952. Combination Antiviral Effects of 2’, 3’-dideoxy-3’fluoroguanosine with Nucleos(t)ide Analogs Against Hepatitis B Virus In-vitro

L. Lu; C. Hui; H. Zhang; Y. Yueng; K. Cheung; J. M. Luk; G. K. Lau

 

Background and Aims

The nucleoside analogue 2’, 3’-dideoxy-3’fluoroguanosine (FLG) acts as an inhibitor of viral DNA polymerase and a chain terminator of viral reverse transcription. FLG has been shown to be active against wild-type and drug-resistant HBV in the in vitro models (Jacquard et al 2006). In this study, we investigated the in vitro antiviral efficacy of combinations of FLG with L-nucleoside analogs (lamivudine and clevudine), acyclic phosphate nucleotide analogues (PMEA, adefovir and PMPA, tenofovir) and carbocyclic analogues (entecavir).

 

Methods

Using the HepAD38 cell line that expresses high levels of wild-type HBV as the in vitro cell model, we assayed the antiviral activities and cytotoxities of each drug alone and in combination with FLG (Tibotec Pharmaceuticals Ltd). Real time PCR was used for the measurement of HBV DNA generated by HepAD38, and the cytotoxicity was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Each combination experiment included 24 samples, 13 of which were controls (five doses of each drug alone plus three untreated wells) and 11 of which were drug combinations. Of the five concentrations tested for each drug, the middle dose was equal to the IC50; two higher doses (corresponding to three and six times the IC50) and two lower doses (corresponding to 0.16 and 0.33 times the IC50) were also tested.

The combination data were analyzed through the Bliss independence model which is defined by the equation Exy= Ex+Ey-(ExEy), where ExEy is the additive effect of drugs x and y as predicted by their individual effects Ex and Ey. The MacSynergy II program, version 1.0 (M. N. Prichard, University of Alabama) was used to evaluate antiviral data according to the Bliss independence model. MacSynergy II uses a nonparametric three-dimensional approach to quantify areas where observed effects are significantly greater (synergy) or less (antagonism) than those predicted from single-drug control data.

 

Results

Analysis using the Bliss independence model indicated that FLG exerted significant synergistic antiviral effects when combined with lamivudine (V=41.51 μM2) or tenofovir (V=95.07μM2) and additive effects when combined with adefovir (V<25 μM2). However, minor antagonism effects were displayed when FLG combined with entecavir (V=-32.12 μM2) or clevudine (V=-60.25 μM2). There was no evidence of cytotoxicity with any of the drugs when used alone or in combination at the tested doses.

 

Conclusion

FLG exerted different kinds of combination antiviral effects when it was used with other nucleos(t)ide analogs. Future clinical study on FLG-related combination therapy is warranted.

 


110.  Induction of Antigen-specific Humoral and Cellular Immune Responses by Antigen-pulsed Human Blood Dendritic Cells in Hepatitis B Vaccine Nonresponders and Patients with Chronic Hepatitis B

S. Akbar; O. Yoshida; S. Furukawa; Y. Hiasa; N. Horiike; M. Onji

 

Purpose

Dendritic cells (DCs) loaded with HBV-related antigens induce HBV-specific immunity in HBV-transgenic mice. However, the utility of antigen-pulsed DCs have not been assessed in human in the context of HBV infection. This study was conducted (1) to prepare hepatitis B surface antigen (HBsAg)-pulsed human blood DCs, (2) to evaluate their safety and (3) to assess their prophylactic and therapeutic efficacies against HBV.

 

Methods

Six hepatitis B vaccine nonresponders, who never developed antibody to HBsAg (anti-HBs) due to immunization with commercial vaccines for 6-9 times and 5 patients with chronic hepatitis B were enrolled in this study. Immature blood DCs were prepared by culturing peripheral blood mononuclear cells with human grade granulocyte-macrophage colony stimulating factor and interleukin-4 for 7 days. HBsAg-pulsed DCs were prepared by culturing immature DCs with HBsAg (Hepatvex II, Banyu Pharmaceutical Co, Tokyo, Japan) for 8 hours. Five million HBsAg-pulsed DCs were administered to each individual and parameters of generalized inflammation, liver functions, kidney functions, and autoantibodies were checked at day 0, 1, 3, 7, 14, 28, 90, and 180 and after 2 years. HBsAg-specific T cell responses and anti-HBs were assessed in all subjects before and after administration of HBsAg-pulsed DCs.

 

Results

HBsAg-pulsed human blood DCs expressed DC-related markers such as HLA DR, CD86 and CD40. In vitro, HBsAg-pulsed DCs induced proliferation of HBsAg-specific T cells and anti-HBs from HBsAg-specific B cells. Hepatitis B vaccine nonresponders or patients with chronic hepatitis B did not develop any adverse effects due to administration of HBsAg-pulsed DCs.

 

All vaccine nonresponders and patients with chronic hepatitis B did not show anti-HBs or HBsAg-specific T cells before administration of HBsAg-pulsed DCs. Anti-HBs and HBsAg-specific T cells were detected in the peripheral blood of all vaccine nonresponders within 28 days after single administration of HBsAg-pulsed DCs. HBsAg-pulsed DCs were safe for all patients with chronic hepatitis B. Three and 2 of these patients exhibited HBsAg-specific T cells and anti-HBs in the peripheral blood, respectively.

 

Conclusions

This study has opened a new field of immune intervention strategy against HBV infection in human in which human consumable HBsAg-pulsed DCs have been prepared in vitro and their safety and efficacy have been confirmed in vivo. The principle of this study may be used for development of cell-based prophylactic and therapeutic vaccines against hepatitis C virus and other viruses when those cannot be achieved by traditional approaches.

 


111. Powerful inhibition of hepatitis B virus replication by CpG-induced cytokines in combination with lamivudine in vitro

I. E. Vincent; J. Lucifora; I. Chemin; D. Durantel; F. Zoulim; C. Trepo

 

Background and Aims

CpG oligodesoxynucleotides (CpG ODN) are synthetic agonists of Toll-like receptor 9 (TLR9) and potent inducers of innate and acquired immunity. Antiviral mechanisms of CpG ODN include induction of antiviral and pro-inflammatory cytokines (INF-α, IL-6), activation of natural killer cells (INF-γ), thereby promoting strong TH1, CD8 T cell and humoral responses. Current therapy of chronic hepatitis B infection, either interferon alpha or nucleoside analogs, fail to control hepatitis B virus (HBV) replication in most patients. This limitation is mainly due to the slow kinetic of viral clearance, HBV genetic variability and weak or absent anti-viral cellular immunity. This provides the rational for the development of immunotherapeutic approaches combining nucleosid analogs and TLR9 agonists to accelerate viral clearance and restore anti-HBV specific immune responses.

 

Our aim was therefore to evaluate the antiviral effect of CpG-induced cytokines combined with lamivudine on HBV replication in vitro.

 

Methods

HepaRG cells were infected with purified HBV virions or transduced with HBV recombinant baculovirus. Peripheral blood mononuclear cells from naïve individuals were stimulated with CpG ODN and cytokine secretion (INF-α, INF-γ, IL-6) determined by Elisa. HBV-infected or -transduced HepaRG cells were treated with CpG-induced cytokines in combination with lamivudine. Antiviral effects were evaluated 48h post-treatment by quantification of antigen secretion (HBsAg, HBeAg) and HBV DNA intermediates of replication by Southern blot.

 

Results

CpG-induced cytokines inhibited HBsAg and HBeAg production from infected HepaRG cells. The antiviral effect was demonstrated by up to 90% inhibition of intracellular HBV DNA intermediates in HBV transduced-cells. More importantly, the combination of CpG-induced cytokines with lamivudine reduced by one hundred fold the lamivudine IC50. Overall, combination was more effective than CpG-induced cytokines or lamivudine treatment alone.

 

Conclusions

CpG-induced cytokines with lamivudine represent a powerful combination to inhibit HBV replication in vitro, most likely by inhibiting different steps of viral replication. The use of TLR9 agonists combined with nucleoside analogs should be evaluated in vivo in order to assess restoration and duration of anti-HBV specific immune responses. The development of such novel immunotherapeutic combinations provides promising approaches to decrease the risk of liver disease progression in chronic hepatitis B patients.

 


1121. Long-Term Inhibition of Hepatitis B Virus Replication Using the Non-viral Episomal Vector pEPI-1

A. C. Jenke; V. Orth; H. Lipps; S. Wirth

 

Introduction and Aim

In the past, it has been shown that RNA interference (RNAi) has the potential to suppress hepatitis B virus (HBV) replication. For further gene therapy an optimal vector system free of common side effects as for example induction of mutagenesis has still to be found. The aim of this study was to test the non viral episomal vector system pEPI-1 as a tool for inhibition of HBV replication.

 

Methods

Recombinant vectors pEPI/HBV1 and pEPI/NonSense were constructed and transfected into HepG2.2.15 cells. After selection of transfected clones, cells were grown for 3 months in the absence of selection pressure and harvested at 2 and 12 weeks. The episomal status of the plasmid was confirmed by Southern analysis. The level of HBsAg mRNA was measured by RT-PCR and the concentration of HBsAg in the supernatant was determined using ELISA.

 

Results

Transfection of HepG2.2.15 cells with pEPI/HBV1 resulted in more than 90% suppression of HBsAg mRNA 2 weeks after transfection compared with the control plasmid pEPI/NonSense. This effect was even more pronounced after 12 weeks, when almost no HBsAg mRNA could be detected. Likewise, the secretion of HBsAg was inhibited by more than 95%. Also the number of DNA copies within the culture medium was significantly decreased.

 

Conclusion

The non viral episomal vector system pEPI-1 allows efficient long term suppression of HBV replication in the absence of selection pressure and without the risk for insertional mutagenesis or expression of additional viral proteins.

 


113. Adeno-associated Virus Rep78 Protein Inhibits Human Hepatitis B Virus Replication in Human Cells by Binding to X Promoter and C Promoter of HBV

T. Liu; H. You; M. Cong

 

Background and Aim

Rep78, the large rep gene product of adeno-associated virus (AAV), is a highly multifunctional protein, which is required for AAV DNA replication and the trans-regulation of AAV gene expression. Apart from these essential functions prerequisite for the life cycle of AAV, Rep78 is able to inhibit the replication and gene expression of some DNA virus by binding to the promoter, such as HPV and HIV. The aim of this study was to determine whether Rep78 can bind to the promoter of HBV DNA and then inhibit the replication of HBV DNA.

 

Methods and Results

Here, we demonstrated that Rep78, as a chimeric with the maltose binding protein, binds to both the full length of HBV X promoter and the full length of HBV C promoter, as analyzed by electrophoretic mobility shift assay. To map the key region of binding, sequentially smaller substrates from the full length of promoter were synthesized and tested for recognition by Rep78. It is demonstrated that the binding between Rep78 and the full length of HBV X promoter was specific, but not with Rep78 and the smaller substrates from the full length. It was therefore possible that multiple binding sites were needed or the secondary structure of the full length of HBV X promoter was needed for specifically binding with Rep78. The smaller substrates from the full length, B1, the active region of HBV C promoter can bind to Rep78 specifically. Furthermore, investigating the biological implications of these interactions, Rep78 inhibited both the HBV X promoter and HBV C promoter activity in vitro transcription assays in Hela nuclear extracts, and the inhibition of X promoter was stronger than that of C promoter.

 

In addition, to investigate whether the binding between Rep78 and HBV DNA promoters could inhibit the replication of HBV DNA, we constructed the plasmid containing the AAV2 rep78 gene and transfected the plasmid to the HepG2.2.15 cell line, the concentration of HBV cccDNA was decreased indicating that the replication of HBV was inhibited.

 

Conclusions

The ability of Rep78 to interact with the HBV X promoter and HBV C promoter and down-regulate corresponding transcription and replication suggests the mechanism by which AAV may modulate the HBV life cycle.

 


114. Construction of the shRNA Plasmid of Human fgl2 Prothrombinase Gene and Its Effect on hfgl2 Expression In-vitro

D. Xi; S. Gao; L. Li; C. Zhu; J. Guo; X. Luo; Q. Ning

 

Background and Aims

Our previous studies have shown an anti-sense plasmid to mouse fgl2 prothrombinase (mfgl2) gene significantly reduced mfgl2 gene expression in vivo, markedly ameliorated inflammatory infiltration, fibrin deposition and hepatocyte necrosis, prolonged the survival time period and elevated the survival rate in Balb/cJ mice with murine hepatitis virus type 3 (MHV-3) induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in the inhibitory application of human fgl2 prothrombinase (hfgl2) expression, which has been reported to be involved in a variety of disease development including fulminant viral hepatitis, acute rejection of allo/xeno transplantation and fetal loss syndrome.

 

Methods

A plasmid named p-hfgl2shRNA complementary to the sequence of hfgl2 was constructed, meanwhile irrelevant shRNA plasmid with a random combination of the hfgl2shRNA sequence was used as control. A plasmid named pEGFP-hfgl2 expressing hfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-hfgl2shRNA on the hfgl2 expression. By cotransfection of p-hfgl2shRNA and pEGFP-hfgl2 or pcDNA3.1-hfgl2 expression construct into CHO cells, the inhibition of hfgl2 expression by hfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, real time PCR and immunohistochemistry staining. Results: The experiments showed the significant inhibitory effect of p-hfgl2shRNA on hfgl2 expression at 48h post-transfection by observation of green fluorescent cells, FACS, real time PCR and immunohistochemistry staining as well in CHO cell lines with the inhibitory efficiency reached as high as 85.5%.

 

Conclusions

The study demonstrated that the construct of p-hfgl2shRNA successfully interferred hfgl2 expression in vitro and this provides the foundation for further investigation of this construct’s application in vivo and further more as a therapeutic strategy for a targeting intervention in the disease control to which the gene fgl2 contributed. This work was supported by the NSFC30571643,30672380 and National Key Basic Research Program of China(2005CB522901).

 


985. Hepatitis B Surface Antigen Titer Was Decreased During Clevudine Therapy

K. Byun; B. Yoo; K. Lee; Y. Chung; S. Ryu; M. Cho; J. Park; B. Kim; H. Lee; J. Han; S. Hwang; H. Kim; K. Yoo; Y. Lee; Y. Lee; C. Chon; S. Cho; J. Yang; Y. Kim; S. Choi; C. Han; M. Lee; J. Choi; H. Lee; J. Kim

 

Background

Clevudine therapy in chronic HBV patients resulted in a significant reduction of serum HBV DNA level. The purpose of this abstract is to determine if reduction of HBV DNA with clevudine therapy may lead to decrease of serum hepatitis B surface antigen (HBsAg).

 

Methods

A total of 50 naïve patients with chronic hepatitis B received clevudine 30 mg daily for 24 weeks followed by clevudine 10 mg daily for another 24 weeks. Serum HBV DNA was determined by Digene hybrid capture II assay (limit of detection of 4700 copies/mL) and Amplicor PCR assay (limit of detection of 300 copies/ml). Serum HBsAg was quantified by the ARCHITECT HBsAg assay.

 

Results

Based on the level of HBsAg titer at baseline, patients were stratified into four groups: group I (n = 18), < 10,000 IU/mL; group II (n = 15), 10,000 – 20,000; group III (n = 9), 20,000 – 40,000; group IV (n = 8), > 40,000). Median baseline HBV DNA were 6.1, 7.1, 8.1 and 9.0 log10 copies/mL and baseline HBsAg levels were 2922, 14577, 25200 and 102605 IU/mL in the group I, II, III and IV, respectively. During first 24 weeks of clevudine treatment, HBsAg titers of group II, III and IV except group I showed a rapid decrease which showed the similar pattern of HBV DNA suppression. At the end of 1-year treatment, median HBV DNA reduction from baseline was 3.6, 4.6, 5.6 and 4.9 log10 copies/mL and the change of HBsAg level from baseline of 1061, -2887, -5573, and –83909 IU/mL in the group I, II, III, and IV, respectively. Particularly, median serum HBsAg level in group IV at week 48 dropped to 18.2% of pretreatment level. Combined data of HBsAg reduction from group II, group III and group IV at week 48 showed statistically significant decrease (P = 0.002).

 

Conclusion

Strong viral suppression with clevudine therapy led to a significant decrease in serum HBsAg titer in patients who had more than 10,000 IU/mL of HBsAg titer before treatment.

 


1004. The Effects of α-galactosylceramide on Chronic Hepatitis B Infection in a Randomized Placebo-Controlled Phase I/II Trial

M. J. ter Borg; D. Sprengers; A. M. Woltman; B. E. von Blomberg; K. C. Van Nieuwkerk; W. C. Tielemans; R. Binda; R. van der Molen; H. L. Janssen

 

Background

The glycosphingolipid alpha-galactosylceramide (α-GalCer) has been shown to have an immunomodulatory effect mediated via the activation of natural killer T (NKT) cells and is able to induce a powerful antiviral immune response through the production of pro-inflammatory cytokines, such as IL-6. α-GalCer activated NKT cells are able to inhibit hepatitis B virus replication in HBV transgenic mice.

 

Aim

The aim of this dose-escalating randomized placebo-controlled phase I/II trial was to investigate the antiviral activity and safety of α-GalCer as a novel class of treatment for chronic hepatitis B patients.

 

Methods

Twenty-seven patients were allocated to a α-GalCer dose of 0.1 μg/kg (n=8), 1 μg/kg (n=6), 10 μg/kg (n=6) or to placebo (n=7). All patients had an HBV DNA level above 105 copies/ml at screening and ALT values >1.2 times the upper limit of normal. The treatment period consisted of intravenous injections with α-GalCer at 0, 4 and 8 weeks. Thereafter, patients were monitored for an additional 16 weeks.

 

Results

Although some patients showed an HBV-DNA decrease after administration, no clinically relevant suppression of viral replication was observed. Four patients exhibited a more than 0.5 log10 copies/mL decrease in HBV DNA following the first administration (1 μg/kg n=2, 10 μg/kg n=2) with a median decline in the first week of 1.1 log10 copies/mL (range 0.5 – 3.0) from baseline, but this decline was not sustained and also not observed after the second and third administration of α-GalCer. There were no clear changes in ALT levels. In three patients of the highest dose level, a rise in IL-6 production in serum was observed four hours after drug administration. In four patients (1 μg/kg n=1, 10 μg/kg n=3) α-GalCer was discontinued prematurely because of an episode of fever and severe rigors shortly after drug administration. Otherwise there were no significant side effects.

 

Conclusion

α-GalCer used as monotherapy for chronic hepatitis B infection at the doses (0.1-10 μg/kg) used in this trial had no clear effect on HBV DNA and ALT levels, was poorly tolerated, and is unlikely to provide an alternative as monotherapy for the current treatment of PEG-IFN and nucleos(t)ide analogs.