Persistent Hepatitis B Virus Infection Research Articles

483. Epigenetic Silencing of Hepatitis B cccDNA In Vitro and In Vivo Using AAV-Delivered Engineered Repressor Transcription Activator-Like Effector

Hepatitis B virus (HBV) infection is hyper-endemic (>8 % chronic carriers) to parts of Asia and sub-Saharan Africa. Persistent HBV infection predisposes to liver diseases such as cirrhosis and hepatocellular carcinoma. Current anti-HBV therapies face several limitations. RNA interference-based therapies lack the robustness and specificity required for therapeutic effect while interferon-α and nucleotide/side analogs function post-transcriptionally and thus allow for the persistence of the covalently closed circular (cccDNA). cccDNA may persist indefinitely and enables re-initiation of HBV replication after withdrawal of treatment. Disabling the cccDNA is essential for the successful treatment of chronic HBV. Our group previously described effective anti-HBV transcription activator-like effector (TALE) nucleases (TALENs). Although potentially useful to counter HBV replication, one drawback is that viral sequences integrated into the host genome may be susceptible to digestion by the TALENs. Transcriptional silencing, rather than cleavage, of cccDNA may therefore be preferable to avoid causing undesirable mutations in the host. To this end, TALE binding domains designed to target the viral preS2 promoter and the basic core promoter/enhancer II regions were fused to a Kruppel-associated box repressor domain to generate repressor-TALEs (rTALEs). These rTALEs were shown to inhibit viral replication in vitro and in vivo without inducing significant toxicity. In an in vivo murine model using hydrodynamic transfection with an HBV replication-competent plasmid, a reduction in secreted HBV surface antigen (HBsAg) of 97% and 98% was seen at day 3 and 93% and 96% at day 5 for P1L and P1R respectively. To develop this approach as a feasible therapy, rTALE-encoding sequences were incorporated into recombinant adeno-associated viruses (rAAVs) and assessed in the HepG2.hNTCP-C4 cell line. These cells overexpress the HBV receptor, human sodium taurocholate co-transporting peptide (hNTCP). The HepG2.NTCP-C4 cell line is infectable with HBV and viral gene expression is dependent on formation of cccDNA. Measurement of viral markers of replication may thus be used as an indicator of inhibitory effects on cccDNA. The anti-HBV efficacy of the rTALEs and epigenetic modification of the targeted HBV DNA was characterized. Chromatin immunoprecipitation assays determined the binding of the rTALEs to the cccDNA and induction of epigenetic markers of viral gene suppression. Mobility shift assays confirmed specificity of rTALE binding. In vivo efficacy of rAAV-delivered rTALEs was evaluated in transgenic HBV mice. Our study provides valuable information on the potential therapeutic utility of rTALEs and demonstrates the feasibility of the approach for treatment of HBV.

Hepatitis B virus infection: An insight into infection outcomes and recent treatment options.

Hepatitis B virus (HBV) currently infects an estimated population of 2billion individuals in the world, including 400million people with chronic HBV infection. HBV virology, replication and the host's immune response to HBV infection contribute to different infection outcomes. Acute hepatitis HBV infection is self-limiting but it leaves a residual infection that can become active in an individual during immunosuppression. In chronic HBV infection, the virus persistently replicates in hepatocytes leading to immune mediated hepatocellular damage. Despite the inability to remove the virus in more than 70% of patients, current treatments for chronic HBV infection, interferon alpha and antiviral nucleotide/nucleoside analogues, aim to reduce viral replication to prevent or at least delay the progression to cirrhosis and hepatocellular carcinoma. In both self resolved acute and persistent HBV infection, the long term existence of chromatinised covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes cannot be targeted by current treatments to eliminate these templates to eradicate the viral persistence. Identifying the mechanisms involve in the removal of infected hepatocytes will be useful as treatment options. In this context, DNA based novel therapeutic and immunization strategies might help to remove stable cccDNA and thus viral persistence.

IL-18 polymorphisms in hepatitis B virus related liver disease

Interleukine-18 (IL-18) was originally called interferon (INF-γ) inducing factor and plays a critical dual role in Th1 polarization and viral clearance. We aimed to explore whether single-nucleotide promoter polymorphisms (SNPs) are associated with the outcome of hepatitis B virus (HBV) infection. 271 HBV infected patients were recruited in this study out of these 109 were spontaneously recovered and 162 were diagnosed to be having persistent HBV infection which includes 48 chronic hepatitis, 84 liver cirrhosis, 30 HCC cases and were compared with 280 healthy controls. IL-18 promoter genotyping was performed with sequence-specific primers. The results demonstrated the significant involvement of genotype AA at position -607 in healthy controls (38.6%) when compared to cases (26.0%) (OR=0.54 (0.385–0.797)) and also associated with spontaneous clearance (37.6%) compared to persistent HBV infections (17.9%) (OR=2.76 (1.582–4.832)). Whereas, genotype CC at position -607 in cases (18.0%) when compared to healthy controls (6.7%) (OR=3.03 (1.734–5.303)) also associated with persistent HBV infections (24.1%) compared to spontaneous clearance (9.2%) (OR=0.31 (0.151–0.67)). And genotype GC at position -137 in cases (49.5%) compared to healthy controls (38.5%) (OR=1.55 (1.11–2.18)). Whereas, genotype GG at position -137 in healthy controls (56.8%) compared to cases (45.4%) (OR=0.63 (0.451–0.885)). No significant difference at position -137 was observed between spontaneous clearance and persistent HBV infections. These polymorphisms of the IL-18 gene promoter region at position -607 and -137 could be associated with different outcomes of HBV infection. The people with allele A at position -607 may be protected against HBV infection; moreover AA genotype is associated with spontaneous clearance.

Significant roles of regulatory T cells and myeloid derived suppressor cells in hepatitis B virus persistent infection and hepatitis B virus-related HCCs.

The adaptive immune system, including type1 helper T cells (Th1 cells), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs), plays an important role in the control of hepatitis B virus (HBV). On the other hand, regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) suppress the immune reaction in HBV and hepatocellular carcinoma (HCC). Excessive activation of immune suppressive cells could contribute to the persistent infection of HBV and the progression of HCC. The frequency and/or function of Tregs could affect the natural course in chronic hepatitis B patients and the treatment response. In addition to the suppressive function of MDSCs, MDSCs could affect the induction and function of Tregs. Therefore, we should understand in detail the mechanism by which Tregs and MDSCs are induced to control HBV persistent infection and HBV-related HCC. Immune suppressive cells, including Tregs and MDSCs, contribute to the difficulty in inducing an effective immune response for HBV persistent infection and HBV-related HCC. In this review, we focus on the Tregs and MDSCs that could be potential targets for immune therapy of chronic hepatitis B and HBV-related HCC.

A -819 C/T polymorphism in the interleukin-10 promoter is associated with persistent HBV infection, but -1082 A/G and -592A/C polymorphisms are not: a meta-analysis.

Single-nucleotide polymorphisms (SNPs) in the interleukin-10 (IL10) gene promoter have been associated with persistent hepatitis B virus (HBV) infection. In particular, the -1082A/G, -819 C/T and -592 A/C polymorphisms have most often been implicated. We performed a meta-analysis of available data to determine the relative importance of these SNPs in persistent HBV infection. We searched available articles in NCBI PubMed, EMBASE, the Chinese National Knowledge Infrastructure (CNKI), and the Chinese Biomedical Literature Database (CBM) and identified 24 studies for inclusion in our meta-analysis. Our results indicated that the presence of the IL10 -819 C allele significantly increased the risk for persistent HBV infection (CC+CT vs. TT: OR = 1.283, 95 % CI 1.023-1.610, P = 0.031; C vs. T: OR = 1.183, 95 % CI 1.001-1.399, P = 0.049). Meanwhile, the -1082A/-819T/-592A haplotype (OR = 0.751, 95 % CI 0.640-0.881, P = 0.000) and the -1082A/-819C/-592C haplotype (OR = 1.568, 95 % CI 1.304-1.884, P = 0.000) were observed to be significantly associated with HBV disease progression in Asians. In contrast, the IL10 -1082A/G and -592A/C polymorphisms were not associated with an increased susceptibility to or outcome of HBV infection. Our meta-analysis supports the growing body of evidence that the presence of the IL10 -819 C/T polymorphism is associated with persistent HBV infection and that the -1082A/-819T/-592A haplotype and the -1082A/-819C/-592C haplotype are associated with HBV disease progression in Asians.

Therapeutic vaccines in treating chronic hepatitis B: the end of the beginning or the beginning of the end?

The antiviral treatment of chronic hepatitis B virus (HBV) infection has greatly improved over the last 20 years since it has allowed a disappearance of cirrhosis decompensation and a significant reduction of the incidence of hepatocellular carcinoma. However, a complete HBV cure has not been achieved, and alternative treatments are still needed to optimize the current treatments. Therapeutic vaccination is a promising new strategy for controlling persistent infections and tumors. However, this approach has not been as successful as initially anticipated for chronic hepatitis B. General impairment of the immune responses generated during persistent HBV infection, with exhausted T cells not responding correctly to therapeutic vaccination, is most likely responsible for the poor clinical responses observed to date. We describe here the past approaches of therapeutic vaccination, in the hope that useful lessons will emerge from these previous clinical trials. Intensive research efforts are now focusing on a better understanding of immune responses in liver, on mechanisms by which HBV escapes innate immunity and on an accurate selection of the patients susceptible to benefit of immune therapy, which could increase the efficacy of therapeutic vaccination.

MicroRNA-26b Inhibits Hepatitis B Virus Transcription and Replication by Targeting the Host Factor CHORDC1 Protein

Hepatitis B virus (HBV) causes acute and chronic hepatitis in humans, and HBV infection is a major threat to global health. HBV replication is regulated by a series of host factors, including microRNAs (miRNAs), which are highly conserved small noncoding RNAs that participate in a variety of physiological and pathological processes. Here, we report that a chemically synthesized mimic of miR-26b inhibited HBV antigen expression, transcription, and replication, whereas antisense knockdown of endogenous miR-26b enhanced HBV replication in HepG2 cells. Overexpression and knockdown experiments showed that miR-26b significantly decreased HBV enhancer/promoter activities. We identified the cysteine- and histidine-rich domain containing 1 (CHORDC1) as a novel host factor target of miR-26b. CHORDC1 protein but not mRNA was markedly decreased by miR-26b overexpression via base-pairing with complementary sequences in the 3'UTR of its mRNA. Overexpression and knockdown studies showed that CHORDC1 increased viral antigen expression, transcription, and replication by elevating HBV enhancer/promoter activities. Conversely, HBV infection suppressed miR-26b expression and increased CHORDC1 protein levels in human liver cells. Another mature miRNA of the hsa-miR-26 family, miR-26a, had a similar function as miR-26b in targeting CHORDC1 and affecting HBV production. These results suggest that suppression of miR-26b expression up-regulates its target gene CHORDC1, which increases HBV enhancer/promoter activities and promotes viral transcription, gene expression, and replication. Our study could provide new insights into miRNA expression and the persistence of HBV infection.

Possible role of tocopherols in the modulation of host microRNA with potential antiviral activity in patients with hepatitis B virus-related persistent infection: a systematic review.

Hepatitis B virus (HBV) infection represents a serious global health problem and persistent HBV infection is associated with an increased risk of cirrhosis, hepatocellular carcinoma and liver failure. Recently, the study of the role of microRNA (miRNA) in the pathogenesis of HBV has gained considerable interest as well as new treatments against this pathogen have been approved. A few studies have investigated the antiviral activity of vitamin E (VE) in chronic HBV carriers. Herein, we review the possible role of tocopherols in the modulation of host miRNA with potential anti-HBV activity. A systematic research of the scientific literature was performed by searching the MEDLINE, Cochrane Library and EMBASE databases. The keywords used were 'HBV therapy', 'HBV treatment', 'VE antiviral effects', 'tocopherol antiviral activity', 'miRNA antiviral activity' and 'VE microRNA'. Reports describing the role of miRNA in the regulation of HBV life cycle, in vitro and in vivo available studies reporting the effects of VE on miRNA expression profiles and epigenetic networks, and clinical trials reporting the use of VE in patients with HBV-related chronic hepatitis were identified and examined. Based on the clinical results obtained in VE-treated chronic HBV carriers, we provide a reliable hypothesis for the possible role of this vitamin in the modulation of host miRNA profiles perturbed by this viral pathogen and in the regulation of some cellular miRNA with a suggested potential anti-HBV activity. This approach may contribute to the improvement of our understanding of pathogenetic mechanisms involved in HBV infection and increase the possibility of its management and treatment.

In vitro stimulation with HBV therapeutic vaccine candidate Nasvac activates B and T cells from chronic hepatitis B patients and healthy donors

Hepatitis B virus (HBV) chronic infections remain a considerable health problem worldwide. The standard therapies have demonstrated limited efficacy, side effects or need life-long treatments. Nowadays therapeutic vaccination is a promising option. Recently, we developed a new vaccine formulation called Nasvac, based on the combination of surface and core antigens from HBV. Clinical trials already performed showed good efficacy in virus control. However, the exact mode of action of Nasvac formulation remains unclear. So far the functional impairment of DCs during persistent HBV infection is a controversial issue. On the other hand, it is known that B cells may function as antigen presenting cells (APC) activating T cells. The hepatitis B core antigen contained in Nasvac vaccine is able to bind and activate a high frequency of naive human B cells. In the present study the surface expression of activation and exhaustion markers on B cells and the subsequent activation of T cells after in vitro stimulation with Nasvac antigens were evaluated in chronic HBV patients and healthy donors. B- and T-cell phenotype and proliferation were assessed by flow cytometry. Our results indicate that in contrast to exhaustions markers B cell activation markers were increased on both study groups after Nasvac stimulation. A shift toward an activation phenotype was observed for both B and T cells. The present work suggests that B cells could act as efficient APCs for Nasvac antigens in humans, which might suggest the use of activated B cells as immunotherapeutic strategy for chronic hepatitis B.

HLA-DPB1 Variant Effect on Hepatitis B Virus Clearance and Liver Cirrhosis Development Among Southwest Chinese Population.

Background:Previous studies have shown that genetic variants in HLA-DP genes affect disease progression in hepatitis B virus (HBV) infection.Objectives:We aimed to evaluate possible association between HLA-DPB1 rs9277534 polymorphism and different clinical complications of hepatitis B virus (HBV) infection.Materials and Methods:Snapshot assay was used to investigate the association of rs9277534 polymorphism in 342 patients with persistent HBV infection and 342 age and gender-matched HBV spontaneous clearance controls. Patients were categorized into asymptomatic HBV carriers (AsC, n = 104), chronic hepatitis B (CHB, n = 116), and liver cirrhosis (LC, n = 122) subgroups.Results:There was a significantly higher proportion of the rs9277534 minor allele A in HBV spontaneous clearance control than that in HBV persistent infection group (OR = 0.58, 95%CI = 0.46-0.73, P < 0.0001). Genotypic analysis showed that GA and AA genotypes were associated with HBV spontaneous clearance (GA: OR = 0.56, 95%CI = 0.40-0.79, P = 0.019; AA: OR = 0.24, 95%CI = 0.14-0.44, P < 0.0001). A significant difference was found between AsC and LC groups in the distribution of AA genotype (OR = 9.32, 95%CI = 1.293-67.14, P = 0.027).Conclusions:Variant at rs9277534 could affect both the spontaneous clearance of HBV infection and progression from asymptomatic HBV carriers to HBV-related liver cirrhosis in Southwest Han Chinese population.

Advances and challenges in the development of therapeutic DNA vaccines against hepatitis B virus infection.

Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Because very weak and functionally impaired virus-specific immune responses play a key role in the persistence of HBV infection, the stimulation of these responses appears to be of particular importance for virus clearance. In this regard DNA-based vaccination has emerged as novel, promising therapeutic approach for chronic hepatitis B. This review provides an update of preclinical studies in animal models (mouse, chimpanzee, duck, woodchuck), which evaluated the ability of DNA vaccines targeting hepadnaviral proteins to induce potent and sustained immune responses in naïve animals and to enhance virus clearance and break immune tolerance in chronic virus-carriers. Different strategies have been developed and evaluated in these models to optimize DNA vaccine including genetic adjuvants, combination with antiviral drugs, prime-boost regimens and plasmid delivery. The delivery of DNA by in vivo electroporation appears to be of particular interest for increase of vaccine potency in both small and large animal models. Based on the promising results generated in preclinical studies, first clinical trials of DNA vaccines have been initiated, although effective therapy of chronic hepatitis B awaits further improvements in vaccine efficacy.

Induction of hepatitis B virus surface antigen‐specific cytotoxic T lymphocytes can be up‐regulated by the inhibition of indoleamine 2, 3‐dioxygenase activity

Cytotoxic T lymphocytes (CTLs) are thought to be major effectors involved in viral clearance during acute infections, including hepatitis B virus (HBV) infection. A persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which may be reflective of tolerance to HBV. Efficient induction of HBV-specific CTLs leads to the clearance of HBV in patients with a chronic HBV infection. Previously, we reported that α-galactosylceramide (α-GalCer), a specific natural killer T (NKT) cell agonist, enhanced the induction of HBV surface antigen (HBsAg)-specific CTLs. In the present study, we found that inhibition of indoleamine 2,3-dioxygenase (IDO) activity enhanced the induction of HBsAg-specific CTLs after immunization with HBsAg and α-GalCer. The administration of HBsAg and α-GalCer increased the production of interleukin-2 and interleukin-12b, which are crucial for the induction of HBsAg-specific CTLs. The production of these cytokines was more strongly enhanced in IDO knockout mice compared with wild-type mice. In addition, α-GalCer induced the production of IDO in CD11b(+) cells, and these cells inhibited proliferation of HBsAg-specific CTLs. Our results lead to strategies for improving the induction of HBsAg-specific CTLs.

Polymorphisms in CISH gene are associated with persistent hepatitis B virus infection in Han Chinese population.

Background and AimCytokine-inducible SRC homology 2 domain protein (CISH) is the first member of the suppressors of cytokine signaling (SOCS) protein family. An association between multiple CISH polymorphisms and susceptibility to infectious diseases has been reported. This study aimed to investigate the possible association of these single nucleotide polymorphisms (SNPs) in CISH gene with different outcomes of Hepatitis B virus (HBV) infection.Methods1019 unrelated Chinese Han subjects, including 240 persistent asymptomatic HBV carriers, 217 chronic hepatitis B patients, 137 HBV-related liver cirrhosis patients, and 425 cases of spontaneously recovered HBV as controls, were studied. Four SNPs (rs622502, rs2239751, rs414171 and rs6768300) in CISH gene were genotyped with the snapshot technique. Transcriptional activity of the CISH promoter was assayed in vitro using the dual-luciferase reporter assay system.ResultsAt position rs414171, A allele and AA genotype frequencies were significantly higher in the HBV-resolved group as compared to the persistent HBV infection group. At position rs2239751, TT genotype was further observed in the HBV-resolved group. Using asymptomatic HBV carriers as controls, our results indicated that the rs414171 and rs2239751 polymorphisms were unrelated to HBV progression. The other two SNPs (rs622502 and rs6768300) showed no association with persistent HBV infection. Haplotype analysis revealed that the GGCA haplotype was associated with spontaneous clearance of HBV in this population. Moreover, luciferase activity was significantly higher in the PGL3-Basic-rs414171T construct as compared to the PGL3-Basic-rs414171A construct (p<0.001).ConclusionTwo SNPs (rs414171 and rs2239751) in the CISH gene were associated with persistent HBV infection in Han Chinese population, but not with HBV progression.

A genome-wide association study on chronic HBV infection and its clinical progression in male Han-Taiwanese.

It is common to observe the clustering of chronic hepatitis B surface antigen (HBsAg) carriers in families. Intra-familial transmission of hepatitis B virus (HBV) could be the reason for the familial clustering of HBsAg carriers. Additionally, genetic and gender factors have been reported to be involved. We conducted a three-stage genome-wide association study to identify genetic factors associated with chronic HBV susceptibility. A total of 1,065 male controls and 1,623 male HBsAg carriers were included. The whole-genome genotyping was done on Illumina HumanHap550 beadchips in 304 healthy controls and HumanHap610 beadchips in 321 cases. We found that rs9277535 (HLA-DPB1, P = 4.87×10−14), rs9276370 (HLA-DQA2, P = 1.9×10−12), rs7756516 and rs7453920 (HLA-DQB2, P = 1.48×10−11 and P = 6.66×10−15 respectively) were significantly associated with persistent HBV infection. A novel SNP rs9366816 near HLA-DPA3 also showed significant association (P = 2.58×10−10). The “T-T-G-G-T” haplotype of the five SNPs further signified their association with the disease (P = 1.48×10−12; OR = 1.49). The “T-T” haplotype composed of rs7756516 and rs9276370 was more prevalent in severe disease subgroups and associated with non-sustained therapeutic response (P = 0.0262). The “G-C” haplotype was associated with sustained therapeutic response (P = 0.0132; OR = 2.49). We confirmed that HLA-DPB1, HLA-DQA2 and HLA-DQB2 loci were associated with persistent HBV infection in male Taiwan Han-Chinese. In addition, the HLA-DQA2 and -DQB2 complex was associated with clinical progression and therapeutic response.

High prevalences of hepatitis B and C virus infections among adults living in Binh Thuan province, Vietnam.

Vietnam is one of the countries with the highest mortality from liver cancer, which is mostly attributed to hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. For planning preventive strategies against these infections, we investigated prevalences of HBV and HCV infections among adults living in Binh Thuan, Vietnam. Our study consisted of a serological survey for HBV and HCV infections and a questionnaire survey on their risk factors. The sample size was calculated based on anticipated rate of hepatitis B surface antigen (HBsAg). Subjects were randomly sampled using a multistage method. Confirmation and family-tree surveys were conducted to examine persistent HBV infection and intrafamilial HBV transmission, respectively. A total of 509 adults, comprised of 230 men (45.2%) and 279 women (54.8%), were enrolled. Prevalences of HBsAg, hepatitis B surface antibody and hepatitis B core antibody were 15.3%, 60.3% and 71.7%, respectively. Most HBV DNA positive sera were classified as genotype B (75.3%) and C (11.7%). Of HBsAg positive subjects, 96.7% were persistently infected and one acutely HBV infected person was identified. Family-tree surveys suggested that horizontal extrafamilial HBV transmission might have been frequent. Prevalences of anti-HCV and HCV RNA were 3.3% and 1.8%, respectively. HCV genotype 6a was prominent (55.6%). In Binh Thuan, prevalences of HBV and HCV infections are high, HBV genotype B and HCV genotype 6a are predominant, and horizontal HBV transmission may still occur. Therefore, raising the coverage of a universal HBV vaccination program may be an effective liver cancer control in Vietnam.

Downregulation of interleukin-18-mediated cell signaling and interferon gamma expression by the hepatitis B virus e antigen.

The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

Amino acid polymorphism in the reverse transcriptase region of hepatitis B virus and the relationship with nucleos(t)ide analogues treatment for preventing mother-to-infant transmission.

A high maternal serum hepatitis B virus (HBV) DNA level is associated with vaccine failure. The administration of nucleos(t)ide analogues (NAs) in pregnancy for decreasing serum HBV is regarded as an effective and safe method to reduce HBV perinatal transmission. However, the effect of NAs treatment is closely related to amino acid polymorphisms in the HBV reverse transcriptase (RT) region. The full-length RT coding region of 334 HBV isolates from untreated Chinese women of childbearing age with persistent HBV infection were sequenced and amino acid polymorphic analysis was performed to evaluate its impact on NAs treatment for decreasing HBV perinatal transmission. Of the 334 isolates, 36 (10.8%) harbored NAs resistance (NAr) mutations which were mainly putative drug mutations related to lamivudine. The primary drug mutation rtA181T/V was detected in three HBeAg-negative women with an HBV DNA level of <4 log IU/ml. These NAr mutations were rarely detected in women with an HBV DNA level of ≥7 log IU/ml (P = 0.014) or in women younger than 35 years (P = 0.001). The NAr mutation rate among young women (<35 years) who had a high HBV DNA level (≥7 log IU/ml) was significantly lower than in women who had lower HBV DNA levels (<7 log IU/ml) or who were older (≥35 years; P = 0.017). These results suggest that younger women with a high HBV DNA level harbor fewer NAr mutations and that this population may respond to NAs treatment for the prevention of mother-to-infant transmission.

Modulation of HBV replication by microRNA-15b through targeting hepatocyte nuclear factor 1α

Hepatitis B virus (HBV) infection remains a major health problem worldwide. The role played by microRNAs (miRNAs) in HBV replication and pathogenesis is being increasingly recognized. In this study, we found that miR-15b, an important miRNA during HBV infection and hepatocellular carcinoma development, directly binds hepatocyte nuclear factor 1α (HNF1α) mRNA, a negative regulator of HBV Enhancer I, to attenuate HNF1α expression, resulting in transactivation of HBV Enhancer I, in turn causing the enhancement of HBV replication and expression of HBV antigens, including HBx protein, finally leading to the down-regulated expression of miR-15b in both cell lines and mice in a long cascade of events. Our research showed that miR-15b promotes HBV replication by augmenting HBV Enhancer I activity via direct targeting HNF1α, while HBV replication and antigens expression, particularly the HBx protein, then repress the expression of miR-15b. The reciprocal regulation between miR-15b and HBV controls the level of HBV replication and might play a role in persistent HBV infection. This work adds to the body of knowledge concerning the complex interactions between HBV and host miRNAs.

Serum IL-21 levels associated with chronic hepatitis B and hepatitis B-related liver failure

The aim of the present study was to investigate the role of interleukin (IL)-21 in chronic hepatitis B virus (HBV) infection. IL-21 stimulates T and B cell responses and plays a role in the control of chronic viral infections. Serum IL-21 levels were measured by enzyme immunoassay in 109 patients with chronic HBV infection at various clinical stages, as well as in 19 healthy controls (HCs). The proportion of T cells producing IL-21 in the peripheral blood was assessed by intracellular cytokine staining and flow cytometry. Mean serum IL-21 levels in patients with chronic hepatitis B (CHB) and the HCs were 303.54±152.77 pg/ml and 68.24±9.06 pg/ml, respectively (P=0.003). In addition, the mean serum IL-21 level in patients with hepatitis B-related acute-on-chronic liver failure (HB-ACLF) was 455.38±412.38 pg/ml, which exhibited a statistically significant difference when compared with the HCs (P=0.000). Serum IL-21 levels were highest in the patients with HB-ACLF (455.38±412.38 pg/ml) and exhibited a significant difference when compared with the CHB patients (P=0.04). The mean serum IL-21 levels in patients with cirrhosis also increased, but there was no statistically significant difference when compared with the HCs (P=0.82). The frequency of IL-21+CD4+ cells also increased compared with the HCs and correlated with the number and percentage of lymphocytes in the peripheral blood. Serum IL-21 levels increased in CHB and HB-ACLF patients. Relatively low serum IL-21 levels in CHB may have a causal role in the persistence of HBV infection. Higher serum levels in HB-ACLF may activate T and B cells to eliminate the virus or injure the liver via the release of inflammatory cytokines.