• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.2 no.2
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• pp.169-177
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• 1999

Purpose: To evaluate the efficacy of interferon alpha therapy with or without prednisolone in children with chronic hepatitis B. Methods: Twenty-eight children (22 boys, 6 girls, mean age 130 months) had seropositive results for HBsAg, HBeAg and HBV DNA; 11 had chronic persistent hepatitis and 17 had chronic active hepatitis. The patients were divided into two groups depending upon their inflammatory activity on liver biopsy, pretreatment serum ALT levels and HBV DNA levels. Fourteen children (group 1: chronic active hepatitis, ALT ${\geq}$ 100 IU/L and HBV DNA ${\leq}$ 100 pg/$300\;{\mu}L$) received interferon alpha 2a 5 $MU/m^2$ of body surface three times weekly for 6 months. Fourteen children (group 2: chronic persistent hepatitis or chronic active hepatitis with ALT < 100 IU/L or HBV DNA > 100 pg/$300\;{\mu}L$) received prednisolone in decreasing daily doses of 60 mg/$m^2$, 40 mg/$m^2$, and 20 mg/$m^2$, each for 2 weeks, followed after 2 weeks by interferon alpha 2a on the same schedule. At the end of therapy, 3 end points were analyzed: HBeAg seroconversion, serum ALT normalization rate and clearance of serum HBV DNA. Results: At the end of treatment, HBe antigen-to antibody seroconversion was higher but not more significant in group 1 than group 2 (71.4% vs. 50.0%). Only one patient in group 2 who lost HBeAg, also cleared HBsAg. ALT normalization was similar in both groups (64.3% in group 1 vs. 55.6% in group 2). Clearance of serum HBV DNA was observed in 78.6% of patients in group 1 and 64.3% in group 2, but no significant differences. Complete response was similarly achieved in both groups (57.1% in group 1 vs. 50.0% in group 2). Interferon alpha therapy with prednisolone priming was well tolerated and all children finished therapy. Conclusion: The combined therapy with prednisolone followed by interferon alpha may be safe and effective in inducing a serological and biochemical remission of the disease in approximately 50% of children with chronic hepatitis B and with a high level of viral replication and less active disease. However, a controlled study should be performed to confirm these results.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.2
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• pp.173-177
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• 2006

In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PH A-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.

• IMMUNE NETWORK
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• v.4 no.1
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• pp.16-22
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• 2004

Background: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. Methods: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. Results: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of $4.46{\times}10^9cfu$ was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of $4.5{\times}10^{-7}M$ and $1.9{\times}10^{-7}M$, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. Conclusion: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.1 no.1
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• pp.79-89
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• 1998

Purpose: Though many antiviral or immunomodulatory agents have been used in patients with chronic HBV hepatitis, interferon is considered to be the only effective therapeutic agent so far. Among immunomodulatory agents, thymodulin, the oral form of thymosin, is currently in clinical trial. We compared the efficacy of alfa-interferon therapy alone with a combined therapy of alfa-interferon and thymodulin in children with chronic active hepatitis B. Method: Twenty three children aged 4.4~13.7 years who were known to be positive for HBsAg and HBeAg in serum for at least 6 months and who had biopsy-proven chronic active hepatitis were given either combined therapy of alfa-interferon and thymodulin or alfa-interferon alone, and all children were HBV DNA positive in their serum at the beginning. Follow-ups have been done for at least 1 year after a 6 month course of therapy and clearance of viral replication markers has been evaluated. Results: 1) During follow up period, 11 (48%) children were seroconverted to anti-HBe and were cleared of HBV DNA from their serum. However, 2 of them relapsed after discontinuance of interferon therapy. 2) Seroconversion occurred more frequently among those who had not been vertically transmitted, had elevated serum ALT levels and low HBV DNA levels before interferon therapy. 3) There was no significant advantage of the combined therapy with thymodulin compared to interferon therapy alone. Conclusion: Combined therapy of alfa-interferon and thymodulin failed to demonstrate synergistic effect. We think that combination therapies of alfa-interferon with other antiviral or immunomodulatory agents need to be studied in order to achieve better therapeutic responses.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.416-423
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• 2009

Hepatitis B core antigen(HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus(HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.3 no.1
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• pp.56-62
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• 2000

Purpose: The aim of this study was to evaluate the efficacy and histologic changes of interferon-alfa therapy on chronic hepatitis B virus infection in children. Patients and Methods: Thirty five children aged 3~16 years who were seropositive for HBV DNA, HBsAg and HBeAg were enrolled. Interferon-alfa 2a ($3.4\;MU/m^2$) were given for 6 months. Serologic markers of viral replication was evaluated 1 year after therapy. Post treatment liver biopsy was performed in 18 patients who showed serologic response. Results: Serum HBeAg and viral DNA became negative in 22 (63%) of treated children at 12 months after therapy. Serum aminotransferase levels normalized in all of the responders and HBsAg became negative in one responder. Horizontal transmission, serum aminotransferase levels more than twice normal, and active inflammation on liver biopsy were predictive factors for response to interferon therapy. Periportal piecemeal necrosis, lobular activity, portal inflammation, fibrosis, and total histologic activity index were reduced in responders. Conclusion: In children with chronic hepatitis B, interferon alfa promotes loss of viral replication and improves aminotransferase. Serologic response is associated with improvement in hepatic histology.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.47-51
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• 2006

Sodium butyrate (NaBu) is used as an enhancer for the production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, NaBu is well-known for its cytotoxic effect, thereby inducing apoptosis. CHO cells which had been engineered to express a humanised anti-HBV antibody were cultured using serum-free medium, Ex-cell 301. From a seeding density of $2{\times}10^5$ cells/ml, CHO cells grown with serum-free medium reached a maximum cell density of $1.3{\times}10^6$ cells/ml after 9 days in culture and produced a maximal antibody concentration of 130 mg/l after 13 days in culture. In the perfusion culture system, CHO cells producing anti-HBV antibody grown in an 7.5 1 bioreactor seeded with $2{\times}10^5$ cells/ml reached a maximal antibody concentration of 85 mg/1 after 720 h in culture. The addition of 0.3 mM NaBu and lowering culture temperature to $33^{\circ}C$ elongated the culture period to 60 days and increased the production yield by 2-fold, compared to control culture.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• Clinical and Experimental Pediatrics
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• v.51 no.11
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• pp.1165-1171
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• 2008

Purpose : This study aimed to identify the true extent of non-responsiveness in full-term infants born from HBsAg-negative or HBsAg-positive mothers and vaccinated against hepatitis B virus (HBV) at 0, 1, and 6 months of age and to evaluate the effect of revaccination among non-responders. Methods : The study included 716 full-term infants born in 2004-2007. Of 716, 662 infants (A group) were born to HBsAg-negative mothers and 54 infants (B group: 50, except HBsAg-positive infants) were born to HBsAg-positive mothers. All infants were administered DNA recombinant vaccines at 0, 1, and 6 months of age. B group infants received hepatitis B immunoglobulin at birth. Anti-HBs titers were tested at 7-12 and 9-15 months in A and B groups, respectively. Three revaccination doses were administered to non-responders whose anti-HBs titers were under 10 mIU/ml; revaccinated infants were retested at 1-3 months after last vaccination. The association between HBeAg seropositivity of mother and the failure of HBV immunoprophylaxis was evaluated. Results : The seroconversion rates after primary hepatitis B vaccination were higher in A group (94.1%) than in B group (78%, P<0.001). The seroconversion rates were high in revaccinated infants (A group non-responders: 96.9%, B group non-responders: 87.5%). The failure of HBV immunoprophylaxis was significantly associated with maternal HBeAg seropositivity (P<0.001). Conclusion : The seroconversion rates after primary hepatitis B vaccination were low in B group infants. Revaccination of non-responders in B group was very effective. Therefore, anti-HBs testing and revaccination of B group is very important. Revaccination of non-responders in A group was also very effective. Thus, testing the immune status of infants born to HBsAg-negative mothers even after primary hepatitis B vaccination should be considered. However, to realize this, further studies on the cost-effectiveness of anti-HBs testing in healthy full-term infants are necessary.