AU780978B2 - Viral variants and methods for detecting same - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION

SEC

NAME OF APPLICANT(S): 104 North Wastcrn Health Care Network ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

*e INVENTION TITLE: "Viral variants and methods for detecting same" The following statement is a full description of this invention, including the best method of performing it known to us: Q\OPER\TDO\2453309 divfile.doc 21t9/01 P.\OPER\TDO\2453309 DIV.DOC- 2119/01 -1- VIRAL VARIANTS AND METHODS FOR DETECTING SAME The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B variants exhibiting complete or partial resistance to nucleoside analogues and/or reduced interactivity with antibodies to viral surface components. The present invention further contemplates assays for detecting such viral variants which assays are useful in monitoring anti-viral therapeutic regimes.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the oooo nucleotide and amino acid sequences referred to in the specification are defined following

I..

15 the bibliography.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common oooo• general knowledge in Australia.

S•Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising" or the term "includes" or variations thereof, will be understood to imply the inclusion of a stated element or integer or group of elements i or integers but not the exclusion of any other element or integer or group of elements or 25 integers. In this regard, in construing the claim scope, an embodiment where one or more features is added to any of claim is to be regarded as within the scope of the invention given that the essential features of the invention as claimed are included in such an embodiment.

Specific mutations in an amino acid sequence are represented herein as "XaalnXaa 2 where Xaal is the original amino acid residue before mutation, n is the residue number and Xaa 2 is the mutant amino acid. The abbreviation "Xaa" may be the three letter or single letter amino acid code. The amino acid residues for Hepatitis B virus DNA polymerase are numbered P.\OPER\TDO\2453309 DIVDOC- 2119/01 -2with the residue methionine in the motif Tyr Met Asp Asp (YMDD) being residue number 550. In the priority document, Australian Patent Application No. P03519, filed 8 November 1996, the same methionine was designated residue 530. The amino acid residues for the DNA polymerase referred to in this specification have been re-numbered accordingly.

Hepatitis B Virus (HBV) can cause debilitating disease conditions and can lead to acute liver failure. HBV is a DNA virus which replicates via an RNA intermediate and utilizes reverse transcription in its replication strategy The HBV genome is of a complex nature having a partially double stranded DNA structure with overlapping open reading frames encoding surface, core, polymerase and X genes. The complex nature of the HBV genome is represented in Figure 1.

The presence of an HBV DNA polymerase has led to the proposition that nucleoside analogues could act as effective anti-viral agents. Examples of nucleoside analogues 15 currently being tested are penciclovir and its oral form famciclovir 3, 4, 5) and S.lamivudine There is potential for such agents to be used in the treatment of chronic HBV infection.

Peniciclovir has been recently shown to have potent inhibitory activity against duck HBV 20 DNA synthesis in vitro and has been shown to inhibit HBV DNA polymerase-reverse transcriptase activity in vitro Similarly, oral famiciclovir has been demonstrated to inhibit intra-hepatic replication of duck HBV virus in vivo In man, famciclovir has been shown to reduce HBV DNA replication in a patient with severe hepatitis B following orfiotopic liver transplantation (OLT) (11).

In work leading up to the present invention, nucleoside analogue antiviral therapy was used to control severe post-OLT recurrence of HBV infection Long term therapy is mandatory where patients are immunosuppressed and the rate of HBV replication is very high. However, under such conditions, as with any long term chemotherapy of infectious agents, there is a potential for development of resistance or reduced sensitivity to the therapeutic agents employed.

P.\OPER\TDO\2453309 DIV.DOC- 2 1/9/01 -3- In accordance with the present invention, the inventors have identified variants of HBV with mutations in the HBV DNA polymerase gene which to varying extents reduce the sensitivity of HBV to nucleoside analogues. The identification of these HBV variants is important for the development of assays to monitor nucleoside analogue therapeutic regimes and to screen for agents which can mask the effects of the mutation. In addition, since the HBV genome comprises a series of overlapping open reading frames, a nucleotide mutation in one open reading frame can affect translation products in another open reading frame. In further accordance with the present invention, the inventors have observed mutations which reduce the interactivity of immunological reagents, such as antibodies and immune cells, to viral surface components. Such viral variants are referred to herein as "escape mutants" since they potentially escape existing immunological memory.

Accordingly, one aspect of the present invention is directed to a variant of an isolated DNA virus which replicates via an RNA intermediate wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase.

oooo Another aspect of the present invention provides a variant of an isolated DNA virus which •replicates via an RNA intermediate wherein said variant comprises a nucleotide mutation in a gene encoding a viral surface component resulting in at least one amino acid addition, substitution and/or deletion in said viral surface component.

Still a further aspect of the present invention is directed to a variant of an isolated DNA virus which replicates via an RNA intermediate at least wherein said variant comprises a nucleotide mutation in an overlapping portion of at least two open reading frames resulting in an amino acid addition, substitution and/or deletion to translation products of said open reading frames.

Preferably, the DNA virus is a hepatitis virus or a related virus and is most preferably HBV.

A "related virus" in accordance with the present invention is one related at the genetic, immunological, epidemiological and/or biochemical levels.

P:\OPER\TDO\2453309 DIV.DOC- 21/9/01 -4- Preferably, the mutation in the DNA polymerase results in decreased sensitivity of the HBV to a nucleoside analogue.

Preferably, the mutation in the viral surface component reduces the interactivity of immunological reagents such as antibodies and immune cells to the viral surface component.

Most preferably, the viral surface component is a viral surface antigen. The reduction in the interactivity of immunological reagents to a viral surface component generally includes the absence of immunological memory to recognise or substantially recognise the viral surface component.

A viral variant may, in accordance with a preferred aspect of the present invention, carry mutation only in the DNA polymerase or the surface antigen or may carry a mutation in both molecules. The term "mutation" is to be read in its broadest context and includes a silent mutation not substantially affecting the normal function of the DNA polymerase or surface antigen or may be an active mutation having the effect of inducing nucleoside analogue resistance or an escape mutant phenotype. Where multiple mutations occur in accordance i 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 as nucleoside analogue resistance or reduced immunological interactivity to the surface antigen.

Regions of the HBV polymerase show amino acid similarity with other RNA-dependent DNA polymerases and RNA-dependent polymerases In this specification, reference is made to the conserved regions defined by Poch et al (13) as domains B and C.

Preferably, the mutation results in an altered amino acid sequence in the B domain and/or C domain or regions proximal thereto of the HBV DNA polymerase. The present invention does not extend to a mutation alone in the YMDD (SEQ ID NO:30) motif of the C domain of the HBV DNA polymerase although such a mutation is contemplated by the present invention if it occurs in combination with one or more mutations in another location.

P: OPER\TDO2453309 DIV.DOC- 2119/01 The mutation in the viral surface component is preferably in one or more amino acid residues within the major hydrophilic regions of the protein, in particular within the amino acid sequence 118-169 of the HBV viral surface antigen and also the regions from amino acids sequence 169 to 207 which are on the external surface of the protein.

According to a preferred aspect of the present invention, there is provided an HBV variant comprising a mutation in the nucleotide sequence encoding a DNA polymerase resulting in an amino acid addition, substitution and/or deletion in said DNA polymerase in its B domain and/or C domain or in a region proximal thereto, provided said mutation is not in the YMDD motif of the C domain alone, and wherein said variant exhibits decreased sensitivity to a nucleoside analogue.

Another preferred aspect of the present invention contemplates an HBV variant comprising a mutation in the nucleotide sequence encoding a viral surface component resulting in an amino acid addition, substitution and/or deletion in said viral surface component in a region corresponding to the B domain and/or C domain of HBV DNA polymerase or a region proximal to the B domain and/or C domain of HBV DNA polymerase and wherein said variant exhibits decreased interactivity of immunological reagents to said viral surface component.

Yet another preferred aspect of the present invention relates to an HBV variant comprising a mutation in the nucleotide sequence encoding a viral surface component resulting in an amino acid addition, substitution and/or addition in said viral surface component in a region defined by amino acids 118 to 169 and also 169 to 207 of the HBV surface antigen or 25 functionally equivalent region wherein said variant exhibits decreased interactivity of immunological reagents to said viral surface component.

Still yet another aspect of the present invention is directed to an HBV variant comprising a mutation in an overlapping open reading frame in its genome wherein said mutation is in the B and/or C domain of DNA polymerase provided that it is not in the YMDD motif of the C domain alone; and in the overlapping region corresponding to amino acids 118 to 169 and also 169 to 207 or equivalent of HBV surface antigen and wherein said variant exhibits P.\OPER\TDO\2453309 DIVDOC- 21/9/01 -6decreased sensitivity to a nucleoside analogue and exhibits decreased interactivity to immunological reagents specific to HBV surface antigens.

The viral variant exhibiting reduced interactivity to immunological reagents is an escape mutant since antibodies or other immunological response to HBV from a prior exposure to the virus or following vaccination are no longer effective in targeting a viral surface component since the mutation has altered a B- and/or T-cell epitope on the surface antigen.

The nucleoside analogues contemplated by the present invention include penciclovir and its oral form famciclovir as well as lamivudine (3TC). Different variants may be resistant to different nucleoside analogues. For example, in one embodiment, a variant in the B domain of HBV DNA polymerase may be resistant to famciclovir whereas a variant in the C domain may be resistant to 3TC.

15 The B domain is considered to comprise amino acid residues 505 to 529 of HBV DNA polymerase. This sequence is represented as follows: S/A H PI I/ LGFRK I/L PMG V/G GLSPFLLAQF. (SEQ ID NO:24) Reference to the B domain includes reference to proximal regions which includes up to about 20 amino acids on either side of the domain. Preferably, the mutation is in one or more of the following amino acids: Q/K T Y/F G R/W KLHL Y/L S/A HPI I/V LGFRK I/L PMG V/G GLSPFLLAQFTSAI C/L S (SEQ ID S* 25 The C domain comprises amino acids 546 to 556 as follows: A/V F S/A YMDD V/L/M VLG (SEQ ID NO:26) This includes the YMDD (SEQ ID NO:30) domain in which the methione residue is considered residue 550 (formally regarded as residue number 530). The residue numbering in this specification has been adjusted according to the new numbering system where the methione of YMDD is 550.

P:AOPERWDO2453309 DIV.DOC 21/9/01 -7- Reference to the C domain includes proximal regions of up to 20 amino acids either side of V the domain.

The term "resistance" is used in its most general sense and includes total resistance or partial resistance or decreased sensitivity to a nucleoside analogue.

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

Preferred mutations in the HBV DNA polymerase include one or more of Arg/Trp499Glu, Thr530Ser, Ile509Val, Phe512Leu, Val519Leu, Pro523Leu, Leu526Met, Ile533Leu, 15 Met550Val/Ile and/or Ser559Thr. Preferred mutations in the HBV surface antigen include one or more of Aspl44Glu and/or Glyl45Arg. These correspond to positions 498 and 499 of DNA polymerase, respectively. More preferably, the variants contain two or more of the above-mentioned mutations.

One particular mutant HBV has the nucleotide sequence set forth in SEQ ID NO:17 and exhibits a multiphenotypic mutation rendering the DNA polymerase resistant to nucleoside analogues and an altered surface antigen such that it has reduced interactivity with antibodies to HBV surface antigen. The mutation is R/W499E in the DNA polymerase open reading frame as D144E and G145R in the surface antigen. This results from a double 25 mutation in nucleotide numbers 226 and 227 of SEQ ID NO:17 to G and A. The polymerase protein of HBV is also similar to the DNA polymerase of Herpes Simplex Virus (HSV) (see Figure 3 for alignment). A mutation (Gly841Cys) in the HSV polymerase gene was selected for in the presence of famciclovir The present invention extends to the nucleotide sequence set forth in SEQ ID NO: 17 as well as a nucleotide sequence having at least 60% similarity thereto and which carries a double mutation in the amino acid sequence of DNA polymerase and the HBV surface antigen.

P:\OPER\TDO\2453309 DIVDOC- 21/9/01 -8- Accordingly, the present invention is directed to an HBV having the nucleotide sequence as set forth in SEQ ID NO: 17 or a derivative thereof having a single or multiple nucleotide addition, substitution and/or deletion thereto such as a nucleotide sequence having at least similarity to SEQ ID NO:17. A derivative includes parts, fragments, portions and homologues of SEQ ID NO:17. This aspect of the present invention also extends to a nucleotide sequence capable of hybridizing under low stringency conditions at 42 0 C to SEQ ID NO: 17.

Reference herein to a low stringency at 42 C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions.

Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for 15 hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.

Accordingly, another aspect of the present invention contemplates a variant HBV exhibiting reduced sensitivity to a nucleoside analogue and reduced interactivity to an antibody to wildtype HBV surface antigen, said HBV variant characterised by one or more of the following characteristics: 25 a nucleotide sequence of its genome as set forth in SEQ ID NO:17 or a sequence having at least 60% similarity thereto; (ii) a nucleotide sequence capable of hybridising to SEQ ID NO: 17 under low stringency conditions at 42"C; (iii) a mutation in an overlapping portion of open reading frames for DNA polymerization and HBV surface antigen; and P oFPERM 2453309 DIVD- 21/9/01 -9a mutation in the B and/or C domain of HBV DNA polymerase and is a region corresponding to amino acids 118 to 169 and also 169 to 207 of HBV surface antigen.

According to another aspect of the present invention, there is provided a variant HBV comprising a nucleotide sequence which encodes a DNA polymerase having the amino acid sequence:

X

1

HPIX

2

LGX

3

RKX

4

PMGX

5

GLSX

6

FLX

7 AQFTSAXsX9 (SEQ ID NO:27)

X

i oFXIIYX1 2

DDXI

3

VLGAXI

4

XI

5 (SEQ ID NO:28) wherein XI is S or A;

X

2 is I or V;

X

3 is F or L;

X

4 is I or L; 15 X 5 is L or V or G;

X

6 is P or L;

X

7 is L or M;

X

8 is I or L;

SX

9 is C or L; Xlo is A or V; X, I is S or A;

X

12 is M or I or V;

X

1 3 is V or L or M;

X

14 is K or R; and/or 25 Xis S orT; and wherein said variant exhibits reduced sensitivity to a nucleoside sensitivity to a nucleoside analogue, such as famciclovir (penciclovir) and/or lamivudine (3TC).

Another embodiment of the present invention is directed to a variant HBV comprising a nucleotide sequence which encodes a surface antigen having at least one amino acid substitution, addition and/or deletion to amino acid residue numbers 118 to 169 and also 169 P.\OPER\TDO\2453309 DIV.DOC- 21/9/01 to 207 of said surface antigen which corresponds to a DNA polymerase having the amino acid sequence:

X

1 6

TX

1 7

X

1 8 X 19

KLHLX

2 0

X

2 1

HPIX

22

LGX

3

RKX

4

PMGX

5

GLSX

6

FLX

7

AQFTSAX

8

X

9 (SEQ ID NO:42).

X

10

FX

1 1

YX

12

DDXI

3 VLGAX1 4

XI

5 (SEQ ID NO:43) wherein: X 1 6 is Q or K;

X

17 is Y or F;

X

18 is G;

X

1 9 is R or W or E;

X

2 0 is Y or L;

X

21 is S or A;

X

22 is I or V;

X

3 is For L;

:X

4 is I or L; V X 5 is Lor Vor G;

X

6 is P or L;

X

7 is Lor M;

X

8 is Ior L; Xg is Cor L; X* X 1 is Aor V; X, 1 is Sor A;

X

12 is Mor Ior V; o.o: 25 X 13 is Vor Lor M;

X

14 is K or R; and/or Sor T; and wherein said variant exhibits reduced interactivity with immunological reagents, such as an antibody, to said surface antigen.

P: \OPER\TDO\2453309 DIV.DOC- 2119/01 -11- Examples of preferred variants comprise the amino acid sequences shown in Figure 4. An example of a particularly preferred mutant is shown in Figure 6 (SEQ ID NO:17).

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

Accordingly, another aspect of the present invention contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside analogue, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in the B domain or C domain or a region proximal thereto of said DNA polymerase wherein the presence of such a mutation is 15 an indication of the likelihood of resistance to said nucleoside analogue.

A further aspect of the present invention provides a method for determining the potential for an HBV to exhibit reduced interactivity to antibody to HBV surface antigen, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a 20 mutation in the nucleotide sequence encoding HBV surface antigen resulting in at least one amino acid substitution, deletion and/or addition in amino acids 118 to 169 and/or 169 to 207 of said surface antigen or a region proximal thereto of said surface antigen wherein the presence of such a mutation is an indication of the likelihood of reducing interactivity of i said antibodies to said mutated surface antigen.

Preferably, the assay determines a mutation resulting in a GluVal5 19Leu substitution and/or a Leu526Met substitution and/or a Pro523Leu substitution and/or a Ser559Thr substitution, and/or Arg/Trp499Glu substitution.

The DNA or corresponding RNA may be assayed or alternatively the DNA polymerase or surface antigen may be screened for the mutation.

P:\OPER\TDO2453309 DIV.DOC- 21/9/01 -12- The detection according to this aspect of the invention may be any nucleic acid-based detection means, for example nucleic acid hybridisation techniques or polymerase chain reaction (PCR). The invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others.

The present invention extends to a range of immunologically based assays to detect variant HBV DNA polymerase or surface antigen. These assays are based on antibodies directed to naturally occurring HBV DNA polymerase or surface antigen which do not, or substantially do not, interact with the variant HBV DNA polymerase or surface antigen. Alternatively, 15 antibodies to a variant HBV DNA polymerase or surface antigen are used which do not or substantially do not, interact with naturally occurring HBV DNA polymerase or surface antigen.

Monoclonal or polyclonal antibodies may be used although monoclonal antibodies are preferred as they can be produced in large quantity and in a homogenous form. A wide range of immunoassay techniques are available such as described in U.S. Patent Nos.

4,016,043, 4,424,279 and 4,018,653.

The detection of amino acid variants of DNA polymerase is conveniently accomplished by 25 reference to the consensus amino acid sequence shown in Figure 4. The polymorphisms shown represent the variations shown in various data bases for active pathogenic HBV strains. Where an HBV variant comprises an amino acid different to what is represented, then such an isolate is considered a putative HBV variant having an altered DNA polymerase activity.

Accordingly, another aspect of the present invention contemplates a method for determining whether an HBV isolate encodes a variant DNA polymerase, said method comprising P:\OPER\TDO\2433309 DIV.DOC- 21t9/01 13 determining the amino acid sequence of its DNA polymerase directly or via a nucleotide sequence and comparing same to the amino acid sequence below: DOMAIN A 421 430 440 450 S DLSWLSLD VSAAFYH pPL HPAAMPHLLiv GSSGLSDRYVA 460 470 480 490 RLSS NSRNNI*N NHQ HGR***DNLH DNSyCSR NLYVS L MLLY QT FG w DOMAIN B 500 510 520 530 KLH L S AHPI LGFRK LPMG G GLSPFLLAQF TSAIC AVV RCR DOMAIN C 540 550 560 F LA S V KS VQ L S FTA AF pHC v vF AY MDD LMVLGA R T G QEHSRE FL Y AS DOMAIN D DOMAIN E S570 580 590 600 V cNsF vLLSD LGI HLNP QKTKRW GYSLNFMGY VI G (SEQ ID NO:29) where the presence of a different amino acid from the consensus sequence indicates a putative HBV variant.

The present invention further contemplates agents which mask the nucleoside analogue resistance mutation. Such agents will be particularly useful in long term treatment by nucleoside analogues. The agents may be DNA or RNA or proteinaceous or nonproteinaceous chemical molecules. Natural product screening such as from plants, coral and microorganisms is also contemplated as a useful potential source of masking agents. The P-\OPEfRTDO\24533O DIVDOC- 21/9/01 -14agents may be in isolated form or in the form of a pharmaceutical composition and may be administered sequentially or simultaneously with the nucleoside analogue.

The subject invention extends to kits for assays for variant HBV. Such kits may, for example, contain the reagents from PCR or other nucleic acid hybridisation technology or reagents for immunologically based detection techniques.

The present invention is further described by the following non-limiting figures and examples.

o o a P:\OPER\TDO\2453309 DIV.DOC- 21/9/01 In the figures: Figure 1 is a diagrammatic representation showing the partially double stranded DNA HBV genome showing the overlapping open reading frames encoding surface core polymerase and X gene.

Figure 2 is a graphical representation showing serum biochemical (ALT) and virological (HBV DNA) profile in the transplant patient and the responses following the introduction of various antiviral treatment programs. Treatment GCV PFF, GCV and FCV[I] and FCV[II] are described in detail in the examples. Treatment GCV PFF is ganciclovir plus 'foscarnet combination treatment GCV is parenteral ganciclovir maintenance therapy and treatment FCV[I] and FCV[II] is oral famciclovir therapy at a dose of 250 mg or 500 mg twice daily, respectively. The day each therapy commenced is shown in brackets. The ALT and the HBV DNA responses are plotted against time from the commencement 15 of antiviral therapy at 6 months post-OLT. The five key time points for the sequence analysis, pre-treatment (PRE-) and days 87, 600, 816 and 1329 post antiviral treatment are shown.

Figure 3 is a representation showing amino acid alignment of the RNA dependent DNA 20 polymerase sequence motifs from HBV, pre-treatment with famciclovir and 370 days posttreatment (total antiviral therapy of 816 days), with the woodchuck hepatitis virus (WHV), human immunodeficiency virus (HIV), and the comparable regions with the DNA polymerase of herpes simplex virus (HSV) (13, 14) (SEQ ID NOs:31-35 and 50-57). The conserved asparagine and glycine residues within the polymerase motifs are in bold 25 type and the amino acid changes found after famciclovir treatment are in bold type and underlined. The location of the mutated amino acid residues within HBV polymerase are shown. The bold face underlined glycine residue in the HSV polymerase becomes a cysteine during penciclovir treatment Figure 4 is a representation showing conserved regions of domain A to E (underlined) of HBV. M in YMDD is designated amino acid number 550. indicates greater than three amino acid possibilities at this position of the consensus sequence (SEQ ID NO:29) P:\OPER\TDO\2453309 DIV DOC- 21/9/01 -16- Figure 5 is a representation showing amino acid alignment of the RNA dependent DNA polymerase sequence motifs from HBV, noting the amino acid changes which have been selected for in the presence of famciclovir and 3TC (SEQ ID NOs:36-41 and 45-49). HBV concensus sequence was derived from published sequences in Genebank/Entrez. The conserved asparagine and glycine residues within the polymerase motifs are in bold type. The amino acid changes found after famciclovir treatment are in bold green type and underlined and after 3TC are in bold blue type and are underlined. The amino acid sequence is shown of the HBV isolated from patient A and patient B, during famicilovir treatment and from Patient C who did not respond to famciclovir and was later treated with 3TC and in which a resistance mutation was selected. The published 3TC changes detected by Ling et al (16) is shown in 3TC 1.

Figure 6 is a representation showing the nucleotide sequence of an HBV variant and *o corresponding amino acid sequences for HBV DNA polymerase and HBV surface antigen showing in bold mutations R/W499E in the polymerase and D144E and G145R in the surface antigen.

9999* o P:\OPER\TDO\2453309 DIV.DOC- 21/9/01 -17- EXAMPLE 1 CASE STUDY 1. PATIENT A The inventors sequenced the HBV polymerase and X open reading frames from a series of isolates from a patient who received antiviral therapy for almost 4 years following post liver transplant recurrence of HBV infection (Figure 2).

The patient (male, aged 42 years) was transplanted because of end-stage liver failure due to chronic HBV infection. The initial post transplant course was unremarkable but by months there was evidence of recurrent infection and very high levels of viral replication and deteriorating liver function The histological picture was consistent with fibrosing cholestatic hepatitis. Antiviral treatment was commenced approximately 6 months post- OLT. Initially, the patient received intravenous (iv) ganciclovir (GCV; 10 mg/kg/day) in combination with iv foscarnet (PFF; 50-125 mg/kg/day; the dose depending on renal function) This is the treatment ofGCV+ PFF described in Figure 2 which lasted for 86 days. Maintenance iv GCV (3.3-6.7 mg/kg/day) three times per week was commenced on day 87 of antiviral treatment (GCV in Figure Oral famciclovir (250 mg, twice daily) was commenced on day 446 of therapy (FCV in Figure 2) which was increased to 500 mg twice daily (FCV [II] in Figure 2) on day 500. The patient is currently on this treatment regime. The clinical and virological details of this patient preceding famciclovir therapy have been reported (12).

Serum samples were routinely collected and stored at -70°C. Informed consent was S 25 obtained from the patient to use these samples for research purposes. Figure 2 shows the alanine amino transferase (ALT) and HBV DNA levels over the entire course of antiviral treatment. The 5 samples chosen for additional studies cover a period of almost four years.

2. PATIENT B Patient B was retransplanted for pre-core mutant associated HBV-related allograph loss 14 months after the initial liver transplant. Antiviral treatment with GCV (7.5 mg/kg/day) was P:OPER\TDO\2453309 DIV.DOC- 21/9/01 -18given for 10 months and then ceased. This was followed by oral famciclovir therapy given (500 mg 3 times/day).

From patient B the entire HBV polymerase gene was sequenced from a serum HBV sample taken post-transplantation after 850 days FCV therapy. The regions encompassing the catalytic domains of the HBV polymerase were sequenced from a serum sample pretransplant prior to FCV treatment.

3. PATIENT C This patient did not respond to famciclovir and was later treated with lamivudine (3TC) (6, 7) in which a resistance mutation was selected.

4. PATIENT D This patient is treated with famciclovir in which resistance mutation is selected.

EXAMPLE 2 VIRAL MARKERS IN SERUM *e Hepatitis B surface antigen (HbsAg), hepatitis B e antigen (HbeAg), anti-HBe, hepatitis B core antigen (HbcAg) specific IgG and IgM, hepatitis A specific IgM, hepatitis delta antigen and antibody, and anti-hepatitis C virus antibody were measured using commercially available immunoassays (Abbott Laboratories, North Chicago, IL). Only the HBV markers were positive. Hepatitis B viral DNA levels were measured and quantified using a capture hybrtidisation assay according to the manufacturer's directions (Digene Diagnostics Inc., 25 Beltsville, MD). This patient was infected with a pre-core HBV mutant pre-OLT (12) and S. this status did not change post-OLT.

EXAMPLE 3 SEQUENCING AND CLONING OF HBV DNA 1. Extraction of DNA from sera: Aliquots of 50 Al of sera were mixed with 150 4l TE (10 mmol/L Tris-HCl (pH 2 mmol/L EDTA), 1% w/v sodium dodecyl sulfate and 1 P \OPER\TDO2453309 DIVDOC- 21/9/01 -19mg/ml pronase and incubated at 37'C for 2 hours. DNA was deproteinised by phenol/chloroform, precipitated with isopropanol and dissolved in 25 ,ul nuclease-free water.

2. Amplification of the viral polymerase and X genes by polymerase chain reaction (PCR): Oligonucleotides were synthesised by Bresatec, Adelaide, Australia. For amplification of the polymerase gene, the sense primer was GGA GTG TGG ATT CGC ACT CC [SEQ ID NO:1] (nucleotides [nt] -40 to -21) and the antisense primer was GCT CCA AAT TCT TTA TA [SEQ ID NO:2] (nt 2831 to 2847). For amplification of the X gene, the sense primer was 5'-CCT TTA CCC CGT TGC CCG GC [SEQ ID NO:3] (nt 2055 to 2074) and the antisense primer GCT CCA AAT TCT TTA TA -3' [SEQ ID NO:4] (nt 2831 to 2847). All nt are numbered from the start of the polymerase gene. Each reaction was carried out using 5 /l of the extracted DNA as template, 1.5 U of Taq polymerase (Perkin Elmer Cetus, Norwalk, CT), 1 tmol/L of sense and antisense primers, 200 pmol/L each of deoxynucleoside triphosphates, 50 mmol/L Kcl, 3.5 mmol/L 15 MgCl, 10 mmol/L Tris-Hcl (pH 8.3) and 0.01% w/v gelatin. Amplification was achieved by 40 cycles of denaturation (94°C for 1 min), annealing (55 0 C for 1 min) and extension (72°C for 1.5 min), followed by a final extension of 7 min (Perkin-Elmer Cetus, Norwalk, CT).

The PCR product was analysed by gel electrophoresis through 1.5 w/v agarose and visualised by UV irradiation after staining with ethidium bromide.

3. Sequencing of the polymerase and X genes of HBV DNA: The specific amplified product was purified using Geneclean II (BIO 101 Inc., La Jolla, CA) and directly sequenced using Sequenase version 2.0 (United States Biochemical Corp., Cleveland, OH).

The PCR primers were used as sequencing primers and internal primers were additionally 25 synthesised to sequence the internal regions of the PCR products. The following internal and sequencing primers were used GCC GCG TCG CAG AAG ATC TCA AT [SEQ ID NO:5] (nt 104-126), GGT TCT ATC CTA ACC TTA CC [SEQ ID NO:6] (nt 341-360), GCC TCA TTT TGT GGG TCA CCA TA [SEQ ID NO:7] (nt 496-518), TGG GGG TGG AGC CCT CAG GCT [SEQ ID NO:8] (nt 731-751), CAC AAC ATT CCA CCA AGC TC [SEQ ID NO:9] (nt 879-899), AAA TTC GCA GTC CCC AAC [SEQ ID NO:10] (nt 1183-1195), GTT TCC CTC TTC TTG CTG T -3' [SEQ ID NO:11] (nt 1429-1447), TTT TCT TTT GTC TTT GGG TAT [SEQ ID P \OPER\TDO\245330 9 DIV DOC- 21/9/01 NO:12] (nt 1683-1703) 5'-CCA ACT TAC AAG GCC TTT CTG-3' [SEQ ID NO:13] (nt 1978-1999), 5'-CAT CGT TTC CAT GGC TGC TAG GC-3' [SEQ ID NO:14] (nt 2239- 2262).

4. Cloning of the HBV polymerase gene into pUC18: Due to the small amount of HBV DNA in the samples, the region encompassing nt 1429 to 1703 from the HBV polymerase gene were amplified by PCR using the primers TCC CTC TTC TTG CTG T-3' [SEQ ID NO:15] (nt 1429-1447) and 5' ATA CCC AAA GAC AAA AGA AAA- 3' [SEQ ID NO:16] (nt 1703-1683), before cloning. The DNA was purified with Geneclean II and ligated using T4 DNA ligase (New England Biolabs, Beverly, MA) into a Sma I digested dephosphorylated pUC 18 plasmid (Pharmacia Biotech, NJ). Clones were directly sequence as above.

EXAMPLE 4 DNA POLYMERASE ASSAY Samples of serum (100 pl) were applied to a 20% w/v sucrose cushion in TNE (20 mmol/L Tris-HCI pH 7.4, 150 mmol/L NaCl 2 1 mmol/L EDTA) and centrifuged at 200,000 g for 3 hr at 10"C using an SW41 rotor in a Beckman Model L8 ultracentrifuge. The pellet was 20 resuspended in 50 mmol/L TRIS-HCI pH 7.5 containing 1.5% v/v Triton-X100, 100 mmol/L Kcl and 0.01% v/v 2-mercaptoethanol and allowed to stand overnight at 4 2 C. Small aliquots of the suspension were assayed for endogenous HBV DNA polymerase activity essentially as described by Price et al Each assay was performed in a total volume of 30 p1 which contained 20 l of the partly purified HBV and (final concentrations) 30 mmol/L Tris-HCI o* 25 pH 7.5, 30 mmol/L MgCl 2 10 /pmol/L each dATP, dTTP and dGTP, and 0.01 pM [a- 32

P]-

dCTP (3,000 Ci/mmol) (Dupont NEN, Boston, MA). To test for penciclovir triphosphate (PCV-TP) sensitivity, paired assays were performed on each sample, with an excess (100 pmol/L penciclovir-triphosphate included in the reaction mixture in one assay of each pair.

After 2 hr at 37C, reactions were stopped by spotting 20 pl aliquots of each reaction mix onto 25mm diameter glass fibre discs (Advantex, Tokyo, Japan) which had been pre-soaked in 10% w/v trichloroacetic acid (TCA). Discs were dried before washing in ice-cold w/v TCA containing 10 mmol/L sodium pyrophosphate. Three further 10 min washes in P\OPER\TDOU2453309 DIV DOC- 21/9/01 -21 cold 5% v/v TCA followed. The washed discs were finally rinsed in absolute ethanol, air dried, and counted for radioactivity. Inhibition of HBV DNA polymerase activity by PCV- TP was expressed as the percentage difference in activity in the assay mix containing PCV- TP compared to the matched control. Because of limited sample amounts, it was not possible to standardize enzyme activity or to perform replicate assays. Despite the inherent variability of the assay, a general time related decrease in sensitivity of the HBV DNA polymerase to PCV-TP was evident (see Table 1).

EXAMPLE EFFECT OF ANTIVIRAL THERAPY Upon commencement of the antiviral treatment strategy GCV+ PFF, the level of HBV DNA post-OLT decreased from over 100,000 pg/ml to 10,800 pg/ml by day 87 (Figure This reduction in viraemia was associated with clinical, biochemical and histological improvement Maintenance famciclovir therapy (treatment GCV) resulted in fluctuating levels of HBV DNA over the ensuring 359 days with two peaks of HBV DNA observed. The switch to oral famciclovir on day 446 was also associated with a rise in HBV DNA, but this was likely to have been the result of insufficient dosing (FCV[I] in Figure 2) rather than a breakthrough in treatment. Following dose increase to FCV [II] on day 500, 20 there was a decrease in HBV DNA. However, the level of HBV DNA gradually rose over time from 3,000 pg/ml on day 600 (154 days of famciclovir), to 8,800 pg/ml on day 816 (370 days famciclovir), peaking at 29,000 pg/ml on day 1302 (856 days of famciclovir), then stabilising at around 12,000 pg/ml on day 1329 (883 days of famciclovir). A students test of the DNA levels during the treatment period from days 816 to days 1329, revealed statistically significant rise. There was a 1.5 to 2 fold rise in ALT levels over the same time interval (Figure 2) and no change in clinical status.

EXAMPLE 6 NUCLEOTIDE CHANGES The X and the polymerase genes of HBV were sequenced at five time points (Figure 2).

During almost 4 years of the antiviral therapy there were no changes in the X gene PAOPER\TDO\2453309 DIV.DOC- 21/9/01 -22compared to the pretreatment sequence. However, there were 5 nt changes detected in the polymerase gene from day 816 and day 1329 samples (Table These changes were detected in separate independent PCR amplifications; furthermore the mutations were detected by sequencing both strands and are therefore unlikely to be the result of PCR generated errors. The nt changes in the polymerase gene were first detected after 816 days of treatment, when the patient had been treated with famciclovir for 370 days. However, only two nt changes, at positions 1498 and 1519 resulted in amino acid changes, Val 519- Leu and Leu 526- Met, respectively. These two nt changes appeared concurrently. At 816 days, three different nt were detected at position 1498 (all of which would result in a Val to Leu change). After 1329 days post-treatment, thymidine was the dominant species at nt 1498. The amino acid changes at 816 and 1329 days post treatment coincided with reduced serum HBV DNA polymerase sensitivity to PCV-TP (Table These nt changes were not found in 6 patients with post-OLT recurrent HBV infection who were not undergoing FCV therapy.

The region encompassing the nt mutations which gave rise to amino acid changes from the sample taken at 1329 days was cloned and sequenced. Three quasi-species were detected.

Seventy-five percent (15/20) of the clones contained both the 1498 and 1519 mutations which occurred together. Pretreatment sequences were detected in 3/20 of the clones. A further mutation at nt 1511, which would result in a proline to leucine change at position 523, was detected in 2/20 of the clones. This mutation was not detected with the two dominant mutations, 1498 (Val 519-Leu) and 1519 (Leu 526-Met), nor was it detected by direct PCR sequencing, indicating it probably occurs at a low frequency. Viral DNA from the sample obtained at 600 days (150 days of FCV treatment) was also cloned and sequenced; however, only the pre-treatment sequences were detected.

EXAMPLE 7 NUCLEOTIDE CHANGES IN PATIENTS B, C AND D The amino acid changes in HBV isolated from patients B and C are shown in Figure 5, and from patient D is shown in Figure 6. In Figure 5, patient A is the same as shown in Figure 3.

P .OPER\TDO\2453309 DIV.DOC- 21/9/01 -23- Patient B was undergoing long term famciclovir treatment (>850 days). The amino acid change selected during famciclovir treatment is shown as HBV (patient B) in Figure Patient C did not respond to famciclovir and was later treated with 3TC (lamivudine The HBV isolated during FCV treatment from patient C, is shown as HBV (patient C-FCV).

All 3TC resistance mutations which developed during treatment with 3TC is shown as HBV (patient C-3TC). The sequence analysis showed a mutation (Thr-Ser substitution) in the HBV polymerase gene near the C domain but no mutation was initially detected in the YMDD motif. A mutation of Met 550 to Ile in the YMDD motif was detected from HBV isolated 32 days (333 days post treatment) after the HBV containing the Thr-Ser substitution was isolated.

EXAMPLE 8 ESCAPE MUTANTS Using the method hereinbefore described, HBV variants are screened for escape mutations.

These are mutations in surface components such as the HBV surface antigen which reduce the interactivity of the surface component to antibodies or other immunological reagents.

Given the overlapping open reading frame of HBV genome, a single mutation may have 20 multiphenotypic consequences. For example, a mutation in the HBV DNA polymerase may also have an affect on the HBV surface antigen.

Preferred mutations in the HBV surface antigen are in amino acids 118 to 169 and/or 169 to 207 such as D144E or G145R. These correspond to DNA polymerase mutations R/W499E.

A particularly preferred escape mutant and nucleoside analogue resistant mutant has a nucleotide sequence set forth in Figure 6 with corresponding amino acid sequences for the DNA polymerase and surface antigen.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also P .OPER\TDO\2453309 DIV.DOC- 21/9/01 -24includes 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.

r r P.opedoU24533(di doc-21.A9A) I TABLE 1 Nucleotide mutations in the polymerase gene and the resulting amino acid changes during antiviral therapy eo *oo o POLYMERASE GENE Days of Days post nt 297 nt 1498 nt 1511* nt 1519 nt2008 nt2331 Inhibition of antiviral famciclovi HBV DNA treatment r reatment Polymerase by

PCV-TP**

Pretreatment T G C C C G 87 600 NR 816 154 1329 370 G,T,C A 0% 883 C T T A A A 0% Amino acid change None Val 519- Pro 523- Leu 526- None None Leu Leu Met The dashes indicate no change from the pre-treatment nucleotide.

The mutation was only detected after cloning the PCR product after 1329 days of antiviral treatment. It occurred at a low frequency and was present in only 10% of clones.

The percentage inhibition of HBV DNA polymerase by PCV-TP in the in vitro assay as described in the Methods section.

NR not relevant NA not assessable P.oedd\433P bdoc-2AN9A) I 26

BIBLIOGRAPHY:

1. Summers J, Mason W. Cell (1982) 29: 403-415.

2. Vere Hodge R.A. Antiviral Chem Chemother (1993) 4:67-84.

3. Boyd MR et al Antiviral Chem Chemother. (1987) 32: 358-363.

4. Kruger T et al Hepatology (1994) 22: 219A.

Main J et al. J Viral Hepatitis (1996) 3:211-215.

6. Severini A et al Antimicrobial Agents Chemother (1995) 39: 1430-143 7. DienstagJLetal New England JMed(1995) 333: 1657-1661.

8. Shaw T,et al. Antimicrobiol Agents Chemother (1994) 38:719-723.

9. Shaw T, et al. Hepatology (1996) 24: in press.

Tsiquaye KN, et al. J Med Virol (1994) 42: 3 06-3 11. Boker KHW, et al. Transplantation (1994) 57: 1706-1708.

12. Angus P, et al. J Gastroenterol Hepatol (1993) 8: 353-357.

:13. Poch0, et al. EMBO]J (1989) 8: 3 867-3 874.

14. Delarue M, et al. Protein Engineering (1990) 3: 461-467.

V 15. Chiou HC, et al. Antiviral Chem Chemother (1995) 6: 28 1-288.

16. Ling R, etal. Hepatology (1996) 24: 711-713.

17. Price PM, et al. Hepatology 1992 16: 8-13.

EDITORIAL NOTE APPLICATION NUMBER 75560/01 The following Sequence Listing pages 1 to 26 are part of the description. The claims pages follow on pages 27 to PAOPERTDOU2JS33oq div q d-2 IANM I <110> <120> <130> <140> <141> <150> <151> SEQUENCE LISTING NORTH WESTERN HEALTH CARE NETWORK.

VIRAL VARIANTS AND METHODS FOR DETECTING SAME.

104 PCT AU DIVISIONAL 2 00 1-0 9-2 1 P0 3519 1996-11-08 57 <170> Patentln Ver. 2 <210> 1 <211> <212> DNA <213> Hepatitis B vir~ <400> 1 ggagtqtgga ttcgcactcc 1 uIs <210> 2 <211> 17 <212> DNA <213> Hepatitis B virus <400> 2 qctccaaatt ctttata <210> 3 <211> <212> DNA <213> Hepatitis B virus <400> 3 cctttacccc gttgcccggc <210> 4 <211> 17 <212> DNA <213> Hepatitis B virus <400> 4 gctccaaatt ctttata <210> <211> 23 <212> DNA P.OPERITDO\4533.9 div q doc-21/ I9 I -2- <213> Hepatitis B virus <400> gccgcgtcgc agaagatctc aat 23 <210> 6 <211> <212> DNA <213> Hepatitis B virus <400> 6 ggttctatcc taaccttacc <210> 7 <211> 23 <212> DNA <213> Hepatitis B virus <400> 7 gcctcatttt gtgggtcacc ata 23 <210> 8 <211> 21 <212> DNA <213> Hepatitis B virus <400> 8 tgggggtgga gccctcaggc t 21 <210> 9 <211> <212> DNA <213> Hepatitis B virus <400> 9 Scacaacattc caccaagctc <210> <211> 18 <212> DNA <213> Hepatitis B virus <400> aaattcgcag tccccaac 18 <210> 11 <211> 19 <212> DNA <213> Hepatitis B virus <400> 11 P:lOPERMTDO24533f9 di %sq doc.2 1/0910 -3gtttccctct tcttgctgt 19 <210> 12 <211> 21 <212> DNA <213> Hepatitis B virus <400> 12 ttttcttttg tctttgggta t 21 <210> 13 <211> 21 <212> DNA <213> Hepatitis B virus <400> 13 ccaacttaca aggcctttct g 21 <210> 14 <211> 23 S <212> DNA <213> Hepatitis B virus <400> 14 catcgtttcc atggctgcta ggc 23 <210> <211> 19 <212> DNA *<213> Hepatitis B virus <400> gtttccctct tcttgctgt 19 <210> 16 <211> 21 <212> DNA <213> Hepatitis B virus <400> 16 atacccaaag acaaaagaaa a 21 <210> 17 <211> 550 <212> DNA <213> Hepatitis B virus <220> <221> CDS <222> P.%OPERITDOU153309 di, q doc.IA)9MI <400> 17 tct too aat ttg tcc Ser Ser Asn Leu Ser 1 5 tgg tta tcg ctg Trp Leu Ser Leu gat gtg tct gcg qcg ttt tat Asp Vai Ser Ala Ala Phe Tyr 10 cat att cct His Ile Pro tct gga tta Ser Gly Leu ctt Leu cat cct gct got His Pro Ala Ala atg Met 25 cct cat ctt ctt Pro His Leu Leu att ggt tct Ile Gly Ser tcc agg atc Ser Arg Ile tca agg tat gtt Ser Arg Tyr Val ogt ttg tcc tct Arg Leu Ser Ser aat Asn aac aao Asn Asn aao atg caa aac Asn Met Gin Asn ctg Leu cac gac tcc tgo His Asp Ser Cys agg caa otc tac Arg Gin Leu Tyr gtt Va1 tcc otc atg ttg Ser Leu Met Leu tao aaa acc tac Tyr Lys Thr Tyr gga Gly 75 gag aaa ttg cac Glu Lys Leu His ctg Leu tat too cat ccc Tyr Ser His Pro ato Ile gto ctg ggo ttt Val Leu Gly Phe aaa ata oct atg Lys Ile Pro Met gga gtg Gly Val ggc otc agt Gly Leu Ser gtg gtt cgt Val Val Arg 115 ccg Pro 100 ttt otc ttg got Phe Leu Leu Ala ttt act agt gc Phe Thr Ser Ala att tgt toa Ile Cys Ser 110 tat atg gat Tyr Met Asp agg got ttc ccc Arg Ala Phe Pro cac His 120 tgt ttg got tto Cys Leu Ala Phe ago Ser 125 gat gtg Asp Val 130 gta ttg ggg gc Val Leu Gly Ala aag Lys 135 tct gta cag cat Ser Val Gin His cgt Arg 140 gag gc ctt tat Glu Ala Leu Tyr got gtt aco aat Ala Val Thr Asn ott ttg tot ctg Leu Leu Ser Leu ggt Gly 155 ata cat tta aac Ile His Leu Asn cct Pro 160 432 480 528 aac aaa aca aaa Asn Lys Thr Lys aga Arg 165 tgg qgt tat too Trp Gly Tyr Ser aac ttc atg ggt Asn Phe Met Gly tao ata Tyr Ile 175 att gga agt Ile Gly Ser tgg Trp 180 gga aca ttg c 550 Gly Thr Leu <210> 18 <211> 183 <212> PRT <213> Hepatitis B virus <400> 18 Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr P WOERkTDO\.2453309 dni uqd-211A) 1 1 His Ser Asn Val1 Tyr Gly Val1 Asp Thr 145 Asn Ile Ile Gly As n Ser Ser Leu Val1 Val1 130 Ala Lys Gly Pro Leu As n Leu His Ser Arg 115 Val1 Val1 Thr Se r Leu Ser Met Met Pro Pro 100 Arq Leu Thr Lys T rp 180 5 His Arg Gin Leu Ile Phe Al a Gly As n Arg 165 Gly Pro Tyr Asn Leu 70 Val1 Leu Phe Al a Phe 150 Trp Thr Al a Al a 40 His Lys Gly Al a His 120 Ser Leu 10 Pro Leu Ser T yr Arg 90 Phe Leu Gin Leu Leu Ser Ser Giu Ile Ser Phe Arg 140 Ile Leu Asn Arg Lys Pro Al a Ser 125 Giu His Ile Ser Gin Le u Met Ile 110 T yr Ala Leu Giy Arg Leu His Gly Cys Met Leu Asn Ser Ile T yr Leu Val Ser Asp Tyr Pro 160 Tyr Ser Leu Asn Phe Met Gly Tyr Ile <210> 19 <211> 550 <212> DNA <213> <220> <221> <222> <220> <221> <222> <220> <221> <222> <220> <221> <222> Hepatitis B virus

CDS

.(472)

CDS

(476) (508)

CDS

(512). (526)

CDS

(530) (550) P.OPERTDOU24533O9 dm- sq doc-2 lV901I -6- <400> 19 t ctt cca att tgt cct ggt tat cgc tgg atg tgt ctg cgg cgt ttt atc 49 Leu Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile ata ttc ctc Ile Phe Leu ctg gat tat Leu Asp Tyr ttc Phe atc ctg ctg cta Ile Leu Leu Leu ctc atc ttc tta Leu Ile Phe Leu ttg gtt ctt Leu Val Leu cca gga tca Pro Gly Ser caa ggt atg ttg Gin Giy Met Leu ccc Pro gtt tgt cot ota Vai Cys Pro Leu aca aca Thr Thr aca tgc aaa acc Thr Cys Lys Thr acg act cct gct Thr Thr Pro Ala caa Gin ggc aao tct acg Gly Asn Ser Thr ccc tca tgt tgc Pro Ser Cys Cys tgt Cys aca aaa cct acg Thr Lys Pro Thr aga aat tgc acc Arg Asn Cys Thr att ccc atc cca Ile Pro Ile Pro tcc tgg gct ttc Ser Trp Aia Phe r r r gca Ala 90 aaa tac cta tgg Lys Tyr Leu Trp gag tgg Glu Trp gcc tca gtc Ala Ser Vai tgg ttc gta Trp Phe Vai 115 cgt Arg 100 ttc tot tgg otc Phe'Ser Trp Leu tta ota gtg oca Leu Leu Val Pro ttt gtt cag Phe Vai Gin 110 ata tgg atg Ile Trp Met ggg ott too ccc Gly Leu Ser Pro act Thr 120 gtt tgg ott tca Vai Trp Leu Ser atg tgg Met Trp 130 tat tgg ggg oca Tyr Trp Gly Pro ctg tao ago ato Leu Tyr Ser Ile gtg Va1 140 agg ccc ttt ata Arg Pro Phe Ile ccg po aca Thr 160 ctg tta oca att Leu Leu Pro Ile aaa caa aaa gat Lys Gin Lys Asp ttc Phe 150 ggg Gly 165 ttt tgt otc tgg Phe Cys Leu Trp gta tao att taa aco cta Val Tyr Ile Thr Leu 155 r r gtt att ccc Val Ile Pro taa act tca Thr Ser 170 tgg gtt aca taa Trp Vai Thr ttg gaa gtt ggg gaa cat Leu Giu Val Giy Glu His 175 <210> <211> 157 <212> PRT <213> Hepatitis B virus tgc 550 Cys 180 <400> P)OPERMTDO243339 &sv doc-22M910 I Leu Pro Ilie Cys Pro Gly Tyr Arg Trp Ilie Leu Th r Ph e Ilie Ala Trp Met Pro 145 Phe Asp Th r Pro Pro Ser Phe Trp 130 Leu Leu Tyr Thr Ser Ilie Val1 Val1 115 Tyr Leu Phe Gin Cys Cys Pro Arg 100 Giy T rp Pro Ile Gly Lys Cys Ser Phe Leu Gi y Ile Leu Met Thr Cys 70 Ser Ser Ser Pro Phe 150 Le u Leu Cys 55 Thr Trp Trp Pro Ser 135 Phe Leu Pro 40 Thr Lys Aia Leu Thr 120 Le u Cys Cys 25 Val Thr Pro Phe Ser 105 Val Tyr Leu Met Le u Cys Pro Th r Ala 90 Leu Trp Ser T rp Le u Phe Le u Gin Arg Tyr Vali Se r Val1 140 Tyr Arg be u Ile Gly As n Leu Pro Ala 125 Arg Ile Arg be u Pro Asn Cys Trp Phe 110 Ile Pro Phe Val1 Gly Ser Thr Giu Val1 Trp Phe Ile Leu Ser Thr Cys Trp Gin Met Ile

V

<210> 21 <211> 11 <212> PRT <213> Hepatitis B virus <400> 21 Thr Leu Thr Lys Gin Lys Asp Gly Vai Ile Pro 1 5 <210> 22 <211> <212> PRT <213> Hepatitis B virus <400> 22 Thr Ser Trp Vai Thr 1 <210> 23 <211> 7 <212> PRT <213> Hepatitis B virus <400> 23 P.OPERTDOUj5339 dw sq doc-2 1M9l -8- Leu 1 <210 <211 <212 <213 <220 <221 <222 <223 <220 <221 <222 <223 <220 <221 <222 <223 Glu Val Gly Glu His Cys 24

PRT

Hepatitis B virus

VARIANT

(1) Xaa Ser or Ala

VARIANT

Xaa Ile or Val

VARIANT

(11) Xaa Ile or Leu r r <220> <221> <222> <223>

VARIANT

Xaa Val or Gly <400> 24 Xaa His Pro Ile 1 Xaa Leu Gly Phe Arg Lys Xaa Pro Met Gly Xaa Gly 5 10 Leu Leu Ala Gin Phe Leu Ser Pro <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> 41

PRT

Hepatitis B virus

VARIANT

(1) Xaa Gin or Lys

VARIANT

(3) Xaa Tyr or Phe <220> <221> VARIANT <222> PA\OPERUIDOU453309 fi, do-21AWAI) -9- <223> Xaa Arg or Trp <220> <221> VARIANT <222> <223> Xaa Tyr or Leu <220> <221> VARIANT <222> (11) <223> Xaa Ser or Ala <220> <221> VARIANT <222> <223> Xaa Ile or Val <220> <221> VARIANT <222> (21) <223> Xaa Ile or Leu <220> <221> VARIANT <222> <223> Xaa Val or Gly <220> <221> VARIANT <222> <223> Xaa Cys or Leu <400> 'e"ot Xaa Thr Xaa Gly Xaa Lys Leu His Leu Xaa Xaa His Pro Ile Xaa Leu 1 5 10 Gly Phe Arg Lys Xaa Pro Met Gly Xaa Gly Leu Ser Pro Phe Leu Leu 20 25 Ala Gin Phe Thr Ser Ala Ile Xaa Ser <210> 26 <211> 11 <212> PRT <213> Hepatitis B virus <220> <221> VARIANT <222> (1) <223> Xaa Ala or Val <220> <221> VARIANT <222> (3) P.NOPERMTDOU2JS33) das- m doc-21~AI~ <223> Xaa =Ser or Ala <220> <221> <222> <223>

VARIANT

(8) Xaa Val or Leu or Met <400> 26 Xaa Phe Xaa 1 Tyr Met Asp Asp Xaa Val Leu Gly a <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 27

PRT

Hepatitis B virus

VARIANT

(1) Xaa =Ser or Ala VAR IANT Xaa Ile or Val

VARIANT

(8) Xaa Phe or Leu

VARIANT

(11) Xaa =lie or Leu a

VARIANT

Xaa Leu or Val or Gly <220> <221> <222> <223> <220> <221> <222> <223>

VARIANT

(19) Xaa Pro or Leu-

VARIANT

(22) Xaa Leu or Met

VARIANT

(29) Xaa Ile or Leu P OPERTDO\2453309di% wcoc.21lA) I -11 I- <220> <221> <222> <223>

VARIANT

Xaa Cys or Leu <400> 27 Xaa His Pro Ile 1 Leu Ser Xaa Phe Xaa Leu 5 Gly Xaa Arq Lys Xaa Pro Met Gly Xaa Gly 10 Leu Xaa Ala Gin Phe Thr Ser Ala Xaa Xaa 25 <210> 28 <211> 14 <212> PRT <213> Hepatitis B virus 6.

<220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>

VARIANT

(1) Xaa Ala or Val

VARIANT

(3) Xaa Ser or Ala

VARIANT

Xaa Met

VARIANT

(8) Xaa Val or Ile or Val or Leu or Met

VARIANT

(13) Xaa Lys or Arg

VARIANT

(14) Xaa Ser or Thr <400> 28 Xaa Phe Xaa Tyr 1 Xaa Asp Asp Xaa Val Leu Giy Ala Xaa Xaa <210> 29 PAOPERUDtO\53309 dn w dwc-21I09MI 12- <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 181

PRT

Hepatitis

VARIANT

(2) Xaa Asn or Asp

VARIANT

(17) Xaa le or Pro

VARIANT

(29) Xaa Ile or Val

VARIANT

Xaa =Ser or Asp

VARIANT

(44) Xaa Thr or Asn

VARIANT

(46) Xaa Arg or Asn

VARIANT

(47) Xaa Asn or Ile

VARIANT

(48) Xaa more than 3 B virus different AA possible

VARIANT

Xaa Asn orTyr or His

VARIANT

(52) Xaa His or Tyr <220> <221> VARIANT <222> (53) P:OPE\TDOU453309 d, sae doc-21/09MI 13- <223> Xaa Gly or Arg <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>

VARIANT

(54) Xaa more

VARIANT

Xaa more

VARIANT

(56) Xaa more than 3 different AA possible than 3 different AA possible than 3 different AA possible <220> <221> VARIANT <222> (57) <223> Xaa Asp or Asn a.

a a *a a <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>

VARIANT

Xaa Asp or Asn

VARIANT

(61) Xaa Ser or Tyr

VARIANT

Xaa Asn or Gin

VARIANT

(71) Xaa Leu or Met

VARIANT

Xaa Lys or Gin

VARIANT

(77) Xaa Tyr or Phe <220> <221> VARIANT <222> (79) <223> Xaa Arg or Trp P.NOPERM)O\2453309 div wq doc-? IAMOI1 14 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>

VARIANT

(84) Xaa Tyr or Leu

VARIANT

Xaa Ser or Ala

VARIANT

(89) Xaa Ie or Val

VARIANT

Xaa Ilie or Leu

VARIANT

(99) Xaa Val or Gly a <220> <221> VARIANT <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> '2 <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> (114) Xaa =Cys or Leu

VARIANT

(115) Xaa Ala or Ser

VARIANT

(116) Xaa Val or Met

VARIANT

(117) Xaa Val or Thr VARIANT (118) Xaa Arg or Cys

VARIANT

(122) Xaa Phe or Pro <220> <221> VARIANT P.%OPERkTDO\24S339 dr d~-2Ai9A)19 <222> (125) <223> Xaa Leu or Val <220> <221> VARIANT <222> (126) <223> Xaa Ala or Val <220> <221> VARIANT <222> (128) <223> Xaa Ser or Ala <220> <221> VARIANT <222> (133) <223> Xaa Val or Leu or Met <220> <221> VARIANT <222> (138) <223> Xaa Lys or Arg <220> <221> VARIANT <222> (139) <223> Xaa Ser or Thr <220> <221> VARIANT <222> (140) <223> Xaa Val or Gly <220> <221> VARIANT <222> (141) <223> Xaa Gin or Glu <220> <221> VARIANT <222> (143) <223> Xaa Leu or Ser or Arg <220> <221> VARIANT <222> (145) <223> Xaa Ser or Phe <220> <221> VARIANT <222> (147) <223> Xaa Phe or Tyr <220> <221> VARIANT <222> (148) <223> Xaa Thr or Ala P.%OPERTDOU4533O9 div w.doc-21"M~J -16- <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>

VARIANT

(149) Xaa Ala or Ser

VARIANT

(150) Xaa Val or Ile *f

S

S

S

*5 a

S

S.

VARIANT

(151) Xaa Thr or Cys

VARIANT

(152) Xaa =Asn or Ser

VARIANT

(153) Xaa Phe or Val

VARIANT

(156) Xaa =Ser or Asp

VARIANT

(157) Xaa Leu or Val <220> <221> VARIANT <222> (164) <223> Xaa Asn or Gin <220> <221> <222> <223> <220>

VARIANT

(179) Xaa Vai or Ile <400> 29 Ser Xaa Leu Ser 1 Trp Leu Ser Leu Asp Val Ser Aia Ala Phe Tyr His 5 10 Xaa Pro Leu His Pro Ala Ala Met Pro His Leu Leu Xaa Giy Ser Ser 25 Giy Leu Xaa Arg Tyr Val Aia Arq Leu Ser Ser Xaa Ser Xaa Xaa Xaa 40 P.%OPERXThOX2433O9 d, wq doc-2 IA)AI1 -17- Asn Xaa Gin Xaa Xaa Xaa Xaa Xaa Xaa Leu His Xaa Xaa Cys Ser Arg 55 Xaa Leu Tyr Val Ser Leu Xaa Leu Leu Tyr Xaa Thr Xaa Gly Xaa Lys 70 75 Leu His Leu Xaa Xaa His Pro Ile Xaa Leu Giy Phe Arg Lys Xaa Pro 90 Met Gly Xaa Gly Leu Ser Pro Phe Leu Leu Ala Gin Phe Thr Ser Ala 100 105 110 Ilie Xaa Xaa Xaa Xaa Xaa Arq Ala Phe Xaa His Cys Xaa Xaa Phe Xaa 115 120 125 Tyr Met Asp Asp Xaa Val Leu Gly Ala Xaa Xaa Xaa Xaa His Xaa Giu 130 135 140 Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Xaa Xaa Gly Ile His 145 i50 155 160 Leu Asn Pro Xaa Lys Thr Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met *165 170 175 *Gly Tyr Xaa Ile Gly ~:180 <210> <211> 4 <212> PRT 5<213> Hepatitis B virus <400> Tyr Met Asp Asp Se 1 <210> 31 <211> 11 *<212> PRT <213> Hepatitis B virus <400> 31 Ala Phe Ser Tyr Met Asp Asp Val Val Leu Giy 1 5 <210> 32 <211> 11 <212> PRT <213> Hepatitis B virus P.%OPERITDOA2A339 di se do-21A)9AOI 18- <400> 32 Ala Phe Ser 1 Tyr Met Asp Asp Val Val Leu Gly 5 <210> 33 <211> 11 <212> PRT <213> Hepatitis B virus <400> 33 Val Phe Ala Tyr Met Asp Asp Leu Val Leu Gly <210> 34 <211> 11 <212> PRT <213> Hepatitis B virus <400> 34 Ile Tyr Gin Tyr

S

5s S 0 0* 6O 0

S

Sq S 055 S

S.

5555

S

OSSSSS

S

S.S

S

S* .8 S S 55 5 0

S

S

00 S S S *5 Met Asp Asp Leu Tyr Val Gly <210> <211> 33 <212> PRT <213> Hepatitis B virus <400> Thr Thr Ile Gly 1 Ala Arg Trp Ala 20 Arg Giu 5 Met Leu Leu Ala Thr Arg Giu Tyr 10 Val His Ala Phe Giu Gin Leu Ala Asp Phe Pro Giu Ala <210> 36 <211> 41 <212> PRT <213> Hepatitis B virus <400> 36 Gin Thr Phe Giy Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Leu Gly Leu Ser Leu 25 Phe Leu Met P.OPERVTDOU453309 div saq doc-2 lH91 -19- T Ala Gin Phe Thr Ser Ala Ile Cys Ser <210> 37 <211> 41 <212> PRT <213> Hepatitis B virus <400> 37 Gin Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu 1 5 10 Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Met 25 Ala Gin Phe Thr Ser Ala Ile Cys Ser <210> 38 <211> 41 <212> PRT <213> Hepatitis B virus <400> 38 Gin Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu 1 5 10 Gly Leu Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Met 20 25 Ala Gin Phe Thr Ser Ala Ile Cys Ser <210> 39 <211> 41 <212> PRT <213> Hepatitis B virus <400> 39 Gin Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Val Leu 1 5 10 Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu 25 Ala Gin Phe Thr Ser Ala Leu Cys Ser <210> P.OPERITDOU433309 ldoc-2 IA9ffiI 20 <211> <212> <213> 14

PRT

Hepatitis B virus <400> Ala Phe Ser Tyr 1 Met Asp Asp Val Val Leu Gly Ala Lys Thr <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 41 31

PRT

Hepatitis B virus

VARIANT

(1) Xaa Ser or Ala VAR IANT Xaa =lie or Val

VARIANT

(11) Xaa Ile or Leu

VARIANT

Xaa Val or Gly <400> 41 Xaa His Pro Ile 1 Xaa Leu Gly Phe Arg Lys Xaa Pro Met Gly Xaa Gly 5 10 Leu Leu Ala Gin Phe Thr Ser Ala Ile Cys Ser Leu Ser Pro <210> <211> <212> <213> <220> <221> <222> <223> 42

PRT

Hepatitis B virus

VARIANT

(1) Xaa Gin or Lys <220> <221> VARIANT <222> (3) P PPER\TDOU453309 div sq doc-2 IA)91 -21- <223> Xaa Tyr or Phe <220> <221> VARIANT <222> <223> Xaa Arg or Trp or Glu <220> <221> VARIANT <222> <223> Xaa Tyr or Leu <220> <221> VARIANT <222> (11) <223> Xaa Ser or Ala <220> <221> VARIANT <222> <223> Xaa Ile or Val <220> <221> VARIANT <222> (18) <223> Xaa Phe or Leu <220> <221> VARIANT <222> (21) <223> Xaa Ile or Leu <220> <221> VARIANT <222> <223> Xaa Leu or Val or Glu <220> <221> VARIANT <222> (29) <223> Xaa Pro or Leu <220> <221> VARIANT <222> (32) <223> Xaa Leu or Met <220> <221> VARIANT <222> (39) <223> Xaa Ile or Leu <220> <221> VARIANT <222> <223> Xaa Cys or Leu P.%OPFRTDOU453309 di% mq dw.2A)9A 22 <400> 42 Xaa Thr Xaa 1 Gly Xaa Arq Ala Gin Phe Gly Xaa Lys Leu His Leu Xaa Xaa His Pro Ile Xaa Leu Xaa Pro Met Gly Xaa Gly Leu Ser Xaa Phe Leu Xaa Thr Ser Ala Xaa <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> -22 3 <220> <221> <222> <223>

VARIANT

(3) Xaa Ser

VARIANT

Xaa =Met

VARIANT

(8) Xaa Val or Ala 43 14

PRT

Hepatitis B virus

VARIANT

(1) Xaa Ala or Val or Ile or Val or Leu or Met

VARIANT

(13) Xaa Lys or Arq

VARIANT

(14) Xaa Ser or Thr <400> 43 Xaa Phe Xaa Tyr Xaa Asp Asp Xaa Val Leu Gly Ala Xaa Xaa <210> <211> <212> <213> 44 31

PRT

Hepatitis B virus P OPERTDOU453309 d. q do.21ID I~ 23 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>

VARIANT

(1) Xaa Ser or Ala

VARIANT

Xaa Ile or Val

VARIANT

(11) Xaa Ile or Leu

VARIANT

Xaa Val or Gly

VARIANT

Xaa Cys or Leu <400> 44 Xaa His Pro Ile 1 Leu Ser Pro Phe 20 Xaa Leu Gly Phe Arg Lys Xaa Pro Met Gly Xaa Gly Leu Leu Ala Gin Phe Thr Ser Ala Ile Xaa Ser 25 <210> <211> <212> <213> <220> <221> <222> <223> 14

PRT

Hepatitis B virus

VARIANT

(1) Xaa Ala or Val <220> <221> VARIANT <222> (3) <223> Xaa Ser or Ala <220> <221> <222> <223>

VARIANT

(8) Xaa Val or Leu or Met <220> <221> VARIANT P:'OPER\TDOU453309 d~ ~q doc-2 O39/ I -24- <222> (13) <223> Xaa <220> <221> <222> <223> Lys or Arg

NT

Ser or Thr

VARIAN

(14) Xaa <400> Xaa Phe Xaa Tyr 1 Met Asp Asp Xaa Val Leu Gly Ala Xaa Xaa 5 <210> 46 <211> 11 <212> PRT <213> Hepatitis B virus <400> 46 Ala Phe Ser Tyr 1 Met Asp Asp Val Val Leu Gly 5 <210> 47 <211> 11 <212> PRT <213> Hepatitis B virus *t e o 6 <400> 47 Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly <210> 48 <211> 11 <212> PRT <213> Hepatitis B virus <220> <221> VARIANT <222> <223> Xaa Val or Ile <400> 48 Ala Phe Ser Tyr 1 Xaa Asp Asp Val Val Leu Gly 5 <210> 49 <211> 11 <212> PRT <213> Hepatitis B virus P.\OPER\TDO\2453309 div q doc-21/D9/01 <400> 49 Ala Phe Ser 1 Tyr Met Asp Asp Val Val Leu Gly 5 <210> <211> 16 <212> PRT <213> Hepatitis B virus <400> Ser Asp Leu Ser 1 Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His 5 10 <210> 51 <211> 16 <212> PRT <213> Hepatitis B virus <400> 51 Ser Asp Leu Ser 1 Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His 5 10 <210> 52 <211> 16 <212> PRT <213> Hepatitis B virus <400> 52 Thr Asp Leu Gin Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His 10 <210> 53 <211> 16 <212> PRT <213> Hepatitis B virus <400> 53 Lys Lys Lys Ser 1 Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser 5 10 <210> 54 <211> 41 <212> PRT <213> Hepatitis B virus <400> 54 Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu P.OPER\TDO2453309 d s q doc-2 IA9MI -26- 1 5 10 Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu 25 Ala Gin Phe Thr Ser Ala Ile Cys Ser <210> <211> 41 <212> PRT <213> Hepatitis B virus <400> Gin Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu 1 5 10 Gly Phe Arg Lys Ile Pro Met Gly Leu Gly Leu Ser Leu Phe Leu Met 25 Ala Gin Phe Thr Ser Ala Ile Cys Ser <210> 56 <211> 41 <212> PRT <213> Hepatitis B virus <400> 56 Lys Thr Tyr Gly Arg Lys Leu His Leu Leu Ala His Pro Phe Ile Met S1 5 10 Gly Phe Arg Lys Leu Phe Met Gly Val Gly Leu Ser Pro Phe Leu Leu 25 Ala Gin Phe Thr Ser Ala Leu Ala Ser <210> 57 <211> 27 <212> PRT <213> Hepatitis B virus <400> 57 Arg Tyr Gin Tyr Asn Val Leu Pro Gin Gly Trp Lys Gly Ser Pro Ala 1 5 10 Ile Phe Gin Ser Ser Met Thr Lys Ile Leu Glu

Claims (19)

1. A method for determining whether an HBV isolate exhibits decreased sensitivity to famciclovir and/or lamivudine, said method comprising screening for the presence of at least one amino acid at the position shown in parentheses in HBV DNA polymerase selected from the list comprising Glu (499), Val (509), Leu (512), Leu (519), Leu (523), Met (526), Ser (530), Leu (533), Val or Ile (550), and Thr (559) wherein the presence of at least one of the aforementioned amino acid residues in said DNA polymerase is indicative of an HBV exhibiting decreased sensitivity to famciclovir and/or lamivudine with the proviso that: when said isolate comprises a Val or Ile at position 550, it contains at least one other of the amino acids listed above; or (ii) when said isolate exhibits reduced sensitivity to lamivudine, the isolate does not have a Leu at position 512 in combination with Ile at position 550; a Met at position 526 in combination with a Val at position 550 or an Ile at position 550 in combination with an Ile at position 553.

2. The parentheses

3. The parentheses

4. The parentheses The parentheses method of Claim 1 wherein the amino acid at the position shown in is Glu (499). method of Claim 1 wherein the amino acid at the position shown in is Val (509). method of Claim 1 wherein the amino acid at the position shown in is Leu (512). method of Claim 1 wherein the amino acid at the position shown in is Leu (519). PAOPER'Ejh\Eljbhmcndd claimz245339 mdad d5ims w7O5 I 5 2 doc-2&VIAOS -28-

6. The method of Claim 1 wherein the amino acid at the position shown in parentheses is Leu (523).

7. The method of Claim 1 wherein the amino acid at the position shown in parentheses is Met (526).

8. The method of Claim 1 wherein the amino acid at the position shown in parentheses is Ser (530).

9. The method of Claim 1 wherein the amino acid at the position shown in parentheses is Leu (533). The method of Claim 1 wherein the amino acid at the position shown in parentheses is Val or Ile (550).

11. The method of Claim 1 wherein the amino acid at the position shown in parentheses is Thr (559).

12. A method for determining whether an HBV isolate exhibits decreased interactability to an antibody specific for HBV surface antigen, said method comprising screening for the presence of at least one amino acid at the position shown in parentheses in HBV DNA polymerase, the coding region of which overlaps with the coding region for said HBV surface antigen, selected from the list comprising Glu (499), Val (509), Leu (512), Leu (519), Leu (523), Met (526), Ser (530), Leu (533), Val or Ile (550), and Thr (559) wherein the presence of at least one of the aforementioned amino acid residues in said DNA polymerase is indicative of an HBV exhibiting decreased interactability to an antibody with the proviso that: when said isolate comprises a Val or Ile at position 550, it contains at least one other of the amino acids listed above; or POPER\Ejh\Ejh\ rd ducl.n12453309 =md dmi w75560-01 vZ dom-28MIhV3 -29- (ii) when said isolate exhibits reduced sensitivity to lamivudine, the isolate does not have a Leu at position 512 in combination with Ile at position 550; a Met at position 526 in combination with a Val at position 550 or an Ile at position 550 in combination with an Ile at position 553.

13. The method of Claim 12 wherein the amino acid at the position shown in parentheses is Glu (499).

14. The method of Claim 12 wherein the amino acid at the position shown in parentheses is Val (509). The method of Claim 12 wherein the amino acid at the position shown in parentheses is Leu (512).

16. The method of Claim 12 wherein the amino acid at the position shown in parentheses is Leu (519).

17. The method of Claim 12 wherein the amino acid at the position shown in parentheses is Leu (523).

18. The method of Claim 12 wherein the amino acid at the parentheses is Met (526).

19. The method of Claim 12 wherein the amino acid at the parentheses is Ser (530). The method of Claim 12 wherein the amino acid at the parentheses is Leu (533).

21. The method of Claim 12 wherein the amino acid at the parentheses is Val or Ile (550). position shown in position shown in position shown in position shown in PAOPER Ej\E~mmdd elaims\245339 maoded dms =7560-01 v2do-2&VUVS

22. The method of Claim 12 wherein the amino acid at the position shown in parentheses is Thr (559).

23. A method for determining whether an HBV isolate has reduced sensitivity to famciclovir or lamivudine as defined in Claim 1 or reduced interactability to an antibody as defined in Claim 12 substantially as hereinbefore with reference to the figures and/or Examples. DATED this 2 8 th day of January, 2005 Melbourne Health by its Patent Attorneys DAVIES COLLISON CAVE