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. 2018 Jan 1;8(2):549-562.
doi: 10.7150/thno.20047. eCollection 2018.

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

Jun-Fang Zhang  1   2   3 , Hua-Long Xiong  1   2 , Jia-Li Cao  1   2 , Shao-Juan Wang  1   2 , Xue-Ran Guo  1   2 , Bi-Yun Lin  4 , Ying Zhang  1   2 , Jing-Hua Zhao  1   2 , Ying-Bin Wang  1   2 , Tian-Ying Zhang  1   2 , Quan Yuan  1   2 , Jun Zhang  1   2 , Ning-Shao Xia  1   2
Affiliations

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

Jun-Fang Zhang et al. Theranostics. .

Erratum in

Abstract

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.

Keywords: Hepatitis B virus X protein; antibodies for intracellular targets; antibody-mediated intracellular immunity; cell-penetrating peptide; hepatitis B virus.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Characterization of the 9D11 mAb specific for HBx protein. Immunofluorescence (A) and Western blot (B) detection of intracellular HBx of genotypes A, B, C, and D in Huh7 cells transfected with HBx-expressing plasmids using the 9D11 mAb. For Western blots, GFP (derived from co-transfected plasmid of pcDNA3.1-GFP) served as a transfection control and β-tubulin served as a sample loading control. Relative HBx-expressing levels of various plasmids were determined by ImageJ and normalized with β-tubulin. The scale bar is 20 µm for the immunofluorescence images. (C) Immunohistochemical analyses of HBx (by 9D11) and HBsAg (by 83H12) in liver tissue samples from patients with CHB related HCC. A total of 3 matched pair tissue sections (both adjacent paracancerous and tumor tissue derived from the same patient) of HBV-positive HCC patients were analyzed. The scale bar is 50 µm. Red arrows indicate representative HBx-stained cells. (D) Western blot analyses of intracellular HBx (by 9D11) and HBsAg (by 83H12) in 5 human CHB-related liver tumor samples and their surrounding non-tumor liver samples. T represents tumor samples; NT represents their surrounding non-tumor samples. Wt-HBx represents the full-length HBx; HBx-ΔC represents C-terminal truncated HBx. The unglycosylated small-HBsAg is p24 and its N-glycosylated form is gp27. (E) Western blot analyses of antibody expressions of Huh7 cells transfected with 9D11 and Ctr-Ab expressing plasmids. Relative expressing levels of antibody H-Chain and L-Chain of the two plasmids were determined by ImageJ and normalized with β-tubulin. (F) Analyses of HBV suppression effect of plasmid transfection-mediated intracellular expression of 9D11 mAb in HBV-transfected Huh7 cells. The levels of various viral antigens (HBsAg, HBeAg, and HBcAg) in supernatants and cell lysates were quantitatively measured by commercial assays. The results represented the fold changes of viral antigen levels from the mAb expressing plasmids-transfected cells compared with those from the vector-transfected cells. The data represented mean ± SD from three independent experiments.
Figure 2
Figure 2
Tat-mediated efficient intracellular delivery of HBx mAb via the endocytic pathway. (A) Schematic structure of Tat-modified antibody. (B) HBx-binding activities of various Tat-modified recombinant 9D11 mAbs. A total of 4 Tat-modified mAbs were generated via fusion expression of the Tat sequence (12 aa) on the N- or C-terminus of the heavy chain (H) or light chain (L) of 9D11. The HBx-binding activities to recombinant HBx were measured by chemiluminescence enzyme immunoassay (CLEIA). The data represent the mean values from three independent experiments. Native 9D11 mAb was used as the control. (C) Immunofluorescence images of intracellular antibodies of Huh7 cells treated with 9D11-Tat and 9D11 at a concentration of 200 µg/mL. The scale bar is 20 µm. (D) Compounds that inhibit various endocytic pathways, including clathrin-mediated endocytosis inhibited by CPZ; lipid raft inhibited by MβCD; clathrin-independent endocytosis inhibited by FLP; and phagocytosis and micropinocytosis inhibited by CYTD. (E) Inhibition of intracellular delivery of 9D11-Tat by CPZ (10 µg/mL), MβCD (1.0 µg/mL), FLP (5.0 mM) and CYTD (4.0 µg/mL) treatments in Huh7 cells. The scale bar is 20 µm.
Figure 3
Figure 3
Intracellular delivery of 9D11-Tat mAb suppressed HBV transcription and replication. (A) Time-course monitoring of internalization of 9D11-Tat and 9D11 in Huh7 cells. Cells were incubated with 9D11-Tat or 9D11 at 200 µg/mL for 0.5, 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0 h. At the end of incubation, cells were washed to remove residual mAbs and collected by trypsinization. The intracellular mAb levels of cell lysates were determined by CLEIA. The data represent mean ± SD from three independent experiments. (B) Immunofluorescence confocal laser scanning microscopy to assess the intracellular localizations of internalized 9D11-Tat and HBx. Huh7 cells transfected with HBV48-WT plasmid were incubated with 9D11-Tat mAb. 6 h after the incubation, the cells were fixed and immunofluorescence detections were performed. The internalized 9D11-Tat was detected by using an Alexa Fluor® 488 labelled goat anti-human antibody, whereas HBx was detected using Alexa Fluor® 594 conjugated anti-HBx antibody 20F3, which targets a different HBx epitope. (C) Intracellular HBx levels of HBV48-WT-transfected Huh7 cells after treatments of 9D11-Tat, Ctr-Ab-Tat, and 9D11 mAbs (200 µg/mL). The internalized mAbs were indicated by the heavy chains. β-tubulin served as a sample loading control. Relative levels of HBx and antibody H-Chain were determined by ImageJ and normalized with β-tubulin. (D) Northern blot and (E) Southern blot assays to assess the effect of mAb treatments on HBV RNA transcription and DNA replication. Relative levels of HBV RNAs and DNAs were determined by ImageJ and normalized with GAPDH. RC: relaxed-circular DNA; DSL: double-strand linear DNA; SS: single-stranded DNA.
Figure 4
Figure 4
Intracellular delivery of 9D11-Tat mAb suppressed the expression of HBV proteins. Quantitative analyses for extracellular HBsAg (A), extracellular HBeAg (B), intracellular HBsAg (C) and HBcAg (D) of the HBV plasmid-transfected Huh7 cells treated with 9D11-Tat, Ctr-Ab-Tat, and 9D11 mAbs (200 µg/mL). HBV48-X null is an HBx-defective HBV mutant. The suppression effects of the mAbs were also investigated in HepG2-NTCP cells that support in vitro HBV infection. The secreted HBsAg (E), secreted HBeAg (F), and intracellular HBcAg (G) of HBV-infected HepG2-NTCP cells treated with 9D11-Tat, Ctr-Ab-Tat, and 9D11 mAbs (200 µg/mL) were analyzed at day 8 post infection (2 days after treatment). The data represented mean± SD from three independent experiments. p values were calculated using a two-sided unpaired t test, ** indicates p<0.01 and ns indicates p>0.05.
Figure 5
Figure 5
9D11-Tat suppressed HBV by TRIM21-dependent pathway (A) Immunofluorescence confocal laser scanning microscopy to assess the intracellular localizations of internalized 9D11-Tat and TRIM21. (B) 9D11-Tat-mediated inhibitions on HBx (B), HBsAg (C), and HBeAg (D) were reduced by TRIM21 knockdown. (C) The interaction between 9D11-Tat and TRIM21 was HBV-independent, and could be abolished via introduction of mutations in the CH3 domain of the 9D11 Fc region (9D11-Mut-Tat). (D) TRIM21 binding-defected 9D11-Tat mutants (9D11-Mut-Tat and 9D11-CH3-/--Tat) lost HBV suppression effects. For Western blots, relative levels of all detection targets were determined by ImageJ and normalized with β-tubulin. For (C), (D), and (F), the data represent mean ± SD from three independent experiments. p values were calculated using a two-sided unpaired t test, ** indicates p<0.01, * indicates p<0.05 and ns indicated p>0.05.
Figure 6
Figure 6
MG132 decreased the HBV suppression effects of 9D11-Tat. Proteasome inhibitor (MG132) treatment (2 µM) reduced the HBV suppression effects of 9D11-Tat in HBV-transfected Huh7 cells on the levels of intracellular HBx (A), secreted HBsAg (B), secreted HBeAg (C), and intracellular HBcAg (D). Relative HBx levels in (A) were determined by ImageJ and normalized with β-tubulin. For (B), (C), and (D), the data represent mean ± SD from three independent experiments. p values were calculated using a two-sided unpaired t test, ** indicates p<0.01, * indicates p<0.05, and ns indicates p>0.05.
Figure 7
Figure 7
9D11-Tat suppressed HBV in mice. Evaluation of the HBV suppression effects of 9D11-Tat in mice on the levels of serum HBsAg (A) and HBeAg (B) at 4 days after hydrodynamic injections of HBV48-WT and HBV-X null plasmids. Antibodies were used at a dosage of 10 mg/kg. The data are expressed as the mean ± SD (n=4 per group). (C) Southern blot assays for intrahepatic levels of HBV DNA after hydrodynamic injection of HBV48-WT; mice received mAb treatments (4 days after mAb infusion). (D) Semi-quantitative analyses of HBV replicative intermediates from data in (C), normalized by the input DNA band. (E-I) Effects of mAb treatment in HBV transgenic mice (n=4 per group). Serum HBV DNA (E), HBsAg (F), creatinine (G), and ALT (H) profiles of HBV-Tg mice after a single infusion of mAbs (9D11-Tat and Ctr-Ab-Tat, 10 mg/kg) or daily oral regimen of entecavir (3.2 mg/kg). The data are expressed as the mean± SD.
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