Effect of Lamivudine Therapy on the Serum Covalently Closed-Circular (ccc) DNA of Chronic Hepatitis B Infection
The American Journal of Gastroenterology
Man-Fung Yuen, M.D. 1 , Danny Ka-Ho Wong, Ph.D. 1 , Simon Siu-Man Sum, M.Phil. 1 , He-Jun Yuan, Ph.D. 1 , John Chi-Hang Yuen, B.Sc. 1 , Annie On-On Chan, M.D. 1 , Benjamin Chun-Yu Wong, M.D. 1 , and Ching-Lung Lai, M.D. 1
Before reporting on this study here are links to additional reports on cccDNA in HBV:
PegIFN+Adefovir Combination for HBV: cccDNA, seroconversion
http://www.natap.org/2005/EASL/easl_9.htm (EASL Report, April 2005)
Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir therapy (Gastroenterology journal)
http://www.natap.org/2004/HBV/062204_09.htm - 22 Jun 2004
New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA (Journal of Hepatology, March 2005)
http://www.natap.org/2005/HBV/021605_03.htm - 16 Feb 2005
Adefovir Reduced HBV Viral Reservoir & HBsAg in Parallel
http://www.natap.org/2004/DDW/ddw_07.htm - 20 May 2004 (DDW Report, May 2004)
Hepatitis B Report: adefovir; Pegasys (EASL 2002)
http://www.natap.org/2002/easl/day3.htm -- 04 Aug 2004
Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients (Hepatology 2004)
http://www.natap.org/2004/HBV/090304_03.htm - 03 Sep 2004
BACK TO STUDY- Lamivudine & cccDNA
OBJECTIVE: To determine the effect of 1-yr lamivudine treatment on serum covalently closed-circular DNA (cccDNA) level.
PATIENTS AND METHOD: Serum total HBV DNA and cccDNA levels at baseline, week 24, and week 52 were measured in 82 lamivudine-treated patients, 17 of whom received 1-yr placebo and acted as controls.
RESULTS: There was a significant reduction in the cccDNA levels from baseline (median 3.0 x 106 copies/ml) to week 24 (33,476 copies/ml) and week 52 (48,694 copies/ml) (p< 0.001 for both). The median reduction in cccDNA level at week 24 and 52 were 2.21 and 2.12 logs, respectively, which were significantly greater than those of controls (0.31 log, p< 0.001; 0.2 log, p< 0.001, respectively). Fifteen patients (18.3%) developed YMDD mutations by week 52. Compared to patients without YMDD mutations, patients with YMDD mutations had significantly less median reduction of total HBV DNA level (4.44 vs 3.65 logs, respectively, p = 0.02) and cccDNA level (2.27 vs 1.65 logs, respectively, p = 0.016) at week 24 and significantly less median reduction of cccDNA at week 52 (2.35 vs 0.8 logs respectively, p< 0.001).
CONCLUSIONS: One-year lamivudine treatment decreased serum cccDNA level by 2 logs. The chance of YMDD mutations at week 52 was related to the magnitude of viral suppression at week 24.
The replication cycle for hepadnaviurses has been fully characterized. Once hepatitis B virus (HBV) enters hepatocytes, the relaxed-circular DNA (rcDNA) of the HBV will convert into covalently closed-circular DNA (cccDNA) inside the nuclei of the infected hepatocytes (1). This process takes place within 24 h upon the inoculation of HBV in the duck model (2, 3). HBV replicates through reverse transcription of the pregenomic RNA produced from the cccDNA template to form a partially double-stranded rcDNA. This in turn either replenishes the depleted cccDNA pool or is secreted outside the hepatocyte as a complete virion after further processing at the endoplasmic reticulum. Since cccDNA does not undergo semiconservative replication, all the cccDNA inside the hepatocytes is derived from viral DNA produced in the cytoplasm. The production of cccDNA is controlled by a negative feedback mechanism dependent on the amount of envelope proteins inside the hepatocytes (4-6). Therefore, complete eradication of HBV not only relies on the inhibition of the active viral replication that will prevent infection of new hepatocytes, but also requires a total elimination of cccDNA in order to prevent the synthesis of new HBV. In cell cultures with undividing hepatocytes as well as in duck HBV studies, cccDNA is found to have high stability (7, 8).
Studies have shown that cccDNA is detectable in the culture medium of 2.2.15 cells-derived from HEPG2 cells, serum of HBV infected rats and serum of HBV patients (9-11). Recently, we have confirmed that cccDNA can be measured in serum of chronic hepatitis B (CHB) patients by using an invader assay (12). The invader assay has also been fully and carefully validated in our previous study (12). More importantly, serum cccDNA levels are found to correlate well with intrahepatic cccDNA levels. This allows for serial assessment of intrahepatic cccDNA levels without the necessity of repeated liver biopsies.
One-year lamivudine treatment is associated with 3 logarithmic reductions of total HBV DNA levels (13, 14). According to studies in woodchuck and duck models, lamivudine in combination with another nucleoside analog or immunomodulator can reduce the cccDNA levels (8, 15). The reduction of cccDNA by lamivudine alone in another woodchuck study is demonstrated to be caused by the loss of hepatocytes rather than an actual decrease in cccDNA content inside the hepatocytes (7). To date, the effect of lamivudine therapy on cccDNA in man is unknown. Yet, clinical relapse of CHB after cessation of therapy is probably due to the reactivation of viral replication from the residual cccDNA inside the hepatocytes (16).
We sought to determine the effect of 1-yr lamivudine treatment on the serum cccDNA levels that may reflect its effect on the intrahepatic cccDNA.
It is likely that the majority of cccDNA in the serum originates from infected hepatocytes, which upon cell death release the cccDNA into the circulation. Some other contributions to serum cccDNA may be coming from extrahepatic infected cells such as peripheral blood lymphocytes (19-21).
There were no significant changes in the serum cccDNA levels over a period of 52 wk in the patients receiving placebo. This suggests that the HBV infection was in an equilibrium state in which the rate of replenishment and depletion of cccDNA from hepatocyte cell death were more or less equal.
Studies with the duck and woodchuck models demonstrate that cccDNA levels decrease with lamivudine therapy (8, 15). The decline in cccDNA level is the result of cell death of the hepatocytes in which the active viral replication is halted by lamivudine. Other studies also suggest that cccDNA inside the hepatocytes will also be lost during the process of mitosis (7, 22). To our knowledge, the present study is the first human study to demonstrate that serum cccDNA levels decreased in CHB patients who received lamivudine. Lamivudine probably disrupts the equilibrium state of cccDNA by decreasing the rate of replenishment through inhibition of viral replication in infected hepatocytes. Moreover, nucleoside analogues are also effective in suppressing viral replication in extrahepatic infected cells (23). Irrespective of the sources of serum cccDNA, inhibition of viral replication can be achieved by lamivudine resulting in a significant reduction of serum cccDNA level.
The present study showed that, over a treatment period of 52 wk, the serum cccDNA level decreased by approximately 2 logs. This probably reflects a similar trend of reduction of intrahepatic cccDNA content after 52 wk of lamivudine treatment since we have demonstrated in our previous study that serum cccDNA levels correlate very well with intrahepatic cccDNA levels (12). The measurement of serum cccDNA together with the conventional total serum HBV DNA gives a clearer picture of the decline of HBV replication with nucleoside analogue therapy. Studying the levels of both total HBV DNA and cccDNA in the serum would allow some insight into the decline of both the replicative and nonreplicative form of the virus without the necessity of performing liver biopsies. Since clearance of cccDNA is one of the ultimate aims of the treatment of CHB, estimation of serum cccDNA may provide some indications for the duration of therapy.
The serum total HBV DNA levels increased when YMDD mutations developed though to levels lower than the pretreatment levels. Similarly serum cccDNA levels at week 52 in the 15 patients with YMDD mutations also increased. The median cccDNA level at week 52 for these 15 patients (173,539 copies/ml) was lower than the median level at baseline (924,671 copies/ml), though this was not statistically significant probably due to the small number of patients. This finding is in agreement with the fact that YMDD mutants are less replication competent than YMDD wild type (24, 25). The 67 patients who did not develop the YMDD mutants continued to have further decrease in HBV DNA and cccDNA levels.
Patients with less viral suppression by lamivudine as reflected by lower logarithmic reduction of both the total HBV DNA and cccDNA at week 24 (Table 2& Fig. 2) are associated with a significantly higher chance of development of YMDD mutations at week 52. According to our previous study, high viral load (total HBV DNA level) at 24 wk of lamivudine treatment is associated with a higher chance of subsequent development of YMDD mutation (12). Nucleotide analogue treatment for HBV should target at an early maximal viral suppression to reduce the chance of subsequent development of drug-resistant mutations in the future.
In conclusion, the present study demonstrated that cccDNA could be quantified in serum with significant changes during lamivudine therapy as well as after the development of YMDD mutations. Lamivudine resulted in a decrease in the cccDNA levels by a magnitude of 2 logs. The chance of emergence of YMDD mutations at week 52 was closely related to the magnitude of viral suppression at week 24 of lamivudine therapy. Serum cccDNA monitoring is a potential tool for monitoring antiviral therapy in the future. A reduction to undetectable levels may signify total, or near total, eradication of HBV, though this may require confirmation by intrahepatic cccDNA in liver biopsies.
PATIENTS and METHODS
A total of 82 patients with CHB infection were recruited in the present study. These patients were from three previous clinical trials NUCB3009, NUCB3018, and NUCB4003, sponsored by GlaxoSmithKline research laboratories. These 3 trials randomized a total of 187 patients to different regimes including lamivudine 25 mg daily for 1-2 yr, famciclovir 500 mg three times daily for 12 wk followed by lamivudine 100 mg daily, or lamivudine 100 mg daily at entry into the trials. Seventeen patients were randomized to receive placebo for 1 yr followed by lamivudine 100 mg daily. The 82 patients recruited in the present study were selected because they were either given lamivudine 100 mg daily at the entry into the trials or placebo for 1 yr followed by lamivudine 100 mg daily. These selection criteria were chosen in order to obtain a homogenous population who received either lamivudine 100 mg daily or placebo. The entry criteria for trial NUCB3009/3018 were: patient >=16 yr old, positive hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) for at least 6 months, HBV DNA levels >=1.4 x 106 copies/ml by solution-hybridization assay (Abbott Diagnostics, Chicago, IL) and ALT levels <= 10 x upper limit of normal (ULN) (13). The entry criteria for trial NUCB4003 were: patient >= 16 yr old, positive HBsAg and HBeAg for at least 6 and 3 months, respectively, detectable HBV DNA levels by branched DNA assay (Bayer Corporation, NJ) (lower limit of detection 0.7 x 106 copies/ml), and ALT levels between 1.3-10 x ULN (17). All three trials were approved by the Ethics Committee of the University of Hong Kong, Hong Kong, and were conducted in the Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong. Sixty-five of these patients were randomized on lamivudine 100 mg daily at the beginning of the trial. The remaining 17 patients were randomized to receive placebo during the first 52 wk and subsequently received lamivudine 100 mg daily. Total HBV DNA and cccDNA levels of these 17 patients during the initial 52 wk on placebo acted as control for comparison with the values obtained during 52 wk of lamivudine treatment (that is the second year data of these 17 patients receiving lamivudine 100 mg for the first time after week 52 together with the initial 52-wk data from the other 65 patients).
The clinical profiles and liver biochemistry of the patients were monitored. Serum taken and stored at -70¡C at baseline (before the first dose of lamivudine), week 24, and week 52 were thawed to measure total HBV DNA and cccDNA levels.
Measurement of Serum Total and cccDNA Levels
The serum total HBV DNA levels were measured by the Cobas Amplicor HBV Monitor test (Roche Diagnostics, Branchburg, NJ) (lower limit of detection of 200 copies/ml).
The serum cccDNA was measured by the Invader¨ HBV DNA assay (Third Wave Technologies, Inc., Madison, WI), which has a lower limit of detection of 104 copies/ml. The detailed methodology was described in a previous paper (12).
Determination of YMDD Mutations
The mutations of the tyrosine, methionine, aspartate, aspartate (YMDD) motif [methionine either substituted by isoleucine (rtM204I) or valine (rtM204V)] in the C domain and leucine substituted by methonine (rtL180M) in the B domain of the HBV DNA polymerase gene were determined in all the lamivudine-treated patients at week 52 by the line probe assay (INNO-LiPA HBV DR, Innogenetics NV, Belgium) as described in our previous study (18).
The data was analyzed by the Mann-Whitney test for the continuous ordinal data, chi 2 test with Yates' correction, and Fisher exact test for the association between two qualitative variables. The differences in paired samples were tested by Wilcoxon signed ranks test. p-Values of less than 0.05 were considered as statistically significant.
The demographic data and the liver biochemistry of the 82 patients (65 on lamivudine 100 mg daily from the start of the trials, and 17 on placebo for 52 wk followed by lamivudine 100 mg daily) are listed in Table 1. There were no differences in the parameters between the two groups of patients (all p = NS).
Total HBV and cccDNA Levels on Placebo
For the 17 patients on placebo for 52 wk, there were no significant changes in the median total HBV DNA level and cccDNA level from baseline to week 24 and to week 52 [total HBV DNA: 7.7 x 107 (range 1.7 x 105 to 3.5 x 1011) vs 3.2 x 106 (range 18,500 to 2.9 x 1011) and vs 5.1 x 108 (range 17,000 to 5.1 x 1011) respectively, p = 0.41 and 0.49 respectively; cccDNA: 4.9 x 106 (range 0 to 7.3 x 107) vs 1.8 x 105 (range 0 to 3.7 x 107) and vs 8.6 x 105 (range 0 to 2.6 x 107) respectively, p = 0.10 and 0.11, respectively].
Total HBV and cccDNA Levels of Patients on Lamivudine for 52 Wk
For the 82 patients receiving lamivudine, there was a significant reduction in the total HBV DNA levels from a median level of 1.58 x 109 (range 17,600 to 5.3 x 1011) copies/ml at baseline to a median level of 66,600 (range <200 to 2.52 x 1010) copies/ml at week 24 (p< 0.001) and a median level of 169,000 (range <200 to 2.45 x 1010) copies/ml at week 52 (p< 0.001). There was also a significant reduction in the cccDNA level from a median level of 3.0 x 106 (range <10,000 to 6.15 x 107) copies/ml at baseline to a median level of 33,476 (range <10,000 to 1.63 x 107) copies/ml at week 24 (p< 0.001) and a median level of 48,694 (range <10,000 to 5.6 x 106) copies/ml at week 52 (p< 0.001).
Comparisons of HBV DNA and cccDNA Levels between Patients on Lamivudine and Placebo
The median total HBV DNA and cccDNA levels at baseline, week 24, and week 52 in lamivudine treated patients and control patients are plotted in logarithmic scale in Figure 1. Patients receiving lamivudine had a significantly greater reduction of median logarithmic total HBV DNA levels at week 24 and week 52 compared to those of control patients [4.4 (range -0.55 to 7.07) vs 0.64 (range -0.78 to 2.86), respectively, p< 0.001 at week 24; 4.1 (range -0.76 to 7.64) vs 0.13 (range -3.63 to 2.19), respectively, p< 0.001 at week 52]. Similarly, lamivudine treated patients had a significantly greater reduction of median logarithmic cccDNA levels at week 24 and week 52 compared to those of control patients [2.21 (range -2.1 to 7.45) vs 0.31 (range -0.84 to 7.87) respectively, p< 0.001 at week 24; 2.12 (-0.93-7.35) vs 0.20 (range -1.07 to 3.05) respectively, p< 0.001 at week 52].
Effects of YMDD Mutations on cccDNA Levels
Fifteen patients (18.3%) developed YMDD mutations by 52 wk. Of these, six patients (40%) also had the concomitant mutations of rtL180M. The median logarithmic reduction of cccDNA at week 52 was significantly less in patients with YMDD mutations compared to patients without YMDD mutations [0.80 (range -0.58 to 6.17) vs 2.35 (range -0.64 to 7.35), respectively, p< 0.001] (Fig. 2). Though the median cccDNA level at week 52 in the 15 patients with YMDD mutations was less than that at baseline [173,539 (range <10000 to 3,551,992) vs 924,671 (range 108,149-31,209,948) copies/ml respectively], the difference was not statistically significant (p = 0.36). Patients with subsequent development of YMDD mutations by week 52 in fact already had a lower median logarithmic reduction of cccDNA at week 24 than patients without YMDD mutations [1.65 (range -0.27 to 6.17) vs 2.27 (range -0.21 to 7.35) respectively, p = 0.015] (Fig. 2).
Effects of Total HBV and cccDNA Reduction at 24 Wk on the Development of YMDD Mutations
Compared to patients without YMDD mutations at week 52, patients with YMDD mutations at week 52 had a significantly lower median logarithmic reduction of total HBV DNA levels and cccDNA levels at week 24 [total HBV DNA: 4.44 (range -0.24 to 7.07) vs 3.65 (-0.55 to 6.84), respectively, p = 0.02; cccDNA: 2.27 (-0.21 to 7.45) vs 1.65 (range -0.07 to 6.17), respectively, p = 0.016)]. The chances of YMDD mutations at week 52 for patients with respect to the different magnitude of logarithmic reduction in the total HBV DNA levels and cccDNA levels at week 24 are listed in Table 2. These data suggest that greater viral suppression as reflected by total HBV DNA and cccDNA at week 24 was associated with a lower chance of YMDD mutations at week 52.
1. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev 2000;64: 51-68.
2. Mason WS, Halpern MS, England JM, et al. Experimental transmission of duck hepatitis B virus. Virology 1983;131: 375-84.
3. Tagawa M, Omata M, Okuda K. Appearance of viral RNA transcripts in the early stage of duck hepatitis B virus infection. Virology 1986;152: 477-82.
4. Summers J, Smith PM, Horwich AI. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J Virol 1990;64: 2819-24.
5. Summers J, Smith PM, Huang MJ, et al. Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus. J Virol 1991;65: 1310-7.
6. Lenhoff RJ, Luscombe CA, Summers J. Competition in vivo between a cytopathic variant and a wild-type duck hepatitis B virus. Virology 1988;251: 85-95.
7. Moraleda G, Saputelli J, Aldrich CE, et al. Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus. J Virol 1997;71: 9392-9.
8. Addison WR, Walters KA, Wong WWS, et al. Half-life of the duck hepatitis B virus covalently closed circular DNA pool in vivo following inhibition of viral replication. J Virol 2002;76: 6356-63.
9. Liu MC, Yu M, Zhang NL, et al. Dynamic analysis of hepatitis B virus DNA and its antigens in 2.2.15 cells. J Viral Hepat 2004;11: 124-9.
10. Wu CH, Ouyang EC, Walton CM, et al. Human hepatocytes transplanted into genetically immunocompetent rats are susceptible to infection by hepatitis B virus in situ. J Viral Hepat 2001;8: 111-9.
11. Chen Y, Sze J, He ML. HBV cccDNA in patients' sera as an indicator for HBV reactivation and an early signal of liver damage. World J Gastroenterol 2004;10: 82-5.
12. Wong DKH, Yuen MF, Yuan HJ, et al. Quantification of serum and intrahepatic total hepatitis B virus DNA and covalently closed circular DNA in chronic hepatitis B patients. Hepatology 2004;40: 727-37.
13. Lai CL, Chien RN, Leung NWY, et al. A one-year trial of lamivudine for chronic hepatitis B. N Engl J Med 1998;339: 61-8.
14. Dienstag JL, Schiff ER, Wright TL, et al. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999;341: 1256-63.
15. Zhou T, Guo JT, Nunes FA, et al. Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections. J Virol 2000;74: 11754-63.
16. Yokosuka O, Omata M, Imazeki F, et al. Changes of hepatitis B virus DNA in liver and serum caused by recombinant leukocyte interferon treatment: Analysis of intrahepatic replicative hepatitis B virus DNA. Hepatology 1985;5: 728-34.
17. Lai CL, Yuen MF, Hui CK, et al. A comparison of the efficacy of lamivudine and famciclovir in Asian patients with chronic hepatitis B: Results of 24 weeks of therapy. J Med Virol 2002;67: 334-8.
18. Yuen MF, Sablon E, Hui CK, et al. Factors predicting hepatitis B virus DNA breakthrough in patients receiving prolonged lamivudine therapy. Hepatology 2001;34(4 Pt 1):785-91.
19. Stoll-Becker S, Repp R, Gleve D, et al. Transcription of hepatitis B virus in peripheral blood mononuclear cells from persistently infected patients. J Virol 1997;71: 5399-407.
20. Torii N, Hasegawa K, Joh R, et al. Configuration and replication competence of hepatitis B virus DNA in peripheral blood mononuclear cells from chronic hepatitis B patients and patients who have recovered from acute self-limited hepatitis. Hepatol Res 2003;25: 234-3.
21. Cabrerizo M, Bartolom J, Caramelo C, et al. Molecular analysis of hepatitis B virus DNA in serum and peripheral blood mononuclear cells from hepatitis B surface antigen-negative cases. Hepatology 2000;32: 116-23.
22. Zhu Y, Yamamoto T, Cullen J, et al. Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol 2001;75: 311-22.
23. Tsiquaye KN, Slomka MJ, Maung M. Oral famciclovir against duck hepatitis B virus replication in hepatic and nonhepatic tissues of ducklings infected in ovo. J Med Virol 1994;42: 306-10.
24. Melegari M, Scaglioni PP, Wands JR. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. Hepatology 1998;27: 628-33.
25. 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: 939-45.