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A cDNA clone of silkworm (Bombyx mori) larval hemolymph antitrypsin (sw-AT) has been isolated from a fat body cDNA library. The cDNA has an open reading frame which codes a 392-amino acid residue polypeptide comprising a 16-residue signal peptide and a 376-residue mature sw-AT of Mr 41,805. The reactive site of sw-AT for inhibition of bovine trypsin [Sasaki, T. et al. (1987) J. Biochem. 102, 433-441] was identified as Lys343-Val344. Alignment of the sw-AT amino acid sequence with those of 11 members of the serpin superfamily of proteins clearly confirmed the homology of sw-AT with serpins. The amino acid sequence of sw-AT is 56% identical with that of the proteinase inhibitor from a lepidopteron, Manduca sexta [Kanost, M.R. et al. (1989) J. Biol. Chem. 264, 965-972], but the sequence around the reactive site shows no homology and the inhibitory specificity for proteinases is very different.

Although nucleot(s)ide analogues and pegylated interferon alpha 2a (PEG-IFN-␣2a) 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-␤ (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 Ͻ 0.001 and P Ͻ 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-␣2a treatment both in vitro and in vivo (P ϭ 0.004 and P ϭ 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-␣2a treatment in vivo (P ϭ 0.0495). cDNA microarrays and ELISA for CXCL10 production revealed significant differences between TRK-560 and PEG-IFN-␣2a 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-␣2a. 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 H epatitis 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

The cDNA of silkworm (Bornbyx rnori) antichymotrypsin (sw-Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature swAchy begins with Phel and ends with Phe384, with a preceding 16-amino-acid signal peptide. The amino-acid sequence similarities of sw-Achy with the serine-proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human a-1-antitrypsin and human a-1 -antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw-Achy. sw-Achy is thought to be a new member of the serpin family. Multiple alignment of sw-Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw-Achy with a-chymotrypsin was identified as Thr343-Ser344 by direct aminoacid sequence analysis of cleaved and purified protein.