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表达牛病毒性腹泻病毒E2糖蛋白的复制子颗粒免疫及抗体反应评估

Replicon Particle Expressing the E2 Glycoprotein of Bovine Viral Diarrhea Virus Immunization and Evaluation of Antibody Response.

作者信息

Hossain Mohammad M, Rowland Raymond R

机构信息

Department of Diagnostic Medicine/Pathobiology, Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), College of Veterinary Medicine, Kansas State University , Manhattan, Kansas.

出版信息

Viral Immunol. 2018 Jan/Feb;31(1):55-61. doi: 10.1089/vim.2017.0053. Epub 2017 Jul 7.

Abstract

The objective of this study was to develop a new antigen delivery system using an alphavirus replicon particle (RP) to induce humoral antibody responses against bovine viral diarrhea virus (BVDV) recombinant antigen produced from envelope glycoprotein E2. An alphavirus RP expressing the E2 glycoprotein of BVDV was used for immunization of pigs. A fluorescent microsphere immunoassay (FMIA) has been applied to detect BVDV E2 antigen-specific antibody isotype in pig immunized with alphavirus RP. Full-length BVDV E2 (aa 1-375) was cleaved into several pieces, eight E2 DNA fragments, including full-length DNA, were cloned into expression vector pHUE, and the recombinant proteins expressed in BL-21 (DE3) Escherichia coli. After successful conjugation of purified proteins with microsphere beads, a multiplex FMIA platform was constructed, and BVDV E2 alphavirus-based RP-immunized animal serum samples were tested in the presence of bead-bound antigen targets. The results were represented as mean fluorescence intensity (MFI); the MFI values were converted to sample value/positive value (S/P) ratios. BVDV E2 (aa 1-183) showed the highest MFI values of eight recombinant E2 fragments when the specific activity of each fragment was tested. In immunized animals, data for BVDV E2-specific IgA, IgG, and IgM in serum and only IgG and IgA in oral fluids were recorded. The MFI values for the positive serum sample showed a 100-fold increase compared with the negative serum sample. Antibody isotype to BVDV E2 antigens showed that IgG > IgM > IgA in serum, whereas IgG > IgA > IgM in oral fluids. The data presented in this study suggested that boosting with the same doses of alphavirus RP in 3-week intervals may potentially enhance antibody response. The experimental results demonstrate that alphavirus RP-expressing BVDV E2 antigen induces antibody response in pig.

摘要

本研究的目的是开发一种新的抗原递送系统,该系统使用甲病毒复制子颗粒(RP)来诱导针对由包膜糖蛋白E2产生的牛病毒性腹泻病毒(BVDV)重组抗原的体液抗体反应。一种表达BVDV E2糖蛋白的甲病毒RP被用于猪的免疫接种。荧光微球免疫测定法(FMIA)已被应用于检测用甲病毒RP免疫的猪体内BVDV E2抗原特异性抗体亚型。全长BVDV E2(氨基酸1 - 375)被切割成几个片段,八个E2 DNA片段,包括全长DNA,被克隆到表达载体pHUE中,并在BL-21(DE3)大肠杆菌中表达重组蛋白。在将纯化的蛋白与微球珠成功偶联后,构建了一个多重FMIA平台,并在存在与珠结合的抗原靶标的情况下检测基于BVDV E2甲病毒的RP免疫动物血清样本。结果以平均荧光强度(MFI)表示;MFI值被转换为样本值/阳性值(S/P)比率。当测试每个片段的比活性时,BVDV E2(氨基酸1 - 183)在八个重组E2片段中显示出最高的MFI值。在免疫动物中,记录了血清中BVDV E2特异性IgA、IgG和IgM以及口腔液中仅IgG和IgA的数据。阳性血清样本的MFI值与阴性血清样本相比增加了100倍。针对BVDV E2抗原的抗体亚型显示,血清中IgG>IgM>IgA,而口腔液中IgG>IgA>IgM。本研究中呈现的数据表明,以3周的间隔用相同剂量的甲病毒RP进行加强免疫可能会增强抗体反应。实验结果表明,表达BVDV E2抗原的甲病毒RP在猪中诱导了抗体反应。

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