Li Zhuang, Cheng Yue, Xi Hualong, Gu Tiejun, Yuan Ruosen, Chen Xiaoxu, Jiang Chunlai, Kong Wei, Wu Yongge
National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China.
National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China.
Mol Immunol. 2015 Dec;68(2 Pt A):168-75. doi: 10.1016/j.molimm.2015.06.027. Epub 2015 Aug 29.
Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.
暴露后使用狂犬病免疫球蛋白（RIG）进行预防性治疗（PEP）可有效预防致命的狂犬病。单链可变片段（scFv）由可变重链（VH）和可变轻链（VL）通过肽接头连接而成，有可能用于替代RIG。然而，在我们之前的研究中，一种针对狂犬病病毒（RV）G蛋白的scFv（scFV57S）由于稳定性较低和肽组装模式改变，其中和效力低于其亲本IgG。在单克隆抗体中，VH和VL通过非共价相互作用，而在scFv中，VH通过人工接头与VL共价连接。在本研究中，我们在大肠杆菌中构建并表达了两种肽57VL-JUN-HIS和57VH-FOS-HA。利用著名的Fos和Jun亮氨酸拉链使VH和VL二聚化，类似于IgG对应物。这两种肽在体外组装形成zipFv57S。由于与IgG在结构上有更大的相似性，zipFv57S蛋白表现出更高的结合能力和亲和力，导致其体外中和活性比相应的scFv有显著提高。还发现zipFv57S蛋白更稳定，在接受致死剂量RV攻击的小鼠中显示出与RIG相似的保护率。我们的结果不仅表明zipFv57S是PEP中RIG的理想替代品，还提供了一种新颖且高效的VH和VL异源二聚化模式，从而提高了中和效力。
