EP1144693A2 - Detektion der resistenz gegen anti-hepatitis b-medikamente - Google Patents

Detektion der resistenz gegen anti-hepatitis b-medikamente

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Publication number
EP1144693A2
EP1144693A2 EP00951337A EP00951337A EP1144693A2 EP 1144693 A2 EP1144693 A2 EP 1144693A2 EP 00951337 A EP00951337 A EP 00951337A EP 00951337 A EP00951337 A EP 00951337A EP 1144693 A2 EP1144693 A2 EP 1144693A2
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EP
European Patent Office
Prior art keywords
hbv
probes
seq
patient
mutations
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EP00951337A
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English (en)
French (fr)
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EP1144693A3 (de
Inventor
Lieven Stuyver
Geert Maertens
Caroline Van Geyt
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Fujirebio Europe NV SA
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Innogenetics NV SA
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Priority to EP00951337A priority Critical patent/EP1144693A3/de
Publication of EP1144693A2 publication Critical patent/EP1144693A2/de
Publication of EP1144693A3 publication Critical patent/EP1144693A3/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis

Definitions

  • the present invention relates to the field of Hepatitis B virus (HBV) diagnosis. More particularly, the present invention relates to the field of genetic monitoring of anti- HBV drug resistance during HBV therapy.
  • HBV Hepatitis B virus
  • Hepatitis B virus is a small, enveloped DNA virus of approximately 3200 bp long that belongs to the Hepadnaviridae, characterized by a significant hepatotropism and species specificity.
  • the HBV genome is of complex nature having a partially double stranded DNA structure with overlapping open reading frames encoding surface, core, polymerase and X genes.
  • HBV replicates via an RNA intermediate and utilizes reverse transcription in its replication strategy (Summers and Mason, 1982).
  • HBV causes major medical problems, such as chronic liver disease and hepatocellular carcinoma (Schroder and Zentgraf, 1990).
  • HBV-DNA In chronic Hepatitis B virus (HBV)-infected patients with active wild-type virus replication, successful antiviral therapy is characterized by clearance of HBV-DNA from the blood circulation, followed by clearance of Hepatitis B e antigen (HBeAg), and seroconversion to anti-HBe antibodies.
  • HBeAg Hepatitis B e antigen
  • HBeAg quantitative HBeAg measurements were suggested to have predictive value for the outcome of antiviral therapy (Heijtink et al., 1997).
  • interferon-alpha (IFNV) and lamivudine (3TC) are the two licensed agents for treatment of chronic Hepatitis B.
  • IFN-V has antiviral and immunomodulatory properties.
  • Lamivudine (3TC) is a (-) enantiomer of 3' thiacytidine, a 2' 3'- dideoxynucleoside analogue, and is known to be a potent inhibitor of HBV replication through inhibition of the reverse transcriptase (RT) activity of the HBV polymerase.
  • Lamivudine treatment can result in histological improvements in chronic hepatitis patients, and, when given pre- and post-liver transplantation, it can prevent graft reinfection (Honkoop et al, 1995; Naoumov et al., 1995). Both drugs may be given in mono-therapy or in combination therapy (Lai et al., 1998; Mutimer et al., 1998). Another compound with direct antiviral effect, famciclovir (9-[4-hydroxy-3 hydroxymethyl-but-1-yl] guanine), is currently in phase III clinical evaluation.
  • V555I XI 99 in HBsAg
  • Pichoud et al. (1999) showed that the V555I mutant has a decreased replication capacity, does not produce HBsAg, and is resistant to penciclovir but sensitive to lamivudine.
  • these mutations are generally considered as the cause of viral non-responsiveness and treatment failure, the detection of these mutations is of clinical importance for the monitoring of anti-HBV drug resistance during HBV therapy. Therefore, a rapid, reliable and very specific method for the detection of these mutations is needed. This will allow a fast and precise monitoring of HBV drug resistance and provide a quicker and more efficient design of anti-HBV treatment strategies.
  • V/L/M555I in the DNA polymerase of a HBV strain V/L/M555I in the DNA polymerase of a HBV strain.
  • V/L/M555I in the DNA polymerase of a HBV strain V/L/M555I in the DNA polymerase of a HBV strain.
  • DNA polymerase of a HBV strain present in a biological sample of a patient receiving anti-HBV treatment.
  • DNA polymerase of a HBV strain present in a biological sample of a patient receiving anti-HBV treatment. It is another aim of the present invention to provide a Line Probe Assay for the simultaneous detection of at least the mutations L528M and M552V/I in the DNA polymerase of a HBV strain.
  • FIG. 1 Alignment of new HBV DNA polymerase sequences from serum samples obtained from patients before and during HBV therapy, as described in example 1. Target sequences that can be used for probe design are boxed.
  • Figure 2 Design of a HBV drug resistance LiPA strip. Conj.cont.: conjugate control; Amp.cont.: amplification control. The strip contains a total of 19 probe lines with a total of 38 probes; n probes equals the total amount of probes on each line. The specific probes applied on each line are indicated in table 6. Interpretation: Polym. or HBsAg: amino acid for the polymerase or HBsAg open reading frame, applied on this particular probe line.
  • HBV drug resistance LiPA strip results illustrating reactivity of each independent probe line. Results are obtained with 16 recombinant clones of the reference panel. Reactive lines are indicated with their number. A strip showing the relative position of all lines and the codons and amino acids detected on that line, is indicated on the right. Interpretation per codon (cd) is given below each strip.
  • FIG. 4 Monitoring of two HBV infected patients with anti-HBV drug resistance. Left: patient A; right: patient C. Days of follow-up are indicated in X-axis. The interpretation of the reactivity pattern on each strip is given for the three codons (indicated as cd). Treatment schedules are shown on top of the graphs.
  • Figure 7 Detailed analysis of selected amplicons on individual LiPA probes. Days of follow up are indicated above each strip. Left panel: samples taken during lamivudine treatment; right panel: samples taken after withdrawal of 3TC therapy; middle panel: genetic composition of the relevant sequences applied on the indicated position. At day 524, a weak hybridization is also observed on a probe containing TAA (stop codon) at codon 551. Probes for codon 5521 and codon 555 were not applied. Control lines are not shown.
  • Figure 8 Analysis of the HBV polymerase variability at three time points (X-axis). The number of clones at each time point is indicated on the Y-axis. The genetic composition of the clones is indicated in the legend.
  • INNO-LiPA strip set up for simultaneous detection of mutations in codon 528, 552 and 555 of the HBV DNA polymerase. De amino acids detected in the indicated HBV DNA polymerase codon and in the corresponding HBsAg codons are indicated. LiPA strip production and use are explained in example 2.
  • Table 8 Analysis of mutation at codon 528 of HBV polymerase. Comparision of data obtained by LiPA versus data obtained by sequencing.
  • Table 9 Analysis of mutation at codon 552 of HBV polymerase. Comparison of data obtained by LiPA versus data obtained by sequencing.
  • the present invention relates to a method for the monitoring of anti-HBV drug resistance in a patient by genetic detection of at least one of the mutation L528M, M552V/I and/or V/L/M555I in the DNA polymerase of the HBV strains present in a biological sample of said patient, comprising the following steps:
  • step (v) inference, from the hybridization signal obtained in step (iv), on the presence or absence of the L528M, M552V/I and/or V/L/M555I mutation in the DNA polymerase and on possible anti-HBV drug resistance of the HBV strains present in said biological sample.
  • the method described above allows to determine whether a HBV strain is susceptible or resistant to a certain anti-HBV drug by the genetic detection of a mutation in at least one of the codons 528, 552 and/or 555 of the HBV DNA polymerase gene.
  • the isolation of a large number of new HBV DNA polymerase gene sequences has allowed the inventors to develop a reference panel of target sequences on which base a very specific and very sensitive hybridization assay for detection of the above mentioned mutations, could be developed.
  • the mutation L528M means that in codon 528 of the HBV DNA polymerase gene the genetic code for leucine is substituted by the genetic code for methionine.
  • the mutation M552V/I means that in codon 552 of the HBV DNA polymerase gene the genetic code for methionine is substituted by the genetic code for valine or isoleucine.
  • the mutation V/L/M555I means that in codon 555 of the HBV DNA polymerase gene the genetic code for valine, leucine or methionine is substituted by the genetic code for isoleucine.
  • genetic detection of a mutation means that a mutation in an amino acid sequence is detected by determination of the corresponding nucleic acid sequence.
  • the mutations in codons 528, 552 and/or 555 of the HBV DNA polymerase are detected by hybridization of the nucleic acids present in the patients biological sample, with one or more probes that are capable of specifically hybridizing with a target sequence in the HBV DNA polymerase gene as shown in Figure 1.
  • the term "to hybridize specifically” means that said probe forms a duplex with part of its target sequence or with the entire target sequence under the experimental conditions used, and that under those conditions said probe does not form a duplex with other sequences of the polynucleic acids present in the sample to be analyzed.
  • target sequence of a probe is a sequence within the HBV DNA polymerase gene that comprises a mutated or a wild type nucleic acid sequence of the codon encoding amino acid 528, 552 and/or 555 of the HBV DNA polymerase and to which the probe is completely complementary or partially complementary (i.e. with up to 20%, more preferably 15%, more preferably 10% or most preferably 5% mismatches). It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases.
  • the target sequences depicted in figure 1 were obtained from serum and plasma samples of various patients in follow up studies and cross sectional studies, and have not been previously disclosed.
  • probes that are designed to specifically hybridize to a target sequence of a nucleic acid, may fall within said target sequence or may to a large extent overlap with said target sequence (i.e. form a duplex with nucleotides outside as well as within said target sequence).
  • probe refers to a single stranded sequence-specific oligonucleotide that has a sequence that is complementary to the target sequence of the HBV DNA polymerase gene.
  • the probe is about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. Particularly preferred lengths of probes include 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 ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine, or nucleotides containing modified groups that do not essentially alter their hybridization characteristics.
  • the probe used in a method of the invention is selected from tables 2, 3 and/or 4, wherein:
  • the probes specifically hybridizing with the L528M target sequences are selected from the following list: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468, HBPr468-l (SEQ ID NO:
  • the probes specifically hybridizing with the M552V/I target sequences are selected from the following list: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-l, HBPr407, HBPr488, HBPr433, HBPr433-l, HBPr465, HBPr456, HBPr380,
  • HBPr453, HBPr485, HBPr486, HBPr487, HBPr488 SEQ ID NO 11 to SEQ ID NO 29, SEQ ID NO 97 to SEQ ID NO 100;
  • the probes specifically hybridizing with the V/L/M555I target sequences 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:
  • the mutations at codon 528, 552 and/or 555 are detected by hybridization with at least one probe, preferably at least 2, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, 15, 16, 17, 18, 19 or more oligonucleotide probes.
  • the present invention relates to a method as indicated above, further characterized in that said probes are optimized for simultaneous hybridization to their target regions under the same hybridization and wash conditions (for instance in a LiPA format, see below) allowing the detection of a number of polymo ⁇ hic regions at the same time.
  • the present invention relates to a method as described above characterized further in that the probes used in step (iii) are at least one of the following combination of probes: - for the detection of the L528M mutation, probes: HBPr270, HBPr293,
  • HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461 and HBPr468- 1 SEQ ID NO 1 to SEQ ID NO 10;
  • probes HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463,
  • HBPr315, HBPr363-l, HBPr407, HBPr488, HBPr433-l, HBPr465, HBPr456, HBPr380 and HBPr453 SEQ ID NO 11 to SEQ ID NO 29;
  • probes HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299 and HBPr419 (SEQ ID NO 30 to SEQ ID NO 40).
  • the present invention also relates to the olignonucleotides used as probes to perform any method as described above.
  • the present invention relates to the oligonucleotides as depicted in tables 2, 3 and 4.
  • Probe sequences are represented throughout the specification as single stranded DNA oligonucleotides form the 5' to the 3' end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U). Since the current application requires the detection of single base pair mismatches, stringent conditions for hybridization of probes are required, allowing only hybridization of exactly complementary sequences.
  • probes may be adapted accordingly by adding or deleting one or more nucleotides at their extremities. It should be understood that these concomitant adaptations should give rise to the same results, namely that the probes still hybridize specifically to their respective type- specific target sequences. Such adaptations may also be necessary if the amplified material is RNA and not DNA as is the case in the NASBA system.
  • the stability of the [probe: target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and %GC result in a Tm about 2-10°C higher than the temperature at which the final assay will be performed.
  • the base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher
  • G-C content will be more stable at higher temperatures.
  • ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that the degree of hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohol's, that disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5 °C below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.
  • the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid.
  • the degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid.
  • probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another that differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability.
  • oligonucleotide probes of different lengths and base composition may be used, preferred oligonucleotide probes of this invention are between about 5 to 50 (more particularly 10-25) bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.
  • hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self-complementarity. Such structures can be avoided through careful probe design.
  • the probes according to the invention can be prepared by cloning recombinant plasmids containing inserts including the corresponding nucleotide sequence, if need be by excision of the latter from the cloned plasmids by use of the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight.
  • the probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method.
  • biological sample refers to any biological material (tissue or fluid) taken either directly form the infected human being, or after culturing (enrichment) and containing HBV nucleic acid sequences. Biological material may be e.g.
  • biological sample refers to blood serum or plasma samples.
  • the nucleic acids are released, concentrated and/or isolated from the biological sample by any method known in the art.
  • various commercial kits are available such as the QIAamp Blood Kit from Qiagen (Hilden, Germany) for the isolation of nucleic acids from blood samples and the 'High pure PCR template preparation Kit' (Roche Diagnostics, Brussels, Belgium).
  • Other well-known procedures for isolation of DNA or RNA from a biological sample are available (Maniatis et al., 1989).
  • the nucleic acids in the sample to be analyzed may be either DNA or RNA, e.g. genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are also termed polynucleic acids.
  • the HBV DNA polymerase gene or part thereof, present in said biological sample can be amplified by 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 by means of Q ⁇ replicase (Lomeli et al., 1989) or by any other suitable method known in the art, that allows the amplification of nucleic acid molecules.
  • TMA Guatelli et al, 1990
  • bDNA Sanchez-Pescador et al., 1988; Urdea et al., 1991
  • primer refers to a single stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product or amplification product that is complementary to the nucleic acid strand to be copied.
  • the length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products.
  • 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 primers is about 20-25 nucleotides. Specific length and sequence will depend on the complexity of the required DNA or RNA target, as well as on the conditions at which the primer is used, such as temperature and ionic strength.
  • primer set refers to a pair of primers allowing the amplification of the HBV DNA polymerase gene or part thereof.
  • a primer set always consists of a forward primer (sense primer or 5' primer) and a reverse primer (antisense primer or 3' primer).
  • the present invention relates to a method as described above, characterized further in that at least one of primers used in step (ii) is selected from table 5 (HBPrl34, HBPrl35, HBPrl35A, HBPrl35B, HBPr75, HBPr94, HBPr94A, HBPr94B, HBPr94C and/or HBPr 105; SEQ ID NO 41 to SEQ ID NO 50). More particularly, the present invention relates to a method as described above characterized further in that the set of primers consists of the following 2 primers:
  • HBPr 134 as forward primer and HBPrl35 as reverse primer
  • - HBPr75 as forward primers and HBPr94 as reverse primer
  • - HBPr75 as forward primers and HBPr 105 as reverse primers.
  • these primers may be adapted by addition or deletion of one or more nucleotides at their extremities. Such adaptations may be required, for instance, if the conditions of amplification are changed, if the amplified material is RNA instead of DNA, as is the case, for example, in the NASBA system.
  • amplification primers do not have to match .exactly with the corresponding target sequence in the template to warrant proper amplification is amply documented in the literature (Kwok et al., 1990). However, when the primers are not completely complementary to their target sequence, it should be taken into account that the amplified fragments will have the sequence of the primers and not of the target sequence.
  • the primers and/or probes of the invention may be labeled. Labeling may be carried out by any method known to the person skilled in the art. The nature of the label may be isotopic ( 32 P, 35 S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
  • oligonucleotides used as primers or probes may also contain or consist of nucleotide analogues such as phosphorothiates (Matsukura et al., 1987), alkylphosphorothiates
  • the present invention relates to a method, as indicated above, further characterized in that said probes are capable of simultaneously hybridizing to their respective target regions under appropriate hybridization conditions and wash conditions allowing the detection of more than one wild type codon and/or mutated codon at the same time.
  • the present invention relates to a method as described above characterized further in that both mutations L528M and M552V/I are detected in one step.
  • the present invention also relates to a method as described above, characterized further in that both mutations L528M and V/L/M555I are detected in one step. More specifically, the present invention relates to a method as described above characterized further 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 characterized further in that the three mutations L528M, M552V/I and V/L/M555I are detected in one step.
  • the method of the present invention can be used to screen for mutations in codon 528, 552 and/or 555 present before initiating anti-HBV therapy and/or arising during the course of anti-HBV drug therapy (i.e. monitoring of drug therapy) and conferring resistance to lamivudine, famciclovir and/or penciclovir. Accordingly, the present invention relates to a method characterized further in that the HBV strain present in the biological sample shows resistance to lamivudine, famciclovir and/or penciclovir.
  • the method of the invention may also be used to determine resistance to anti-HBV drugs other than the above-mentioned drugs, provided that resistance to these other drugs is linked to one or more of the three mutations that are detected by this method.
  • anti-HBV drug refers to any anti-HBV nucleoside analog or any other DNA polymerase inhibitor that causes a reduction of the viral DNA in the patient.
  • anti-HBV drugs include but are not limited to 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.
  • the method of the invention can also be used in combination with a method for the detection of one or more other mutations that possibly confer resistance to other anti-HBV drugs.
  • probes that allow the detection of other mutations can be added in the method of the invention.
  • the present invention also relates to a composition, comprising at least one probe of the invention.
  • composition as used in the present invention relates to a mixture of probes in an appropriate buffer used to carry out the method of the invention.
  • present invention also relates to the use of the probes and the composition as defined above, for in vitro monitoring of anti-HBV drug resistance in a patient.
  • the present invention also relates to a diagnostic kit for the monitoring of anti-HBV drug resistance in a patient by genetic detection of at least one of the mutations L528M, M552V/I and/or V/L/M555I in the HBV DNA polymerase of the HBV strains present in a biological sample of said patient, comprising the following components:
  • hybridization buffer means a buffer allowing a hybridization reaction between the probes and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions.
  • wash solution means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
  • any assay method that relies on the formation of a hybrid between the nucleic acids of the biological sample and the oligonucleotide probes according to the present invention, may be used.
  • the hybridization can be accomplished using a Southern blot,
  • Northern blot or dot blot format the unlabelled amplified sample being bound to a membrane, the membrane being inco ⁇ orated with at least one labeled probe under suitable hybridization and wash conditions, and the presence of bound probe being monitored.
  • An alternative is a "reverse" format, in which the amplified sequence contains a label. In this format, the selected probes are immobilized to certain locations on a solid support and the amplified polynucleic acids are labeled in order to enable the detection of the hybrids formed.
  • solid support can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low.
  • the solid substrate will be a microtiter plate (e.g. in the DEIA technique), a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip.
  • a membrane e.g. nylon or nitrocellulose
  • a microsphere bead
  • a chip e.g. a chip
  • modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.
  • the present invention relates to a line probe assay for the monitoring of antiviral drug resistance in a patient by the genetic detection of at least one of the mutations L528M, M552V/I and/or V/L/M555I in the HBV DNA polymerase of the
  • HBV strains present in a biological sample of said patient comprising the following components:
  • HBPr488, fixed to a solid support and/or
  • the selected set of probes is immobilized to a membrane strip in a line fashion. Said probes may be immobilized individually or as mixtures to the delineated locations.
  • the amplified HBV DNA polymerase polynucleic acids or part thereof can be labeled with biotin, and the hybrid can then, via a biotin-streptavidine coupling, be detected with a non-radioactive color developing system.
  • the present invention also relates to novel sequences as depicted in figure 1 (SEQ ID NO:
  • the present invention relates to an isolated nucleic acid consisting of or comprising a nucleotide sequence as indicated above.
  • nucleic acid refers to a single stranded or double stranded nucleic acid sequence, which may contain from 10 nucleotides to the complete nucleotide sequence as shown in figure
  • a nucleic acid may consist of deoxyribonucleotides, ribonucleotides, nucleotide analogues or modified nucleotides. It is to be understood that also the complement of the above mentioned nucleic acids forms part of the present invention.
  • Serum or plasma samples were collected from 41 patients during follow up studies and from 80 patients in a cross sectional study.
  • the patients in the follow up studies were as follows: 18 patients in a follow up study by Dr. F. Zoulim (INSERM, Lyon, France), 5 patients in a follow up study by Dr. D. Pillay (Public Health Laboratory Service, Birmingham, UK), 3 patients in a follow up study by Dr. G. Leroux (University Hospital, Gent, Belgium) and 15 patients in a follow up study by Dr. D. Lau (NTH, Bethesda, MD, US).
  • the cross sectional study was carried out by Dr. J. Lau (Schering Plough, Madison, NJ, US).
  • HBV DNA was isolated from the serum or plasma samples by using the commercially available >High pure PCR template preparation Kit' (Boehringer-Mannheim, Brussels, Belgium). Purified DNA was amplified for the HBV polymerase region using a nested PCR approach: 10 ⁇ l purified DNA was mixed with 5 ⁇ l 10 x buffer, 0.4 ⁇ l 10 mM dXTPs, 10 pmol of the sense primer, 10 pmol of antisense primer, 1 Unit Taq polymerase (Stratagene Europe, Amsterdam, The Netherlands), and completed to 50 ⁇ l with HPLC-grade H 2 O. PCR consisted of annealing at 45°C, extension at 72°C, and denaturation at 94°C, each time for 30 sec.
  • outer PCR contained 40 cycles; the nested reaction contained 35 cycles.
  • the HBV polymerase region was amplified with the following primer combination: outer sense HBPrl34: 5'-TGCTGCTATGCCTCATCTTC-3' (SEQ ID NO 41); outer anti-sense HBPrl35: 5'- CAG/AAGACAAAAGAAAATTGG -3' (SEQ ID NO 42); nested sense HBPr75: 5'- CAAGGTATGTTGCCCGTTTGTCC -3' (SEQ ID NO 45); nested anti-sense HBPr 94: 5'-GG(T/C)A(A/T)AAAGGGACTCA(C/A)GATG -3' (SEQ ID NO 46).
  • Nested amplification products were (primers included) 341 bp long, analyzed on a 2% agarose gel, and visualized by ethidium bromide.
  • primers were provided at their 5' ends with a biotin group.
  • a reference panel for probe design was developed simultaneously with the evaluation of the probes.
  • a few probes specific for the detection of the presence or absence of the mutations L528M, M552V/I and/or V/L/M555I were designed and obtained after considering parameters of percentages of GC, probe length, ionic strength of the hybridization buffer, and temperature of incubation. These specific probes were evaluated by applying them to nitrocellulose membranes followed by reverse hybridization of the biotinylated PCR fragments generated from the plasma or serum samples (in a LiPA format), streptavidine-alkaline phosphatase incubation, and color development. Details on the probe optimization phase, LiPA strip production and reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • Double-stranded sequences were obtained from biotinylated PCR products or, in 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 a LiPA for monitoring drug-resistance in HBV-infected patients
  • Probes were pooled according to their ability to detect the different wild type and mutant codons in the HBV DNA polymerase, but also according to relevant information from the overlapping HBsAg reading frame, and applied on a strip.
  • probes designed for different nucleotide polymo ⁇ hisms but not introducing an amino acid change were pooled together and applied on one line. This finally resulted in a strip with 19 different probe lines with in total 38 specific probes ( Figure 2; Table 6). Details on the probe optimization phase and LiPA strip production are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • Example 3 Use of the LiPA for monitoring anti-HBV drug resistance in two patients receiving HBV therapy
  • the LiPA was used to test follow up samples of 2 patients taken before the start of therapy, during therapy and during therapy but with viral rebound. Evidence of treatment failure was shown by viral load and ALT levels (Figure 4).
  • Patient A had been infected with HBV genotype A, patient C with genotype C.
  • the LiPA reactivity obtained with these follow up samples is also shown in figure 4.
  • patient A treated with lamivudine, there was a mixture of V555I (line 14 and 19) transiently present at day 360, 420, 570 and weakly at day 630.
  • a reactivity at line 19 correlates with a translational stop at HBsAg codon 199. This motif disappeared after the emergence of a mutant at codon 552 and 528.
  • Example 4 Use of the LiPA for monitoring the dynamics of emergence and disappearance of lamivudine-associated mutations in Hepatitis B virus.
  • HBV DNA levels were undetectable by conventional assays (not PCR), and ALT levels normalized.
  • HBV purification and amplification HBV DNA was extracted from 50 ⁇ l of plasma using the Tri-Reagent LS protocol for DNA. Nested PCR was used to amplify the HBV polymerase regions A-E. First round PCR combined: 3 ⁇ l of the purified DNA template, 10 ⁇ l 10X buffer with MgCl 2 , 2 ⁇ l dNTP (120 mM stock), 1 ⁇ l each of forward (HBV-8For 5'-CATCAGGATTCCTAGGACC-3'; SEQ ID NO 87) and reverse (HBV-9Rev 5'-ATACTTTCCAATCAATAGGCC-3'; SEQ ID NO 88) primer (30 pmol/ ⁇ l stock), 0.5 ⁇ l Taq DNA polymerase and brought to 100 ⁇ l with HPLC- grade H 2 O.
  • the amplification program consisted of: denaturation at 94°C (45 sec), annealing at 49.8°C (1 min), extension at 72°C (2 min) for 40 cycles.
  • the 810 bp product was purified with the Qiagen PCR Purification Kit and eluted into 30-50 ⁇ l H 2 O.
  • Nested PCR reaction combined 5 ⁇ l of first round PCR product, 5 ⁇ l 10X buffer II, 3.7 ⁇ l MgCl 2 , 1 ⁇ l dNTP (10 mM stock), 1 ⁇ l each of forward (HBV-2For 5'- CGCTGGATGTGTCTGCGGCG-3'; SEQ ID NO 89) and reverse (HBV-R3 5'- CCAACTTCCAATTACATAACCC-3'; SEQ ID NO 90) primers (30 pmol/ ⁇ l stocks), 0.5 ⁇ l Taq DNA polymerase and brought to 50 ⁇ l with HPLC-grade H 2 O.
  • Amplification program consisted of: denaturation at 94°C (45 sec), annealing at 55°C
  • 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 A.L.F. sequencing protocols were followed and the A.L.F. analytical program was set to scan for sequence mutations at the highest stringency (homologous).
  • HBPr75-94 amplification product Two ⁇ l of the HBPr75-94 amplification product (see example 1) was ligated into the pretreated pGem plasmid vector (Promega, Leusden, The Netherlands) and transformed into competent E. coil cells. Single recombinants were selected and the plasmid DNA purified with the High Pure Plasmid Isolation Kit (Boehringer Mannheim, Brussels, Belgium). Inserts from recombinant clones were PCR amplified with either the plasmid-derived primers or the nested HBV primers. All products were analysed by means of LiPA.
  • Samples selected before, during and after (day 331, 338, 345, 633, 715, 726, 738, 752, 779, and 802) lamivudine therapy were sequenced over the HBV polymerase region between amino acid (aa) 473 and 561.
  • This region included domain B (HBV polymerase aa 508 to 530) and domain C (HBV polymerase aa 548 to 558).
  • variability was observed from day 633 onwards, with the detection of M528 and V552, but from day 779 onwards, wild type motifs L528 and M552 re-appeared (Figure 5). There were no other amino acid changes observed in these follow up samples.
  • Lamivudine resistance mutations were detected as complex mixtures as early as day 524 (L and M at 528; M, V and I at 552). The 1552 variant could not be further detected at day 548, but all other mixtures remained. From day 633 pure mutant was detected. This co ⁇ esponded with an increase in viral load. Until day 835, mixtures of wild type and mutant variants at both codon positions were detected. Finally, at day 1041 only pure wild type virus was apparent. Re-initiation of lamivudine therapy occurred at day 1105, and a 139 days later, a complex mixture of wild types and mutant strains at codon 552 were present (nearly comparable with day 524). But since M528 could not be detected, L528 + M552 and L528 + 1552 combinations are the most likely combinations selected (day 1244 and day 1259).
  • HBV amplicons from day 442, 468, and 524 were cloned in plasmids, and a total of 54 individual clones were further analyzed ( Figure 8). Six kinds of clones were detected in which different combinations of motifs at codon 528, 550 and 552 were present.
  • Example 5 Comparison of data obtained with the LiPA for monitoring drug- resistance versus data obtained by sequencing.
  • Plasma or serum samples were collected at two centers: Prof. Dr. S. Locarnini, Research and Molecular Development, Contemporary Infectious Diseases Reference Laboratory, North Melbourne, Australia (9 patients) and Prof. Dr. A. S. F. Lok, University of Michigan Medical Center, Ann Arbor, MI, USA (9 patients). All patients were HBV-infected individuals under lamivudine treatment. The first sample to be included had to correspond with the 'baseline' (time point just before lamivudine treatment started). The second and third sample had to correspond respectively with a time point before and after redectable viral load levels. For some patients also additional sequential samples were obtained.
  • HBV isolation and amplification was done as described above. Sequencing was carried out by standard methods (Sambrook et al., 1989). The LiPA was used as described in example 2.
  • the LiPA detected the mixture wild type/mutant (L/M), while sequencing only detected the mutant (M).
  • the LiPA showed a mixture wild type/mutant (L/M), while sequencing only detected the wild type (L).
  • Colacino JM and Staschke KA (1998) The identification and development of antiviral agents for the treatment of chronic hepatitis B virus infection. Prog Drug Res 50: 259- 322.
  • Kwoh D Davis G, Whitfield K, Chappel H, Dimichele L and Gingeras T (1989) Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad Sci USA 86: 1173-1177. Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C and Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18: 999- 1005.
  • Pillay D, Bartholomeusz A, Cane P, Mutimer D, Schinazi RF, and Locarnini S (1998) Mutations in the Hepatitis B virus DNA polymerase associated with antiviral resistance. Int Antiviral News 6: 167-169. Saiki RK, Gelfland DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, and Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491.

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