Development of a Novel Site-Specific Pegylated Interferon Beta for Antiviral Therapy of Chronic Hepatitis B Virus
ABSTRACT
Although nucleot(s)ide analogues and pegylated interferon alpha 2a (PEG-IFN-α2a) can suppress hepatitis B virus (HBV) replication, it is difficult to achieve complete HBV elimination from hepatocytes. A novel site-specific pegylated recombinant human IFN-β (TRK-560) was recently developed. In the present study, we evaluated the antiviral effects of TRK-560 on HBV replication in vitro and in vivo. In vitro and in vivo HBV replication models were treated with antivirals including TRK-560, and changes in HBV markers were evaluated. To analyze antiviral mechanisms, cDNA microarray analysis and an enzyme-linked immunoassay (ELISA) were performed. TRK-560 significantly suppressed the production of intracellular HBV replication intermediates and extracellular HBV surface antigen (HBsAg) (P < 0.001 and P < 0.001, respectively), and the antiviral effects of TRK-560 were enhanced in combination with nucleot(s)ide analogues, such as entecavir and tenofovir disoproxil fumarate. The reduction in HBV DNA levels by TRK-560 treatment was significantly higher than that by PEG-IFN-α2a treatment both in vitro and in vivo (P = 0.004 and P = 0.046, respectively), and intracellular HBV covalently closed circular DNA (cccDNA) reduction by TRK-560 treatment was also significantly higher than that by PEG-IFN-α2a treatment in vivo (P = 0.0495). cDNA microarrays and ELISA for CXCL10 production revealed significant differences between TRK-560 and PEG-IFN-α2a in the induction potency of interferon-stimulated genes. TRK-560 shows a stronger antiviral potency via higher induction of interferon-stimulated genes and stronger stimulation of immune cell chemotaxis than PEG-IFN-α2a. As HBsAg loss and HBV cccDNA eradication are important clinical goals, these results suggest a potential role for TRK-560 in the development of more effective treatment for chronic hepatitis B infection.
KEYWORDS: HBV, antiviral effect, gene expression, human hepatocyte chimeric mouse, pegylated interferon beta
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INTRODUCTION
Hepatitis B virus (HBV) infection is a serious global health problem. More than 500,000 people per year die due to HBV-related liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). To prevent the progression of liver diseases, antiviral therapies based on interferon (IFN) and/or nucleos(t)ide analogues (NAs) have been used in the treatment of chronic HBV infection (2,–4). Although these antiviral therapies can strongly suppress viral replication, it is difficult to achieve complete HBV elimination from hepatocytes. The main impediment to viral clearance is the presence of HBV covalently closed circular DNA (cccDNA) in the nucleus. HBV cccDNA is a minichromosome composed of viral DNA supplemented with histone and nonhistone host proteins (5,–10). In general, cccDNA is refractory to IFN and NA therapy.
[size=15.9991px]Currently, loss of the HBV surface antigen (HBsAg) is considered a crucial goal of antiviral therapy for preventing the progression to advanced liver diseases and for decreasing the mortality related to chronic hepatitis B infection (11). To achieve HBsAg loss, several clinical trials using pegylated interferon alpha 2a (PEG-IFN-α2a) in combination with NAs have been performed, and some positive results have been reported (12,–16). In one clinical trial using tenofovir disoproxil fumarate (TDF) and PEG-IFN-α2a, the cumulative HBsAg seroclearance rate in patients with combination therapy using TDF and PEG-IFN-α2a was significantly higher than in patients treated with either TDF or PEG-IFN-α2a monotherapy (13). However, the HBsAg seroclearance rate was inadequate (∼9.1%), and it is necessary to improve the success rate for treatment of chronic hepatitis B. Interferon beta (IFN-β) is a type I interferon that has been used in antiviral therapy for HBe antigen-positive chronic hepatitis B patients (17). IFN-β binds to IFN receptors IFNAR1 and IFNAR2, which are the same receptors used by IFN-α, and induces activation of interferon-stimulated genes (ISGs) via JAK/STAT signaling (18). Because IFN-β is administered intravenously, the blood concentration of IFN-β increases immediately; however, the concentration rapidly decreases to levels insufficient for antiviral effects. Therefore, only 20 to 50% of chronic hepatitis B patients were able to achieve HBV DNA reduction and alanine aminotransferase (ALT) normalization during IFN-β monotherapy, and ALT reelevation or HBV DNA rebound sometimes occurred after completion of the therapy in some responders (19). However, prolonged stimulation with IFN-α has been shown to induce a state of refractoriness, desensitizing cells to further interferon stimulation; on the other hand, Makowska et al. reported that repeated stimulation of hepatocytes with IFN-β or IFN-λ, unlike treatment with IFN-α, does not lead to refractoriness, at least in the case of hepatitis C virus infection (20). Therefore, we considered that the improved potency of IFN-β might suppress HBV replication or reduce HBV cccDNA more effectively than IFN-α. To improve the potency of IFN-β, the blood concentration of IFN-β should be maintained at a sufficient level for a longer period of time, and ISGs should be induced continuously without desensitizing the interferon signaling pathway. We have recently developed a novel site-specific pegylated recombinant human IFN-β (PEG-IFN-β; TRK-560) that contains a single polyethylene (PEG) molecule (43 kDa) covalently linked to the amino group of lysine (Lys) amino acid 134 of recombinant human IFN-β produced in Escherichia coli and has a longer blood half-life without loss of activity than conventional IFN-β (21). To avoid interfering with binding to IFN receptors due to pegylation and to avoid inducing neutralizing antibodies, in TRK-560 polyethylene glycol was site-specifically introduced into a single optimal amino acid within the epitope region of IFN-β. In the present study, we evaluated the antiviral effects of TRK-560 using in vitro and in vivo HBV replication models.
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RESULTS
Induction of ISGs by novel pegylated interferon-β, TRK-560.To verify the effects on interferon signaling by the novel pegylated IFN-β, TRK-560, Huh7 cells were treated with either PEG-IFN-α2a (100 IU/ml, or 7.14 ng/ml) or TRK-560 (100 IU/ml, or 0.887 ng/ml) for 4 h, and then cDNA microarray analysis was performed using the cells. The expression profiles of interferon-stimulated genes (ISGs) were compared between cells treated with TRK-560 and cells treated with PEG-IFN-α2a. As shown in Fig. 1A, most ISGs were regulated similarly by both TRK-560 and PEG-IFN-α2a treatment. However, although the cells were treated with the same unit dose (100 IU/ml) of PEG-IFN-α2a or TRK-560, the induction levels of several ISGs differed between treatment with PEG-IFN-α2a and TRK-560. To verify these differences, we also analyzed the induction levels of specific ISG products (MxA and CXCL10) by Western blotting and enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 1B, the concentration-response curve of TRK-560-induced CXCL10 production was shifted to the left compared with that of PEG-IFN-α2a. A difference in potency was also observed for MxA production (Fig. 1C). These results indicate that TRK-560 could activate interferon signaling and may have a higher potency for ISG induction.
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发表于 2018-2-28 21:20
