Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system
Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system
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DOI：
10.1128/iai.69.9.5709-5715.2001
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发表时间：
2001-09-01
影响因子：
3.1
通讯作者：
Smith, JF
中科院分区：
文献类型：
--
作者：
Lee, JS;Pushko, P;Smith, JF
利用委内瑞拉马脑炎（VEE）病毒复制子载体研制了A型肉毒神经毒素（BoNT/A）候选疫苗。该疫苗载体由含有所有VEE非结构基因和顺式作用元件的自我复制RNA以及位于亚基因组26 S启动子下游的异源免疫原基因（代替病毒结构基因）组成。在这项研究中，无毒的50 kDa的BoNT/A重链的羧基末端片段（H-C）克隆到复制子载体（H-C-复制子）。用H-C-复制子和两种辅助RNA分子（后者编码所有VEE结构蛋白）体外共转染BHK细胞，导致繁殖缺陷型H-C VEE复制子颗粒（H-C-VRP）的组装和释放。用H-C-VRP感染的细胞有效地表达这种蛋白质时，通过免疫荧光或Western印迹分析。为了评价H-C-VRP的免疫原性，在不同的时间间隔用不同剂量的H-C-VRP接种小鼠。皮下接种H-C-VRP的小鼠可免受高达100，000 50%致死剂量单位BoNT/A的腹膜内攻击。保护作用与血清酶联免疫吸附测定对BoNT/A的滴度直接相关。在疫苗接种后6个月和1年时检测获得的免疫力的持续时间，在这些时间点激发的小鼠仍然对BoNT/A激发不敏感。
A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H-C) of the BoNT/A heavy chain was cloned into the replicon vector (H-C-replicon). Cotransfection of BHK cells in vitro with the H-C-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H-C VEE replicon particles (H-C-VRP). Cells infected with H-C-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H-C-VRP, mice were vaccinated with various doses of H-C-VRP at different intervals. Mice inoculated subcutaneously with H-C-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.