International Association of Physicians in AIDS Care, February 2000 Journal
Hubert G.M. Niesters, PhD
Molecular Diagnostics, Department of Virology, University Hospital Rotterdam,
Rotterdam, The Netherlands
| Introduction Two Targets of HBV Replication Lamivudine and Resistance HBV Antivirals and Viral Load Measurements Molecular Diagnostics and Quantitation in Clinical Virology Conclusions References |
Understanding of the pathogenesis of HIV/AIDS has advanced considerably in the last few years. Reliable laboratory testing and accurate measurement of viral RNA by polymerase chain reaction (PCR) or branched DNA (bDNA) hybridization assays have revealed much about the relation between disease progression and viral replication. Furthermore, tremendous advances in the development of potent antiretroviral drugs have yielded combinations that can reduce and even block viral replication. Clinical trials of this highly active antiretroviral therapy (HAART) demonstrate that long-term suppression of replication is possible, indicated by low or undetectable levels of HIV RNA in plasma.
These developments have also had their impact on advances in antiviral therapy targeting other viruses, like the hepatitis viruses and herpesviruses. Research established that hepatitis B virus (HBV) and HIV have more similarities than one would at first assume from their replication strategies. Chronic HBV infection affects approximately 5 percent of the population worldwide, and as many as 20 percent of patients infected with HIV.1 The largest percentage of chronic HBV carriers have been identified in parts of Africa and Southeast Asia.
Up to 90 percent of intravenous drug users are chronically infected with another hepatitis virus, hepatitis C virus (HCV), and some of these individuals are coinfected with HIV. However, because most clinical studies of antivirals for hepatitis have involved HBV, this article will focus primarily on that virus.
Two major targets for inhibition of HBV replication have been identified,2 virus-specific post-transcriptional events of the HBV replication cycle and viral DNA polymerase. Interferon is the major antiviral agent directed against the first target. It acts via the 2'-5'oligoadenylate synthetase pathway and by immune modulation. In most countries interferon is the only licensed treatment for chronic hepatitis B infection.
The second target, viral DNA polymerase, can be attacked by chain terminator molecules, exactly as nucleoside analogs inhibit replication of HIV. These molecules are incorporated into the growing DNA chain of the HBV genome and thereby block further elongation. Most progress in HBV treatment over the last few years has involved this second group of molecules.3 To date, only one antiretroviral drug has proved effective against HBV. Lamivudine (2',3'-dideoxy-3'-thiacytidine) reduces replication of both HIV and HBV by inhibiting the reverse transcriptase step during viral replication.4-8 This is remarkable, since HBV and HIV belong to different groups of viruses. HBV belongs to the hepadnaviridae, a group of circular DNA viruses that replicate using a reverse transcriptase step. The genome is partly double-stranded and contains four overlapping open reading frames, the largest of which encodes for the viral DNA polymerase gene.
This HBV polymerase contains both RNA-dependent DNA polymerase or reverse transcriptase activity, as well as DNA-dependent DNA polymerase activity. After the virus enters the cell, a completely double-stranded DNA molecule is synthesized in the nucleus from the partly double-stranded genome in the virion. This molecule is also known as the cccDNA of the virus, or covalently closed circular DNA. The cccDNA acts as a minichromosome containing supercoiled DNA. From this cccDNA, a more than full-length RNA molecule is synthesized. This RNA molecule is packaged with the polymerase molecule in a new HBV virion. Within this virion, the reverse transcriptase activity of the polymerase generates a new DNA genome molecule. This cccDNA minichromosome remains within the infected cell and so can trigger HBV viral replication at any moment.
Lamivudine inhibits HBV viral replication effectively in most patients; in more than 80 percent, a hybridization assay cannot detect viremia. However, the detection level of these assays is approximately 1 to 5 million copies/mL. And problems with HBV resistance to lamivudine are similar to those with HIV resistance to this nucleoside analog. Lamivudine monotherapy results in the emergence of drug-resistant HBV strains in 149 to 3910 percent of patients.
| Mutation | Domain | Effect on HBsAg |
|---|---|---|
| Group 1 | ||
| M552V + L528M |
C B |
I195M No change |
| Group 2 | ||
| M552I |
C | W195S W196L |
| The effect on HBsAg (hepatitis B surface antigen) is caused by the overlapping gene organization. |
||
Part of the mutation pattern involved in lamivudine resistance to HBV is identical to that found in lamivudine resistance to HIV. These mutations are located in the so-called YMDD motif of the polymerase, which acts as the catalytic site of the enzyme.11-13 This site is also called the C domain of the HBV DNA polymerase. A number of polymerase molecules have similar sites. In the YMDD region methionine (M) changes to isoleucine or valine (YIDD and YVDD). We and others have described a second lamivudine-induced mutation that lies upstream in the B domain and is related to the YVDD mutation.14,15 This mutation at position 528 changes a leucine (L) into methionine (M). Other mutations that could contribute to resistance against lamivudine have also been described (Table 1).
|
Other antivirals now being studied are good candidates to inhibit HBV replication (Table 2). Whether they can also reduce the HBV DNA load of lamivudine-resistant strains is not fully analyzed. Mutants resistant to certain antivirals, such as famciclovir, are predominantly located in the B domain, which is the template-binding domain of the polymerase. A dominant famciclovir-selected mutation at position 528 has also been found in lamivudine-resistant viruses that have a YVDD motif.16 Therefore, it is not surprising that famciclovir is not very effective against some lamivudine-resistant strains.17,18
Resistance patterns and incidence against lamivudine have been well described. Honkoop and colleagues found a 39 percent actuarial cumulative incidence of lamivudine resistance in their study population.10 This incidence is much higher than that determined by Lai and coworkers, who found only 14 percent resistant strains after one year of treatment.9 Easy-to-use techniques that can detect all possible HBV resistance variants at an early stage are urgently needed. The few techniques available now are mostly limited to research laboratories.
DNA sequencing is the most informative resistance mutation technique. However, this technique generally cannot detect fewer than 15 to 20 percent of variants in a viral population. Schuurman and colleagues demonstrated that standardization of resistance assays for HIV remains a great problem (Schuurman R. Unpublished data, 1999). But one must remember that sequencing gives information on a large part of the gene, which is important at this stage of research since not all mutations involved in resistance against antivirals are known.
Assays based on restriction fragment length polymorphisms (RFLPs) apparently can detect the major variants in the YMDD motif. This assay is easy to use and could contribute to the understanding of the occurrence of the mutant HBV strains.14,19 Other techniques, like the point mutation assay, the line probe assay, and specific molecular beacons, are currently available for research use only and are not implemented in routine diagnostics.
Besides resistance attributable to mutant viral variants, two other problems have emerged with lamivudine therapy. The first is the occasional inability of this antiviral to reduce HBV viral load significantly even though resistance-related mutations do not appear or are not detected. In some patients lamivudine reduces HBV DNA levels only by a few logs, to around 1,000,000 copies/mL. One theoretical explanation is that the phosphorylated form of the drug did not reach high enough levels within the target cell, the hepatocyte. But there are no data to support this hypothesis.
This incomplete suppression of viremia raises the question of what the goal of antiviral treatment should be in patients infected with HBV. In HIV infection the goal is absolute reduction of viral load to plasma levels less than 50 copies/mL. With HBV infection, on the other hand, patients in whom the infection resolves, as measured by anti-HBeAg levels, may still have HBV DNA levels up to 10,000 copies/mL in serum.
A second problem with lamivudine treatment of HBV infection is hepatic flare after drug withdrawal. When lamivudine treatment is stopped, more than 10 percent of HBV-infected patients experience this phenomenon, characterized by high-level wild-type HBV replication and increased hepatitis. Monitoring patients after lamivudine withdrawal by measuring viral load is therefore necessary and is routine in our hospital.20
The introduction of more potent antivirals for viruses like HIV, the hepatitis viruses HBV and HCV, and the herpesviruses cytomegalovirus and Epstein-Barr virus have made molecular amplification assays important tools in monitoring treatment effects. But the most useful assay results are quantitative rather than qualitative.
| Virus | Dynamic range (geq/mL) |
|---|---|
| HIV-1 | Up to 1 x 106 |
| HCV | Up to 5 x 107 |
| HBV | Up to 1 x 1010 |
| CMV* | Up to 1 x 105 |
| EBV* | Up to 5 x 106 |
| CMV = cytomegalovirus; EBV = Epstein-Barr virus; geq/mL = genome equivalents/mL. * Measured in kidney and bone marrow transplant patients. |
|
The introduction of commercially available assays simplified clinical use of these techniques, but differences between assays are still noted, for three reasons: The assays are not standardized, they cannot detect different genotypes of viruses with equal sensitivity, and viruses can be present in plasma over a great dynamic range (Table 3). The assays available generally cannot detect viral DNA or RNA over this whole dynamic range, so different techniques would have to be used to monitor the total range of any antiviral effect. However, standardization remains the most important issue to be addressed.21-23
Over the last few years considerable effort has been expended on the development of quantitative molecular amplification techniques for hepatitis viruses. These techniques can largely be divided into two groups: target amplification and signal amplification assays.
Signal amplification assays like the bDNA technique (Bayer Diagnostics) and the hybrid capture technique (Digene Corporation) are based on hybridization of nucleic acid onto target sequences, after which the hybrid is detected. This signal can be amplified in numerous ways (chemically, by more nucleic acid input), so the detection limit for these techniques is more sensitive than hybridization. Signal amplification assays can easily detect fewer than 500 copies/mL of plasma. These techniques can also detect large number of viruses (up to 100 million copies/mL) without saturation of the detection system.
The PCR technique (Roche Molecular Diagnostics) has been the most successful target amplification assay. PCR assays are based on quantitative competition amplification. The target sequence is detected together with a fixed amount of known synthetic standard, which has the same primer sequences as the target but can be detected using a different probe. Thus, because this technique measures relative amplification related to this standard and can monitor effects of inhibitors in clinical samples, it may lead to inaccurate determinations of the amount of target sequences. PCR assays can measure accurately only over a small range; measurements above 1,000,000 copies tend not to be accurate.
Both target and signal amplification assays can be performed semiautomatically, so the hands-on time needed to perform these assays is limited. Both types of assays can easily be interchanged if the correct standardization procedures are used, and neither assay excludes the other's use in measuring viruses over a large dynamic range. Target and signal amplification assays can have similar detection levels with excellent agreement. When the same Eurohep standards for HBV are used,22 the Digene assay has a detection limit of 6000 copies/mL, whereas the standard PCR-based assay has a detection limit of 1000 copies/mL.
The next generation of assays will most likely be based on the so called TaqMan technology or real-time PCR, which permits measurement of a single sample over the whole dynamic range.24-27 This technique is currently being developed for a number of viral targets. When coupled with automated DNA and RNA isolation, real-time PCR should make possible a fully automated process from sampling to quantitative results.
New antivirals against the hepatitis viruses will become available in the next few years. These agents should be effective against drug-resistant strains and should be able to reduce cccDNA levels in the liver to yield a long-term treatment benefit. The strategy that has proved effective against HI V infection--combination therapy--must be developed for hepatitis viruses as well. Furthermore, new techniques must be devised to monitor for drug-resistant strains. Meanwhile, assays will soon be available to measure the antiviral effect on viral DNA or RNA over the whole dynamic range.
1. Ockenga J, Tillmann HL, Trautwein C, et al. Hepatitis B and C in HIV-infected patients: prevalence and prognostic value. J Hepatol 1997;27:18-24.
2. Locarnini S, Birch C. Antiviral chemotherapy for chronic hepatitis B infection: lessons learned from treating HIV-infected patients. J Hepatol 1999;30:536-550.
3. Schalm SW, de Man RA, Heijtink RA, Niesters HG. New nucleoside analogues for chronic hepatitis B. J Hepatol 1995;22(suppl): 52-56.
4. Boucher CA, Cammack N, Schipper P, et al. High-level resistance to (-)enantiomeric 2'-deoxy-3'-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother 1993;37:2231-2234.
5. Dienstag JL, Perrillo RP, Schiff ER, et al. A preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med 1995;333:1 657-1661.
6. Grimm I. Lamivudine: a magic bullet for chronic hepatitis B? Gastroenterology 1996;111:262-264.
7. Keulen W, Back NK, van Wijk A, et al. Initial appearance of the 184Ile variant in lamivudine-treated patients is caused by the mutational bias of human immunodeficiency virus type 1 reverse transcriptase. J Virol 1997;71:3346-3350.
8. Thibault V, Benhamou Y, Seguret C, et al. Hepatitis B virus (HBV) mutations associated with resistance to lamivudine in patients coinfected with HBV and human immunodeficiency virus. J Clin Microbiol 1999;37:3013-3016.
9. Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med 1998;339:61-68.
10. Honkoop P, Niesters HG, de Man RA, et al. Lamivudine resistance in immunocompetent chronic hepatitis B: incidence and patterns. J Hepatol 1997;26:1393-1395.
11. Bartholomew MM, Jansen RW, Jeffers LJ, et al. Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation [see comments]. Lancet 1997;349:20-22.
12. Ono-Nita SK, Kato N, Shiratori Y, et al. YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: a study by in vitro full-length viral DNA transfection. Hepatology 1999;29:939-945.
13. Tisdale M, Kemp SD, Parry NR, Larder BA. Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase. Proc Natl Acad Sci USA 1993;90:5653-5656.
14. Allen MI, Deslauriers M, Andrews CW, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 1998;27: 1670-1677.
15. Niesters HG, Honkoop P, Haagsma EB, et al. Identification of more than one mutation in the hepatitis B virus polymerase gene arising during prolonged lamivudine treatment. J Infect Dis 1998;177: 1382-1385.
16. Tillmann HL, Trautwein C, Bock T, et al. Mutational pattern of hepatitis B virus on sequential therapy with famciclovir and lamivudine in patients with hepatitis B virus reinfection occurring under HBIg immunoglobulin after liver transplantation. Hepatology 1999; 30:244-256.
17. de Man RA, Bartholomeusz AI, Niesters HG, et al. The sequential occurrence of viral mutations in a liver transplant recipient re-infected with hepatitis B: hepatitis B immune globulin escape, famciclovir non-response, followed by lamivudine resistance resulting in graft loss.J Hepatol 1998;29:669-675.
18. Wolters LM, Honkoop P, Niesters HG, de Man RA. 1998. Efficacy of famciclovir treatment in chronic hepatitis B patients with different mutations at position 552 of the DNA polymerase gene. J Hepatol 28:909-910.
19. Allen MI, Gauthier J, Deslauriers M, et al. Two sensitive PCR-based methods for detection of hepatitis B virus variants associated with reduced susceptibility to lamivudine. J Clin Microbiol 1999; 37:3338-3347.
20. Honkoop P, de Man RA, Heijtink RA, Schalm SW. Hepatitis B reactivation after lamivudine. Lancet 1995;346:1156-1157.
21. Damen M, Cuypers HT, Zaaijer HL, et al. International collaborative study on the second Eurohep HCV-RNA reference panel. J Virol Methods 1996;58:175-185.
22. Heermann KH, Gerlich WH, Chudy M, et al. Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations. Eurohep Pathobiology Group. J Clin Microbiol 1999;37:68-73.
23. Quint WGV, Heijtink RA, Schirm J, et al. Reliability of methods for hepatitis B virus DNA detection. J Clin Microbiol 1995;33:225-228.
24. Abe A, Inoue K, Tanaka T, et al. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J Clin Microbiol 1999;37:2899-2903.
25. Hawrami K, Breuer J. Development of a flurogenic polymerase chain reaction assay (TaqMan) for the detection and quantitation of varicella zoster virus. J Virol Methods 1999;79:33-40.
26. Kawai S, Yokosuka O, Kanda T, et al. Quantification of hepatitis C virus by TaqMan PCR: comparison with HCV Amplicor Monitor assay. J Med Virol 1999;58:121-126.
27. Lewin SR, Vesanen M, Kostrikis L, et al. Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J Virol 1999;73:6099-6103.
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This information is designed to support, not replace, the relationship that exists between you and your doctor.