靶向乙型肝炎病毒表面蛋白的大肠杆菌产生的单链可变片段

StephenW

Antiviral Res. 2018 Dec 29. pii: S0166-3542(18)30207-9. doi: 10.1016/j.antiviral.2018.12.019. [Epub ahead of print]
An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion.
Li C1, Wang Y1, Liu T1, Niklasch M2, Qiao K1, Durand S3, Chen L1, Liang M4, Baumert TF5, Tong S1, Nassal M2, Wen YM1, Wang YX6.
Author information

1
    Key Laboratory of Medical Molecular Virology, Ministry of Education and Ministry of Health, Shanghai Medical College of Fudan University, Shanghai, China.
2
    University Hospital Freiburg, Department of Internal Medicine II/Molecular Biology, Freiburg, Germany.
3
    Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.
4
    Key Laboratory for Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
5
    Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France; Université de Strasbourg, Strasbourg, France; Institut Hospitalo-Universitaire, Pôle 5 Hépato-digestif, Nouvel Hôpital Civil, Strasbourg, France.
6
    Key Laboratory of Medical Molecular Virology, Ministry of Education and Ministry of Health, Shanghai Medical College of Fudan University, Shanghai, China. Electronic address: [email protected].

Abstract

Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface-exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge-dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000-fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single-chain variable fragment (scFv) homolog of IgG12, G12-scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12-scFv to a conformational S epitope shared with IgG12. Despite 20-fold lower affinity, G12-scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50 = 1.8 nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50 = 1.25 nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications.

PMID:
    30599174
DOI:
    10.1016/j.antiviral.2018.12.019

StephenW

抗病毒药物2018年12月29日.pii：S0166-3542（18）30207-9。 doi：10.1016 / j.antiviral.2018.12.019。 [提前打印]
靶向乙型肝炎病毒表面蛋白的大肠杆菌产生的单链可变片段（scFv）有效地抑制病毒体分泌。
Li C1，Wang Y1，Liu T1，Niklasch M2，Qiao K1，Durand S3，Chen L1，Liang M4，Baumert TF5，Tong S1，Nassal M2，Wen YM1，Wang YX6。
作者信息

1
    复旦大学上海医学院医学分子病毒学教育部重点实验室，上海，上海。
2
    弗莱堡大学医院，内科II /分子生物学，德国弗赖堡。
3
    Inserm，U1110，Institut de Recherche sur les Maladies ViralesetHépatiques，Strasbourg，France。
4
    中国疾病预防控制中心国家病毒病预防控制所，NHFPC医学病毒学重点实验室，北京，中国。
五
    Inserm，U1110，Institut de Recherche sur les Maladies ViralesetHépatiques，Strasbourg，France;法国斯特拉斯堡斯特拉斯堡大学; Institut Hospitalo-Universitaire，Pôle5Hépato-digestif，NouvelHôpitalCivil，法国斯特拉斯堡。
6
    复旦大学上海医学院医学分子病毒学教育部重点实验室，上海，上海。电子地址：[email protected]。

抽象

乙型肝炎病毒（HBV）包膜以及空的亚病毒颗粒在其脂质膜中携带小（S），中（M）和大（L）表面蛋白，统称为乙型肝炎表面抗原（HBsAg）。由于它们的共同S结构域，所有三种蛋白质共享表面暴露的亲水性抗原环（AGL），其具有复杂的二硫键桥依赖性结构。 AGL对HBV感染性和病毒粒子分泌至关重要，因此是中和抗体的主要靶标。以前，靶向AGL中的构象表位IgG12的人单克隆抗体（mAb）表现出比乙型肝炎免疫球蛋白（HBIG）高1000倍的中和活性。在这里，我们设计了IgG12，G12-scFv的单链可变片段（scFv）同源物，其可以在大肠杆菌SHuffle细胞的细胞质中以可溶形式有效产生。独立的体外测定证实了G12-scFv与与IgG12共有的构象S表位的特异性结合。尽管亲和力低20倍，G12-scFv但不是无关的scFv有效中和易感肝细胞瘤细胞的HBV感染（IC50 = 1.8nM）。引人注目的是，低浓度的G12-scFv阻断了HBV产生细胞的病毒粒子分泌（IC50 = 1.25nM）而没有干扰细胞内病毒的复制，而细胞外HBsAg仅在高于> 100倍时降低，尽管仍然是无毒浓度。抑制作用与S结合特异性相关，并且可能还有G12-scFv内化到细胞中。这些数据共同表明G12-scFv是一种高度特异且易于获得的新型工具，适用于基础，诊断和未来可能的治疗应用。

结论：
    30599174
DOI：
    10.1016 / j.antiviral.2018.12.019

StephenW

令人鼓舞的国际研究.

newchinabok

StephenW 发表于 2019-1-3 13:31
令人鼓舞的国际研究.

感谢分享，确实鼓舞，但是是十年后了