Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection

BACKGROUND AND AIMS: The expression of hepatitis B viral (HBV) antigens in liver tissue reflects the replicative status of chronic HBV infection. We have previously recognized a novel marginal pattern of hepatitis B surface antigen (HBsAg) in hepatocytes, which usually clusters in groups and emerges at the late non-replicative phase. This study was designed to investigate whether the marginal-type HBsAg represented the gene product of a specific HBV-surface mutant. METHODS: Microdissection of cirrhotic nodules homogeneously expressing marginal HBsAg was performed on two of 12 resected livers from HBsAg-seropositive patients with hepatocellular carcinoma. The gene presumably encoding marginal HBsAg was polymerase chain reaction (PCR)-cloned, sequenced and analysed. In vitro transfection and expression of the cloned surface mutant plasmids were performed on the Huh7 cell line to illustrate intrahepatic HBsAg expression. RESULTS: Immunohistochemical staining revealed that the marginal HBsAg was positive for pre-S1 and thus contained large surface proteins. The PCR cloning and sequencing of the genes presumably encoding marginal-type HBsAg in both cases revealed the same deletion at the 5' terminus (nt 2-55) of pre-S2. A point mutation on the small-surface (S) antigen was also found in one case. The pre-S2 deletion sequence and the mutation sites of the S gene coincide with human lymphocyte antigen-restricted T- and/or B-cell epitopes. In vitro transfection of the mutant plasmid revealed a blot-like retention or accumulation of HBsAg in the cytoplasm or at the periphery of hepatocytes, accompanied by a decreased secretion of HBsAg in the culture supernatant, mimicking intrahepatic expression. CONCLUSION: A natural pre-S2 deletion mutant was identified in hepatocytes expressing a novel marginal pattern of HBsAg, which probably contains mutant, large, surface proteins. The biological significance of the pre-S2 deletion mutant should be interesting in view of the clustering proliferation of hepatocytes expressing marginal HBsAg.     

Hepatitis B surface antigen clearance in inactive hepatitis B surface antigen carriers treated with peginterferon alfa-2a

AIM: To examine the association between interferon(IFN) therapy and loss of hepatitis B surface antigen(HBs Ag) in inactive HBs Ag carriers. METHODS: This was a retrospective cohort study in inactive HBs Ag carriers, who were treatment-naive, with a serum HBs Ag level 100 IU/m L and an undetectable hepatitis B virus(HBV) DNA level( 100 IU/m L). All the 20 treated patients received subcutaneous PEG-IFN alfa-2a 180 μg/wk for 72 wk and were then followed for 24 wk. There were 40 untreated controls matched with 96 wk of observation. Serum HBs Ag, HBV DNA, and alanine aminotransferases were monitored every 3 mo in the treatment group and every 3-6 mo in the control group. RESULTS: Thirteen(65.0%) of 20 treated patients achieved HBs Ag loss, 12 of whom achieved HBs Ag seroconversion. Mean HBs Ag level in treated patients decreased to 6.69 ± 13.04 IU/m L after 24 wk of treatment from a baseline level of 26.22 ± 33.00 IU/m L. Serum HBV DNA level remained undetectable( 100 IU/m L) in all treated patients during the study. HBs Ag level of the control group decreased from 25.72 ± 25.58 IU/m L at baseline to 17.11 ± 21.62 IU/m L at week 96(P = 0.108). In the control group, no patient experienced HBs Ag loss/seroconversion, and two(5.0%) developed HBV reactivation.CONCLUSION: IFN treatment results in HBs Ag loss and seroconversion in a considerable proportion of inactive HBs Ag carriers with low HBs Ag concentrations.     

Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma

BACKGROUND: Pre-S deletion mutant of hepatitis B virus (HBV) affects the expression of middle and small surface proteins, resulting in intracellular accumulation of large surface protein. The correlation between pre-S deletion mutant and risk of hepatocellular carcinoma (HCC) in hepatitis B virus carriers remains unclear. METHODS: Using molecular assays, pre-S deletion mutant of HBV were determined in 266 patients with chronic HBV genotype B or C infection. They included 202 asymptomatic carriers and 64 HCC patients. RESULTS: The overall prevalence of pre-S deletion mutant was 16.5%. Hepatocellular carcinoma (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.23-8.48, P = 0.02) and genotype C (OR, 3.19; 95%CI, 1.54-6.62, P = 0.002) were independently associated with the presence of pre-S deletion mutant. The prevalence of pre-S deletion mutant was comparable between HCC patients with genotype B and C infection. Nevertheless, in asymptomatic carriers, patients with genotype C infection were significantly associated with the presence of pre-S deletion mutant compared to those with genotype B infection (20.8% vs 7.2%, P = 0.007). Compared with age- and genotype B-matched asymptomatic carriers, young HCC patients (<50 years of age) had a significantly higher frequency of pre-S deletion (3.4% vs 20%, P = 0.04). CONCLUSIONS: Pre-S deletion mutant is more frequent in HBV carriers with genotype C infection, and those with pre-S deletion mutant may be associated with the development of HCC, irrespective of HBV genotype.     

Progressive and sufficient decrease of hepatitis B core antibody can predict the disappearance of hepatitis B virus DNA in Japanese patients with hepatitis B surface antigen clearance

The aim of this study was to elucidate the relationships among serum levels of hepatitis B virus (HBV) DNA, periods after hepatitis B surface (HBs) antigen clearance, and the titer of hepatitis B core (HBc) antibody in 200-fold diluted serum. Twelve patients who had clearance of HBs antigen from serum were studied. Five patients had not received any treatment (group A), and seven had received prednisolone withdrawal therapy. The patients in groups A and B were followed up for 86 months and 108 months (median), respectively. Serum HBV was measured by the nested polymerase chain reaction method. In both groups, serum HBV tended to become gradually undetectable after HBs antigen clearance. The positive rate of HBV in the sera 5 years or more after HBs antigen clearance was significantly lower than that in the sera at less than 5 years, both in group A (P = 0.004) and group B (P = 0.010). In both groups, the titer of HBs tended to decline every year after HBs antigen clearance. HBV was still detectable in the sera of some patients for a long period of time after they showed seroconversion to HBs antibody. The results suggest that detection of HBV was difficult in sera with an HBc titer of 30% or lower and at more than 5 years after HBs antigen clearance in both groups. It is important to note that HBV DNA rarely exists in the serum, even when HBs antigen and HBc are both negative. Received: November 22, 1999 / Accepted: April 28, 2000     

核苷类似物干预下乙型肝炎病毒前S抗原消长与DNA复制水平的关系

目的 监测核苷类似物干预下HBV前S1抗原(PreS1)、前S2抗原(PreS2)的动态变化,探讨PreS1、PreS2与HBV DNA复制水平的相关性.方法 选取拉米夫定治疗有效应答后出现病毒学反跳的慢性乙型肝炎患者12例,以及持续5年阿德福韦酯抗病毒治疗的慢性乙型肝炎患者20例,动态监测PreS1、PreS2及其他HBV标志物.根据不同资料分别选择t检验、X2检验、线性相关与回归分析或方差分析.结果 12例拉米夫定治疗的患者,在HBV DNA发生剧烈变化的4个时间点,均未观察到PreS1、PreS2滴度随HBV DNA复制水平的一致变化;20例阿德福韦酯治疗的患者中,仅1例患者PreS1、PreS2随HBV DNA和HBeAg一起阴转,其余均无显著变化;采用不等距重复测量设计方差分析绘制变化趋势轮廓图,未观察到PreS1、PreS2滴度随HBV DNA水平一致的变化趋势.结论 在核苷类似物干预下PreS1、PreS2滴度不能反映HBV DNA复制水平以及HBeAg的血清学转换.     

Changes of pre-Sl and pre-S2 antigens in sera of patients with hepatitis B virus infection

Polypeptides encoded by the pre-Sl and pre-S2 genes of hepatitis B virus (HBV) (pre-Sl antigen and pre-S2 antigen) were detected by enzyme-linked immunosorbent assay (ELISA) in 137 serum samples of patients with HBV infection. The HBV-DNA level closely correlated with the titer of pre-S antigens. However, HBV-DNA levels more closely correlated with the titer of the pre-Sl antigen [HBV-asymptomatic carrier (ASC): n=40, r=0.800, P<0.01; chronic hepatitis B (CH): n=60, r=0.730, P<0.01] than with the titer of the pre-S2 antigen [ASC: r=0.675, P<0.01; CH: r=0.575,P<0.01]. Thirty patients with CH, in whom hepatitis e antigen (HBeAg) was cleared after acute exacerbation (AE) [alanine aminotransferase (ALT) level> 200 IU/L] and the ALT level normalized, were followed for 12 months and classified into two groups: Group 1, those in whom HBeAg reappeared with an elevated ALT level within 12 months, and Group 2, those in whom HBeAg was persistently cleared from the serum and a normal ALT level continued. Of the 30 patients, 24 (80%) were classifed into Group 1 and 6 (20%) were classified into Group 2. Changes in serum levels of HBV markers a month before and after AE were observed. The HBV-DNA level and DNA-P activity became negative after AE in both groups. The titer of pre-Sl antigen also decreased after AE, and no significant differences were observed between Group 1 and Group 2. In contrast, the titer of pre-82 antigen and pAR activity decreased more significantly in Group 2 than in Group 1. These results suggest that the detection of the pre-S2 antigen and pAR activity in sera seems to be more useful as a prognostic test for patients with HBV infection than the detection of pre-Sl antigen, HBV-DNA, DNA-P or HBeAg.     

Hepatocyte hepatitis B surface antigen expression in chronic hepatitis B virus carriers in Singapore: Correlation with viral replication and liver pathology

The hepatocyte hepatitis B surface antigen (HBsAg) expression in 149 liver biopsies from 124 chronic hepatitis B virus (HBV) carriers was correlated with serum HBV DNA status and histologic activity. Hepatocyte HBsAg was stained by the peroxidase-antiperoxidase method and serum HBV DNA was determined by dot blot hybridization. Sixty-five biopsies (44%) showed minimal changes (MC), 82 biopsies (55%) showed chronic liver disease (CLD) and 2 biopsies (1%) showed hepatocellular carcinoma. Hepatocyte HBsAg was found in 144 biopsies (97%). It was present in the cytoplasm of 141 specimens (95%) and/or plasma membrane of 48 specimens (32%). Approximately half (45%) of the cytoplasmic HBsAg-positive biopsies showed discrete distribution, while the other half (55%) were grouped. Fifty-five per cent (77 of 141) of cytoplasmic HbsAg-positive biopsies had CLD, while 44% (62 of 141) showed MC. There was no relationship between the presence of cytoplasmic HBsAg or its topographic distribution with disease activity. Membrane HBsAg distribution was similar for both groups of patients (MC vs CLD: 25 of 65 (38%) vs 23 of 82 (28%); P = NS). Serum HBV DNA was detected in 98 patients (66%) and was seen mostly in association with CLD (CLD vs MC: 61% vs 39%, P less than 0.001). It was also detected more often in the sera of patients with membrane HBsAg than in those with cytoplasmic HBsAg staining (41 of 48 (85%) vs 97 of 141 (67%); P less than 0.02). However, discrete distribution of cytoplasmic HBsAg was associated with positive serum HBV DNA when compared with grouped distribution (52 of 63 (83%) vs 43 of 78 vs (55%); P less than 0.005).     

Novel hepatitis B virus surface antigen mutations associated with occult genotype B hepatitis B virus infection affect HBsAg detection

The causative factors of occult hepatitis B infection are complicated and not yet been fully elucidated. Mutations in hepatitis B virus (HBV) S gene are one of the factors may contributing to occult infection. In this study, 89 blood donors with genotype B occult HBV infection were investigated. Fifty‐seven hepatitis B surface antigen (HBsAg)‐positive/HBV DNA‐positive blood donors served as control group for comparison. Occult HBV‐related mutations with a high incidence (P < .05) in the S gene were identified. To further verify these occult infection‐related mutations, a conservative full‐gene expression vector of HBV B genotype (pHBV1.3B) was constructed. Then, the mutant plasmids on the basis of pHBV1.3B were constructed and transfected into HepG2 cells. Extracellular as well as intracellular HBsAg was analysed by electrochemical luminescence and cellular immunohistochemistry. Ten occult infection‐related mutations (E2G, Q101R, K122R, M133T, D144E, G145R, V168A, S174N, L175S and I226S) were significantly more frequent in the occult infection group (P < .05). Five of the ten mutations (E2G, D144E, G145R, V168A and S174N) strongly decreased extracellular HBsAg level (P < .05) in the transfection system. Notably, the E2G mutation had the most significant impact on the ratio of extracellular HBsAg (3.8% vs pHBV1.3B) and intracellular HBsAg (239.3% vs pHBV1.3B) (P < .05), and the fluorescence density of E2G mutant HBsAg was significantly higher than that of pHBV1.3B (P < .0001). Hence, ten mutations were associated with genotype B occult HBV infection; E2G and V168A were novel mutations which we confirmed significantly affect HBsAg detection. E2G might cause HBsAg secretion impairment that results in intracellular accumulation and a decrease in HBsAg secretion.     

Analysis of hepatitis B surface antigen (HBsAg) using high‐sensitivity HBsAg assays in hepatitis B virus carriers in whom HBsAg seroclearance was confirmed by conventional assays

Aim

We investigated the utility of high‐sensitivity hepatitis B surface antigen (HBsAg) assays compared with conventional HBsAg assays.

Methods

Using serum samples from 114 hepatitis B virus (HBV) carriers in whom HBsAg seroclearance was confirmed by conventional HBsAg assays (cut‐off value, 0.05 IU/mL), the amount of HBsAg was re‐examined by high‐sensitivity HBsAg assays (cut‐off value, 0.005 IU/mL). Cases negative for HBsAg in both assays were defined as consistent cases, and cases positive for HBsAg in the high‐sensitivity HBsAg assay only were defined as discrepant cases.

Results

There were 55 (48.2%) discrepant cases, and the range of HBsAg titers determined by high‐sensitivity HBsAg assays was 0.005–0.056 IU/mL. Multivariate analysis showed that the presence of nucleos(t)ide analog therapy, liver cirrhosis, and negative anti‐HBs contributed to the discrepancies between the two assays. Cumulative anti‐HBs positivity rates among discrepant cases were 12.7%, 17.2%, 38.8%, and 43.9% at baseline, 1 year, 3 years, and 5 years, respectively, whereas the corresponding rates among consistent cases were 50.8%, 56.0%, 61.7%, and 68.0%, respectively. Hepatitis B virus DNA negativity rates were 56.4% and 81.4% at baseline, 51.3% and 83.3% at 1 year, and 36.8% and 95.7% at 3 years, among discrepant and consistent cases, respectively. Hepatitis B surface antigen reversion was observed only in discrepant cases.

Conclusions

Re‐examination by high‐sensitivity HBsAg assays revealed that HBsAg was positive in approximately 50% of cases. Cumulative anti‐HBs seroconversion rates and HBV‐DNA seroclearance rates were lower in these cases, suggesting a population at risk for HBsAg reversion.     

Changes in levels of hepatitis B virus markers in patients positive for low-titer hepatitis B surface antigen

Aim: Recently, patients positive for the low-titer hepatitis B surface antigen (HBsAg) have been found occasionally owing to the increase in the accuracy of detection methods. The aim of this study is to clarify the clinical status of acute hepatitis B virus (HBV) infection in patients positive for low-titer HBsAg. Method: Eight patients, who were positive for HBsAg at low titers and diagnosed as having acute HBV infection, were enrolled in this study. Assays of HBsAg, hepatitis B core antibody (anti-HBc), hepatitis B e-antigen (HBeAg), hepatitis B e-antibody (anti-HBe), hepatitis B surface antibody (anti-HBs) and HBV DNA, and biochemical tests were basically conducted every 4 weeks for at least 24 weeks. Result: The average cut-off index of HBsAg was 8.7 ± 9.6 (range, 1.0–25.7). All the patients were negative for anti-HBc, HBeAg, anti-HBe and HBV DNA on their initial visit. The genotype of HBV could be determined in four patients: two were infected with genotype B/HBV, one was infected with genotype A/HBV, and the remaining patient was infected with genotype C/HBV. Although HBsAg clearance was observed within 4 months in all the patients, none of the other HBV markers seroconverted during the observation period. Conclusion: HBV infection terminating with seronegativity for HBV markers may occur in transient HBV infection.     

Clinical significance of pre-S mutations in patients with genotype C hepatitis B virus infection

We investigated the overall and site-specific prevalence of pre-S mutations and its clinical significance in patients with genotype C hepatitis B virus (HBV) infection. Three hundred subjects were included: 50 asymptomatic carriers (AC), 87 chronic hepatitis (CH), 91 liver cirrhosis (LC) and 72 hepatocellular carcinoma (HCC). Pre-S mutations were determined by nucleotide sequence analysis. Possible correlations between pre-S mutations and clinical/virological parameters were examined. Pre-S mutations were detected in 82 cases (27.3%); it was more frequently found in HCC (43.1%) and LC (35.2%) group than in the CH (20.7%) and AC (2.0%) group. Pre-S2 deletion was the most commonly found mutation (10.7%), followed by pre-S2 start codon mutation (9.7%), pre-S1-S2 deletion (3.0%) and both pre-S2 deletion and start codon mutation (2.7%). Pre-S2 deletion and pre-S2 start codon mutation were more frequently detected in advanced diseases (LC and HCC). Pre-S mutations were associated with older age and higher rates of positive HBV DNA (>/=0.5 pg/mL). Advanced disease and positive HBV DNA were shown to be independent predictors of pre-S mutations by logistic regression analysis. These findings suggest that pre-S mutations, especially pre-S2 deletions and pre-S2 start codon mutations, are common in patients with genotype C HBV infection and are associated with advanced liver disease and active viral replication.     

Spontaneous hepatitis B surface antigen clearance in a long-term follow-up study of patients with chronic type B hepatitis. Lack of correlation with hepatitis C and D virus superinfection

We investigated the frequency of HBsAg clearance and the possible role of viral superinfection in a long-term follow-up of 184 patients with chronic hepatitis B (CHB). Our subjects were 184 patients with chronic hepatitis B and the follow-up was 12–216 months (mean 66.2±53.7 months). The investigative methods used were: immunoenzymatic assays for HBV, HCV, HDV, and HIV markers; polymerase chain reaction (PCR) for HBV DNA; and liver biopsy and immunoperoxidase. During the follow-up, 20 of the 184 patients cleared serum HBsAg. A comparison of patients with persistent HBsAg (group I) and of those who cleared this marker (group II) showed a significant difference in mortality (P=0.002) between the two groups and a tendency to a more severe exacerbation (flare) in group II (P=0.07). Antibodies to hepatitis C and D virus as well as antibodies to HIV were equally distributed in both groups. Thirteen patients (7.9%) from group I, but none from group II, subsequently developed hepatocellular carcinoma. These results suggest that the frequency of spontaneous clearance of HBsAg during chronic HBV infection is low. No determinant factor for the clearance was found, including the presence of liver cirrhosis. Serum HBV DNA was undetectable by PCR after clearance in 16 out of 17 patients. Some of these findings were presented at the Biennial Scientific Meeting of the International Association for the Study of the Liver, held in Brighton, UK, in June 1992.     

Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance

Summary. Seroconversion from hepatitis B surface antigen (HBsAg) to antibodies against HBsAg (anti‐HBs) usually indicates resolution of hepatitis B virus (HBV) infection. Here, two HBV‐infected patients with seroconversion to anti‐HBs were found to be persistently positive for HBeAg and HBV DNA. Immunohistology of liver biopsies confirmed the expression of HBV proteins in the liver of one patient. The neutralizing ability of anti‐HBs in patient sera was demonstrated by blocking HBV infection of primary tupaia hepatocytes. Analysis of the HBsAg‐encoding region of HBV isolates from patients indicated the coexistence of heterogeneous HBV genomes in patients. The majority of recombinant variant HBsAg was reactive in HBsAg assays and was able to bind to anti‐HBs. Circulating immune complexes (CIC) of HBsAg in patient sera could be detected by polyethylene glycol precipitation and trypsin digestion. Thus, neutralizing anti‐HBs may appear in chronic HBV carriers for long periods but does not necessarily lead to complete viral clearance.     

Long-term outcome after hepatitis B surface antigen seroclearance in patients with chronic hepatitis B

Purpose

The aim of this study was to elucidate the long-term outcome after hepatitis B surface antigen (HBsAg) seroclearance in a large number of Japanese patients.

Methods

We studied the biochemical, virologic, histologic, and prolonged prognoses of 231 Japanese patients with HBsAg seroclearance (median follow-up, 6.5 years). Serum alanine aminotransferase, serum hepatitis B virus (HBV) markers, liver histology, and clinical aspects were monitored. HBV-DNA levels were measured with the qualitative polymerase chain reaction assay. The mean age of patients with HBsAg seroclearance was 52 years.

Results

After HBsAg seroclearance, 203 patients (87.9%) had normal alanine aminotransferase levels 1 year after HBsAg seroclearance. HBV-DNA showed positive results in 4 patients (1.7%) 1 year after HBsAg seroclearance. Thirteen patients were examined for histologic changes of the liver after HBsAg seroclearance. All patients showed marked improvement of necroinflammation of the liver, but only 2 of the 13 patients showed no liver fibrosis. Liver cirrhosis and hepatocellular carcinoma did not develop in any of the 164 patients without evidence of liver cirrhosis at the time of HBsAg seroclearance. Hepatocellular carcinoma developed in 2 of the 67 patients with liver cirrhosis at the time of HBsAg seroclearance. During the observation period, 15 patients died. However, the cause of death of these 15 patients was not related to liver disease, such as hepatocellular carcinoma, decompensated liver cirrhosis, and rupture of esophageal varices.

Conclusion

Our results suggest that HBsAg seroclearance confers favorable long-term outcomes in patients without hepatocellular carcinoma or decompensated liver cirrhosis at the time of HBsAg seroclearance     

Immunohistochemical study of pre-S(2) antigen in liver and serum of patients with chronic hepatitis B

To evaluate the significance of pre-S(2) in the liver, the authors studied the relationship between serum and intrahepatic pre-S(2) in 27 patients with chronic hepatitis B. Intrahepatic expression of preS(2) was observed under light and electron microscopy and compared with that of HBsAg and HBcAg. All patients were positive for hepatic pre-S(2), and 25 for serum pre-S(2). Intrahepatic pre-S(2) expression was membranous or both membranous and cytoplasmic in 18 cases and was cytoplasmic in 9. The serum levels of pre-S(2) were significantly higher in cases whose intrahepatic pre-S(2) was membranous than in the cytoplasmic cases. These findings indicate that membranous expression of pre-S(2) reflects its secretion into the circulation. Intrahepatic pre-S(2) expression was found almost completely to resemble that of HBsAg on both light and electron microscopy. Membranous pre-S(2) was seen in the liver of 16 of 25 cases who were positive for intrahepatic HBcAg. In detailed studies of membranous pre-S(2) and HBcAg, both membranous pre-S(2) and HBcAg appeared in the liver of all 7 cases before or at the peak of acute exacerbation, but after it, membranous pre-S(2) was not found in 4 of 8 cases. These results suggest that disappearance of membranous pre-S(2) and HBsAg may be related to hepatic cell necrosis in some cases with acute exacerbation.     

Profound suppression of chronic hepatitis C following superinfection with hepatitis B virus.

BACKGROUND: Hepatitis B virus (HBV) and hepatitis C virus (HCV) share similar transmission routes; thus, coinfection is frequent. The consequences of acute HBV infection in patients with chronic hepatitis C are unknown. METHODS: We describe a 47-year-old male with chronic hepatitis C who acquired HBV and then spontaneously and apparently completely cleared HCV but developed chronic hepatitis B. Five serum samples collected over 14 months and lymphoid cells obtained after acquiring HBV were tested for HCV and HBV by both standard assays and ultra-sensitive polymerase chain reaction/nucleic acid hybridization (PCR/NAH) (sensitivity approximately 2 IU/ml). RESULTS: After superinfection with HBV, HBV surface antigen-positive chronic hepatitis developed with readily detectable HBV DNA. All sera collected after acquisition of HBV, which tested negative for HCV RNA by standard laboratory assay, were positive for HCV genomes when analysed by PCR/NAH. Peripheral lymphoid cells carried HBV DNA and covalently closed circular DNA, but were negative for HCV. CONCLUSIONS: This is the first reported case of profound suppression of chronic hepatitis C after superinfection with HBV and establishment of chronic hepatitis B. It is hypothesized that HBV infection precipitated generalized and/or virus-specific cellular immune responses that profoundly suppressed HCV replication and yet failed to inhibit progression to chronic hepatitis B.     

Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B

The immunopathogenesis of hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-gamma (IFN-gamma) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with overlapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182-191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-gamma producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-gamma assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained.     

Mutations associated with occult hepatitis B virus infection result in decreased surface antigen expression in vitro

Summary. Occult hepatitis B virus (HBV) infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild‐type HBsAg expression vectors were constructed from genotype‐matched chronic HBV sequences. Site‐directed mutagenesis was then utilized to introduce three genotype A mutations – M103I, K122R and G145A – associated with occult HBV infection in vivo, alone and in combination, into the wild‐type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all three mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.     

Hepatitis B surface antigen seroconversion is associated with favourable long-term clinical outcomes during lamivudine treatment in HBeAg-negative chronic hepatitis B patients

The aims of this study were to assess hepatitis B surface antigen (HBsAg) seroconversion and to determine its impact on the natural course of the disease in patients with HBeAg-negative chronic hepatitis B (CHB) during lamivudine (LMV) treatment. A total of 183 consecutive patients with HBeAg-negative CHB who were treated with LMV were included in the study. Data were retrospectively collected from outpatient visit charts. The primary endpoint was HBsAg seroconversion to anti-HBs. The secondary endpoint was to determine the development of cirrhosis. Loss of HBsAg was confirmed in 10 patients and seroconversion to anti-HBs in nine patients during LMV treatment or after its discontinuation. HBsAg seroconversion was achieved on-treatment in four patients after a median treatment duration of 30 months and off-treatment in the remaining five patients in a median 61 months after LMV discontinuation. The cumulative probability of HBsAg seroconversion increased from 0.6% at 1 year and 1.9% at 5 years to 21.5% at 10 years of LMV during and after LMV treatment. HBsAg clearance was preceded by undetectable serum hepatitis B virus (HBV) DNA. The majority of the patients responding to treatment had undetectable HBV DNA levels at 24 weeks of treatment. The cumulative probability of LMV resistance increased from 2.2% at 1 year to 37.3% at 5 years. No baseline parameter predicting either HBsAg seroconversion or the emergence of LMV resistance was identified. None of the patients with HBsAg seroconversion experienced virological breakthrough or disease progression during the follow-up period. These results indicate that HBsAg seroclearance can occur in patients with HBeAg-negative CHB under LMV therapy and predicts better clinical outcome.