AU2012203487A1 - Viral variants detection and application - Google Patents

Abstract

C \RPonbl\DCC\AAR\3%705.LDOC-14AW2012 -76 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 virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to these antibodies. The present invention further contemplates assays for detecting such viral variants, which assays are useful in monitoring anti-viral therapeutic regimens and in developing new or modified vaccines directed against viral agents and in particular 1-11V variants. The present invention also contemplates the use of the viral variants to screen for and/or develop or design agents capable of inhibiting infection, replication and/or release of the virus.

Description

P/00/0l Regulation 3 2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT (ORIGINAL) Name of Applicant: Melbourne Health of Level 6-6 East, Royal Melbourne Hospital, Grattan Street, Parkville, Victoria 3050; and Austin Health of Studley Road, Heidelberg, Victoria 3081; and Southern Health of Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168; and Alfred Health of Commercial Road, Melbourne, Victoria 3004 and St. Vincent's Hospital (Melbourne) Ltd. trading as St. Vincent's Hospital Melbourne of Victoria Parade, Fitzroy, Victoria 3065 Actual Inventors: Angeline Ingrid BARTHOLOMEUSZ, Stephen Alister LOCARNINI, Anna AYRES, Lily Ka Wai YUEN, Joseph John SASADEUSZ, Peter William ANGUS, William SIEVERT, Sharon Ruth LEWIN, Paul Vincent DESMOND, Sally Jane BELL Address for Service: DAVIES COLLISON CAVE, Patent & Trademark Attorneys, of I Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM Invention Title: Viral variants detection and application The following statement is a full description of this invention, including the best method of performing it known to us:- C:\NRPonhIDCC\AAR\436705_fDOC-146/2012 'VIRAL VARIANTS DETECTION AND APPLICATION This application is a divisional of Australian Application No. 2004285983, the entire contents of which are incorporated herein by reference. 5 BACKGROUND OF THE INVENTION FIELD OF THE INVENTION 10 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 virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to 15 these antibodies. The present invention further contemplates assays for detecting such viral variants, which assays are useful in monitoring anti-viral therapeutic regimens and in developing new or modified vaccines directed against viral agents and in particular HBV variants. The present invention also contemplates the use of the viral variants to screen for and/or develop or design agents capable of inhibiting infection, replication and/or release 20 of the virus. DESCRIPTION OF THE PRIOR ART Bibliographic details of the publications referred to in this specification are also collected 25 at the end of the description. 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 general knowledge in any country. 30 C:\NRPonb\DCC\AAR\41%705_1 DOC-14/06/2012 -2 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 (Summers and Mason, Cell 29: 403-415, 1982). The HBV genome is of a complex nature having a partially double-stranded DNA 5 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 polymerase consists of four functional regions, the terminal protein (TP), spacer, reverse transcriptase (rt) and ribonuclease (RNAse). 10 The polymerase gene of HBV overlaps the envelope gene, mutations in the catalytic domain of the polymerase gene can also affect the nucleotide and the deduced amino acid sequence of the envelope protein and vice versa. In particular, the genetic sequence for the neutralization domain of HBV known as the 'a' determinant, which is found within the HBsAg and located between amino acids 99 and 169, actually overlaps the major catalytic 15 regions of the viral polymerase protein and in particular domains A and B. The presence of an HBV DNA polymerase has led to the proposition that nucleoside or nucleotide analogs could act as effective anti-viral agents. Examples of nucleoside or nucleotide analogs currently being tested are penciclovir and its oral form (FCV) IVere 20 Hodge, Antiviral Chem Chemother 4: 67-84, 1993; Boyd et at., Antiviral Chem Chemother. 32: 358-363, 1987; Kruger et al., Hepatology 22: 219A, 1994; Main et al., J. Viral Hepatitis 3: 211-215, 1996], Lamivudine [(-)-p-2'-deoxy-3'-thiacytidine]; (3TC or LMV) [Severini et al., Antimicrobial Agents Chemother. 39: 430-435, 1995; Dienstag et al., New England]J Med 333: 1657-1661, 1995]. New nucleoside or nucleotide analogs 25 which have already progressed to clinical trials include the pyrimidines Emtricitabine, ((-) p-L-2'-3'-dideoxy-5-fluoro-3'-thiacydidine; FTC), the 5-fluoro derivative of 3TC, and Clevudine (1-(2-fluoro-5-methyl-p-L-arabino-furanosyl) uracil; L-FMAU), a thymidine analog. Like 3TC, these are pyrimidine derivatives with an unnatural "L"- configuration. Several purine derivatives have also progressed to clinical trials; they include Entecavir 30 (BMS-200, 475; ETV), a carbocyclic deoxyguanosine analog, diaminopurine dioxolane (DAPD), an oral pro-drug for dioxolane guanine ((-)-p-D-2-aminopurine dioxolane; DXG) C NRPorbr\DCC\AAR\439670_ I DOC. 14M62012 -3 and Adefovir dipivoxil, an oral prodrug for the acyclic deoxyadenosine monophosphate nucleoside or nucleotide analog Adefovir (9-[phosphonyl-methoxyethyl]-adenine; PMEA). Other drugs in pre-clinical and clinical trials include FLG [Medivir], ACH-126,443 (L d4C) [Archillion Pharmaceuticals], ICN 2001-3 (ICN) and Racivir (RCV) [Pharmassett]. 5 Whilst these agents are highly effective in inhibiting HBV DNA synthesis, there is the potential for resistant mutants of HBV to emerge during long term antiviral chemotherapy. In patients on prolonged LMV therapy, key resistance mutations are selected in the rt domain within the polymerase at rtM204I/V +/- rtL180M as well as other mutations. The 10 nomenclature used for the polymerase mutations is in accordance with that proposed by Stuyver et al., 2001, supra. LMV is a nucleoside or anucleotide analog that has been approved for use against chronic HBV infection. LMV is a particularly potent inhibitor of HBV replication and reduces HBV DNA titres in the sera of chronically infected patients after orthotopic liver transplantation (OLT) by inhibiting viral DNA synthesis. LMV 15 monotherapy seems unlikely to be able to control HBV replication in the longer term. This is because emergence of LMV-resistant strains of HBV seems almost inevitable during monotherapy. Adefovir dipivoxil (ADV: formerly, bis-pom PMEA) is an orally available prodrug of the 20 acyclic deoxyadenosine monophosphate analog adefovir (formerly, PMEA) (Figure 2). ADV is also a potent inhibitor of HBV replication and has recently been given FDA approval for use against chronic HBV infection. Adefovir dipivoxil differs from other agents in this class in that it is a nucleotide (vs. nucleoside) analog and as such bypasses the first phosphorylation reaction during drug activation. This step is often rate-limiting. 25 Adefovir dipivoxil has demonstrated clinical activity against both wild-type and lamivudine-resistant strains of HBV and is currently in phase III clinical Testing (Gilson et al., J Viral Hepat 6: 387-395, 1999; Perrillo et al., Hepatology 32: 129-134, 2000; Peters el al., Transplantation 68: 1912-1914, 1999; Benhamou et al., Lancet 358: 718-723, 2001). During phase II studies a 30 mg daily dose of adefovir dipivoxil resulted in a mean 30 4 logo decrease in viremia over 12 weeks (1-leathcote et al., Hepatology 28: A620, 1998).

C.\NRPorthhDCC\AAR\43,715_. DOC-141IW2012 -4 ADV is a substituted acyclic nucleoside phosphonate. This class of compounds also includes tenofovir disoproxil fumarate (also referred to as tenofovir DF, or tenofovir, or (TFV) or 9-R-(2-phosphonomethoxypropyl)adenine (PMPA) and is marketed as Viread by Gilead sciences). 5 TV has antiviral activity against both 1-BV and HIV (Ying ei al., J Viral Hepal. 7(2). 161-165, 2000; Ying et al., J. Viral Hepat. 7(1): 79-83, 2000; Suo et al., J Biol Chem. 273(42): 27250-27258. 1998). 10 FTC has activity against HBV and HIV (Frick el al., Antimicrob Agents Chemother 37: 2285-2292, 1993). Nucleoside or nucleotide analog therapy may be administered as monotherapy or combination therapy where two or more nucleoside or nucleotide analogs may be 15 administered. The nucleoside or nucleotide analogs may also be administered in combination with other antiviral agents such as interferon or hepatitis B immunoglobulin (HBIG). There is a need to monitor for the emergence of nucleoside/nucleotide-analog- or antibody 20 resistant strains of HBV and to develop diagnostic protocols to detect these resistant viruses and/or to use them to screen for and/or develop or design agents having properties making them useful as anti-viral agents. Defective forms of these resistant strains or antigenic components therefrom are also proposed to be useful in the development of therapeutic vaccine compositions as are antibodies directed to viral surface components.

C .NRForhCC\AAR\115_ .DOC144I62012 -5 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 number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 10 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims. Specific mutations in an amino acid sequence are represented herein as "XaainXaa 2 " where Xaa is the original amino acid residue before mutation, n is the residue number and 15 Xaa 2 is the mutant amino acid. The abbreviation "Xaa" may be the three letter or single letter (i.e. "X") code. An "rt" before "XaainXaa 2 " means "reverse transcriptase". An "s" means an envelope gene. The amino acid residues for HBV DNA polymerase are numbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD) being residue number 204 (Stuyver el al., Hepatology 33: 751-757, 2001). The amino acid 20 residues for hepatitis B virus surface antigen are number according to Norder el al. (.J Gen. Virol. 74. 341-1348, 1993). Both single and three letter abbreviations are used to define amino acid residues and these are summarized in Table 2. In accordance with the present invention, the selection of HBV variants is identified in 25 patients (Patients A to L) with chronic HBV infection treated with ADV. Patient E is a nonresponder to ADV. Other variants of HBV are identified during a combination ADV and LMV treatment or a combination of TFV and LMV treatment (eg. Patent M) or a combination of TFV and FTC or treatment with TFV alone with mutations in the HBV DNA polymerase gene which reduce the sensitivity of HBV to nucleoside or nucleotide 30 analogs. Consequently, HBV rt variants are contemplated which are resistant to, or which exhibit reduced sensitivity to, ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; C \NRPortb\DCC\AAR\41%705_ I DOC-14/lW'2112 -6 LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof . Corresponding mutations in 5 the surface antigen also occur. The identification of these HBV variants is important for the development of assays to monitor ADV, LMV, FTC and/or TFV resistance and/or resistance to other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and to screen for agents which are useful as alternative therapeutic agents. 10 Reference herein to "anti-HBV agents" includes nucleoside and nucleotide analogs as well as immunological reagents (e.g. antibodies to HBV surface components) and chemical, proteinaceous and nucleic acid agents which inhibit or otherwise interfere with viral replication, maintenance, infection, assembly or release. 15 The detection of such HBV variants is particularly important in the management of therapeutic protocols including the selection of appropriate agents for treating HBV infection. The method of this aspect of the present invention is predicated in part on monitoring the development in a subject of an increased HBV load in the presence of a 20 nucleoside or nucleotide analog or other anti-HBV agents or combinations thereof. The clinician is then able to modify an existing treatment protocol or select an appropriate treatment protocol accordingly. Accordingly, one aspect of the present invention is directed to an isolated Hepatitis B virus 25 (HBV) variant wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase and wherein said variant exhibits decreased sensitivity to one or more nucleoside or nucleotide analogs selected from the list consisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and 30 TFV; FTC and LMV; ADV and LMV and TFV; ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; ADV and FTC and LMV and TFV, and other C\NRPonblDCC\AARWI67051_ DOC-14/A6/20I2 -7 nucleoside or nucleotide analogs, and/or anti-HBV agents. The variant IBV comprises a mutation in an overlapping open reading frame in its genome in a region defined by one or more of domains F and G and domain A through to E of HBV DNA polymerase. 5 Another aspect of the present invention provides an isolated HBV variant comprising a nucleotide mutation in the S gene resulting in at least one amino acid addition, substitution and/or deletion to the surface antigen and which exhibits decreased sensitivity to one or more nucleoside or nucleotide analogs selected from the list consisting of ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC 10 and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV and other nucleoside or nucleotide analogs and/or anti-HBV agents. Useful mutants in the rt region include, in one embodiment, rtT38K, rtR55H and rtA 181V; 15 in another embodiment include rtS/T78S, rtV80L, rtN/SI18N, rtN/K139K, rtEl42V, rtA/TI81A rtI204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and rtY245H; in yet another embodiment include rtN238T; and yet another embodiment include rt I22V and rtA181T; in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, rtH237H/P, rtV253G, in yet another embodiment include rtT128N and rtN236T, in yet 20 another embodiment include tL180M, rtM204V rtQ215S, in yet another embodiment include rtT128S rtL180M, rtM204Vand rtQ215S, in yet another embodiment include rtI80L, rtl204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another embodiment include rtIl87V, in yet another embodiment include rtN123N/l rtSl35Y, rtV214A/V and rtQ215Q/P/Stop/S,or a combination thereof or an equivalent mutation. 25 Other HBV variants are also contemplated with mutations rtT38K (in the F domain of the DNA polymerase), rtR55H (located between the F and A domains), rtS/T78S, rtV80L (these are located within the A domain), rtN/SII8N, rt1l22V, rtN123N/l, rtSl35Y, rtN/K139K, rtE142V (located between the A and B domains, rtAl8lV, rtA181T (these 30 are located in the B domain),; rt I87V (located between the B and C domains), rtA/V200V (Located in the C Domain), rtV214A, rtV214A/V and rtQ215Q/P/Stop/S, C:\NRPonMb\DCC\AAR\46705.I DOC.4/06/2012 -8 rtQ/P/S/Stop2l5S, rtQ215S, rt E/K218E (located between the C and D domains), rtn236T, rtH237H/P rtN/H238H, rtN238T, rtN238T/A (these are located in the D domain), rtY245H (located between the D and E domains), and rtV253G (located in the E Domain) or a combination thereof or an equivalent mutation. 5 Useful mutations in the S gene include, in one embodiment include sQ30K. sE44G, sA47T, sI126T, sA159V and sL173F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sI/VI 101, sN/T131N, sNI34Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sV14A, sL95W, sV96G, and sl208T/I; and in yet another 10 embodiment include sT47A and sWI72stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F192F, sI195M, and in yet another embodiment include sS53L, and in yet another embodiment include sP120T, and in yet another embodiment include sN40S, sS207R, and in yet another embodiment include sQ10R, sl195M, sS207R, and in yet another embodiment include sL95W and sL196W, and in yet another embodiment 15 include sV14A, in yet another embodiment include sL42R, sQI02Q/R, sT15T/S, sS207S/R, sL215R and sL216Stop, or a combination thereof or an equivalent mutation. The present invention further contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to ADV, LMV,TFV or FTC; ADV and LMV; ADV 20 and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof by isolating DNA or corresponding mRNA from the HBV and screening for a mutation in the nucleotide 25 sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and G and domains A through to E or a region proximal thereto of the DNA polymerase and associated with resistance or decreased sensitivity to ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or 30 ADV and FTC and TFV; 'fFV and FTC and LMV; ADV and LMV and FTC; and/or ADV CANRPonb\DCC\AAR\43%7"5 1 DOC-14/O(/2012 -9 and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-1-BV agents or combination thereof. The presence of such a mutation is an indication of the likelihood of resistance to ADV, 5 LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. 10 The present invention also provides a composition comprising a variant HBV resistant to ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV 15 and TFV, ADV and LMV and FTC and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof or an HBV surface antigen from the variant HBV or a recombinant or derivative form thereof or its chemical equivalent and one or more pharmaceutically acceptable carriers and/or diluents. 20 Yet another aspect of the present invention provides a use of the aforementioned composition or a variant HBV comprising a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to the DNA polymerase and a decreased sensitivity to ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; 25 ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof in the manufacture of a medicament for the treatment and/or prophylaxis of hepatitis B virus infection. 30 C:NRPonbl\CC\AARW3%967)5l .DOC-14/6/2012 -10 The present invention also contemplates a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or other anti-l-BV agents or by isolating DNA or corresponding mRNA from the HIBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase wherein the presence of the 5 following mutations in the rt region: in one embodiment, rtT38K, rtR55H and rtA 18 IV; in another embodiment include rtS/T78S, rtV80L, rtN/Sll8N, rtN/K139K, rtEl42V, rtA/TI81A rtI204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and rtY245H; in yet another embodiment include rtN238T; and yet another embodiment include rtll22V and rtAl8lT; in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, 10 rtH237H/P, rtV253G, in yet another embodiment include rtTl28N and rtN236T, in yet another embodiment include tL180M, rtM204V rtQ215S, in yet another embodiment includes rtT128S rtL180M, rtM204Vand rtQ215S, in yet another embodiment includes rtl80L, rtI204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another embodiment include rtIl87V in yet another embodiment include rtNl23N/l rtSl35Y, 15 rtV214A/V and rtQ215Q/P/Stop/S, or combinations thereof or an equivalent one or more other mutation is indicative of a variant which exhibits a decreased sensitivity to ADV. LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV 20 and/or optionally other nucleoside or nucleotide analogs or other anti-H-BV agents or combination thereof. Still a further methodology comprises screening for a mutation in the nucleotide sequence encoding the envelope genes (s) wherein the presence of the following mutations in the s 25 gene: in one embodiment include sQ30K. sE44G, sA47T, slI26T, sA159V and sL173F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sI/V1101, sN/T131N, sNI34Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sV14A, sL95W, sV96G, and sI208T/I; and in yet another embodiment include sT47A and sWI72stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F192F, 30 sIl195M, and in yet another embodiment include sS53L, and in yet another embodiment include sPl20T, and in yet another embodiment include sN40S, sS207R, and in yet C \NRPonbl\DCC\AAR\41%7'5_ ItDOC-14/21112 - 11 another embodiment include sQIOIR, sI195M, sS207R, and in yet another embodiment include sL95W and sL196W, and in yet another embodiment include sV14A, in yet another embodiment include sL42R, sQI02Q/R, sTI15T/S, sS207S/R, sL215R and sL216Stop or combinations thereof or an equivalent one or more other mutation is 5 indicative of a variant which exhibits a decreased sensitivity to ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV, and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. 10 Preferably, the variants are in an 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 15 variant HBV. The detection of HBV or its components in cells, cell lysates, cultured supernatant fluid and bodily fluid may be by any convenient means including any nucleic acid-based detection means, for example, by nucleic acid hybridization techniques or via one or more polymerase chain reactions (PCRs). The term "bodily fluid" includes any fluid derived from the blood, lymph, tissue or organ systems including serum, whole blood, 20 biopsy and biopsy fluid, organ explants and organ suspension such as liver suspensions. Another aspect of the present invention is directed to a variant HBV comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to a surface antigen from a reference or 25 wild type HBV and wherein an antibody generated to the reference or wild type surface antigen exhibits an altered immunological profile relative to the HBV variant. One altered profile includes a reduced capacity for neutralizing the HBV. More particularly, the surface antigen of the variant HBV exhibits an altered immunological profile compared to a pre-treatment HBV where the variant HBV is selected for by a nucleoside or nucleotide 30 analog or other anti-HBV agents of the HBV DNA polymerase. The variant IBV of this aspect of the invention may also comprise a nucleotide sequence comprising a single or C.\NRPorbl\DCCAAR\U%705_1 DOC-14/n612012 - 12 multiple nucleotide substitution, addition and/or deletion compared to a pre-treatment HBV. The present invention further contemplates a method for detecting a variant HBV 5 exhibiting an altered immunological profile said method comprising isolating an HBV from a subject exposed to a nucleoside or nucleotide analog or combination of analogs selected from the listed consisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; 10 ADV and FTC and LMV and TFV, and then contacting said HBV with a panel of one or more antibodies to a surface antigen and screening for any change in binding affinity or binding spectrum. In a related invention, the present invention provides a method for detecting a variant HBV 15 exhibiting an altered immunological profile said method comprising isolating a serum sample from a subject exposed to a nucleoside or nucleotide analog selected from the listed consisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; ADV and FTC and LMV 20 and TFV, and then contacting the serum with a panel of HBV surface antigens or antibody binding fragments thereof and screening for any change in binding affinity or binding spectrum. The present invention extends to an isolated HBsAg or a recombinant form thereof or 25 derivative or chemical equivalent thereof corresponding to the variant HBV. Generally, the HBsAg or its recombinant or derivative form or its chemical equivalent comprises an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to an HBsAg from a reference HBV and wherein an antibody directed to a reference HBV exhibits an altered immunological profile to an IBV 30 carrying said variant HBsAg. In one embodiment, the altered immunological profile comprises a reduction in the ability to neutralize the variant HBV.

C :NRPonblDCC\AAR\U%70S_ .DOC-N'I 2012 - 13 Another aspect of the present invention contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV by generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in a 5 plasmid vector and then transfecting said cells with said construct, contacting the cells, before, during and/or after transfection, with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agents; and the subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral 10 component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent. In a preferred embodiment, the plasmid vector in a baculovirus vector and the method comprises generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in or fused to an amount of a baculovirus 15 genome effective to infect cells and then infecting said cells with said construct, contacting the cells, before, during and/or after infection, with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or 20 viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent. In connection with these methods, the plasmid vector may include genes encoding part or 25 all of other viral vectors such as baculovirus vectors or adenovirus vectors (see Ren and Nassal,.J. Virol. 75(3). 1104-1116, 2001). In an alternative embodiment, the method comprises generating a continuous cell line comprising an infectious copy of the genome of the HBV in a replication competent 30 effective amount such that said infectious HBV genome is stably integrated into said continuous cell line such as but not limited to the 2.2.15 or AD cell line, contacting the C.\NRPonbl\DCCAAR\I%.70_JI.DOC-14/A4W20I2 - 14 cells with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to the agent and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to 5 determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent. In an alternative embodiment, the present invention also contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV polymerase in an in vitro 10 polymerase assay. The HBV polymerase activity can be examined using established assays (Gaillard et al., Antimicrob Agents Chemother. 46(4): 1005-1013, 2002; Xiong et al., Hepatology. 28(6): 1669-73, 1998). The HBV polymerase may be a wild-type or reference HBV polymerase or mutant HBV polymerase. 15 The identification of viral variants enables the production of vaccines comprising particular recombinant viral components such as polymerases or envelope genes PreS1, PreS2, S encoding for L, M, S proteins as well as therapeutic vaccines comprising defective HBV variants. Rational drug design may also be employed to identify or generate therapeutic molecules capable of interacting with a polymerase or envelope genes 20 PreS1, PreS2, S encoding for L, M, S proteins or other component of the HBV. Such drugs may also have diagnostic potential. In addition, defective HBV variants may also be used as therapeutic compositions to generate an immune response against the same, similar or homologous viruses. Alternatively, antibodies generated to the HBV variants or surface components thereof may be used in passive immunization of subjects against infection by 25 HBV variants or similar or homologous viruses. Furthermore, agents such as nucleoside or nucleotide analogs, RNAi or siRNA molecules (both DNA-derived or synthetic), antisense or sense oligonucleotides, chemical or proteinaceous molecules having an ability to down regulate the activity of a component of HBV and inhibit replication, maintenance, infection, assembly or release are contemplated by the present invention. 30 C:\NRPorbl\DCC\AAR\43%7')5_I DOC-14/)6/2012 - 15 A summary of the abbreviations used throughout the subject specification are provided in Table 3. A summary of sequence identifiers used throughout the subject specification is provided in 5 Table 1. TABLE I Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1 Formula 1 2 Formula II 3 OSI primer 4 TTA3 primer 5 JM primer 6 TTA4 primer 7 OS2 primer 8 sense primer 9 antisense primer 10 internal regions primer S1I internal regions primer 12 PCI forward primer 13 PC2 reverse primer 14 HBV-specific molecular beacon primer 10 C :NRPonbiKDCCAAR4196705._ .DOC-14/ 12012 - 16 TABLE2 Single and three letter amino acid abbreviations Amino Acid Three-letter Abbreviation One-letter symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutanic acid Glu E Glycine Gly G Histidine His H Isoleucine lIle Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine The T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X C:\NRPorbl\DCC\AAR396705_ DOC.-14/062012 - 17 TABLE3 Abbreviations ABBREVIATION DESCRIPTION 3TC (LMV); (-)-p-2'-deoxy-3'-thiacytidine ADV adefovir dipivoxil DAPD diaminopurine dioxalone DXG dioxolane guanine ETV entecavir FAM famciclovir FCV famciclovir FTC emtricitabine HBIG hepatitis B immunoglobulin HBsAg hepatitis B surface antigen H1BV hepatitis B virus LMV lamividuine PMEA 9-[phosphonyl-methoxyethyl]-adenine; adefovir PMPA 9-R-(2-phosphonomethoxypropyl)adenine RNase ribonuclease rt ("rt" before "Xaai nXaa 2 " means reverse transcriptase reverse transcriptase) s (as used in a mutation, envelope gene e.g. sF134V) TFV tenofovir disoproxil fumarate YMDD Tyr Met Asp Asp-a motif in the polymerase protein; where the Met residue is designated residue number 204 of the reverse transcriptase 5 C NRPorb\DCC\AAR\41%6705_. DOC-14/06/2012 - 18 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagrammatic representation showing the partially double stranded DNA HBV genome showing the overlapping open reading frames encoding surface (S), core 5 (C), polymerase (P) and X gene. Figure 2 is a diagrammatic representation of the chemical structure of ADV. Figure 3 is a diagrammatic representation of a computer system for determining the value 10 of a variant HBV. Figure 4 is a representation showing comparison of the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in sequential samples from Patient A during ADV treatment. 15 Figure 5 is a representation showing comparison of the deduced amino acid sequence of the catalytic region of the polymerase gene in sequential samples from Patient A during ADV therapy. 20 Figure 6 is a representation showing comparison of the deduced amino acid sequence of the envelope gene in sequential samples from Patient A during ADV therapy. Figure 7 is a representation showing comparison of the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in sequential samples from Patient B 25 during ADV and LMV treatment. Figure 8 is a representation showing comparison of the deduced amino acid sequence of the catalytic region of the polymerase gene in sequential samples from Patient B during ADV and LMV therapy. 30 CkNRPorbl\DC(AAR\1%6705_,IDOC.-I4AW2012 -19 Figure 9 is a representation showing comparison of the deduced amino acid sequence of the envelope gene in sequential samples from Patient B during ADV and LMV therapy. Figure 10 is a representation the HBV nucleotide sequence encoding the catalytic region 5 of the polymerase gene in samples from Patient C during ADV treatment. Figure 11 is a representation the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient C during ADV therapy. 10 Figure 12 is a representation the deduced amino acid sequence of the envelope gene in samples from Patient C during ADV therapy. Figure 13 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient D during ADV treatment. 15 Figure 14 is a representation the deduced amino acid sequence of the catalytic region of the polymerase gene in sequential samples from Patient D during ADV therapy. Figure 15 is a representation showing comparison of the deduced amino acid sequence of 20 the envelope gene in sequential samples from Patient D during ADV therapy. Figure 16 is a representation showing the HBV nucleotide sequence encoding the catalytic .region of the polymerase gene in samples from Patient E during ADV treatment. 25 Figure 17 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient E during ADV therapy. Figure 18 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient E during ADV therapy. 30 C XNRPobl\DCC\AAR\-3%705_ I DOC.14/-'21112 - 20 Figure 19 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient F during ADV treatment. Figure 20 is a representation showing the deduced amino acid sequence of the catalytic 5 region of the polymerase gene in samples from Patient F during ADV therapy. Figure 21 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient F during ADV therapy. 10 Figure 22 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient G during ADV treatment. Figure 23 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient G during ADV therapy. 15 Figure 24 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient G during ADV therapy. Figure 25 is a representation showing the HBV nucleotide sequence encoding the catalytic 20 region of the polymerase gene in samples from Patient H during ADV treatment. Figure 26 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient 1H during ADV therapy. 25 Figure 27 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient H during ADV therapy. Figure 28 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient I during ADV treatment. 30 C :NRPorb\CC\AAR"43%705_tDOC-14AW6/2012 -21 Figure 29 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient I during ADV therapy. Figure 30 is a representation showing the deduced amino acid sequence of the envelope 5 gene in samples from Patient I during ADV therapy. Figure 31 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient J during ADV treatment. 10 Figure 32 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient J during ADV therapy. Figure 33 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient J during ADV therapy. 15 Figure 34 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient K during ADV treatment. Figure 35 is a representation showing the deduced amino acid sequence of the catalytic 20 region of the polymerase gene in samples from Patient K during ADV therapy. Figure 36 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient K during ADV therapy. 25 Figure 37 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient L during ADV treatment. Figure 38 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient L during ADV therapy. 30 C:\NRPonbI\DCC\AARW396705_1.DOC-14/06/2012 -22 Figure 39 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient L during ADV therapy. Figure 40 is a representation showing the HBV nucleotide sequence encoding the catalytic 5 region of the polymerase gene in samples from Patient M during TFV treatment. Figure 41 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in samples from Patient M during TFV therapy. 10 Figure 42 is a representation showing the deduced amino acid sequence of the envelope gene in samples from Patient M during TFV therapy. Figure 43 is a representation showing the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in another sample from Patient M during TFVtreatment. 15 Figure 44 is a representation showing the deduced amino acid sequence of the catalytic region of the polymerase gene in another sample from Patient M during TFV therapy. Figure 45 is a representation showing the deduced amino acid sequence of the envelope 20 gene in another sample from Patient M during TFV therapy.

C \NRPortbl\DCC\AAR\41967h5_I DOC-14/06/2012 - 23 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated in part on the identification and isolation of nucleoside or nucleotide analog-resistant variants of HBV following treatment of patients with either 5 ADV or LMV or more particularly ADV and LMV or TFV and LMV, or optionally other nucleoside analogs or nucleotide analogs or other anti-HBV agents such as TFV or FTC. In particular, ADV or ADV and LMV treated patients gave rise to variants of H13V exhibiting decreased or reduced sensitivity to ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and 10 TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV . Reference herein to "decreased" or "reduced" in relation to sensitivity to ADV and/or LMV and/or FTC and/or TFV includes and encompasses a complete or substantial resistance to the nucleoside or nucleotide analog or other anti-HBV agents as well as partial resistance and includes a replication rate or 15 replication efficiency which is more than a wild-type in the presence of a nucleoside or nucleotide analog or other anti-H1BV agents. In one aspect, this is conveniently measured by an increase in viral load during treatment, or alternatively, there is no substantial decrease in HBV DNA viral load from pre-treatment HBV DNA levels during treatment (i.e., non-response to treatment). 20 Accordingly, one aspect of the present invention contemplates an isolated Hepatitis B virus (HBV) variant wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase and wherein said variant exhibits decreased sensitivity to one or 25 more nucleoside or nucleotide analogs selected from the list consisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; ADV and FTC and LMV and TFV, and other nucleoside or nucleotide analogs, and/or anti-HBV agents. 30 C:\NRPortb1\DCC\AAR\3%6705_ .DOC-1410612012 - 24 Another aspect of the present invention provides a method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside or nucleotide analog selected from ADV, LMV, TFV and FTC or a combination thereof or optionally other nucleoside or nucleotide analogs, said method comprising isolating DNA or corresponding mRNA from 5 said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and A through E or a region proximal thereto of said DNA polymerase and associated with resistance or decreases sensitivity to one or more of ADV, LMV, TFV and/or FTC wherein the presence of such a mutation is an indication of the 10 likelihood of resistance to said one or more of ADV, LMV, TFV and/or FTC. Before describing the present invention in detail, it is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulations of components, manufacturing methods, dosage regimens, or the like, as such may vary. It is 15 also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the subject specification, the singular forms "a". "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for 20 example, reference to "a nucleoside or nucleotide analog" includes a single analog, as well as two or more analogs; reference to "an HBV variant" includes reference to two or more HBV variants; and so forth. In describing and claiming the present invention, the following terminology is used in 25 accordance with the definitions set forth below. The terms "analog", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a chemical compound that induces a desired effect such as inhibit viral replication, infection, 30 maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. The terms also encompass pharmaceutically acceptable and pharmacologically active C RPonbl\DCC\AAR\3%7CI5_I DOC. 14AW2012 -25 ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "analog", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent 5 per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The present invention contemplates, therefore, compounds useful in inhibiting HBV replication, infection, maintenance, assembly and/or the function of an enzyme such as 10 HBV DNA polymerase. Reference to an "analog", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" such as ADV, LMV, FTC and/or TFV includes combinations of two or more actives such as ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and 15 FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV. A "combination" also includes a two-part or more such as a multi-part anti-HIBV therapeutic composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation. 20 The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of the agent to provide the desired therapeutic or physiological effect of inhibiting HBV replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. Furthermore, an "effective HBV-inhibiting amount" or "effective symptom-ameloriating amount" of an 25 agent is a sufficient amount of the agent to directly or indirectly inhibit replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount 30 required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible CANRPobl\DCCAAR\UA705_1 DOC- I A4/0122 - 26 to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. 5 By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emusifying agents, pH 10 buffering agents, preservatives, and the like. Similarly, a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable. 15 The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage in relation to HBV infection. Thus, for example, "treating" a 20 patient involves prevention of HBV infection as well as treatment of a clinically HBV symptomatic individual by inhibiting HBV replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. Thus, for example, the present method of "treating" a patient with HBV infection or with a propensity for one to develop encompasses both prevention of IBV infection as well as treating H3V infection 25 or symptoms thereof. In any event, the present invention contemplates the treatment or prophylaxis of HBV infection. "Patient" as used herein refers to an animal, preferably a mammal and more preferably a primate including a lower primate and even more preferably, a human who can benefit 30 from the formulations and methods of the present invention. A patient regardless of whether a human or non-human animal may be referred to as an individual, subject, C \NRPornb\DCC\AAR\41 705_1 DOC-141/21112 - 27 animal, host or recipient. The compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry. For convenience, an "animal" includes an avian species such as a poultry bird (including ducks, chicken, turkeys and geese), an aviary bird or game bird. 5 The condition in a non-human animal may not be a naturally occurring HBV infection but HBV-like infection may be induced. As indicated above, the preferred animals are humans, non-human primates such as marmossets, baboons, orangatangs, lower primates such as tupia, livestock animals, 10 laboratory test animals, companion animals or captive wild animals. A human is the most preferred target. However, non-human animal models may be used. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or 15 animal model as do primates and lower primates. Livestock animals include sheep, cows, pigs, goats, horses and donkeys. Non-mammalian animals such as avian species, zebrafish, amphibians (including cane toads) and Drosophila species such as Drosophila melanogaster are also contemplated. Instead of a live animal model, a test system may also comprise a tissue culture system. 20 An "anti-HBV agent" includes a nucleoside or nucleotide analog, protein, chemical compound, RNA or DNA or RNAi or siRNA oligonucleotide (either DNA-derived or synthetic). 25 Preferably, the decreased sensitivity is in respect of ADV. Alternatively, the decreased sensitivity is in respect of LMV. Alternatively, the decreased sensitivity is in respect of TFV. Alternatively, the decreased sensitivity is in respect of FTC. Alternatively, the decreased sensitivity is in respect of ADV and LMV. Alternatively, the decreased sensitivity is in respect of ADV and TFV. Alternatively, the decreased sensitivity is in 30 respect of LMV and TFV. Alternatively, the decreased sensitivity is in respect of ADV and FTC. Alternatively, the decreased sensitivity is in respect to FTC and TFV. Alternatively, C :NRPonbl\DCC\AAR\4%7I)05L.DOC-14/U6W2012 -28 the decreased sensitivity is in respect of FTC and LMV. Alternatively, the decreased sensitivity is in respect of ADV and LMV and TFV. Alternatively, the decreased sensitivity is in respect to ADV and TFV and FTC. Alternatively, the decreased sensitivity is in respect to LMV and TFV and FTC. Alternatively, the decrease sensitivity is in respect 5 of ADV and LMV and FTC. Alternatively, the decreased sensitivity is in respect of ADV and FTC and TFV and LMV. Reference herein to "anti-HBV agents" includes nucleoside and nucleotide analogs as well as immunological reagents (e.g. antibodies to HBV surface components) and chemical, 10 proteinaceous and nucleic acid agents which inhibit or otherwise interfere with viral replication, maintenance, infection, assembly or release. Reference herein to "nucleic acid" includes reference to a sense or antisense molecule, RNA or DNA, oligonucleotides and RNAi and siRNA molecules and complexes containing same. 15 In addition to a mutation in the gene encoding DNA polymerase, due to the overlapping nature of the HBV genome (Figure 1), a corresponding mutation may also occur in the gene encoding the S gene encoding the surface antigen (HBsAg) resulting in reduced interactivity of immunological reagents such as antibodies and immune cells to HBsAg. The reduction in the interactivity of immunological reagents to a viral surface component 20 generally includes the absence of immunological memory to recognize or substantially recognize the viral surface component. The present invention extends, therefore, to an HBV variant exhibiting decreased sensitivity to ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and 25 LMV and FTC; and/or ADV and FTC and LMV and TFV or a reduced interactivity of an immunological reagent to HBsAg wherein the variant is selected for following ADV and/or LMV combination or sequential treatment. The term "sequential" in this respect means ADV followed by LMV and/or TFV, and /or FTC, LMV followed by ADV and/or TFV, and /or FTC, or multiple sequential administrations of each of ADV, LMV and/or 30 TFV, and /or FTC.

C:\NRPorbl\DCCAAR\1%705_. DOC-14/062112 - 29 A viral variant may, therefore, carry A mutation only in the DNA polymerase gene or both in the DNA polymerase gene and the S gene. The term "mutation" is to be read in its broadest context and includes multiple mutations. 5 The present invention extends to a mutation and any domain of the HBV DNA polymerase and in particular regions F and G, and domains A through to E provided said mutation leads to decreased sensitivity to ADV and/ or LMV and/or TFV or combinations thereof. In this specification, reference is particularly made to the conserved regions of the DNA 10 polymerase as defined by domains A to E. Regions A to E are defined by the amino acid sequence set forth in Formula II in Australian Patent No. 734831. Preferably, the mutation results in an altered amino acid sequence in any one or more of domains F and G, and domains A through to E or regions proximal thereto of the HBV 15 DNA polymerase. Another aspect of the present invention provides an HBV variant comprising a mutation in an overlapping open reading frame in its genome wherein said mutation is in a region defined by one or more of domains F and G, and domains A through to E of HBV DNA 20 polymerase and wherein said variant exhibits decreased sensitivity to ADV, LMV,TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; and/or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents. 25 Another preferred aspect of the present invention contemplates an HBV variant comprising a mutation in the nucleotide sequence encoding HBsAg resulting in an amino acid addition, substitution and/or deletion in said H-BsAg wherein said variant exhibits decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; 30 LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or C \NRPonbl\DCC\AAR\43%6705_ I DOC-14AW6/212 - 30 ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-1-BV agents or combination thereof. More particularly, the present invention provides a variant HBV comprising a surface 5 antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to a surface antigen from a reference or wild-type HBV and wherein an antibody generated to the reference or wild-type surface antigen exhibits reduced capacity for neutralizing said HBV variant, said variant selected by exposure of a subject to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; 10 LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. 15 The term "combination therapy" means that both combinations of ADV, LMV, FTC and/or TFV are co-administered in the same composition or simultaneously in separate compositions. The term "sequential therapy" means that the two agents are administered within seconds, minutes, hours, days or weeks of each other and in either order. Sequential therapy also encompasses completing a therapeutic course with one or other of ADV, 20 LMV, FTC or TFV and then completing a second or third or subsequent therapeutic courses with the other of ADV, LMV, FTC or TFV. Accordingly, another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple 25 amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant IBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. 30 C \NRPonlrDCC\AAR\43%705_L1,DOC.14/16121)12 - 31 Another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment IBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to 5 the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to LMV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. Yet another aspect of the present invention contemplates an HBV variant comprising a 10 surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to FTC therapy or therapy by one or more other nucleoside or nucleotide 15 analogs or other anti-HBV agents. Still another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and 20 wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. 25 Even yet another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HIBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure 30 of a subject to ADV and LMV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-IBV agents.

CANRPonbl\DCC\AAR\41%75_ I DOC-14/06/2012 - 32 Even still another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment IBV and 5 wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. 10 A further aspect of the present invention contemplates an IBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure 15 of a subject to LMV and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. Another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, 20 addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV and FTC therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. 25 Yet another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment IBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile 30 compared to the pretreatment HBV where the said variant HBV is selected for by exposure C:\NRPorbl\DCC\AARi4967l5. I DOC. 14/02012 -33 of a subject to TFV and FTC therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. Still another aspect of the present invention contemplates an HBV variant comprising a 5 surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to FTC and LMV therapy or therapy by one or more other nucleoside or 10 nucleotide analogs or other anti-HBV agents. Even yet another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and 15 wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, LMV and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. 20 Even still another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure 25 of a subject to ADV, LMV and TFV therapy or therapy by one or more other nucleoside or neuleotide analogs or other anti-HBV agents. A further aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid 30 substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile C .NRPenbIDCC\AAR\4%705_ .DOC.4m 2012 - 34 compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, LMV and FTC therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. 5 Another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject 10 to FTC, LMV and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. Yet another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid 15 substitution, addition and/or deletion or truncation compared to the pretreatment -IBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, FTC and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. 20 Still yet another aspect of the present invention contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment IBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile 25 compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, LMV, FTC and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents. Preferably, the variants are in isolated form such that they have undergone at least one 30 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 C \NRPonbl\DCC\AAR\43967(l5_. DOC- 14/A/2012 - 35 contemplates infectious molecular clones comprising the genome or parts thereof from a variant HBV. Furthermore, the present invention provides isolated components from the variant HBVs such as but not limited to an isolated HBsAg. Accordingly, the present invention provides an isolated HBsAg or a recombinant form thereof or derivative or 5 chemical equivalent thereof, said HBsAg being from a variant HBV selected by exposure of a subject to one or more of ADV, LMV, FTC and/or TFV or optionally one or more nucleoside or nucleotide analogs or other anti-HBV agents. More particularly, yet another aspect of the present invention is directed to an isolated 10 variant HlBsAg or a recombinant or derivative form thereof or a chemical equivalent thereof wherein said HBsAg or its recombinant or derivative form or its chemical equivalent exhibits an altered immunological profile compared to an HBsAg from a reference HBV, said HBsAg being from a variant HBV selected by exposure of a subject to one or more of ADV, LMV, FTC and/or TFV or optionally one or more nucleoside or 15 nucleotide analogs or other anti-HBV agents. Even more particularly, the present invention provides an isolated variant HBsAg or a recombinant or derivative form thereof or a chemical equivalent thereof wherein said HBsAg or its recombinant or derivative form or its chemical equivalent comprises an 20 amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to an HBsAg from a reference HBV and wherein a neutralizing antibody directed to a reference HBV exhibits no or reduced neutralising activity to an HBV carrying said variant HBsAg, said HBsAg being from a variant HBV selected by exposure of a subject to one or more of ADV, LMV, FTC and/or TFV or 25 optionally one or more nucleoside or nucletoide analogs or other anti-HBV agents. Preferred mutations in the HBV DNA polymerase include variants selected from patients with HBV recurrence following ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV 30 and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV treatment. Nucleoside or nucleotide analogs or other C NRPonbl\DCCAAR\41175_IDOC-14AW2012 - 36 anti-HBV agents may be indicated during , after or prior to a transplantation procedure (e.g. bone marrow transplantation (BMT) or OLT) or following treatment of patients diagnosed with hepatitis. Following selection of variants, viral loads are obtainable at levels similar to pre-treatment levels or increase while on therapy. 5 Useful mutants in the rt region include, in one embodiment, rtT38K, rtR55H and rtA]81V; in another embodiment include rtS/T78S, rtV80L, rtN/S1l8N, rtN/K139K, rtEl42V, rtA/TI81A rtl204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and rtY245H; in yet another embodiment include rtN238T; and yet another embodiment include rtll22V and 10 rtAl81T; in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, rt-1237H/P, rtV253G, in yet another embodiment include rtTl28N and rtN236T, in yet another embodiment include tL180M, rtM204V rtQ215S, in yet another embodiment include rtTl28S rtLl80M, rtM204Vand rtQ215S, in yet another embodiment includes rtl80L, rtl204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another 15 embodiment include rtIl87V, in yet another embodiment include rtN123N/I rtS135Y, rtV214A/V and rtQ215Q/P/Stop/S, or a combination thereof or an equivalent mutation Such HBV variants are proposed to exhibit a decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; 20 FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. It should be noted that the nomenclature system for amino acid positions is based on the methionine residues in the YMDD motif being designated codon rtM204. This 25 numbering system is different to that in Australian Patent No. 734831 where the methionine residue in the YMDD motif within the polymerase gene is designated codon 550. In this regard, rtL180M and rtM204V correspond to L526M and M550V, respectively, in Australian Patent No. 734831. Corresponding mutations may also occur in envelope genes such as in one or more of PreS 1, PreS2 and S. 30 C \NRPonbI\DCCAAR\43%705 I DOC-14)6/24112 - 37 Another potential mode of action of ADV and other acyclic nucleoside phosphonates is that of immune stimulation (Calio et al., Antiviral Res. 23: 77-89, 1994). A number of mutations resulted in changes in the envelope gene detected in HBV variants which may be associated with immune escape. These changes include in one embodiment include in 5 one embodiment include sQ30K. sE44G, sA47T, s1126T, sA159V and sL173F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sl/V1OI, sN/T1 31N, sN134Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sVl4A, sL95W, sV96G, and sl208T/I; and in yet another embodiment include sT47A and sW172stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F192F, 10 sIl95M, and in yet another embodiment include sS53L, and in yet another embodiment include sP120T, and in yet another embodiment include sN40S, sS207R, and in yet another embodiment include sQIO1R, sI195M, sS207R, and in yet another embodiment include sL95W and sL196W, and in yet another embodiment include sVl4A, in yet another embodiment include sL42R, sQ102Q/R, sTi15T/S, sS207S/R, sL215R and 15 sL216Stop, or a combination thereof or an equivalent mutation. 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 20 monitor vaccination protocols, or develop new or modified vaccine preparations. Still another aspect of the present invention contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; 25 or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents, said method comprising isolating DNA or corresponding mRNA from said lBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino 30 acid substitution, deletion and/or addition in any one or more of domains F and G, and A domains through to E or a region proximal thereto of said DNA polymerase and associated C NRPrbI\DCC\AARI%1967I15_. DOC. 14/6/21I12 - 38 with resistance or decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or 5 nucleotide analogs or other anti-HBV agents wherein the presence of such a mutation is an indication of the likelihood of resistance to said ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or 10 nucleotide analogs or other anti-HBV agents. Accordingly, another aspect of the present invention provides a method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside or nucleotide analog selected from ADV, LMV, TFV and FTC or optionally other nucleoside or nucleotide 15 analogs, said method comprising isolating DNA or corresponding mRNA from said IBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and A through E or a region proximal thereto of said DNA polymerase and associated with resistance or decreases sensitivity to one or more of ADV, LMV, TFV 20 and/or FTC wherein the presence of such a mutation is an indication of the likelihood of resistance to said one or more of ADV, LMV, TFV and/or FTC. Preferably, the assay detects one or more of the following mutations: in one embodiment, rtT38K, rtR55H and rtA181V; in another embodiment include rtS/T78S, rtV80L, 25 rtN/SII8N, rtN/K139K, rtE142V, rtA/TI81A rt1204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and rtY245H; in yet another embodiment include rtN238T; and yet another embodiment include rtIl22V and rtA181T; in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, rtH237H/P, rtV253G, in yet another embodiment include rtT128N and rtN236T, in yet another embodiment include tLI80M, rtM204V rtQ215S, in 30 yet another embodiment include rtTI28S rtL180M, rtM204Vand rtQ215S, in yet another embodiment include rtl80L, rtI204M, rtN238T, in yet another embodiment include C.\NRPonbI\DCC\AAR\41%705_ DOC.14/W'/2112 -39 rtN238T/A, in yet another embodiment include rtIl87V, in yet another embodiment include rtN123N/I rtS135Y, rtV214A/V and rtQ215Q/P/Stop/S, or combinations thereof or an equivalent one or more other mutation is indicative of a variant wherein said variant exhibits a decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV 5 and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. 10 Accordingly, another aspect of the present invention produces a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or other anti-HBV agents, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase and/or a corresponding region of the S gene, wherein the presence of a 15 mutation selected from, in one embodiment include sQ30K. sE44G, sA47T, sII26T, sAI59V and sL173F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sI/Vi101, sN/T131N, sN134Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sV14A, sL95W, sV96G, and sl208T/l; and in yet another embodiment include sT47A and sWI72stop, and in yet another embodiment include PreS2 20 T6S, sT47A, sP62L, sL/F192F, s1195M, and in yet another embodiment include sS53L, and in yet another embodiment include sPI20T, and in yet another embodiment include sN40S, sS207R, and in yet another embodiment include sQ101R, sl195M, sS207R, and in yet another embodiment include sL95W and sL196W, and in yet another embodiment include sVl4A, in yet another embodiment include sL42R, sQI02Q/R, sTlI15T/S, 25 sS207S/R, sL215R and sL216Stop and in yet another embodiment include sT47A and sW172stop, in even still another embodiment, rtT38K, rtR55H and rtAI81V; in another embodiment include rtS/T78S, rtV80L, rtN/S1l8N, rtN/K139K, rtEI42V, rtA/T181A rt1204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and rtY245H; in yet another embodiment include rtN238T; and yet another embodiment include rtlI22V and rtAl8 IT; 30 in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, rtl-12371-/P, rtV253G, in yet another embodiment include rtT128N and rtN236T, in yet another C\NRPcnbnEDCC\AAR\43%'705.1 DOC-1/W2I12 -40 embodiment include tL180M, rtM204V rtQ215S, in yet another embodiment include rtTl128S rtL180M, rtM204Vand rtQ215S, in yet another embodiment includes rtI80L, rtI204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another embodiment include rtIl87V, in yet another embodiment include rtNl23N/I rtSl35Y, 5 rtV214A/V and rtQ215Q/P/Stop/S, or combinations thereof or an equivalent one or more other mutation is indicative of a variant which exhibits a decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV 10 and/or optionally other nucleoside or nucleotide analogs or other anti--IBV agents or combination thereof. A further aspect of the present invention produces a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or other anti 15 HBV agents, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase and/or a corresponding region of the S gene, wherein the presence of a mutation selected from, in one embodiment, sQ30K. sE44G, sA47T, sI 26T, sA159V and sLI73F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sI/Vl101, 20 sN/T131N, sN134Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sV14A, sL95W, sV96G, and sl208T/1; and in yet another embodiment include sT47A and sWl72stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F192F, s1195M, and in yet another embodiment include sS53L, and in yet another embodiment include sP120T, and in yet another embodiment include 25 sN40S, sS207R, and in yet another embodiment include sQ101R, sll95M, sS207R, and in yet another embodiment include sL95W and sL196W, and in yet another embodiment include sV14A, in yet another embodiment include sL42R, sQI02Q/R, sTI15T/S, sS207S/R, sL215R and sL216Stop and in yet another embodiment include sT47A and sW172stop, in even still another embodiment, rtT38K, rtR55H and rtAl81V; in another 30 embodiment include rtS/T78S, rtV80L, rtN/S1 18N, rtN/K139K, rtEl42V, rtA/T181A rtl204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and rtY245H; in yet another C.kNRPornhRDC0AAR\U11,71)5_1 DOC-14IW 12 -41 embodiment include rtN238T; and yet another embodiment include rtIl22V and rtA181T; in yet another embodiment includes rtL180M, rtA/V200V, rtM204V, rtV2l4A, rtH237H/P, rtV253G, in yet another embodiment include rtT128N and rtN236T, in yet another embodiment include tL180M, rtM204V rtQ215S, in yet another embodiment 5 include rtT128S rtLl80M, rtM204Vand rtQ215S, in yet another embodiment include rt180L, rtI204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another embodiment include rtI187V, in yet another embodiment include rtN123N/ rtS135Y, rtV214A/V and rtQ215Q/P/Stop/S, combinations thereof or an equivalent one or more other mutation is indicative of a variant which exhibits a decreased sensitivity to ADV, 10 LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti--IBV agents or combination thereof. 15 The detection of HBV or its components in cells, cell lysates, cultured supernatant fluid and bodily fluid may be by any convenient means including any nucleic acid-based detection means, for example, by nucleic acid hybridization techniques or via one or more polymerase chain reactions (PCRs). The term "bodily fluid" includes any fluid derived 20 from the blood, lymph, tissue or organ systems including serum, whole blood, biopsy and biopsy fluid, organ explants and organ suspension such as liver suspensions. The invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification 25 and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others. Other forms of detection include Northern blots, Southern blots, PCR sequencing, antibody procedures such as ELISA, Western blot and immunohistochemistry. A particularly useful assay includes the reagents 30 and components required for immobilized oligonucleotide- or oligopeptide-mediated detection systems.

C \NRPonbl\DCCAAR\4396705 . DOC-14/AW2012 - 42 One particularly useful nucleic acid detection system is the reverse hybridization technique. In this technique, DNA from an HBV sample is amplified using a biotin or other ligand-labeled primer to generate a labeled amplificon. Oligonucleotides 5 immobilized to a solid support such as a nitrocellulose film are then used to capture amplified DNA by hybridization. Specific nucleic acid fragments are identified via biotin or the ligand. Generally, the labeled primer is specific for a particular nucleotide variation to be detected. Amplification occurs only if the variation to be detected is present. There are many forms of the reverse hybridization assay and all are encompassed by the present 10 invention. Another aspect contemplated by the present invention provides a method for detecting a variant HBV exhibiting an altered immunological profile said method comprising isolating an HBV from a subject exposed to a nucleoside or nucleotide analog selected from the 15 listed consisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; ADV and FTC and LMV and TFV, and then contacting said HBV with a panel of one or more antibodies to a surface antigen and screening for any change in binding affinity or binding spectrum. 20 In a related embodiment, the present invention contemplates a method for detecting a variant HBV exhibiting an altered immunological profile said method comprising isolating a serum sample from a subject exposed to a nucleoside or nucleotide analog selected from the listed consisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV 25 and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ADV and LMV and TFV; ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; ADV and FTC and LMV and TFV, and then contacting the serum with a panel of HBV surface antigens or antibody-binding fragments thereof and screening for any change in binding affinity or binding spectrum. 30 Detecting HBV replication in cell culture is particularly useful.

C \NRPonbl\DCC\AAR\4V%705_ IDOC-14/06/2012 - 43 This and other aspects of the present invention is particularly amenable to microarray analysis such as to identify oligonucleotides including sense and antisense molecules, RNAi or siRNA molecules or DNA or RNA-binding molecules which down-regulate 5 genomic sequences or transcripts of HBV. Microarray analysis may also be used to identify particular mutations in the HBV genome such as within the HBV DNA polymerase-coding region or the HBsAg-coding region. Another aspect of the present invention contemplates a method for detecting an agent 10 which exhibits inhibitory activity to an HBV by: generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in a plasmid vector and then transfecting said cells with said construct; 15 contacting the cells, before, during and/or after transfection, with the agent to be tested; culturing the cells for a time and under conditions sufficient for the HBV to 20 replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agents; and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, 25 expressed genetic material and/or assembled and/or been released in the presence of the agent. In a preferred embodiment, the plasmid vector may include genes encoding part or all of other viral vectors such as baculovirus or adenovirus (Ren and Nassal, 2001, supra) and 30 the method comprises: C \NRPorlDCCAAR\J9%705_ .DOC-14WI 21112 -44 generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in or fused to an amount of a baculovirus genome or adenovirus genome effective to infect cells and then infecting said cells with said construct; 5 contacting the cells, before, during and/or after infection, with the agent to be tested; culturing the cells for a time and under conditions sufficient for the HBV to 10 replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, 15 expressed genetic material and/or assembled and/or been released in the presence of the agent. In an alternative embodiment, the method comprises: 20 generating a continuous cell line comprising an infectious copy of the genome of the HBV in a replication competent effective amount such that said infectious IBV genome is stably integrated into said continuous cell line such as but not limited to 2.2.15 or AD; 25 contacting the cells with the agent to be tested; culturing the cells for a time and under conditions sufficient for the I-IBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to the agent; and 30 C:\NRPodbKDCCAAR\41%75) DOC- 14/KW2012 - 45 then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent. 5 The above-mentioned methods are particularly useful in identifying or developing agents against HBV variants such as those carrying mutations, in one embodiment, rtT38K, rtR55H and rtAl81V; in another embodiment include rtS/T78S, rtV80L, rtN/SI18N, rtN/K139K, rtE142V, rtA/T181A rtI204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and 10 rtY245H; in yet another embodiment include rtN238T; and yet another embodiment includes rtII22V and rtA181T; in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, rtH237H/P, rtV253G, in yet another embodiment include rtTI28N and rtN236T, in yet another embodiment include tL180M, rtM204V rtQ215S, in yet another embodiment include rtT128S rtL180M, rtM204Vand rtQ215S, in yet another embodiment 15 include rtl80L, rt1204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another embodiment includes rtl187V, in yet another embodiment includes rtNI23N/I rtS135Y, rtV214A/V and rtQ215Q/P/Stop/S, or a combination thereof or an equivalent mutation; in a further embodiment, sQ30K. sE44G, sA47T, sIl126T, sA159V and sL173F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sI/V1101, sN/T131N, 20 sN134Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sV14A, sL95W, sV96G, and sl208T/l; and in yet another embodiment include sT47A and sW172stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F192F, sl195M, and in yet another embodiment include sS53L, and in yet another embodiment include sP120T, and in yet another embodiment include sN40S, sS207R, and in yet 25 another embodiment include sQlO1R, s1l95M, sS207R, and in yet another embodiment include sL95W and sLl96W, and in yet another embodiment include sVI4A, in yet another embodiment include sL42R, sQI02Q/R, sTI15T/S, sS207S/R, sL215R and sL216Stop, or a combination thereof or an equivalent mutation. 30 Accordingly, another aspect of the present invention contemplates a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or C \NRPorbl\DCC\AAR\43%705_1 DOC-14'261112 - 46 nucleotide analog or other potential anti-HBV agent, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence of the envelope genes or DNA polymerase gene selected from, in one embodiment, in one embodiment, rtT38K, rtR55H and rtAl81V; in another embodiment 5 includesrtS/T78S, rtV80L, rtN/SI18N, rtN/K139K, rtE142V, rtA/T181A rtl204M, rtQ/P/S/Stop2l5S, rtE/K218E, rtN/H238H and rtY245H; in yet another embodiment include rtN238T; and yet another embodiment include rtIl22V and rtAI8lT; in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, rtH237H/P, rtV253G, in yet another embodiment include rtT128N and rtN236T, in yet another 10 embodiment include tL180M, rtM204V rtQ215S, in yet another embodiment include rtT128S rtL180M, rtM204Vand rtQ215S, in yet another embodiment include rtl8OL, rt1204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another embodiment include rtII87V, in yet another embodiment include rtN123N/I rtSl35Y, rtV214A/V and rtQ215Q/P/Stop/S, or a combination thereof or an equivalent mutation; in 15 a further embodiment, sQ30K. sE44G, sA47T, sI126T, sA159V and sL173F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sI/VI 101, sN/T131N, sN134Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sVI4A, sL95W, sV96G, and sl208T/; and in yet another embodiment include sT47A and sWI72stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F I 92F, 20 sl195M, and in yet another embodiment include sS53L, and in yet another embodiment include sP120T, and in yet another embodiment include sN40S, sS207R, and in yet another embodiment include sQIO1R, sl195M, sS207R, and in yet another embodiment include sL95W and sL196W, and in yet another embodiment include sV14A, in yet another embodiment include sL42R, sQ102Q/R, sTl 15T/S, sS207S/R, sL215R and 25 sL216Stop, or combinations thereof or an equivalent one or more other mutation is indicative of a variant wherein said variant exhibits a decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV 30 and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof.

C.\NRPonbhDCC\AAR\43%,705_1 DOC- I1/6/2) IZ - 47 The detection of amino acid variants of DNA polymerase is conveniently accomplished by a range of amino acid detection techniques. Where an HBV variant comprises an amino acid change, then such an isolate is considered a putative HBV variant having an altered 5 DNA polymerase activity. The present invention further contemplates agents which inhibit ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC 10 and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV resistant H1BV variants. Such agents are particularly useful if long term treatment by ADV, LMV, FTC and/or TFV and/or optionally other nucleoside or nucleotide analogs such as TFV is contemplated by the clinician. The agents may be DNA or RNA or proteinaceous or non proteinaceous chemical molecules. Natural product screening such as from plants, coral 15 and microorganisms is also contemplated as a useful potential source of masking agents as is the screening of combinatorial or chemical libraries. The agents may be in isolated form or in the form of a pharmaceutical composition or formulation and may be administered in place of or sequentially or simultaneously with a nucleoside or nucleotide analog. Furthermore, rationale drug design is contemplated including solving the crystal or NMR 20 structure of, for example, HBV DNA polymerase and designing agents which can bind to the enzyme's active site. This approach may also be adapted to other HBV components. Accordingly, another aspect of the present invention contemplates a method for detecting an agent which exhibits inhibitory activity to an HIBV which exhibits resistance or 25 decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof, said method comprising: 30 C\INRPorbhDCC\AAR\U%(705_1. DOC- 14/16W21112 -48 generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in a plasmid vector and then transfecting said cells with said construct; 5 contacting said cells, before, during and/or after transfection, with the agent to be tested; culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like 10 particles if resistant to said agent; and subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent. 15 Still another aspect of the present invention provides a method for detecting an agent which exhibits inhibitory activity to an HBV which exhibits resistance or decreased sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or 20 ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof, , said method comprising: generating a genetic construct comprising a replication competent-effective 25 amount of the genome from said HBV contained in or fused to an amount of a baculovirus genome effective to infect cells and then infecting said cells with said construct; contacting said cells, before, during and/or after infection, with the agent to be tested; 30 C:\NRPorbthlDCC\AAR\439675_I DOC-14AWQ2012 - 49 culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and 5 subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent. Preferably, the HBV genome is stably integrated into the cells' genome. 10 Particularly useful cells are 2.2.15 cells (Price et al., Proc. Nail. Acad. Sci. USA 86(21): 8541-8544, 1989 or AD cells (also known as HepAD32 cells or HepAD79 cells [Ying el al., Viral Hepat. 7(2). 161-165, 2000. 15 Whilst the baculovirus vector is a particularly useful in the practice of the present invention, the subject invention extends to a range of other vectors such as but not limited to adenoviral vectors. The present invention further extends to cell lines (e.g. 2.2.15 or AD cells) carrying genetic 20 constructs comprising all or a portion of an HBV genome or a gene or part of a gene therefrom. The present invention also provides for the use of the subject HBV variants to screen for anti-viral agents. These anti-viral agents inhibit the virus. The term "inhibit" includes 25 antagonizing or otherwise preventing infection, replication, assembly and/or release or any intermediate step. Preferred anti-viral agents include nucleoside or nucleotide analogs or anti-HBV agents, however, the present invention extends to non-nucleoside molecules. In addition, rational drug design is also contemplated to identify or generate chemical 30 molecules which either mimic a nucleoside or which interact with a particular nucleotide sequence or a particular nucleotide. Combinatorial chemistry and two hybrid screening are C.\NRPonb\DMCC\AAR\41%705 DOC-14WW21112 -50 some of a number of techniques which can be employed to identify potential therapeutic or diagnostic agents. In one example, the crystal structure or the NMR structure of polymerase or the surface 5 antigen is used to rationally design small chemical molecules likely to interact with key regions of the molecule required for function and/or antigenicity. Such agents may be useful as inhibitors of polymerase activity and/or may alter an epitope on the surface antigen. 10 Several models of the HBV polymerase have been prepared due to the similarity with reverse transcriptase from HIV (Das et al., J. Virol. 75(10): 4771-4779, 2001; Bartholomeusz el al., Intervirology 40(5-6). 337-342 1997; Allen et al., Hepatology 27(6): 1670-1677, 1998). The models of the HBV polymerase can be used for the rational drug design of new agents effective against HBV encoding the resistant mutations as well as 15 wild type virus. The rational drug that is designed may be based on a modification of an existing antiviral agent such as the agent used in the selection of the HBV encoding the mutations associated with resistance. Viruses or clones expressing -IBV genomic material encoding the mutations may also be used to screen for new antiviral agents. 20 In an alternative embodiment, the present invention also contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV polymerase in an in vitro polymerase assay. The HBV polymerase activity can be examined using established assays (Gaillard el al., Antimicrob Agents Chemother. 46(4). 1005-1013, 2002; Xiong ei al., Hepatology 28(6): 1669-1673, 1998). 25 As indicated above, microarray technology is also a useful means of identifying agents which are capable of interacting with defined HBV internal or external components. For example, arrays of HBV DNA polymerase or peptide fragments thereof carrying different amino acid variants may be used to screen for agents which are capable of binding or 30 otherwise interacting with these molecules. This is a convenient way of determining the differential binding patterns of agents between HBV variants. Arrays of antibodies may C \NRPortbklDCC\AAR\4 %705_1 DOC-14A6/2012 -51 also be used to screen for altered HBsAg molecules. Microarrays are also useful in proteomic analysis to identify molecules such as antibodies, interferons or cytokines which have an ability to interact with an HBV component. Microarrays of DNA and RNA molecules may also be employed to identify sense and antisense molecules for genetic 5 regions on the HBV genome or transcripts thereof. The above methods are particularly useful in identifying an inhibitor of an HBV resistant to or exhibiting reduced sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and 10 LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. The present invention extends, therefore, to compositions of the inhibitors. The inhibitors may also be in the form of antibodies or genetic molecules such as ribozymes, antisense molecules and/or sense 15 molecules for co-suppression or the induction of RNAi or may be other nucleoside or nucleotide analogs or other anti-HBV agents or derivatives of known analogs. Reference to RNAi includes reference to short, interfering RNAs (siRNA) and all RNAi-type molecules may be DNA-derived or synthetic. 20 The term "composition" includes a "pharmaceutical composition" or a formulation. The inhibitor is referred to below as an "active ingredient" or "active compound" and may be selected from the list of inhibitors given above. 25 The composition may include an antigenic component of the HBV, a defective HBV variant or an agent identified through natural product screening or rational drug design (including combinatorial chemistry). Pharmaceutically acceptable carriers and/or diluents include any and all solvents, 30 dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active C \NRPonb\DCC\AAR\467)5_I DOC 14/)6/2012 - 52 substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. 5 The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of encoding an aspartyl protease inhibitor. The vector may, for example, be a viral vector. 10 Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, 15 polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it 20 will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. 25 Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the active ingredient and optionally other active ingredients as required, followed by filtered sterilization or other appropriate means of sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and the freeze-drying 30 technique which yield a powder of active ingredient plus any additionally desired ingredient.

C.\NRPonbDCC\AA R\3%75_i DOC-14/W20) 12 - 53 When the active ingredient is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral 5 therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight 10 of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 pg and 200 mg of active compound. Alternative dosage amounts include from about 1 ig to about 1000 mg and from about 10 ptg to about 500 mg. These 15 dosages may be per individual or per kg body weight. Administration may be per hour, day, week, month or year. The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as 20 dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be 25 present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavouring. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non 30 toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

C \RPenbl\DCC\AAR'%6705_I DOC-14AM/2012 - 54 As stated above, the present invention further extends to an isolated HBsAg from the HBV variants herein described. More particularly, the present invention provides an HBsAg or a recombinant form thereof or derivative or chemical equivalent thereof. The isolated 5 surface component and, more particularly, isolated surface antigen or its recombinant, derivative or chemical equivalents are useful in the development of biological compositions such as vaccine formulations. Yet another aspect of the present invention provides a composition comprising a variant 10 -IBV resistant to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or an HBV surface antigen from said variant HBV or a recombinant or 15 derivative form thereof or its chemical equivalent and one or more pharmaceutically acceptable carriers and/or diluents. Such a composition may be regarded as a therapeutic composition and is useful in generating an immune response including a humoral response. Generally, the -IBV variants are "defective" and in themselves are unable to cause a sustained infection in a subject. 20 As indicated above, antibodies may be generated to the mutant HBV agents and used for passive or direct vaccination against infection by these viruses. The antibodies may be generated in humans or non-human animals. In the case of the latter, the non-human antibodies may need to be deimmunized or more specifically humanized prior to use. 25 Deimmunized may include, for example, grafting complimentarity determining regions (CDRs) from the variable region of a murine or non-human animal anti-HBV antibody onto a human consensus fragment antibody binding (Fab) polypeptide. Alternatively, amino acids defining epitopes in the variable region of the antibody may be mutated so that the epitopes are no longer recognized by the human MHC II complex. 30 C \NRPonb\)CCAAR\41%705_ IDDC.14A16/2012 - 55 Insofar as ribozyme, antisense or co-suppression (RNAi) or siRNA or complexes thereof repression is concerned, this is conveniently aimed at post-transcription gene silencing. DNA or RNA may be administered or a complex comprising RNAi or a chemical analog thereof specific for HBV mRNA may be employed. 5 All such molecules may be incorporated into pharmaceutical compositions. In another embodiment, the present invention provides a biological composition comprising a variant HBV or an HBsAg or L, M or S proteins from said variant HBV or a 10 recombinant or derivative form thereof or its chemical equivalent. Generally, if an HBV is used, it is first attenuated. The biological composition according to this aspect of the present invention generally further comprises one or more pharmaceutically acceptable carriers and/or diluents. 15 The biological composition may comprise HBsAg or like molecule from one HBV variant or the composition may be a cocktail of HbsAgs or L, M or S proteins or like molecules from a range of ADV- and/or LMV- and/or, FTC- and/or TFV-resistant IHBV variants. Similar inclusions apply where the composition comprises an HBV. 20 The present invention is further directed to the use of defective HBV variants in the manufacture of therapeutic vaccines to vaccinate individuals against infection by H1BV strains having a particular nucleotide sequence or encoding a particular polymerase or surface antigen or L, M or S proteins. 25 Examples of suitable vaccine candidates are defective forms of HBV variants comprising a mutation selected from, in one embodiment, in one embodiment, rtT38K, rtR55H and rtAI81V; in another embodiment include rtS/T78S, rtV80L, rtN/S1l8N, rtN/K139K, rtE142V, rtA/T181A rt1204M, rtQ/P/S/Stop215S, rtE/K218E, rtN/H238H and rtY245H; in 30 yet another embodiment include rtN238T; and yet another embodiment include rtlI22V and rtA181T; in yet another embodiment include rtLl80M, rtA/V200V, rtM204V, C ANRPonbPDCC\AAR\41%705_ LDOC.14/6/20 12 - 56 rtV214A, rtH237H/P, rtV253G, in yet another embodiment include rtTI28N and rtN236T, in yet another embodiment include tLI80M, rtM204V rtQ215S, in yet another embodiment include rtT128S rtL180M, rtM204Vand rtQ215S, in yet another embodiment include rtl80L, rt1204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another 5 embodiment include rtIl87V, in yet another embodiment include rtN123N/I rtSl35Y, rtV214A/V and rtQ215Q/P/Stop/S, or a combination thereof or an equivalent mutation; in a further embodiment, sQ30K. sE44G, sA47T, sIl26T, sA159V and sL173F, in another embodiment include sF55S, sC/Stop69C, sC/Y76Y, sl/Vl101, sN/TI3IN, sNl34Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sVl4A, 10 sL95W, sV96G, and sl208T/; and in yet another embodiment include sT47A and sW172stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F192F, slI95M, and in yet another embodiment include sS53L, and in yet another embodiment include sP120T, and in yet another embodiment include sN40S, sS207R, and in yet another embodiment include sQ101R, sl195M, sS207R, and in yet another embodiment 15 include sL95W and sL196W, and in yet another embodiment include sVl4A, in yet another embodiment include sL42R, sQ102Q/R, sTI15T/S, sS207S/R, sL215R and sL216Stop, or a combination thereof or an equivalent mutation. In one embodiment, for example, an HBV variant may be identified having a particular 20 mutation in its polymerase conferring resistance or decreased sensitivity to a nucleoside or nucleotide analog. This variant may then be mutated to render it defective, i.e. attenuated or unable to cause infection. Such a defective, nucleoside or nucleotide analog-resistant virus may then be used as a therapeutic vaccine against virulent viruses having the same mutation in its polymerase. 25 The subject invention extends to kits for assays for variant HBV resistant to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV. 30 Such kits may, for example, contain the reagents from PCR or other nucleic acid hybridization technology or reagents for immunologically based detection techniques. A C\NRPonbl\DCC\AAR\43%67'05_I DOC-14I(J2MI2 - 57 particularly useful assay includes the reagents and components required for immobilized oligonucleotide- or oligopeptide-mediated detection systems. Still another aspect of the present invention contemplates a method for determining the 5 potential for an HBV to exhibit reduced sensitivity to ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof, said 10 method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and G, and domains A through to E or a region proximal thereto of said DNA polymerase and associated with resistance or decreased sensitivity to ADV, LMV, TFV, or FTC; or 15 ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV, wherein the presence of such a mutation is an indication of the likelihood of resistance to said ADV, LMV, TFV, or FTC; or ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; 20 FTC and LMV; or ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and FTC; or ADV and FTC and LMV and TFV. An assessment of a potential viral variant is important for selection of an appropriate therapeutic protocol. Such an assessment is suitably facilitated with the assistance of a 25 computer programmed with software, which inter alia adds input codes for at least two features associated with the viral variants to provide a value corresponding to the resistance or sensitivity of a viral variant to a particular chemical compound or immunological agent. The value can be selected from (a) the ability to exhibit resistance for reduced sensitivity to a particular compound or immunological agent; (b) an altered 30 DNA polymerase from wild-type HBV; (c) an altered surface antigen from wild-type HBV; or (d) morbidity or recovery potential of a patient. Thus, in accordance with the C WRPonbKlDCC\AAR\4396705_1 DOC-14/0W2012 - 58 present invention, the values for such features are stored in a machine-readable storage medium, which is capable of processing the data to provide a value for a particular viral variant or a biological specimen comprising same. 5 Thus, in another aspect, the invention contemplates a computer program product for assessing the likely usefulness of a viral variant or biological sample comprising same for determining an appropriate therapeutic protocol in a subject (Figure 3), said product comprising: 10 (1) code that receives as input code for at least two features associated with said viral agents or biological sample comprising same, wherein said features are selected from: (a) the ability to exhibit resistance for reduced sensitivity to a 15 particular compound or immunological agent; (b) an altered DNA polymerase from wild-type HBV; (c) an altered surface antigen from wild-type -IBV; or (d) morbidity or recovery potential of a patient; 20 (2) code that adds said input code to provide a sum corresponding to a value for said viral variants or biological samples; and (3) a computer readable medium that stores the codes. 25 In a related aspect, the invention extends to a computer for assessing the likely usefulness of a viral variant or biological sample comprising same in a subject, wherein said computer comprises: (1) a machine-readable data storage medium comprising a data storage 30 material encoded with machine-readable data, wherein said machine readable data comprise input codes for at least two features associated C:\NRPorbtlDCC\AAR\3705_I DOC.4Af6/2012 - 59 with said viral variant or biological sample; wherein said features are selected from: (a) the ability to exhibit resistance for reduced sensitivity to a 5 particular compound or immunological agent; (b) an altered DNA polymerase from wild-type HBV; (c) an altered surface antigen from wild-type HBV; or (d) morbidity or recovery potential of a patient; 10 (2) a working memory for storing instructions for processing said machine readable data; (3) a central-processing unit coupled to said working memory and to said machine-readable data storage medium, for processing said machine 15 readable data to provide a sum of said input code corresponding to a value for said compound(s); and (4) an output hardware coupled to said central processing unit, for receiving said value. 20 Any general or special purpose computer system is contemplated by the present invention and includes a processor in electrical communication with both a memory and at least one input/output device, such as a terminal. Figure 3 shows a generally suitable computer system. Such a system may include, but is not limited, to personal computers, workstations 25 or mainframes. The processor may be a general purpose processor or microprocessor or a specialized processor executing programs located in RAM memory. The programs may be placed in RAM from a storage device, such as a disk or pre-programmed ROM memory. The RAM memory in one embodiment is used both for data storage and program execution. The computer system also embraces systems where the processor and memory 30 reside in different physical entities but which are in electrical communication by means of a network.

C\NRPorbl\DCC\AAR\41'%705. DOC-14/N21112 - 60 In an alternative embodiment, the program screens for a mutation selected from, in one embodiment, in one embodiment, rtT38K, rtR55H and rtAI81V; in another embodiment include rtS/T78S, rtV80L, rtN/SI18N, rtN/K139K, rtE142V, rtA/TI8IA rtl204M, 5 rtQ/P/S/Stop2l5S, rtE/K218E, rtN/1238H and rtY245H; in yet another embodiment include rtN238T; and yet another embodiment include rtIl22V and rtAI81T; in yet another embodiment include rtL180M, rtA/V200V, rtM204V, rtV214A, rtH2371-/P, rtV253G, in yet another embodiment include rtT128N and rtN236T, in yet another embodiment include tL180M, rtM204V rtQ2l5S, in yet another embodiment include 10 rtT128S rtLl80M, rtM204Vand rtQ215S, in yet another embodiment include rtl80L, rtl204M, rtN238T, in yet another embodiment include rtN238T/A, in yet another embodiment include rtIl87V, in yet another embodiment include rtN123N/I rtSl35Y, rtV214A/V and rtQ215Q/P/Stop/S, or a combination thereof or an equivalent mutation; in a further embodiment, sQ30K. sE44G, sA47T, sI126T, sA159V and sL173F, in another 15 embodiment include sF55S, sC/Stop69C, sC/Y76Y, sI/VI101, sN/TI3IN, sN134Y, sStop/W172W, sStop/W196W, sS/R207R; in yet another embodiment include sVI4A, sL95W, sV96G, and sI208T/I; and in yet another embodiment include sT47A and sWI72stop, and in yet another embodiment include PreS2 T6S, sT47A, sP62L, sL/F192F, sll95M, and in yet another embodiment include sS53L, and in yet another embodiment 20 include sP120T, and in yet another embodiment include sN40S, sS207R, and in yet another embodiment include sQIIR, s1195M, sS207R, and in yet another embodiment include sL95W and sLI96W, and in yet another embodiment include sVl4A, in yet another embodiment include sL42R, sQ102Q/R, sTI 15T/S, sS207S/R, sL215R and sL216Stop, or a combination thereof or an equivalent mutation. 25 The present invention is further described by the following non-limiting Examples.

C-\NRPorl\DCC\AAR\43%71)5_I DOC-14/)6/2012 - 61 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-SI and Pre-S2, and partially overlaps the X and core (C) genes. The HBV envelope comprises small, middle and large proteins HBV surface antigens. The large protein component is referred to as the HBV surface antigen (HBsAg) and is encoded by the S gene sequence. The Pre-Sl and Pre-S2 gene sequences encode the other envelope components. 10 EXAMPLE 2 Patients on ADV Treatment and Analysis of HBV DNA Patient A: During ADV treatment, unique HBV mutations were detected by 15 sequencing (Table 4). This includes the unique mutation at rtT38K, and rtAl8IV. A number of other changes were also detected in the polymerase rtR55H and in the overlapping envelope gene (Table 4, Figures 4, 5 and 6 ). The changes in the -IBsAg include sQ30K, sE44G, sA47T, sI126T, sA159V and sLI73F. These unique changes were compared to reference sequences from each of the seven genotypes A-G as well as a 20 consensus sequence from pretreatment samples to determine unique changes Patient B: The HBV mutations during ADV treatment are listed in Table 5 and Figures 7, 8, and 9. The unique changes in the rt region of the HBV DNA polymerase include rtY245H. Other changes in the HBV polymerase while on ADV treatment include 25 rtS/T78S, rtV80L, rtN/S1 18N, rtN/K139K, rtEI42V, rtA/T181A rt1204M, rtQ/P/S/Stop2l5S,rt E/K218E, rtN/H238H. The changes in the HBsAg while on ADV treatment include sF55S, sC/Stop69C, sC/Y76Y, sI/V1101, sN/T131N, sN134Y, sStop/W 172W, sStop/W 196W, sS/R207R. 30 Patient C : The HBV mutations prior to ADV treatment and during ADV treatment are listed in Table 6 and Figures 10, 11 and 12. The unique changes in the rt region of the C:\NRPonbl\DCC\AR\ 3%705_I DOC.14/ft)d2112 - 62 HBV DNA polymerase while on ADV treatment include rtN238T. The unique changes in the HBsAg include sV14A, sL95W, sV96G, and s1208T/l Patient D: The HBV mutations during ADV treatment is listed in Table 7 and Figures 13, 5 14 and 15. The unique changes in the HBV DNA polymerase include rtll22V and rtA18 1T. The unique changes in the surface include sT47A and sW172stop. Patient E. This patient was previously treated with lamivudine and selected the unique mutations rtH237H/P while on LMV. This patient did not respond to ADV treatment 10 Changes in the polymerase on ADV treatment include rtLI80M, rtA/V200V, rtM204V, rtV214A, rtH237H/P and rtV253G. The unique changes in the surface include PreS2 T6S, sT47A, sP62L, sL/F192F, slI95M. NB: Patient on TFV and responded Changes on ADV listed in Table 8 and Figures 16, 17 and 18. 15 Patient F: Unique changes during ADV treatment include the polymerase mutations at rtN238T and envelope mutations at sS53L. Changes on ADV listed in Table 9 and Figures 19, 20 and 21. Patient G: Unique mutations while on ADV treatment include changes in the polymerase 20 rtT128N and rtN236T and a change in envelope sP120T. Changes on ADV listed in Table 10 and Figures 22, 23 and 24. Patient H: Mutations in the polymerase gene while on ADV treatment include rtLl80M, rtM204V rtQ215S. Changes in envelope gene includesN40S, sS207R. Changes on ADV 25 listed in Table I 1 and Figures 25, 26 and 27. Patient 1: Mutations in the polymerase gene while on ADV treatment include rtT128S rtL180M, rtM204Vand rtQ215S, while mutations in the envelope gene included sQIOIR, sl195M, sS207R. Changes on ADV listed in Table 12 and Figures 28, 29 and 30. 30 C :NRPorbKlDCC\AAR\U1%705_ .DOC-14AW2012 -63 Patient J: Mutations in the polymerase gene included rtl80L, rtI204M, rtN238T and mutations in envelope sL95W and sL196W during ADV treatment. Changes on ADV listed in Table 13 and Figures 31, 32 and 33. 5 Patient K: Mutations in the polymerase gene at rtN238T/A was detected during Adv treatment. No changes in envelope were detected during treatment. Changes on ADV listed in Table 14 and Figures 34, 35 and 36. Patient L: Mutations in the polymerase gene at rtl187V was detected during ADV 10 treatment. A mutation in the envelope gene at sV I 4A was also detected. Changes on ADV listed in Table 15 and Figures 37, 38 and 39. Patient M is an HIV HBV co-infected patient treated with tenofovir and lamivudine as part of anti-retroviral therapy for HIV. These two agents are also useful in the treatment of 15 IBV. This patient has not been treated with ADV. Three sequential samples were sequenced while the patient was on antiviral treatment: In one sample there were unique changes in the polymerase at rtN123N/I, rtS135Y, and rtQ215Q/P/ stop/S. Unique changes in the envelope gene were detected at sL42R, sQI02Q/R, sT1 15T/S. These changes are listed in Table 16 and Figures 40, 41 and 42. The remaining two samples were identical to 20 each other in the polymerase and evelope 9 (HBsAg) gene. Unique changes in the polymerase were detected at rtSI35Y and rtV214A/V, and rtS207S/R and unique changes in the envelope gene were detected at sQ102Q/R, sL215R and sL216Stop while on TFV and LMV treatment. These changes are listed in Table 16 and Figures 43, 44 and 45. 25 The changes at rtQ215Q/P/Stop/S and rtV214A/V have been previously detected in ADV treated patients. Thus, these mutations are important mutations and may result in potential cross-resistance to both ADV and TFV.

C WRPorblCC\AAR43%705_l .DOC-I4f6/2012 - 64 EXAMPLE 3 Detection of Viral Markers Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), anti-IBe and 5 hepatitis B core antigen (HBcAg) specific IgG and IgM were measured using commercially available immunoassays (Abbott Laboratories, North Chicago, IL, USA). Hepatitis B viral DNA levels were measured using a capture hybridization assay according to the manufacturer's directions (Digene Hybrid Capture II, Digene Diagnostics Inc., Beltsville, MD). The manufacturers stated cut-off for detecting HBV viremia in clinical 10 specimens was 0.7x 106 copies/ml or 2.5 pg/ml, [Hendricks ei al., Am J Clin Pathol 104: 537-46, 1995]. H1BV DNA levels can also be quantitated using other commercial kits such as Cobas amplification HBV monitor kit (Roche). EXAMPLE 4 15 Sequencing of HB V DNA HBV DNA was extracted from 100 pl of serum as described previously by Aye ei al., J. Hepatol. 26. 1148-1153, 1997. Oligonucleotides were synthesized by Geneworks, Adelaide, Australia. Amplification of the HBV polymerase gene has been described by 20 Aye et al., 1997, supra. The specific amplified products were purified using PCR purification columns from MO BIO Laboratories Inc (La Jolla, CA) and directly sequenced using Big Dye terminator Cycle sequencing Ready Reaction Kit (Perkin Elmer, Cetus Norwalk, CT). The PCR 25 primers were used as sequencing primers, OSI 5'- GCC TCA TTT TGT GGG TCA CCA TA-3' (nt 1408-1430) [SEQ ID NO:3], TTA3 5'-AAA TTC GCA GTC CCC AAA 3'(nt2128-2145) [SEQ ID NO:4], JM 5'-TTG GGG TGG AGC CCT CAG GCT 3'(nt1676-1696) [SEQ ID NO:5], TTA4 5'-GAA AAT TGG TAA CAG CGG -3' (nt 2615 2632) [SEQ ID NO:6], OS2 5' TCT CTG ACA TAC TTT CCA AT 3' (nt 2798-2817) 30 [SEQ ID NO:7], to sequence the internal regions of the PCR products.

C-\NRPonbl\DCC\AARM3%705_ IDOC-14/W 2112 - 65 EXAMPLE 5 A defovir Dipivoxil (AD ) 5 ADV (formerly Bis-pom PMEA)) is a potent inhibitor of -IBV replication. The structure of ADV is shown in Figure 2 and its synthesis is described by Benzaria et al., J Med Chem. 39: 4958-4965, 1996). Those skilled in the art will appreciate that the invention described herein is susceptible to 10 variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features 15 C \NRPonbrDCCOAARU396705_1 DOC.14AW211 2 - 66 Table 4: Patient A HB V Polymerase and envelope mutations detected during AD V therapy Treatment Date HBV viral load HBV RT HBsAg Mutations Mutations ADV 22/10/02 8.77E+06 - sT47A sTl 3 IT/I ADV 14/01/03 1.21 E+09 - ADV 8/7/03 7.92 E+07 rtT38K sL173F rtA 181 V Table 5: Patient B R T and Polymerase mutations detected during AD V therapy Treatment Date HBV RT Mutations HBsAg Mutations ADV 13/02/03 rtV80L sC76Y/C rtNI 18N/S s I OV/1 rtN139N/K sN131N/T rtVl42E sY I 34N rtA 181 A/T sW 172Stop/W sStop l 96W rtl204M sR/S207S rtQ/P/S/Stop215Q rtE218K/E rtN238N/H ADV 21/03/03 rtS78T/S sS55F rtV80L sC69Stop rtNI18N/S sC76Y/C rtNl39N/K sI OV/I sN131N/T rtV I 42E rtA 181 A/T sY I 34N sW172Stop/W rt1204M sStop/W] 96W rtQ215 Q/P/S/Stop sS207S/R rtE218K/E rtN238N/H C \NRPorbl\DCC\AAR\41,705_.DOC.1416/2D12 - 67 ADV 22/07/03 rtS/T78S sF55S rtV80L sC/Stop69C rtN/S1 18N sC/Y76Y rtN/K139K sI/V I101 sN/T131N rtEI42V rtA/T181A sN134Y sStop/W 1 72W rt1204M sStop/W 1 96W rtQ/P/S/Stop21 sS/R207R rt5SE/K218E rtN/-1238H rtY245H Table 6: Patient C HR V Polymerase and envelope mutations detected during AD V therapy Treatment Date HBV RT HBsAg Mutations Mutations ADV 18/08/03 rtN238T sVI4A sL95W sV96G sI208T/ Table 7: Patient D H BV Polymerase and envelope mutations detected during ADV therapy Treatment Date HBV RT HBsAg Mutations Mutations ADV 20/08/03 rtI I 22V sT47A rtA181T sW172Stop C \NRPonbRlDCC\AAR\43%703_1 DOC-14/OW2DI2 - 68 Table 8: Patient E HBV Polymerase and envelope mutations detected during AD V therapy Treatment Date HBV RT HBsAg Mutations Mutations LMV 23/05/02 rtL180M rtA200V/A sL192L/F rtM204V sI195M rtV214A rtP237H ADV 17/07/03 - PreS2 T6S - sT47A rtL18OM sP62L rtA/V200V sL/F192F rtM204V sIl95M rtV214A rtH237H/P rtV253G Table 9: Patient F HBV Polymerase and envelope mutations detected during ADV therapy Treatment Date HBV RT HBsAg Mutations Mutations ADV 16/10/03 rtN238T sS53L Table 10: Patient G HB V Polymerase and envelope mutations detected during AD V therapy Treatment Date I-IBV RT 1-IBsAg Mutations Mutations ADV 12/11/03 rtT128N sP120T rtN236T C:\NRPorblDCC\AAR\ 9675_ I.DOC.14/1/2012 -69 Table 11: Patient H HBVPolymerase and envelope mutations detected during A DV therapy Treatment Date HBV RT HBsAg Mutations Mutations ADV 05/11/03 rtL180M sN40S rtM204V sI195M rtQ215S sS207R Table 12: Patient I HBVPolymerase and envelope mutations detected during ADV therapy Treatment Date HBV RT IBsAg Mutations Mutations ADV 05/02/03 - sQ1iR rtT128S rtL18OM rtM204V sI195M rtQ215S sS207R Table 13: Patient J HR V Polymerase and envelope mutations detected during AD V therapy Treatment Date HBV RT HBsAg Mutations Mutations ADV 18/11/03 rtI80L sL95W rtI204M sL196W rtN238T Table 14: Patient K HBVPolymerase and envelope mutations detected during ADV therapy Treatment Date I-IBV RT HBsAg Mutations Mutations ADV 31/03/03 rtN238T/A No changes C .NRPenbl\DCC\AAR\43%70"5_L.DOC-14/A 20112 - 70 Table 15: Patient L HBVPolymerase and envelope mutations detected during ADV therapy Treatment Date HBV RT HBsAg Mutations Mutations ADV 17/09/03 rtI I 87V sV14A Table 16: Patient M HBV Polymerase and envelope mutations detected during TFV and LMV therapy Treatment Date Viral load HBV RT HBsAg Mutations copie/ml Mutations TFV LMV 18/06/04 2.76E+06 - sL42R, sQ102Q/R, sTI 15T/S rtN123N/I rtS135Y rtQ215Q/P/stop/S sS207S/R TFV LMV 9/7/04 4.31 E+07 - sQ102Q/R rtS135Y rtV214A/V sL215R sL216*. TFV LMV 22/08/04 >l.OOE+08 - sQ102Q/R rtS135Y rtV214A/V sL215R sL216*.

C-\NRPorb\DCC\AAR\43%1705S_ DOC-14/l6/2112 -71 BIBLIOGRAPHY Allen et al., Hepatology 27(6): 1670-1677, 1998 Aye et al., J. Hepatol. 26: 1148-1153, 1997 Bartholomeusz et al., Intervirology 40(5-6): 337-342 1997 Benhamou et al., Lancet 358: 718-723, 2001 Benzaria et al., J Med Chem. 39: 4958-4965, 1996 Boyd et al., Antiviral Chem Chemother. 32: 358-363, 1987 Calio et al., Antiviral Res. 23: 77-89, 1994 Das et al., .1 Virol. 75(10). 4771-4779, 2001 Delaney et al., Antimicrob Agents Chemother 45(6): 1705-1013, 2001 Dienstag et al., New England J Med 333: 1657-1661, 1995 Frick et al., Antimicrob. Agents Chemother.3 7: 2285-2292, 1993 Gaillard et al., Antimicrob Agents Chemother. 46(4): 1005-1013, 2002 Gilson et al., J Viral Hepat 6: 387-395, 1999 Heathcote et al., Hepatology 28: A620, 1998 1-endricks et al., A m J Clin Pathol 104: 537-46, 1995 Kruger et al., Hepatology 22: 219A, 1994 Main et al., J Viral Hepatitis 3: 211-215, 1996 Norder et al.,((J. Gen. Virol. 74: 341-1348, 1993 C.NRPorbl\DCCAAR\413%7)5_I DOC-140,/2012 - 72 Perrillo et al., Hepatology 32: 129-134, 2000 Peters et al., Transplantation 68: 1912-1914, 1999 Price et al., Proc. Natil. Acad Sci. USA 86(21): 854 1-8544, 1989 Ren and Nassal, J. Virol. 75(3): 1104-1116, 2001 Severini et al., Antimicrobial Agents Chemother. 39: 430-435, 1995 Stuyver et al., Hepatology 33: 751-757, 2001 Summers and Mason, Cell 29: 403-415, 1982 Suo et al., JBiol Chem. 273(42): 27250-27258. 1998 Vere Hodge, Antiviral Chem Chemother 4: 67-84, 1993 Xiong et al., Hepatology. 28(6): 1669-73, 1998 Ying et al., J Viral Hepat. 7(2). 161-165, 2000 Ying et al., J. Viral Hepat. 7(1). 79-83, 2000 Ying et al., Viral Hepat. 7(2). 161-165, 2000

Claims (12)

1. An isolated Hepatitis B virus (HBV) variant wherein said variant comprises a nucleotide mutation in a gene encoding a reverse transcriptas (rt) portion of a DNA polymerase resulting in at least one amino acid substitution to said rt portion of the DNA polymerase and wherein said variant exhibits decreased sensitivity to adefovir (ADV) and wherein the amino acid substitution is selected from the list consisting of a substitution from an alanine (A) to a valine (V) at codon 200 (rtA200V); a substitution from a V to an A at codon 214 (rtV214A); a substitution from a glutamine (Q) to a serine (S) at codon 215 (rtQ215S); a substitution from a histidine (H) to a proline (P) at codon 237 (rtH237P); a substitution from a V to a glycine (G) at codon 253 (rtV253G); a substitution from asparagine (N) to an H or threonine (T) at codon 238 (rtN238H/T); and a substitution from isoleucine (I) to a methionine (M) at codon 204 (rtI204M).

2. The isolated HBV variant of Claim 1 wherein the variant further exhibits reduced sensitivity to lamivudine (LMV).

3. The isolated HBV variant of Claim I or 2 wherein the variant further exhibits reduced sensitivity to tenofovir (TFV).

4. A method for determining the potential for an HBV to exhibit reduced sensitivity to ADV, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding an rt portion of an HBV DNA polymerase resulting in an amino acid substitution selected from the list consisting of rtA200V, rtV214A, rtQ215S, rtH237P, rtV253G, rtN238H/T and rtl204M wherein the presence of the mutation is an indication of ADV resistance and the absence of the mutation is an indication of sensitivity to ADV.

5. The method of Claim 4 wherein the HBV also exhibits reduced sensitivity to LMV. C\NRPorbPDCC\AAR\96325_ IDOC.14W/6/2W2 - 74

6. The method of Claim 4 or 5 wherein the IHBV also exhibits reduced sensitivity to TFV.

7. A method for detecting an agent which exhibits inhibitory activity to an HBV variant which exhibits resistance or decreased sensitivity to ADV said method comprising: generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in a plasmid vector and then transfecting said cells with said construct; contacting said cells, before, during and/or after transfection, with the agent to be tested; culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent wherein the HBV variant comprises a substitution mutation selected from the list consisting of rtA200V, rtV214A, rtQ215S, rtH237P, rtV253G, rtN238H/T and rtI204M.

8. The method of Claim 7 wherein the HBV variant is further resistant to LMV and/or TFV.

9. Use of an HBV variant of any one of Claims I to 3 in the manufacture of a medicament for the treatment or prophylaxis of HBV infection.

10. A kit for an assay for variant HBV resistant to ADV when used in the method of Claim 4, said kit comprising genetic agents capable of detecting a mutation rt portion of a DNA polymerase gene wherein the mutation is a substitution selected from the list consisting of rtA200V, rtV214A, rtQ215S, rtH237P, rtV253G, rtN238H/T and rtI204M. C:NRPolbRDCCAARWl%7 5_ I DOC-lA/6J2I12 - 75

11. The kit of Claim 10 further comprising reagent for PCR.

12. An isolated HBV variant according to any one of Claims I to 3 or a method of Claim 4 to 8 or a use of Claim 9 or a kit of Claim 10 or I I substantially as herein described with reference to the Figures and/or Examples.