Viral variants and methods for detecting same

Abstract

The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B variants exhibiting complete or partial resistance to nucleoside analogues and/or reduced interactivity with antibodies to viral surface components. The present invention further contemplates assays for detecting such viral variants which assays are useful in monitoring anti-viral therapeutic regimes.

1. An isolated HBV mutant, comprising a mutation in the gene encoding the HBV DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type HBV, wherein said mutation results in at least one amino acid addition, substitution, and/or deletion in the B domain corresponding to amino acid residues 495-535 of a wild-type HBV polymerase, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       2. An isolated HBV mutant, comprising a mutation in the gene encoding the HBV DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type HBV, wherein said mutation results in at least one amino acid addition, substitution, and/or deletion in the B domain corresponding to amino acid residues 505-535 of a wild-type HBV polymerase, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       3. An isolated HBV mutant, comprising a mutation in the gene encoding the HBV DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type HBV, wherein said mutation results in at least one amino acid addition, substitution, and/or deletion in the B domain corresponding to amino acid residues 505-529 of a wild-type HBV polymerase, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       4. An isolated HBV mutant exhibiting, relative to an isolated wild-type HBV, reduced sensitivity to a nucleoside analogue and reduced interactivity to an antibody to a wild-type HBV surface antigen, said HBV mutant comprising at least one of:
 (i) a nucleotide sequence of its genome as set forth in SEQ ID NO:17 or a sequence having at least 60% similarity thereto;  
 (ii) a nucleotide sequence capable of hybridising to SEQ ID NO:17 under low stringency conditions at 42° C.;  
 (iii) a mutation in an overlapping portion of open reading frames for DNA polymerase and HBV surface antigen; and  
 (iv) a mutation in a region corresponding to amino acids 118 to 169 and/or 169 to 207 of HBV surface antigen,  
 wherein said mutant contains a Trp/Arg499Glu amino acid substitution in the DNA polymerase and an Asp144Glu and Gly145Arg amino acid substitution in the surface antigen.  
 
     
     
       5. The isolated HBV mutant according to any of  claim 1  or  4  wherein said nucleoside analogue is selected from the group consisting of famciclovir, penciclovir and lamivudine. 
     
     
       6. The HBV mutant according to any of  claim 1  or  4  wherein said mutation is selected from the group consisting of Arg/Trp499Glu, Phe512Leu, Val519Leu, Pro523Leu, Leu526Met, Thr530Ser, and Ile533Leu. 
     
     
       7. The HBV mutant according to  claim 5  wherein said mutation is selected from the group consisting of Arg/Trp499Glu, Phe512Leu, Val519Leu, Pro523Leu, Leu526Met, Thr530Ser, and Ile533Leu. 
     
     
       8. An isolated HBV mutant exhibiting, relative to an isolated wild-type HBV, reduced sensitivity to a nucleoside analogue, said mutant comprising at least one mutation in its genome wherein said at least one mutation produces at least one amino acid substitution in the DNA polymerase selected from the group consisting of Trp/Arg499Glu, Phe512Leu and Val519Leu, said amino acid substitution in the DNA polymerase resulting in a concurrent amino acid substitution in the overlapping open reading frame of the HBV surface antigen, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       9. An isolated HBV mutant exhibiting, relative to an isolated wild-type HBV, reduced sensitivity to a nucleoside analogue, said mutant comprising at least one mutation in its genome wherein said at least one mutation produces at least one amino acid substitution in the DNA polymerase selected from the group consisting of Trp/Arg499Glu, Phe512Leu, Val519Leu and Ser559Thr, said amino acid substitution in the DNA polymerase resulting in a concurrent amino acid substitution in the overlapping open reading frame of the HBV surface antigen, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       10. An isolated mutant according to  claim 8  or  9  wherein said nucleoside analogue is selected from the group consisting of famciclovir, penciclovir and lamivudine. 
     
     
       11. A method for determining the potential for an HBV to exhibit, relative to an isolated wild-type HBV, reduced sensitivity to at least one of lamivudine, penciclovir and famciclovir, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in a nucleotide sequence encoding the B domain of HBV polymerase, corresponding to amino acid residues 495-535 of a wild-type HBV polymerase, with said mutation resulting in at least one amino acid substitution, deletion and/or addition in said B domain,
 wherein the presence of such a mutation is an indication of the potential of reduced sensitivity of said HBV to at least one of lamivudine, penciclovir and famciclovir.  
 
     
     
       12. A method for determining the potential for an HBV to exhibit, relative to an isolated wild-type HBV, reduced sensitivity to at least one of lamivudine, penciclovir and famciclovir, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in a nucleotide sequence encoding the B domain of HBV polymerase corresponding to amino acid residues 505-535 of a wild-type HBV polymerase, with said mutation resulting in at least one amino acid substitution, deletion and/or addition in said B domain,
 wherein the presence of such a mutation is an indication of the potential of reduced sensitivity of said HBV to at least one of lamivudine, penciclovir and famciclovir.  
 
     
     
       13. A method for determining the potential for an HBV to exhibit, relative to an isolated wild-type HBV, reduced sensitivity to at least one of lamivudine, penciclovir and famciclovir, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in a nucleotide sequence encoding the B domain of HBV polymerase corresponding to amino acid residues 505-529 of a wild-type HBV polymerase, with said mutation resulting in at least one amino acid substitution, deletion and/or addition in said B domain,
 wherein the presence of such a mutation is an indication of the potential of reduced sensitivity of said HBV to at least one of lamivudine, penciclovir and famciclovir.  
 
     
     
       14. A method for determining the potential for an HBV to exhibit, relative to an isolated wild-type HBV, reduced sensitivity to at least one of lamivudine, penciclovir and famciclovir, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in a nucleotide sequence encoding HBV DNA polymerase, wherein the screening detects at least one mutation selected from the group consisting of Arg/Trp499Glu, Phe512Leu, Val 519Leu, Pro523Leu, Leu526Met, Thr530Ser, Ile533Leu, Met550Val and Met550Ile,
 such that when said at least one amino substitution in the DNA polymerase is Met550Val or Met550Ile, said method detects at least one amino acid substitution other than Phe512Leu, Leu526Met or Val553Ile,  
 wherein the presence of such a mutation is an indication of the potential of reduced sensitivity of said HBV to at least one of lamivudine, penciclovir and famciclovir.  
 
     
     
       15. A method for determining the potential for an HBV to exhibit, relative to an isolated wild-type HBV, reduced sensitivity to at least one of penciclovir and famciclovir, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in a nucleotide sequence encoding HBV DNA polymerase, wherein the screening detects at least one mutation selected from the group consisting of Arg/Trp499Glu, Phe512Leu, Val 519Leu, Pro523Leu, Leu526Met, Thr530Ser, Ile533Leu, Met550Val and Met550Ile,
 wherein the presence of such a mutation is an indication of the potential of reduced sensitivity of said HBV to at least one of penciclovir and famciclovir.  
 
     
     
       16. The method according to any of  claim 11 ,  14  or  15 , wherein the screening for a mutation comprises sequencing said isolated HBV DNA or corresponding mRNA. 
     
     
       17. The method according to any of  claim 11 ,  14  or  15 , wherein the screening for a mutation comprises a PCR method or PCR-based method. 
     
     
       18. The method according to any of  claim 11 ,  14  or  15 , wherein the screening for a mutation comprises a hybridization method. 
     
     
       19. An isolated Hepadnavirus mutant, comprising a mutation in the gene encoding the DNA polymerase, resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type Hepadnavirus, wherein said mutation results in at least one amino acid addition, substitution, and/or deletion in the B domain corresponding to amino acid residues 495-535 of a wild-type HBV polymerase, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       20. An isolated Hepadnavirus mutant, comprising a mutation in the gene encoding the DNA polymerase, resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type Hepadnavirus, wherein said mutation results in at least one amino acid addition, substitution, and/or deletion in the B domain corresponding to amino acid residues 505-535 of a wild-type HBV polymerase, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       21. An isolated Hepadnavirus mutant, comprising a mutation in the gene encoding the DNA polymerase, resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type Hepadnavirus, wherein said mutation results in at least one amino acid addition, substitution, and/or deletion in the B domain corresponding to amino acid residues 505-529 of a wild-type HBV polymerase, and said mutant contains an unmutated YMDD motif in the C domain. 
     
     
       22. An isolated mutant according to  claim 19 , wherein the nucleoside analogue is selected from the groups consisting of famciclovir, penciclovir and lamivudine. 
     
     
       23. An isolated mutant according to  claim 19  or  22 , wherein the Hepadhavirus is woodchuck hepatitis virus. 
     
     
       24. An isolated mutant according to  claim 19  or  22 , wherein the Hepadnavirus is duck hepatitis virus. 
     
     
       25. A method for determining the potential for an HBV to exhibit, relative to an isolated wild-type HBV, reduced sensitivity to an anti-viral agent, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in a nucleotide sequence encoding the B domain of HBV polymerase corresponding to amino acid residues 495-535 of a wild-type HBV polymerase, with said mutation resulting in at least one amino acid substitution, deletion and/or addition in said B domain,
 wherein the presence of such a mutation is an indication of the potential of reduced sensitivity of said HBV to an anti-viral agent.  
 
     
     
       26. An isolated HBV mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type HBV, wherein the mutation consists of an amino acid addition, substitution or deletion in the B domain corresponding to amino acid residues  495 - 535  of a wild - type HBV polymerase.   
     
     
       27. An isolated HBV mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type HBV, wherein the mutation consists of an amino acid addition, substitution or deletion in the B domain corresponding to amino acid residues  505 - 535  of a wild - type HBV polymerase.   
     
     
       28. An isolated HBV mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type HBV, wherein the mutation consists of an amino acid addition, substitution or deletion in the B domain corresponding to amino acid residues  505 - 529  of a wild - type HBV polymerase.   
     
     
       29. An isolated HBV mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type HBV and affecting the interactivity of an antibody to a wild - type HBV surface antigen, wherein said HBV mutant comprises at least one of    ( i )  a nucleotide sequence of its genome as set forth in SEQ ID NO: 17  or a sequence having at least  60   %  similarity thereto;      ( ii )  a nucleotide sequence capable of hybridising to SEQ ID NO: 17  under low stringency conditions at  42 ° C.;      ( iii )  a mutation in an overlapping portion of open reading frames for DNA polymerase and HBV surface antigen; and      ( iv )  a mutation in a region corresponding to amino acids  118  to  169  and/or  169  to  207  of HBV surface antigen,        wherein said mutant contains a Trp/Arg 499 Glu amino acid substitution in the DNA polymerase and an Asp 144 Glu and Gly 145 Arg amino acid substitution in the surface antigen.     
     
     
       30. The isolated HBV mutant according to  claim 26  wherein said nucleoside analogue is selected from the group consisting of famciclovir, penciclovir and lamivudine. 
     
     
       31. The HBV mutant according to  claim 26  wherein said mutation is selected from the group consisting of Arg/Trp 499 Glu, Phe 512 Leu, Val 519 Leu, Pro 523 Leu, Leu 526 Met, Thr 530 Ser, and Ile 533 Leu. 
     
     
       32. An isolated HBV mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue selected from the group consisting of famciclovir, penciclovir and lamivudine, compared to a wild- type HBV, wherein the mutation is selected from the group consisting of Arg/Trp 499 Glu, Phe 512 Leu, Val 519 Leu, Pro 523 Leu, Leu 526 Met, Thr 530 Ser, and Ile 533 Leu.   
     
     
       33. An isolated Hepadnavirus mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type Hepadnavirus, wherein said mutation consists of an amino acid addition, substitution or deletion in the B domain corresponding to amino acid residues  495 - 535  of a wild - type HBV polymerase.   
     
     
       34. An isolated Hepadnavirus mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type Hepadnavirus, wherein said mutation consists of an amino acid addition, substitution or deletion in the B domain corresponding to amino acid residues  505 - 535  of a wild - type HBV polymerase.   
     
     
       35. An isolated Hepadnavirus mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type Hepadnavirus, wherein said mutation consists of an amino acid addition, substitution or deletion in the B domain corresponding to amino acid residues  505 - 529  of a wild - type HBV polymerase.   
     
     
       36. An isolated mutant according to  claim 33 , wherein the nucleoside analogue is selected from the groups consisting of famciclovir, penciclovir and lamivudine. 
     
     
       37. An isolated mutant according to  claim 33  or  38 , wherein the Hepadhavirus is woodchuck hepatitis virus. 
     
     
       38. An isolated mutant according to  claim 33  or  38 , wherein the Hepadnavirus is duck hepatitis virus. 
     
     
       39. A method for determining the potential for an HBV to exhibit, relative to an isolated wild- type HBV, reduced sensitivity to an anti - viral agent, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in a nucleotide sequence encoding the B domain of HBV polymerase corresponding to amino acid residues  495 - 535  of a wild - type HBV polymerase, with said mutation resulting in at least one amino acid substitution, deletion and/or addition in said B domain,      wherein the presence of such a mutation is an indication of the potential of reduced sensitivity of said HBV to an anti - viral agent.     
     
     
       40. An isolated HBV mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue selected from the group consisting of famciclovir, penciclovir and lamivudine, compared to a wild- type HBV and affecting the interactivity of an antibody to a wild - type HBV surface antigen, wherein said HBV mutant comprises at least one of    ( i )  a nucleotide sequence of its genome as set forth in SEQ ID NO: 17  or a sequence having at least  60   %  similarity thereto;      ( ii )  a nucleotide sequence capable of hybridising to SEQ ID NO: 17  under low stringency conditions at  42 ° C.;      ( iii )  a mutation in an overlapping portion of open reading frames for DNA polymerase and HBV surface antigen; and      ( iv )  a mutation in a region corresponding to amino acids  118  to  169  and/or  169  to  207  of HBV surface antigen,        wherein said mutant contains a Trp/Arg 499 Glu amino acid substitution in the DNA polymerase and an Asp 144 Glu and Gly 145 Arg amino acid substitution in the surface antigen.     
     
     
       41. An isolated HBV mutant with a single mutation affecting the normal function of the DNA polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild- type HBV and affecting the interactivity of an antibody to a wild - type HBV surface antigen, wherein said HBV mutant comprises at least one of    ( i )  a nucleotide sequence of its genome as set forth in SEQ ID NO: 17  or a sequence having at least  60   %  similarity thereto;      ( ii )  a nucleotide sequence capable of hybridising to SEQ ID NO: 17  under low stringency conditions at  42 ° C.;      ( iii )  a mutation selected from the group consisting of Phe 512 Leu, Val 519 Leu, Pro 523 Leu, Leu 526 Met, Thr 530 Ser, and Ile 533 Leu; and      ( iv )  a mutation in a region corresponding to amino acids  118  to  169  and/or  169  to  207  of HBV surface antigen,        wherein said mutant contains a Trp/Arg 499 Glu amino acid substitution in the DNA polymerase and an Asp 144 Glu and Gly 145 Arg amino acid substitution in the surface antigen.