Inhibition of HBV gene expression and replication by stably expressed interferon-alpha1 via adeno-associated viral vectors

Abstract

Background: Interferon-alpha2 (IFNalpha2) is routinely used for anti-hepatitis B virus (HBV) treatment. However, the therapeutic efficiency is unsatisfactory, particularly in East Asia. Such inefficiency might be a result of the short half-life, relatively low local concentration and strong side-effects of interferons. Frequent and repeated injection is also a big burden for patients. In the present study, a single dose of vector-delivered IFNalpha1 was tested for its anti-HBV effects.

Methods: Adeno-associated viral vector (AAV-IFNalpha1) was generated to deliver the IFNalpha1 gene into hepatocytes. IFNalpha1, hepatitis B surface (HBsAg) and e (HBeAg) antigens were measured by enzyme-linked immunosorbent assay and/or western blotting. The level of viral DNA was measured by quantitative real-time polymerase chain reaction.

Results: AAV-IFNalpha1 effectively transduced HBV-producing cells (HepAD38) and mouse hepatocytes, where IFNalpha1 was expressed in a stable manner. Both intracellular and extracellular HBsAg and HBeAg were significantly reduced in vitro. In the HBV-producing mice, the concentration of IFNalpha1 in the liver was eight-fold higher than that in plasma. Compared with control groups, HBeAg/HBsAg antigen levels were reduced by more than ten-fold from day 1-5, and dropped to an undetectable level on day 9 in the AAV-IFNalpha1 group. Concurrently, the level of viral DNA decreased over 30-fold for several weeks.

Conclusions: A single dose administration of AAV-IFNalpha1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo. Therefore, the use of AAV-IFNalpha1 might be a potential alternative strategy for anti-HBV therapy.

Figure 1

IFNα1 expression after rAAV‐IFNα1 transduction of HepAD38 cells. IFNα1 protein was detected both in the culture media (A) and intracellular extracts (B) by western blotting. Cells were transduced with AAV‐EGFP (lanes 1, 2 and 3) or AAV‐IFNα1 (lanes 4, 5 and 6). The samples were collected at 24 h (lanes 1 and 4), 48 h (lanes 2 and 5) and 72 h (lanes 3 and 6) post‐transduction, respectively. (C) The IFN‐α1 protein level in the culture media was measured by ELISA

Figure 2

Reduction of HBsAg and HBeAg in the culture media. HepAD38 cells were transduced with AAV‐EGFP, AAV‐IFNα1 viruses or nontransduced (Mock). Relative HBsAg (A) and HBeAg (B) levels were measured by ELISA. Values are expressed as the mean ± SD (sample to cut‐off ratio) at each time point (n = 5, *p < 0.01). The experiments were performed at least three times, and consistent results were obtained

Figure 3

Significant reductions of HBV antigens and viral loads in the plasma after administration of AAV vectors. Both (A) HBsAg level and (B) HBeAg levels were determined by ELISA. The viral DNA was isolated from plasma and the viral load was measured by qPCR (C). *p < 0.01 indicates statistical significance

Figure 4

Highly effective gene transfers by rAAV and rAAV‐mediated high‐level gene expression in hepatocytes. Liver sections were examined under a fluorescence microscope (upper and middle panels) or a light microscope (lower panels). Upper panel: mice were administrated with PBS (control); middle panels: mice were administrated with AAV‐EGFP (left) and AAV‐IFNα1 (right). EGFP was directly detected under fluorescence microscopy; whereas IFNα1 was detected by indirect immunostaining with Cy3‐cojugated secondary antibody. Lower panel, liver sections were subjected to H&E staining

Figure 5

IFNα1, ALT and AST levels in plasma. Total proteins were extracted to measure: (A) the IFNα1 level and (B) ALT and AST levels in the plasma, after administration of AAV vectors (n = 5, *p < 0.01)

Figure 6

Local expression of IFNα1 reduced HBsAg level in the liver. The HBsAg was demonstrated by Cy3 fluorescence (middle panels); the position of cell nucleus was revealed by trypan blue staining (top panels)