An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion.
An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion.
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DOI：
10.1016/j.antiviral.2018.12.019
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发表时间：
2019-03
影响因子：
7.6
通讯作者：
Wang YX
中科院分区：
文献类型：
--
作者：
Li C;Wang Y;Liu T;Niklasch M;Qiao K;Durand S;Chen L;Liang M;Baumert TF;Tong S;Nassal M;Wen YM;Wang YX
B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B型肝炎表面抗原（HBsAg）。由于它们共同的S结构域，所有三种蛋白质共享具有复杂的二硫桥依赖性结构的表面暴露的亲水性抗原环（AGL）。AGL对于HBV感染性和病毒体分泌至关重要，因此代表中和抗体的主要靶标。以前，靶向AGL中构象表位的人单克隆抗体（mAb）IgG 12表现出比B型肝炎免疫球蛋白（HBIG）高1，000倍的中和活性。我们设计了一种IgG 12的单链可变区（single-chain variable fragment，scFv）同源物G12-scFv，它能在大肠杆菌胞质中以可溶性形式高效表达。coli SHuffle细胞。独立的体外测定验证了G12-scFv与IgG 12共有的构象S表位的特异性结合。尽管亲和力低20倍，但G12-scFv而不是无关的scFv有效地中和易感肝癌细胞的HBV感染（IC 50 =1.8 nM）。引人注目的是，低浓度的G12-scFv阻断了HBV产生细胞的病毒体分泌（IC 50 =1.25 nM），而不干扰细胞内病毒复制，而细胞外HBsAg仅在>100倍的浓度下降低，但仍然无毒。抑制作用与S结合特异性相关，并且推测也与G12-scFv内化到细胞中相关。总之，这些数据表明G12-scFv是一种高度特异性但易于获得的新型工具，可用于基础、诊断和可能的未来治疗应用。
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 1,000-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.
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影响因子：
4.9
作者：
Ladner, SK;Otto, MJ;King, RW
通讯作者：
King, RW
影响因子：
5.3
作者：
Li, Zhe;Krippendorff, Ben-Fillippo;Shah, Dhaval K.
通讯作者：
Shah, Dhaval K.
DOI：
10.2174/138620709789104915
发表时间：
2009-09-01
影响因子：
1.8
作者：
Concepcion, Joy;Witte, Krista;Tan, Hong
通讯作者：
Tan, Hong
DOI：
10.1089/153685902761022742
发表时间：
2002-10-01
期刊：
HYBRIDOMA AND HYBRIDOMICS
影响因子：
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作者：
Kim, SH;Park, SY
通讯作者：
Park, SY
影响因子：
5.4
作者：
Kwei, Karen;Tang, Xiaoli;Tong, Shuping
通讯作者：
Tong, Shuping