West Nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome-associated membrane protein: increased cellular concentration of the transgene product, targeting to the MHC II compartment, and enhanced neutralizing antibody response
West Nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome-associated membrane protein: increased cellular concentration of the transgene product, targeting to the MHC II compartment, and enhanced neutralizing antibody response
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DOI：
10.1016/j.virol.2004.11.022
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发表时间：
2005-02-05
期刊：
影响因子：
3.7
通讯作者：
August, JT
中科院分区：
文献类型：
--
作者：
Anwar, A;Chandrasekaran, A;August, JT
利用西尼罗病毒（WN）前膜包膜（WN preM-E）基因序列与溶酶体相关膜蛋白（LAMP）的跨膜和羧基末端结构域编码为嵌合体，合成了西尼罗病毒（WN）的遗传疫苗。LAMP序列用于将抗原蛋白引导到转染的专业抗原呈递细胞（APCs）的主要组织相容性II类（MHC II）囊泡室。在pVAX1和pITR质粒主干上合成了编码天然WN preM-E和WN preM-E/LAMP嵌合体的疫苗构建体。人成纤维细胞293和猴肾COS-7细胞的提取液中含有的E含量明显高于编码天然WN preM-E的细胞。转染细胞中天然E和E/LAMP嵌合体的浓度差异归因于天然E的分泌。与E蛋白相比，细胞提取物中preM蛋白的含量以及DNA和RNA转录物的水平在转染WN preM-E和转染WN preM-E/LAMP的细胞之间没有差异。此外，转染B细胞的共聚焦和免疫电镜分析显示，WN preM-E/LAMP嵌合体定位在含有内源性LAMP、MHC II和H2-M的囊泡腔室中，而缺乏LAMP序列的天然病毒preM-E分布在细胞囊泡网络中，与LAMP或MHC II的关联很小。用表达WN preM-E/LAMP抗原的DNA构建体免疫小鼠可诱导显著的抗体和长期中和效价，而用表达非靶向抗原的质粒免疫小鼠则可诱导最低效价和短期中和效价。这些结果强调了LAMP将WN包膜靶向MHC II区室在设计遗传性WN疫苗中的实用性。(C) 2004爱思唯尔公司版权所有。
A genetic vaccine for West Nile virus (WN) has been synthesized with the WN premembrane-envelope (WN preM-E) gene sequences encoded as a chimera with the transmembrane and carboxyl terminal domains of the lysosome-associated membrane protein (LAMP). The LAMP sequences are used to direct the antigen protein to the major histocompatibility class II (MHC II) vesicular compartment of transfected professional antigen-presenting cells (APCs). Vaccine constructs encoding the native WN preM-E and WN preM-E/LAMP chimera were synthesized in pVAX1 and pITR plasmid backbones. Extracts of human fibroblast 293 and monkey kidney COS-7 cells transfected with the WN preM-E/LAMP chimera constructs contained much greater amounts of E than did the cells transfected with constructs encoding the native WN preM-E. This difference in the concentration of native E and the E/LAMP chimera in transfected cells is attributed to the secretion of native E. The amount of preM protein in cell extracts, in contrast to the E protein, and the levels of DNA and RNA transcripts, did not differ between WN preM-E- and WN preM-E/LAMP-transfected cells. Additionally, confocal and immunoelectron microscopic analyses of transfected B cells showed localization of the WN preM-E/LAMP chimera in vesicular compartments containing endogenous LAMP, MHC II, and H2-M, whereas native viral preM-E lacking the LAMP sequences was distributed within the cellular vesicular network with little LAMP or MHC II association. Mice immunized with a DNA construct expressing the WN preM-E/LAMP antigen induced significant antibody and long-term neutralization titers in contrast to the minimal and short-lived neutralization titer of mice vaccinated with a plasmid expressing the untargeted antigen. These results underscore the utility of LAMP targeting of the WN envelope to the MHC II compartments in the design of a genetic WN vaccine. (C) 2004 Elsevier Inc. All rights reserved.