WO2001064959A1

WO2001064959A1 - Detection of hepatitis b virus rna

Info

Publication number: WO2001064959A1
Application number: PCT/EP2001/002143
Authority: WO
Inventors: Jaap Goudsmit; Sol Carl Yates; Maarten Tjerk Penning; Lambertus Henricus Maria Weijer Van De
Original assignee: Akzo Nobel N.V.
Priority date: 2000-03-02
Filing date: 2001-02-26
Publication date: 2001-09-07
Also published as: AU2001254670A1

Abstract

The present invention is concerned with a method for monitoring anti-viral therapy wherein HBV RNA is detected and oligonucleotides that can be used in the detection of Hepatitis B RNA. Today's tools for the monitoring of anti HBV therapy are either based on immunologic markers or the detection of the HBV DNA in virus particles. However, the latest therapies are more and more targeting the viral polymerase and therefore these therapies directly affect the replication of the virus. Consequently, a need exists for tools that can be used to monitoring the efficacy of the treatment. Such monitoring tools should then actually directly measure the replication of the virus and immediately indicate the emergence of resistant virus strains. The present invention provides such a tool. It provides a method for detecting HBV RNA and it provides insight in how to interpret the results in order to predict the outcome of the therapy. A more efficient therapy can then be given to the patient concerned.

Description

Detection of Hepatitis B virus RNA

The present invention is concerned with oligonucleotides that can be used as in the detection of Hepatitis B mRNA. Furthermore a method for the diagnosis of Hepatitis B infection is provided.

The virus that causes hepatitis B or serum hepatitis appears to infect only man and chimpanzees. Hepatitis B virus (HBV) infection in humans is widespread. The hepatitis infection is transmitted by three general mechanisms: (1 ) by parenteral inoculation of infected blood or body fluids, either in large amounts as in blood transfusions or in minute amounts as through an accidental skinprick; (2) by close family or sexual contact; and (3) by some mothers, who transmit the virus to their new-born children. Under natural conditions, HBV is not highly contagious. Transmission by inhalation occurs rarely, if ever. The transmission route through contaminated blood or blood products is a major threat to the human health.

Infection with HBV often results in subclinical or acute self-limited liver disease or can result in chronic long-term infection. Chronic HBV infection elicits a spectrum of disease entities ranging from the most severe form of chronic active hepatitis to less severe chronic persistent hepatitis to the asymptomatic carrier state. An array of diagnostic assays have recently been developed to aid the clinician in differentiating hepatitis B virus infections from other forms of viral hepatitis (i.e., hepatitis A, hepatitis C or hepatitis E). However, the ability to distinguish between an acute hepatitis B infection and symptomatic chronic hepatitis B infection is still problematic. This is especially true since chronic active hepatitis and chronic persistent hepatitis patients often demonstrate a cyclic pattern of hepatitis characterised by acute exacerbation of liver injury alternating with normal liver function.

After infection with HBV, large quantities of the virus and associated particles are present in the serum. During symptomatic phases of infection, both acute and chronic HBV patients have elevated liver enzyme levels, possess the hepatitis B surface antigen (HBsAg) in their serum, and produce antibodies to the nucleocapsid antigen (HBcAg). Antibodies specific for the HBsAg or the hepatitis B e antigen (HBeAg) are not detected. The appearance of antibody to HBsAg is usually not observed until approximately two months following disappearance of circulating HBsAg. The viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid, known as the core antigen (HBcAg). Antibody production of HBcAg occurs early in the course of the acute phase of HBV infection and can persist for many years, and chronically infected patients can produce high titers of anti-HBc antibodies.

HBsAg is established as the most important marker of acute or chronic hepatitis B infection, detectable in serum of infected individuals. HBsAg screening of donor blood for example, is essential to avoid transmission of hepatitis B. It is clear that sensitivity is of utmost importance in diagnostic HBV assays.

HBV is the smallest DNA virus known, and its genome shows a highly compacted organization. A unique aspect in the replication cycle of HBV is that a pregenomic mRNA serves as a template for the synthesis of the first viral DNA strand ^-4. The RNAse H activity of the HBV DNA polymerase subsequently removes the mRNA and the complementary DNA strand is then synthesized, generating a partially double stranded DNA molecule for packaging in virions. Upon successful virus entry, the partially double stranded DNA molecule is converted into a fully double stranded DNA molecule.

HBV DNA can be detected in the blood of infected hosts who are HBsAg and HBeAg positive in more than 90% of cases. The state of the art method of measuring the quantity of infectious particles is by measuring the quantity of viral DNA in serum or plasma, because it most reliably reflects the amount of replicating virus. Several assays are available for this purpose, such as the branched DNA (bDNA) assay⁵, DNA hybridization assays and quantitative PCR '7 Most of these assays, however, have only limited sensitivity. Kόck et al. ° found that a considerable number of HBV virus particles circulating in the serum may contain viral mRNA rather than DNA. Although some other reports imply that HBV mRNA may be packaged in virion particles, its nature, quantity and clinical significance remain unknown. Kόck et al. ° describe an assay for the detection of this HBV RNA in in vitro infected cells and in patient's serum, however the publication is silent about the clinical and diagnostic relevance of this assay.

Therapy of chronic HBV infected patients usually involves treatment with interferon. However, only a limited number of patients respond to interferon therapy with complete virus elimination. It is difficult to predict whether an infection will be self-limiting or chronic and which patients will eventually respond to interferon therapy. Therefore a need exists for reliable methods for the prognosis of hepatitis B. If such a method is available it can be established whether a patient should be treated with anti-viral agents and the effect of such treatment can be predicted or monitored.

The monitoring of antiviral therapy plays an important role in the management of HBV patient care. A close monitoring of the efficacy of the therapy is necessary in order to adjust antiviral therapy to the needs of the patient Typical problems that are encountered during the course of therapy are the emergence of resistant virus strains and undesired co-effects of the drugs used.

As detailed above, today's tools for the monitoring of anti HBV therapy are either based on immunologic markers or the detection of the HBV DNA in virus particles. However, the latest therapies are more and more targeting the viral polymerase and therefore these therapies directly affect the replication of the virus. Consequently, a need exists for monitoring tools that can be used to follow the efficacy of the treatment. Such monitoring tools should then actually directly measure the replication of the virus and immediately indicate the emergence of resistant virus strains. The present invention provides such a tool. As will be shown below, the HBV RNA assay as presented here has several advantages over methods known in the art such as conventional HBV DNA assays. First, the HBV RNA assay is able to detect HBV RNA earlier than HBV DNA in the course of HBV infection which would allow an earlier start of the treatment as well as an earlier exclusion of such patients from giving blood for transfusion. Secondly, the HBV RNA assay can distinguish between patients that will go into a chronic course of the disease and those with a transient course. Thirdly, HBV RNA is detectable in higher quantities than HBV DNA after therapy is stopped and therefore is a more sensitive marker of recurrent disease.

Various techniques for amplifying nucleic acid are known in the art. One example of a technique for the amplification of a DNA target segment is the so-called "polymerase chain reaction" (PCR). With the PCR technique the copy number of a particular target segment is increased exponentially with a number of cycles. A pair of primers is used and in each cycle a DNA primer is annealed to the 3' side of each of the two strands of the double stranded DNA-target sequence. The primers are extended with a DNA polymerase in the presence of the various mononucleotides to generate double stranded DNA again. The strands of the double stranded DNA are separated from each other by thermal denaturation and each strand serves as a template for primer annealing and subsequent elongation in a following cycle. The PCR method has been described in Saiki et al., Science 230, 135, 1985 and in European Patents no. EP 200362 and EP 201184.

Another technique for the amplification of nucleic acid is the so-called transcription based amplification system (TAS). The TAS method is described in International Patent Appl. no. WO 88/10315. Transcription based amplification techniques usually comprise treating target nucleic acid with two oligonucleotides one of which comprises a promoter sequence, to generate a template including a functional promoter. Multiple copies of RNA are transcribed form said template and can serve as a basis for further amplification. An isothermal continuous transcription based amplification method is the so-called NASBA process ("NASBA") as described in European Patent no. EP 329822. NASBA includes the use of T7 RNA polymerase to transcribe multiple copies of RNA from a template including a T7 promoter.

Other more or less equivalent assays for the amplification of RNA and DNA are known in the art. Many use a primer set and suitable amplification reagents for that purpose, however, amplification methods employing only one primer are known in the art. The skilled person would know how to select an amplification technique particularly suitable for the desired purpose.

We designed an amplification assay in which HBV RNA can be amplified by RT- PCR from culture medium of in vitro HBV infected cells (Table 1 , example 1 ). This assay is highly specific for RNA since samples containing only DNA gave negative test results (Example 1 ).

TABLE 1

Table 1. Results of RT PCR for HBV RNA. HBV RNA could be detected in duplicate experiments in Culture medium from HBV infected cells not in water and also not when no reverse transcription (RT) step was performed when using culture medium. See Example 1 for details of the procedure.

When this assay was applied to 6 samples from patients known to be infected with HBV, 4 samples tested positive for HBV RNA, whereas all 6 tested positive for HBV DNA (table 2). TABLE 2

Table 2. Amplification of HBV mRNA on 5 serum samples and 1 plasma sample, tested positive for HBV with serological markers (HbsAg, HbcAg) and bDNA. RT-PCR was performed in duplicate. POS/POS = both duplicates positve, POS/NEG one of duplicates negative the other positive, NEG/NEG = both duplicates negative.

Yet another indication for the notion that our assay was very specific for HBV RNA and did not detect DNA came from an experiment where nucleic acid (RNA and DNA) was extracted from a patient's sample and wherein subsequently DNA was degraded by DNAse I treatment. The DNAse treated sample did not contain any DNA anymore (as indicated by a negative result in a HBV DNA PCR reaction) but still produced a strong signal when tested in the HBV RNA assay (Table 3, Example 3).

TABLE 3

Table 3. Results from DNA and RNA HBV assays on nucleic acid (RNA+DNA) extracted from patients serum, before and after DNAse I treatment We also found that the HBV RNA circulating in serum from HBV infected patients was at least 750 nucleotides in length. This was concluded from experiments performed with primers located 640 and 750 nucleotides away from the poly-A tail of the mRNA (Table 5). Primers located at relative positions 640 and 750 gave positive results in the HBV RNA assay, whereas primers located at relative positions 850, 950 and 1650 tested negative (Table 4).

TABLE 4

Table 4. Results with HBV RNA amplification assay performed on a serum sample when using different primers: HBV 340, HBV 1050, HBV 1160, HBV 2537, HBV 2674 which are situated 1650, 950, 850, 750 and 640 nucleotides away respectively from the poly-A strectch of the HBV mRNA.

TABLE 5

Table 5. Nucleotide sequence of primers used in determination of length of HBV mRNA.

We also found that the mRNA was associated with virus particles. Evidence for this comes from experiments detailed in Example 3. It was shown there that RNA isolated form human serum could not withstand incubation in plasma in its isolated form. It is hypothesized that the RNA becomes rapidly degraded when exposed to the RNAses which naturally occur in serum and plasma. The same phenomenon was observed in culture medium, which is known to contain a relatively high concentration of RNAses originating from one of its constituents, the fetal calf serum. TABLE 6

Table 6 Results of HBV RNA and DNA assays on samples where naked HBV RNA + DNA was added to either water, normal plasma or RNAse A.

If naked RNA cannot survive in serum, yet be isolated in high titers from serum of HBV infected patients, it must be protected there from degradation. We postulate therefore that the RNA in blood is protected by an RNAse resistant coat, such as for instance a virus-like particle. This notion is corroborated by experiments where extra RNAse A was added to serum from HBV patients and culture medium from HBV infected cells. Even this extra RNAse A was not able to degrade the RNA while it was present in its natural environment, whereas the equivalent amount of RNAse A added to naked nucleic acid rapidly degraded the HBV RNA.

Additional evidence that the HBV RNA was associated with a particle the size of a virion comes from ultracentrifugation experiments described in Example 3. HBV RNA isolated from culture medium is 10 times enriched in the pellet fraction upon ultracentrifugation while no longer detectable in the supernatant (table 7).

TABLE 7

Table 7. Results of HBV RNA and DNA assay on culture medium before ultracentrifugation, and on pellet and supernatant fractions after ultracentrifugation in serial dilutions

In a CsCI gradient analysis (Example 3) particles containing HBV RNA were found to sediment in fractions with densities of 1.36 g/ml and 1.26 g/ml. It is at these densities that the majority of HBV virion particles is expected (Figure 1 and 2). Taken together these data prove that HBV RNA is present in particles, possibly co-packaged together with HBV DNA into the same particle.

The absolute sensitivity (expressed in copy numbers of molecules) of the HBV RNA assay appeared to be less than that of the HBV DNA assay. In a limiting- dilution experiment, 10⁶ copies of RNA still could be detected whereas the HBV DNA assay could detect as little as 2.4. 10⁴ copies of DNA (Table 8A and 8B).

TABLE 8A

Table 8A. Semi-quantitative analysis of the sensitivity of the HBV RNA assay. TABLE 8B

Table 8B. Semi-quantitative analysis of the sensitivity of the HBV DNA assay.

When using these semi-quantitative assays to determine the ratio of RNA/DNA in patient samples, it appeared that RNA was present in excess of DNA in ratio's ranging from 23 to 6700 (Table 9).

TABLE 9

Table 9. Quantitation of HBV DNA and RNA by limiting dilution PCR and nested RT-PCR in serum samples

A similar phenomenon was observed with cultured cells: in their supernatant RNA was detected in a copy number of 10¹⁰ per ml, whereas DNA was present in approximately 10⁷ copies per ml, resulting in a RNA DNA ratio of approximately 10³ (Table 10). TABLE 10

Table 10. Duplicate quantification of HBV RNA by limiting dilution PCR and nested RT-PCR in cell pellet (Cells) and culture supernatant (Medium) of the hepatoma cell line (Example 5). Median values are presented as copies per 10^s cells or per ml medium.

A significant correlation was observed between the DNA and RNA copy number, strongly suggesting that RNA was produced in the replicative cycle of HBV.

Next, we developed an HBV RNA NASBA assay. Herein a set of specific primer sequences is used. As in every NASBA assay, the P1 primers (P1.4 and P1.11 ) contained a T7 RNA promoter sequence (Table 11 ). In addition to that, they contained an initiation transcription site with the sequence AGAAGG. Primer P1.4 detected only RNA whereas P1.11 detected both RNA and DNA (Example 6). Hence the invention relates to a method for monitoring anti-viral therapy wherein HBV nucleic acid is detected, said method comprising the following steps: a) obtaining a sample from an individual suspected of HBV infection and optionally extracting nucleic acid from that sample b) amplifying a target nucleic acid sequence in that sample using at least one oligonucleotide primer and suitable amplification reagents c) detecting the amplified sequence.

a preferred embodiment is a method according to the invention wherein the nucleic acid is amplified using the NASBA amplification method. We now found that the assay even better distinguished between HBV RNA and HBV DNA when a so-called anchor sequence was inserted in the P1 primer immediately after the oligo-T sequence. This anchor sequence should consist of 4 to 10 nucleotides hybridisable to the sequence of the HBV mRNA adjacent to the poly-A tail which is 5' GCA TGGACA TTG ACC CTT ATA AAG AAT TTG GAG CT polyA 3'. The anchor sequence therefore consists of at least nucleotides AGCT and at most of nucleotides AGCTCCAAAT (SEQ ID NO 1 ). Example 7 shows a comparison of primers with and without such an anchor sequence and the results shown in Figure 3 and 4 clearly indicate that a primer with an anchor sequence has an improved sensitivity. P1 primers with less than 4 nucleotides did not show this improved performance, whereas P1 primers with more than 10 nucleotides of anchor sequence were found to amplify DNA as well. The invention therefore relates to an oligonucleotide primer comprising a T7 RNA promoter sequence followed by a transcription initiation site followed by an oligo dT stretch followed by an anchor sequence consisting of at least the first 4 nucleotides of SEQ ID NO 1 optionally followed by the remaining nucleotides of SEQ ID NO 1 in consecutive order. The invention also relates to a pair of oligonucleotide primers for the amplification of HBV RNA comprising a first oligonucleotide according to the invention and a second oligonucleotide capable of hybidising to HBV RNA at such a position that amplification of the region between the first and the second oligonucleotide may occur.

TABLE 11

Table 11 Primers and probes used in example 6. The T7 RNA promoter sequence (including the first 3 G residues of the RNA that is being made) that is part of the P1 primers is shown in italics, the inserted transcription initiation site is shown underlined. When this HBV RNA NASBA was applied to patients samples obtained during different stages of infection with HBV (longitudinal samples) it appeared that this assay allowed improved detection of HBV infection. In some samples it detected HBV infection earlier than conventional HBV DNA assays or HBV immunoassays (Example 8). In patients that were chronically infected with HBV, the HBV RNA NASBA assay remained positive whereas a conventional HBV DNA assay became negative after HbcAg seroconversion. In a patient with a transient HBV infection, the HBV RNA NASBA assay remained negative whereas a conventional HBV DNA assay became positive (Example 8). Table 12 shows an overview of possible diagnoses dependent on the outcome of the HBV RNA assay

Table 12

Table 12. Diagnostic potential of HBV RNA assay.

A possible commercial embodiment of the invention is a test kit for the diagnosis of HBV infection comprising: a) one or more oligonucleotides according to the invention or a pair of oligonucleotides according to the invention b) a suitable probe optionally provided with a detectable label, c) optionally suitable amplification reagents Such kits would have the advantage that they provide quality controlled reagents suitable for use together as well as controls for their performance

Legends to the figures

Figure 1. CsCI gradient analysis of HBV-positive cell line sample. • designates the density as determined by refractometry, o HBsAg titer, v HBV DNA level, and

_* HBV mRNA level. HBV DNA and mRNA are both found to peak at 1.36 g/ml, where nude core particles representing the majority of HBV DNA containing particles are located. Note that there is another NA peak at the density expected to contain the HBV Dane particle population (1.26 g/ml) and the peak of HBsAg at lower density (around 1.23 g/ml) representing empty HBV envelope (sAg) particles.

Figure 2. CsCI gradient analysis of HBV-positive cell line sample. • designates the density as determined by refractometry, ₀ HBsAg titer, v HBV DNA level, and

* HBV mRNA level. HBV DNA and mRNA are both found to peak at 1.28 g/ml, where Dane particles, representing the majority of HBV DNA particles, are located. Note the peak of HBsAg at lower densities (around 1.23 g/ml) representing empty HBV envelope (sAg) particles.

Figure 3 and 4: NASBA amplifications with two different P1 primers.

Figure 5 - 7 Samples of an HBV infected individual analysed for different markers at several time points before and after HbcAg seroconversion

Figure 8: Samples of an HBV infected individual analysed for HBV RNA and DNA markers at several time points during the infection and treatment thereof. Treatment regimes are indicated by the dotted areas. Definitions

The term "oligonucleotide" as used herein refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides such as primers and probes. The term "primer" as used herein refers to an oligonucleotide either naturally occurring (e.g. as a restriction fragment) or produced synthetically, which is capable of acting as a point of initiation of synthesis of a primer extension product which is complementary to a nucleic acid strand (template or target sequence) when placed under suitable conditions (e.g. buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization, such as DNA dependent or RNA dependent polymerase. A primer must be sufficiently long to prime the synthesis of extension products in the presence of an agent for polymerization. A typical primer contains at least about 10 nucleotides in length of a sequence substantially complementary (P1) or homologous (P2) to the target sequence, but somewhat longer primers are preferred. Usually primers contain about 15-26 nucleotides but longer primers may also be employed. Normally a set of primers will consist of at least two primers, one 'upstream' and one 'downstream' primer which together define the amplificate (the sequence that will be amplified using said primers). The oligonucleotides according to the invention may also be linked to a promoter sequence. The term "promoter sequence" defines a region of a nucleic acid sequence that is specifically recognized by an RNA polymerase that binds to a recognized sequence and initiates the process of transcription by which an RNA transcript is produced. In principle any promoter sequence may be employed for which there is a known and available polymerase that is capable of recognizing the initiation sequence. Known and useful promoters are those that are recognized by certain bacteriophage RNA polymerases such as bacteriophage T3, T7 or SP6. It is understood that oligonucleotides consisting of the sequences of the present invention may contain minor deletions, additions and/or substitutions of nucleic acid bases, to the extent that such alterations do not negatively affect the yield or product obtained to a significant degree. An oligonucleotide sequence used as detection-probe may be labeled with a detectable moiety. Various labeling moieties are known in the art. Said moiety may, for example, either be a radioactive compound, a detectable enzyme (e.g. horse radish peroxidase (HRP)) or any other moiety capable of generating a detectable signal such as a colorimetric, fluorescent, chemiluminescent or electrochemiluminescent signal. Preferred analysis systems wherein said labels are used are electrochemiluminescence (ECL) based analysis or enzyme linked gel assay (ELGA) based analysis.

EXAMPLES

Example 1

In this example we show the amplification of HBV RNA from HBV positive serum samples and the culture medium of in vitro HBV infected cells. Nucleic acid for analysis of RNA was isolated from the samples according to the method described by Boom et al. (1990)¹⁶. For detection of DNA nucleic acid was isolated by a slightly modified method described by Boom et al (1991 )¹⁷. The RT-PCR was performed by mixing 5.0 μl total RNA and 3.5 μl primer T17R1 R0 (=700 ng, sequence 5' TTT TTT TTT TTT TTT TTG ACA TCG ATA ATA CGA CAA GGA TCC GTC GAC ATC 3') followed by an 5 minutes 65°C incubation and chilled on ice. Subsequently 1.8 μl CMB1 (100mM TRIS pH 8.3, 500 mM KCI, 1 % Triton), 3.6 μl dNTPs (4.5 mM each dNTP), 2.7 μl MgCI2 (13.5 mM), 1.4 μl H2O, 0.5 μl 100 mmol/l dithiothrietol (DTT), 0.5 μl Rnasin (40 U/μl), 1.0 μl Superscript II reverse transcriptase (200 U/μl) was added and the reaction mix incubated 45 min at 42°C. The cDNA was PCR amplified with the following protocol: 2.5 μl cDNA, 5.0 μl 10x PCR buffer, 0.75 μl MgCI2 (1.5 mM), 0.25 μl Taq DNA polymerase (2.5 U), 0.4 μl dNTPs (0.2 mM each), 0.5 μl primer R0 (50 ng, sequence 5' AA GGA TCC GTC GAC ATC 3'), 0.5 μl primer HBV2659+ (50 ng, sequence 5' GCT GCT AGG CTG TGC TGC CAA 3') and 40.1 μl H2O were added together in an eppendorf tube and incubated 3 minutes at 95°C in PE9700 thermocycler. The PCR was performed by applying 40 cycles of 30 seconds 95°C, 20 seconds 53°C and 45 seconds 72°C. The last cycle was followed by a 10 minutes 72°C incubation. The amplification products of this first PCR amplification were further amplified in a subsequent PCR reaction, i.e. the nested PCR. One 1 μl of PCR product was mixed with 5.0 μl 10x PCR buffer, 0.5 μl R1 (50 ng, sequence 5' GAC ATC GAT AAT ACG AC 3'), 0.5 μl primer HBV2674+ (50 ng, sequence 5' TGC CAA CTG GAT CCT GCG CGG 3'), 0.4 μl dNTPs (50 μmol each dNTP), 1.0 μl MgCI2 (2.0 mM), 0.25 μl Taq DNA polymerase (2.5 U) and 41.4 μl H2O. The reaction mix was incubated 3 minutes at 95°C in PE9700 thermocycler. The nested PCR was performed by applying 30 cycles of 30 seconds 95°C, 20 seconds 53°C and 45 seconds 72°C. The last cycle was followed by a 10 minutes 72°C incubation. The HBV specific primers used in this protocol have been described by Sallie et al.¹⁴

The primers are designed such that the chance of DNA amplification is minimal due to the application of a poly A stretch in the downstream primer. The specific RNA amplification is confirmed by the fact that without the use of reverse transcriptase in the method there is no amplification (see table 1 ).

In 6 patient samples that were tested positive for HBV with immunological assays and bDNA, 4 samples were found positive with the RT-PCR specific for HBV RNA (see table 2). The HBV identity of the amplified RNA was confirmed by dideoxysequencing of the DNA amplicons of the RT-PCR reaction.

In another experiment, extracted HBV nucleic acids derived from a high titer plasma were subjected to Dnase I treatment (30 minutes at 37°C, 2 units per μl final Dnase I concentration, 50 mM Tris/HCI pH 7.5, 10 mM MgCI₂, 100 mM NaCl and 1 mM dithioerythritol). The Dnase I treated sample contained no amplifiable HBV DNA, but still contained HBV mRNA detectable by RT-PCR. Both HBV DNA and mRNA were detectable in the corresponding untreated sample (table 3). This result strongly suggests that the primers used in the HBV mRNA nested RT- PCR are specific for mRNA and not for polyadenylated DNA. Example 2

The length of the RNA molecule that is amplified by the nested RT-PCR was investigated by the subsequent use of 5' sense primers that were each time located further away from the 3' anti-sense primer containing the poly A stretch using the protocol described in example 1. The primers used in this study are shown in table 1. Only sequences smaller than approximately 800 bp were amplifiable by both HBV-positive plasma and HBV-positive cell culture medium. This suggests that the RNA has a size of 800 nucleotides (see table 4). In HBV- positive cells and plasmid-derived nucleic acids, all primer combinations gave positive results. When detecting sequences in a plasmid sample, a different antisense primer was used (Rcpro) in a normal DNA PCR, because the HBV plasmid construct does not contain an poly-A tail.

In the light of these results it seems unlikely that the HBV mRNA present in patient material and cell medium is much longer than 800 bp. However, the possibility cannot be excluded that the RNA is larger in size, up to 3 kb, due to the fact that the amplification method may amplify long sequences less efficiently. Further studies to determine the length of the HBV-associated mRNA are underway.

Example 3

In this example we proof the association of the detected HBV RNA with virus particles and more in particular that the RNA is encapsulated in the viral particle and thereby protected from degradation by RNases in the medium. The methods used for isolation of nucleic acid and RT-PCR are described in example 1. When HBV nucleic acids purified from a plasma containing a high titer (10⁹ copies per ml) of HBV was added to HBV-negative cell line culture medium or HBV-negative human plasma, no HBV mRNA could be detected due the presence of high RNase concentration in the medium (as part of the fetal calf serum) and plasma. The addition of extra RNase A to HBV-positive culture medium or plasma had no influence on the amplification of particle associated HBV RNA (see table 6). Any HBV RNA that was amplified from cell culture medium or plasma therefore has to be protected by a RNase resistant coat, i.e. the viral particle.

To further determine whether the HBV mRNA was virion associated, 60 ml HBV- transfected cell line (2.2.15) culture medium was centrifuged in a table-top centrifuge for 10 min. at 2800 RPM to pellet cell debris, and the resulting supernatant was then passaged through a 0.45 μM filter. Subsequently, the sample was ultracentrifuged using a SW41TI rotor for 20 hours at 30K and room temperature. The resulting supernatant was collected for analysis and the corresponding pellet was resuspended in 1 ml PBS for further analysis. Analysis was performed by extraction (as described in example 1 ) followed by RT-PCR. These data (see table 7) show that the HBV mRNA is probably associated with a particle, possibly the HBV Dane particle.

Finally, both HBV-positive culture medium and plasma were subjected separately to CsCI gradient analysis. 1 ml of sample was loaded on top of 9 ml of a 20% (w/w) CsCI in PBS solution. Subsequently samples were spun for 72 hours at 30000 RPM and 20 °C using a Beckman SW41Ti swingout rotor and a Beckman ultracentrifuge. After that, samples were divided into fractions of approximately 250μl by removing supernatant starting from the top. Prior to dialysis in 5 liters of PBS o/n, the density of each fraction was detemined by refractometry. All fractions were then tested for HBsAg, HBV DNA and HBV mRNA using the methods described in example 1. mRNA was found to peak at the same density as HBV DNA in both cell culture medium (see figure 1 ) and plasma sample (see figure 2), at 1.36 g/ml and 1.28 g/ml, respectively. These densities correspond to the expected densities at which the majority of HBV DNA containing particles can be found. For cell culture medium, these are the nude core particles, while for plasma they are the Dane particles. This will be confirmed by electron microscopy analyses of these fractions. Taken together, these data strongly suggest that the HBV mRNA is co-packaged with the HBV DNA into the same particle. Example 4

We wanted to investigate whether the quantity of HBV mRNA is related to the quantity of HBV DNA in sera and the culture medium of the HBV transfected hepatoma cell line mentioned before. Therefore, we developed limiting dilution assays using the previously described nested RT-PCR (see example 1 ) and a normal DNA PCR (same protocol as the second round PCR of 35 cycles only) specific for HBV core DNA using the primers HBC1 (5' TTG CCT TCT GAC TTC

TTT CC 3') and HBC2 (5' TCT GCG AGG CGA GGG AGT TCT 3')¹⁵. PCRs were performed in duplo on 10-fold and 2-fold dilutions of extracted nucleic acids on multiple occasions. Copy numbers in samples were calculated using the

QUALITY statistical software¹⁸.

The sensitivity of these assays were determined by usage of an in vitro generated HBV mRNA (640 bp) and a plasmid containing the full genome of HBV in tandem (pHBTIII-6). Detection was by visualization on an ethidium bromide agarose gel following (nested RT-) PCR. The results are shown in table 8A and 8B.

Example 5

Here we investigate whether quantity of HBV mRNA associated to viral particles is related to the quantity of HBV DNA associated with viral particles in sera and the culture medium of the HBV transfected hepatoma cell line mentioned before. DNA was measured using the previously described limiting dilution PCR (example 4). RNA was measured by the limiting dilution nested RT-PCR. In the in vitro cultured hepatoma cell line (see table 10), RNA/DNA ratio was about 1x1 θ5 in cells and 10^ in culture medium.

In serum samples (see table 9), RNA/DNA ratio was on average about 10^ , comparable to culture medium.

Plotting of the quantitative RNA and DNA data from tables 9 and 10 showed that there is a significant correlation between HBV DNA and mRNA load and that the latter is considerably higher than the DNA load. Example 6

In this example nucleic acid extracted from the serum of an HBV infected individual was amplified with NASBA using different primer sets for HBV DNA and RNA amplification. The nucleic acid was extracted and purified from 6 aliquots of 200 μl serum of the same patient using the "Boom" method (Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J, 1990. Rapid and simple method for purification of nucleic acids. J Clin Microbiol; 28(3):495-503). After the extraction all isolated nucleic acid was pooled and subsequently aliquoted into 3 batches of 100 μl each. Before using this nucleic acid in the amplification the 3 nucleic acid batches were treated with RNase A (addition of 10 μl RnaseA [50 mg/ml], 1 hour incubation at 37°C and subsequently the addition of 1 μl Proteinase K [20 mg/ml] followed by an incubation of 15 minutes at 37°C) or DNase I (addition of 3 μl DNasel [10 U/μl, Pharmacia] and 2 μl MgCI₂ [0.5 M] and incubated for 1 hour at 37°C followed by a 10 minutes incubation at 70°C to inactivate the DNase I). As a control a non- treated nucleic acid solution was incubated at 37° for 1 hour. After the treatments the nucleic acid samples were re-extracted with the Boom method (Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J, 1990. Rapid and simple method for purification of nucleic acids. J Clin Microbiol; 28(3):495-503) the nucleic acid extracted in 50 μl water. For amplification by NASBA 5 μl of this nucleic acid solution were used directly and 5 μl of a ten-fold dilution were used as input for the amplification reactions. The primers and molecular beacon probes (For reference see: Leone G, van Schijndel H, van Gemen B, Kramer FR, Schoen CD [1998] Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids Res May 1 ;26(9):2150-2155) that were used in the experiment are described in table 11. Special feature of the P1 primers used in this example is the insertion of an purine rich transcription initiation sequence in the primers between the T7 RNA promoter sequence and the target sequence that needs to be amplified (see table 11 ). P1 primer PolA P1.4 is specific for the amplification of HBV RNA, while P1 primer PolA P1.11 amplifies both HBV DNA and RNA (see table 11 for sequences). The molecular beacon probe that is used in this experiment (MB WT1 , see table 11 ) is labeled with fluoresceine (the label) at its 5' end and DABCYL (the quencher) at its 3' end. The NASBA reactions (Tris-HCI 40 mM, pH=8.5, MgCI₂ 12 mM, KCI 70 mM, DTT 5 mM, dNTP's (each) 1 mM, rATP 2 mM, rUTP 2 mM, rCTP 2 mM, rGTP 1.5 mM, ITP 0.5 mM, EDTA 0.75 mM, DMSO 15% v/v, oligonucleotide P1 0.2 μM, oligonucleotide P2 0.2 μM, molecular beacon probe 0.2 μM and Sorbitol 0.375 M) were incubated at 65°C for 5 minutes and subsequently at 41 °C for 5 minutes. Than the enzyme mix was added (BSA 2.1 mg, RNase H 0.01 units, T7 RNA Polymerase 37 units, AMV-RT 7.5 units) and after gentle mixing by tapping the reactions were incubated at 41 °C in a fluorimeter (Cytofluor 4000, Perkin Elmer) for 90 minutes with continues measurement of the fluorescent signal.

From the results it is clear that P1 primer PolA P1.4 is only capable of amplifying HBV RNA since no signal could be observed after RNase A treatment, whereas signals obtained after DNAse I treatment were identical with those obtained with untreated sample. In contrast, P1 primer PolA P1.11 can amplify both RNA and DNA indicated by the positive results after both RNase A and DNase I treatment, showing amplification of DNA and RNA, respectively.

Example 7

In this example nucleic acid extracted from the serum of an HBV infected individual was amplified with NASBA using different primer sets for HBV RNA amplification. The nucleic acid was in vitro generated by transcription using a plasmid as template. For amplification by NASBA 5 μl of this RNA solution or the appropriate dilution were used.

The primers and probes that were used in the experiment are described in table 13. Special feature of the P1 primers used in this example is the insertion of an purine rich transcription initiation sequence in the primers between the T7 RNA promoter sequence and the target sequence that needs to be amplified (see table 13). Table 13

Table 13, Primers and probes used in example 7

The T7 RNA promoter sequence (including the first 3 G residues of the RNA that is being made) that is part of the P1 primers is shown in italics, the inserted transcription initiation site is shown underlined.

The detection probe WT-1d was labeled at the NH2 group with an electrochemiluminescence (ECL) label for detection pruposes. The NASBA reactions (Tris-HCI 40 mM, pH=8.5, MgCI₂ 12 mM, KCI 70 mM, DTT 5 mM, dNTP's (each) 1 mM, rATP 2 mM, rUTP 2 mM, rCTP 2 mM, rGTP 1.5 mM, ITP 0.5 mM, EDTA 0.75 mM, DMSO 15% v/v, oligonucleotide P1 0.2 μM, oligonucleotide P2 0.2 μM, and Sorbitol 0.375 M) were incubated at 65°C for 5 minutes and subsequently at 41 °C for 5 minutes. Then the enzyme mix was added (BSA 2.1 mg, RNase H 0.01 units, T7 RNA Polymerase 37 units, AMV-RT 7.5 units) and after gentle mixing by tapping the reactions were incubated at 41 °C in a waterbath for 90 minutes. After the amplification the amplicons were hybridized with the capture and detection probe and bound to magnetic particles covered with streptavidin (that binds the biotin group of the capture probe). After hybridization the ECL signals were measured in an Origin ECL reader (Igen Inc), the signal strength corresponds with the amount of amplicons that were made during the amplification. The results of the experiment are shown in figures 3 and 4. The results as depicted in figures 3 and 4 clearly show that the primer P1.4 with four additional specific nucleotides at the 3' end (adjacent to the poly T stretch) has better sensitivity and performance compared to a primer that only has the poly T stretch (P1.3).

Example 8

The following experiments were all performed with the HBV NASBA method as described in examples 6 and 7. In this example several different patients were analyzed and the added value of HBV RNA detection and quantification is shown here.

In figure 5 it is clearly shown that HBV RNA is the first nucleic acid marker that can be measured in the course of a HBV infection. Therefore HBV RNA can be used as a marker for early diagnosis of HBV infection.

In figure 6 it is clearly shown that HBV RNA remains detectable in patient samples after the HBV DNA detection has become negative. This persistent HBV RNA positivity is a marker for disease development and a chronic course of the infection.

In figure 7 it is clearly shown that HBV RNA remains undetectable in patient samples after the HBV DNA detection has become positive. We argue that this persistent HBV RNA negativity is a marker for absence of future disease development and a transient course of the infection.

In the experiment shown in figure 8 the patient was treated with antiviral therapy (interferon and later 3TC, see figure 8) and HBV DNA and RNA measured at several time points. After stopping of interferon therapy the amount of HBV RNA in serum increases much more than the amount of DNA, at least a factor 10, see figure 8 for details. It is clear from this result that RNA is a better marker for measuring the effect of stopping of therapy on the replication of the HBV virus. This result also indicates that RNA probably is also a better marker for measuring the breakthrough to therapy of resistant viruses because HBV RNA is a better market for active replication than HBV DNA.

References

1. Chu CM, Liaw YF, 1997. Natural history of chronic hepatitis B virus infection: an immunopathological study. Journal of Gastroenterology & Hepatology 12: S218- 222.

2. Summers J, Mason WS, 1982. Replication of the genome of a hepatitis B— like virus by reverse transcription of an RNA intermediate. Cell 29: 403-415.

3. Nassal M, Schaller H, 1996. Hepatitis B virus replication-an update. Journal of Viral Hepatitis 3: 217-226.

4. Kann M, Lu X, Gerlich WH, 1995. Recent studies on replication of hepatitis B virus. Journal of Hepatology 22: 9-13. 5. Hendricks DA, Stowe BJ, Hoo BS, Kolberg J, Irvine BD, Neuwald PD, Urdea MS, Perrillo RP, 1995. Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay. Am J Clin Pathol 104: 537-546.

6. Pawlotsky JM, Bastie A, Lonjon I, Remire J, Darthuy F, Soussy CJ, Dhumeaux D, 1997. What technique should be used for routine detection and quantification of HBV DNA in clinical samples? Journal of Virological Methods 65: 245-253.

7. Zaaijer HL, ter Borg F, Cuypers HT, Hermus MC, Lelie PN, 1994. Comparison of methods for detection of hepatitis B virus DNA. J Clin Microbiol 32: 2088-2091.

8. Kock J, Theilmann L, Galle P, Schlicht HJ, 1996. Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus. Hepatology 23: 405-413.

9. Gunther S, Sommer G, Iwanska A, Will H, 1997. Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA. Virology 238: 363-371.

10.Terre S, Petit MA, Brechot C, 1991. Defective hepatitis B virus particles are generated by packaging and reverse transcription of spliced viral RNAs in vivo. Journal of Virology 65: 5539-5543. H .Terre S, Rosmorduc O, Kremsdorf D, Petit MA, Brechot C, 1991. Reverse transcription and packaging of hepatitis B virus (HBV)-RNA generates in vivo defective serum HBV particles. Journal of Hepatology 13 Suppl 4: S42-48.

12. Sommer G, Gunther S, Sterneck M, Otto S, Will H, 1997. A new class of defective hepatitis B virus genomes with an internal poly(dA) sequence. Virology 239: 402-

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17. Boom R, Sol CJ, Heijtink R, Wertheim-van Dillen PM, van der Noordaa J, 1991 , Rapid purification of hepatitis B virus DNA from serum. J Clin Microbiol 29(9):1804-11

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Claims

1 ) Method for monitoring anti-viral therapy wherein HBV mRNA is detected, said method comprising the following steps: a) obtaining a sample from an individual suspected of HBV infection and optionally extracting nucleic acid from said sample b) amplifying a target nucleic acid sequence in that sample using at least one oligonucleotide primer and suitable amplification reagents c) detecting the amplified sequence.

2) Method of claim 1 wherein said amplifying a target nucleic acid sequence is performed using a transcription based amplification technique.

3) Method according to claim 2 wherein the transcription based amplification technique is NASBA

4) Oligonucleotide primer comprising a T7 RNA promoter sequence followed by a transcription initiation site followed by an oligo dT stretch followed by an anchor sequence consisting of at least the first 4 nucleotides of SEQ ID NO 1 optionally followed by the remaining nucleotides of SEQ ID NO 1 in consecutive order.

5) Oligonucleotide primer according to claim 4 wherein said T7 RNA promoter sequence comprises SEQ ID NO 3 and/or said transcription initiation site comprises SEQ ID NO 4.

6) Oligonucleotide primer according to claim 5 comprising SEQ ID NO 2.

7) Pair of oligonucleotide primers for the amplification of HBV RNA comprising a first oligonucleotide according to claims 4-6 and a second oligonucleotide capable of hybidising to HBV RNA at such a position that amplification of the region between the first and the second oligonucleotide may occur. 8) Use of any of the oligonucleotides according to claims 1 to 3 in a nucleic acid amplification reaction.

9) Test kit for the diagnosis of HBV infection comprising:

a) one or more oligonucleotides according to claims 4 to 6 or a pair of oligonucleotides according to claim 7 b) a suitable probe optionally provided with a detectable label, c) optionally suitable amplification reagents