IBMC, Miguel Hernandez University, 03202-Elche, Spain.
Vaccine. 2012 Sep 7;30(41):5983-90. doi: 10.1016/j.vaccine.2012.07.013. Epub 2012 Jul 20.
DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.
DNA 疫苗在控制和预防病毒疾病方面开辟了一个新时代，因为 DNA 疫苗在某些常规疫苗失败的情况下非常有效，就像针对鱼类虹彩病毒的疫苗一样。然而，对于具有免疫相关细胞的体外模型测定，用于初步筛选体内 DNA 疫苗候选物，存在很大的缺乏。为了解决这个问题，早期原代培养的虹鳟鱼肾细胞被两种质粒 DNA 构建体转染，一种编码绿色荧光蛋白（GFP），另一种编码病毒性出血性败血症病毒（VHSV）糖蛋白 G（G（VHSV）） - 这是唯一赋予体内保护的病毒抗原。在评估 GFP 和 G（VHSV）表达细胞的转录和蛋白水平后，评估了转染肾细胞的免疫反应。在转染后 24 小时，G（VHSV）上调了 migm 和 tcr 转录物的表达，表明 B 和 T 细胞被激活，同时也观察到 tnfα 基因的高度上调。转染后 72 小时，我们检测到 mx 和 tnfα 基因转录物和 Mx 蛋白的上调，这与诱导抗 VHSV 状态相关。总的来说，我们已经成功地用 pAE6G 转染了肾细胞，这与体内保护结果相关，并且比体内测定更耗时且更快。因此，这一结果为使用早期原代培养的肾细胞进行初步筛选鱼类 DNA 疫苗以及进一步研究这些细胞在 DNA 免疫后鱼类免疫反应协调中的功能开辟了可能性。
