The Investigation of Hepatitis B Vaccine Immune Responses in Occult Hepatitis B Virus-Infected Patients.

Objectives: There is no effective treatment for occult hepatitis B virus infection (OBI) patients, and immunotherapy may be one of the most promising options. We aim to investigate the underlying mechanism and therapeutic potential of hepatitis B vaccine immunotherapy for OBI patients. Methods: Outpatient OBI patients were screened and randomly divided into treatment (Group A) and control (Group B) groups. At weeks 0, 4, and 24, patients in Group A received a subcutaneous/intramuscular injection of hepatitis B vaccine (Engerix-B, 20 µg/time) according to the standard vaccination schedule; patients in Group B served as blank control. The patients were followed for 36 weeks, with clinical, biochemical, virological, immunological, and imaging data collected and analyzed at weeks 0, 12, 24, and 36, respectively, and the relation between the virology and immunology results was analyzed. Results: Of the 228 OBI patients, 28 were excluded, and 200 were enrolled for observation. In the end, 44 patients were included in Group A and 39 in Group B after excluding lost cases. At week 0 (baseline), some patients in two groups had liver disease symptoms, HBV-related liver function damage, and liver fibrosis. 86.36% (38/44) and 82.05% (32/39) patients were positive for serum hepatitis B surface antibodies (anti-HBs) in Group A and Group B, respectively, with the median (quartile) of 42.47 (16.85, 109.1) and 39.27 (16.06, 117.4) mIU/ml, respectively. Reduced peripheral blood CD4+T, CD8+T, and B lymphocytes were found in some patients in two groups. These results were not statistically different between Group A and Group B (P>0.05). At week 36, all patients were serum anti-HBs (+) in Group A, with a median (quartile) of 1000 (483.9, 1000) mIU/ml, which was significantly higher than that at week 0 (P<0.05) and that in Group B (P<0.05). Compared to week 0, the number of CD8+ T and B lymphocytes increased significantly and were significantly higher than Group B at the same point. Two patients in Group B were found to have hepatitis B virus reactivation from week 12 to week 36. Correlation Analysis: Anti-HBs in Group A patients were positively correlated with B lymphocytes (r=0.3431, 0.3087, and 0.3041, respectively) and positively correlated with CD8+ T lymphocytes (r=0.4954, 0.3054, and 0.3455, respectively) at weeks 12, 24, and 36. Conclusion: Virological reactivation is a risk for OBI patients. Serum hepatitis B surface antibodies were significantly increased after hepatitis B vaccine treatment, the same as the numbers of peripheral blood B and CD8+ T lymphocytes; changes in hepatitis B surface antibody levels were positively correlated with the changes in peripheral blood B and CD8+ T lymphocytes.

Reactivation of Occult Hepatitis B Virus Infection During Long-Term Entecavir Antiviral Therapy.

Up to now, it has not been clear whether occult hepatitis B virus (HBV) infection (OBI) can be treated with antiviral therapy whether OBI can develop drug resistance gene mutation or not. We report a middle-aged female patient with OBI who showed HBV reactivation (HBVr) during more than 3 years of intermittent entecavir (ETV) antiviral therapy: seropositive HBV surface antigen (HBsAg), increased e antigen (HBeAg), and repeatedly elevated serum HBV DNA. Genotype analysis showed that the patient was infected with HBV type B. Genetic sequencing of HBV showed the mutants of S143T, D144G, and G145R in the S gene region, and the mutant of site 1896 in the pre-Core region coexisted with the wild type (G1896A/G). No mutation was found in other HBV gene segments. Drug resistance gene analysis found RtL229W mutant, resistant to lamivudine but sensitive to ETV and other nucleoside analogs. This case of OBI provides us with the following clinical experiences: Firstly, it is necessary to detect HBV genotype, mutation, and drug-resistant genes at the initial diagnosis, which can be helpful for reasonable treatment. Secondly, identifying the risk factors and mechanisms associated with HBVr could help quantify the risk of HBVr and manage the clinical consequences. Thirdly, the OBI patients with hepatitis B e antigen-positive, HBV DNA > 1 × 103 IU/ml should be recommended regular and continuous antiviral therapy as soon as possible to prevent the occurrence of hepatocirrhosis and hepatocellular carcinoma (HCC).

The Serious Challenge of Occult Hepatitis B Virus Infection-Related Hepatocellular Carcinoma in China.

BACKGROUND: It is unknown how many people in China have chronic occult hepatitis B virus (HBV) infection (OBI) [chronic HBV infection with negative serum hepatitis B surface antigen (HBsAg) (N-HBsAg)]. Their clinical and virological characteristics, especially the correlation between the OBI and hepatocellular carcinoma (HCC), are still elusive and need to be investigated, including prevention, early diagnosis, and treatment strategies. METHODS: 138 patients with HCC related to OBI were screened from 698 patients of HCC associated with HBV infection, their characteristics of epidemiology, clinical, biochemistry, virology, diagnostics, and therapeutics were analyzed retrospectively. Furthermore, the correlation between virological features and clinical features was investigated. RESULTS: It was found that 19.8% (138/698) of patients with HBV-related HCC were OBI, of which 79.7% (110/138) were men, and 20.3% (28/138) were women. Most of the patients with OBI-related HCC were older men, and the median age was 63.2 years. In total 78.3% (108/138) of the patients had apparent right upper abdomen discomfort and/or pain and then sought medical examination, while 21.7% (30/138) of the patients were identified by health examination. A total of 10.9% (15/138) of the patients were admitted with chronic infection of HBV, and 2.2% (3/138) of the patients were admitted with a family history of hepatitis B. The alpha-fetoprotein (AFP) serum-positive rate was 39.1% (54/138). Tumor lesions >5.0 cm, with intrahepatic and/or extrahepatic metastasis, were found in 72.5% (100/138) of the patients. The diameter of the tumor in the Group of hepatitis B core antibody-positive [HBcAb(+)] and hepatitis B surface antibody-positive [HBsAb(+)] was 7.03 ± 3.76 cm, which was much smaller than 8.79 ± 4.96 cm in the Group of HBcAb(+) and HBsAb(-) (P = 0.035). CONCLUSION: It is estimated that at least 21 million OBI patients live in China. HBcAb(+) was not only the evidence of chronic HBV infection but also a dangerous mark for surface antigen-negative patients. A semi-annual or annual medical checkup is essential for all OBI patients to identify HCC as early as possible. The hypothesis underlying our analysis was that hepatitis B surface antibody would prevent the progress of HCC and facilitate the clearance of HBV in patients with OBI. Thereby, the hepatitis B vaccine could be used to prevent severe disease consequences.

[Effective antiviral therapy with entecavir in chronic hepatitis B virus carriers].

OBJECTIVE: To evaluate the short-term effect and safety of entecavir for the treatment of chronic hepatitis B (CHB) virus carriers. METHODS: Ninety-three cases of CHB virus infection (hepatitis B surface antigen (HBsAg)-positive, hepatitis B e antigen (HBeAg)-positive, hepatitis B core antibody (HBcAb)-positive, HBV DNA≥1x10(5) copies/mL) were divided into two groups: CHB virus carrier (47 cases) and CHB (46 cases). All of the 93 cases were given 0.5 mg entecavir orally once a day for 48 weeks. Virology, serology and biochemistry tests were perrmed at treatment weeks 0, 4, 12, 24 and 48. Side effects of entecavir and the incidence of liver cirrhosis and hepatocellular carcinoma were recorded. RESULTS: The CHB virus carrier and CHB group had complete virological response rates of 14.9% and 17.4% at week 4, 51.1% and 63.0% at week 12, 76.6% and 89.1% at week 24, and 97.9% and 100% at week 48, respectively; there was no significant difference between the two groups (P>0.05). The CHB virus carrier and CHB group had partial virological response rates of 42.6% and 47.8% at week 4, 57.44% and 65.2% at week 12, 85.0% and 89.1% at week 24, and 100% and 100% at week 48, respectively; there was no significant difference between the two groups (P>0.05). No cases in either group experienced virologic breakthrough during the treatment course. The CHB virus carrier and CHB group had serological response (HBeAg-negative) rates of 0 and 4.3% at week 4, 2.1% and 8.7% at week 12, 4.3% and 13.0% at week 24, and 8.5% and 21.7% at week 48, respectively; there was no significant difference between the two groups (P>0.05). The CHB virus carrier and CHB group had HBeAg seroconversion rates of 0 and 0 at week 4, 0 and 4.4% at week 12, 2.1% and 10.9% at week 24, and 6.4% and 17.4% at week 48, respectively; there was no significant difference between the two groups (P>0.05). No case in either group showed HBsAg-negativity and seroconversion during the treatment course. The CHB group had a biochemical response (alanine aminotransferase normalization) rate of 26.1% at week 4, 65.2% at week 12, 91.3% at week 24, and 97.8% at week 48.No case in either group showed biochemical breakthrough during the treatment course. There were no cases of liver cirrhosis or hepatocellular carcinoma in either group. There were no side effects of the entecavir treatment experienced in either group. CONCLUSION: Antiviral therapy with entecavir is effective, safe and well tolerated in CHB virus carriers.

Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA.

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs). METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19 x 10(12) copies of linear HBV DNA/1 x 10(7) PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group. RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive > or =2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group. CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species. HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

[Study on the replication of Hepatitis B Virus in primary hepatocytes from heterogenous species].

OBJECTIVES: By studying the possibility and degree of the replication and expression of human hepatitis B virus (HBV) genes in normal liver cells from heterogenous species, such as primary duck hepatocytes (PDH) and primary rat hepatocytes (PRH), to investigate the species-specificity of HBV infection and replication. METHODS: PDH and PRH isolated by in situ perfusion with low concentration collagenase were transfected with complete HBV genome by electroporation (transfection group, about 1.19 10(12) copies of linear HBV DNA/1 10(7) PRH/PDH). From 1 day to 15 days after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH/PDH were measured with IMX System, HBcAg was assayed with western blotting, Immunol dot blotting and Immunocytochemistry. Moreover, HBV S-mRNA and X-mRNA were tested with RT-PCR. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PRH/PDH electroporated only was used as control group. RESULTS: HBsAg in the lysates of transfected PDH group were 15.24 (1day), 14.55 (3 days) and 5.13 ( 5 days; P/N values, positive>or=2.1), HBeAg all was negative (<2.1), and both were negative in the supernatants of transfected group. Viral antigen production in transfected rat hepatocytes: HBsAg in the lysates of transfected hepatocytes was positive, P/N values ranging from 2.17 to 93.41, The average P/N values was 14.74+/-31.82, and could be maintained for 15 days after transfection. Whereas, HBsAg in the supernatants of transfected group was only found positive on 1 day after transfection, which P/N value was 6.66. HBeAg and HBcAg in the lysates of PRH of transfection group were positive during the first 3 days following transfection, P/N values was around 2.8. The total amount of HBV DNA in the transfected PDH and PRH groups was strongly positive by dot blotting, whereas that of the control group was negative. Southern blot analysis of intracellular total HBV DNA indicated that there were relaxed circular (RC), covalently closed circular (ccc) and single-stranded (SS) HBV DNA replicative intermediates in the transfected PDH and PRH groups, there was no integrated HBV DNA in the cellular genome. Control groups were negative at all. CONCLUSION: Expression of HBV genes and production can occur in hepatocytes from nonmammalian species or mammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

Effects of electroporation on primary rat hepatocytes in vitro.

AIM: To investigate the effects of electroporation on primary rat hepatocyte and to optimize the electroporation conditions introducing foreign genes into primary hepatocytes. METHODS: A single-pulse procedure was performed at low voltage (220-400 V) but with high capacitance (500-950 microF). Hepatocytes were divided into 4 groups according to the electroporation conditions: group I, 220 V and 500 microF; group II, 220 V and 950 microF; group III, 400 V and 950 microF,and group IV. The control group was freshly isolated hepatocytes and directly cultured under the same conditions as those of electroporation groups. The effects of electroporation on primary rat hepatocytes were detected by trypan blue exclusion (TBE) and MTT analysis. Besides, albumin (Alb), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in the supernatants of cultured hepatocytes were measured by biochemical assay. RESULTS: Between day 1 and day 15 after incubation, primary rat hepatocytes of each electroporation group appeared normal, being the same with those of control group. TBE staining showed that slight hepatocyte damage and high survival rate were found in the electroporation groups and the control group. Cultured for 3, 7, 11 and 15 days, hepatocyte viability was approximately 92.6+/-2.5 %, 89.5+/-3.3 %, 82.0+/-3.5 % and 74.3+/-1.2 %, respectively. MTT analysis indicated that the viabilities of hepatocytes had no significant difference between each electroporation group, and those were similar to that of control group. At the 36th hour after electroporation, Alb, ALT and LDH in the supernatants of control group were 5.3+/-0.1 g x L(-1), 183.7+/-8.4 nkat x L(-1) and 896.8+/-58.5 nkat x L(-1); those of group II were 5.7+/-0.1 g x L(-1), 215.4+/-16.7 nkat x L(-1) and 1063.8+/-51.8 nkat x L(-1); and those of group III were 5.8+/-0.2 g x L(-1), 217.1+/-8.4 nkat x L(-1) and 1063.8+/-10.0 nkat x L(-1). Statistically, the proteins of group II and group III were significantly higher than those of control group (P<0.05), whereas the protein production of group I, Alb, ALT and LDH were 5.3+/-0.2 g x L(-1), 205.4+/-3.3 nkat x L(-1) and 1035.4+/-116.9 nkat x L(-1), were similar to those of control group. At the same time, TBE and MTT analysis indicated that there was no significant cell viability difference between electroporation groups and control group. CONCLUSION: This single-pulse electroporation procedure performed at low voltage (220-400 V) but with high capacitance (950 microF) is one of the optimal choices to introduce foreign genes into primary rat hepatocyte.

[Replication and transfection of hepatitis B virus DNA into primary duck hepatocytes].

OBJECTIVE: By studying the possibility of obtaining expression of human hepatitis B virus (HBV) genes and production in normal liver cells from heterologous species like normal primary duck hepatocytes (PDH), to investigate the species-specificity of HBV infection and replication. METHODS: Two days after transfecting the complete HBV genome into PDH by electroporation (transfected group), HBsAg and HBeAg in the supernatants and lysates of PDH were measured by the IMX system. Meanwhile, replication of HBV in PDH was analyzed by Southern blotting and dot blotting procedures. PDH was electroporated as control. RESULTS: HBsAg in the lysate of transfected group was 9.10 (P/N values, positive?2.1), HBeAg was 1.0 (negative?2.1), both were negative in the supernatants of transfected group. dot blotting revealed that transfected group was strongly positive, whereas the control group was negative. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (RC), covalently closed circular (CCC) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, and there was no integrated HBV DNA in the cellular genome. Control groups were negative. CONCLUSIONS: Replication of HBV can occur in hepatocytes from nonmammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.