ROSSAU RUDI (BE)
MAERTENS GEERT (BE)
STUYVER LIEVEN (BE)
ROSSAU RUDI (BE)
MAERTENS GEERT (BE)
| WO1994012670A2 | 1994-06-09 |
| EP0569237A2 | 1993-11-10 | |||
| EP0229701A2 | 1987-07-22 |
| 1. | Method for detection and/or genetic analysis of HBV in a biological sample, comprising: (i) if need be releasing, isolating or concentrating the polynucleic acids present in said sample; (ii) if need be amplifying the relevant part of a suitable HBV gene present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with a combination of at least two nucleotide probes, with said combination hybridizing specifically to a mutant target sequence chosen from the HBV RT pol gene region and/or to a mutant target sequence chosen from the HBV preCore region and/or to a mutant target sequence chosen from the HBsAg region of HBV and/or to a HBV genotypespecific target sequence, with said target sequences being chosen from Figure 1 , and with said probes being applied to known locations on a solid support and with said probes being capable of hybridizing to the polynucleic acids of step (i) or (ii) under the same hybridization and wash conditions, or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T of said target sequence is replaced by U; (iv) detecting the hybrids formed in step (iii); (v) inferring the HBV genotype and/or mutants present in said sample from the differential hybridization signal(s) obtained in step (iv) . |
| 2. | Method according to claim 1 , characterized further in that in step (iii) a combination of at least two oligonudeotide probes is used and that said combination of probes hybridizes specifically to at least two of the following groups of target sequences: a mutant target sequence chosen from the HBV RT pol gene region, a mutant target sequence chosen from the HBV preCore region, a mutant target sequence chosen from the HBsAg region of HBV, a HBV genotypespecific target sequence. |
| 3. | Method according to claim 1 , characterized further in that in step (iii) a combination of at least three oligonudeotide probes is used and that said combination of probes hybridizes specifically to at least three of the following groups of target sequences: a mutant target sequence chosen from the HBV RT pol gene region, a mutant target sequence chosen from the HBV preCore region, a mutant target sequence chosen from the HBsAg region of HBV, a HBV genotypespecific target sequence. |
| 4. | Method according to claim 1 , characterized further in that in step (iii) a combination of at least four oligonudeotide probes is used and that said combination of probes hybridizes specifically to all of the following groups of target sequences: a mutant target sequence chosen from the HBV RT pol gene region, a mutant target sequence chosen from the HBV preCore region, a mutant target sequence chosen from the HBsAg region of HBV, a HBV genotypespecific target sequence. |
| 5. | Method according to any of claims 1 to 4, characterized further in that the oligonudeotide probes used in step (iii) are selected from Table 1 , wherein: the probes hybridizing specifically to mutant target sequences chosen from the RT pol region of HBV are selected from the following list: SEQ ID 1 14, SEQ ID NO 1 1 5, SEQ ID NO 1 1 6, SEQ ID NO 1 1 7, SEQ ID NO 1 27, SEQ ID NO 1 28, SEQ ID NO 1 29, SEQ ID NO 1 30, SEQ ID NO 1 31 , SEQ ID NO 132, SEQ ID NO 1 33, SEQ ID NO 227, SEQ ID NO 228, SEQ ID NO 229, SEQ ID NO 230, SEQ ID NO 231 , SEQ ID NO 232, SEQ ID NO 233, SEQ ID NO 234, SEQ ID NO 243, SEQ ID NO 244, SEQ ID NO 245, SEQ ID NO 246, SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO 249, SEQ ID NO 250, SEQ ID NO 251 , SEQ ID NO 252, SEQ ID NO 253, SEQ ID NO 254, SEQ ID NO 255, SEQ ID NO 256, SEQ ID NO 257, SEQ ID NO 258, SEQ ID NO 259, SEQ ID NO 260, SEQ ID NO 261 , SEQ ID NO 262, SEQ ID NO 263, SEQ ID NO 264, SEQ ID NO 265, SEQ ID NO 266, SEQ ID NO 267, SEQ ID NO 268, SEQ ID NO 269, SEQ ID NO 270, SEQ ID NO 271 , SEQ ID NO 272, SEQ ID NO 273, SEQ ID NO 274, SEQ ID NO 275, SEQ ID NO 276, SEQ ID NO 277, SEQ ID NO 278, and/or the probes hybridizing specifically to mutant target sequences chosen from the preCore region of HBV are selected from the following list: SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91 , SEQ ID NO 1 18, SEQ ID NO 1 19, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 1 25, SEQ ID NO 126, and/or the probes hybridizing specifically to mutant target sequences chosen from the HBsAg region of HBV are selected from the following list: SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 136, SEQ ID NO 1 37, SEQ ID NO 1 38, and/or the probes hybridizing specifically to genotypespecific target sequences of HBV are selected from the following list: SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79 SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 1 53, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 1 57, SEQ ID NO 158, SEQ ID NO 1 59, SEQ ID NO 160, SEQ ID NO 1 61 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 1 66, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181 , SEQ ID NO 182, SEQ ID NO 1 83, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186, SEQ ID NO 187, SEQ ID NO 188, SEQ ID NO 189, SEQ ID NO 1 90, SEQ ID NO 191 , SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194. |
| 6. | Method according to any of claims 1 to 5, wherein the oligonudeotide probes of step (iii) are characterized in that they specifically hybridize to target sequences in the RT pol region of HBV and permit detection of mutations that confer resistance to lamivudine, with said probes being for instance SEQ ID NO 1 14, SEQ ID NO 1 15, SEQ ID NO 1 16, SEQ ID NO 1 17, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 227, SEQ ID NO 228, SEQ ID NO 229, SEQ ID NO 230, SEQ ID NO 231 , SEQ ID NO 232, SEQ ID NO 233, SEQ ID NO 234, SEQ ID NO 243, SEQ ID NO 244, SEQ ID NO 245, SEQ ID NO 246, SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO 249, SEQ ID NO 250, SEQ ID NO 251 , SEQ ID NO 252, SEQ ID NO 253, SEQ ID NO 254, SEQ ID NO 255, SEQ ID NO 256, SEQ ID NO 257, SEQ ID NO 258, SEQ ID NO 259, SEQ ID NO 260, SEQ ID NO 261 , SEQ ID NO 262, SEQ ID NO 263, SEQ ID NO 264, SEQ ID NO 265, SEQ ID NO 266, SEQ ID NO 269, SEQ ID NO 270, SEQ ID NO 271 , SEQ ID NO 272, SEQ ID NO 275, SEQ ID NO 276, SEQ ID NO 277, SEQ ID NO 278. |
| 7. | Method according to any of claims 1 to 5, wherein the oligonudeotide probes of step (iii) are characterized in that they specifically hybridize to target sequences in the RT pol region of HBV and permit detection of mutations that confer resistance to penciclovir, with said probes being for instance SEQ ID NO 267, SEQ ID NO 268, SEQ ID NO 269, SEQ ID NO 270, SEQ ID NO 271 , SEQ ID NO 272, SEQ ID NO 273, SEQ ID NO 274, SEQ ID NO 275, SEQ ID NO 276, SEQ ID NO 277, SEQ ID NO 278. |
| 8. | Method according to any of claims 1 to 5, wherein the oligonudeotide probes of step (iii) are characterized in that they specifically hybridize to target sequences in the RT pol region of HBV and permit detection of at least one of the mutations that give rise to a change of the following amino acids: F at position 51 4, V at position 521 , P at position 525, L at position 527, M at position 552, V at position 555, with said probes being for instance SEQ ID NO 1 14, SEQ ID NO 1 1 5, SEQ ID NO 1 16, SEQ ID NO 1 17, SEQ ID NO 1 27, SEQ ID NO 1 28, SEQ ID NO 1 29, SEQ ID NO 1 30, SEQ ID NO 1 31 , SEQ ID NO 1 32, SEQ ID NO 1 33, SEQ ID NO 227, SEQ ID NO 228, SEQ ID NO 229, SEQ ID NO 230, SEQ ID NO 231 , SEQ ID NO 232, SEQ ID NO 233, SEQ ID NO 234, SEQ ID NO 243, SEQ ID NO 244, SEQ ID NO 245, SEQ ID NO 246, SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO 249, SEQ ID NO 250, SEQ ID NO 251 , SEQ ID NO 252, SEQ ID NO 253, SEQ ID NO 254, SEQ ID NO 255, SEQ ID NO 256, SEQ ID NO 257, SEQ ID NO 258, SEQ ID NO 259, SEQ ID NO 260, SEQ ID NO 261 , SEQ ID NO 262, SEQ ID NO 263, SEQ ID NO 264, SEQ ID NO 265, SEQ ID NO 266, SEQ ID NO 267, SEQ ID NO 268, SEQ ID NO 269, SEQ ID NO 270, SEQ ID NO 271 , SEQ ID NO 272, SEQ ID NO 273, SEQ ID NO 274, SEQ ID NO 275, SEQ ID NO 276, SEQ ID NO 277, SEQ ID NO 278. |
| 9. | Method according to any of claims 1 to 5, characterized further in that the probes of step (iii) hybridize specifically to a genotypespecific target sequence from the HBsAg region, said probes being for instance SEQ ID NO77 SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 1 51 , SEQ ID NO 152, SEQ ID NO 1 53, SEQ ID NO 1 54, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 1 58, SEQ ID NO 159, SEQ ID NO 1 60, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 1 64, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 1 73, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177 SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181 , SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 1 85, SEQ ID NO 186, SEQ ID NO 187, SEQ ID NO 1 88, SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191 , SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 214, SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO 219. |
| 10. | Method according to any of claims 1 to 5, characterized further in that the probes of step (iii) hybridize specifically to a genotypespecific target sequence from the preSI region, said probes being for instance SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 92, SEQ ID NO 93. 1 1 . A composition comprising at least one probe as defined in any of claims 1 to 1 0. 1 2. A composition comprising at least one probe as defined in claim 5. 1 3. Use of a composition of probes as defined in claims 1 1 and/or 1 2 for in vitro diagnosing and/or monitoring HBV mutants and/or genotypes present in a biological sample. |
| 11. | 14 Assay kit for the detection and/or the genetic analysis of HBV mutants and/or genotypes present in a biological sample according to the method of any of claims 1 to 10, comprising the following components : (i) when appropriate, a means for releasing, isolating or concentrating the polynucleic acids present in said sample; (ii) when appropriate, at least one suitable primer pair; (iii) at least one of the probes according to claim 1 1 and/or 1 2, possibly fixed to a solid support; (iv) a hybridization buffer, or components necessary for producing said buffer; (v) a wash solution, or components necessary for producing said solution; (vi) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization. (vii) when appropriate, a means for attaching said probe to a known location on a solid support. |
The present invention relates to the field of Hepatitis B virus (HBV) diagnosis. More particularly, the present invention relates to the field of HBV genotyping and/or determination of the presence of HBV mutants in test samples. The present invention relates particularly to a method for the rapid and reliable detection of HBV mutants and/or genotypes occuring in a test sample using specific sets of probes optimized to function together in a reverse- hybridisation assay.
Hepatitis B virus is a small enveloped DNA virus of approximately 3200 bp long. Historically it has been characterized on the basis of immunological reaction of the HBsAg with sets of monoclonal antibodies. Isolates were described as a, indicating the common determinant for all different subtypes, followed by subtype- specific combinations: dw, dr, yw, or yr. The latter are mutually exiusive pairs of determinants, covering the HBsAg amino acids 1 22 (<_/ = lys, y = arg) and 1 60 ( = lys, r= arg). Several subdeterminants for w exist and can be ascribed to the appeareance of certain amino acid variants at codon 127. More recently, a genetic classification has been proposed, based on molecular analysis of the virus. This kind of analysis showed that in total six different genotypes exist, indicated from A to F, with a maximum genetic divergence of 8% when comparing complete genomes (reviewed by Magnius and Norder, 1 995) .
The genetic variability of HBV might be clinically important, indeed, the genome variability might include some mechanisms by which HBV avoids immune clearance, and hence induces chronic infection. An important protein marker in inducing immune tolerance, virus elimination, and chronic infection, is HBeAg. The expression of this protein is strictly controled both at the transcriptional and translational level (Li et al., 1 993; Okamoto et al., 1 990; Yuan et al., 1 995; Sato et al., 1 995) . Therefore, in the natural course of HBV infection, a well characterized stage of the disease is indicated as HBe-negative chronic hepatitis B (reviewed by Hadziyannis S.J., 1 995). This phase is mostly due to the appeareance of preCore translational stop codon mutations. The overal genetic
variability determines the frequency and physical location on the viral genome where these translational stop-codon mutations appear. The transcriptional regulation was proposed to be the mechanism for genotype A (and possibly also F), whereas the translational control was more likely to be found in the other genotypes (Li et al.; 1 993; Sato et al. , 1 995). Contradictory to the translational regulation, it was shown that the transcriptional regulation was unable to block the HBeAg expression completely and was therefore proposed to categorize the phenotype of this mutant as HBe-suppressed, rather than as HBe-negative (Takahashi et al., 1 995). In any case, these preCore mutants would lead to a destruction of the pre-existing balance between HBeAg in circulation and the HBc- derived peptides presented by class I HLA molecules on the surface of infected hepatocytes, thereby diminishing the supressive effect of HBeAg on T cells, finally resulting in partial liberation of core-specific CTLs and leading to apoptosis of the infected hepatocytes. In general, after the emergence of the HBe-minus variants, the course of the viral infection is characterized by the progression of chronic hepatitis, which may lead to the development of cirrhosis and hepatocellular carcinoma (Hadziyannis, 1 995).
Another issue for which the genetic variability or genotyping of the virus might be of relevance is in the development of vaccines where the response may be mediated by the virus type. Protection against HBV infection of all subtypes is conferred by antibodies to the common 'a' determinant of the HB surface antigen (HBsAg). It has been shown that this 'a' determinant presents a number of epitopes, and that its tertiary structure is most important for its antigenicity. The most important region lies between amino acid 124 and 147, but can be extended from amino acid 1 14 to 1 50. An adequate anti-HBs response, built up after vaccination, is in principle fully protective. Infection with a HBV strain harboring mutations in the 'a' determinant region might result in vaccine failure, because the vaccine-induced humoral immune response does not recognize the mutant HBsAg. The most common vaccine-associated escape mutants are the substitutions of a glycine at position 145 to an arginine (G 145R), K141 E, and T126N. But a 2-aa insertion between aa position 122 and 123, and 8-aa insertion between aa 1 23
and 1 24 have also been found (Carman et al., 1 990, 1 995; Crawford, 1 990; Waters et al., 1 992) .
Lamivudine is a (-) enantiomer of 3' thiacytidine, a 2'3'-dideoxynucleoside analogue, and is known to be a potent inhibitor of HBV replication through inhibition of the reverse transcriptase (RT) activity of the HBV polymerase.
Lamivudine treatment can result in histological improvements in chronic hepatitis patients, and when given pre- and post-liver transplantation, it can prevent graft reinfection (Honkoop et al., 1 995; Naoumov et al., 1 995) . However, after treatment, a hepatitis flare-up can be observed in most patients, with ALT elevations and HBV DNA that becomes detectable again. This HBV DNA rebound is associated with a new quasi species equilibrium. In a few cases, virus breakthrough during therapy was observed, due to the selection of lamivudine resistent HBV strains. The exact nature of this breakthrough has been ascribed to the accumulation of mutations in the RT part of the Polymerase. A similar mechanism in the HIV RT polymerase has been found, where upon lamivudine treatment, mutations accumulate in the YMDD motif (Gao et al., 1993). This YMDD motif is also present in the RT part of the HBV polymerase, and lamivudine- selected mutations in HBV have been found in this region (Tipples et al., 1 996), as well as in other regions of the RT part of the polymerase (Ling et al., 1 996). Penciclovir is another drug that has been shown to inhibit the reverse transcriptase activity of the HBV polymerase (Shaw et al. , 1 996), and mutations in the HBV polymerase may also be detected upon treatment with this drug.
From all this it can be concluded that the information on the following issues is essential for proper in vitro diagnosis, monitoring and follow-up of HBV infections:
- genotype;
- preCore mutations;
- vaccine escape mutations;
- RT gene mutations selected by treatment with drugs such as lamivudune and penciclovir.
To obtain all this information using existing technologies is complicated, time-
consuming, and requires highly-skilled and experienced personel.
It is thus an aim of the present invention to develop a rapid and reliable detection method for determination of the presence or absence of one or more
HBV genotypes possibly present in a biological sample. More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of the presence or absence of one or more variations in the HBV preS1 region and/or in the HBsAg region representing one or more HBV genotypes possibly present in a biological sample in one single experiment. More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of the presence or absence of one or more HBV mutants possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of one or more mutations in the preCore region of HBV possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of one or more mutations in the HBsAg region of HBV possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of one or more mutations in the polymerase (pol) gene region of HBV possibly present in a biological sample in one single experiment. More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for the simultaneous determination of one or several HBV genotypes in combination with one or several HBV mutants possibly present in a biological sample in one single experiment.
It is also an aim of the present invention to provide a genotyping assay or method which allows to infer the nucleotide sequence at codons of interest and/or the HBV mutants of interest, and/or infer the HBV genotype possibly present in a
biological sample.
Even more particularly it is also an aim of the present invention to provide a genotyping assay allowing the detection of the different HBV mutants and genotypes in one single experimental setup. It is another aim of the present invention to select particular probes able to discriminate one or more HBV mutations in one of the above mentioned regions of the HBV genome and/or able to discriminate one or more HBV genotypes.
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HBV from mutant HBV sequences. It is also an aim of the present invention to select particular probes able to discriminate wild-type and polymorphic variants of HBV from mutant HBV sequences.
It is also an aim of the present invention to select particular probes able to discriminate HBV genotype sequences. It is moreover an aim of the present invention to combine a set of selected probes able to genotype HBV and/or discriminate different HBV mutants possibly present in a biological sample, whereby all probes can be used under the same hybridisation and wash conditions.
It is also an aim of the present invention to select primers enabling the amplification of the gene fragment(s) determining the HBV genomic mutations or variations of interest as discussed above.
The present invention also aims at diagnostic kits comprising said probes useful for developing such a genotyping assay and/or assays for detecting, monitoring or following-up HBV infection and/or assays for detecting HBV mutations.
All the aims of the present invention have been met by the following specific embodiments.
As a solution to the above-mentioned problem that it is essential for proper diagnosis, monitoring and follow-up of HBV infection to have information on the genotype of HBV present, the present invention provides an elegant way to tackle
problems of such complexity which involves residing to a reverse hybridization approach (particularly on Line Probe Assays strips, as described by Stuyver et al. , 1 993). Using this technology it is possible to conveniently obtain all essential data in one test run. To achieve this goal, a set of probes needs to be designed and assembled which can detect all relevant polymorphisms in the HBV gene regions of interest.
The present invention thus particularly relates to a method for determining the presence or absence of one or more HBV genotypes in a biological sample, comprising: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of a suitable HBV gene present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two nucleotide probes hybridizing specifically to a HBV genotype specific target sequence chosen from Figure 1 ; with said probes being applied to known locations on a solid support and with said probes being capable of hybridizing to polynucleic acids of step (i) or (ii) under the same hybridization and wash conditions or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T of said target sequence is replaced by
U; (iv) detecting the hybrids formed in step (iii) ;
(v) inferring the HBV genotype present in said sample from the differential hybridization signal(s) obtained in step (iv) .
The genotype specific target sequences can be any nucleotide variation appearing upon alignment of the different HBV genomes that permits classification of a certain HBV isolate as a certain genotype (see Figure 1 ).
The expression "relevant part of a suitable HBV gene" refers to the part of the HBV gene encompassing the HBV genotype specific target sequence chosen from Figure 1 to be detected.
According to a preferred embodiment of the present invention, step (iii) is performed using a set of at least 2, preferably at least 3, more preferably at least 4 and most preferably at least 5 probes all meticulously designed such that they show the desired hybridization results, when used in a reverse hybridisation assay format, more particularly under the same hybridization and wash conditions implying that each of said probes is able to form a complex upon hybridisation with its target sequence present in the polynucleic acids of the sample as obtained after step (i) or (ii).
The numbering of the HBV gene encoded amino acids and nucleotides is as generally accepted in literature.
More particularly, the present invention relates to a set of at least 2 probes allowing the detection of a genotype specific variation, possibly aiso including one or more probes allowing the detection of a wild-type sequence, a polymorphic or a mutated sequence at any one of the nucleotide positions showing a sequence diversity upon alignment of all known or yet to be discovered HBV sequences as represented in Figure 1 for all complete HBV genomes found in the EMBL/NCBI/DDBJ/Genbank.
The sets of probes according to the present invention have as a common characteristic that all the probes in said set are designed so that they can be used together in a reverse-hybridization assay, more particularly under similar or identical hybridization and wash conditions as indicated above and below.
Selected sets of probes according to the present invention include probes which allow to differentiate any of the HBV genotype specific nucleotide changes as represented in Figure 1 , preferably in the preS1 or HBsAg region of HBV. Said probes being characterized in that they can function in a method as set out above.
In order to solve the above-mentioned problem of obtaining information on the possible presence of HBV mutants in a given sample, the present invention provides an elegant way to tackle this problem which involves residing to a reverse hybridisation approach (particularly on Line Probe Assays strips, as described by Stuyver et al., 1 993). Using this technology it is possible to conveniently obtain all essential data in one test run. To achieve this goal, a set of probes needs to be
designed and assembled which can detect all relevant mutations and possibly also wild-type sequences or polymorphisms in the HBV gene regions of interest.
Another particularly preferred embodiment of the present invention thus is a method for determining the presence or absence of one or more HBV mutants in a biological sample, comprising:
(i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample;
(ii) if need be amplifying the relevant part of a suitable HBV gene present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two nucleotide probes hybridizing specifically to a HBV mutant target sequence chosen from Figure 1 , with said probes being applied to known locations on a solid support and with said probes being capable of hybridizing to the polynucleic acids of step (i) or (ii) under the same hybridization and wash conditions, or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T of said target sequence is replaced by U and with said set or probes possibly also comprising one or more wild-type HBV probes corresponding with the respective mutated HBV target sequence; (iv) detecting the hybrids formed in step (iii);
(v) inferring the HBV mutant(s) present in said sample from the differential hybridization signal(s) obtained in step (iv). It is to be understood that the term "mutant target sequence" not only covers the sequence containing a mutation, but also the corresponding wild-type sequence. The HBV mutant target sequence according to the present invention can be any sequence including a HBV mutated codon known in the art or yet to be discovered. Particularly preferred HBV mutant target regions are set out below. In order to solve the problem as referred to above of obtaining information on the essential issues for proper diagnosis of HBV (namely genotype and different mutations particularly mutations in the preCore region, vaccine escape mutations and RT gene mutations selected by treatment with drugs such as lamivudine and
penciclovir), the present invention provides a particularly elegant way to obtain such complex information.
Moreover, careful analysis of the data obtained by the present inventors clearly revealed that combining the information concerning the preCore and escape mutants with data on the genotype is essential to allow adequate interpretation of the results. Hence it is highly advantageous to be able to produce all relevant data simultaneously.
In this method for diagnosing HBV mutants, preferably in combination with HBV genotyping, a set of probes selected as defined above may be used, wherein said set of probes is characterized as being chosen such that for a given HBV mutation, the following probes are included in said set :
-at least one probe for detecting the presence of the mutated nucleotide(s) at said position;
-at least one probe for detecting the presence of the wild-type nucleotide(s) at said position;
-possibly also (an) additional probe(s) for detecting wild-type polymorphisms at positions surrounding the mutation position. Inclusion of the latter two types of probes greatly contributes to increasing the sensitivity of said assays as demonstrated in the examples section. Selected sets of probes according to the present invention include at least one probe, preferably at least two probes, characterizing the presence of a HBV mutation at nucleotide positions chosen from the preCore region of HBV, more particularly from the following list of codons susceptible to mutations in the HBV preCore region, such as codon 1 5 in genotype A, and for all genotypes: codon 28, codon 29, and codon 28 and 29, or in the preCore promoter region (see Figure 1 ).
Said probes being characterized in that they can function in a method as set out above.
An additional embodiment of the present invention includes at least one probe, preferably at least two probes, characterizing the presence of a vaccine escape mutation in codon positions chosen from the HBsAg region of HBV, more particularly from the list of codons susceptible to mutations in the HBV HBsAg
region, such as at codons 1 22, 1 26, 141 , 143, 144 or 1 45 (see Figure 1 ) .
An additional embodiment of the present invention includes at least one probe, preferably at least two probes, characterizing the presence of a mutation in the RT pol gene region of HBV, that gives rise to resistance to drugs such as lamivudine and penciclovir, for instance mutation of M to V or to I at position 552
(in the YMDD motif), mutation of V to I at position 555, mutation of F to L at position 514, mutation of V to L at position 521 , mutation of P to L at position 525 and mutation of L to M at position 528 (see Figure 1 ).
In a selected embodiment, a combination of at least two oligonudeotide probes is used and said combination of probes hybridizes specifically to at least two of the following groups of target sequences: a mutant target sequence chosen from the HBV RT pol gene region, a mutant target sequence chosen from the HBV preCore region, a mutant target sequence chosen from the HBsAg region of HBV, a HBV genotype-specific target sequence.
For instance, an embodiment involves hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from Figure 1 and at least one nucleotide probe hybridizing specifically to a HBV mutant target sequence chosen from Figure 1 . Another selected embodiment involves, for instance, hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from Figure 1 and at least one nucleotide probe hybridizing specifically to a HBV mutant target sequence chosen from the RT pol gene region as represented in Figure 1 . Another selected embodiment involves, for instance, hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from Figure 1 and at least one nucleotide probe hybridizing specifically to a HBV mutant target sequence chosen from the preCore region as represented in Figure 1 . Another selected embodiment involves, for instance, hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence
chosen from Figure 1 and at least one nucleotide probe hybridizing specifically to a HBV vaccine escape mutant target sequence within the HBsAg region as represented in Figure 1 .
In a selected embodiment, a combination of at least three oligonudeotide probes is used and said combination of probes hybridizes specifically to at least three of the following groups of target sequences: a mutant target sequence chosen from the HBV RT pol gene region, a mutant target sequence chosen from the HBV preCore region, a mutant target sequence chosen from the HBsAg region of HBV, a HBV genotype-specific target sequence.
For instance, an embodiment involves hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from Figure 1 , and at least one nucleotide probe hybridizing specifically to a HBV mutant target sequence chosen from the preCore region as represented in Figure 1 , and at least one nucleotide probe hybridizing specifically to a HBV vaccine escape mutant target sequence chosen from the HBsAg region as represented in Figure 1 .
For instance, another embodiment involves hybridizing with at least one probe hybridizing specifically to a mutant target sequence from the HBV RT pol gene region of HBV, and at least one probe hybridizing specifically to a mutant target sequence from the HBsAg region of HBV, and at least one probe hybridizing specifically to a genotype-specific target sequence from the HBsAg region of HBV. According to this embodiment, the relevant part of the HBV genome can be amplified by use of one primer pair, for instance HBPr 75 and HBPr 94. in a selected embodiment, a combination of at least four oligonudeotide probes is used and said combination of probes hybridizes specifically to all of the following groups of target sequences: a mutant target sequence chosen from the HBV RT pol gene region, a mutant target sequence chosen from the HBV preCore region, a mutant target sequence chosen from the HBsAg region of HBV, a HBV genotype-specific target sequence.
Particularly preferred embodiments of the invention thus include a set of probes as set out above comprising at least one, preferably at least two, at least three, at least four or more probe(s) for targeting one, preferably two, three or more nucleotide changes appearing in the alignment of HBV genomes as represented in Figure 1 .
Even more preferred selected sets of probes according to the present invention include probes derived from two of the same or different regions of HBV bearing HBV mutated nucleotides, or in addition also a third (set of) probe(s) characterizing the presence of a third HBV mutation at any of the positions shown in Figure 1 , or particular combinations thereof.
Particularly preferred is also a set of probes which allows simultaneous detection of HBV mutations at codons 1 5, 28 and 29 in the preCore region, possibly in combination with mutations in the preCore promoter regions, in combination with mutations at codons 1 22, 1 26, 141 , 143, 144, 145 in the HBsAg region, possibly also in combination with mutations in the HBV pol gene at codons 514, 521 , 525, 528, 552 or 555.
In the instances where the alignment of HBV genomes of Figure 1 is referred to in this invention, it should be construed as referring to an alignment of all existing and future HBV genomes. The existing HBV genome sequences can be deduced from any database, such as the EMBL/NCBI/DDBJ/GENBAN database.
A preferred set of preCore, preSI , HBsAg and RT pol gene probes of this invention are the probes with SEQ ID NO 1 to 278 of Table 1 (see also Figure 1 ).
Particularly preferred sets of probes in this respect are shown in Figure 2 and in Figure 4. The probes in Figure 2 and in Figure 4 were withheld after a first selection for preCore, preSI , HBsAg and RT pol probes.
The probes of the invention are designed for obtaining optimal performance under the same hybridization conditions so that they can be used in sets of at least 2 probes for simultaneous hybridization. This highly increases the usefulness of these probes and results in a significant gain in time and labour. Evidently, when other hybridization conditions would be preferred, all probes should be adapted accordingly by adding or deleting a number of nucleotides at their extremities. It
should be understood that these concomitant adaptations should give rise to essentially the same result, namely that the respective probes still hybridize specifically with the defined target. Such adaptations might also be necessary if the amplified material should be RNA in nature and not DNA as in the case for the NASBA system.
The selection of the preferred probes of the present invention is based on a reverse hybridization assay format using immobilized oligonudeotide probes present at distinct locations on a solid support. More particularly the selection of preferred probes of the present invention is based on the use of the Line Probe Assay (LiPA) principle which is a reverse hybridization assay using oligonudeotide probes immobilized as parallel lines on a solid support strip (Stuyver et al. 1 993; international application WO 94/1 2670). This approach is particularly advantageous since it is fast and simple to perform. The reverse hybridization format and more particularly the LiPA approach has many practical advantages as compared to other DNA techniques or hybridization formats, especially when the use of a combination of probes is preferable or unavoidable to obtain the relevant information sought. It is to be understood, however, that any other type of hybridization assay or format using any of the selected probes as described further in the invention, is also covered by the present invention. The reverse hybridization approach implies that the probes are immobilized to certain locations on a solid support and that the target DNA is labelled in order to enable the detection of the hybrids formed.
The following definitions serve to illustrate the terms and expressions used in the present invention. The term "genetic analysis" refers to the study of the nucleotide_sequence of the genome of HBV by any appropriate technique.
The term "HBV mutant" refers to any HBV strain harbouring genomic variations with serological, genetical or clinical consequences.
The term "vaccine escape mutant" is reviewed in the introduction section and in Example 7. The most important region lies between amino acid 1 24 and 147 of the HBsAg region, but can be extended from amino acid 1 14 to 1 50.
The term "mutant resistant to drugs such as lamivudine and penciclovir" is reviewed in the introduction section and in Example 8.
The term "HBV genotype" refers to HBV strains with an intergenotype variation of 8% or more based on a comparison of complete genomes. The target material in the samples to be analyzed may either be DNA or
RNA, e.g. genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are also termed polynucleic acids.
It is possible to use genomic DNA or RNA molecules from samples susceptible of containing HBV in the methods according to the present invention. Well-known extraction and purification procedures are available for the isolation of RNA or DNA from a sample (f.i. in Maniatis et al. , Molecular Cloning:
A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press (1 989)).
The term "probe" refers to single stranded sequence-specific oligonudeotides which have a sequence which is complementary to the target sequence to be detected.
The term "target sequence" as referred to in the present invention describes the nucleotide sequence of a part of wild-type, polymorphic or mutant HBV gene sequence to be specifically detected by a probe according to the present invention. The polymorphic sequence may encompass one or more polymorphic nucleotides; the mutant sequence may encompass one or more nucleotides that are different from the wild-type sequence. It is to be understood that the term "mutant target sequence" not only covers the sequence containing a mutation, but also the corresponding wild-type sequence. Target sequences may generally refer to single nucleotide positions, codon positions, nucleotides encoding amino acids or to sequences spanning any of the foregoing positions. In the present invention said target sequence often indudes one, two or more variable nucleotide positions. In the present invention polynucleic acids detected by the probes of the invention will comprise the target sequence against which the probe is detected.
It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases. The target sequences as defined in the present invention provide sequences which should at least be complementary to
the central part of the probe which is designed to hybridize specifically to said target region. In most cases the target sequence is completely complementary to the sequence of the probe.
The term "complementary" as used herein means that the sequence of the single stranded probe is exactly the (inverse) complement of the sequence of the single-stranded target, with the target being further defined as the sequence where the mutation to be detected is located.
Since the current application requires the detection of single basepair mismatches, stringent conditions for hybridization are required, allowing in principle only hybridization of exactly complementary sequences. However, variations are possible in the length of the probes (see below). It should also be noted that, since the central part of the probe is essential for its hybridization characteristics, possible deviations of the probe sequence versus the target sequence may be allowable towards head and tail of the probe when longer probe sequences are used. These variations, which may be conceived from the common knowledge in the art, should however always be evaluated experimentally, in order to check if they result in equivalent hybridization characteristics as the exactly complementary probes.
Preferably, the probes of the invention are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. Particularly preferred lengths of probes include 10, 1 1 , 12, 1 3, 14, 1 5, 1 6, 17, 18, 1 9, 20, 21 , 22, 23, 24 or 25 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonudeotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridisation characteristics.
Probe sequences are represented throughout the specification as single stranded DNA oligonudeotides from the 5' to the 3' end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U). The probes according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide
sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method. The term "solid support" can refer to any substrate to which an oligonudeotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip. Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopoiymer tailing, coupling with different reactive groups such as aliphatic groups, NH 2 groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins. The term "labelled" refers to the use of labelled nucleic acids. Labelling may be carried out by the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1 988) or Bej et al. ( 1 990) or labelled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic ( 32 P, 35 S, etc.) or non- isotopic (biotin, digoxigenin, etc.) .
The term "primer" refers to a single stranded oligonudeotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nudeic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.
The expression "suitable primer pair" in this invention refers to a pair of primers allowing the amplification of part or all of the HBV gene for which probes are immobilized.
The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al. , 1 990) .
The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1 988), ligase chain reaction (LCR; Landgren et al., 1 988; Wu &
Wallace, 1 989; Barany, 1 991 ), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1 990; Compton, 1 991 ), transcription-based amplification system (TAS; Kwoh et al., 1 989), strand displacement amplification (SDA; Duck, 1 990; Walker et al., 1 992) or amplification by means of Qβ repiicase (Lizardi et al., 1 988; Lomeli et al., 1 989) or any other suitable method to amplify nucleic acid molecules known in the art.
The oligonudeotides used as primers or probes may also comprise nucleotide analogues such as phosphorothiates (Matsukura et al., 1 987), alkylphosphorothiates (Miller et al., 1 979) or peptide nucleic acids (Nielsen et al., 1 991 ; Nielsen et al., 1 993) or may contain intercalating agents (Asseline et al.,
1 984).
As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptions with respect to the conditions under which the oligonudeotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridisation will be essentially the same as those obtained with the unmodified oligonudeotides.
The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonudeotide molecules, etc.
The "sample" may be any biological material taken either directly from the infected human being (or animal), or after culturing (enrichment). Biological material may be e.g. expectorations of any kind, broncheolavages, blood, skin tissue, biopsies, sperm, lymphocyte blood culture material, colonies, liquid cultures, faecal samples, urine etc.
The sets of probes of the present invention will include at least 2, 3, 4, 5,
6, 7, 8, 9, 10, 1 1 , 1 2, 1 3, 14, 1 5, 1 6, 1 7, 18, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may be applied in two or more (possibly as many as there are probes) distinct and known positions on a solid substrate. Often it is preferable to apply two or more probes together in one and the same position of said solid support.
For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.
Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.
The stability of the [probe : target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and %GC result in a Tm about 2- 1 0°C higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.
Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic
oligonudeotide probes of about 10-50 bases in length occurs approximately 5 C C below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity. It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the nontarget nudeic acid. In the present case, single base pair changes need to be detected, which requires conditions of very high stringency.
The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonudeotide probes of different lengths and base composition may be used, preferred oligonudeotide probes of this invention are between about 5 to 50 (more particularly 1 0-25) bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.
Regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to
participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interaction.
However, in certain instances, it may not be possible to avoid this type of interaction.
Standard hybridization and wash conditions are disclosed in the Materials & Methods section of the Examples. Other conditions are for instance 3X SSC
(Sodium Saline Citrate) , 20% deionized FA (Formamide) at 50°C.
Other solutions (SSPE (Sodium saline phosphate EDTA), TMACI (Tetramethyl ammonium Chloride), etc.) and temperatures can also be used provided that the specificity and sensitivity of the probes is maintained. If need be, slight modifications of the probes in length or in sequence have to be carried out to maintain the specificity and sensitivity required under the given circumstances. In a more preferential embodiment, the above-mentioned polynucleic acids from step (i) or (ii) are hybridized with at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the above-mentioned target region specific probes, preferably with 5 or 6 probes, which, taken together, cover the "mutation region" of the relevant HBV gene.
The term "mutation region" means the region in the relevant HBV gene sequence where at least one mutation encoding a HBV mutant is located in a preferred part of this mutation region is represented in figure 1 .
Apart from mutation regions as defined above the HBV wild-type or mutant genomes may also show polymorphic nucleotide variations at positions other than those referred to as genotype specific or mutant specific variated positions as shown in Figure 1 . Since some mutations may be more frequently occurring than others, e.g. in certain geographic areas or in specific circumstances (e.g. rather dosed
communities) it may be appropriate to screen only for specific mutations, using a selected set of probes as indicated above. This would result in a more simple test, which would cover the needs under certain circumstances.
In order to detect HBV genotypes and/or HBV mutants with the selected set of oligonudeotide probes, any hybridization method known in the art can be used
(conventional dot-blot, Southern blot, sandwich, etc.) .
However, in order to obtain fast and easy results if a multitude of probes are involved, a reverse hybridization format may be most convenient.
In a preferred embodiment the selected set of probes are immobilized to a solid support in known distinct locations (dots, lines or other figures). In another preferred embodiment the selected set of probes are immobilized to a membrane strip in a line fashion. Said probes may be immobilized individually or as mixtures to delineated locations on the solid support.
A specific and very user-friendly embodiment of the above-mentioned preferential method is the LiPA method, where the above-mentioned set of probes is immobilized in parallel lines on a membrane, as further described in the examples.
The invention also provides for a set of primers allowing amplification of the region of the respective HBV gene to be detected by means of probes. Examples of such primers of the invention are given in Table 1 and Figure 1 .
Primers may be labelled with a label of choice (e.g. biotine). Different primer- based target amplification systems may be used, and preferably PCR-amplification, as set out in the examples. Single-round or nested PCR may be used.
The invention also provides a kit for detection and/or genetic analysis of HBV genotypes and/or HBV mutants present in a biological sample comprising the following components: (i) when appropriate, a means for releasing, isolating or concentrating the polynucleic acids present in said sample; (ii) when appropriate, at least one suitable primer pair; (iii) at least two of the probes as defined above, possibly fixed to a solid support;
(iv) a hybridization buffer, or components necessary for producing said buffer; (v) a wash solution, or components necessary for producing said solution; (vi) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization. (vii) when appropriate, a means for attaching said probe to a known location on solid support.
The term "hybridization buffer" means a buffer enabling a hybridization reaction to occur between the probes and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions. The term "wash solution" means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
As illustrated in the Examples section, a line probe assay (LiPA) was designed for screening for HBV genotypes and/or HBV mutants. The principle of the assay is based on reverse hybridization of an amplified polynucleic acid fragment such as a biotinylated PCR fragment of the HBV gene onto short oligonudeotides. The latter hybrid can then, via a biotine-streptavidine coupling, be detected with a non-radioactive colour developing system.
The following examples only serve to illustrate the present invention. These examples are in no way intended to limit the scope of the present invention.
FIGURE AND TABLE LEGENDS
Figure 1 : Alignment of 35 complete HBV genomes. Isolates belonging to genotype A are: HBVXCPS, HBVADW, HVHEPB, S50225, HPBADWZCG; genotype B: HPBADW3, HPBADWZ, HPBADW1 , HPBADW2; genotype C: HPBCGADR, HBVADRM, HPBADRA, HPBCG, HEHBVAYR, HBVADR, HBVADR4, HPBADR1 C,
HPBADRC, HBVPREX, HPBETNC, HHVBC, HHVCCHA; genotype D: HBVAYWMCG, HBVAYWC, HBVAYWCI, HBVAYWE, HBVDNA, HPBHBVAA, XXHEPAV, HBVORFS; genotype E: HHVBE4, HHVBBAS; and genotype F: HHBF, HHVBFFOU, HBVADW4A. To preserve alignment, several gaps were created in the alignment and are indicated with /. Positions of start and end of the different HBV encoded genes is indicated: HBsAg: hepatitis B surface antigen (small surface antigen); HBx: hepatitis B X protein; HB Pol: hepatits B polymerase protein, encoding a terminal protein, a spacer, a RT/DNA polymerase region, and an RNAse H activity; HBeAg: hepatitis B Core antigen; HBpreSI Ag: hepatitis B preSI antigen (large surface antigen); HBpreS2Ag: hepatitis B preS2 antigen (middle surface antigen). The position of the PCR primers is indicated with a large box over all 35 sequences. The polarity of the PCR primer can be deduced from the position of the name above these boxes: left = antisense primer; right = sense primer. LiPA probes are indicated with small boxes, the numbers of the probes are indicated next to the probes or to the right of the alignment, and correspond to the probe numbers in Table 1 .
Figure 2: LiPA HBV design. The content of a HBV LiPA strip is detailed. For each line number, the region on the viral genome is indicated, together with the genotype that is detected, the probe number that corresponds with the boxes from the alignment in Figure 1 , and the sequence of the probe.
Figure 3: Combined result of genotype determination in the preSI region and preCore scanning on 24 samples. The interpretation of each sample is given under each strip. Probe reactivities on lines 3 to 14 are obtained from the preSI PCR
fragment, probe reactivities on lines 1 5 to 27 are due to the preCore PCR fragment. Genotypes are indicated from A to F. The interpretation for the preCore region is as follows: W = wild type; M = mutant; I = indeterminate, meaning that no reactivity is observed, which is due to mutations that could not yet be detected with the selected probes; mix = mixture of wild type and mutant; interpretation of codon 1 5 is only relevant for genotype A, the absence of reactivity on HBPr 45 for genotypes B to F is of no use as is indicated with - (not applicable). Since the presence or absence of preCore mutations has effect on the serological HBeAg status, this is also indicated.
Figure 4: Probes used in HBV LiPA. Probes were designed for genotyping in the
HBsAg region and for detection of drug resistance mutations in the YMDD motif (see also Figure 5), as well as for detection of mutations in the pre Core region (see also Figure 6) .
Figure 5: Example of a LiPA assay combining HBV genotyping in the HBsAg region and detection of drug resistance mutations in the YMDD motif. Genotypes are indicated from A to F. The design of the strip is shown to the right, with the numbers of the probes corresponding to the numbers in Table 1 and in Figure 4. The genotypes and mutant motifs to which each probe hybridizes are written to the outer right. The combination of reactive probes allows the determination of a unique genotype.
Figure 6: Example of the determination of preCore mutations by the LiPA technique. The design of the strip is shown to the right, with the numbers of the probes corresponding to the numbers in Table 1 . The mutant target sequences to which the probes hybridize are indicated to the outer right. Motif M2 corresponds to a mutation in codon 28, M4 corresponds to a mutation in codon 29. M2/M4 has mutations in both 28 and 29.
Figure 7: Detection of a mutation in the YMDD motif of HBV pol upon treatment
with lamivudune. The graph shows a time course of the viral load during lamivudine treatment. To the right LiPA strips are shown, corresponding to assays at the beginning of the treatment (5/95), 1 0 months of treatment (2/96) and 1 4 months of treatment (6/96) . The assay shows that during treatment the YMDD motif mutates to YVDD.
Table 1 : Overview of all primers and probes referred to in the Figures with an indication of their respective SEQ ID NO and the region of the HBV genome they are designed for. Primers from the PreSI region include 1 , 106, 2 (sense primers) and 4, 1 07 and 3 (antisense primers). Primers from the HBsAg region include 75 and 1 04 (sense primers) and 76, 94 and 105 (antisense primers) . Primers from the PreCore region include 5, 6, 69, 70, 84, 86, 87 and1 08 (sense primers) and 7, 8, 85 and 1 09 (antisense primers) . The remaining oligonudeotides are probes from the PreCore, PreSI , HBsAg and RT pol gene regions of HBV as indicated. The YMDDV motif and its mutants consist of amino acids 551 to 555 of the RT pol protein; the sequence MGVGL and its mutant consist of amino acids 51 9 to 523 of the RT pol protein; the sequence SPFLL and its mutants and genotypic variants consist of amino acids 524 to 528 of the RT pol protein.
Table 1 : HBV probe and primer design
Name Sequence SEQ ID NO Region
HBPrl GGGTCΛCCΛTATTCTTGGG 1 preSI primer sense
HBPr2 GAACAAGAGCTACAGCATGGG 2 preSI primer sense
HBP 3 CCACTGCΛTGGCCTGΛGGΛTG 3 preSI primer anti-sense
HBP GTTCCT/GGΛΛCTGGΛGCCΛCCΛG 4 preSI primer an i-sense
HBPr5 TCTTTGTΛTTΛGGAGGCTGTΛG 5 preCore primer sense
HBPr6 GCTGTAGGCΛTAΛΛTTGGTCTG e preCore primer sense
HBPr7 CTCCΛCΛGT/ΛΛGCTCCΛΛΛτTC 7 preCore primer anti-sense
HBPrB GΛΛGGAΛAGΛΛGTCΛGΛΛGGC 8 preCore primer anti-sense
HBPr9 TGGCTTTGGGGCΛTGG 9 preCore
HBPrl0 TGGCTTTAGGGCATGG 10 preCore
HBPrll TGGCTTTΛGGΛCΛTGG 11 preCore
HBPrl2 ΛAGTTGCATGGTGCTG 12 preCore
HBPrl3 CACCTCTGCCTΛΛTCΛT 13 preCore
HBPrl. TGGGGTGGAGCCCTCAG 14 preSI
IIBPrl5 GCCAGCAGCCΛΛCCΛG 15 preSI
HBPrl6 CCCATGGGGGACTGT 16 preSI
HBPrl7 ΛACCCCAΛCAΛGGΛTG 17 preSI
HBPrl8 TCCΛCCAGCΛΛTCCT 18 preSI
HBPr19 TGGGGGAAGAATATTT 19 preSI
HBPr20 ΛAATTCCAGCΛGTCCC 20 preSI
HBPr21 GTTCCCAΛCCCTCTGG 21 preSI
HBPr22 AΛCCTCGCΛΛΛGGCΛT 22 preSI
HBPr23 TGCΛTTCΛΛΛσCCΛΛC 23 preSI
.HBPr24 TΛCTCΛCΛΛCTGTGCC 24 preSI
HBPr25 ACCCTGCGTTCGGAGC 25 preSI
HBPr26 CΛGGAAGΛCΛGCCTΛC 26 preSI
HBPr27 GATCCΛGCCTTCΛGΛG 27 preSI
HBPr28 ATGCTCCAGCTCCTΛC 28 preSI
HBPr29 GCTTTCTTGGΛCGGTC 29 preSI
HBPr30 CTΛCCCCΛΛTCΛCTCC 30 presi
HBPr31 ΛGCΛCCTCTCTCΛΛCG 31 preSI
HBP 32 CCAΛTGGCAΛΛCΛΛGG 32 preSI
HBPr33 CTGΛGGGCTCCΛCCCCΛ 33 preSI
HBPr34 ΛTGCAΛCTTTTTCΛCC 34 preCore
HBPr35 ATCTCTTGTACΛTGTC 35 preCore
HBPr36 ATCTCATGTTCΛTGTC HBPr37 CAGTGGGACΛTGTΛCΛ HBPr3B CAGTAGGΛCΛTGΛΛCΛ HBPr39 CTGTTCAAGCCTCCΛΛ HBPr40 ΛGCCTCCΛΛGCTGTGC HBPr41 AAAGCCACCCΛΛGGCΛ HBPr42 TGGCTTTAGGACATGGA HBPr GACΛTGTACΛΛGAGΛTGΛ HBPr44 GΛCΛTGΛΛCΛTGΛGΛ_GΛ HBPr45 TGTACΛTGTCCCΛCTGTT HBPr 6 TGTTCATGTCCTΛCTGTT HBPr47 ΛCTGTTCAAGCCTCCΛΛG HBPr48 GGCACAGGCTTGGΛGGC1T HBPr49 AAAGCCACCCAAGGCACA HBPr50 CCCAGAGGGTTGGGAAC HBPr51 CAGCATGGGGCAGΛΛTCT
HBPr52 TCCACCΛGCΛATCCTCTG
HBPr53 GGΛTCCAGCCTTCΛGΛGC
HBPr54 1CΛGGΛΛGΛCΛGCCrΛC
HBPr55 TTCAΛCCCCΛΛCΛΛGGΛTC
HBPr56 ΛΛTGCTCCAGCTCCTΛC
IIBPr57 CTGCATTCΛΛΛGCCΛΛCl
HBPr58 CCCCATGGGGGΛCTGTTG
HBPr59 CΛTACTCACAΛCTGTGCCA
HBPr60 GGGCTTTCTTGGΛCGGTCC
HBPr61 CTCTCGAAIGGGGGAAGA
HBPr62 CCTACCCCAATCΛCTCCΛ
HBPr63 AGCACCTCTCTCΛΛCGΛCΛ
HBPr6 GCΛAATTCCAGCΛG1CCCG
HBPr65 GCCAATGGCAAACAAGGTA
HBPr66 GACΛTGAACATGΛGATG
HBPr67 GGACATGAACAΛGΛGΛT
HBPr68 GACATGTACΛAGΛGΛTG
HBPr69 ACATAAGΛGGΛCTCTTGGΛC
HBPr70 TACTTCAΛΛGACTGTGTGTTTΛ
HBPr71 ACAAAGACCTTTAAC/TCT
HBPr72 ACΛΛAGATCΛTTΛΛC/TCr
HBPr73 TTCCACCΛGCΛΛTCCTC
HBPr74 GATCCAGCCTTCAGAGC
HBPr75 CΛΛGGTATGTTGCCCGTTTGTCC 75 HBsAg primer sense IIBPr76 CCΛΛΛCΛGrGGGGGΛΛΛGCCC 76 HBsAg primer anti-sense HBPr77 CTACGGΛTGGΛΛΛTTGC 77 HBsAg codon 145 wild type HBPr78 TACGGACGGΛΛACTGC 78 HBsAg codon 145 wild type HBP179 TTCGGACGGAAACTGC 79 HBsAg codon 145 wild type HBPr80 CTTCGGACGGAAΛTTGC 80 HBsAg codon 145 wild type HBPr81 CTACGGΛTΛGΛΛΛTTGC 81 HBsAg codon 145 mutant IIBPr82 CTTCGGΛCΛGΛΛΛITGC 82 HBsAg codon 145 mutant HBPr83 CTATGGGAG GGGCClCAGr/CC 83 HB Pol HBPr84 GCTGTΛGGCATΛΛATTGGTCTG 84 preCore primer sense HBPr85 CTCCΛCAGT/AΛGCTCCΛΛΛITC 85 preCore primer anti-sense HBPr86 ACΛTAΛGAGGΛCrCTTGGΛC 86 preCore primer sense HBPr87 TACTTCAAAGACTGTGTGTTTA 87 preCore primer sense HBPrββ TΛGGTTAAΛGGTCTTTGT 88 preCore promoter
HBPr89 TAGGTTΛΛTGATCTTTGT 89 preCore promoter HBPr90 CΛTGTCCCACTGTTCΛΛ 90 preCore
HBPr91 CATGTCCTΛCTGTTCΛΛ 91 preCore
HBPr92 TTCTGCCCCATGCTGTA 92 preSI
HBPr93 TTCTGCCCCΛTGCTGTΛG 93 preSI CD
HBPr94 GGTAA/TAAAGGGACrCAC/AGATG 94 HBsAg primer anti-sense
HBPr95 ICΛGCTATATGGΛIGΛΓ 95 HB Pol
HBPr96 CAGCTΛTΛΓGGΛTGΛT 96 HB Pol
HBPr97 TTCAGCTΛTΛTGGΛTG 97 HB Pol
HBPr98 TCAGTTΛTATGGΛTGΛT 98 HB Pol
HBPr 9 TTTCAGTTATATGGATG 99 HB Pol
HBPrlOO TTTΛGTTΛTATGGΛTGΛ 100 HB Pol
HBPrlOl TCAGCTATGTGGATGAT 101 HB Pol
HBPrl02 TCAGTTATGTGGATGAT 102 HB Pol
HBPrl03 TTTCAGCTATGTGGATG 103 HB Pol
HBPrl04 CAAGGTATGTTGCCCGTTTGTCC 104 HBsAg primer sense
HBPrl05 GGT/CAA/TAAAGGGACTCAC/AGATG 105 HBsAg primer anti-sense
HBPrl06 GGGTCΛCCATATTCTTGGG 106 preSI primer sense
HBPrl07 GTTCCT/GGAΛCTGGAGCCΛCCAG 107 preSI primer anti-sense
HBPrlOβ CCGGAAAGCTTGAGCTCTTCl TTTTCACCTCTGCCTAATC 108 preCore primer sense
HBPr109 CCGGAAAGCTTGAGCTCTTCAAAAAGTTGCATGGTGCTGG 109 preCore primer anti-sense
HBPrl10 CCTCTGCCGATCCATACTGCGGAAC 110 preX primer sense
HBPr111 CTGCGAGGCGAGGGAGTTCTTCTTC 111 HB Core primer anti -sense
HBPr112 TGCCΛTTTGTTCΛGTGGTTCGTΛGGGC 112 HBsAg primer sense
HBPrll3 CCGGCAGATGAGAAGGCACAGACGG 113 HBX primer antisense
HBPrll4 TiCΛGClΛTΛTGG rGΛl' 114 YMDD motif
HBPr115 TCAGCTATATGGATGATG 115 YMDD motif
HBPr116 TTCΛGCTΛTGTGGΛTGΛT 116 YMDD moti
HBPrll7 TCΛGCTATGTGGΛTGΛTG 117 YMDD motif
HBPrllβ GGCTTTGGGGCΛTGG 118 preCore codon 2B wild type
HBPrl19 TGGCTTTGGGGCΛTG 119 preCore codon 28 wild type
HBPr120 GTGGCTTTGGGGCATG 120 preCore codon 28 wild type
HBPrl21 GGCTTTGGGGCΛTGGΛ 121 preCore codon 28 wild type
HBPrl22 TGGCTTTGGGΛCΛTGG 172 preCore codon 28 wild type, codon 29 mutant
HBPr123 GGCTTTGGGΛCΛTGG 123 preCore codon 28 wild type, codon 29 mutant
HBPrl24 TGGCTTTGGGACATG 124 preCore codon 28 wild type, codon 29 mutant
HBPrl25 GTGGCTTTGGGΛCΛTG 125 preCore codon 28 wild type, codon 29 mutant
HBPrl26 GGCTTTGGGACATGGA 126 preCore codon 28 wild type, codon 29 mutant
HBPrl27 TCΛGTTATATGGΛTGΛTG 127 YMDD genotype D, wild tpye
HBPrl2B TTCAGTTΛTΛTGGΛTGAT 128 YMDD genotype D, wild tpye
HBPr129 TTTCAGTTATATGGΛTGΛT 129 YMDD genotype D, wild tpye
HBPrl30 TCAGTTATGTGGATGATG 130 YMDD genotype D, mutant
HBPrl31 TTCAGTTATGTGGΛTGΛT 131 YMDD genotype D, mutant
HBPrl32 TTTCΛGTTATGTGGΛTGΛT 132 ts
YMDD genotype D, mutant
HBPr133 TTTCAGTTATGTGGATGΛ 133 YMDD genotype D, mutant
HBPrl34 TGCTGCTATGCCTCΛTCTTC 134 outer HBsAg primer sense
HBPr1 5 CA(G/Λ)ΛGΛCAAΛAGΛΛΛΛlTGG 135 outer HBsΛg primer anti-sense
HBPrl36 CTATGGATGGAAΛTTGC 136 HBsAg mutant codon 143
HBP 137 CCTΛTGGATGGΛAΛTTG 137 HBsAg mutant codon 143
HBPR138 ACCTATGGATGGΛΛΛTT 138 HBsAg mutant codon 143
HBPr139 CT CAA GGC ΛΛC TCT ΛTG TGG 139 HBsAg, genotype Λ
HBPr140 CT CAΛ GGC ΛΛC TCT ΛTG GG 140 HBsAg, genotype Λ
HBPr141 T CAA GGC ΛΛC TCT ΛTG TTG 141 HBsAg, genotype Λ
HBPrl42 ATC CCA TCA TCT TGG G 142 HBsAg, genotype B
HBPrl43 ATC CCA TCΛ TCT TGG GCG G 143 HBsAg, genotype B
HBPrl44 TC CCA TCΛ TCT TGG GCG G 144 HBsAg, genotype B
HBPr145 C CCA TCA TCT TGG GCT GG 145 HBsAg, genotype B
HBPr146 TTC GCA AAΛ TΛC CTΛ TGG 146 HBsAg, genotype B
HBPr147 T TTC GCA AAA TΛC CTΛ TG 147 HBsAg, genotype B
HBPrl48 CT TTC GCA ΛΛA TAC CTA TG 148 HBsAg, genotype B
HBPrl49 TC GCA AAΛ TΛC CTΛ TGG G 149 HBsAg, genotype B
HBPrl50 T CTA CTT CCA GGΛ ΛCΛ T 150 HBsAg, genotype C
HBPrlSl T CTΛ CTT CCA GGΛ ΛCΛ TC 151 HBsΛg, genotype C
HBPrl52 CT CTΛ CTT CCΛ GGΛ ΛCΛ T 152 HBπΛg, genotype C
HBPrl53 CT CTΛ CTT CCA GGΛ ΛCΛ G
HBPrl54 C TGC ΛCG ATT CCT GCT
HBPrl55 TGC ACG ATT CCT GCT CΛ
HBPrl56 C TGC ΛCG ATT CCT GCT C
HBPrl57 TGC ACG ATT CCT GCT CΛΛ
HBPrlSB TTC GCA ΛGΛ TTC CTΛ TG
HBPrl59 CT TTC GCΛ ΛGΛ TTC CTΛ T
HBPrlβO CT TTC GCA ΛGΛ TTC CTΛ
HBPrlδl CT TTC GCΛ ΛGΛ TTC CTΛ TG
HBPrl62 C TCT ΛTG TAT CCC TCC T
HBPrl63 TCT ATG TAT CCC TCC TG
HBPrl64 C TCT ΛTG TAT CCC TCC TGG
HBPrl65 CC TCT ATG TAT CCC TCC T
HBPrl66 C TGT ACC ΛΛΛ CCT TCG G
HBPrl67 C TGT ACC ΛAΛ CCT ICG
HBPrlββ GC TGT ACC AΛΛ CCT TCG G
HBPrl69 TGT ACC AAΛ CCT TCG GAG
IIBPrl70 GGA CCC TGC CGΛ ΛCC T
U
HBPrl71 GGΛ CCC TGC CGΛ ΛCC G
HBPrl72 G GGA CCC TGC CGΛ AC
HBPrl73 GGA CCC TGC CGΛ AC
HBPrl74 GT TGC TGT TCΛ AΛΛ CCT T
HBPrl75 GT TGC TGT TCΛ ΛΛA CCT G
HBPrl76 TGT TGC TGT TCA AAΛ CCT G
HBPrl77 A TGT TGC TGT TCΛ ΛΛΛ CCT G
HBPrl78 GA TCC ΛCG ΛCC ΛCC A
HBPrl79 GGA TCC ACG ACC ACC Λ
HBPrlβO GGΛ TCC ΛCG ΛCC ΛCC
HBPrlβl GΛ TCC ACG ΛCC ΛCC ΛGG
HBPrl82 TGT TCC ΛΛA CCC TCG G
HBPrl83 C TGT TCC ΛAA CCC TCG
HBPrl84 C TGT TCC ΛΛA CCC TCG G
HBPrl85 GT TCC AΛΛ CCC TCG GΛT
HBPrl86 G CCA AΛT CTG TGC ΛGC
HBPrl87 CCA ΛAT CTG TGC ΛGC ΛT
HBPrlββ G CCA ΛΛT CTG TGC ΛGC ΛG
HBPrl89 GG CCΛ ΛΛT CTG TGC ΛGC
HBPrl90 A TCΛ ΛCΛ ΛCΛ ΛCC ΛGT Λ
HBPrl91 GΛ TCΛ ΛCΛ ACΛ ΛCC ΛGT
HBPrl92 GΛ TCΛ ACΛ ΛCA ΛCC ΛGT A 192 HBsAg, genotype A
HBPrl93 GGA TCA ΛCΛ ΛCΛ ΛCC ΛGT 193 HBsAg, genotype A
HBPrl94 T CΛΛ GGC AΛC TCT ΛTG TGG 194 HBsΛg, genotype Λ
HBPrl95 ΛGG TTA ΛΛG GIC TTT GT 195 promoter genotype Λ wild type
HBPrl96 T ΛGG TTA ΛΛG GTC TTT GG 196 promoter genotype Λ wild type
HBPrl97 TT ΛGG TTA ΛΛG GTC TTT 197 promoter genotype A wild type
HBPrl98 GG TTA ΛΛG GTC TTT GTΛ GG 198 promoter genotype Λ wild type
HBPrl99 AGG TTA ATG ATC TTT GT 199 promoter genotype A mutant
HBPr200 T AGG TTΛ ΛTG ΛTC TTT GG 200 promoter genotype A mutant
HBPr201 CT TTC GCΛ ΛGΛ TTC CTΛ TGG 201 IIBsΛg genotype C codon 160
HBPr202 GCT TTC GCA AGA TTC CTA TG 202 HBsΛg genotype C codon 160
HBPr203 GCT TTC GCΛ AGΛ TTC CTΛ TGG 203 HBsΛg genotype C codon 160
HBPr204 CT TTC GCΛ AGΛ TTC CTΛ TGG G 204 HBsΛg genotype C codon 160
HBPr205 GC TGT ACC AΛΛ CCT TCG GΛG 205 HBsAg genotype D codon 140
HBP 206 TGC TGT ACC ΛΛΛ CCT TCG G 206 HBsΛg genotype D codon 140
HBPr207 TGC TGT ΛCC ΛΛΛ CCT TCG GΛG 207 HBsAg genotype D codon 140
HBPr208 GC TGT ΛCC ΛΛΛ CCT TCG GΛT 208 HBsΛg genotype D codon 140
HBPr209 TGG TTC GCC GGG CTT T 209 HBsΛg genotype E codon 184
UJ
HBPr210 G TGG TTC GCC GGG CTT G 210 HBsΛg genotype E codon 184
HBPr211 GG TTC GCC GGG CTT TC 211 HBsAg genotype E codon 184
HBPr212 TGG TTC GCC GGG CTT TC 212 HBsΛg genotype E codon 184
HBPr213 AG TGG TTC GCC GGG CTG G 213 HBsΛg genotype E codon 184
HBPr214 A GGA TCC ACG ACC ΛCC AGG 214 IIBsΛg genotype F
HBPr215 A GGA TCC ACG ΛCC ΛCC ΛGT 215 IIBsΛg genotype F
HBPr216 CA GGA TCC ACG ΛCC ΛCC AGG 216 HBsΛg genotype F
HBPr217 C TGT TCC ΛΛΛ CCC TCG GΛG 217 HBsΛg genotype F
HBPr218 C TGT TCC ΛΛΛ CCC TCG GΛT 218 HBsΛg genotype F
HBPr219 GC TGT TCC ΛΛA CCC TCG GΛG 219 HBsΛg genotype F
IIBPr220 CTGΛΛCCTTTΛCCCCGTTGC 220 enhancer primer
HBPr221 CTCGCCAΛCTTACΛΛGGCCTTTC 221 enhancer primer
HBPr222 AGAATGGCTTGCCTGΛGTGC 222 Core primer anti-sense
HBPr223 GCT TTC GCΛ ΛGΛ TTC CTA TGG G 223 HBsΛg genotype C codon 160
HBPr224 G GCT TTC GCΛ ΛGΛ TTC CTA TGG 224 HBsAg genotype C codon 160
HBPr225 G GCT TTC GCΛ ΛGΛ TTC CTA TGG G 225 HBsAg genotype C codon 160
HBPr226 G GCT TTC GCΛ ΛGΛ TTC CTA TGG GΛ 226 IIBsΛg genotype C codon 160
HBPr227 C ΛGC TΛT ΛTG GΛT GΛT GTG 227 YMDDV motif
HBPr228 AGC TΛT ΛTG GΛT GΛT GTG GG 228 YMDDV motif
HBPr229 GC TAT ΛTG GAT GΛT GTG GT 229 YMDDV motif
HBPr230 ΛGC TΛT ΛPG GΛT GΛT GTG GT 730 YMDDV motif
HBPr231 C AGC TAT ATG GΛT GΛT ATA
HBPr232 ΛGC TAT ATG GAT GAT ΛTΛ GG
HBPr233 GC TΛT ΛTG GΛT GAT ΛTΛ GT
HBPr234 ΛGC TΛT ATG GΛT GΛT ΛTA GT
HBPr235 CCΛ TCΛ TCT TGG GCT TG
HBPr236 CΛ TCΛ TCT TGG GCT 11
HBPr237 CCA TCA TCT TGG GCT TT
HBPr23β CCA TCA TCT TGG GCT TTC
IIBPr239 CCC ACT GTC TGG CTT TC
HBPr240 CC ACT GTC TGG CTT IC
HBPr241 CC ACT GTC TGG CTT T
HBPr242 CCC ACT GTC TGG CTT G
HBPr243 TΛT ATG GAT GΛT GIG GTA
HBPr2 4 TAT GTG GAT GΛT GTG GTA
HBPr245 TAT ATA GAT GΛT GTG GTΛ
HBPr246 TΛT ΛTT GΛT GAT GIG GTA
HBPr247 TAT GTA GΛT GAT GTG GTA
HBPr248 TAT GTT GΛT GAT GTG GTΛ
HBPr249 TΛT ΛTG GΛT GΛT Λ1Λ GTΛ r
HBPr250 TΛT ΛTG GΛT GΛT ATC GTΛ
HBPr251 TΛT GTG GAT GΛT ΛTA GTΛ
HBPr252 TAT GTG GΛT GAT ATC GTA
HBPr253 TAT ATA GΛT GΛT ΛTΛ GTΛ
HBPr254 TAT ATΛ GAT GΛT AlC GTΛ
HBPr25S TAT ATT GAT GΛT ΛTΛ GTΛ
HBPr256 TAT ΛTT GΛT GAT ΛIC GTA
HBPr257 TΛT GTA GΛT GΛT ATΛ GTA
HBPr258 TΛT GTΛ GΛT GΛT ΛIC GTΛ
HBPr259 TAT GTT GΛT GΛ1 ΛTΛ GTA
HBPr260 TAT GTT GAT GΛT ATC GTΛ
HBPr261 TAT ATG GΛT GΛT CTG GTA
HBPr262 TAT GTG GAT GAT CTG GTA
HBPr263 TAT ATA GΛT GAT CTG GTΛ
HBPr264 TAT ATT GAT GAT CTG GTA
HBPr265 TAT GTA GAT GAT CTG GTΛ
HBPr266 TΛT GTT GΛT GAT CTG GTA
HBPr267 T ATG GGA GTG GGC CTC ΛG
HBPr26B T ATG GGA TTG GGC CTC ΛG
HBPr?69 C ΛGT CCG TIT CTC TIG GC
EXAMPLES
Example 1. HBV DNA preparation and PCR amplification
Serum samples were collected from HBsAg-positive individuals and stored at minus 20°C until use in 0.5 ml aliquots. To prepare the viral genome, 1 8 μl serum was mixed with 2 μ\ 1 N NaOH and incubated at 37°C for 60 minutes. The denaturation was stopped and neutralized by adding 20 μ\ of 0.1 N HCI. After a 1 5 minutes centrifugation step, the supernatant was collected and the pellet discarded. PCR was carried out on this lysate as follows: 32 μ\ H 2 O was mixed with 5 μ\ of 10x PCR buffer, 1 /I 10 mM dXTPs, 1 μ\ of each biotinylated primer ( 10 pmol/ l) , 1 0 μ\ of serum lysate, and 2 U Taq enzyme. The amplification scheme contained 40 cycles of 95 °C 1 min, annealing at 45 °C for 1 min, and extension at 72°C for 1 min. Amplification products were visualized on 3% agarose gel. The outer primer set for preSI has the following sequence: outer sense: HBPr 1 : 5 * -bio-GGGTCACCATATTCTTGGG- 3" outer antisense HBPr 4: 5'-bio-GTTCC(T/G)GAACTGGAGCCACCAG-3'
The outer primer set for preCore has the following sequence: outer sense: HBPr 69: 5'-bio-ACATAAGAGGACTCTTGGAC-3' outer antisense: HBPr 8: 5'-bio-GAAGGAAAGAAGTCAGAAGGC-3' The outer primer set for HBsAg has the following sequence: outer sense: HBPr 1 34: 5'-bio-TGCTGCTATGCCTCATCTTC-3' outer antisense: HBPr 1 35: 5'-bio-CA(G/A)AGACAAAAGAAAATTGG-3' . Samples that were negative in the first round PCR were retested in a nested reaction composed of the following: μ\ H 2 0, 5 μ\ l Ox Taq buffer, 1 μ\ 1 0 mM dXTPs, 1 μ\ of each nested primer (10 pmol/μl), 1 μ\ of the first round PCR product, and 2 U Taq polymerase. The amplification scheme was identical as for the first round PCR. The sequence of the nested primers were as follows, for the preSI region: nested sense HBPr 2: 5'-bio-GAACAAGAGCTACAGCATGGG- 3' nested antisense HBPr 3: 5'-bio-CCACTGCATGGCCTGAGGATG-3' ;
and for the preCore region: nested sense HBPr 70: 5'-bιo-TACTTCAAAGACTGTGTGTTTA-3' nested antisense HBPr 7: 5'-bιo- CTCCACAG(T/A)AGCTCCAAATTC-3' In a second reaction the HBsAg region can be amplified in a similar protocol by using the following primers: HBPr 75: 5'-bιo-CAAGGTATGTTGCCCGTTTGTCC-3' in combination with either HBPr 76: 5'-bιo-CCAAACAGTGGGGGAAAGCCC-3'; or with HBPr 94: 5'-bιo-GGTA(A/T)AAAGGGACTCA(C/A)GATG-3\
Example 2. Preparation of the Line Probe Assays
Probes were designed to cover the universal, genotypic and mutant motifs. In principle only probes that discriminate between one single nucleotide variation were retained. However, for certain polymorphisms at the extreme ends of the probe, cross-reactivity was tolerated. Specificity was reached experimentally for each probe individually after considering the % (G + C), the probe length, the final concentration, and hybridization temperature. Optimized probes were provided enzymatically with a poly-T-tail using the TdT (Pharmacia) in a standard reaction condition. Briefly, 400 pmol probe was incubated at 37 °C in a 30 μ\ reaction mix containing 5.3 mM dTTP, 25 mM Tπs.HCL pH 7.5, 0.1 M sodium cacodylate, 1 M CoCI 2 , 0.1 M DTT and 1 70 U terminal deoxynucleotidyl transferase (Pharmacia) . After one hour incubation, the reaction was stopped and the tailed probes were precipitated and washed with ice-cold ethanol Probes were dissolved in 6x SSC at their respectively specific concentrations and applied as horizontal lines on membrane strips in concentrations between 0.2 and 2.5 pM/ml Biotinylated DNA was applied alongside as positive control (LiPA line 1 ) The oligonudeotides were fixed to the membrane by baking at 80°C for 1 2 hours. The membrane was than sliced into 4 mm strips. The design of this strip is indicated in Figure 2.
Example 3. LiPA test performance
Equal volumes (1 0 μ\ each) of the biotinylated PCR fragment and of the denaturation solution (DS; 400 mM NaOH/1 0 M EDTA) were mixed in test
troughs and incubated at room temperature for 5 minutes. Then, 2 ml of the 37 °C prewarmed hybridization solution (HS, 3x SSC/0.1 % SDS) was added, followed by the addition of one strip per test trough. Hybridisation occured for 1 hour at 50 ± 0.5°C in a closed shaking water bath. The strips were washed twice with 2 ml of stringent wash solution (3x SSC/0.1 % SDS) at room temperature for 20 seconds, and once at 50 °C for 30 minutes. Following this stringent wash, strips were rinsed two times with 2 ml of the Innogenetics standard Rinse Solution (RS). Strips were incubated on a rotating platform with the alkaline phosphatase-labelled streptavidin conjugate, diluted in standard Conjugate Solution for 30 minutes at room temperature (20 to 25 °C). Strips were than washed twice with 2 ml of RS and once with standard Substrate Buffer (SB), and the colour reaction was started by adding BC1P and NBT to the SB. After maximum 30 minutes at room temperature, the colour reaction was stopped by replacing the colour compounds by distilled water. Immediately after drying, the strips were interpreted. Reactivities were considered positive whenever the reactivity was stronger than the reaction on the negative control. Strips can be stored on a dry dark place. The complete procedure described above can also be replaced by the standardized Inno-ϋPA automation device (auto-UPA).
Example 4. Selection of reference material. PCR fragments were prepared, derived from members of the different genotypes, the different preCore wild type and mutant sequences, drug resistant motifs and vaccine escape mutants. The PCR fragments were amplified with primers lacking the biotine group at their 5'-end and cloned into the pretreated EcoRV site of the pGEMT vector (Promega) . Recombinant clones were selected after α-complementation and restriction fragment length analysis, and sequenced with plasmid primers. Other biotinylated fragments were directly sequenced with a dye-terminator protocol (Applied Biosystems) using the amplification primers. Alternatively, nested PCR was carried out with analogs of the primers, in which the biotine group was replaced with the T7- and SP6-primer sequence, respectively. These amplicons were than sequenced with an SP6- and T7-dye-primer procedure.
By doing so, a reference panel of recombinant clones was prepared, which is necessary for optimizing LiPA probes.
Example 5: Genotyping HBV-infected serum samples.
Only after creating a sequence alignment as shown in Figure 1 , it became clear which regions could be useful for HBV genotyping. The preSI region seems to be suitable because of the high degree of variability. Probes were therefore designed to cover most of these variable regions as shown in Table 1 . Only a limited selection of probes was retained because of their specific reaction with the reference panel. The most important ones are indicated as boxed regions in Figure 1 . These selected probes were then applied in a LiPA format indicated in Figure 2, as line number 2 to 14. Some of the probes could be applied together in one line, because of their universal character, while others need to be applied separately. With the selection of probes thus obtained, serum samples collected in different parts of the world (Europe, South-America, Africa, Middle-East) were tested. The upper part of Figure 3 shows the reactivity of a selection of samples on these probes. Genotyping of these samples is straightforward, with samples 2 to 8 belonging to genotype A, samples 9 and 1 0 belonging to genotype B, samples 1 1 and 1 2 belonging to genotype C, samples 1 3 to 1 9 belonging to genotype D, samples 20 to 23 belonging to genotype E, and sample 24 belonging to genotype F.
Genotyping can also be performed in the HBsAg region. Again, probes were designed to cover most of the variable regions shown in Fig. 1 . Only a limited selection of probes were retained. These probes are boxed in Fig.1 and are listed in Figure 4. A LiPA strip was prepared carrying these probes and samples belonging to the different genotypes were characterized, as shown in Fig. 5.
Example 6. Scanning the preCore region for mutations.
HBeAg expression can be regulated at the transcriptional and translational level. It is postulated that a transcriptional regulation exists due to the presence of a dinucleotide variation in the promoter region of the preCore mRNA. Probes
covering the wild type (e.g. probe HBPr 88) and the mutant (e.g. HBPr 89) motif were selected and their positions are indicated in the alignment shown in Figure 1 , and applied on the LiPA strip as line 1 5 and 1 6 (Figure 2) .
At the translational level, much more mutations might arise, all possibly resulting in abrogation of the HBeAg expression: any mutations at codon 1 (ATG) destroying translation initiation, codon 2 (CAA to TAA), codon 7 (TGC to TGA), codon 1 2 (TGT to TGA) , codon 1 3 in genotype B, C, D, E, F (TCA to TGA or TAA), codon 1 4 (TGT to TGA), codon 1 8 (CAA to TAA), codon 21 (AAG to TAG), codon 23 (TGC to TGA), codon 26 (TGG to TAG or TGA), codon 28 (TGG to TAG or TGA) . However, due to secondary contrain of the encapsidation signal, most of the mutations occur at codon 28 (TGG to TAG). Along with the mutation at codon 28, a second mutation at codon 29 (GGC to GAC) is often observed. In the case of genotype A and again as a consequence of the secondary constrain, stop codon mutations at codon 28 are only likely to occur after selection of a codon 1 5 mutation (CCC to CCT). Hence, correct interpretation of preCore mutations is genotype dependent. In addition to the above mentioned stop codons, a huge amount of different deletion- or insertion-mutations in the preCore open reading frame might give essentially the same result.
In order to develop a sensitive assay to detect the relevant mutations and the hypothetical mutations, a probe scanning procedure was developed. Partially overlapping probes were designed and applied in a LiPA format (Figure 2, line 17 to 27). In this assay format, wild type sequences over the complete preCore region, together with the codon 1 5 variation for genotype A versus non-A genotypes, and the most common mutations at codon 28 (TAG) , at codon 29 ( GAC) and the combination of codon 28 and 29 (TAGGAC) are positively recognized. Absence of reactivity at one of the other probes is always indicative for the presence of a variation. The exact nature of this variation can then be revealed by sequence analysis or with further designed LiPA probes.
Figure 3 shows the reactivity of the selected genotyped samples on the probes for the preCore region. Samples were previously tested for the presence of HBeAg or for anti-HBe. The interpretation of the reactivity on the LiPA probes for
each sample is indicated below each strip. This approach allowed for the simultaneous screening of a sample for preCore mutations and the characterization of the viral genotype.
Figure 6 also shows a panel of samples with mutations in the preCore region, as well as wild type samples. The probes used in this assay are listed in
Figure 4. This assay includes a codon 29 mutant (M4 motif), which was not present in the experiment in Figure 3.
Example 7. Detection of mutants in the HBsAg region.
Vaccine escape mutants have been described. The most commonly found mutant is the variation at codon 145 of HBsAg (G 145R or GGA to AGA). LiPA probes are designed to detect wild type and mutant probes. Genotypic variations are present in the vicinity of codon 145. Therefore, genotype A is covered by probe 77, genotype B by probe 78, genotype C by probe 79, and genotype D/E by probe 80. Hence, in principle, it is possible to genotype and detect the wild type strains of the virus in one single experiment. Mutant target sequences are covered by probe 81 and 82 for genotype A and D, respectively. Probe 83 can be used as a positive control in these experiments. Further detection of mutants in the a determinant region is possible by means of a probe scanning approach. Herefore, probes are designed to cover the wild type sequence of the different genotypes over the HBsAg epitope region and applied in a LiPA format. Again here, absence of staining at one of these probes is indicative for the presence of a mutant strain. The exact nature of this variant is then determined by sequencing analysis.
Example 8. Detection of HBV strains resistant to lamivudine.
Through analogy with HIV and the resistance against the anti-viral compound 3TC (lamivudine or (-)-β-1 -2',3'-dideoxy-3'-thiacytidine), it was predicted that upon treatment of HBV-infected patients with 3TC, viral strains would be selected showing resistance at the YMDD motif in the HB pol gene. The YMDD motif is physically located in the HBsAg region, but is encoded in another reading frame. Hence, this part of the HBV pol region is amplified with th.e primer combination
HBPr 74-HBr 94, but not with the combination HBPr 74-HBr 76. Probes covering the wild type YMDD motif and YVDD mutant motif are indicated in Figure 1 , respectively probes 95 to 1 00 and 101 to 1 03, as well as probes 1 1 5, 1 1 6, 1 27 and 1 32, the latter probes yielding the best results in the LiPA assay. Such an assay was used to determine the presence of mutations in the YMDD motif in serum of a HBV-infected patient during treatment with lamivudine. Fig. 7 shows that in the first phase of the treatment (May 1 995) no mutations were detected. During the treatment, the viral load decreased, reaching a level of approximately 1 0 4 during November and December 1 995, whereafter a breakthrough was observed, resulting in a level as high as during the first months of the treatment by
June 1 996 . interestingly, a LiPA assay performed in February 1 996 indicated that the majority of virus present, possessed a mutation in the YMDD motif, which had changed to YVDD. in June 1 996, no more wild type motif, but only mutant YVDD could be detected. With this assay, resistant HBV strains can thus easily be detected. Furthermore, the combined detection of the YMDD motif and preCore mutants might be clinically important in prediction and prognosis of further treatment.
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