KR20010075005A - Detection of anti-hepatitis b drug resistance - Google Patents

Abstract

The present invention genetically detects one or more 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, thereby preventing the patient's anti-HBV drug resistance. It relates to a method for monitoring. The present invention provides novel HBV DNA polymerase sequences to be used for the design of novel probes capable of highly specific and sensitive detection of anti-HBV drug resistance.

Description

Detection of anti-hepatitis drug resistance {DETECTION OF ANTI-HEPATITIS B DRUG RESISTANCE}

Hepatitis B virus is a small, enveloped DNA virus of about 3200 bp in length that belongs to Hepadnaviridae, and is characterized by high liver affinity and species specificity. The HBV genome is of a complex nature with a double-stranded DNA structure with overlapping frameworks encoding the surface, core, polymerase and X gene. HBV replicates using RNA intermediates and reverse transcription in its replication strategy (Summers and Mason, 1982). HBV causes major health problems, such as chronic liver disease and hepatocellular sarcoma (Schroder and Zentgraf, 1990).

In patients with chronic hepatitis B virus (HBV) infection, in which wild-type viruses are actively replicating, successful antiviral therapies include removal of HBV-DNA from the blood circulation, followed by removal of hepatitis B e antigen (HBeAg) and anti-HBe antibodies. Is characterized by a seroconversion of. Unfortunately, the loss of HBV-DNA is not always followed by HBeAg seroconversion, and it has been suggested that quantitative HBeAg measurements may have expectations for the results of antiviral therapy (Heijtink et al., 1997).

Currently, interferon-alpha (IFN∀) and lamivudine (3TC) are two licensed drugs for the treatment of chronic hepatitis B. IFN∀ has antiviral and immunomodulatory properties. Lamivudine (3TC) is the (-) enantiomer of 3 'thiacytidine, a 2' 3'-dideoxynucleoside analog, and is potent in HBV replication through inhibition of reverse transcriptase (RT) activity of HBV polymerase. Known as inhibitors. Lamivudine treatment can induce tissue improvement in chronic hepatitis patients and prevent transplant rejection when administered before and after liver transplantation (Honkoop et al., 1995; Naoumov et al., 1995). Both drugs can be administered alone or in combination therapy (Lai et al., 1998; Mutimer et al., 1998). Another compound with direct antiviral effect, famciclovir (9- [4-hydroxy-3hydroxymethyl-but-1-yl] guanine), is currently in phase III clinical trials. Other antiviral compounds, lobucavir (Colacino and Staschke, 1998) and adefovir (adefovir; Heathcote et al., 1998) have been shown to be safe and effectively inhibit viral replication, but are still in clinical trials. Penciclovir has been shown to inhibit the reverse transcriptase activity of HBV polymerase (Shaw et al., 1996).

However, in some patients, hepatitis triggers are observed during or after treatment, increasing ALT and making HBV DNA detectable again. The HBV DNA repair is associated with equilibrium of new species. Several independent reports have exemplified the development of famciclovir- and lamivudine-resistant HBV strains (as described in Bartholmeusz et al., 1998). The exact nature of this resistance was due to the accumulation of mutations in the RT portion of the polymerase. Treatment schedules of antiviral compounds such as lamivudine and famcyclovir result in the accumulation of various mutations in the HBV polymerase. Sequence analysis revealed the emergence of specific mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV polymerase gene, in which methionine was replaced with isoleucine or valine (Ling et al., 1996; Tipples et al., 1996). In lamivudine treatment, a similar mechanism in HIV RT polymerase was also found in which mutations accumulate in the YMDD motif (Gao et al., 1993). Another important mutation site is located 24 amino acids upstream from the YMDD motif, where methionine (M) is substituted with leucine (L) (WO 98/21317 to Locarnini et al.). V555I (X199 in HBsAg) mutations may also be clinically relevant. Pichoud et al. (1999) showed that the V555I variant had reduced replication ability, did not produce HBsAg, was resistant to fencyclovir, but susceptible to lamivudine. The results of the schematic and viral adaptation of mutations observed to date after lamivudine or famciclovir treatment can now be found (Ling et al., 1996; Tripples et al., 1996; Honkoop et al., 1997; Bartholomeusz et al) , 1998; Buri et al., 1998; Chayama et al., 1998; Melagari et al., 1998; Multimer et al., 1998; Niester et al., 1998; Pillay et al., 1998; Hunt et al. , 2000). Mutations in HBV polymerase are also detected when treated with fencyclovir and other anti-HBV drugs.

Since the mutations are generally considered to be the cause of non-reactive and therapeutic failure of the virus, the detection of these mutations is clinically important for the monitoring of anti-HBV drug resistance during HBV therapy. Thus, there is a need for a rapid, reliable and very specific method for the detection of such mutations. This will enable fast and accurate monitoring of HBV drug resistance and provide a faster and more efficient anti-HBV treatment strategy.

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

1 is an alignment of novel HBV DNA polymerase sequences from serum samples obtained from patients before and during HBV therapy, as described in Example 1. FIG. Boxed target sequences can be used to design probes.

2 is a design of HBV drug resistant LiPA strips. Conj.cont .: binding control; Amp.cont .: amplification control. The strip includes a total of 19 probe lines with a total of 38 probes; The probe is equal to the total amount of the probe on each line. Specific probes attached to each line are shown in Table 6. Interpretation: Polym. Or HBsAg: Amino acid of the polymerase or HBsAg open reading frame, attached to this particular probe line.

3 is the result of an HBV drug resistant LiPA strip showing the reactivity of each independent probe line. Results were obtained with 16 recombinant clones of the reference panel. Reactive lines are indicated by their numbers. Strips are shown on the right showing the relative positions of all lines and the codons and amino acids detected in that line. The interpretation per codon (cd) is given at the bottom of each strip.

4 shows anti-HBV drug resistance of two HBV infected patients. Left: Patient A, Right: Patient C. Follow-up days are shown on the X-axis. The interpretation of the reactive pattern on each strip is given for three codons (indicated by cd). The treatment schedule is shown at the top of the scheme.

5 is a patient treatment schedule according to viral load and ALT data as described in Example 4. FIG. This graph represents the traced sample analyzed. At the bottom of the data is genetic information for codons 528, 552 and 555. Cd: codon; Seq: data of sequencing; LiPA: data obtained from LiPA analysis. Amino acids are indicated by the Hangul code. The mix is shown in lowercase: lm = leucine + methionine, mvi = methionine + valine + isoleucine, mi = methionine + isoleucine, vm = valine + methionine. 3TC = lamivudine; LT: liver transplantation; HBlg: Hepatitis B Immunoglobulin.

FIG. 6 is a LiPA analysis of follow-up patients over 1259 days as described in Example 4. FIG. Sampling date is shown at the top of each strip. Treatment schedules are also shown: 3TC: lamivudine, Fam: famciclovir, HBlg: hepatitis B immunoglobulin. Conj cont: binding control; Ampl. Cont .: HBV amplification control.

7 Detailed analysis of selected amplicons on individual LiPA probes. Follow-up days are shown at the top of each strip. Left panel: samples taken during lamivudine treatment; Right panel: samples taken after rejection of 3TC therapy; Middle panel: genetic composition of the relevant sequence at the indicated position. At day 524, weak hybridization was also observed on probes containing TAA at codon 551. Probes for codon 552I and codon 555 were not attached. Control lines are not shown.

Figure 8 Analysis of HBV polymerase mutations at three time points (X-axis). The clone count at each time point is shown on the Y-axis. The genetic composition of the clones is shown in the explanatory table.

Detailed description of the invention

The present invention genetically detects one or more 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, thereby preventing the patient's anti-HBV drug resistance. A method of monitoring, comprising the following steps:

(i) if necessary, ejection, separation and / or concentration of the polynucleic acid present in the biological sample;

(ii) if necessary, amplification of the HBV DNA polymerase gene or portion thereof in the biological sample with one or more suitable primer pairs;

(iii) one capable of hybridizing specifically to the target nucleic acid or complementary sequence in the HBV DNA polymerase gene selected from the target sequences shown in FIG. 1 with the polynucleic acid obtained in steps (i) and / or (ii). Hybridization of the probe above;

(iv) detection of the hybrid formed in step (iii);

(v) from the hybridization signal obtained in step (iv), relating to the presence or absence of L528M, M552V / I and / or V / L / M555I mutations in DNA polymerase and to the anti HBV strain present in the biological sample. -Estimation of the possibility of HBV drug resistance.

The method described above determines whether the HBV strain is sensitive or resistant to certain anti-HBV drugs by genetic detection of mutations in one or more of the codons 528, 552, and / or 555 of the HBV DNA polymerase gene. To be able. By separating a number of novel HBV DNA polymerase gene sequences, the inventors were able to develop a reference panel of target sequences, which was the basis for developing a very specific and highly sensitive hybridization assay for the detection of the aforementioned mutations.

In the present application, the codon number of the HBV polymerase gene was taken from genotype A type. A summary of the numbers corresponding to codon positions 528, 552, and 555 in other genotypes is shown in Table 1.

The mutation L528M means that the genetic code of leucine is replaced with the genetic code of methionine at codon 528 of the HBV DNA polymerase gene. The mutation M552V / I means that the genetic code of methionine is replaced with the genetic code of valine or isoleucine at codon 552 of the HBV DNA polymerase gene. The mutation V / L / M555I means that the genetic code of valine, leucine or methionine is replaced with the genetic code of isoleucine at codon 555 of the HBV DNA polymerase gene.

As used herein, the term "genetic detection of mutations" means that mutations in amino acid sequences are detected by determination of the corresponding nucleic acid sequence.

In the method of the present invention, mutations in codons 528, 552 and / or 555 of HBV DNA polymerase are selected from the nucleic acid present in the biological sample of the patient and the target sequence in the HBV DNA polymerase gene as shown in FIG. Detected by hybridization with one or more probes that can specifically hybridize. The term “specifically hybridizes” means that under the experimental conditions used, the probe pairs with some or all of the target sequence, and under such conditions the polynucleic acid is present in the sample to be analyzed. It does not form a pair with other sequences of.

According to the invention, the term "target sequence" of a probe comprises a mutated or wild type nucleic acid sequence of a codon encoding amino acids 528, 552 and / or 555 of an HBV DNA polymerase and the probe By a sequence in the HBV DNA polymerase gene that is fully complementary or partially complementary (ie less than 20%, more preferably 15%, even more preferably 10% or most preferably 5% mismatch). It is to be understood that the complementary sequence of the target sequence is also a suitable target sequence in some cases. The target sequences shown in FIG. 1 were obtained from serum and plasma samples from various patients in follow-up and crossover studies and have not been disclosed previously. The use of the novel polymorphic nucleotide sequence of the HBV DNA polymerase gene, which forms part of the present invention, also allows the design of novel probes that allow highly specific and sensitive detection of anti-HBV drug resistance. . Probes designed to specifically hybridize to a target sequence of a nucleic acid should be understood to be included in the target sequence or may overlap many parts with the target sequence (ie, not only in the target sequence but also outside the nucleotide sequence). Form a double strand).

The term “probe” refers to a single stranded sequence-specific oligonucleotide having a sequence complementary to the target sequence of the HBV DNA polymerase gene. Preferably, the probe is about 5-50 nucleotides in length, more preferably about 10-25 nucleotides. Particularly preferred lengths of the probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. Nucleotides used in the probes of the present invention may be modified nucleotides such as ribonucleotides, deoxynucleotides and inosine, or nucleotides comprising modified groups that do not essentially change their hybridization characteristics.

In specific embodiments, the probes used in the methods of the present invention are selected from Tables 2, 3 and / or 4, as follows:

Probes that specifically hybridize with the L528M target sequence are selected from the following list: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468, HBPr468-1 (SEQ ID NOs: 1 to SEQ ID NO: 10);

Probes that hybridize specifically with M552V / I target sequences are selected from the following list: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr488, HBPr488 HBPr433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487, HBPr488 (SEQ ID NO: 11-SEQ ID NO: 29, SEQ ID NO: 97-SEQ ID NO: 100);

Probes that hybridize specifically with V / L / M555I target sequences are selected from the following list: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr49, HBPr491 HBPr494, HBPr495, HBPr496 (SEQ ID NO: 30 to SEQ ID NO: 40, SEQ ID NO: 101 to SEQ ID NO: 106).

Mutations in codons 528, 552 and / or 555 may comprise one or more probes, preferably two or more probes, more preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Detected by hybridization with 13, 14, 15, 16, 17, 18, 19 or more oligonucleotide probes.

In a preferred embodiment the invention provides that the probes are optimized to hybridize simultaneously to their desired sites under the same hybridization and washing conditions that can simultaneously detect multiple polymorphic sites (see LiPA format, see below). It is further characterized by the method described above.

More specifically, the invention relates to the method described above, further characterized by the probe used in step (iii) being a combination of one or more of the following probes:

Probes for detection of L528M mutation: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461 and HBPr468-1 (SEQ ID NOs: 1 to 10);

Probes for the detection of M552V / I mutations: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1r, HBPr465, HBPr453 (SEQ ID NOs: 11 to 29);

Probes for detection of V / L / M555I mutations: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299 and HBPr419 (SEQ ID NOs: 30 to SEQ ID NO: 40).

The invention also relates to oligonucleotides used as probes to perform any of the methods described above.

In a preferred embodiment, the present invention relates to oligonucleotides as shown in Tables 2, 3 and 4.

Probe sequences are shown throughout the specification in the form of single stranded DNA oligonucleotides at the 5 'to 3' end. It will be apparent to those skilled in the art that any of the probes specified below can be used as such or in their complementary form, or in their RNA form (with T substituted for U).

Since the present application requires the detection of single base pair mismatches, stringent conditions are required for hybridization of the probe, which allows only hybridization of the exact complementary sequence. However, it should be noted that, since the core of the probe is essential for its hybridization characteristics, possible modifications of the probe sequence to the target sequence may be acceptable for the ends of the probe when using long probe sequences. If other hybridization conditions are desired, the probes can therefore be modified by adding or deleting one or more nucleotides at their ends. Incidental modifications above should yield the same result, ie probes still hybridize specifically to their relative type-specific target sequences. Such modifications may also be necessary when the amplified material is RNA rather than DNA, such as in NASBA systems. However, such modifications and variations, conceivable from common knowledge in the art, should always be tested experimentally to ensure that they exhibit exactly the same hybridization characteristics as in the complementary probe.

For the design of probes with the intended characteristics, the following useful guidelines known to those skilled in the art can be applied. Since the degree and specificity of the hybridization reaction as described above is influenced by a number of factors, manipulation of one or more of those factors will determine whether or not the exact sensitivity and specificity of a particular probe is completely complementary to its purpose or not. .

The importance and effects of various assay conditions are further explained below:

[Probe: Purpose] The stability of the nucleic acid hybrid should be selected to suit the analytical conditions. This can be achieved by avoiding long AT-rich sequences, with the end of the hybrid as a G: C base pair, and by devising a probe with the appropriate Tm. The start and end points of the probe should be chosen so that the length and% GC have a Tm about 2-10 ° C higher than the temperature at which the final analysis will be performed. The base composition of the probe is important because G-C base pairs exhibit higher thermodynamic stability compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving higher G-C content complementary nucleic acids will be more stable at higher temperatures.

Conditions such as the ionic strength and reaction temperature at which the probe will be used should also be taken into account when designing the probe. It is known that as the ionic strength of the reaction mixture increases, the degree of hybridization will also increase, and as the ionic strength increases, the thermal stability of the hybrid will also increase. On the other hand, chemical reagents such as formamide, urea, DMSO and alcohol that interfere with hydrogen bonding will increase the stringency of hybridization. Destabilization of hydrogen bonds due to the reagents can greatly reduce Tm. In general, optimal hybridization of synthetic oligonucleotide probes of about 10-50 bases long occurs below about 5 ° C. of the melting temperature of a given pair of strands. Reactions at temperatures below optimum can hybridize mismatched sequences and thus reduce specificity.

It is desirable to have a probe that hybridizes only under conditions of high stringency. Under high stringency conditions only very complementary nucleic acid hybrids will be formed. Hybrids without sufficient complementarity will not be formed. Thus, the stringency of the assay conditions determines the amount of complementarity required between two nucleic acid strands forming a hybrid. The degree of stringency is chosen to maximize the difference in stability between the desired and untargeted nucleic acids and the hybrids formed. In the case of the present invention, since a single base pair change must be detected, this requires very high stringency conditions.

The length of the probe sequence may also be important. In some cases, there may be several sequences of varying positions and lengths from a specific site that will produce a probe with the intended hybridization characteristics. In other cases, only a single base may be much better for one sequence than the other. Nucleic acids that are not completely complementary can hybridize, but usually the longest range of fully complementary base sequences will primarily determine the stability of the hybrid. Although oligonucleotide probes of different lengths and base compositions can be used, preferred oligonucleotide probes of the present invention have a sufficient range within about 5 to 50 (more specifically, 10-25) bases in length and sequences completely complementary to the desired nucleic acid sequence. Have

Sites within the target DNA or RNA, known to form strong internal structures that inhibit hybridization, are less preferred. Likewise, probes with broad self-complementarity should be excluded. As described above, hybridization is a bond in which two single strands of complementary nucleic acids form a hydrogen bonded double strand. If one of the two strands is involved in the hybrid in whole or in part, it means that it is unlikely that it will participate in the formation of a new hybrid. If there is sufficient self-complementarity, intramolecular and intermolecular hybrids can be formed in the molecule of the probe in some form. This structure can be ruled out through careful probe design. 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. It is possible to retrieve this type of interaction with computer programs. However, in certain cases, this type of interaction may not be ruled out.

Standard hybridization and washing conditions are disclosed in the materials and methods part of the examples. Other conditions include, for example, 3 × SSC (sodium citrate saline), 20% desalted FA (formamide) at 50 ° C. Other solutions (SSPE (sodium phosphate saline EDTA), TMAC (tetramethyl ammonium chloride), etc.) and temperatures can also be used if the specificity and sensitivity of the probe is maintained. If necessary, local modification of the probe in length or sequence should be performed to maintain the required specificity and sensitivity under the given circumstances.

Probes according to the invention can be used to clone recombinant plasmids having an insert comprising the corresponding nucleotide sequence, to cut the latter from the cloned plasmids using suitable nucleases, if necessary, and fractions according to molecular weight, for example. By recovery, it can be produced by recovery. Probes according to the invention can also be synthesized chemically, for example by conventional phospho-triester methods.

The term "biological sample" as used herein refers to any biological material (tissue or body fluid) obtained directly from an infected human or after culture (concentration) and containing HBV nucleic acid sequences. Biological substances can be, for example, all kinds of objects, bronchial lavage, blood, skin tissue, biopsies, sperm, lymphocyte blood culture material, colonies, liquid cultures, aliquots, urine, hepatocytes and the like. More specifically, "biological sample" means a sample of blood serum or plasma.

The nucleic acid is ejected from the biological sample, concentrated and / or isolated by any method known in the art. Recently, various commercial kits such as QIAamp blood kits from Qiagen (Hilden, Germany) and 'High pure PCR template preparation kit' (Roche Diagnostics, Brussels, Belgium) for the separation of nucleic acids from blood samples are commercially available. Other known methods can also be used for the isolation of DNA or RNA from biological samples (Maniatis et al., 1989).

The nucleic acid in the sample to be analyzed may be DNA or RNA, ie genomic DNA, messenger RNA, viral RNA or amplified form of the above. The molecules are also called polynucleic acids.

The HBV DNA polymerase gene, or a portion thereof, present in the biological sample may be 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), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA); Duck, 1990) or amplification with Qβ replicaase (Lomeli et al., 1989) or any other suitable method known in the art that can amplify nucleic acid molecules. TMA (Guatelli et al., 1990) or bDNA (Sanchez-Pescador et al., 1988; Urdea et al., 1991) techniques can also be used in the methods of the present invention.

The term "primer" refers to a single stranded oligonucleotide sequence that can serve as a starting point for the synthesis of primer extensions or amplification products complementary to the nucleic acid strand to be copied. The length and sequence of the primers should be such that they allow to facilitate the synthesis of the extended product. Preferably, the primer is about 5-50 nucleotides in length. More preferably, the length of the primer is about 10-30 nucleotides. Most preferably, the length of the primer is about 20-25 nucleotides. Specific lengths and sequences will be based on the complexity of the DNA or RNA purpose required, as well as the conditions under which the primers are used, such as temperature and ionic strength.

The expression "primer set" refers to a primer pair capable of amplifying the HBV DNA polymerase gene or a portion thereof. The primer set always consists of the front primer (sense primer or 5 'primer) and the back primer (antisense primer or 3' primer).

In a preferred embodiment, the present invention provides that at least one primer used in step (ii) is prepared according to Table 5 (HBPr134, HBPr135, HBPr135A, HBPr135B, HBPr75, HBPr94, HBPr94A, HBPr94B, HBPr94C and / or HBPr105; SEQ ID NO: 41-SEQ ID NO: And a method as described above, which is further characterized in that it is selected from. More specifically, the invention also relates to a method as described above, wherein the primer set consists of the following two primers:

HBPr134 as the front primer and HBPr135 as the rear primer; And / or

HBPr75 as the front primer and HBPr94 as the rear primer; And / or

HBPr75 as the front primer and HBPr105 as the back primer.

The skilled person will understand that the primers (SEQ ID NOs: 41-50) can be modified by adding or deleting one or more nucleotides at their ends. For example, such modifications may be necessary if the conditions of amplification change or if the amplified material is RNA instead of DNA, such as for example in a NASBA system. It is described in detail in the literature that amplification primers do not have to exactly match the corresponding target sequences in the template to ensure proper amplification (Kwok et al., 1990). However, it should be considered that if the primers are not completely complementary to their target sequence, the amplified fragments will have the sequence of the primer and not the target sequence.

The primers and / or probes of the invention can be labeled. Labeling can be carried out by any method known to those skilled 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 include phosphorothiate (Matsukura et al., 1987), alkylphosphoroate (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al. , 1993) or may comprise or contain a nucleotide analogue (Asseline et al., 1984). Introduction of such modifications may be advantageous to positively affect properties such as the dynamics of hybridization, the reversibility of hybrid-forming, the biological stability of oligonucleotide molecules, and the like.

Since most other variations or modifications are introduced into the native DNA sequences of the primers and probes, these variations will require modification depending on the conditions in which oligonucleotides should be used to obtain the required specificity and sensitivity. However, the final result of primers or hybridization with the modified oligonucleotides should be essentially identical to that obtained with unmodified oligonucleotides.

In a preferred embodiment, the invention is further characterized in that the probes are capable of simultaneously hybridizing to their respective target sites under suitable hybridization and washing conditions capable of simultaneously detecting one or more wild type codons and / or mutated codons. And a method as described above.

More specifically, the present invention relates to a method as described above, characterized in that both mutations L528M and M552V / I are detected in one step.

More specifically, the invention relates to a method as described above, characterized in that both the mutations L528M and V / L / M555I are detected in one step.

More specifically, the invention relates to a method as described above, characterized in that both mutations M552V / I and V / L / M555I are detected in one step.

More specifically, the present invention relates to a method as described above, wherein three mutations L528M, M552V / I and V / L / M555I are detected in one step.

The methods of the invention are present prior to initiation of anti-HBV therapy and / or appear during the course of anti-HBV drug therapy to confer resistance to lamivudine, famcyclovir and / or fencyclovir to codons 528, 552 and / or 555 It can be used to screen for mutations within. Accordingly, the present invention relates to a method wherein the HBV strain present in the biological sample is further characterized by resistance to lamivudine, famcyclovir and / or phencyclovir. The method of the present invention can also be used to determine resistance to anti-HBV drugs other than the drugs mentioned above when resistance to the other is associated with one or more of the three mutations detected by the method. . The term “anti-HBV drug” refers to any anti-HBV nucleoside analog or any other DNA polymerase inhibitor that induces a reduction in viral DNA in a patient. Other anti-HBV drugs include adefovir, BMS 200475, foscarnet, fiacitabine, fialuridine, (-)-FTC, ganciclivor, GEM 132, interferon, L-FMAU, lobucavir, n-docosanol, ribavirin, sorivudine, vidarabine or compounds mentioned in WO 98/18818 Include, but are not limited to. The methods of the invention can also be used in combination with methods for the detection of one or more other mutations that can confer resistance to other anti-HBV drugs. Thus, probes capable of detecting other mutations can also be added to the methods of the invention.

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

The invention also provides anti-HBV drug resistance in a patient by genetic detection of one or more of the mutations L528M, M552V / I and / or V / L / M555I in the HBV DNA polymerase of the HBV strain present in the patient's biological sample. A diagnostic kit for monitoring, consisting of the following components:

(i) means for ejecting, separating or enriching the polynucleic acid, if appropriate, present in the biological sample;

(ii) where appropriate, one or more suitable primer pairs;

(iii) one or more probes as shown above, which may be attached to a solid support;

(iv) hybridization buffers, or components necessary for the preparation of such buffers;

(v) wash solution, or ingredients necessary for the preparation of said solution;

(vi) means for detecting a hybrid resulting from the preceding hybridization, where appropriate;

(vii) means, if appropriate, to attach the probe to a known position on a solid support.

The term "hybridization buffer" means a buffer that can hybridize between the probe and the polynucleic acid present in the sample or amplified product under appropriate stringent conditions.

The term "washing solution" means a solution capable of washing a hybrid formed under appropriately stringent conditions.

Any assay based on the formation of a hybrid between a nucleic acid of a biological sample and an oligonucleotide probe according to the invention can also be used. For example, hybridization is a Southern blot, northern blot, or dot, in which an unlabeled and amplified specimen is bound to the membrane, attaches one or more labeled probes to the membrane under suitable hybridization and washing conditions, and monitors for the presence of the bound probe. This can be done using a blot format. An alternative is the "inverse" format, in which the amplified sequence contains a label. In this format, the selected probe is fixed at a specific location on the solid support and labeled to enable detection of the hybrid with the amplified polynucleic acid formed. The term "solid support" can refer to any substrate to which an oligonucleotide probe can be bound so long as its hybridization properties are maintained and the basic level of hybridization is kept low. Typically the solid substrate will be a microtiter plate (eg in the DEIA technique), a membrane (eg nylon or nitrocellulose) or microspheres (beads) or chips. It may be convenient to modify the nucleic acid probes to facilitate immobilization or to improve hybridization efficiency before applying or immobilizing on the membrane. Such modifications may include bonding with different reactive groups, such as homopolymer tailings, aliphatic groups, NH ₂ groups, SH groups, carboxylic groups, or with biotin, hapten or protein.

Thus, the present invention provides antiviral drug resistance in a patient to at least one of the mutations L528M, M552V / I and / or V / L / M555I in the HBV DNA polymerase of the HBV strain present in the patient's biological sample. It relates to a line probe assay for monitoring by detection entirely and consists of the following components:

(i) means for ejecting, separating or enriching the polynucleic acid, if appropriate, present in the patient's biological sample;

(ii) where appropriate, one or more suitable primer pairs;

(iii) one or more probes that specifically hybridize to the target sequence of L528M shown in Table 1 selected from HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461 and HBPr468 attached to a solid support; And / or

(iv) HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1, HBPr465r H45r456 H45r456, HBPr456 attached to the solid support. At least one probe selected from HBPr486, HBPr487 and HBPr488 and specifically hybridizes with the target sequence of M552V / I shown in Table 1; And / or

(v) HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBPr492, HBPr4995 and HBPr495, attached to a solid support, are selected from One or more probes that specifically hybridize to a target sequence of V / L / M555I;

(vi) hybridization buffers, or components necessary for the preparation of such buffers;

(vii) a wash solution, or ingredients necessary for the preparation of said solution;

(viii) means for detecting hybrids resulting from the preceding hybridization, where appropriate.

In this embodiment, the selected primer set is fixed to the membrane strip in a line manner. The probes can be fixed in position described 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 with a non-radioactive chromogenic system via biotin-streptavidin binding.

The invention also relates to the novel sequences shown in Table 1 (SEQ ID NOS: 51-86 and SEQ ID NOs: 107 to 109), or fragments thereof, wherein the fragments are 10 or more, preferably 15, as shown in Table 1. , More preferably 20 contiguous nucleotides, wherein said fragment comprises one of the codons encoding wild type or mutated amino acids 528, 552 and / or 555 of the HBV DNA polymerase. Accordingly, the present invention relates to an isolated nucleic acid consisting of or comprising a nucleotide sequence as indicated above. The term "nucleic acid" refers to a single stranded or double stranded nucleic acid sequence, which may comprise from 10 nucleotides to the entire nucleotide sequence as shown in FIG. 1. Nucleic acids can be composed of deoxyribonucleotides, ribonucleotides, nucleotide analogs or modified nucleotides. Complement of the above-mentioned nucleic acid should also be understood to form part of the present invention.

Throughout this specification and claims, changes such as the words “comprise” and “comprises” and “comprising” are described as integers or steps, unless the context otherwise requires. It will be understood that the inclusion of the described integers or groups of steps is not meant to mean the exclusion of other integers or steps or groups of integers or steps.

Purpose of the Invention

It is an object of the present invention to provide a rapid, reliable and accurate method for the monitoring of anti-HBV drug resistance in a patient.

Another object of the present invention is to provide a rapid, reliable and accurate method for the monitoring of anti-HBV drug resistance in patients undergoing anti-HBV treatment.

Another object of the present invention is to provide a rapid, reliable and accurate method for the genetic detection of one or more of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of the HBV strain. .

Another object of the invention is to detect the genetic detection of one or more of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of the HBV strain present in a biological sample of a patient undergoing anti-HBV treatment. Is to provide a quick, reliable and accurate way.

Another object of the present invention is to provide a rapid, reliable and accurate method for the simultaneous detection of at least the mutations L528M and M552V / I in the DNA polymerase of HBV strains.

Another object of the present invention is to provide a rapid, reliable and accurate method for the simultaneous detection of at least the mutations L528M and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a rapid, reliable and accurate method for the simultaneous detection of at least the mutations M552V / I and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a rapid, reliable and accurate method for the simultaneous detection of at least the mutations L528M, M552V / I and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a rapid, reliable and accurate method for the monitoring of lamivudine resistance in a patient.

It is yet another object of the present invention to provide a rapid, reliable and accurate method for the monitoring of famciclovir resistance in a patient.

It is another object of the present invention to provide a rapid, reliable and accurate method for the monitoring of pencyclovir resistance in a patient.

Another object of the present invention is to provide one or more probes for use in the method described above.

Another object of the present invention is to provide a composition containing at least one of the probes described above.

It is an object of the present invention to provide a diagnostic kit for monitoring anti-HBV drug resistance of a patient.

Another object of the present invention is to provide a diagnostic kit for the monitoring of anti-HBV drug resistance in patients undergoing anti-HBV treatment.

Another object of the present invention is to provide a diagnostic kit for the genetic detection of one or more of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the invention is to detect the genetic detection of one or more of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of the HBV strain present in a biological sample of a patient undergoing anti-HBV treatment. It is to provide a diagnostic kit for.

Another object of the present invention is to provide a diagnostic kit for the simultaneous detection of at least the mutations L528M and M552V / I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a diagnostic kit for the simultaneous detection of at least the mutations L528M and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a diagnostic kit for the simultaneous detection of at least the mutations M552V / I and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a diagnostic kit for the simultaneous detection of at least the mutations L528M, M552V / I and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a diagnostic kit for the monitoring of lamivudine resistance in a patient.

Another object of the present invention is to provide a diagnostic kit for the monitoring of famciclovir resistance in a patient.

It is yet another object of the present invention to provide a diagnostic kit for the monitoring of pencyclovir resistance in a patient.

It is an object of the present invention to provide a Line Probe Assay for the monitoring of anti-HBV drug resistance in a patient.

It is another object of the present invention to provide a line probe assay for the monitoring of anti-HBV drug resistance in patients undergoing anti-HBV treatment.

Another object of the present invention is to provide a line probe assay for the genetic detection of one or more of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the invention is to detect the genetic detection of one or more of the mutations L528M, M552V / I and / or V / L / M555I in the DNA polymerase of the HBV strain present in a biological sample of a patient undergoing anti-HBV treatment. To provide a line probe analysis method.

Another object of the present invention is to provide a line probe assay for the simultaneous detection of at least the mutations L528M and M552V / I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a line probe assay for the simultaneous detection of at least the mutations L528M and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a line probe assay for the simultaneous detection of at least the mutations M552V / I and V / L / M555I in the DNA polymerase of the HBV strain.

Another object of the present invention is to provide a line probe assay for the simultaneous detection of at least the mutations L528M, M552V / I and V / L / M555I in the DNA polymerase of the HBV strain.

It is another object of the present invention to provide a line probe assay for the monitoring of lamivudine resistance in a patient.

It is another object of the present invention to provide a line probe assay for the monitoring of famciclovir resistance in a patient.

It is yet another object of the present invention to provide a line probe assay for the monitoring of pencyclovir resistance in a patient.

It is an object of the present invention to provide a novel sequence of the HBV DNA polymerase gene.

Example 1 Isolation of a Novel HBV DNA Polymerase Gene Sequence

a. patient

Serum or plasma samples were collected from 41 patients in follow-up and 80 patients in cross-test. Patients under follow-up are: 18 patients being tracked by Dr. F. Zoulim (INSERM, Lyon, France), 5 patients being followed by Dr. D. Pillay (Public Health Laboratory Service, Birmingham, UK). Three patients with follow-up by Dr. Leroux (University Hospital, Ghent, Belgium) and 15 patients with follow-up by Dr. D. Lau (NIH, Besseda, Mass., USA). The crossover test was performed by Dr. J. Lau (Schering Plow, Madison, NJ).

b. HBV DNA Purification and Amplification

HBV DNA was isolated from serum or plasma samples using a commercially available> high purity PCR template preparation kit (Berlinger Mannheim, Brussels, Belgium). The isolated DNA was amplified for the HBV polymerase site using nested PCR method: 10 μl of purified DNA was added 10 × buffer 5 μl, 10 mM dXTPs 0.4 μl, sense primer 10 pmol, antisense primer 10 pmol, 1 unit of Taq polymerase (Stratagene Europe, Amsterdam, Netherlands) was mixed and 50 μl were made with HPLC-grade H ₂ O. PCR consisted of homologous binding at 45 ° C., polymerization extension at 72 ° C., and denaturation at 94 ° C. for 30 seconds each. External PCR included 40 times and nested reactions included 35 times. HBV polymerase sites were amplified with a combination of the following primers: external sense HBPr134: 5'-TGCTGCTATGCCTCATCTTC-3 '(SEQ ID NO: 41); External antisense HBPr135: 5′-CAG / AAGACAAAAGAAAATTGG-3 ′ (SEQ ID NO: 42); Nested sense HBPr75: 5'-CAAGGTATGTTGCCCGTTTGTCC-3 '(SEQ ID NO: 45); Nested antisense HBPr94: 5'-GG (T / C) A (A / T) AAAGGGACTCA (C / A) GATG-3 '(SEQ ID NO: 46). Nested amplification products were 341 bp in length (including primers), analyzed on 2% agarose gels and visualized with ethidium bromide. For LiPA experiments, primers with biotin groups at their 5 'ends were used.

c. Plasmid Cloning and DNA Purification

2 μl of the amplification product was mixed with 1 μl of the pretreated EcoRV pGemT vector (Promega, Leiden, Netherlands) and ligated with> Ready to Go = T4 ligase (Pharmacia, Leusden, The Netherlands). After transformation into a water-soluble E. coli strain, single recombinant clones were screened to purify the plasmid DNA with a> high purity plasmid separation kit (Beringer Mannheim, Brussels, Belgium) or the 'Qia prep 96 turbo biorobot kit (Qiagen, Hilden, Germany). It was. Inserts of the recombinant clones were PCR-amplified with primers from plasmids or nested HBV primers.

d. Development of the reference panel and design of the probe

A reference panel for probe design was developed simultaneously with the evaluation of the probe. In the first step, specific for detection of the presence or absence of mutations L528M, M552V / I and / or V / L / M555I, taking into account variables of GC percent, probe length, ionic strength of hybridization buffer, and reaction temperature, A few probes were designed and obtained. The specific probes are attached to nitrocellulose membranes, followed by reverse hybridization (in LiPA format) with biotinylated PCR fragments in plasma or serum samples, reaction with streptavidin-alkaline phosphatase, and color development. Was evaluated. For details on probe optimization steps, LiPA strip preparation and reverse hybridization, see Stuyver et al., (1996), Stuyver et al., (1997) and Van Geyt et al. (1998).

In the analysis of serum and plasma samples with the LiPA strip of the first stage, many of those PCR products did not show reactivity to the probes selected in the first stage. Sequencing of the non-reactive PCR products revealed new motifs in which corresponding probes were designed. The newly designed probe on the LiPA strip reduced the non-reactivity of the sample. Plasma and serum samples were then further used to analyze the reactivity with the new probe set, sequencing all PCR-products that were unreactive with the selected probe set, adding new motifs to the reference panel, and devising new probes. Finally, a total of 35 selected clones were obtained and sequenced in this manner as a reference panel. From biotinylated PCR products, or in the case of recombinant clones, see Stuyver et al. (1996) double-stranded sequences were obtained using vector-derived sequencing primers as described. The newly obtained sequences are shown in FIG. 1 (SEQ ID NOS: 51-86 and SEQ ID NOs: 107-109).

Example 2: Design and Testing of LiPA for the Surveillance of Drug-Resistence in HBV-Infected Patients

a. Design of LiPA for Monitoring Drug Tolerance

By using the sequences in the reference panel, probes specific to codon positions 528, 552, and 555, and for different genotypes and polymorphisms, covering both wild type and mutant motifs, were designed and evaluated. Probes were pooled and attached to the strips according to their ability to recognize different wild-type and mutant codons in HBV DNA polymerase, as well as the appropriate information in the overlapping HBsAg reading frame. Several probes designed for different nucleotide polymorphisms but no amino acid changes were put together and attached on one line. This finally produced a strip with 19 different probe lines with a total of 38 specific probes (FIG. 2; Table 6). Details of the probe optimization step and LiPA strip preparation are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).

b. Evaluation of LiPA Using Clones from the Reference Panel

LIPA strips were then obtained from the recombinant clones and reacted with the biotinylated PCR product in a reference panel. Details on inverse hybridization can be found in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998). 3 shows the specific reactivity of some PCR products and probes on the strip. The choice of probe was very specific for the detection of the corresponding amplicons. Simultaneous determination of wild type codons 528, 552 and 555 or mutant codons was possible in one single experiment.

Example 3: Use of LiPA to Monitor Anti-HBV Drug Tolerance in Two Patients Undergoing HBV Therapy

a. Analysis of Patient Follow-up Samples Using HBV LiPA for Drug Tolerance

LiPA was used to test follow-up specimens taken before initiation of therapy, during therapy, and during virus relapse therapy in two patients. Evidence of treatment failure was shown by viral load and ALT level (FIG. 4). Patient A was infected with HBV of genotype A and patient C was infected with HBV of genotype C. The LiPA reactivity obtained in the trace samples is also shown in FIG. 4. In patient A treated with lamivudine, a mix of V555I (lines 14 and 19) was briefly present on days 360, 420, 570 and weak on day 630. As can be inferred from FIG. 2, the reactivity at line 19 was associated with the stop translation of HBsAg codon 199. The motif was lost after the appearance of mutations at codons 552 and 528. On day 630, mixing was also observed on two codons 528 (line 1 + 3) and 552 (line 7 + 8). Wild-type motifs L528 and M552 were lost at day 750, when the virus suddenly increased. In patient C treated with famciclovir followed by lamivudine, sequential selection of the M528 mutation followed by the M528 + V552 double mutation was observed. On day 325 wild type L528 (line 1) coexists with mutant M528 (line 3), but from day 407 the wild type motif was no longer detected. Gradual take over to mutant virus (line 8) against wild type virus (line 7) was observed at codon 552 from day 450. From day 542 onwards, only pure double mutation M528 + V552 was present. In patient C, no codon changes at codon 555 were observed. Currently selected probes have been shown to be useful for monitoring the emergence of drug resistance during antiviral therapy.

b. Clone Analysis of Trace Samples

From patient A, a total of seven plasma samples were obtained at different time points over the 750 day period of antiviral treatment. The amplification products were cloned and 158 recombinant clones were obtained for LiPA analysis (Table 7). Except for I555, there was no evidence of mutation selection at codons 528 and 552 from day 1 to 570 in a total of 99 clones. At day 630, most of the clones were already in the double mutant group of M528 + V552. However, there were some single mutations of L / V / V and M / M / V, which could be interpreted as an intermediate form residue for double-mutant selection. This indicated that the appearance of resistance occurred within the 60 day period (between 570 days and 630 days).

In patient C, another 72 clones were analyzed over 7 different time points during HBV treatment. Due to the treatment schedule of famciclovir, at 325 days a single mutation of M528 was present as a large population. Only after administration of lamivudine (day 325), the M528 + V552 mutation was detected as a majority population (FIG. 4 and Table 7). Selection of the M552 mutation occurred within a period of up to 69 days (between days 450 and 519). The pressure created on the single M528 mutant virus (from day 325) by lamivudine therapy resulted in the rapid selection of the double mutation on day 450. .

Example 4 Use of LiPA for Surveillance and Disappearance Kinetics of Lamivudine-Related Mutations in Hepatitis B Virus

a. Patient history

A 52-year-old Caucasian male was first diagnosed with chronic hepatitis B in 1985 when he was found to have abnormal serum aminotransferase (ALT) levels during routine laboratory tests. The patient remained asymptomatic but visited a doctor since 1995. At that point, he was found to have a slightly increased ALT level. Serum HBV DNA measured by liquid phase hybridization assay (Abbott Diagnostics, Chicago, Ill.) Was 0.7 pg / ml. The patient had evidence of mild interstitial insufficiency manifested by thrombocytopenia (platelet = 64000) and reduced serum albumin (3.1 g / dL) and slightly delayed prothrombin time (17.1 seconds). The patient was closely monitored for several months by regularly checking his HBV serotype profile. In December 1995, he was highly positive for HBV DNA and showed abnormal serum ALT levels (FIG. 5). Lamivudine (3TC) therapy (150 mg per day) was initiated in patients (day 331) to induce rapid loss of HBV DNA and normalization of serum ALT activity. At day 633 (after 302 days of 3 TC therapy), serum ALT and viral load rapidly increased and HBV DNA became detectable again. Lamivudine therapy was discontinued at day 663 and replaced with famciclovir (FCV) (500 mg three times daily). The therapy was discontinued at day 779 because it had no effect on HBV DNA levels. Three months later (on day 856), the patient developed ascites, worsening of coagulopathy and varicose veins bleeding. He was on the list for liver transplantation in August 1997 (about 950 days). While waiting for the transplant, the patient did not receive antiviral therapy but experienced a spontaneous, transient decrease in HBV DNA. When HBV was reactivated (day 1105), 3TC was restarted to quickly reduce HBV DNA to unmeasurable levels. The patient received a donor's liver at day 1223 and started hepatitis B immunoglobulin (HBIg) treatment to prevent reinfection. On day 1259, HBV DNA levels were not measurable by conventional assays (not PCR) and ALT levels were normalized.

b. DNA manipulation

HBV purification and amplification. HBV DNA was extracted from 50 μl of plasma using the LS protocol for the DNA of the Tri-reagent. Nested PCR was used to amplify the HBV polymerase site AE. Primary PCR was formulated with the following: 3 μl purified DNA template, 10 μl of 10 × buffer containing MgCl ₂ , ₂ μl of dNTP (120 mM stock), each anterior (HBV-8For 5′-CATCAGGATTCCTAGGACC-3 ′ SEQ ID NO: 87) and posterior (HBV-9Rev 5'-ATACTTTCCAATCAATAGGCC-3 '; SEQ ID NO: 88) 1 μl of primer (30 pmol / μl stock), 0.5 μl of Taq DNA polymerase and 100 μl with HPLC-grade water Made with. The amplification program consisted of: denaturation at 94 ° C. (45 seconds) for 40 times, homology at 49.8 ° C. (1 min), extension of polymerization at 72 ° C. (2 min). 810 bp of product was purified by Qiagen PCR purification kit and eluted with 30-50 μl of H ₂ O. Nested PCR reactions consisted of 5 μl of primary PCR product, 5 μl of 10 × buffer II, 3.7 μl of MgCl ₂ , 1 μl of dNTP (10 mM stock), each anterior (HBV-2For 5′-CGCTGGATGTGTCTGCGGCG-3 ′; No. 89) and 0.5 μl of Taq DNA polymerase were combined in 1 μl each of the rear (HBV-R3 5′-CCAACTTCCAATTACATAACCC-3 ′; SEQ ID NO: 90) primer (30 pmol / μl stock) and 50 mL with HPLC-grade water. Made to μl. The amplification program consisted of: denaturation at 94 ° C. (45 seconds) for 35 times, homology at 55 ° C. (1 min) and extension of polymerization at 72 ° C. (2 min). Final section size was 533 bp.

Sequencing. Pharmacia (Sweden, Uppsala) ALF sequencing was performed using a set of the following primers labeled with fluorescein: HBV-8ALF (5'-CGTTCCACCAAACTCTTCAAG-3 '; SEQ ID NO: 91), HBV-10ALF (5 '-CAAGGTATGTTGCCCGTTTGTCCTC-3'; SEQ ID NO: 92), HBV-12ALF (5'-CCCATCCCATCATCTTGGGC-3 '; SEQ ID NO: 93), HBV-14ALF (5'-GGGTATGTAATTGGAAGTTGG-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 ALF sequencing protocols were followed and the ALF analysis program was tailored to search for sequence mutations at the highest (corresponding) stringency.

Clone analysis. 2 μl of HBPr75-94 amplification product (see Example 1) was ligated into the pretreated pGem plasmid vector (Promega, Leusden, The Netherlands) and transformed into soluble E. coli cells. A single recombinant was selected to purify the plasmid DNA with a high purity plasmid separation kit (Beringer Mannheim, Brussels, Belgium). Inserts of recombinant clones were PCR amplified with plasmid-derived primers or nested HBV primers. All products were analyzed using LiPA.

c. Variation in HBV DNA Polymerase Drug-Resistant Codons 528, 552, and 555

Before, during and after lamivudine therapy (Days 331, 338, 345, 633, 715, 726, 738, 752, 779 and 802), selected samples were selected for the HBV polymerase site between amino acids (aa) 473-561. Sequencing was performed. This region included domain B (HBV polymerase aa 508-530) and domain C (HBV polymerase aa 548-558). Compared with the 331th sequence, mutations were observed by the detection of M528 and V552 after day 633, but the wild type motifs L528 and M552 were re-expressed from day 779 (FIG. 5). No other amino acid changes were observed in the follow-up sample.

All obtainable samples were also analyzed on LiPA (FIGS. 5 and 6). Lamivudine resistant mutations were detected as complex mixtures as early as day 524 (L and M at 528; M, V and I at 552). The I552 variant could no longer be detected from day 548, but all other mix remained. From day 633, pure mutations were detected. This was consistent with an increase in viral load. By day 835, mixing of wild type and mutant variants at two codon positions was detected. Finally, on day 1041 only pure wild type virus was significant. Lamivudine therapy was re-initiated at day 1105 and after 139 days there was a complex mix of wild type and mutant strains at codon 552 (almost similar to day 524). However, since M528 could not be detected, the L528 + M552 and L528 + I552 combinations were the most selected combinations (day 1244 and 1259).

d. Viral Evolution During Antiviral Therapy: Analysis of Variants

Another hybridization experiment was performed to better understand the evolution of the virus at the codon position (FIG. 6), which initially shows nucleotide changes during therapy. Thus, LiPA probes, which were otherwise collected (as described previously in Example 2), were individually attached onto strips and reacted with amplicons affixed with biotin obtained from plasma virus (FIG. 7). Two significant observations were made: (i) the selection of the M528 and V552 variants took place via intermediate nucleotide sequences that did not affect aa levels; And (ii) the loss of the mutant virus followed the same intermediate form. For codon 528: Wild type L (TTG) evolved to L (CTG) and finally became resistant variant M (ATG). After discontinuation of 3TC therapy, retrograde from L (CTG) to L (TTG) was observed from mutant M (ATG). For codon 552: An intermediate form was observed that changed from codon 550 to serine (from AGC to AGT). However, the material disappeared when V552 (GTG) appeared. Observation for codon 528 followed by regression from codon 552 to wild type, again with transient detection of codon 550 (AGT) mutations, compared to discontinuation of 3TC therapy.

To further detail the intermediate form, HBV amplicons at days 442, 468 and 524 were cloned into the plasmid and a total of 54 individual clones were further analyzed (FIG. 8). Six types of clones with different combinations of motifs were detected in codons 528, 550 and 552. L528 (CTG) + M552 (phosphorus AGT at 550) clones existed as an important group prior to the selection of resistant motifs. In addition, two clones of M528 + M552 were also found, suggesting that this single group of mutations is also an intermediate form for double mutations. However, no single mutation I552 was found in 19 clones isolated from day 524 amplicons. Despite the omission of the mutation in the cloning results, as shown in FIG. 6, the mutation was clearly present in the group (line 11 on day 524).

Example 5 Comparison of Data Obtained by LiPA versus Data Obtained by Sequencing for Monitoring Drug Tolerance

a. Patient history

Plasma or serum samples were collected at two centers: Professor S. Locarnini (Research and Molecular Development, Victorian Infectious Disease Reference Laboratory, North Melbourne, Australia (9 patients)) and Dr. Locarnini Prof. A. S. F. Lok (University of Michigan Medical Center, MI Ann Arbor, USA (9 patients)). All patients were HBV-infected people under lamivudine treatment. The first sample included corresponds to the 'baseline' (time point just before lamivudine treatment was initiated). The second and third samples corresponded before and after the time point at which viral load levels could be remeasured, respectively. Additional sequential samples were also obtained for some patients.

b. DNA manipulation

HBV isolation and amplification was performed as described above. Sequencing was performed by standard methods (Sambrook et al. 1989). LiPA was used as described in Example 2.

c. result

Analysis of the possibility of mutation at codon 528 by LiPA and by sequencing is shown in Table 8. For most samples the data obtained by LiPA versus the data obtained by sequencing were consistent: 44 samples showed the presence of wild type (L) virus and 23 samples showed the presence of mutant (M) virus. It was. In 15 samples, sequencing detected only mutations (M), while LiPA detected a mix of wild type / mutants (L / M). In one sample, sequencing detected only wild type (L), whereas LiPA detected a mix of wild type / mutant (L / M).

Analysis of the possibility of mutation at codon 552 by LiPA and by sequencing is shown in Table 9. For most samples the data obtained by LiPA vs. the data obtained by sequencing were consistent: 45 samples showed the presence of wild type (M) virus and 17 samples showed the presence of mutant (V) virus. , 8 specimens showed the presence of mutant (I) virus. In one sample, sequencing detected only mutations (V), whereas LiPA detected a mix of wild type / mutations (M / V). In two samples, sequencing detected only wild type (M), whereas LiPA detected a mix of wild type / mutant (M / V). In four samples, sequencing detected only mutation (I), while LiPA detected a mix of wild type / mutant (M / I). In two samples, sequencing detected mutation V only, while LiPA detected mutation V / I.

Analysis of the possibility of mutation at codon 555 by LiPA and by sequencing indicated the presence of wild type codon V555 in all samples.

From this test it became clear that the data obtained by LiPA for monitoring drug resistance is consistent with the data obtained by sequencing. In addition, the fact that no mixing was detected by sequencing while a higher number of mixed sequences were detected at codons 528 and / or 552 by LiPA strongly suggests that higher sensitivity is obtained by LiPA.

references

Claims (16)

How to monitor anti-HBV drug resistance of a patient by genetically detecting one or more 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 Thus, the method consists of the following steps: (i) if necessary, ejection, separation and / or concentration of the polynucleic acid present in the biological sample; (ii) if necessary, amplifying the HBV DNA polymerase gene or portion thereof in the biological sample with one or more suitable primer pairs; (iii) is selected from the polynucleic acid obtained in steps (i) and / or (ii) with the target sequences shown in FIG. 1 and specifically hybridized with the target sequence within the HBV DNA polymerase gene or its complementary sequence. Hybridization of one or more probes; (iv) detection of the hybrid formed in step (iii); (v) From the hybridization signal obtained in step (iv), the presence or absence of the L528M, M552V / I and / or V / L / M555I mutations in the DNA polymerase and the possibility of the HBV strain present in the biological sample. Estimation for anti-HBV drug resistance. The method of claim 1, wherein the probe used in step (iii) is selected from Tables 1, 2 and / or 3 as follows: Probes that specifically hybridize with the L528M target sequence are selected from the following list: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468, HBPr468-1 (SEQ ID NOs: 1 to SEQ ID NO: 10); Probes that hybridize specifically with M552V / I target sequences are selected from the following list: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr488, HBPr488 HBPr433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487, HBPr488 (SEQ ID NO: 11 to SEQ ID NO: 29 or SEQ ID NO: 97 to SEQ ID NO: 100); Probes that hybridize specifically with V / L / M555I target sequences are selected from the following list: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr49, HBPr491 HBPr494, HBPr495, HBPr496 (SEQ ID NO: 30 to SEQ ID NO: 40 or SEQ ID NO: 101 to SEQ ID NO: 106) The method according to claim 1 or 2, characterized in that the probe used in step (iii) is a combination of one or more of the following probes: Probes for detection of L528M mutations: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468 and HBPr468-1; Probes for the detection of M552V / I mutations: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433, HBPr433-1 H380 And HBPr453; Probes for detection of V / L / M555I mutations: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299 and HBPr419. The method according to any one of claims 1 to 3, wherein the primer set used in step (ii) is selected from (Table 5): HBPr134, HBPr135, HBPr135A, HBPr135B, HBPr75, HBPr94, HBPr94A, HBPr94B, HBPr94C and / or HBPr105 (SEQ ID NOs: 41 to 50). The method of claim 4, wherein the primer set consists of two primers: HBPr134 as the front primer and HBPr135 as the rear primer; And / or HBPr75 as front primer and HBPr94 as rear primer; And / or HBPr75 as front primer and HBPr105 as back primer. The method according to any one of claims 1 to 5, wherein two mutations L528M and M552V / I are detected in one step. The method according to any one of claims 1 to 5, wherein two mutations L528M and V / L / M555I are detected in one step. 6. The method of claim 1, wherein two mutations M552V / I and V / L / M555I are detected in one step. 7. The method of any one of claims 1 to 5, wherein three mutations L528M, M552V / I and V / L / M555I are detected in one step. 10. The method of claim 1, wherein the HBV strain present in the biological sample is resistant to lamivudine, famciclovir, and / or phencyclovir. 11. For use in the monitoring of antiviral drug resistance in patients and for the genetic detection of mutations L528M, M552V / I and / or V / L / M555I in DNA polymerases of HBV strains present in the patient's biological samples. A probe as defined in any of claims 1 to 10. A composition consisting of one or more probes as defined in claim 11. Use of the probe composition according to claim 12 for in vitro monitoring of antiviral drug resistance of a patient. To monitor the antiviral drug resistance of the patient by genetically detecting one or more of the mutations L528M, M552V / I and / or V / L / M555I in the HBV DNA polymerase of the HBV strain present in the patient's biological sample. Diagnostic kit, containing the following ingredients: (i) means for ejecting, separating or enriching the polynucleic acid, if appropriate, present in the biological sample; (ii) where appropriate, one or more suitable primer pairs; (iii) at least one probe according to claim 11 attachable to a solid support; (iv) hybridization buffers, or components necessary for the preparation of such buffers; (v) wash solution, or ingredients necessary for the preparation of said solution; (vi) means for detecting a hybrid resulting from the preceding hybridization, where appropriate; (vii) Where appropriate, means for attaching the probe to a known position on a solid support. The following components for monitoring antiviral drug resistance of a patient by genetically detecting the mutations L528M, M552V / I and / or V / L / M555I in the HBV DNA polymerase of the HBV strain present in the patient's biological sample. Line Probe Assay, Containing: (i) means for ejecting, separating or enriching the polynucleic acid, if appropriate, present in the patient's biological sample; (ii) where appropriate, one or more suitable primer pairs; (iii) one or more probes selected from HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468 attached to a solid support and specifically hybridize with the target sequence of L528M shown in FIG. 1; And / or (iv) HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1, HBPr465r H45r456 H45r456, HBPr456 attached to the solid support. At least one probe selected from HBPr486, HBPr487, HBPr488 and specifically hybridizing with the target sequence of M552V / I shown in FIG. 1; And / or (v) HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBPr492, HBPr4995, attached to the solid support, are shown in Fig. One or more probes that specifically hybridize to the target sequence of V / L / M555I. (vi) hybridization buffers, or components necessary for the preparation of such buffers; (vii) a wash solution, or ingredients necessary for the preparation of said solution; (viii) means for detecting a hybrid resulting from the preceding hybridization, where appropriate; An isolated nucleic acid consisting of or comprising a sequence as shown in FIG. 1 (SEQ ID NOs: 50-86 and SEQ ID NOs: 107 to 109), or a fragment thereof, wherein the fragment has at least 10 contiguous nucleotides as shown in FIG. And a nucleic acid containing one of the codons encoding amino acids 528, 552 or 555 of the HBV DNA polymerase.