C C X 52 K P X 53

X

54 G N C T C I P I P S X 55 W A X 56

X

57

X

5 8 X 59 L W E X 60

X

61 S X 62 R X 63 S W L X 64

LLX

65

X

66 F V Q X 67

X

6 8 X 6 9 X 7 o L X 71 P 25 X 72 V W X 73

X

74

X

75 I W X 76

X

77 W X 78 W X 79 P X 80

X

81

X

8 2 X83 I X 8 4 X 85 P F X 86 P L L P I F X8 7

X

8 8 L X8 9 X90 X 91 I wherein: 30 X 1 isEorGorD;

X

2 is N or S or K; WO 01/57244 PCT/AUO1/00098 -27 X 3 is I or T;

X

4 is T or A;

X

5 is F or L;

X

6 is G or R; 5 X 7 is L or R;

X

8 is G or V;

X

9 is F or C;

X

10 is L or S or W; Xu 1 is R or K; 10 X 12 is L or R;

X

13 is T or K;

X

14 is Q or K;

X

15 is D or H; X16 is G or E or A; 15 X17 is S or A or V or T or L;

X

18 is P or T;

X

1 9 is V or R or T or K or G; X20 is L or P; X21 is Q or L or K; 20 X22 is S or L; X23 is P or Q; X24 is T or 1;

X

25 is N or S;

X

2 6 is S or L; 25 X27 is T or I; X28 is S or C; X29 is I or T; X3o is P or A; X31 is R or Q; 30 X32 is F or C; X33 is Y or C; WO 01/57244 PCT/AUO1/00098 -28

X

34 is P or H or S;

X

35 is I or L; X3 6 is G or R;

X

37 is S or T; 5 X3s is T or S;

X

39 is T or V or A;

X

40 is G or E or Q; X41 is P or A or S;

X

42 is K or R; 10 X43 is T or M;

X

44 is T or I or S or A;

X

45 is P or T or A or I or L;

X

46 is A or V; X47 is N or T; 15 X48 is M or K or L; X49 is F or Y or I; X50 is S or Y;

X

51 is C or S; X52 is T or I or S; 20 X53 is T or S; X54 is D or A; X55 is S or T; X56 is F or L; X57 is A or G or V; 25 X58 is K or R or T; X59 is Y or F; X6o is W or G; X61 is A or G; X62 is V or A; 30 X63 is F or L; X64 is S or N; WO 01/57244 PCT/AUO1/00098 -29 X 65 is V or A;

X

66 is P or Q;

X

67 is W or C or S;

X

68 is F or C; 5 X 69 is V or D or A;

X

7 o is G or E;

X

7 1 is S or F;

X

72 is T or I;

X

73 is L or P; 10 X 74 is S or L;

X

75 is A or V;

X

7 6 is M or I;

X

77 is M or I;

X

78 is Y or F; 15 X 79 is G or E;

X

8 o is S or N or K;

X

81 is L or Q;

X

82 is Y or F or H or C;

X

8 3 is S or G or N or D or T; 20 X8 4 is V or L;

X

5 is S or N; X8 6 is I or M or L;

X

87 is F or C;

X

88 is C or Y; 25 X 89 is W or R;

X

90 is V or A; and

X

91 is Y or I or S. Accordingly, another aspect of the present invention provides a method for detecting a 30 variant HBV having an altered sensitivity to an agent, the said variant HBV comprising a surface antigen having an amino acid sequence with a single or multiple amino acid WO 01/57244 PCT/AUO1/00098 -30 substitution, addition and/or deletion or a truncation compared to a surface antigen from a reference HBV such that an antibody generated to the reference surface antigen exhibits altered capacity for neutralizing said HBV variant said method comprising: 5 generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting said cells with said construct; contacting said cells, before, and/or during, and/or after infection, with the 10 agent to be tested; culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences or assemble if resistant to said agent; and 15 subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled in the presence of said agent. The amino acid sequence of the HBsAg of the reference HBV is as set forth in Formula II 20 above. Generally, the method of the present invention is capable of detecting an HBV variant which may be regarded as an escape mutant which it is substantially incapable of being adversely effected by chemical therapy directed against the HBV polymerase or vaccine 25 therapy directed against the surface antigen. The term "escape" mutant also encompasses reduced susceptibility to chemical or vaccine therapy directed to the reference HBV. More particularly, another aspect of the present invention provides a method for detecting a variant HBV which is capable of replicating in the presence of an agent which inhibits or 30 reduces replication of a reference HBV, said reference HBV comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition WO 01/57244 PCT/AUO1/00098 -31 and/or deletion or a truncation compared to a surface antigen from a reference HBV such that an antibody generated to the reference surface antigen exhibits altered capacity for neutralizing said HBV variant said method comprising: 5 generating a genetic construct comprising a replication competent amount of said variant HBV genome contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting cells with the construct; contacting said cells, before and/or during, and/or after infection, with the 10 agent to be tested; culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic material or assemble if resistant to said agent; and 15 subjecting the cells or cell lysates to viral- or viral component-detection means to determine whether or not the variant virus has replicated, expressed genetic material or assembled in the presence of said agent. The present invention also extends to changes in the HBsAg following immunological 20 selection based on vaccines comprising HBsAg or a derivative thereof or an HBV comprising same and wherein the HBsAg comprises an amino acid sequence substantially as set forth in Formula II. An "agent" therefore extends to a chemical agent (e.g. nucleotide and nucleoside 25 analogues and non-iucleoside analogues), an immunological agent (e.g. antibodies or cytokines) or other therapeutic molecule. One group of agents contemplated by the present invention are non-nucleoside analogue reverse transcriptase inhibitors (such as but not limited to AT-61 a phenylpropenamide derivative; 16) and non-nucleoside analogue DNA dependent DNA polymerase inhibitors. A group of nucleoside analogues contemplated 30 herein comprises 3TC, PMEA and PCV and related molecules.

WO 01/57244 PCT/AUO1/00098 -32 Reference to an altered immunological profile in accordance with the present invention in relation to the surface antigen includes reference to an altered humoral or T cell response. Examples of an altered immunological profile include altered specificity to antibodies, altered amino acid sequences of an epitope or within the "a" determinant, an altered 5 capacity to induce proliferation of T cells primed to an HBsAg from a reference HBV. Preferably, the altered immunological profile means that neutralizing antibodies which are capable of substantially neutralising or otherwise reducing serum or blood levels of the reference HBV are substantially incapable of or exhibit reduced capacity to neutralize and/or clear the variant HBV. 10 The HBsAg mutations of the present invention may also be defined in terms of a corresponding mutation in the HBV DNA polymerase. A mutation in the HBV DNA polymerase may be in amino acids 421-431, 426-436, 431-441, 436-446, 441-451, 446 456, 451-461, 456-466, 461-471, 466-476, 471-481, 476-486, 481-491, 486-496, 491-501, 15 496-506, 501-511, 506-516, 511-521, 516-526, 521-531, 526-536, 531-541, 536-546, 541 551, 546-556, 551-561, 556-566, 561-571, 566-576, 571-581, 576-586, 581-591, 586-596, 591-601, 596-601 (referring to the amino acid numbering in Figure 2). Preferred HBV DNA polymerase mutations include L4261/V, L428I/V, Q476, N480G, 20 N485K, K495R, R499Q, G499E, W499Q, F512L, 1515L, V519L, L526M, M550V, M5501, V5531, S565P. Useful multiple mutants include L526M/M550I, L526M/M550V, V519L/L526M/M550V and V519L/L526M/M550I. Preferred mutations in the amino acid sequence of HBsAg are amino acid substitutions, 25 deletions and/or additions or truncations in amino acids 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, 70-80, 75-85, 80-90, 85-95, 90-100, 95-105, 100-110, 105-115, 110-120, 115-125, 120-130, 125-135, 130-140, 135 145, 140-150, 145-155, 150-160, 155-165, 160-170, 165-175, 170-180, 175-185, 180-190, 185-195, 190-200, 195-205, 200-210, 205-215, 210-220, 215-225, 220-226 (referring to 30 the numbering of Formula II) of HBsAg. Particularly useful mutations are G112R, T123P WO 01/57244 PCT/AUO1/00098 -33 Y/F134S, D144E, G145R, A157D, E164D, F170L, M1951, W196L, W196S, W196STOP, M198I, W199S, S204T and S210R. The term "stop" means a stop codon. Even more preferred mutations are D144E, G145R, A157D, E164D, M1951, W196L, 5 W196S, W196STOP, M1981, W199S and S210R. Particularly preferred precore gene and BCP mutations are selected from A1814T, C1856T, G1896A, G1897A, G1898A, G1899A, G1896A/ G1899A, A1762T/ G1764A, T1753C, G1757A and C1653T (where the numbering is from the unique EcoRl site in 10 HBV). The present invention extends to combinations of two or more of the above mutations such as but not limited to a precore mutation and a mutation in the DNA polymerase and optionally the overlapping HBsAg nucleotide sequences resulting in, for example, reduce 15 sensitivity to LAM. Examples of the latter include but are not limited to a precore mutation such as G1896A with L526M+M550V [M1951] or M550I [W196L, W196S or W196STOP]. The altered HBsAg molecules of the HBV variants of the present invention may also be 20 defined at the nucleotide level. The nucleotide sequence encoding the HBsAg from a reference HBV is set forth below in Formula III: FORMULA III 25 A C N1 A A A C C T N2 N3 G G A N4 G G A A A N 5 T G C A C N 6 T G T A T T C C C ATCCC AT C N 7 T CN 8 T GG GC TT TC GN 9

AAN

1 0 ATN C C TAT GGG A G N 12 G G G C C T C A G N 13 C C G T T T C T C N 14 T G G C T C A G T T T A C T AGT GC CATTT GTT CAGT GGTT C GN 15 A GGGCT TT C C C C CAC T GTN 16 T GGC TTT CAGN 17 TATAT GGAT GAT GT GGTNs TT GG 30 GGGC CAAGT C T GTACAN19 CAT CN 2 0TGAGTC CC TTTN21 TN 22 WO 01/57244 PCT/AUO1/00098 -34 C CN 23

CTN

24 TT A C CA AT TTTCTTN 2 5 TGT C TN 26

TGGGN

27 AT A C ATT wherein: 5

N

1 is A or C;

N

2 is T or A;

N

3 is C or T;

N

4 is C or T; 10 N 5 is C or T;

N

6 is C or T;

N

7 is A or G;

N

8 is T or C;

N

9 is C or G; 15 Nio is G or A;

N

1 1 is T or A;

N

12 is T or G;

N

13 is T or C;

N

14 is C or T; 20 N 15 is T or C;

N

16 is T or C;

N

17 is T or C; Nis is A or T;

N

19 is A or G; 25 N 20 is T or G;

N

2 1 is A or T;

N

22 is A or G;

N

23 is T or G;

N

24 is A or G; 30 N 25 is T or C;

N

26 is T or C; and WO 01/57244 PCT/AUO1/00098 -35

N

27 is T or C. Accordingly, another aspect of the present invention provides an assay for an HBV variant which an altered sensitivity to an agent, said variant HBV comprising a nucleotide 5 sequence comprising a single or multiple nucleotide substitution, addition and/or deletion to the nucleotide sequence set forth in Formula III and which HBV variant has a surface antigen exhibiting an altered immunological profile relative to a surface antigen defined by Formula II said method comprising: 10 generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and infecting said cells with said construct; contacting said cells, before, and/or during, and/or after infection, with the 15 agent to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; 20 culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences or assemble 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 variant virus has 25 replicated, expressed genetic material or assembled in the presence of said agent. Generally, the effective amount of HBV genome required to be inserted into the baculovirus genome is functionally equivalent to but comprises more than 100% of an HBV genome. For example, constructs containing approximately 1.05, 1.1, 1.2, 1.28, 1.3, 30 1.4, 1.5 and 1.6-1.9, 2.0 and 3.0 times the HBV genome are particularly useful.

WO 01/57244 PCT/AUO1/00098 -36 Any cells which are capable of infection by baculovirus may be used in the practice of the present invention. The hepatoblastoma cell line, HepG2, or its derivatives, is particularly useful and is capable of in vitro cell culture. Huh-7 cells may also be used. Alternatively, any hepatocyte cell line and primary hepatocyte cell culture may be used. 5 For convenience, a genetic construct comprising an HBV genome and an infection effective amount of baculovirus genome is referred to herein as "HBV baculovirus", "recombinant HBV baculovirus" and "HBV baculovirus vector". Recombinant HBV baculovirus is an efficient vector for the delivery of HBV genetic information to human 10 cells and can be used to initiate HBV gene expression and replication in the cells. HBV transcripts, intracellular and secreted HBV antigens are produced and replication occurs as evidenced by the presence of high levels of intracellular, replicative intermediates and protected HBV DNA in the medium. Viral CCC DNA can be detected indicating that, in this system, HBV core particles are capable of delivering newly synthesized HBV 15 genomes back into the nucleus of infected cells. Strong HBV gene expression can be detected as early as one day post-infected (p.i.) High levels of HBV replicative intermediates, extracellular DNA, and CCC DNA persist through at least 11 days p.i. Endogenous HBV enhancers and promoters may be used to obtain high levels of HBV expression and replication in the cells. 20 The level of HBV expression and replication in the cells infected with HBV baculovirus can be altered over a considerable range simply by changing the moi. Furthermore, co-infection or superinfection may occur using the genomes from two or 25 more types of HBV such as two or more HBV variants or a variant and a wild-type strain. In this regard, a number of quasi-species of HBV are generally isolated from subjects infected with HBV which consists of two or more HBV variants, or a variant (or variants) and a wild-type strain. It is important, in some circumstances, to test for these quasi species and to determine the altered sensitivity or resistance of the quasi-species to the therapeutic 30 agents. Nucleic acid or antibody detection systems may be used to detect relative amounts of the different HBVs. This is important for the development of therapeutic protocols WO 01/57244 PCT/AUO1/00098 -37 which will need to be effective against HBV variants of a single species as well as multiple HBV variants including the wild type strain. Reference to "HBV" or its "components" in relation to the detection assay includes 5 reference to the presence of RNA, DNA, antigenic molecules or HBV-specific activities. Conveniently, the assay is conducted quantitively, partially quantitively or qualitively. Most preferably, total HBV RNA or DNA is detected which provides an amount of RNA or DNA in the presence of a particular agent. When the HBV variant is more resistant to a particular agent relative to a wild-type strain, then a graphical representation of total RNA 10 or DNA versus concentration of agent is likely to result in a reduced gradient of inhibition and/or an increase in the concentration of agent required before inhibition of RNA or DNA generation. Another aspect of the present invention provides a method for the detection of polymerase 15 activity from HBV particles isolated from variant HBV baculovirus infected cells and determining the sensitivity of said polymerase to nucleoside triphosphate analogues or non-nucleoside analogues reverse transcriptase inhibitors or non-nucleoside analogues DNA dependent DNA polymerase inhibitors. The HBV particles can be collected from variant HBV cell culture fluid, cell lysates or infected cells. This assay will determine the 20 effect of the said nucleoside triphosphate analogues or non-nucleoside analogues reverse transcriptase inhibitors or non-nucleoside analogues DNA dependent DNA polymerase inhibitors on the reference HBV and variant HBV. Accordingly, this aspect of the present invention provides a method for detecting a variant 25 HBV comprising DNA polymerase which exhibits an altered sensitivity to an agent, said method comprising: generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus 30 genome capable to infect cells and then infecting said cells with said construct; WO 01/57244 PCT/AUO1/00098 -38 optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant 5 HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus like particles; and subjecting the cells, cell lysates or culture supernatant fluid or HBV particles purified therefrom to a HBV DNA polymerase assay in the presence or absence 10 of nucleoside triphosphate analogues or non-nucleoside analogues reverse transcriptase inhibitors or non- nucleoside analogues DNA dependent DNA polymerase inhibitors. A further aspect of the present invention provides a method for detecting a variant HBV comprising DNA polymerase which exhibits an altered sensitivity to an agent said method 15 comprising: generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting said cells with said construct; 20 optionally contacting said cells, before and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a 25 genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus like particles if resistant to said agent; and 30 WO 01/57244 PCT/AUO1/00098 -39 subjecting the cells, cell lysates or culture supernatant fluid or virus purified therefrom to HBV DNA polymerase assay in the presence or absence of nucleoside triphosphate analogues or non-nucleoside analogues reverse transcriptase inhibitors or non nucleoside analogues DNA dependent DNA polymerase inhibitors. 5 Yet a further aspect of the present invention contemplates a method of detecting DNA polymerase activity in the presence of an antiviral agent, said method comprising: generating a genetic construct comprising a replication competent amount 10 of a genome from said an HBV capable of producing said DNA polymerase, said genome contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting said cells with said construct; contacting said cells, before and/or after infection, with the antiviral agent 15 to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV strain; 20 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 strain; and subjecting the cells, cell lysates or culture supernatant fluid or virus purified 25 therefrom to HBV DNA polymerase assay in the presence or absence of nucleoside triphosphate analogues or non-nucleoside analogues reverse transcriptase inhibitors or non nucleoside analogues DNA dependent DNA polymerase inhibitors. The present invention is further described by the following non-limiting Examples.

WO 01/57244 PCT/AUO1/00098 -40 EXAMPLE 1 Overlapping genome of HB V The overlapping genome of HBV is represented in Figure 1. The gene encoding DNA 5 polymerase (P), overlaps the viral envelope genes, Pre-S1 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-S1 and Pre-S2 gene sequences encode the other envelope components. 10 EXAMPLE 2 Amino acid consensus sequence of HB VDNA polymerase The amino acid consensus sequence for HBV DNA polymerase protein from genotypes A 15 through G is shown in Figure 2. EXAMPLE 3 Consensus sequence of HBsAg 20 The nucleotide sequence from various strains of HBV encoding the surface antigen is shown in Figure 3. The amino acid sequence of the surface antigen beginning at amino acid 108 is shown above the nucleotide sequence. EXAMPLE 4 25 HB V variants produced by site directed mutagenesis Table 1 provides a summary of some of the HBV variants produced by site directed mutagenesis.

WO 01/57244 PCT/AUO1/00098 -41 TABLE 1 HB V variants produced by site directed mutagenesis Nucleotide analogue selected polymerase Corresponding surface (S) mutation mutations 1. L426V No change 2. L4261 No change 3. G499E (B domain) D144E 4. W499Q (B domain) G145R 5. F512L (B domain) A157D 6. V519L (B domain) E164D 7. L526M (B domain) No change 8. M550V (C domain) M1951 9. M550V (C domain) M196L 10. M550I (C domain) W196S 11. V5531 (C domain) M198I 12. V55EI (C domain) W199S 13. S565P S21OR Double polymerase mutations Corresponding surface (S) mutation 14. L526M/M550V M1951 15. L526M/M550I W196S 16. L4261/M5501 W196S 17. L426V/M550I W196S Triple polymerase mutations Corresponding surface (S) mutation 18. V519L/L526M/M550V E164D/M1951/W196L 19. V519L/L526M/M550I E164D/W196S 20. L4261/L526M/M5501 W196S 21. L426V/L526M/M550I W196S Precore and basal core promoter mutations (number from the unique EcoR1 site) A1814T C1856T G1896A G1897A G1898A G1899A G1896A/G1899A A1762T/G1764A WO 01/57244 PCT/AUO1/00098 - 42 Precore and basal core promoter mutations (number from the unique EcoRl site) continued T175C G1757A G1653T EXAMPLE 5 Cell culture 5 Sf21 insect cells were maintained in supplemented Grace's insect medium further supplemented with 10% v/v heat-inactivated fetal bovine serum (Gibco BRL, Gaithersburg, MD) in humidified incubator at 28 0 C with CO 2 . HepG2 cells were maintained in minimal essential medium supplemented with 10% v/v heat-inactivated fetal 10 bovine serum (MEM-FBS). HepG2 cells were grown in humidified 37"C incubators at 5% v/v CO 2 . EXAMPLE 6 PREPARATION OF BA CULO VIRUS TRANSFER VECTOR 15 A recombinant transfer vector was created by excising a fragment containing the required amount of variant HBV genome construct and cloning it into the multiple cloning region of a baculovirus vector such as pBlueBac4.5 (Invitrogen, Carlsbad, CA). Figures 4A and 4B show a representation of the plasmid encoding the recombinant transfer vector 1.28 and the 20 1.5 HBV genome construct, respectively. Similar beculovirus transfer vectors may also be constructed from an HBV 1.3 construct. A diagrammatic representation of the recombinant transfer vector HBV 1.3 is shown in Figure 1 of International Patent Application No. PCT/US99/01153 [WO 99/37821]. Analysis of recombinant transfer vector by restriction mapping demonstrated the presence of only one copy of the HBV genome portion in the 25 construct. The nucleotide sequence of the plasmid and the point mutations generated by site directed mutagenesis were confirmed by sequencing using the ABI Prism Big Dye WO 01/57244 PCT/AUO1/00098 -43 Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer's specifications (Perkin Elmer, Cetus Norwalk, CT). EXAMPLE 7 5 The sequence of 1.28 HBVgenome and the 1.5 HBVgenome The sequence of the 1.28 and the 1.5 HBV genome (Figures 5A and 5B, respectively) were elucidated using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer's specifications (Perkin Elmer, Cetus Norwalk, CT). 10 EXAMPLE 8 Generation of recombinant baculoviruses containing the 1.28, 1.5 or 1.3 HBV construct 15 Purified recombinant transfer vector and linear AcMNPV baculovirus DNA were co transfected into Sf21 cells using the BacNBlue transfection kit from Invitrogen (Carlsbad, CA); recombinant viruses were isolated by plaque assay according to the manufacturer's instructions. A series of recombinant viruses were amplified from isolated plaques by infecting 100-mm dishes of Sf21 cells. Viral DNA was extracted from amplified viruses 20 using standard procedures. Purified viral DNA was digested with restriction enzymes and then fractionated by electrophoresis in a 1.0% v/v agarose gel. Southern blotting was performed to determine which virus isolates contained the intact 1.28, 1.5 or 1.3 HBV construct. A Boehringer Mannheim Random Prime DNA Labeling kit (Indianapolis, IN) was used to generate [P 32 ]-radiolabeled probes. A full-length double-stranded HBV 25 genome was used as a template for all radiolabeled probes. Viral DNA sequence was confirmed by PCR amplification of the polymerase catalytic region using the sense primer 5'-GCC TCA TTT TGT GGG TCA CCA TA-3' [<400>1], (nucleotide 1408 to 1430 according to HBV Genebank Accession number M38454) and the antisense primer 5'-TCT CTG ACA TAC TTT CCA AT-3' [<400>2] (nucleotides 2817 to 2798 according to HBV 30 Genebank Accession number M38454). The following primers were utilized for the sequencing of internal regions 5'-TGC ACG ATT CCT GCT CAA-3' [<400>3] WO 01/57244 PCT/AU01/00098 - 44 (nucleotides 2345-2362 according to HBV Genbank Accession number M38454) and 5' TTT CTC AAA GGT GGA GAC AG-3' [<400>4] (nucleotides 1790-1810 according to HBV Genbank Accession number M38454). 5 EXAMPLE 9 Preparative baculovirus amplification and purification Baculoviruses were amplified by infecting suspension cultures of Sf21 cells in log phase at a multiplicity of infection (moi) of 0.5 pfu/cell. Infections were allowed to proceed until a 10 majority of the cells in the flasks showed visible signs of infection (four to five days). Virions were concentrated from infected Sf21 medium by centrifugation at 80,000 x g and purified through a 20-60% w/v sucrose gradient. Purified virus was titrated in quadruplicate in Sf21 cells by end-point dilution. An aliquot of each high titre stock was used for DNA extraction. The polymerase gene was amplified and sequenced to confirm 15 the presence of the site-directed mutagenesis as in Example 8. EXAMPLE 10 Infection of HepG2 cells with recombinant HBV expressing baculovirus 20 HepG2 cells were seeded at approximately 20-40% confluency and then were grown for 16-24 hours before infection. On the day of infection, triplicate plates of cells were trypsinized, and viable cell number was determined with a hemocytometer using Trypan blue exclusion. Average cell counts were calculated and used to determine the volume of high-titer viral stock necessary to infect cells at the indicated moi. HepG2 cells were 25 washed one time with serum-free MEM to remove traces of serum. Baculovirus was diluted into MEM without serum to achieve the appropriate moi using volumes of 1.0, 0.5, and 0.25 ml to infect 100-mm, 60 mm, and 35-mm dishes, respectively. Baculovirus was adsorbed to HepG2 cells for one hour at 37"C with gentle rocking every 15 minutes to ensure that the inoculum was evenly distributed. The inoculum was then aspirated and 30 HepG2 cells were washed two times with phosphate-buffered saline and refed MEM-FBS with or without various concentrations of agents.

WO 01/57244 PCT/AUO1/00098 -45 EXAMPLE 11 Analysis of secreted HBV antigens Detection of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) was 5 performed by radioimmunoassay and microparticle enzyme immunoassay using kits purchased from Abbott Laboratories (Abbott Park, IL). Medium from HepG2 cells was collected, centrifuged at 6,000 g to remove cellular debris, transferred to clean tubes, and stored at 20*C until analysis. HBsAg amounts were calculated from a standard curve constructed using known amounts of HBsAg (provided with the kit). HBeAg values are 10 expressed as fold of positive control. Medium samples were diluted appropriately so that radioimmunassay results were within the standard curve (HBsAg) or below positive control values (HBeAg). EXAMPLE 12 15 Detection of intracellular replicative intermediates HBV core particles were isolated from the cytoplasmic fraction of HepG2 cells lysed in 0.5% w/v NP-40. Cytoplasmic extracts were adjusted to 10 mmol/l McC12 and unprotected DNA was removed by an incubation to 500 sg/ml Proteinase K for 1.5 hours 20 at 37"C. Following sequential phenol and chloroform extractions, nucleic acids were recovered by ethanol precipitation. Nucleic acids were resuspended in 50 pd/l TE (10 mmol/l Tris, 1 mmol/l ethylenediaminetetraacetic acid), normalized by OD260, and digested with 100 gtg/ml RNase (Boehringer Mannheim, Indianapolis, IN) for one hour at 37"C before analysis by electrophoresis and Southern blotting. A BioRad GS-670 imaging 25 densitometer and the Molecular Analyst software (BioRad, Hecules California) was used to analyze suitable exposures of Southern blots. Densitometry data was fitted to logistic dose response curves using the TableCurve2D software package from Jandel Scientific. Logistic dose response equations were used to calculate IC 50 and IC 90 values and co efficients of variation.

WO 01/57244 PCT/AUO1/00098 -46 EXAMPLE 13 Extracellular HBVDNA analysis Conditioned medium was collected from HepG2 cells and subjected to centrifugation at 5 6,000 g for five minutes to remove cellular debris. HBV particles were precipitated with 10% w/v PEG 8000 and were concentrated by centrifugation at 12,000 xg. Viral pellets were resuspended in 1 ml of MEM-FBS and divided into two aliquots. One set of aliquots was treated with 750 pIg/ml Pronase for one hour and then with 500 mg/ml DNase 1 for one hour. Both sets of aliquots were then digested with Proteinase K, and extracted with 10 phenol and chloroform. DNA was precipitated with 0.1 volume of 3 mol/l sodium acetate and 1 volume of isopropanol. Ten micrograms of transfer RNA was added as a carrier during precipitation. Pellets were resuspended in 25 pl of TE and digested with 0.5 mg/ml RNase for one hour. DNA was then analyzed by electrophoresis and Southern blotting. 15 EXAMPLE 14 3TC, PMEA and PCV treatments 3TC and PCV were a gift from Klaus Esser, SmithKline Beecham, Collegeville, PA. PMEA was obtained from Gilead (Foster City, California). 3TC was resuspended in sterile 20 water, aliquoted, and frozen at -20'C to avoid repeated freezing and thawing of the drug. PMEA was resuspended in sterile water after the pH was adjusted to 7.0 with NaOH. PCV was resuspended in dimethyl sulphoxide. Medium containing 3TC was prepared daily as needed using fresh aliquots of 3TC. In experiments in which 3TC treatment was initiated after viral infection, HepG2 cells were exposed to the indicated concentration of 3TC 25 immediately after infection with HBV baculovirus. In experiments utilizing pretreatment with 3TC, cells were fed medium containing 3TC 16 hours prior to HBV baculovirus infection, HBV baculovirus infection was also carried out in medium containing 3TC, and cells were refed fresh medium containing 3TC immediately after completion of the infection and washing procedures. Treatment with PMEA and PCV were conducted in a 30 similar manner.

WO 01/57244 PCT/AUO1/00098 -47 EXAMPLE 15 1. Antiviral testing performed with wild-type HBV baculovirus 5 The dose effect of 3TC and PMEA, on wild-type HBV is shown graphically in Figure 6 (A, B,) and a Southern Blot of intracellular HBV replicative intermediates and extracellular virus produced by HepG2 cells transduced with wild-type HBV-baculovirus in the presence of increasing concentrations of PCV is shown in Figure 6C and the IC 50 for each antiviral agent is shown in Table 2. 3TC had the most pronounced effect whereas 10 PCV had a modest effect on wild-type HBV replication.

WO 01/57244 PCT/AUO1/00098 -48 2. Antiviral testing with L526MHBVbaculovirus Figure 7A shows a pronounced resistance of L526M to 3TC compared to the wild-type in Figure 1A in which there was >0.5 log shift in the IC 50 . No significant change was 5 observed with PMEA or PCV (Figures 7B, 7C). 3. Antiviral testing with L526M/M550VHB V baculovirus Figure 8A shows that L526M/M550V is completely resistant to 3TC. There was no 10 substantial change to PMEA or PCV (Figures 8B, 8C). 4. Antiviral testing with M550IHBVbaculovirus M5501 is completely resistant to 3TC (Figure 9A). The M5501 HBV baculovirus had a 15 similar sensitivity to PMEA as the wild-type virus as in Figure 9B.

WO 01/57244 PCT/AUO1/00098 -49 NO C) I CD 00 m 00 Cr4 0'00 00 t0t Ct- 00-(--1 0'** 00 .0r rjrQ owC 06' t 0 ) Nr-0 t r-- ~0 N -H0 00000000 C:) -H 0C: 00 -H WO 01/57244 PCT/AUO1/00098 - 50 .0 0 .5 t4 tj) ;-q rn ~ ~ fr~7:$ w2I 0 4-4 co ;-q -4 , Cd Cd a) bfl D -c-ri)i I-4t C0 a) C)C) 0 Cd S0 a0 4.)-1 Cd 0 d 0 C0 a) -c.n d~ s'.' f 0 '4 02 -l o 9 ) WO 01/57244 PCT/AUO1/00098 - 51 EXAMPLE 16 1. Preparation of HB Vparticles from HBV baculovirus infected cells 5 HepG2 cells were infected with 1.5 HBV baculovirus at an moi of 50-200. Starting on day three post infection, conditioned medium was collected from infected cells, centrifuged at 3000 x g to remove cellular debris, and stored frozen at -20*C. HBV particles were concentrated from conditioned medium by ultra-centrifugation in an SW28 rotor at 27,000 rpm for 5 hours. Pelleted virus was resuspended in a small volume phosphate-buffered 10 saline, aliquoted and frozen at -20*C. 2. Endogenous polymerase assay using HBV particles prepared from HBV baculovirus infected cells 15 Aliquots of virus were thawed and brought to a final concentration of 0.5% NP-40, 2.5 mM Tris pH 7.5, and 6 mM DTT for 10 minutes at room temperature to disrupt the viral envelope. The virus solution was then brought to 150 mM NaCl, 10 mM MgCl 2 , 20 mM KCL and 10 mM dATP, dGTP and dTTP. 10 mC of 32 P-labled dCTP (approximately 0.2 mM) was added and the reaction was incubated at 37"C for 40 minutes to allow the HBV 20 polymerase to extend the viral DNA genome. In order to stop the reaction it was adjusted to 0.5% SDS, 25 mM Tris, and 10 mM EDTA. The reaction mixture was digested with 500 pg/ml of proteinase K overnight at 37*C and HBV DNA was purified from by one extraction with 1:1 phenol:chloroform followed by ethanol precipitation. DNA pellets were resuspended in a small volume of water and electrophoresed through 1% w/v agarose gels 25 for 2 hours. Gels were dried using a Biorad gel dryer and autoradiography of the dried gels was performed to visualize the amount of 32 P-labelled dCTP incorporated into HBV genomes by HBV polymerase.

WO 01/57244 PCT/AUO1/00098 - 52 3. An assay to measure the competition of radio-labelled [a 32 P]-dCTP incorporation by cold dCTP using the endogenous polymerase assay using HBV PARTICLES prepared from baculovirus infected cells 5 Polymerase assays were performed as described above except that cold dCTP was added to each reaction to compete with the incorporation of labelled dCTP. In separate reactions, concentrations of 0.1, 1, 10, 100, and 1000 mM were added at the same time as labelled dCTP and the polymerase reactions were allowed to proceed. After an extraction of viral DNA and analysis by gel electrophoresis and autoradiography, the amount of labelled 10 DNA from each reaction was quantified by densitometery of the audioradiogram. Densitometry data were fitted to logistic dose response curves using Tablecurve2D software. Figure 10 shows the effect of the competition of cold dCTP on 32 P-labelled dCTP. 15 4. An assay which can be used to measure the efficacy of an antiviral agents in an endogenous polymerase assay, using HBV particles prepared from baculovirus infected cells In an analogous manner, to that described in the above example, the efficacy of antiviral 20 agents such as nucleotide triphosphates and non-nucleoside analogue polymerase inhibitors in inhibiting the catalytic activity of the HBV polymerase can be tested using this assay. The nucleoside triphosphates includes compounds such as 3TC-triphosphate, PMEA triphosphate or PCV- triphospahte. Polymerase assays can be performed as described in Example 20 part (2), except that various concentrations of an antiviral agent can be added 25 to each reaction to compete with the incorporation of labelled dCTP or another radiolabelled deoxynucleotide triphosphate. In separate reactions, various concentrations of the agents are added at the same time as labelled dCTP and the polymerase reactions are allowed to proceed. After an extraction of viral DNA and analysis by gel electrophoresis and autoradiography, the amount of labelled DNA from each reaction is quantified by 30 densitometery of the audioradiogram. Densitometry data is fitted to logistic dose response curves using Tablecurve2D software.

WO 01/57244 PCT/AUO1/00098 -53 EXAMPLE 17 1. Antiviral testing of the precore mutant (G1869A) HBVbaculovirus 5 In this study, comparable HBV DNA production by wild-type (1.3 x genomic length HBV) and the precore mutant HBV was found using the recombinant HBV-baculovirus system. The dose effect of lamivudine, adefovir, and penciclovir on the wild-type HBV /bacuolvirus virus and the precore mutant (G1 896A) HBV are shown in Figure 11 (A, B, C) and the calculated IC50 is shown in Table 3. HBV with the specific mutation of 10 G1896A seemed to be at least as sensitive for adefovir, and may be more sensitive to lamivudine and penciclovir compared with wild type HBV. 2. Antiviral testing with the precore mutant (G1869A), anD L526M/M550V or M550I HB V baculovirus 15 Intracellular HBV DNA and extracellular virus produced by HepG2 cells transduced with various recombinant HBV/baculovirus (including L526M/M550V, precore/L526M/L550V, M550I and precore/M550I) are shown in Figure 12. The HBV DNA yield from the various mutants showed that wild-type and precore mutant were 20 comparable, the M5501 and L526M/M550V mutants showed much lower HBV DNA production than wild-type, and the precore/M550I and precore/L526M/552V mutants had similar levels of DNA production (both intracellular and extracellular) as the precore mutant. The presence of M550I and L526M/M550V changes did not seem to diminish their replication fitness compared to the precore mutant. 25 Intracellular HBV DNA and extracellular virus produced by HepG2 cells transduced with various recombinant HBV-baculovirus (including M550I, precore/M550I, L526M/M550V and precore/L526M/L550V) in the presence of adefovir, or lamivudine or penciclovir are shown in Figure 13 (A-F) and the calculated IC50 for adevoir is shown in Table 4. The 30 adefovir concentration required to inhibit HBV replication (intracellular single-stranded DNA) by 50% (IC 5 o) was 0.94 pM and 0.93 pM for the recombinant HBV-baculovirus WO 01/57244 PCT/AUO1/00098 - 54 mutants M550I and L526M/M550V respectively, and 0.28 pLM and 0.47 IM for precore/M5501 and precore/L526M/M550V respectively. The Southern blots of intracellular HBV replicative intermediates. and extracellular virus produced by HepG2 cells transduced with respective recombinant HBV-baculovirus showed that for any 5 HBV/baculovirus variant encoding the mutations at M550I or L526M/M550V changes conferred a high degree of resistance to lamivudine and penciclovir and no dose response could be plotted. TABLE 3 ICso of adefovir, lamivudine and penciclovir for wild-type (WT) orprecore 10 (GI 896A) recombinant HB V-baculovirus Drugs WT Precore adefovir IC SSI 0.235a 0.195b

IC

50 (pM) EC RC 0.0535 0.025' lamivudine IC SS 0.0697e 0

.

0 2 0 7 f

IC

5 0 W(M) EC RC 0.0189 0.0111 penciclovir IC SS 226 85

IC

50 (IM) EC RC 23 6 a average of 2 experiments (0.25 and 0.22 pM) b average of 2 experiments (0.20 and 0.19 pM) 15 average of 2 experiments (0.06 and 0.047 M) d average of 2 experiments (0.02 and 0.03 jM) C average of 3 experiments (0.067, 0.085 and 0.0572 pM) d average of 3 experiments (0.014, 0.014 and 0.032 M) average of 3 experiments (0.022, 0.0293 and 0.0095 M) 20 h average of 3 experiments (0.012, 0.01 and 0.01 [M) intracellular single-stranded HBV DNA extracellular relaxed circular HBV DNA WO 01/57244 PCT/AUO1/00098 - 55 TABLE 4 IC 5 o of adefovir, lamivudine and penciclovir for recombinant HBV baculovirus encoding changes conferring lamivudine resistance with or without the precore (G1896A) mutation 5 Recombinant adefovir lamivudine penciclovir HBV-baculovirus IC 50 (GM) IC 50 (ptM) IC 50 (pM) M5501 IC SS 0.94 NDR NDR ECRC * * * L526M/M550V IC SS 0.93 NDR NDR ECRC * * * Precore/M550I IC SS 0.28 NDR NDR EC RC 0.09 NDR NDR Precore/L526M/M550V IC SS 0.47 NDR NDR EC RC 1.0 NDR NDR NDR no dose response * HBV DNA production to low to be measured. 10 IC SS intracellular single-stranded HBV DNA EC RC extracellular relaxed circular HBV DNA 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 15 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.

WO 01/57244 PCT/AUO1/00098 -56 BIBLOGRAPHY 1. Norder et al. J. Gen. Virol. 74: 341-1348, 1993. 2. Stuyver et al., A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol. 81: 67-74, 2000. 3. Ryder et al. Lancet ii(8400): 449-52, 1984. 4. Beasley et al. Lancet ii; 1159-63, 1981 5. Tiollais et al. Nature 317: 489-495, 1985. 6. Gerlich et al. Viral Hepatitis and Liver Disease. F.B. Hollinger et al. eds Williams Wilkens, Baltimore, MD, ppl2l-134, 1991. 7. Carman et al. Gastroenterology 102: 711-719, 1992. 8. Carman et al. Lancet 336: 325-329, 1990. 9. Okamoto et al. Paediatric Research 32: 264-268, 1992. 10. McMahon et al. Hepatology 15: 757-766, 1992. 11. Fujii et al. Biochem. Biophys. Res. Commun. 184: 1152-1157, 1992. 12. Harrison et al. J. Hepatol. 13: 5105-5107, 1991. 13. Delaney and Isom HC, Hepatology 28: 1134-46, 1998 14. Delaney et al. Antimicrob Agents Chemother 43: 2017-26, 1999 WO 01/57244 PCT/AUO1/00098 -57 15. Angus et al., Hepatology 21: 14-8, 1995 16. King et al., Antimicrob Agents Chemother. 42: 3179-86, 1998

Claims (24)

1. A method for detecting a variant HBV which exhibits an altered sensitivity to an agent said method comprising: generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting said cells with said construct; contacting said cells, before, and/or during, and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant 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 variant virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.

2. A method according to Claim 1 wherein the variant HBV is capable of replicating in the presence of an agent which inhibits or reduces infection, replication or assembly of a reference HBV.

3. A method according to Claim 2 wherein the agent is a nucleoside analogue or a non-nucleoside analogue. WO 01/57244 PCT/AUO1/00098 - 59

4. A method according to Claim 3 wherein the agent is a non-nucleoside analogue reverse transcriptase inhibitor and/or a non-nucleoside analogue DNA dependent DNA polymerase inhibitor.

5. A method according to Claim 3 wherein the nucleoside analogue is 3TC, PMEA or PCV.

6. A method according to Claim 2.wherein the agent is an immunointeractive molecule.

7. A method according to Claim 6 wherein the immunointeractive molecule is an antibody.

8. A method according to any one of Claims 1 to 7 wherein the variant HBV comprises an altered HBV DNA polymerase.

9. A method according to Claim 7 wherein the altered HBV DNA polymerase is selected from L4261/V, L4281/VN480G, N485K, K495R, R499Q, G499E, W499Q, F512L, I515L, V519L, L526M, M550V, M5501, V553I, S565P.

10. A method according to any one of Claims 1 to 7 wherein the altered HBV is a multiple mutant selected from L526M/M550I, L526M/M550V, V519L/L526M/M550V and V519L/L526M/M5501.

11 A method according to any one of Claims 1 to 7 wherein the variant HBV comprises an altered HBV precore gene or basal core promoter.

12. A method according to Claim 11 wherein the altered HBV precore promoter or basal core promoter is selected from A1814T, C1856T, G1896A, G1897A, G1898A, G1899A, G1896A/ G1899A, A1762T/ G1764A, T1753C, G1757A and C1653T (where the numbering is from the unique EcoR1 site in HBV). WO 01/57244 PCT/AUO1/00098 - 60

13. A method according to any one of Claims 1 to 7 wherein the variant HBV comprises an altered HBsAg.

14. A method according to Claim 13 wherein the altered HBsAg is selected from G112R, T123P Y/F134S, D144E, G145R, A157D, E164D, F170L, M1951, W196L, W196S, W196STOP, M1981, W199S, S204T, S210R.

15. A method according to Claim 14 wherein the altered HBsAg is selected from D144E, G145R, A157D, E164D, M1951, W196L, W196S, W196STOP, M1981, W199S and S210R.

16. A method according to any one of Claims 1 to 7 or any one of Claims 8 to 12 wherein the variant HBV comprises an altered HBV precore promoter or basal core promoter and an altered HBV DNA polymerase.

17. A method according to any one of Claims 1 to 7 or any one of Claims 11 or 12 or any one of Claims 13 to 15 wherein the variant HBV comprises an altered HBV precore promoter or basal core promoter and an altered HBV HBsAg.

18. A method according to any one of Claims 1 to 7 or any one of Claims 8 to 10 or any one of Claims 13 to 15 wherein the variant HBV comprises an altered HBV HBsAg and an altered HBV DNA polymerase.

19. A method according to any one of Claims 1 to 7 or any one of Claims 8 to 15 wherein the variant HBV comprises an altered HBV precore promoter or basal core promoter, an altered HBV HBsAg and an altered HBV DNA polymerase.

20. An HBV variant or a recombinant or derivative form thereof or a chemical equivalent thereof or a recombinant or chemical equivalent of a component thereof detected by the method according to any one of Claims 1 to 19. WO 01/57244 PCT/AUO1/00098 -61

21. A method according to any one of Claims 1 to 7 or any one of Claims 8 to 19 wherein the cells are co-infected with multiple combinations of the variant HBV comprises an altered HBV precore promoter or basal core promoter and/or an altered HBV HBsAg and/or an altered HBV DNA polymerase or combinations thereof.

22. A method according to any one of Claims 1 to 7 or any one of Claims 8 to 19 wherein the cells are superinfected with multiple combinations of the variant HBV comprising an altered HBV precore promoter or basal core promoter and/or an altered HBV HBsAg and/or an altered HBV DNA polymerase or combinations thereof.

23. A method for detecting a variant HBV comprising DNA polymerase which exhibits an altered sensitivity to an agent said method comprising: generating a genetic construct comprising a replication competent amount of the genome from said variant HBV contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting said cells with said construct; contacting said cells, before and/or after infection, with the agent to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV variant; culturing said cells for a time and under conditions sufficient for the variant 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 or virus purified therefrom to HBV DNA polymerase assay in the presence or absence of nucleoside WO 01/57244 PCT/AUO1/00098 -62 triphosphate analogues or non-nucleoside analogues reverse transcriptase inhibitors or non nucleoside analogues DNA dependent DNA polymerase inhibitors.

24. A method for detecting DNA polymerase activity in the presence of an antiviral agent said method comprising: generating a genetic construct comprising a replication competent amount of a genome from an HBV capable of producing said DNA polymerase, said genome contained in or fused to an amount of a baculovirus genome capable to infect cells and then infecting said cells with said construct; contacting said cells, before and/or after infection, with the antiviral agent to be tested; optionally further infecting said cells with the same genetic construct or a genetic construct comprising the genome of HBV wild-type or another HBV strain; culturing said cells for a time and under conditions sufficient for the HBV and 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 or virus purified therefrom to HBV DNA polymerase assay in the presence or absence of nucleoside triphosphate analogues or non-nucleoside analogues reverse transcriptase inhibitors or non nucleoside analogues DNA dependent DNA polymerase inhibitors.