Anti‑HBV effect of interferon‑thymosin α1 recombinant proteins in transgenic Dunaliella salina in vitro and in vivo

Zhang,Zhihao; He,Ping; Zhou,Yan; Xie,Xuhua; Feng,Shuying; Sun,Changyu

doi:10.3892/etm.2018.6227

Anti‑HBV effect of interferon‑thymosin α1 recombinant proteins in transgenic Dunaliella salina in vitro and in vivo

Authors:
- Zhihao Zhang
- Ping He
- Yan Zhou
- Xuhua Xie
- Shuying Feng
- Changyu Sun
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Affiliations: Department of Infectious Disease, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China, Medical Research Center, Henan University of Science and Technology, Luoyang, Henan 471023, P.R. China
Published online on: May 29, 2018 https://doi.org/10.3892/etm.2018.6227
Pages: 517-522
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to investigate the anti‑hepatitis B virus (HBV) effect of interferon (IFN)‑thymosin α1 (TA1) in a transgenic Dunaliella salina (TDS) system in vitro and in vivo. The toxicity of TDS in the HepG2.2.15 cell line was assessed using an MTT assay. The effect of TDS on the secretion of HBV early antigen (HBeAg) and HBV surface antigen (HBsAg) in culture supernatants was measured using ELISA. In addition, HBV‑DNA was analyzed using quantitative polymerase chain reaction. Drug treatment experiments were performed in vivo on ducks congenitally infected with duck HBV (DHBV). The drug was administered once daily for 21 continuous days. Blood was drawn from all ducks prior to treatment, following treatment for 7, 14 and 21 days, and following drug withdrawal for 5 days. Serum DHBV‑DNA was determined using quantitative PCR. In addition, the histology of duck liver tissues was assessed using hematoxylin and eosin, and orcein staining. The results demonstrated that TDS suppressed cell viability and HBsAg and HBeAg secretion in HepG2.2.15 cells. Furthermore, the treatment index values for HBsAg and HBeAg following TDS treatment were 2.96 and 3.07 respectively, which were greater than those of the IFN‑α treated group. In addition, the DHBV‑infected duck model experiments indicated that serum DHBV‑DNA levels were significantly decreased in the group of TDS (20 g/kg) following treatment for 7, 14 and 21 days compared with the control group. Following withdrawal of the drug for 5 days, the levels of DHBV‑DNA did not relapse in the medium and high dose groups of TDS (10 and 20 g/kg, respectively). Histological analysis of duck liver also demonstrated that TDS and IFN‑α treatment alleviated inflammation and HBsAg signals in duck livers. In conclusion, TDS markedly suppresses HBV replication in vitro and in vivo and its anti‑HBV effect is greater than that of IFN‑α.

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1	Seeger C and Mason WS: Hepatitis B virus biology. Microbiol Mol Biol Rev. 64:51–68. 2000. View Article : Google Scholar : PubMed/NCBI
2	Choi JG, Chung YH, Kim JA, Jin YJ, Park WH, Lee D, Shim JH, Lee YS, Seo DD, Jang MK, et al: High HBV-DNA titer in surrounding liver rather than in hepatocellular carcinoma tissue predisposes to recurrence after curative surgical resection. J Clin Gastroenterol. 46:413–419. 2012. View Article : Google Scholar : PubMed/NCBI
3	Yang G, Han M, Chen F, Xu Y, Chen E, Wang X, Liu Y, Sun J, Hou J, Ning Q and Wang Z: Hepatitis B virus genotype B and mutations in basal core promoter and pre-core/core genes associated with acute-on-chronic liver failure: A multicenter cross-sectional study in China. Hepatol Int. 8:508–516. 2014. View Article : Google Scholar : PubMed/NCBI
4	Mangano C, Squadrito G, Cacciola I, Carpentieri M, Foti G and Raimondo G: Effectiveness of add-on pegylated interferon alfa-2a therapy in a lamivudine-treated patient with chronic hepatitis B. Ann Hepatol. 10:84–87. 2011.PubMed/NCBI
5	Cho H and Kelsall BL: The role of type I interferons in intestinal infection, homeostasis, and inflammation. Immunol Rev. 260:145–167. 2014. View Article : Google Scholar : PubMed/NCBI
6	Bandurska K, Król I and Myga-Nowak M: Interferons: Between structure and function. Postepy Hig Med Dosw (Online). 68:428–440. 2014.(In Polish). View Article : Google Scholar : PubMed/NCBI
7	Pestka S, Krause CD and Walter MR: Interferons, interferon-like cytokines, and their receptors. Immunol Rev. 202:8–32. 2004. View Article : Google Scholar : PubMed/NCBI
8	Horras CJ, Lamb CL and Mitchell KA: Regulation of hepatocyte fate by interferon-γ. Cytokine Growth Factor Rev. 22:35–43. 2011. View Article : Google Scholar : PubMed/NCBI
9	Gao B, Wang H, Lafdil F and Feng D: STAT proteins-key regulators of anti-viral responses, inflammation, and tumorigenesis in the liver. J Hepatol. 57:430–441. 2012. View Article : Google Scholar : PubMed/NCBI
10	Koumbi L: Current and future antiviral drug therapies of hepatitis B chronic infection. World J Hepatol. 7:1030–1040. 2015. View Article : Google Scholar : PubMed/NCBI
11	Geng D, Wang Y, Wang P, Li W and Sun Y: Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta). J Appl Phycol. 15:451–456. 2003. View Article : Google Scholar
12	Sun Y, Yang Z, Gao X, Li Q, Zhang Q and Xu Z: Expression of foreign genes in Dunaliella by electroporation. Mol Biotechnol. 30:185–192. 2005. View Article : Google Scholar : PubMed/NCBI
13	Feng S, Li X, Xu Z and Qi J: Dunaliella salina as a novel host for the production of recombinant proteins. Appl Microbiol Biotechnol. 98:4293–4300. 2014. View Article : Google Scholar : PubMed/NCBI
14	Wang HH, Yin WB and Hu ZM: Advances in chloroplast engineering. J Genet Genomics. 36:387–398. 2009. View Article : Google Scholar : PubMed/NCBI
15	Barzegari A, Hejazi MA, Hosseinzadeh N, Eslami S, Mehdizadeh Aghdam E and Hejazi MS: Dunaliella as an attractive candidate for molecular farming. Mol Biol Rep. 37:3427–3430. 2010. View Article : Google Scholar : PubMed/NCBI
16	Sells MA, Chen ML and Acs G: Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci USA. 84:1005–1009. 1987. View Article : Google Scholar : PubMed/NCBI
17	Zhu X, Xie C, Li YM, Huang ZL, Zhao QY, Hu ZX, Wang PP, Gu YR, Gao ZL and Peng L: TMEM2 inhibits hepatitis B virus infection in HepG2 and HepG2.2.15 cells by activating the JAK-STAT signaling pathway. Cell Death Dis. 7:e22392016. View Article : Google Scholar : PubMed/NCBI
18	Li L, Lei QS, Zhang SJ, Kong LN and Qin B: Suppression of USP18 potentiates the anti-HBV activity of interferon alpha in HepG2.2.15 cells via JAK/STAT signaling. PLoS One. 11:e01564962016. View Article : Google Scholar : PubMed/NCBI
19	Romani L, Bistoni F, Montagnoli C, Gaziano R, Bozza S, Bonifazi P, Zelante T, Moretti S, Rasi G, Garaci E and Puccetti P: Thymosin alpha1: An endogenous regulator of inflammation, immunity, and tolerance. Ann N Y Acad Sci. 1112:326–338. 2007. View Article : Google Scholar : PubMed/NCBI
20	Billich A: Thymosin alpha1. SciClone Pharmaceuticals. Curr Opin Invest Drugs. 3:698–707. 2002.
21	Feng SY, Xue LX, Liu HT and Lu PJ: Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method. Mol Biol Rep. 36:1433–1439. 2009. View Article : Google Scholar : PubMed/NCBI
22	Meng XP, Yang JY, Qiao XL, Liang LX and Shou-Min XI: Optimization of construction of fusion gene-BTRCP-CypA by using SOE-PCR technique. Biotechnology. 23:51–54. 2013.
23	Pant K, Gupta P, Damania P, Yadav AK, Gupta A, Ashraf A and Venugopal SK: Mineral pitch induces apoptosis and inhibits proliferation via modulating reactive oxygen species in hepatic cancer cells. BMC Complement Altern Med. 16:1482016. View Article : Google Scholar : PubMed/NCBI
24	Mi S: A research for screening anti-hepatitis B virus drugs with the 2.2.15 cell line. Zhonghua Yi Xue Za Zhi. 72:612–615, 640. 1992.(In Chinese). PubMed/NCBI
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
26	Sari M: Effects of production system and gender on liveweight and body measurements in pekin ducks. Atatürk Üniversitesi Vet Bil Derg. 8:112–121. 2013.
27	Low HC: Molecular analysis of acute and chronic duck hepatitis B virus (DHBV) infections in ducks. (unpublished PhD thesis). University of Adelaide, School of Molecular and Biomedical Science; Adelaide, South Australia; 2012
28	Tian Y, Chen WL and Ou JH: Effects of interferon-α/β on HBV replication determined by viral load. PLoS Pathog. 7:e10021592011. View Article : Google Scholar : PubMed/NCBI
29	Guidotti LG, Morris A, Mendez H, Koch R, Silverman RH, Williams BR and Chisari FV: Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice. J Virol. 76:2617–2621. 2002. View Article : Google Scholar : PubMed/NCBI
30	Hansen BE, Buster EH, Steyerberg EW, Lesaffre E and Janssen HL: Prediction of the response to peg-interferon-alfa in patients with HBeAg positive chronic hepatitis B using decline of HBV DNA during treatment. J Med Virol. 82:1135–1142. 2010. View Article : Google Scholar : PubMed/NCBI
31	Lu NF, Huang AL, Zheng RQ, Zhu YB, Xia ZF, Tang N, Yan G, Gao XL and Wu Y: Anti-HBV effect of fusion protein (TA1-IFN) in vitro. Zhonghua Gan Zang Bing Za Zhi. 13:252–254. 2005.(In Chinese). PubMed/NCBI
32	Cho HJ, Kim JK, Nam JS, Wang HJ, Lee JH, Kim BW, Kim SS, Noh CK, Shin SJ, Lee KM, et al: High circulating microRNA-122 expression is a poor prognostic marker in patients with hepatitis B virus-related hepatocellular carcinoma who undergo radiofrequency ablation. Clin Biochem. 48:1073–1078. 2015. View Article : Google Scholar : PubMed/NCBI
33	Ganem D and Prince AM: Hepatitis B virus infection-natural history and clinical consequences. N Engl J Med. 350:1118–1129. 2004. View Article : Google Scholar : PubMed/NCBI
34	Rehermann B and Nascimbeni M: Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol. 5:215–229. 2005. View Article : Google Scholar : PubMed/NCBI
35	Xu WS, Zhao KK, Miao XH, Ni W, Cai X, Zhang RQ and Wang JX: Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro. World J Gastroenterol. 16:2028–2037. 2010. View Article : Google Scholar : PubMed/NCBI
36	Mason WS, Seal G and Summers J: Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J Virol. 36:829–836. 1980.PubMed/NCBI
37	Liu Q, Jia R, Wang M, Huang J, Zhu D, Chen S, Yin Z, Wang Y, Chen X and Cheng A: Cloning, expression and purification of duck hepatitis B virus (DHBV) core protein and its use in the development of an indirect ELISA for serologic detection of DHBV infection. Arch Virol. 159:897–904. 2014. View Article : Google Scholar : PubMed/NCBI