De Paula Sérgio Oliveira, Lima Danielle Malta, de Oliveira França Rafael Freitas, Gomes-Ruiz Alessandra Cristina, da Fonseca Benedito Antônio Lopes
Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, Universidade Federal de Viçosa, Av PH Rolfs, s/n, Viçosa, Minas Gerais, CEP 36570-000, Brazil.
Arch Virol. 2008;153(12):2215-23. doi: 10.1007/s00705-008-0250-3. Epub 2008 Nov 12.
In an effort to develop a suitable DNA vaccine candidate for dengue, using dengue-3 virus (DENV-3) as a prototype, the genes coding for premembrane (prM) and envelope proteins (E) were inserted into an expression plasmid. After selecting recombinant clones containing prM/E genes, protein expression in the cell monolayer was detected by indirect immunofluorescence and immunoprecipitation assays. After selecting three vaccine candidates (pVAC1DEN3, pVAC2DEN3 and pVAC3DEN3), they were analyzed in vivo to determine their ability to induce a DENV-3-specific immune response. After three immunizations, the spleens of the immunized animals were isolated, and the cells were cultivated to measure cytokine levels by ELISA and used for lymphoproliferation assays. All of the animals inoculated with the recombinant clones induced neutralizing antibodies against DENV-3 and produced a T cell proliferation response after specific stimuli. Immunized and control mice were challenged with a lethal dose of DENV-3 and observed in order to assess their survival capability. The groups that presented the best survival rate after the challenge were the animals vaccinated with the pVAC3DEN3 clones, with an 80% survival rate. Thus, these data show that we have manufactured a vaccine candidate for DENV-3 that is able to induce a specific immune response and protects mice against a lethal challenge.
为了开发一种合适的登革热DNA疫苗候选物，以登革热3型病毒（DENV-3）为原型，将编码前膜（prM）和包膜蛋白（E）的基因插入表达质粒中。在筛选出含有prM/E基因的重组克隆后，通过间接免疫荧光和免疫沉淀试验检测细胞单层中的蛋白表达。在筛选出三种疫苗候选物（pVAC1DEN3、pVAC2DEN3和pVAC3DEN3）后，对它们进行体内分析，以确定其诱导DENV-3特异性免疫反应的能力。三次免疫后，分离免疫动物的脾脏，培养细胞以通过ELISA测量细胞因子水平，并用于淋巴细胞增殖试验。所有接种重组克隆的动物均诱导出针对DENV-3的中和抗体，并在特异性刺激后产生T细胞增殖反应。用致死剂量的DENV-3攻击免疫小鼠和对照小鼠，并观察以评估它们的生存能力。攻击后存活率最高的组是接种pVAC3DEN3克隆的动物，存活率为80%。因此，这些数据表明，我们已经制备出一种DENV-3疫苗候选物，它能够诱导特异性免疫反应并保护小鼠免受致死性攻击。
