Transbody against hepatitis B virus core protein inhibits hepatitis B virus replication in vitro☆
Introduction
Infection with hepatitis B virus (HBV), a member of the hepadnavirus family, represents a severe public health problem worldwide. Although the administration of prophylactic vaccine has dramatically reduced the incidence of HBV infection, more than 350 million people are chronically infected with the virus globally, which may progress to chronic liver disease including chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1], [2], [3], [4].
Much effort and progress have been made in the development of antiviral drugs. To date, agents approved for treatment of chronic HBV infection are divided into two main groups, the immunomodulator, interferon-α (IFN-α), and the nucleotide/nucleoside analogues, lamivudine, adefovir dipivoxil, entecavir, telbivudine and tenofovir. However, IFN-α is effective in only 20–40% of patients with chronic HBV and causes numerous side effects [5]. The synthetic nucleot(s)ide analogues induce fewer side effects, but their therapeutic success is hampered by the development of drug-resistant mutants during long-term treatment and the high rate of relapse with the discontinuation of the drugs [6]. Therefore, there still exists a significant unmet medical need for safe and efficacious novel strategies to combat HBV infection.
Intrabody, an antibody or a fragment of an antibody, that is expressed intracellularly and can be directed to a specific target antigen present in various subcellular locations, is a novel approach to manage viral infections [7], [8]. Due to combining exquisite specificity and high antigen-binding affinity, intrabodies that interfere with viral replication have been developed against various viruses including HBV [9], [10], [11]. Nevertheless, there are two major challenges faced with intrabody expression through the use of recombinant DNA technology. First, the stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment [12]. This prevents the formation of intradomain disulfide bonds which in turn affects protein conformational folding. As a result, intrabodies can be non-functional, showing poor expression levels, low solubility, and a reduced active half-life within the cell. The second challenge is the safety concerns associated with the application of transfected recombinant DNA in human clinical therapy [13], which is required to achieve intrabody expression within the cells.
Protein transduction domains (PTDs) are short peptide sequences capable of transducing cargo, including protein, peptide, antisense oligonucleotides, peptide nucleic acids, siRNA, liposome and plasmid across the membrane, allowing cargo to accumulate within the cells [14]. PTDs have many desirable features for cellular delivery, such as efficacy in vivo, applicability in a wide variety of cell types and no apparent cargo size restrictions or immunogenic, antigenic or inflammatory properties. These properties make PTDs ideal for use in a wide range of therapeutic applications [15]. Therefore, a “cell-permeable” antibody, termed transbody, by coupling PTD to antibodies, which is different from the conventional intrabodies expressed within the cell, may provide a new strategy to interfere with viral replication and simultaneously overcome the shortcomings of intrabody.
The hepadnavirus core antigen is essential for not only the nucleocapsid assembly, but also the regulation of the virus replication in the life cycle [16], [17], [18]. Therefore, a strategy that makes the antibodies against core antigen to be able to enter the virus infected cells may represent a novel approach to inhibit the virus replication.
Currently, HepG2.2.15 cell is a widely used cell model for studying HBV in vitro [19], [20]. In this study, HBV core antigen monoclonal antibodies (HBcMAb) were developed into a cell-penetrable formats, HBV transbody, by coupling one of the most commonly used PTD found in the human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (TAT), the TAT protein transduction domain (TAT PTD, residues 47-57:YGRKKRRQRRR) [14], [15], to HBcMAb. The HBV transbody was then tested for the ability to penetrate the cells and to interfere with the replication of HBV in vitro in HepG2.2.15 cells. To our knowledge, this is the first report of “cell-permeable” antibody or transbody-mediated inhibition of hepadnaviruses, showing that this approach potently suppressed the virus replication in vitro and thus may provide a novel and specific therapeutic strategy for the treatment of HBV infection.
Section snippets
Coupling TAT PTD to HBcMAb
The preparation of HBcMAb (Xi'an Hua Guang Biology Engineering Co., Xi'an, China) coupled with TAT PTD (Beijing Scilight Biotechnology Ltd. Co., Beijing, China) was carried out according to Imject Immunogen EDC Conjugation Kits (Pierce, Holmdel, NJ, USA). Briefly, HBcMAb was dissolved in 0.2 mL of deionized water, and the equimolar of TAT PTD with antibody was dissolved in 0.5 mL of conjugation buffer and added to 0.2 mL antibody solution. Then three-fold molar excess of EDC was added to the
Binding of HBcMAb–TAT PTD conjugate to HBcAg
HBcMAb–TAT PTD conjugate was generated as described in the Material and methods section. To verify whether the HBcMAb and TAT were coupled successfully and the conjugate was able to bind to HBcAg, binding of the HBcMAb–TAT PTD conjugate to HBcAg was evaluated by an ELISA using HBcAg precoated plate and mouse anti-TAT-HRP as the detection reagent. With the reduction of the concentration of HBcMAb–TAT PTD conjugate and mouse anti-TAT antibody, the optical density was gradually reduced in a
Discussion
Clinically, most hepatitis B patients produce anti-HBcAg antibody of high titer and this antibody is only used as a diagnostic marker for HBV infection [21]. The anti-HBcAg antibody does not show any antiviral effect owing to the inability to enter the virus infected cells. Antibody against HBcAg may inhibit the virus replication by blocking the functions of HBcAg in multiple steps of the virus life cycle if it can penetrate the HBV infected cells. In fact, intrabodies targeting HBcAg have been
Disclosures
The authors disclose no conflict of interests.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant no. 81101260) and Innovation Project Plan Program of Shaanxi Province (Grant no. 2011KTCL03-16). The authors thank Dr. Ai Feng, Wei Wang, Lin Zhang, Quanli Wang, Guoqing Zhou, Guangde yang, Jing Zhang and Lieting Ma for their help in the study.
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Grant Support: National Natural Science Foundation of China (Grant no. 81101260) and Innovation Project Plan Program of Shaanxi Province (Grant no. 2011KTCL03-16).