Tianying Zhang

HBV life cycle is restricted in mouse hepatocytes expressing human NTCP

Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1–6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1–6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.

Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen

Objective This study aimed to investigate the therapeutic potential of monoclonal antibody (mAb) against HBV as a novel treatment approach to chronic hepatitis B (CHB) in mouse models. Methods Therapeutic effects of mAbs against various epitopes on viral surface protein were evaluated in mice mimicking persistent HBV infection. The immunological mechanisms of mAb-mediated viral clearance were systematically investigated. Results Among 11 tested mAbs, a novel mAb E6F6 exhibited the most striking therapeutic effects in several HBV-persistent mice. Single-dose administration of E6F6 could profoundly suppress the levels of hepatitis B surface antigen (HBsAg) and HBV DNA for several weeks in HBV-transgenic mice. E6F6 regimen efficiently prevented initial HBV infection, and reduced viral dissemination from infected hepatocytes in human-liver-chimeric mice. E6F6-based immunotherapy facilitated the restoration of anti-HBV T-cell response in hydrodynamic injection (HDI)-based HBV carrier mice. Immunological analyses suggested that the Fcγ receptor-dependent phagocytosis plays a predominant role in E6F6-mediated viral suppression. Molecular analyses suggested that E6F6 recognises an evolutionarily conserved epitope (GPCK(R)TCT) and only forms a smaller antibody–viral particle immune complex with limited interparticle crosslinking when it binds to viral particles. This unique binding characteristic of E6F6 to HBV was possibly associated with its effective in vivo opsonophagocytosis for viral clearance. Conclusions These results provided new insight into understanding the therapeutic role and mechanism of antibody against persistent viral infection. The E6F6-like mAbs may provide a novel immunotherapeutic agent against human chronic HBV infection.

Quantitative hepatitis B core antibody levels in the natural history of hepatitis B virus infection

We previously demonstrated that pretreatment quantitative anti-hepatitis B core protein (qAnti-HBc) levels can predict the treatment response for both interferon and nucleoside analogue therapy, but the characteristics of qAnti-HBc during chronic hepatitis B virus (HBV) infection remain poorly understood. To understand this issue, the qAnti-HBc levels were evaluated in individuals with past HBV infection, occult HBV infection and chronic HBV infection in the immune tolerance phase, immune clearance phase, low-replicative phase and hepatitis B e antigen (HBeAg)-negative hepatitis phase. Individuals with hepatitis B surface antigen (n = 598, 3.74 ± 0.90 log10 IU/mL) had significantly higher (p < 0.001, approximately 1000-fold) serum qAnti-HBc levels than those who had occult HBV, and serum qAnti-HBc levels were significantly higher in the occult HBV group than in the past HBV infection group (p < 0.001). qAnti-HBc levels were positively correlated with alanine aminotransferase levels (R = 0.663, p < 0.001), and subjects with an abnormal alanine aminotransferase level had a higher qAnti-HBc level (p < 0.001). Serum qAnti-HBc level varied in different phases of HBV infection, as determined by host immune status. Serum qAnti-HBc level is strongly associated with hepatitis activity in subjects with chronic HBV infection.

The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication.

The CRISPR/Cas9 system is a novel genome editing technology which has been successfully used to inhibit HBV replication. Here, we described a novel gRNA-microRNA (miRNA)-gRNA ternary cassette driven by a single U6 promoter. With an anti-HBV pri-miR31 mimic integrated between two HBV-specific gRNAs, both gRNAs could be separated from the long transcript of gRNA-miR-HBV-gRNA ternary cassette through Drosha/DGCR8 processing. The results showed that the gRNA-miR-HBV-gRNA ternary cassette could efficiently express two gRNAs and miR-HBV. The optimal length of pri-miRNA flanking sequence in our ternary cassette was determined to be 38 base pairs (bp). Besides, HBV-specific gRNAs and miR-HBV in gRNA-miR-HBV-gRNA ternary cassette could exert a synergistic effect in inhibiting HBV replication and destroying HBV genome in vitro and in vivo. Most importantly, together with RNA interference (RNAi) approach, the HBV-specific gRNAs showed the potent activity on the destruction of HBV covalently closed circular DNA (cccDNA). Since HBV cccDNA is an obstacle for the elimination of chronic HBV infection, the gRNA-miR-HBV-gRNA ternary cassette may be a potential tool for the clearance of HBV cccDNA.

Antibody-mediated immunotherapy against chronic hepatitis B virus infection

ABSTRACT The currently available drugs to treat hepatitis B virus (HBV) infection include interferons and nucleos(t)ide analogs, which can only induce disease remission and are inefficient for the functional cure of patients with chronic HBV infection (CHB). Since high titers of circulating hepatitis B surface antigen (HBsAg) may be essential to exhaust the host anti-HBV immune response and they cannot be significantly reduced by current drugs, new antiviral strategies aiming to suppress serum hepatitis B surface antigen (HBsAg) could help restore virus-specific immune responses and promote the eradication of the virus. As an alternative strategy, immunotherapy with HBsAg-specific antibodies has shown some direct HBsAg suppression effects in several preclinical and clinical trial studies. However, most described previously HBsAg-specific antibodies only had very short-term HBsAg suppression effects in CHB patients and animal models mimicking persistent HBV infection. More-potent antibodies with long-lasting HBsAg clearance effects are required for the development of the clinical application of antibody-mediated immunotherapy for CHB treatment. Our recent study described a novel mAb E6F6 that targets a unique epitope on HBsAg. It could durably suppress the levels of HBsAg and HBV DNA via Fcγ receptor-dependent phagocytosis in vivo. In this commentary, we summarize the current research progress, including the therapeutic roles and mechanisms of antibody-mediated HBV clearance as well as the epitope-determined therapeutic potency of the antibody. These insights may provide some clues and guidance to facilitate the development of therapeutic antibodies against persistent viral infection.

Toward the development of monoclonal antibody-based assays to probe virion-like epitopes in hepatitis B vaccine antigen

Prophylactic vaccines against hepatitis B Virus (HBV) infection were produced in different expression systems under different processing conditions. Since the recombinant HBV surface antigen (HBsAg) in these vaccines is a cysteine-rich protein with 14 cysteines among a total of 226 amino acids, the epitopes are dependent on the formation of intra- and intermolecular disulfide bonds. A panel of 22 monoclonal antibodies (mAbs) were developed and evaluated with respect to their sensitivity to disulfide reduction treatment of recombinant HBsAg. Not surprisingly, different mAbs showed different degree of sensitivity to controlled HBsAg disulfide reduction. With a view to exploring the functionality of anti-HBsAg mAbs to be used in HBsAg quality analysis, in vitro neutralization activity for the mAbs was assessed. One of the mAbs tested, 5F11, which showed high sensitivity to the disulfide integrity in HBsAg, was shown also to be highly effective in neutralizing HBV in vitro. Conversely, 42B6, while exhibiting similar neutralization activity, showed comparable binding HBsAg with or without reduction treatment. Based on these mAb characteristics, a sandwich ELISA with 42B6 being the capture Ab and detection Ab was developed to quantify HBsAg (like a “mass” assay) during antigen bioprocessing or in vaccine products. In parallel, when 5F11 was used as the detection Ab (with the same capture Ab), the assay can be used to probe disulfide-dependent and virion-like epitopes in intermediates or final products of hepatitis B vaccine, serving as a surrogate marker for vaccine efficacy to elicit neutralizing antibodies. This approach enables the comparative epitope specific antigenicity analysis of HBsAg antigen preparations from different sources.

A novel therapeutic anti-HBV antibody with increased binding to human FcRn improves in vivo PK in mice and monkeys

Antibody immunotherapy is a well-established therapeutic modality for cancer, acute viral infections (Marasco and Sui, 2007) and persistent viral infection such as HIV (Barouch et al., 2013) and HCMV (Freed et al., 2013). To reduce immunogenicity of rodent antibodies (Abs), approved antibody drugs entering clinical trials are of human origin or are humanized versions of rodent antibodies (Reichert, 2008). Recently, there is a strong drive to improve therapeutic efficacy, reduce cost, and provide convenient dosing to patients by designing next-generation antibodies with improved pharmacokinetic properties and modulated immune effector functions (Grevys et al., 2015). The neonatal Fc receptor (FcRn) is a heterodimer that comprises transmembrane α chains and β2-microglubulin (β2m). Optimizing FcRn-IgG interaction through Fc engineering is an effective strategy to improve pharmacokinetic (PK) or pharmacodynamics (PD) properties of therapeutic antibodies (Datta-Mannan et al., 2007). Increased affinity of the FcRn-IgG interaction at pH 6.0 and/or 7.4 has resulted in improved terminal phase halflife (t1/2) of antibodies in vivo (Dall’Acqua et al., 2002). In this study, five Fc variants known to enhance human FcRn (hFcRn) binding with mutations in the CH2 and/or CH3 domains were constructed on a humanized version of E6F6 (huE6F6), a novel therapeutic mAb against HBV. This mAb binds to an unique epitope on HBsAg and potently suppress levels of HBsAg and HBV DNA for several weeks in HBV transgenic mice (Zhang et al., 2016). All five Fc variants showed binding to hFcRn increased by a factor of up to 60-fold at pH 6.0 when compared to wild-type huE6F6 (WT huE6F6). A competitive binding assay was developed to identify the candidate suitable for further pharmacokinetic studies. Finally, huE6F6 Fc mutant M252Y/S254T/T256E (huE6F6-YTE) showed considerably longer serum half-life than the wild-type antibody in both mouse and cynomolgus monkey models. Taken together, these results provide a PKimproved immunotherapeutic agent, the first Fc-modified humanized antibody against chronic HBV infection (CHB). To obtain huE6F6 IgG1 Fc variants with enhanced PK properties, several Fc-engineered variants were made by substitution of amino acid residues at the CH2-CH3 interface, which have been reported to modulate binding to hFcRn, transplacental transport, and serum half-life. Our preliminary results suggested that only these five Fc mutants, T307A/ E380A/N434A (AAA) (Petkova et al., 2006; Yeung et al., 2010), M252Y/S254T/T256E (YTE) (Dall’Acqua et al., 2006; Zalevsky et al., 2010; Robbie et al., 2013), T250Q/M428L (QL) (Hinton et al., 2005), M428L/N434S (LS) (Zalevsky et al., 2010), and N434S (N/S) (Zalevsky et al., 2010) displayed increased level in hFcRn binding compared with WT huE6F6 (data not shown). HBsAg specific chemiluminescent enzyme immunoassay (CLEIA) of titrated Abs showed that these Fc mutants bound equally well to HBsAg, indicating that the Fc mutations had no effect on HBsAg binding (Fig. 1A). As hFcRn binds human IgG (hIgG) at the lower pH of the early endosome (pH 6.0–6.5) and dissociates at the neutral pH of blood (pH 7.4), we established a CLEIA assay to screen antibodies for hFcRn binding at pH 6.0 and pH 7.4, respectively. As expected, hFcRn was shown to bind WT huE6F6 and Fc mutants in a strictly pH-dependent manner, with strong binding at pH 6.0 but reduced interaction at pH 7.4 (Fig. 1B). Furthermore, a side-by-side comparison of the five Fc-engineered variants revealed that they all bound more strongly to hFcRn than did wild-type (WT) by a factor of 4 to 60 at pH 6.0, with a hierarchy from strongest to weakest binding as follows: YTE > LS > N/S > QL > AAA > WT (Fig. 1B). Significant increased level was detected in YTE variant group as compared with WT huE6F6 group (Fig. 1B, P < 0.05). The binding of Fc variants at pH 7.4 were comparably improved with the same ranking of binding affinity as that at pH 6.0 (Fig. 1C). To compare Fc-engineered antibodies and WT huE6F6 in a flow cytometry-based competitive assay of binding to hFcRn at pH 6.0, we constructed a human FcRn-transfected Madin-Darby canine kidney (MDCK) epithelial cell line. Dylight-594 labeled human IgG was used as competitor. Comparison of the IC50 values indicated that the YTE variant with IC50 values of 24.7 μg/mL performed about 40-fold better than did WT huE6F6 in competitive binding to hFcRn at pH 6.0 (Fig. 1D, P < 0.05), which was used in analyzing

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study.

A cell-penetrating whole molecule antibody targeting intracellular HBx suppresses hepatitis B virus via TRIM21-dependent pathway

Rationale: Monoclonal antibodies (mAbs) mostly targeting extracellular or cell surface molecules have been widely used in the treatment of various diseases. However, mAbs cannot pass through the cell membrane as efficiently as small compounds, thus limiting their use against intracellular targets. Methods to shuttle antibodies into living cells may largely expand research and application in areas based on mAbs. Hepatitis B virus X protein (HBx) is an important intracellular multi-functional viral protein in the life cycle of hepatitis B virus (HBV). HBx plays essential roles in virus infection and replication and is strongly associated with HBV-related carcinogenesis. Methods: In this study, we developed a cell-penetrating whole molecule antibody targeting HBx (9D11-Tat) by the fusion of a cell penetrating peptide (CPP) on the C-terminus of the heavy chain of a potent mAb specific to HBx (9D11). The anti-HBV effect and mechanism of 9D11-Tat were investigated in cell and mouse models mimicking chronic HBV infection. Results: Our results demonstrated that the recombinant 9D11-Tat antibody could efficiently internalize into living cells and significantly suppress viral transcription, replication, and protein production both in vitro and in vivo. Further analyses suggested the internalized 9D11-Tat antibody could greatly reduce intracellular HBx via Fc binding receptor TRIM21-mediated protein degradation. This process simultaneously stimulated the activations of NF-κB, AP-1, and IFN-β, which promoted an antiviral state of the host cell. Conclusion: In summary, our study offers a new approach to target intracellular pathogenesis-related protein by engineered cell-penetrating mAb expanding their potential for therapeutic applications. Moreover, the 9D11-Tat antibody may provide a novel therapeutic agent against human chronic HBV infection.

Role of quantitative hepatitis B core antibody levels in predicting significant liver inflammation in chronic hepatitis B patients with normal or near-normal alanine aminotransferase levels

Chronic hepatitis B (CHB) patients with normal alanine aminotransferase (ALT) levels are not free from significant hepatic lesions. Recently, there has been an improved understanding of the clinical significance of quantitative hepatitis B core antibody levels (qAnti‐HBc) during CHB management. In this cross‐sectional study, we evaluated the utility of qAnti‐HBc in identifying significant liver inflammation in CHB patients.