JP2003505021A - Detection of anti-hepatitis B drug resistance - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for treating a patient by genetically detecting at least one of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of the HBV strain present in the patient's biological sample. The present invention provides a method for monitoring anti-HBV drug resistance in a plant. The present invention provides a novel HBV DNA polymerase sequence that is used in the design of new probes that allow very specific and sensitive detection of anti-HBV drug resistance.

Description

Detailed Description of the Invention

The present invention relates to the field of hepatitis B virus (HBV) diagnosis. More specifically,
The present invention relates to the field of genetic monitoring of anti-HBV drug resistance during HBV treatment.

BACKGROUND OF THE INVENTION Hepatitis B virus is a small, enveloped DN of about 3200 bp in length.
A virus, which belongs to the Hepadnaviridae family and is characterized by significant liver tropism and species specificity. The HBV genome has the complex property of having a partially double-stranded DNA structure with overlapping open reading frames encoding the surface, core, polymerase and X genes. HBV is RNA
It replicates via intermediates and uses reverse transcription in its replication strategy (Summers and Maso
n, 1982). HBV causes serious medical problems such as chronic liver disease and hepatocellular carcinoma (Schroder and Zentgraf, 1990).

In patients with chronic hepatitis B virus (HBV) infection with active wild-type virus replication, successful antiviral therapy is the clearance of HBV-DNA from circulating blood,
It is characterized by subsequent clearance of hepatitis B e antigen (HBeAg) and seroconversion to anti-HBe antibodies. Unfortunately, HBeAg seroconversion does not always occur after HBV-DNA loss, suggesting that quantitative HBeAg measurements are predictive of antiviral treatment outcomes (Heijtink et al., 1997).

Currently, interferon-alpha (IFNα) and lamivudine (3TC)
Are two approved drugs for the treatment of chronic hepatitis B. TFN-α has antiviral and immune modulating properties. Lamivudine (3TC) is 2 '
, Is a (-) enantiomer of 3'thiacytidine, which is a 3'-dideoxynucleoside analogue, and is known to be a potent HBV replication inhibitor by inhibiting the reverse transcription (RT) activity of HBV polymerase. Treatment with lamivudine can cause histological improvement in patients with chronic hepatitis and can prevent transplant rejection if given before or after liver transplantation (Honkoo
p et al., 1995; Naoumov et al., 1995). Both drugs can be administered in monotherapy or in combination therapy (Lai et al., 1998; Mutimer et al., 1998).
). Another compound with a direct antiviral effect, famciclovir (9-
[4-Hydroxy-3-hydroxymethyl-but-1-yl] guanine) is currently in Phase III clinical trials. Other antiviral compounds Robucavir (Colaci
no and Staschke, 1998) and adefovir (Heathcote et al., 1998) have been shown to be safe and effectively inhibit viral replication, but are still in the clinical phase. Penciclovir has been shown to inhibit the reverse transcription activity of HBV polymerase (Shaw et al., 1996).

However, recurrence of hepatitis is observed in some patients during the post-treatment period. ALT rises and HBV DNA becomes detectable again. This HBV DNA
Rebound is associated with a new quasi species equilibrium. Several independent studies have reported famciclovir and lamivudine resistant H
The occurrence of BV strains has been shown (review in Bartholmeusz et al., 1998). The precise nature of this resistance has been attributed to the accumulation of mutations in the RT portion of the polymerase. Treatment schedules with antiviral compounds such as lamivudine and famciclovir caused accumulation of various mutations in HBV polymerase. By sequence analysis, HBV polymerase tyrosine-methionine-aspartate-
The existence of a specific mutation in the aspartic acid (YMDD) motif was revealed, and the mutation replaced methionine with isoleucine or valine (Linge
t al., 1996; Tipples et al., 1996). A similar mechanism has been found in HIV RT polymerase and treatment with lamivudine accumulates mutations in the YMDD motif (Gao et al., 1993). Another important mutation site is located 24 amino acids upstream from the YMDD motif, with leucine (L) replaced by methionine (Locarnini et al. WO 98/21317). The V555I (X199 in HBsAg) mutation may be clinically useful. Pichoud et al. (1999) showed that the V555I mutant has reduced replication capacity, does not produce HBsAg, is resistant to penciclovir, but is sensitive to lamivudine. It is possible to summarize the mutations and viral consequences previously observed with treatment with lamivudine or famciclovir (Ling et al., 1996; Tipples et al., 1996; H
onkoop et al., 1997; Bartholomuesz et al., 1998; Buri et al., 1998; Chay
ama et al., 1998; Melagari et al., 1998; Mutimer et al., 1998; Niesters
et al., 1998; Pillay et al., 1998; Hunt et al., 2000). Mutations in HBV polymerase have also been detected by treatment with penciclovir and other anti-HBV agents.

[0006] It is generally believed that these mutations are responsible for viral unresponsiveness and treatment failure, and detection of these mutations is clinically relevant for monitoring anti-HBV drug resistance during HBV treatment. Is important to. Therefore, there is a need for a rapid, reliable and highly specific method for detecting these mutations. This will allow for rapid and accurate monitoring of HBV drug resistance, resulting in faster and more effective anti-H
Can design BV treatment strategies.

OBJECT OF THE INVENTION It is an object of the present invention to provide a rapid, reliable and accurate method for monitoring anti-HBV drug resistance in a patient. It is another object of the invention to provide a rapid, reliable and accurate method for monitoring anti-HBV drug resistance in patients undergoing anti-HBV therapy. Mutations L528M, M552V / I and // in the DNA polymerase of the HBV strain
Or it is another object of the present invention to provide a rapid, reliable and accurate method for genetically detecting at least one of V / L / M555I. DNA of HBV strain present in biological samples of patients undergoing anti-HBV therapy
Mutations L528M, M552 V / I and / or V / L / M5 in the polymerase
It is another object of the invention to provide a rapid, reliable and accurate method for genetically detecting at least one of 55I. At least the mutations L528M and M552 in the DNA polymerase of the HBV strain
It is another object of the invention to provide a fast, reliable and accurate method for simultaneous detection of V / I. At least the mutations L528M and V / L / in the DNA polymerase of the HBV strain
It is another object of the invention to provide a rapid, reliable and accurate method for simultaneous detection of M555I. At least the mutations M552V / I and V / in the DNA polymerase of the HBV strain
It is another object of the present invention to provide a rapid, reliable and accurate method for simultaneous detection of L / M555I. At least the mutations L528M, M552V / in the DNA polymerase of the HBV strain
It is another object of the invention to provide a rapid, reliable and accurate method for simultaneous detection of I and V / L / M555I. It is another object of the present invention to provide a rapid, reliable and accurate method for monitoring lamivudine resistance in a patient. It is another object of the present invention to provide a fast, reliable and accurate method for monitoring famciclovir resistance in a patient. It is another object of the invention to provide a rapid, reliable and accurate method for monitoring penciclovir resistance in a patient. It is another object of the invention to provide one or more probes for use in the above method. It is another aspect of the present invention to provide a composition comprising at least one of the above probes.
One purpose. It is an object of the present invention to provide a diagnostic kit for monitoring anti-HBV drug resistance in a patient. It is another object of the invention to provide a diagnostic kit for monitoring anti-HBV drug resistance in patients undergoing anti-HBV therapy. Mutations L528M, M552V / I and // in the DNA polymerase of the HBV strain
Alternatively, it is another object of the present invention to provide a diagnostic kit for genetically detecting at least one of V / L / M555I. DNA of HBV strain present in biological samples of patients undergoing anti-HBV therapy
Mutations L528M, M552 V / I and / or V / L / M5 in the polymerase
It is another object of the invention to provide a diagnostic kit for genetically detecting at least one of 55I. At least the mutations L528A and M552 in the DNA polymerase of the HBV strain
It is another object of the invention to provide a diagnostic kit for the simultaneous detection of V / I. At least the mutations L528A and V / L / in the DNA polymerase of the HBV strain
It is another aspect of the present invention to provide a diagnostic kit for the simultaneous detection of M555I.
One purpose. At least the mutations M552V / I and V / in the DNA polymerase of the HBV strain
It is another object of the invention to provide a diagnostic kit for the simultaneous detection of L / M555I. At least the mutations L528M, M552V / in the DNA polymerase of the HBV strain
It is another object of the invention to provide a diagnostic kit for the simultaneous detection of I and V / L / M555I. It is another object of the invention to provide a diagnostic kit for monitoring lamivudine resistance in a patient. It is another object of the invention to provide a diagnostic kit for monitoring famciclovir resistance in a patient. It is another object of the invention to provide a diagnostic kit for monitoring penciclovir resistance in a patient. It is an object of the present invention to provide a Line Probe Assay for monitoring anti-HBV drug resistance in a patient. It is another object of the invention to provide a line probe assay for monitoring anti-HBV drug resistance in patients undergoing anti-HBV therapy. Mutations L528M, M552V / I and // in the DNA polymerase of the HBV strain
Or it is another object of the invention to provide a line probe assay for genetically detecting at least one of V / L / M555I. DNA of HBV strain present in biological samples of patients undergoing anti-HBV therapy
Mutations L528M, M552 V / I and / or V / L / M5 in the polymerase
It is another object of the invention to provide a line probe assay for genetically detecting at least one of 55I. At least the mutations L528M and M552 in the DNA polymerase of the HBV strain
It is another object of the invention to provide a line probe assay for the simultaneous detection of V / I. At least the mutations L528M and V / L / in the DNA polymerase of the HBV strain
It is another object of the invention to provide a line probe assay for simultaneous detection of M555I. At least the mutations M552V / I and V / in the DNA polymerase of the HBV strain
It is another object of the invention to provide a line probe assay for the simultaneous detection of L / M555I. At least the mutations L528M, M552V / in the DNA polymerase of the HBV strain
It is another object of the invention to provide a line probe assay for the simultaneous detection of I and V / L / M555I. It is another object of the invention to provide a line probe assay for monitoring lamivudine resistance in a patient. It is another object of the invention to provide a line probe assay for monitoring famciclovir resistance in a patient. It is another object of the invention to provide a line probe assay for monitoring penciclovir resistance in a patient. It is an object of the present invention to provide a new HBV DNA polymerase gene sequence.

[0008] table

[Table 1]

[0009]

[Table 2]

[0010]

[Table 3]

[0011]

[Table 4]

[0012]

[Table 5]

[0013]

[Table 6]

[0014]

[Table 7]

[0015]

[Table 8]

[0016]

[Table 9]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the inheritance of at least one of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of an HBV strain present in a biological sample of a patient. Of an anti-HBV drug resistance in a patient by biological detection by the following steps: (i) releasing, isolating and isolating polynucleic acid present in the biological sample, if necessary. (Ii) amplifying the HBV DNA polymerase gene or part thereof in the biological sample, if necessary using at least one suitable primer pair; (iii) step (i) and / Alternatively, the polynucleic acid obtained in (ii) is added to HBV
Hybridizing with at least one probe capable of specifically hybridizing with a target sequence in the DNA polymerase gene or its complement, said target sequence being selected from the target sequences shown in FIG. 1; (iv) step Detecting the hybrid formed in (III); (v) L52 in the DNA polymerase of the HBV strain present in the biological sample.
Inferring from the hybridization signal obtained in step (iv) for the presence or absence of the 8M, M552V / I and / or V / L / M555I mutations and possible anti-HBV drug resistance.

The above method involves genetically detecting a mutation in at least one of codons 528, 552 and / or 555 of the HBV DNA polymerase gene to allow HBV to be sensitive or resistant to a particular anti-HBV agent. Allows you to decide whether to have Isolation of many new HBV DNA polymerase gene sequences has allowed the inventor to obtain a reference panel of target sequences, on the basis of which a very specific and very sensitive for detecting the above mutations. A hybridization assay could be developed. In the present invention, the codon numbering of the HBV polymerase gene is adopted from that of genotype A. Codon positions 528, 552 and 55 in other genotypes
The numbers corresponding to 5 are summarized in Table 1. The mutation L528M means that at codon 528 of the HBV DNA polymerase gene, the genetic codon for leucine is replaced by the genetic codon for methionine. The mutation M552V / I is HBV DNA
At codon 552 of the polymerase gene, it means that the genetic codon for methionine has been replaced by the genetic codon for valine or isoleucine. The mutation V / L / M555I means that at codon 555 of the HBV DNA polymerase gene, the genetic codon for valine, leucine or methionine is replaced by the genetic codon for isoleucine. The term "genetic detection of mutations" according to the present invention means that a mutation in an amino acid sequence is detected by determining the corresponding nucleic acid sequence. In the method of the invention, nucleic acids present in a biological sample of a patient are hybridized with one or more probes capable of specifically hybridizing with a target sequence in the HBV DNA polymerase gene shown in FIG. To allow codons 528, 552 and / or 5 of HBV DNA polymerase.
A mutation at 55 is detected. The term "specifically hybridize" refers to
Under the experimental conditions used, the probe forms a double strand with a part of the target sequence or the entire target sequence, and under these conditions, the probe contains another polynucleic acid present in the sample to be analyzed. Means that it does not form a double strand. The "target sequence" of the probe of the present invention is a sequence in the HBV DNA polymerase gene containing a mutation of the codon encoding amino acids 528, 552 and / or 555 of HBV DNA polymerase or a wild-type nucleic acid sequence, for which the probe Are completely complementary or partially complementary (ie
Up to 20%, more preferably up to 15%, even more preferably up to 10%, or most preferably up to 5% mismatch). It should be understood that the complement of the target sequence is also a suitable sequence in some cases. The target sequences shown in Figure 1 were obtained from serum and plasma samples of various patients in a follow-up study, which have not yet been disclosed. H, which also forms part of the invention
By using these novel polymorphic nucleotide sequences of the BV DNA polymerase gene, it becomes possible to design new probes that allow very specific and sensitive detection of anti-HBV drug resistance. A probe designed to specifically hybridize to a target sequence of nucleic acid may be one that matches, or may overlap to a large extent, the target sequence (ie,
It may form a double strand outside the target sequence as well as with the nucleotides in the target sequence). The term "probe" refers to a single-stranded, sequence-specific oligonucleotide having a sequence complementary to the target sequence of HBV DNA polymerase. Preferably, the probe is about 5 to 50 nucleotides in length, more preferably about 10 to 25.
It is the length of the nucleotide. Particularly preferred probe lengths are 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides used in the probes of the present invention may be modified nucleotides such as ribonucleotides, deoxyribonucleotides and inosine, or nucleotides containing modifying groups that do not essentially change the hybridization properties.

In a particular embodiment, the probe used in the method of the present invention is selected from Tables 2, 3 and / or 4, wherein the probe specifically hybridizing to the L528M target sequence is from the list below. Selected: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461,
HBPr468, HBPr468-1 (SEQ ID NO: 1 to SEQ ID NO 10) Probes that specifically hybridize with M552V / I are selected from the following list: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318. , HBPr426, HBPr427,
HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433, HBPr433-1, HBPr4
65, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487, HBPr488 (SEQ I
D NO 11 to SEQ ID NO 29, SEQ ID NO 97 to SEQ ID NO 100) Probes that specifically hybridize with V / L / M555I are selected from the following list: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385,
HBPr289, HBPr299, HBPr419, HBPr 490, HBPr 491, HBPr 492, HBPr 494, HBPr
495, HBPr 496 (SEQ ID NO 30 to SEQ ID NO 40, SEQ ID 101 to SEQ
Up to ID NO 106). At least one probe, preferably at least two probes, more preferably at least 3,4,5,6,7,8,9,10,11,12,13,14
, 15, 16, 17, 18, 19 or more codons 528, 552 and / or 55 by hybridization with oligonucleotide probes.
The mutation in 5 is detected.

In a preferred embodiment of the invention, the invention is such that the probes hybridize simultaneously with their target region under the same hybridization and wash conditions (eg LiPA format, see below). It relates to the above method, which is further characterized in that it is optimized and that many polymorphic regions can be detected simultaneously. More particularly, the invention relates to the above method, further characterized in that the probe used in step (iii) is at least one of the following probe combinations: For detecting the L528M mutation: Probes HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, H
BPr461 and HBPr468-1 (SEQ ID NO 1 to SEQ ID NO 10) To detect M552V / I: probe HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, H
BPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1, HBPr46
5, HBPr456, HBPr380 and HBPr453 (SEQ ID NO 11 to SEQ ID NO 29)
To detect the V / L / M555I mutation: probes HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, H
BPr385, HBPr289, HBPr299 and HBPr419 (SEQ ID NO 30 to SEQ ID NO 40
Until).

The invention also relates to the oligonucleotides used as probes for carrying out the above method. In a preferred embodiment, the invention relates to the oligonucleotides shown in Tables 2, 3 and 4. In the present specification, the probe sequence is defined as a single-stranded DNA oligonucleotide, and 5
Shown from the'end to the 3'end. Any of the following probes can be used as a probe, or their complementary form or their RNA form (where T is U
It is clear to a person skilled in the art that even (a. Since the present invention requires the detection of a mismatch of only one base pair, there is a need for stringent conditions for hybridization of the probe that allow only hybridization of exactly complementary sequences. However, it should be noted that the deviation of the probe sequence from the target sequence in the distal direction of the probe is possible when using longer probes, since the central part of the probe is essential for its hybridization properties. If other hybridization conditions are preferred, the probe may be modified accordingly by adding or deleting one or more nucleotides at their ends. It should be understood that these collateral modifications should produce the same result, ie the probes should also specifically hybridize to their type specific target sequences. Such modifications may be necessary if the amplification material is RNA and not DNA, as is the case with NASBA. However, one should always experimentally evaluate the above changes and mutations that can be envisioned by one of ordinary skill in the art in order to check whether they will give hybridization properties equivalent to the exactly complementary probe. is there. The following useful guidelines known to those skilled in the art can be applied to design probes with desired properties. Since the extent and specificity of a hybridization reaction as described herein is influenced by many factors, manipulating one or more of those factors can affect the precise sensitivity and specificity of individual probes. One would determine sex and see if it is perfectly complementary to its target.

The importance and effect of various assay conditions are further explained: The stability of the [probe: target] nucleic acid hybrid should be chosen to suit the assay conditions. This can be done by avoiding long AT-rich sequences, ending the hybrid at C: G base pairs, and designing the probe to have an adaptive Tm. Depending on the length and the GC content, T than the temperature at which the final assay is performed.
Select the start and end points of the probe so that m is about 2-10 ° C higher. The base composition of the probe is important. This is because the G-C base pair has higher thermal stability than the AT base pair due to hydrogen bonding. Thus, hybridizations involving complementary nucleic acids with high GC content are more thermostable.

Conditions such as ionic strength and incubation temperature at which the probe is used should also be considered in designing the probe. It is known that the degree of hybridization increases as the ionic strength of the reaction mixture increases, as well as the thermal stability of the hybrid increases with increasing ionic strength. On the other hand, chemical reagents such as formamide, urea and alcohols that break hydrogen bonds will increase the stringency of hybridization. Destabilization of hydrogen bonds with such reagents can significantly reduce Tm. Generally, optimal hybridization of synthetic oligonucleotide probes about 10 to 50 bases in length occurs at about 5 ° C below the melting temperature of the duplex. Incubation below the optimum temperature allows hybridization of mismatched base sequences, thus reducing specificity.

Probes that hybridize only under conditions of high stringency are desirable. Under conditions of high stringency, only highly complementary nucleic acid hybrids will form. Hybrids that are not sufficiently complementary will not form. Thus, the stringency of the assay conditions determines the amount of complementarity required between the two nucleic acid strands forming a hybrid. The degree of stringency is chosen to maximize the difference in stability between the hybrid formed with the target nucleic acid and the hybrid formed with the non-target nucleic acid. In the case of the present invention, only one base pair change needs to be detected, and very high stringency is required.

The length of the probe sequence is also important. In some cases, there may be several sequences from a particular region that differ in location and length and that have the desired hybridization characteristics. In other cases, only one base difference may result in one sequence being better than another. Although it is possible to hybridize nucleic acids that are not perfectly complementary, the longest length of a perfectly complementary base sequence usually determines hybrid stability. Although oligonucleotide probes having different lengths and base compositions may be used, the preferred oligonucleotide probe of the present invention has a length of about 5 to 50 bases (more preferably 10 to 25 bases) and has a length relative to the target nucleic acid sequence. Sufficient complete complementarity is present in the chain.

Regions in the target DNA or RNA known to form strong internal structures that interfere with hybridization are less preferred. Similarly, probes that are highly self-complementary should also be avoided. As explained above, hybridization is the association of two single stranded complementary nucleic acids to form a hydrogen bonded double strand. It is implicative that if one of the two strands is completely or imperfectly contained in the hybrid, it will be less able to participate in the formation of a new hybrid. If there is sufficient self-complementarity, there may be intramolecular and intermolecular hybrids formed in certain types of probe molecules. Careful probe design can avoid such structures. By designing the probe so that a substantial portion of the sequence of interest is single-stranded, the rate and extent of hybridization can be greatly increased. Computer programs can be used 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 and Methods section of the Examples. Other conditions are, for example, 3X SSC at 50 ° C.
(Sodium citrate saline), 20% deionized FA (formamide). If the specificity and sensitivity of the probe is maintained, then other solutions (SSPE
(Sodium phosphate saline EDTA), TMAC (tetramethylammonium chloride), etc.) and temperature can also be used. If necessary, minor changes in probe length or sequence will be required to maintain the required specificity and sensitivity under the given conditions.

Cloning the recombinant plasmid containing the insert containing the corresponding nucleotide sequence by excising the corresponding nucleotide sequence from the cloned plasmid, if necessary with sufficient nucleases, and then fractionating, eg by molecular weight. The probe of the present invention can be prepared by For example, the probe of the present invention can be chemically synthesized by a conventional phospho-triester method.

The term “biological sample” according to the present invention is a biological material (tissue or liquid) obtained directly from an infected human or after culture (enrichment), which is HBV
Refers to those containing a nucleic acid sequence. Biological materials include, for example, exudates of any kind, bronchial washes, blood, skin tissue, biopsy samples, semen, lymphocytes, blood cultures, colonies, liquid cultures, fecal samples, urine, liver. It may be a cell or the like. More specifically,
"Biological sample" refers to a serum or plasma sample. Nucleic acid is released, concentrated, and / or isolated from a biological sample by any method known in the art. Currently Qi for isolation of nucleic acids from blood samples
QIAamp Blood Kit and'High pur commercially available from agen (Hilden, Germany)
e PCR template preparation Kit '(Roche Diagnostics, Brussels, Belgium)
Various commercial kits are available, such as Other well known methods for isolating DNA or RNA from biological samples may be utilized (Maniatis et al., 1989.
). The nucleic acid in the sample to be analyzed may be DNA or RNA, for example genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are also called polynucleic acids. Polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (
LCR; Landgren et al., 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), strand displacement amplification (SDA; Duck, 1990) Or amplification using Qβ replicase (Lomeli
et al., 1989) or by any other suitable method known in the art that allows nucleic acid amplification to amplify the HBV DNA polymerase gene or a portion thereof present in the biological sample. TMA method (Guatelli et al., 199
0) or bDNA method (Sanchez-Pescador et al., 1988; Urdea et al., 1991)
Can also be used in the method of the invention.

The term “primer” refers to a single-stranded oligonucleotide sequence that can serve as a starting point for the synthesis of primer extension products or amplification products complementary to the nucleic acid strands to be copied. The length and sequence of the primers must be such that they allow the synthesis of extension products to be initiated. Preferably, the length of the primer is about 5-50 nucleotides. More preferably, the length of the primer is about 10-30 nucleotides. Most preferably, the length of the primer is about 20-25 nucleotides. Individual length and sequence depends on the DNA required
Or it will depend on the complexity of the RNA target and the conditions under which the primer is used, such as temperature and ionic strength. The expression "primer set" refers to a pair of primers that allows amplification of HBV DNA polymerase or a portion thereof. A primer set always consists of a forward primer (sense primer or 5'primer) and a reverse primer (antisense primer or 3'primer). In a preferred embodiment, the invention provides that at least one primer used in step (ii) is as shown in Table 5 (HBPr134, HBPr135, HBPr135A, HBPr135B, HBPr75, HBPr94).
, HBPr94A, HBPr94B, HBPr94C and / or HBPr105 (SEQ ID NO 41 to SE
Q ID NO 50 up to))). More particularly, the invention relates to the above method, further characterized in that the set of primers consists of the following two primers: HBPr134 as the forward primer and HBPr1 as the reverse primer.
35; and / or HBPr75 as the forward primer and HBPr94 as the reverse primer.
And / or HBPr75 as the forward primer and HBPr10 as the reverse primer;
5. Those skilled in the art will appreciate that these primers (SEQ ID NO 41 to 50) may be modified by adding or deleting one or more nucleotides at the ends. For example, if the amplification conditions are changed, N
Such modifications may be necessary when the amplification material is RNA rather than DNA, such as the ASBA system. The fact that amplification primers do not have to match exactly to the corresponding sequences in the template to ensure correct amplification is well documented in the literature (Kwok et al., 1990). However, it should be considered that the amplified fragments will have the sequences of the primers rather than the sequences of the target sequences if the primers are not perfectly complementary to their target sequences. The primers and / or probes of the present invention may be labeled. Labeling can be done by any method known to those of skill in the art. The nature of the label may be isotopic ( ³² P, ³⁵ S, etc.) or non-isotopic (biotin, digoxigenin, etc.). Oligonucleotides used as primers or probes are phosphorothioates (Matsukura et al., 1987), alkylphosphorothioates (Miller et al.
al., 1979) or peptide nucleic acid (Nielsen et al., 1991; Nielsen et al., 199).
It may contain or consist of a nucleotide analogue such as 3) or it may contain an intercalating agent (Asseline et al., 1984). The introduction of these modifications may be advantageous for positively influencing features such as hybridization reaction properties, reversibility of hybridization, biological stability of the oligonucleotide molecule, and the like. Where most other changes or modifications are introduced in the original DNA sequences of the primers and probes, these changes need to be adapted with respect to the conditions under which the oligonucleotide should be used to obtain the desired specificity and sensitivity. Ah The final result of priming or hybridization with these modified oligonucleotides should be essentially the same as that obtained with unmodified oligonucleotides.

In a preferred embodiment, the present invention provides that the above probes are capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and washing conditions, with more than one wild type codon and / or mutated. It relates to the above method, which is further characterized in that the codons can be detected simultaneously. More specifically, the invention relates to a method further characterized in that both mutations L528M and M552V / I are detected in one step. More particularly, the invention relates to a method further characterized in that both mutations L528M and V / L / M555I are detected in one step. More specifically, the invention relates to a method, further characterized in that both the mutations M552V / I and V / L / M555I are detected in one step. More specifically, the invention relates to three mutations L528M, M552V / I and V /
L / M555I is detected in one step. Mutations at codons 528, 552 and / or 555 that are present prior to initiation of anti-HBV treatment and / or during anti-HBV drug treatment and confer resistance to lamivudine, famciclovir and / or penciclovir using the methods of the invention. Can be screened (ie, monitored for drug treatment).
The invention therefore relates to the above method, further characterized in that the HBV strain present in the biological sample is resistant to lamivudine, famciclovir and / or penciclovir. Although the methods of the invention may be used to determine resistance to anti-HBV agents other than those mentioned above, resistance to these other agents may be linked to one or more of the three mutations detected by this method. Is a condition. The term "anti-HBV agent" refers to an anti-HBV nucleoside analog or any other DNA polymerase inhibitor that causes a reduction in viral DNA in a patient. Other anti-HBV agents include adefovir, BMS200475, foscarneto, fiacitabine,
Fiarlysine, (−)-FTC, ganciclovir, GEM132, interferon, L-FAMU, lobukavir, n-docosanol, ribavirin, soribudine, vidarabine or the compounds described in WO98 / 18818,
Not limited to these. The methods of the invention can also be used in combination with methods for detecting one or more mutations that may confer resistance to other anti-HBV agents. Thus, probes that allow the detection of other mutations can be added to the method of the invention.

The invention also relates to compositions comprising at least one probe of the invention.
The term "composition" of the invention is a mixture of probes in a suitable buffer used to carry out the methods of the invention. The invention also relates to the use of the above probes and compositions for the in vitro monitoring of anti-HBV drug resistance in a patient.

The invention also provides the mutations L528M, M552V / I and / or V / L / M55 in the HBV DNA polymerase of the HBV strain present in the biological sample of the patient.
Anti-HBV in patients by genetically detecting at least one of 5I
It also relates to a diagnostic kit for monitoring drug resistance, which kit comprises the following components: (i) for releasing, isolating or concentrating polynucleic acid present in said biological sample, where appropriate. (Ii) at least one suitable primer pair, if appropriate; (iii) at least one of the above probes optionally immobilized on a solid support; (iv) a hybridization buffer, or Components necessary to obtain a buffer; (v) a washing solution, or components necessary to obtain the solution; (vi) if appropriate, a means for detecting the hybrid obtained by the above hybridization; (Vii) Where appropriate, means for attaching the probe to a known location on the solid support. The term "hybridization buffer" means a buffer that allows a hybridization reaction between a probe and a polynucleic acid or amplification product present in a sample under conditions of appropriate stringency. The term "wash solution" means a solution that allows washing of the hybrids formed under conditions of suitable stringency.

Assay methods that rely on the formation of hybrids between the nucleic acid of the biological sample and the oligonucleotide probe of the invention can be used. For example, hybridization can be performed using Southern blot, Northern blot, or dot blot formats, in which the amplified, unlabeled sample is bound to the membrane and the membrane is subjected to at least one hybridization condition under appropriate hybridization and washing conditions. The labeled probe is incorporated and the presence of bound probe is monitored. An alternative is the "reverse" format, where the amplified sequence contains a label. In this format, selective probes are immobilized at specific locations on the solid support and the amplified polynucleic acid is labeled to allow detection of hybridization. The term "solid support" refers to a substrate to which an oligonucleotide probe can be coupled, provided the oligonucleotide probe retains its hybridization properties and the background level of hybridization remains low. Becomes Usually, the solid substrate is a microtiter plate (eg used in the DEIA method), a membrane (eg nylon or nitrocellulose) or microspheres or chips. Prior to membrane application or immobilization, it may be convenient to modify the nucleic acid to facilitate immobilization or increase hybridization efficiency. Such modifications include homopolymer tailing, aliphatic groups, NH ₂ groups, SH
Group, coupling with differently reactive groups such as carboxyl groups, or coupling with biotin, haptens or proteins.

Therefore, the present invention relates to HBV strains of HBV present in a biological sample of a patient.
Mutations L528M, M552 V / I and / or V / L in DNA polymerase
A line probe assay for the monitoring of antiviral drug resistance in a patient by genetically detecting at least one of / M555I, said assay comprising the following components: (i) where appropriate Means for freeing, isolating or concentrating polynucleic acids present in a patient biological sample; (ii) at least one suitable primer pair, where appropriate; (iii) L528M shown in FIG. Probes that hybridize specifically with target sequences and HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355
, At least one probe selected from HBPr415, HBPr461 and HBPr468, which is immobilized on a solid support; and / or (iv) specifically with the M552V / I target sequence shown in FIG. Hybridizing probes and HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr3
18, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBP
at least one probe selected from r433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487 and HBPr488, which is immobilized on a solid support; and / or (v) Probes specifically hybridizing with the V / L / M555I target sequence shown in FIG. 1 and HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBP
r332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBP
at least one probe selected from r492, HBPr494, HBPr495 and HBPr496, which is immobilized on a solid support; (vi) a hybridization buffer, or a component necessary for obtaining the buffer; (Vii) a wash solution, or components necessary to obtain the solution; (viii) where appropriate, means for detecting the hybrids obtained by the above hybridization. In this embodiment, a selected set of probes is immobilized on a linear membrane strip. The probes may be immobilized in place either individually or as a mixture. The amplified HBV DNA polymerase polynucleic acid or a portion thereof can be labeled with biotin, and the hybrid can then be detected by a non-radioactive chromogenic system with biotin-streptavidin coupling.

The present invention relates to the novel sequences shown in FIG. 1 (SEQ ID NO 51 to 86, as well as SEQ ID NO
107 to 109), or a fragment thereof, wherein said fragment is
As shown in FIG. 1, it consists of at least 10, preferably at least 15, more preferably at least 20 contiguous nucleotides, the fragment being H
It contains one of the codons encoding wild-type or mutated amino acids 528, 552 and / or 555 of BV DNA polymerase. Therefore, the present invention relates to an isolated nucleic acid consisting of or containing the above nucleotide sequence. The term "nucleic acid" refers to a single-stranded or double-stranded nucleic acid sequence, shown in FIG.
It may contain from one nucleotide to the complete nucleotide sequence. The nucleic acid may consist of deoxyribonucleotides, ribonucleotides, nucleotide analogs or modified nucleotides. It will be appreciated that the complements of the above nucleic acids also form part of the present invention.

Throughout this specification and the claims that follow, unless otherwise stated,
The terms "comprise" and variations such as "comprises" and "comprising" are meant to include the stated number or steps or the recited number or groups of steps, and other numbers or steps or numbers. Nor does it exclude a group of steps.

Examples Example 1: Isolation of a new HBV DNA polymerase gene sequence a. Patients Serum or plasma samples were obtained from 41 patients during the follow-up period and 80 patients in a cross sectional study. Patients in the follow-up study were: Dr. F. Zoulim (INSERM, Lyon, France
18 patients in a follow-up study by Dr. D. Pillay (Public Health
Laboratory Service, Birmingham, UK) 5 patients in a follow-up study, Dr. G. Leroux (University Hospital, Gent, Belgium) 3 patients in a follow-up study and D. Lau (NIH, Bethesda, 15 patients in a follow-up study by MD, US). Dr. L. Lau (Schering Plow, Madison
, NJ, US).

B. Purification and amplification of HBV DNA Commercially available "High pure PCR template preparation Kit" (Boehringer-Mannheim,
Brussels, Belgium) from serum or plasma samples to HBV DN
A was isolated. Purified DNA was amplified for the HBV polymerase region using the nested PCR method. 10 μl of purified DNA was added to 5 μl of 10x buffer, 0.4
μl of 10 mM dXTPs, 10 pmol of sense primer, 10 pmol
Antisense primer, 1 unit of Taq polymerase (Stratagene Eur
ope, Amsterdam, The Netherlands) and added 50 μl of HPLC grade H ₂ O. PCR annealing at 45 ° C, extension at 72 ° C, then 9
Denaturation was carried out at 4 ° C. for 30 seconds each. 40 cycles of outer PCR and 35 cycles of nested reaction were performed. The HBV polymerase region was amplified using the following primer combination: HBPr134: 5'-TGCTGCTATGCCTCATCTTC-3 '.
(SEQ ID NO 41); Outer Antisense HBPr135: 5'-CAG / AAGACAAAAGAAAATTGG
-3 '(SEQ ID NO 42); Nested Sense HBPr75: 5'-CAAGGTATGTTGCCCGTTTGTCC
-3 '(SEQ ID NO 45); Nested Antisense HBPr 94: 5'-GG (T / C) A (A / T) AAAG
GGACTCA (C / A) GATG -3 '(SEQ ID NO 46). Nested amplification products were 341 bp long (including primers), analyzed on a 2% agarose gel and visualized with ethidium bromide. For LiPA experiments, a biotin group was added to the 5'end of the primer.

C. Plasmid Cloning and DNA Purification 2 μl of amplification product was digested with EcoRV pGemT vector 1
Mix with μl (Promega, Leiden, The Netherlands) and read “Ready to Go = T4 ligas
ligation was performed by using e "(Pharmacia, Leusden, The Netherlands). After transformation in a competent E. coli strain, a single recombinant clone was selected and labeled with" H "
igh pure PCR template preparation Kit ”(Boehringer-Mannheim, Brussels,
Belgium) or “Qia prep 96 Turbo Bio Robot kit” (Qiagen, Hilden, Germ
Any) was used to purify the plasmid DNA. Inserts from recombinant clones were PCR amplified using either plasmid-derived or nested HBV primers.

D. Reference panel development and probe design A reference panel for probe design was developed at the same time as the probe evaluation. In the first stage, the mutations L528M, M552V / I and / or V /
Several probes specific for the detection of the presence or absence of L / M55I were designed, including GC content, probe length, ionic strength of the hybridization buffer,
And obtained after considering the incubation temperature. These probes were applied to nitrocellulose membranes and then biotinylated P produced from plasma or serum samples.
These specific probes were evaluated by reverse hybridization of CR fragments (in LiPA format), streptavidin-alkaline phosphatase incubation, followed by color development. Details of the probe optimization step, production of LiPA pieces and reverse hybridization are described in Stuyver et al.
1996), Stuyver et al. (1997) and Van Geyt et al. (1998). Analysis of serum and plasma samples using this first stage LiPA strip revealed that
Many of their PCR products did not react with the probes selected in these first steps. Sequence analysis of these non-reactive PCR products revealed new motifs for which corresponding probes were designed. The use of these newly designed probes on LiPA strips reduced the non-reactivity of the sample. Then, in a further analysis of the reactivity of the new probe set to plasma and serum samples, all PCR products that were not reactive with the selected probe set were sequenced and the new motif was used as a reference panel. In addition, a new probe was designed. Finally, a total of 35 selected clones were thus retained as a reference panel and sequenced. Double-stranded sequences were obtained from biotinylated PCR products or, in the case of recombinant clones, by using vector-derived sequencing primers as described in Stuyver et al. (1996). The newly obtained sequences are shown in Figure 1 (SEQ ID NOS: 51 to 86 and SEQ ID NOS: 107 to 109).

Example 2: Design and testing of LiPA for monitoring drug resistance in HBV infected patients a. Design of LiPA for monitoring drug resistance By using the sequence in the reference panel, codon positions 528,552
And 555-specific probes that correspond to both wild-type and mutant motifs and also to different genotypes and polymorphisms were designed and confirmed. Depending on their ability to detect wild-type and mutant codons in HBV DNA polymerase, and according to the important information obtained from overlapping HBsAg reading frames, probes were pooled and applied to the strips. Several probes designed for different nucleotide polymorphisms but not introducing amino acid changes were pooled together and applied on the line. A piece was finally obtained with 19 different probe lines containing a total of 38 specific probes (Fig. 2; Table 6). See Stuyver et al. (1996), Stuyver et al. For details on probe optimization steps and LiPA strip production.
(1997) and Van Geyt et al. (1998).

B. Testing of LiPA with clones from the reference panel Then biotinylated P obtained from the recombinant clones in the reference panel
LiPA pieces were incubated with the CR product. For details of reverse hybridization, see Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt.
et al. (1998). FIG. 3 shows the specific reactivity of some PCR products with probes on one piece. The choice of probe was very specific in detecting the corresponding amplicon. Simultaneous detection at wild type or mutated codons 528, 552 and 555 was possible in a single experiment.

[0044] Example 3: Use of LiPA for the anti-HBV drug resistance in two patients undergoing HBV treat monitoring a. Analysis of follow-up samples of patients performed with HBV LiPA for drug resistance Testing follow-up samples of 2 patients taken before treatment, during treatment, and during treatment but during a period of viral rebound LiPA was used for this purpose.
Evidence of treatment failure was shown by viral load and ALT levels (Figure 4). Patient A had HBV genotype A and patient C had genotype C.
The LiPA reactivity obtained with these follow-up samples is also shown in FIG. Mixed V555I in patient A treated with lamivudine (lines 14 and 19)
Appeared briefly on days 360, 420, 570 and was weakly present on day 630.
As can be deduced from FIG. 2, the reactivity at line 19 is associated with translational termination at HBsAg codon 199. This motif disappeared after the appearance of variants at codons 552 and 528. Also, on day 630, codon 528 (line 1 + 3
) And 552 (lines 7 + 8). The wild type motifs L528 and M552 disappeared at day 750, which is the time of the virus breakthrough. M52 in patient C treated with famciclovir followed by lamivudine
Eight mutants were subsequently observed, followed by the M528 + V552 double mutant. 325
On day one wild type L528 (line 1) co-appeared with mutant M528 (line 3), but 4
From day 07 this wild type motif was no longer detected. Wild type virus (
A transfer from line 7) to mutant virus (line 8) was observed from day 450 at codon 552. From day 542, the pure double mutant M528 + V552 was present. No codon change at codon 555 was observed in patient C. The current selection of probes may be useful in monitoring the emergence of drug resistance during antiviral therapy.

B. Analysis of Clones of Follow-Up Samples From Patient A, a total of 7 plasma samples were available at different time points during the 750 day antiviral treatment period. Amplification products were cloned and 158 recombinant clones were taken for LiPA analysis (Table 7). Codons 528 and 55 between days 1 and 570 in all 99 clones except I555.
There was no evidence for selection of mutations in 2. By day 630, most of the clones already belonged to the group of double mutants M528 + V552. However,
There were few single mutants L / V / V and M / M / V, which were interpreted as the remnants of intermediates leading to double mutation selection. This means that the emergence of resistance within 60 days (
(Between days 570 and 630). In Patient C an additional 72 at 7 different time points during the HBV treatment period.
Individual clones were analyzed. 32 due to treatment schedule with famciclovir
At day 5, the single mutant M528 was present as a major population. Only after the introduction of lamivudine (day 325) was the M528 + V552 mutant detected as a major population (Figure 4 and Table 7). Selection of M552 mutants took 69 days (450
From day to day 519) the odor was maximally produced. Pressure on the single mutant virus M528 produced by lamivudine treatment (from day 325) resulted in rapid selection of double mutants at day 450.

[0046] Example 4: B-type emergence of lamivudine related mutations in hepatitis virus and use of LiPA for monitoring Da Ina mix loss a. Patient History A 52-year-old Caucasian male was first diagnosed with chronic hepatitis B in 1985, at which time abnormal serum aminotransferase (ALT) levels were shown in routine laboratory tests. The patient was asymptomatic but went to a doctor in 1995. There was a slightly elevated ALT level at that time. Serum HBV DNA was 0.7 pg / ml as measured by liquid phase hybridization assay (Abbot Diagnostics, Chicago, IL, USA). The patient had thrombocytopenia (platelet = 64000) with evidence of mild liver dysfunction with reduced serum albumin (3.1 g / dl), slightly prolonged prothrombin time (17.1 seconds)
Had. The patient was monitored for extension for several months while regularly testing for HBV serological characteristics. In December 1995 the patient became very positive for HBV DNA and showed abnormal serum ALT levels (Figure 5). The patient was lamivudine (3TC
) Treatment (150 mg daily) was started (Day 331), which resulted in HBV DNA
Rapidly disappeared and the serum ALT activity was normalized. At 633 days (302 days after the start of 3TC treatment), serum ALT and viral load increased sharply, and HBV D
NA was rediscovered. Lamivudine treatment was discontinued on day 663 and changed to famciclovir (FCV) (500 mg tid). This treatment had no effect on HBV DNA levels and was discontinued on day 779. Three months later (day 856), the patient had accumulated ascites, worsening blood clotting abnormalities, and varicose vein bleeding. The patient was 1997, 8
Enrolled in the list of liver transplants monthly (approximately day 950). While waiting for the transplant, the patient did not receive antiviral treatment, but a spontaneous transient reduction of HBV DNA was seen. When HBV is reactivated (day 1105), 3TC is restarted and HBV D
NA decreased sharply to an undetectable level. Donor livers were transplanted on day 1223, and hepatitis B immunoglobulin (H
BIg) treatment. At day 1259, HBV DNA levels were undetectable by conventional assay (no PCR) and ALT levels were also normalized.

B. DNA Handling HBV Purification and Amplification HBV from 50 μl plasma using the Tri-Reagent LS protocol for DNA
The DNA was extracted. HBV polymerase regions AE using nested PCR
Was amplified. The first round of PCR was performed by mixing the following: 3 μl of purified DNA template, 10 μl of MgCl ₂ containing 10 × buffer, 2 μl of dNTPs.
(120 mM stock), forward direction (HBV-8For 5'-CATCAGGATTCCTAGGACC-3 '; SEQ I
D NO 87) and reverse (HBV-9Rev 5'-ATACTTTCCAATCAATAGGCC-3 '; SEQ ID NO 88)
1 μl of each primer (30 pmol / μl stock), 0.5 μl Taq DNA polymerase, and 100 μl of HPLC grade water. Amplification program: denaturation at 94 ° C (
Forty cycles of annealing (1 minute) at 49.8 ° C. and extension (2 minutes) at 72 ° C. were carried out. The 810 bp product was purified with the Qiagen PCR Purification Kit,
It was eluted in 30 to 50 μl of water. Nested PCR reactions were performed by mixing the following: 5 μl of first round PCR product, 5 μl of 10X Buffer I
I, 3.7 μl MgCl ₂ , 1 μl dNTP (10 mM stock), forward (HBV-2For 5 ′
-CGCTGGATGTGTCTGCGGCG-3 '; SEQ ID NO 89) and reverse (HBV-R3 5'-CCAACTTC
CAATTACATAACCC-3 '; SEQ ID NO 90) Primer (30 pmol / μl stocks) 1 μl each
, 0.5 μl Taq DNA polymerase, and 50 μl HPLC grade water. Amplification program: denaturation at 94 ° C (45 seconds), annealing at 55 ° C (1 minute), 7
35 cycles of extension (2 minutes) at 2 ° C were performed. Final fragment size is 5
It is 33 bp. Sequencing Using the set of fluorescein-labeled primers below, Pharmacia (Upsala, Swe
den) ALF sequencing was performed: HBV-8ALF (5'-CGTTCCACCAAACTCTTCAAG-3 '; SEQ
ID NO 91), HBV-10ALF (5'-CAAGGTATGTTGCCCGTTTGTCCTC-3 '; SEQ ID NO 92), HB
V-12ALF (5'-CCCATCCCATCATCTTGGGC-3 '; SEQ ID NO 93), HBV-14ALF (5'-GGGTAT
GTAATTGGAAGTTGG-3 '; SEQ ID NO 94), HBV-3ALF (5'-GCACTAGTAAACTGAGCCA-3';
SEQ ID NO 95) or HBV-6ALF (5'-AGTCTAGACTCGTGGTGGAC-3 '; SEQ ID NO 96). Standard A. L. F. According to the sequencing protocol, A. L. F. An analysis program was set up to scan for sequence variations at the highest stringency. Analysis of clones pG pretreated with 2 μl of HBPr75-94 amplification product (see Example 1)
ligated into the em plasmid vector (Promega, Leusden, The Netherlands)
Transformed into competent E. coli cells. Select a single recombinant, High
Plasmid DNA was purified using the Pure Plasmid Isolation Kit (Boehringer Mannheim, Brussels, Belgium). The inserts from the recombinant clones were digested with either plasmid-derived primers or nested HBV primers.
CR amplified. All products were analyzed by using LiPA.

C. Changes in HBV DNA Polymerase Drug Resistance Codons 528, 552 and 555 Before, During and After Lamivudine Treatment (331, 338, 345, 633,
Samples selected at 715, 726, 738, 752, 779 and 802) were sequenced in the HBV polymerase region between amino acids (aa) 473 and 561. This region contains domain B (HBV polymerase aa 508 to 530) and domain C (HBV polymerase aa 548 to 558). Compared to the sequence at day 331, changes were observed from day 633, and M
528 and V552 were detected, but from day 779, the wild-type motif L52 was detected.
8 and M552 reappeared (FIG. 5). No other amino acid changes were observed in these follow-up samples. All available samples were analyzed by LiPA (Figures 5 and 6). 524
Lamivudine resistance mutations were detected as complex mixtures on day (L and M at 528, M, V and I at 552). The I552 mutant was not detected on day 548, but all other mixtures remained. Pure mutants were detected from day 633. This corresponded to the increased viral load. By day 835, a mixture of wild type and mutant at both codon positions was detected. Finally, on day 1041, pure wild type virus appeared. 139 days after restarting lamivudine treatment on day 1105, wild type and codon 55
There was a complex mixture of mutant strains in 2 (approximately comparable at day 524). However, because M528 could not be detected, L528 + M552 and +5
The combination of 28 + I552 is the most likely selected combination (
1244 and 1259 days).

D. Viral Evolution during Antiviral Treatment: Analysis of quasispecies In order to better understand the evolution of the virus at codon positions that show nucleotide changes early in the treatment period (Figure 6), another hybridization experiment was performed. went. Therefore, separately pooled LiPA probes (same as previously described in Example 2) were applied individually on one side and incubated with biotinylated amplicons obtained from plasma virus (FIG. 7). There were two striking observations: (i) selection of M528 and V552 variants occurred at intermediate nucleotide sequences but had no effect at the amino acid level; and (ii
The elimination of mutant virus goes through the same intermediate. Regarding codon 528: Wild-type L (TTG) evolves through L (CTG) to eventually give rise to the resistant variant M (ATG). After discontinuing 3TC treatment, M (ATG) to L (CTG) to L (T
A retrograde to TG) was observed. Regarding codon 552: Changes regarding serine (A
An intermediate was observed at codon 550 with the presence of GC to AGT). However, the latter disappeared when V552 (GTG) appeared. Similar to the observations for codon 528 and after withdrawal of 3TC treatment, a retrograde to wild type occurred at codon 552 and the change at codon 550 (AGT) was rediscovered. To further explain these intermediates, the 442, 468 and 524 day HBV amplicons were cloned into a plasmid and a total of 54 clones were further analyzed (FIG. 8). Six clones were detected, codons 528 and 550
There were different combinations of motifs in and 552. L528 (C
Clones with TG) + M552 (AGT at 550) were present as a key population prior to resistance motif selection. Furthermore, two clones with M528 + M552 were found, indicating that this single mutant population was also an intermediate of the double mutant. However, 19 isolated from 524 day amplicons
No single mutant I552 was found in this clone. Despite this anomaly in the cloning results, as seen in Figure 6 (lines 11, 52
On day 4), the single mutant I552 is clearly present in the population.

Example 5: Comparison of data obtained with LiPA to monitor drug resistance with data obtained by sequencing a. Patient Samples Plasma or serum samples were collected at two centers: Research a
nd Molecular Development, Victorian Infectious Diseases Reference Labora
Dr. S. Locarnini of Tory, North Melbourne, Australia (9 patients) and University of Michigan Medical Center, Ann Arbor, MI, USA (9 patients)
Dr. ASF Lok (9 patients). All patients were HBV infected individuals receiving lamivudine treatment. The first sample used should correspond to the "baseline" (time point just before the start of lamivudine treatment). The second and third samples should correspond to time points before and after detectable viral load levels, respectively. Additional samples were then obtained for some patients.

B. DNA Handling HBV isolation and amplification was performed as described above. Standard Method (Sambrook
Sequencing was performed according to et al., 1989). LiPA was used as described in Example 2.

C. Results The results of the LiPA and sequencing analysis for possible mutations at codon 528 are shown in Table 8. For most samples, there was a match between the data obtained by LiPA and the results obtained by sequencing. 44 samples were wild type (L
) Showed the presence of virus and 23 samples showed the presence of mutant (M) virus. In 15 samples LiPA detected a mixture of wild type / mutant (L / M), but by sequencing only the mutant (M) was detected. In one sample, LiPA showed a mixture of wild type / variant (L / M), but sequencing showed only wild type (L). The results of the LiPA and sequencing analysis for possible mutations at codon 552 are shown in Table 9. For most samples, there was a match between the data obtained by LiPA and the results obtained by sequencing. 45 samples are wild type (M
) Showed the presence of virus, 17 samples showed the presence of mutant (V) virus, 8 samples showed the presence of mutant (I) virus. L in one sample
Wild-type / mutant (M / V) mixture was detected by iPA, but only mutant (V) was detected by sequencing. In two samples LiPA detected a wild type / mutant (M / V) mixture, but by sequencing only wild type (M) was detected. Wild type / mutant (M / I) by LiPA in 4 samples
Was detected, but only the mutant (I) was detected by sequencing. Mutant V / I was detected by LiPA in two samples, but only mutant V was detected by sequencing. Analysis by LiPA and sequencing for possible mutations at codon 555 showed the presence of wild type codon V555 in all samples. From this study it is clear that the data obtained by LiPA to monitor drug resistance are consistent with the data obtained by sequencing. further,
More mixed sequences at codons 528 and / or 552 were detected by LiPA, but these mixtures were not detected by sequencing, indicating that LiPA provides even greater sensitivity.

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[Brief description of drawings]

FIG. 1 is a side-by-side comparison of new HBV DNA polymerase sequences from serum samples obtained from patients before and during the HBV treatment described in Example 1. Boxed are target sequences that can be used to design the probe.

FIG. 2 shows the design of HBV drug resistant LiPA strips. Conj.cont is the conjugate control; Amp.cont is the amplification control. The strip contains a total of 19 probe lines with a total of 38 probes. n probes equals the total number of probes on each line. The special probes applied to each line are shown in Table 6. Interpretation: Polym. Or HBsAg is the amino acid or HBsAg of the polymerase applied to this particular probe line.
It is an open reading frame.

FIG. 3 shows the results of HBV drug resistant LiPA strips, showing the reactivity of each independent probe line. Results were obtained using 16 recombinant clones in the reference panel. Reactive lines are indicated by their numbers. The relative position of all lines detected on the line and the codons and amino acids are shown on the right. The interpretation for each codon (cd) is shown below each strip.

FIG. 4 shows monitoring of anti-HBV drug resistance in two HBV infected patients. Left is patient A; right is patient C. The number of days of follow-up is shown on the X-axis.
Interpretation of the reactivity pattern of each strip is shown for three codons (designated cd). The treatment schedule is shown above the graph.

FIG. 5 shows a patient treatment schedule using the viral load and ALT data described in Example 4. This graph shows the follow-up samples analyzed. Below the data is shown the genetic information for codons 528, 552 and 555. Cd is a codon; Seq is data by sequence analysis; LiPA is LiP
a is data obtained by analysis. Amino acids are indicated by the one-letter code. The mixture is shown in lower case. im = leucine + methionine, mvi = methionine + valine + isoleucine, mi = methionine + isoleucine, vm = valine + methionine. Three
TC = lamivudine; LT is liver transplantation; HBlg is hepatitis B immunoglobulin.

FIG. 6 shows a LiPA analysis of the patient shown in Example 4 during the 1259 day follow-up period. The number of sampling days is shown above each strip. The treatment schedule is also shown. 3TC is lamivudine, Fam is famciclovir, and HBlg is hepatitis B immunoglobulin. Conj.cont is the conjugate control; Ampl.cont. Is the HBV amplification control.

FIG. 7 shows a detailed analysis for selected amplicons on individual LiPA probes. The number of follow-up days is shown above each strip. The left panel is the sample taken during the treatment period with lamivudine and the right panel is the sample taken after the treatment with 3TC was stopped. The middle panel is the genetic composition of key sequences applied at the indicated positions. Weak hybridization was also observed with the probe containing TAA (stop codon) at codon 551 at day 524. Codon 552I
And no probe for codon 555 was applied. The control line is not shown.

FIG. 8 is an analysis of HBV polymerase diversity at three time points (X-axis). The number of clones at each time point is shown on the Y axis. The genetic composition of the clones is shown in the legend.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Caroline van Hate             Belgium, Be-9000 Ghent, Korfeny             Earth Gang 59 F-term (reference) 4B024 AA01 AA14 BA10 CA01 CA09                       HA12 HA14                 4B063 QA01 QA07 QA19 QQ02 QQ10                       QQ12 QQ28 QR32 QR55 QR62                       QR82 QS34 QX01

Claims (16)

[Claims] 1. By genetically detecting at least one of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of an HBV strain present in a biological sample of a patient. A method of monitoring anti-HBV drug resistance in a patient, comprising the steps of: (i) liberating, isolating and / or concentrating polynucleic acid present in said biological sample, if necessary; (ii) Amplify the HBV DNA polymerase gene or part thereof in the biological sample, if necessary, with at least one suitable primer pair; (iii) obtained in steps (i) and / or (ii) At least one polynucleic acid capable of specifically hybridizing with a target sequence in the HBV DNA polymerase gene or its complement. Hybridized to a lobe, the target sequence being selected from the target sequences shown in Figure 1; (iv) detecting the hybrid formed in step (III); (v) in the biological sample. L52 in the DNA polymerase of the existing HBV strain
A method comprising inferring from the hybridization signal obtained in step (iv) for the presence or absence of the 8M, M552V / I and / or V / L / M555I mutations and possible anti-HBV drug resistance. 2. The method further characterized in that the probe used in step (iii) is selected from those listed in Tables 1, 2 and / or 3, wherein the listed probe is: Item 1. The probe specifically hybridizing with the L528M target sequence is selected from the following list: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461,
HBPr468, HBPr468-1 (SEQ ID NO 1 to SEQ ID NO 10) Probes that specifically hybridize with M552V / I are selected from the following list: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427. ,
HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433, HBPr433-1, HBPr4
65, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487, HBPr488 (SEQ I
D NO 11 to SEQ ID NO 29, SEQ ID NO 97 to SEQ ID NO 100) Probes that specifically hybridize with V / L / M555I are selected from the following list: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385,
HBPr289, HBPr299, HBPr419, HBPr 490, HBPr 491, HBPr 492, HBPr 494, HBPr
495, HBPr 496 (SEQ ID NO 30 to SEQ ID NO 40, SEQ ID 101 to SEQ
Up to ID NO 106). 3. The method according to claim 1, wherein the probe used in step (iii) is at least one of the following probe combinations.
The method according to any one of 1. to detect the L528M mutation: probes HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, H
BPr461 and HBPr468-1 (SEQ ID NO 1 to SEQ ID NO 10) To detect M552V / I: probe HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, H
BPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1, HBPr46
5, HBPr456, HBPr380 and HBPr453 (SEQ ID NO 11 to SEQ ID NO 29)
To detect the V / L / M555I mutation: probes HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, H
BPr385, HBPr289, HBPr299 and HBPr419 (SEQ ID NO 30 to SEQ ID NO 40
Until). 4. The set of primers used in step (ii) is as follows (Table 5): HBPr134, HBPr135, HBPr135A, HBPr135B, HBPr75, HBPr94, HBPr94A, HBPr94B, HBPr94B,
HBPr94C and / or HBPr105 (SEQ ID NO 41 to SEQ ID NO 50)
The method according to any one of claims 1 to 3, further characterized by being selected from: 5. A primer set comprising the following two primers: HBPr134 as a forward primer and HBPr1 as a reverse primer.
35; and / or HBPr75 as the forward primer and HBPr94 as the reverse primer.
And / or HBPr75 as the forward primer and HBPr10 as the reverse primer;
The method of claim 4, further comprising: 6. The method according to any one of claims 1 to 5, further characterized in that both the L528M and M552V / I mutations are detected in one step. 7. The method according to any one of claims 1 to 5, further characterized in that both L528M and V / L / M555I mutations are detected in one step. 8. The method according to any one of claims 1 to 5, further characterized in that both the M552 V / I and V / L / M555I mutations are detected in one step. 9. L528M, M552 V / I and V / L / M555I 3
The method according to any of claims 1 to 5, further characterized in that one mutation is detected in one step. 10. The method according to claim 1, further characterized in that the HBV strain present in the biological sample exhibits resistance to lamivudine, famciclovir and / or penciclovir. 11. L5 in DNA polymerase monitoring of antiviral drug resistance in a patient and HBV strain DNA polymerase present in a biological sample of said patient.
A probe as defined in any of claims 1 to 10 for use in the genetic detection of 28M, M552V / I and / or V / L / M555I. 12. A composition comprising at least one probe as defined in claim 11. 13. Use of the composition of the probe according to claim 12 for monitoring in vitro antiviral drug resistance in a patient. 14. An HBV strain of HBV DN present in a biological sample of a patient.
Mutations L528M, M552 V / I and / or V / L / M in A polymerase
Anti-H in patients by genetically detecting at least one of 555I
A diagnostic kit for monitoring BV drug resistance, comprising the following components: (i) means, where appropriate, for releasing, isolating or concentrating polynucleic acid present in said biological sample; ii) at least one suitable primer pair, if appropriate; (iii) at least one of the above probes, which may be immobilized on a solid support; (iv) a hybridization buffer, or to obtain said buffer (V) Washing solution, or components necessary to obtain the solution; (vi) If appropriate, means for detecting the hybrid obtained by the above hybridization; (vii) Suitable In some cases, a kit comprising means for attaching the probe to a known location on a solid support. 15. The HBV strain of HBV DN present in a patient biological sample.
Mutations L528M, M552 V / I and / or V / L / M in A polymerase
A line probe assay for monitoring antiviral drug resistance in a patient by genetically detecting at least one of 555I,
The following components: (i) where appropriate, means for releasing, isolating or concentrating polynucleic acids present in a biological sample of a patient; (ii) where appropriate at least one set of suitable (Iii) a probe specifically hybridizing with the L528M target sequence shown in FIG. 1 and HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355.
, At least one probe selected from HBPr415, HBPr461 and HBPr468, which is immobilized on a solid support; and / or (iv) specifically with the M552V / I target sequence shown in FIG. Hybridizing probes and HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr3
18, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBP
at least one probe selected from r433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487 and HBPr488, which is immobilized on a solid support; and / or (v) Probes specifically hybridizing with the V / L / M555I target sequence shown in FIG. 1 and HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBP
r332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBP
at least one probe selected from r492, HBPr494, HBPr495 and HBPr496, which is immobilized on a solid support; (vi) a hybridization buffer, or a component necessary for obtaining the buffer; (Vii) a wash solution, or the components necessary to obtain said solution; (viii) an assay comprising means for detecting the hybrids obtained by the above hybridization, where appropriate. 16. The sequence shown in FIG. 1 (SEQ ID NO 50-86 and SEQ ID NO
107-109), or a fragment thereof, or an isolated nucleic acid which comprises at least 10 contiguous nucleotides as shown in Figure 1 and comprises amino acids 528,552 of HBV DNA polymerase.
Or an isolated nucleic acid comprising one of the codons encoding 555.