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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References

Summary

Background

Appearance of immunoglobulin class M antibody against hepatitis B core antigen is a predictor of beneficial response to interferon-alpha therapy in chronic hepatitis B patients, but its relationship with the efficacy of lamivudine therapy remains unclear.

Aim

To investigate the outcome of lamivudine therapy in chronic hepatitis B patients with immunoglobulin class M antibody against hepatitis B core antigen and acute exacerbation.

Methods

Chronic hepatitis B patients with acute exacerbation receiving a national-wide therapeutic trial of 18-month lamivudine monotherapy were enrolled for the analysis. Four consecutive seronegative patients were recruited as individual matching controls of one positive subject. Immunoglobulin class M antibody against hepatitis B core antigen in serum was assayed monthly by an automated microparticle enzyme immunoassay.

Results

Fifteen (8.9%) of 167 chronic hepatitis B patients with acute exacerbation were seropositive for IgM anti-HBc. Thus 60 seronegative patients were consecutively recruited as control group. At the end of therapy, two (13.3%) of the 15 seropositive patients achieved a sustained response, significantly lower than 26 (43.3%) of the control group.

Conclusions

Appearance of immunoglobulin class M antibody against hepatitis B core antigen in chronic hepatitis B patients with acute exacerbation is a predictor of poor response to lamivudine monotherapy. This is clinically relevant to the decision-making in treating chronic hepatitis B patients with acute exacerbation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References

Lamivudine has become a main therapeutic option for treating hepatitis B virus (HBV) infection.1–5 Its antiviral effects against HBV have been established both in vitro and in vivo.6–8 Clinical trials revealed that lamivudine is effective in reducing HBV replication and preventing the progression of chronic liver disease.4, 5, 9, 10 Although complete suppression of viral replication, as determined by negative serum HBV DNA, may be achieved in most patients, clearance of hepatitis B virus e antigen (HBeAg) with antibody (anti-HBe) seroconversion was seen only in a minority of patients.1, 3, 5 The mechanisms of successful lamivudine therapy thus remain to be elucidated.2, 11

Several studies have established the association between serum immunoglobulin class M against hepatitis B core antigen (IgM anti-HBc) and the different phases of HBV infection.12–16 With a quantitative assay, one can identify patients with acute HBV infection or HBV-related chronic hepatitis with IgM anti-HBc in the serum.15–18 It is suggested that a titre of 600 U of IgM anti-HBc by the method of IgM capture assay forms a lower cutoff value for acute hepatitis B, and moderate titres of 30–600 U for chronic hepatitis B (CH-B).15 The appearance of IgM anti-HBc in serum is detected in 14–16% of patients with CH-B.13–17 It indicates the induction of humoral immune response to hepatitis B core antigen (HBcAg) during the course of chronic HBV infection. IgM anti-HBc is a good surrogate marker for hepatocellular damage immunopathologically related to HBV but is not directly involved in the elimination of the infected hepatocytes.18 Some investigators have also demonstrated the correlation of the IgM anti-HBc with hepatic necroinflammatory activities of liver damage.12–14, 16, 17 The appearance IgM anti-HBc in serum is shown to be a predictor of beneficial response to interferon-alpha (IFN-α) therapy,18–20 but its relationship with outcome of lamivudine therapy remains unclear. In this study, we compared the outcome of lamivudine therapy for 18 months in consecutive IgM anti-HBc positive patients with that of negative patients enrolled from a national-wide therapeutic trial for CH-B with acute exacerbation (AE) in Taiwan.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References

Patients

A total of 167 CH-B patients with AE receiving a national-wide therapeutic trial of 18-month lamivudine therapy in Chi-Mei Medical Center (Tainan, Taiwan) were enrolled for the analysis. The 18-month regimen of lamivudine therapy is recommended by the National Health Insurance of Taiwan (NHIT) and the cost is fully covered by NHIT as well. AE or acute flare of CH-B is defined as an abrupt increase of serum alanine aminotransferase (ALT) level to greater than five times the upper limit of normal (ULN = 40 U/L), i.e. ALT >200 U/L.21, 22 Four consecutive patients negative for IgM anti-HBc were recruited as individual matching controls of one positive subject. The study protocol conformed to the 1975 Declaration of Helsinki and approved by the local ethical committee. All patients gave written informed consent and met the following criteria before the enrolment of treatment: presence of hepatitis B surface antigen (HBsAg) and HBeAg in serum for at least 6 months; with AE of CH-B; seropositivity for HBV DNA on at least two occasions 1 month apart before entry; and undetectable tyrosine-methionine-aspartate-aspartate (YMDD) mutants. Concurrent hepatitis C virus (HCV) or hepatitis delta virus (HDV) infections were excluded, and none had human immunodeficiency virus (HIV) infection.

Oral lamivudine (Zeffix; GlaxoWellcome, Greenford, UK) 100 mg per day was administered for 18 months. Sustained response (SR) to the treatment is defined as a sustained loss of HBeAg with seroconversion to anti-HBe, together with the disappearance of serum HBV DNA (referred to as a virological response), and the normalization of ALT levels (referred to as a biochemical response), as well as neither biochemical nor virological relapse at least for 6 months after cessation of lamivudine. A transient normalization of serum ALT levels followed by a relapse either after the end of treatment or during lamivudine therapy is defined as partial response (PR). Non-response (NR) is defined as the lack of either a biochemical or a virological response to the therapy. Patients’ characteristics and treatment outcome of IgM anti-HBc positive patients compared with the control group are displayed in Table 1.

Table 1.  Characteristics of immunoglobulin class M antibody against hepatitis B core antigen (IgM anti-HBc) positive patients compared with control group
DataIgM anti-HBc
positive (n = 15)Control group (n = 60)P-value
  1. SR, sustained response.

  2. a Fisher's exact test.

  3. b Mann–Whitney U-test.

  4. c Digits represent mean ± SD, median (min–max).

  5. d One with dual hepatitis B virus genotype B and C.

  6. e At the end of treatment.

  7. f Also had seroconversion to anti-HBe.

Sex (M/F)8/733/271.000a
Age (y/o)32.7 ± 7.8c 30.5(22–50)40.5 ± 8.9 33.5(18–65)0.766b
Alanine aminotransferase (U/L)720.1 ± 370.6 710 (250–2048)651.2 ± 739.0 690 (230–2574)0.226b
Total bilirubin8.3(2.3–19.9)9.1(1.8–27.7)0.568a
<5 mg/dL836 
>5 mg/dL724 
HBV DNA (pg/mL)5633.7 ± 4341.2 (286–12758)4789.5 ± 3789.7 (240–11748)0.755b
<1.8001.000a
1.8–10000 
100–1000522 
>10001038 
HBV genotype (A-B-C-D-E-F-G)0-9-6-0-0-0-00-30-29-0-0-0-0d0.575a
Ascites3160.747a
Prothrombin time seconds prolonged
 3–511431.000a
 >5417 
Normalization of ALT levelse4290.131a
Loss of HBeAge, f2f280.020a
Undetectable HBVDNAe3300.036a
SR2 (13.3%)26 (43.3%)0.039a

Follow-up and virological assays

Patients with AE were treated with oral lamivudine and followed up weekly or biweekly. Patients with severe AE may be associated with hepatic decompensation including ascites, jaundice, prolonged prothrombin time; they were therefore recommended a hospital care. After the AE episode, they were followed up monthly with continuing lamivudine therapy. Routine follow-up studies included clinical assessment, and conventional liver biochemical tests. Serum IgM anti-HBc was assayed at pre-treatment and each follow-up monthly using a microparticle enzyme immunoassay kit (AxSYM CORE-M; Abbott Laboratories, Abbott Park, IL, USA) according to manufacturer's instructions. The data were expressed as IgM anti-HBc index (=sample rate/index calibrator mean rate). Samples with Index values <0.80 are considered non-reactive for IgM anti-HBc. Samples with Index values in the range of 0.80–1.20 are considered grey zone reactive. Serum HBV DNA was quantified using a Hybrid Capture assay (Digene Hybrid Capture II HBV DNA Test, Digene Corp., Gaithersburg, MD, USA). The detection limit of HBV DNA is 0.5 pg/mL or 1.4 × 105 copies/mL. Serum samples of each follow-up were collected for detection of IgM anti-HBc and HBV DNA. Abdominal ultrasonography was performed when indicated to detect ascites. Biochemical tests were carried out in the clinical pathology laboratories of Chi-Mei Medical Center with routine automated techniques. All patients received a post-treatment assessment at 6 months after the end of 18-month of lamivudine treatment.

HBV genotyping and YMDD mutation assay

HBV genotyping was performed by line probe assay using commercially available kit (INNO-LiPA HBV Genotyping; Innogenetics Technology, Ghent, Belgium) according to the manufacturer's instructions. Tyrosine–methionine–aspartate–aspartate motif (YMDD) mutants were also detected by line probe assay using INNO-LiPA HBV DR Kit (Innogenetics Technology), and were confirmed with DNA sequencing of PCR products from each serum sample using CEQ2000 DNA Analysis System (Beckman Coulter, Inc., Fullerton, CA, USA). To avoid false-positive result, instructions to prevent cross contamination were strictly followed. In addition, an aliquot of HBV-positive serum containing 108 copies/mL of HBV DNA was serially diluted and used as the standard of HBV DNA quantitation and for determination of the sensitivity of HBV genotyping. The sensitivity of these two assays approximated 100 copies of HBV DNA.

Statistical analysis

Statistical analysis was performed with the SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA). Mann–Whitney U-test was used to compare two unpaired continuous data and Fisher's exact test was used to compare categorical data between two groups. Difference with a P-value <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References

The clinical features and laboratory data including gender, range of age, levels of ALT, total bilirubin, prolonged prothrombin time, HBV DNA, distribution of HBV genotypes and development of ascites were comparable between the 15 IgM anti-HBc positive patients and control group (Table 1).

Fluctuations of serum IgM anti-HBc were detected during the course of lamivudine therapy. Mostly, their IgM anti-HBc index values are in gray zone reactive and 13 of the 15 patients become non-reactive in terms of IgM anti-HBc index value <0.80 at the end of 18-month lamivudine therapy (data not shown).

At the end of 18-month lamivudine treatment, two lost HBeAg with anti-HBe seroconversion, 3 with undetectable HBVDNA and four had ALT normalization in IgM anti-HBc positive group; while 28, 29, and 30 of the control group achieved such conditions (Table 1), and with a P-value 0.020, 0.036, and 0.131 respectively (Fisher's exact test).

Totally, two of 15 IgM anti-HBc positive patients (13.3%) achieved a SR to lamivudine therapy while 26 of the control group (43.3%) had a SR, significantly higher than that of IgM anti-HBc positive group (P = 0.039, Fisher's exact test). YMDD mutant virus with YIDD mutation at the end of treatment was detected in one of the control group who did not respond to the therapy (data not shown). Dual HBV genotype B and C infection was also detected in pre-treatment serum of this patient (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References

It has been reported that IgM antibodies to viral antigens frequently persist in chronic viral infections with ongoing viral replication. Persistence of IgM anti-HBc has been identified in CH-B and chronic HDV patients.12–17 During IFN-α treatment of CH-B patients, a significant rise in serum IgM anti-HBc levels is detected in a large proportion of responders, followed by ALT flare-ups and subsequently HBeAg seroconversion.18–20 Thus appearance of IgM anti-HBc is considered as a predictor of beneficial response to IFN-α treatment. In a meta-analysis of 15 clinical trials, the overall response rate was 33% among the patients treated with IFN-α for a course of 4–6 months, as compared with 12% among controls.23 A study by Marinos et al.18 showed that 82.4% of IFN-α responders had a pre-treatment IgM anti-HBc index >0.3 compared with 25% of non-responders, Contrarily, in this study, our data showed that the SR rate to 18-month lamivudine therapy in IgM anti-HBc positive patients was significantly lower than that of negative patients with AE. This indicates that IgM anti-HBc is a predictor of poor response to lamivudine therapy. The reason of these contradictory results is not yet fully understood. Pharmacologically, lamivudine is a reverse-transcriptase inhibitor, which has little effect on the episomal template of HBV, i.e. closed circular (ccc) DNA, a source of viral replication.2, 6 Whereas IFN-α, belonging to type I interferons, may exert widely overlapping pleiotropic effects including inhibition of cell growth, anti-tumour action, involvement in haematopoiesis, and regulatory effects on cellular and humoral immune responses.24–26 In humans, IFN-α antiviral action is mediated by induction of at least three major proteins, 2′-5′-oligoadenylate synthetase, protein kinase R, and MxA protein.24, 25 For example, secretion of HBV viral proteins was profoundly reduced in MxA – expressing HuH7 cells.27 IFN-α likely acts by combining stimulation of the immune response and a direct anti-viral effect.24, 25 These distinct differences in action mechanisms between lamivudine and IFN-α might account for the different treatment outcome in CH-B patients with IgM anti-HBc. Moreover, accumulating evidence suggests that cell-mediated immune responses play an important role in successful lamivudine therapy.28–34 Thus failure to mount an adequate cell-mediated immune response during lamivudine therapy in IgM anti-HBc positive patients might also lead to poor response. The detailed mechanisms warrant further investigations.

The SR rate of control group, i.e. 43.3%, is remarkably higher than that of lamivudine trial in Asia.1, 35, 36 The reason of such a high response rate might be due to that the pre-treatment ALT levels of CH-B patients with AE in this study are higher than those of lamivudine trial patients in Asia. It is reported that CH-B patients on AE with a pre-treatment ALT>5X ULN may achieve a rate up to 60% of sustained HBeAg response to lamivudine therapy.32, 36, 37

In conclusion, the data presented suggest that the appearance of serum IgM anti-HBc in CH-B patients is a predictor of poor response to lamivudine therapy. This result is clinically relevant to the decision-making in treating chronic hepatitis B with AE.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References

The authors do not have a commercial or other association that might pose a conflict of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References

This work was supported by the Chi-Mei Foundation CMFHT9102, Tainan, Taiwan, to S.L. Tsai. We thank Miss Chin-Li Lu, statistician, Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan, for statistical analysis of data.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Acknowledgements
  9. References
  • 1
    Lai CL, Chien RH, Leung NWY, et al. A one-year trial of lamivudine for chronic hepatitis B. N Engl J Med 1998; 339: 618.
  • 2
    Jarvis B, Faulds D. Lamivudine. A review of its therapeutic potential in chronic hepatitis B. Drugs 1999; 58: 10141.
  • 3
    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: 125663.
  • 4
    Liaw YF, Leung NWY, Chang TT, et al. Effects of extended lamivudine therapy in Asian patients with chronic hepatitis B. Gastroenterology 2000; 119: 17280.
  • 5
    Jonas MM, Kelley DA, Mizerski J, et al. Clinical trial of lamivudine in children with chronic hepatitis B. N Engl J Med 2002; 346: 170613.
  • 6
    Doong SL, Tsai CH, Schinazi RF, et al. Inhibition of the replication of hepatitis B virus in vitro by 2′, 3′-Dideoxy-3′-thiacytidine and related analogues. Proc Natl Acad Sci USA 1991; 88: 84959.
  • 7
    Dienstag JL, Perillo RP, Schiff ER, et al. A Preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med 1995; 333: 165761.
  • 8
    Mckenzie R., Fried MW, Sallie R, et al. Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B. N Engl J Med 1995; 333: 1099105.
  • 9
    Liaw YF, Sung JJY, Chow WC, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004; 351: 152131.
  • 10
    Dienstag JL, Goldin RD, Heathcote EJ, et al. Histological outcome during long-term lamivudine therapy. Gastroenterology 2003; 124: 10517.
  • 11
    Dusheiko G, Bertoletti A. Resistance to lamivudine therapy: is there more than meets the eye? Gut 2005; 54: 910.
  • 12
    Zavaglia C, Mondazzi L, Maggi G, et al. Are alanine aminotransferase, hepatitis B virus DNA or IgM antibody to hepatitis B core antigen serum levels predictors of histological grading in chronic hepatitis B? Liver 1997; 17: 837.
  • 13
    Mels GC, Bellati G, Leandro G, et al. Fluctuations in viremia, aminotransferases and IgM antibody to hepatitis B core antigen in chronic hepatitis B patients with disease exacerbations. Liver 1994; 14: 17581.
  • 14
    Smith HM, Lau JY, Davies SE, et al. Significance of serum IgM anti-HBc in chronic hepatitis B virus infection. J Med Virol 1992; 36: 1620.
  • 15
    Gerlich WH, Uy A, Lambrecht F, Thomssen R. Cutoff levels of immunoglobulin M antibody against viral core antigen for differentiation of acute, chronic, and past hepatitis B virus infections. J Clin Microbiol 1986; 24: 28893.
  • 16
    Sjogren M, Hoofnagle JH. Immunoglobulin M antibody to hepatitis B core antigen in patients with chronic type B hepatitis. Gastroenterology 1985; 89: 2528.
  • 17
    Surrenti C, Ambu S, Patussi V, et al. Diagnostic significance of anti-HBc IgM (RIA) in healthy HBsAg carriers and in chronic hepatitis B. J Med Virol 1986; 18: 22934.
  • 18
    Marinos G, Smith HM, Naoumov NV, Williams R. Quantitative assessment of serum IgM anti-HBc in the natural course and during interferon treatment of chronic hepatitis B virus infection. Hepatology 1994; 19: 30311.
  • 19
    Scully LJ, Brown D, Lloyd C, Shein R, Thomas HC. Immunological studies before and during interferon therapy in chronic HBV infection: identification of factors predicting response. Hepatology 1990; 12: 11117.
  • 20
    Chen G, Karayiannis P, McGarvey MJ, et al. Subclasses of antibodies to hepatitis B core antigen in chronic HBV infection: changes during treatment with alpha interferons and predictors of response. Gut 1989; 30: 11238.
  • 21
    Perrillo R. Acute flares in chronic hepatitis B: the natural and unnatural history of an immunologically mediated liver disease. Gastroenterology 2001; 20: 100922.
  • 22
    Tsai SL, Chen PJ, Lai MY, et al. Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. J Clin Invest 1992; 89: 796.
  • 23
    Wong DKH, Cheung AM, ÒRourke K, et al. Effect of alpha-interferon treatment in patients with hepatitis B e antigen-positive chronic hepatitis B: a meta-analysis. Ann Intern Med 1993; 119: 31923.
  • 24
    Stark GR, Kerr IM, Williams BR, et al. How cells respond to interferons. Annu Rev Biochem 1998; 67: 22764.
  • 25
    Levy DE, Marié I, Prakash A. Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity. Curr Opin Immunol 2003; 15: 5258.
  • 26
    Müller U, Steinhoff U, Reis LFL, et al. Functional role of type I and type II interferons in antiviral defense. Science 1994; 264: 191824.
  • 27
    Gordien E, Rosmorduc O, Peltekian C, et al. Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein. J Virol 2001; 75: 268491.
  • 28
    Boni C, Bertoletti A, Penna A, et al. Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B. J Clin Invest 1998; 02: 96875.
  • 29
    Hultgren C, Weiland O, Milich DR, Sallberg M. Cell-mediated immune responses and loss of hepatitis Be antigen (HBeAg) during successful lamivudine and famciclovir combination therapy for chronic replicating hepatitis B virus infection. Clin Infet Dis 1999; 29: 15757.
  • 30
    Boni C, Penna A, Ogg GS, et al. Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy. Hepatology 2001; 33: 96371.
  • 31
    Lin CL, Tsai SL, Lee TH, Chien RN, Liao SK, Liaw YF. High frequency of functional anti-YMDD and – mutant cytotoxic T lymphocytes after in vitro expansion correlates with successful response to lamivudine therapy for chronic hepatitis B. Gut 2005; 54: 15261.
  • 32
    Liaw YF, Tsai SL, Chien RN, et al. Prednisolone priming enhances Th1 response and efficacy of subsequent lamivudine therapy in patients with chronic hepatitis B. Hepatology 2000; 32: 6049.
  • 33
    Tsai SL, Sheen IS, Chien RN, et al. Activation of Th1 immunity is a common immune mechanism for the successful treatment of hepatitis B and C: tetramer assay and therapeutic implications. J Biomed Sci 2003; 10: 12035.
  • 34
    Tsai SL, Lee TH, Chien RN, et al. A method to increase tetramer-staining efficiency of CD8+ T cells with MHC-peptide complexes: therapeutic applications in monitoring cytotoxic T lymphocyte activity during hepatitis B and C treatment. J Immunol Methods 2004; 285: 7187.
  • 35
    Chang TT, Lai CL, Chien RN, et al. Four years of lamivudine treatment in Chinese patients with chronic hepatitis B. J Gastroenterol Hepatol 2004; 19: 127682.
  • 36
    Liaw YF. Results of lamivudine trials in Asia. J Hepatol 2003; 39: S1115.
  • 37
    Chien RN, Yeh CT, Tsai SL, et al. The determinants for sustained HBeAg response to lamivudine therapy. Hepatology 2003; 8: 126773.