Usefulness of the Fibrosis-4 index and alanine aminotransferase at 1 year of nucleos(t)ide analog treatment for prediction of hepatocellular carcinoma in chronic hepatitis B patients.

AIM: Nucleos(t)ide analogs do not completely prevent hepatocellular carcinoma (HCC) in chronic hepatitis B virus infection. This study aimed to evaluate the dynamics of a non-invasive liver fibrosis marker, the Fibrosis-4 (FIB-4) index, for predicting HCC development. METHODS: Among a total of 882 chronically hepatitis B virus infection-infected patients who were treated with nucleos(t)ide analogs, 472 patients without HCC history whose FIB-4 at baseline and 1 year of treatment was obtained were evaluated for the incidence of HCC. RESULTS: The median FIB-4 was 2.00 at baseline and was significantly reduced to 1.58 at 1 year (P < 0.001), but the reduction was small at 2 years or later. When a receiver operating characteristic analysis of FIB-4 was performed to predict HCC within 5 years, the area under the curve of FIB-4 at 1 year was higher than that at baseline (0.676 vs. 0.599). The HCC incidence was significantly higher in patients with FIB-4 ≥1.58 than in those with FIB-4 <1.58 (14.8% vs. 3.6% at 10 years, P < 0.001). Additionally, an abnormal alanine aminotransferase (≥31 U/L) at 1 year was an independent risk for HCC. When a fibrosis and alanine aminotransferase-1 (FAL-1) score was evaluated as an applicable number of FIB-4 ≥1.58, and alanine aminotransferase ≥31 as 0, 1, and 2, the HCC risk in patients with score 2 was significantly higher than in those with score 1 or score 0 (24.1% vs. 9.8% vs. 0.7% at 10 years, P < 0.001). CONCLUSIONS: FIB-4 ≥1.58 and alanine aminotransferase ≥31 at 1 year of nucleos(t)ide analog was an independent risk factor for HCC development, and a score using these factors stratified the risk of HCC.

Switching to Tenofovir Alafenamide Fumarate in Chronic Hepatitis B Patients Who Had Detectable HBV DNA during Treatment with Entecavir.

Nucleos(t)ide analogues (NAs) suppress hepatitis B virus (HBV) replication, but the risk of hepatocellular carcinoma still remains. The presence of detectable HBV DNA in the serum during NA therapies for chronic hepatitis B patients has been reported to be associated with the risk of hepatocellular carcinoma. In this study, we investigated the antiviral effect of switching from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) in chronic hepatitis B patients who had detectable HBV DNA in the serum at least once within a year. Among a total of 77 cases in 7 hospitals that switched NAs from ETV to TAF, 23 patients with detectable HBV DNA in a year before switching were analyzed. When the detection frequencies of HBV DNA in the 1st and 2nd years after switching to TAF were analyzed, they were significantly lower than those in the year before switching (68.8% vs. 34.1% for the 1st year and 21.3% for the 2nd year, P < 0.001 for both). The HBsAg decline tended to be larger after switching than before (-2.5% vs. -3.0% for 1st year and -3.1% for 2nd year), but the difference was not significant. One patient died of a cardiovascular event 11 months after the treatment switch, but no adverse effects due to TAF including renal function were observed. In conclusion, it was suggested that switching from ETV to TAF might be effective to suppress the HBV DNA level further in patients whose HBV DNA is detectable, even if at a very low level.

Non-Achievement of Alanine Aminotransferase Normalization Associated with the Risk of Hepatocellular Carcinoma during Nucleos(t)ide Analogue Therapies: A Multicenter Retrospective Study.

Patients with a chronic hepatitis B virus (HBV) infection who are treated with nucleos(t)ide analogues (NAs) are still at risk for hepatocellular carcinoma (HCC), and it has been clinically questioned whether patients with a high risk of HCC can be identified efficiently. We aimed to clarify the risk factors associated with the development of HCC during NA therapies. A total of 611 chronically HBV-infected patients without a history of HCC, who were treated with NAs for more than 6 months (median 72 months), from 2000 to 2021, were included from 16 hospitals in the Tohoku district in Japan. Incidences of HCC occurrence were analyzed with clinical factors, including on-treatment responses. Alanine aminotransferase (ALT) normalization, based on the criteria of three guidelines, was analyzed with other parameters, including the age−male−ALBI−platelets (aMAP) risk score. During the observation period, 48 patients developed HCC, and the cumulative HCC incidence was 10.6% at 10 years. Non-achievement of ALT normalization at 1 year of therapy was mostly associated with HCC development when ALT ≤ 30 U/L was used as the cut-off (cumulative incidence, 19.9% vs. 5.3% at 10 years, p < 0.001). The effectiveness of the aMAP risk score at the start of treatment was validated in this cohort. A combination of an aMAP risk score ≥ 50 and non-achievement of ALT normalization could stratify the risk of HCC significantly, and notably, there was no HCC development in 103 patients without these 2 factors. In conclusion, non-achievement of ALT normalization (≤30 U/L) at 1 year might be useful in predicting HCC during NA therapies and, in combination with the aMAP risk score, could stratify the risk more precisely.

Comparison of hepatitis B virus genotypes B and C among chronically hepatitis B virus-infected patients who received nucleos(t)ide analogs: A multicenter retrospective study.

AIM: Hepatitis B virus genotype B (HBV/B) has been reported to have less risk of liver cirrhosis and hepatocellular carcinoma (HCC), but long-term observation has rarely been reported. We aimed to clarify the characteristics of HBV/B in nucleos(t)ide analog-treated patients in an area where HBV/B is more prevalent than in other areas of Japan. METHODS: A total of 498 chronically HBV-infected patients treated with nucleos(t)ide analog (lamivudine, entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide fumarate) for >6 months (mean 70.6 months) were included from nine hospitals in northeast Japan. The frequencies of hepatitis B surface antigen loss and HCC occurrence were analyzed. RESULTS: Among 427 patients whose genotype could be determined, 34.0% and 64.4% were infected with HBV/B and genotype C (HBV/C), respectively. The age of patients with HBV/B was significantly older than those with HBV/C (57.7 vs. 48.1). The cumulative rate of hepatitis B surface antigen loss was significantly higher in HBV/B than in HBV/C (3.6% vs. 0.7% at 10 years). Among 480 patients without HCC history, HCC occurrence was found in 40 patients (13.4% at 10 years). There was no cumulative rate difference of HCC occurrence among the genotypes, but after propensity score matching for age/sex, it was significantly lower in HBV/B than in HBV/C (5.3% vs. 18.5% at 10 years). CONCLUSIONS: Although a lower rate of HCC occurrence in HBV/B was shown by an age/sex-matched analysis than that in HBV/C, patients with HBV/B were significantly older and had a comparative risk of HCC occurrence in nucleos(t)ide analog-treated patients.

Shifting hepatitis B virus genotypes of acute hepatitis B patients in northeast Japan.

It has been reported that acute hepatitis B (AHB) patients with genotype A HBV (HBV/A) have been increasing since the 1990s in metropolitan areas in Japan. However, little is known about the trends of HBV genotypes in AHB patients in northeast Japan where genotype B HBV (HBV/B) prevails more than in other areas. In this study, we aimed to clarify the changes in the HBV genotypes and clinical characteristics of AHB patients in this area. HBV genotypes were determined by direct sequencing (n = 125) or enzyme immunoassay (n = 9) using serum samples from AHB patients including fulminant hepatitis in 1987-2014. Among 134 patients, 26 (19%), 33 (25%), and 75 (56%) patients were infected with HBV of genotypes A, B, and C, respectively. HBV/A emerged from 2001 and the percentage was increased gradually up to 48% in 2010-2014, whereas HBV/B was reduced from 40% in 1987-1994 to 10% in 2010-2014. Phylogenetic analysis showed that three major subgenotype A2 strains had come into this area serially. The levels of HBV DNA and prothrombin time were higher in HBV/A patients than other genotypes. This study could not show significant difference in the HBsAg-positive period among genotypes nor the effect of nucleoside analogues to shorten the HBsAg-positive period. A higher level of initial HBV DNA was associated with late disappearance of HBsAg. In conclusion, the percentage of HBV/A in AHB patients has been increasing in northeast Japan since 2001, which is later than metropolitan areas, whereas that of HBV/B is decreasing.

Association between S21 substitution in the core protein of hepatitis B virus and fulminant hepatitis.

BACKGROUND: The viral factors of hepatitis B virus (HBV), such as genotypes and mutations, were reported to affect the development of fulminant hepatitis B (FHB), but the mechanism is still unclear. OBJECTIVES: To investigate HBV mutations associated with FHB, especially in the subgenotype B1/Bj HBV (HBV/B1), which are known to cause FHB frequently in Japan. STUDY DESIGN: A total of 96 serum samples from acute self-limited hepatitis B (AHB) patients and 13 samples from FHB patients were used for full-genome/partial sequencing. A total of 107 chronic infection patients with HBV were also examined for the distribution of mutants. RESULTS: In the analysis of full-genome sequences of HBV/B1 (FHB, n=11; non-FHB, n=35) including those from the databases, mutations at nt 1961 [T1961V (not T)] and nt 1962 [C1962D (not C)], which change S21 in the core protein, were found more frequently in FHB than in non-FHB (100% vs. 20%, 55% vs. 3%, respectively). When our FHB and AHB samples were compared, T1961V and C1962D were significantly more frequent in FHB than in AHB, both in the overall analysis (46% vs. 6%, 39% vs. 3%, respectively) and in HBV/B1 (100% vs. 29%, 100% vs. 14%, respectively). A newly developed PCR system detecting T1961V showed that HBV/B1 and low viral load were independent factors for the mutation among chronic infection patients. CONCLUSIONS: T1961V/C1962D mutations were found frequently in FHB, especially in HBV/B1. The resulting S21 substitution in the core protein may play important roles in the development of FHB.

Enhanced replication of hepatitis B virus with frameshift in the precore region found in fulminant hepatitis patients.

BACKGROUND: The genotype B of hepatitis B virus (HBV) was reported to associate with fulminant hepatitis (FH). We aimed to clarify the characteristics of HBV obtained from FH patients in an area of Japan where genotype B HBV is prevalent. METHODS: Using serum samples of 16 HBV-associated FH patients, partial HBV sequences were determined. The effects of HBV mutation/insertion/deletion were evaluated using an in vitro HBV replication system. RESULTS: Of the 16 HBV isolates, 31% belonged to subgenotype B1/Bj, 38% were subgenotype B2/Ba, and 31% were subgenotype C2/Ce. Notably, the single nucleotide insertion/deletion that resulted in a frameshift of the precore protein was found exclusively in 60% of B1/Bj strains. An in vitro study showed that all of the frameshift mutants had significantly higher amounts of HBV DNA than did the wild type. One of the isolates had a novel insertion of A between nucleotides 1900 and 1901, which resulted in a 3-nucleotide change within the Kozak sequence of the core protein and enhanced the core protein expression in vitro. CONCLUSIONS: The frameshift insertion/deletion in the precore region enhanced HBV replication and might be associated with the development of FH by the subgenotype B1/Bj HBV.

Hepatitis B virus replication could enhance regulatory T cell activity by producing soluble heat shock protein 60 from hepatocytes.

BACKGROUND: HBcAg-specific regulatory T (T(reg)) cells play an important role in the pathogenesis of chronic hepatitis B. Soluble heat shock proteins, especially soluble heat shock protein 60 (sHSP60), could affect the function of T(reg) cells via Toll-like receptor. METHODS: We analyzed the relationship between soluble heat shock protein production and hepatitis B virus (HBV) replication with both clinical samples from HBeAg-positive patients with chronic hepatitis B (n= 24) and HBeAb-positive patients with chronic hepatitis B (n= 24) and in vitro HBV-replicating hepatocytes. Thereafter, we examined the biological effects of sHSP60 with isolated T(reg) cells. RESULTS: The serum levels of sHSP60 in patients with chronic hepatitis B were statistically significantly higher than those in patients with chronic hepatitis C (P<.01), and the levels of sHSP60 were correlated with the HBV DNA levels (r = 0.532; P<.001) but not with the alanine aminotransferase levels. Moreover, the levels of sHSP60 in HBV-replicating HepG2 cells were statistically significantly higher than those in control HepG2 cells. Preincubation of CD4(+) CD25(+) cells with recombinant HSP60 (1 ng/mL) statistically significantly increased the frequency of HBcAg-specific interleukin 10-secreting T(reg) cells. The frequency of IL7R(-)CD4(+)CD25(+) cells, the expression of Toll-like receptor 2, and the suppressive function of T(reg) cells had declined during entecavir treatment. CONCLUSION: The function of HBcAg-specific T(reg) cells was enhanced by sHSP60 produced from HBV-infected hepatocytes. Entecavir treatment suppressed the frequency and function of T(reg) cells; this might contribute to the persistence of HBV infection.

Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis.

A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.

Analysis of the entire nucleotide sequence of hepatitis B causing consecutive cases of fatal fulminant hepatitis in Miyagi Prefecture Japan.

We encountered five consecutive patients with fulminant hepatitis induced by acute hepatitis B virus (HBV) infection in 2000--2001 in Japan. They had not had previous contact each other, and were referred to us from different hospitals. Although a 69-year-old woman could be rescued by intensive internal treatment, the four patients died. We analyzed the partial (nt 278-646) and entire nucleotide sequences of the HBV obtained from them, and their divergences were 0-0.3% and 0-0.2%, respectively. The results suggested that they had been infected with the same HBV isolates. The isolates belonged to genotype B and subgenotype B2 on the phylogenetic tree analysis (AB302942-AB302946). As for the nucleotides sequences of them, previously reported mutations of G1896A, A1762T, and G1764A were present. Amino acid analysis revealed that previously reported Ile97Leu and Pro130Non-Pro in the core region and Trp28Stop in the precore region were present. As for the entire nucleotide sequences among B2, AB302942 showed low divergences with AF121245 and AB073834 (1.7%), and X97850 from patients with fulminant hepatitis (3.2%). We compared the two consensus nucleotides derived from AB302942 and X97850 (fulminant hepatitis) versus AY121245 and AB073834 (non-fulminant hepatitis), which revealed a difference in nt 1,504 located in the P and X region. Nucleotide 1,504 was C for isolates from fulminant hepatitis and G for non-fulminant hepatitis, and it was recognized among most of the isolates belonging to B2 registered on GenBank. Further studies could disclose the mechanism of severe inflammation of liver that finally leads to fulminant hepatitis.

The high incidence of the emergence of entecavir-resistant mutants among patients infected with lamivudine-resistant hepatitis B virus.

Hepatitis B virus (HBV) infection remains to be one of the most prevailing infection in the world, causing chronic liver diseases. Although lamivudine has been effective to suppress HBV replication, longer durations of administration can lead to the emergence of drug-resistant mutant viruses, followed by reactivation of hepatic inflammation (breakthrough hepatitis). Moreover, the optimal period of administration as well as the effects of anti-viral nucleot(s)ide such as lamivudine, adefovir, and entecavir, has not been established. To evaluate the efficacy of the anti-viral effects of entecavir for lamivudine-resistant HBV, we administered entecavir sequentially in four patients with chronic HBV infection, who demonstrated the emergence of lamivudine-resistant HBV and histological active hepatitis. The antiviral effects were evaluated by the serum viral loads and biochemical laboratory data. After follow-up periods of more than 36 months, we found high incidence in the emergence of entecavir resistant mutants (3/4, i.e., 75%). An additional mutation at the 184th amino acid, different from the previously reported lamivudine-resistant mutations (80th, 180th, and 204th), seemed to have a close relationship with the induction of entecavir-resistant mutants at least for Japanese HBV genotype C. Our observation draws attention to the possibility that the usage of entecavir for lamivudine-resistant HBV could promptly induce entecavir-resistant mutations in addition to lamivudine-resistance.

Sustained clinical improvement of a patient with decompensated hepatitis B virus-related cirrhosis after treatment with lamivudine monotherapy.

Hepatitis B virus (HBV) infection, which causes liver cirrhosis and hepatocellular carcinoma, remains a major health problem in Asian countries. Recent development of vaccine for prevention is reported to be successful in reducing the size of chronically infected carriers, although the standard medical therapies have not been established up to now. In this report, we encountered a patient with decompensated HBV-related cirrhosis who exhibited the dramatic improvements after antiviral therapy. The patient was a 50-year-old woman. Previous conventional medical treatments were not effective for this patient, thus this patient had been referred to our hospital. However, the administration of lamivudine, a reverse transcriptase inhibitor, for 23 months dramatically improved her liver severity. During this period, no drug resistant mutant HBV emerged, and the serum HBV-DNA level was continuously suppressed. These virological responses were also maintained even after the antiviral therapy was discontinued. Moreover, both hepatitis B surface antigen and e antigen were observed to have disappeared in this patient. The administration of lamivudine to patients with HBV-related cirrhosis, like our present case, should be considered as an initial medical therapeutic option, especially in countries where liver transplantation is not reliably available.

Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B.

AIM: To study the mechanisms of hyporesponsiveness of HBV-specific CD4+ T cells by testing TH1 and TH2 commitment and regulatory T cells. METHODS: Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4+CD25+ cells. RESULTS: Level of mRNAs for T-bet, IL-12R beta2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4+CD25highCTLA-4+ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4+ cells and CD4+CD25+ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAg-specific TH1 antibody compared with control antibody (P < 0.01, 0.34% +/- 0.12% vs 0.15% +/- 0.04%). Deletion of CD4+CD25+ T cells increased the amount of HBcAg-specific TH1 antibody when compared with lymphocytes reconstituted using regulatory T cells (P < 0.01, 0.03% +/- 0.02% vs 0.18% +/- 0.05%). CONCLUSION: The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.

A case-control study of response to lamivudine therapy for 2 years in Japanese and Chinese patients chronically infected with hepatitis B virus of genotypes Bj, Ba and C.

BACKGROUND/AIMS: In eastern Asian countries, hepatitis B virus (HBV) genotype Ba (HBV/Ba), HBV/Bj and HBV/C are prevalent. The aim was to investigate the response or resistance to lamivudine therapy among patients with different HBV genotypes. METHODS: Of 67 Japanese and Chinese patients with chronic hepatitis B, 18 patients with HBV/Bj, 15 with HBV/Ba and 34 with HBV/C were selected for a case-control study matched according to gender and age. All the patients were treated with lamivudine for 2 years and evaluated the response or emergence of the YMDD mutation at year 2 during the treatment. HBV genotypes were detected by the restriction fragment length polymorphism. The YMDD mutation was detected by the direct sequencing after amplification by PCR. RESULTS: At year 2 during therapy, 44.8% of the patients showed normalization of ALT and undetectable HBV DNA (favorable response), 35.8% developed the YMDD mutation. There was no significant difference of response to the therapy among the three genotype groups. The emergence of the YMDD mutation was associated with HBV/C. By the multiple logistic regression analysis, however, the significant factor of a favorable response was a higher pretreatment ALT level and negative HBeAg status and the significant factor of the emergence of the YMDD mutation was HBV/C. CONCLUSIONS: Higher pretreatment ALT level, HBeAg status or HBV genotype may affect the response or resistance to lamivudine therapy.

Analysis of the entire nucleotide sequence of hepatitis B virus genotype B in the Philippines reveals a new subgenotype of genotype B.

The entire nucleotide sequences were determined for hepatitis B virus (HBV) genotype B (HBV/B) genomes extracted from five patients in the Philippines and designated GenBank AB219426, AB219427, AB219428, AB219429 and AB219430. The serotype of the first four isolates was ayw and that of GenBank AB219430 was adw. Divergences of entire sequences were 1.0-2.0 % between the first four isolates and 3.8-4.2 % between these four and GenBank AB219430. Phylogenetic-tree analysis revealed that, worldwide, HBV/B comprises five subgenotypes: B1, B2, B3, B4 and the new Philippines group, designated B5. Divergences of the entire genome sequences between four isolates in subgenotype B5 and isolates from other countries (subgenotypes) were 4.4-4.8 % with Vietnam (B4), 2.9-3.5 % with Indonesia (B3), 4.7-5.1 % with China (B2) and 5.4-6.0 % with Japan (B1). Similarly, GenBank AB219430 showed the lowest divergences: 3.4 % with the isolate from Indonesia (B3), 5.0 % with Vietnam (B4), 5.4 % with China (B2) and 6.1 % with Japan (B1). This is the first report of entire nucleotide sequences of HBV/B from the Philippines and the results show that these sequences belong to a new subgenotype, B5. The present study identified that HBV/B isolates throughout the world are divided genetically into five subgenotypes, the relationships between geographical distances and the genetic distances of HBV/B being well-correlated.

A patient with clinical features of acute hepatitis E viral infection and autoimmune hepatitis.

Hepatitis E virus (HEV) is one of the major causative agents of acute hepatitis in many developing countries. Recent intensive examination has revealed the existence of non-imported cases in industrialized countries. The patient was a 25-year-old Japanese female with acute hepatitis. Laboratory test demonstrated positive anti-nuclear antibody (ANA), anti-smooth muscle antibody (ASMA) and high level of serum immunoglobulin G (IgG). The patient was negative for serum markers of hepatitis A, B or C virus infection. She demonstrated a clinical course similar to severe autoimmune hepatitis, including response to prednisolone therapy. After a few years, with the availability of tests for the serum antibodies to HEV, we examined the frozen stocked sera of the patient and found her exact diagnosis was acute hepatitis E. Although we could not detect HEV-RNA, which is positive only in limited period of acute phase, serum IgA and IgG antibodies to HEV were positive and the titer of IgA and IgG antibodies were declined with the time course. In conclusion, we must take into consideration of HEV infection for the diagnosis of acute cryptogenic hepatitis including autoimmune hepatitis. Further studies are feasible to understand the pathogenesis of liver injuries induced by HEV infections.

Recovery of functional cytotoxic T lymphocytes during lamivudine therapy by acquiring multi-specificity.

To characterize cytotoxic T lymphocytes (CTLs) that appeared in circulation during lamivudine therapy, we analyzed HBV-specific CTLs using HLA-A24 tetramer and HBcAg-specific Th1 cells in patients receiving lamivudine therapy. Six patients (HLA-A24(+)) with chronic hepatitis B, six patients (HLA-A24(-)) with chronic hepatitis B, and six patients (HLA-A24(+)) with chronic hepatitis C were studied. In addition to known CTL epitopes (C117 and P756), three epitopes were confirmed as CTL epitopes (C23, S89, S226) by chromium release assay and by staining intracellular perforin. CTLs specific for P756 were most frequently found at pre-treatment. During lamivudine therapy, increase in the frequencies of HLA-tetramer(+) cells was found for C117, S89, and S226. Recovery of CTLs was observed earlier in patients with HBeAg(-)/anti-HBe(+) compared with those with HBeAg(+)/anti-HBe(-). HBcAg-specific Th1 cells did not increase significantly up to 8 weeks. T cell lines from patients with chronic hepatitis B had a lower level of proliferation (0- to 24.9-fold expansion by in vitro stimulation) and a higher ability to produce interferon-gamma (0-84% except for S89), while perforin-positive cells showed low frequencies (0-50% except for S89). In conclusion, these results suggest that lamivudine therapy induces mainly CTLs that were less frequent before the therapy. Since recovered CTLs maintained the ability to produce interferon-gamma in response to peptides, these CTLs apparently contribute to the efficacy of lamivudine therapy in patients with hepatitis B.