Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Braunschweig, Germany.
PLoS One. 2012;7(5):e37242. doi: 10.1371/journal.pone.0037242. Epub 2012 May 30.
Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.
委内瑞拉马脑炎病毒（VEEV）属于甲病毒属，该属的几种病毒对人类具有致病性。这些病毒被归类为生物战剂和恐怖主义的潜在制剂，因此需要进行敏感检测以及有效的预防和抗病毒治疗。在这项工作中，我们从非人类灵长类动物（NHP）抗体基因文库中分离出抗 VEEV 的单链片段可变区（scFv），ToR67-3B4。我们报告了它在双价 scFv-Fc 格式中的重新克隆，并进一步对其进行了免疫和生化特性分析。scFv-Fc ToR67-3B4 可识别活病毒以及福尔马林和β-丙内酯（β-Pl）灭活的病毒颗粒，可用于 VEEV 蛋白的免疫印迹分析和 VEEV 感染细胞的免疫组织化学分析。它特异性地检测到 VEEV 的 E1 包膜蛋白，但与还原的病毒糖蛋白制剂没有反应，这表明识别取决于构象表位。该重组抗体能够检测到多种 VEEV 亚型，除了EEEV 之外，与其他甲病毒属的物种仅有轻微的交叉反应性。此外，由于该 scFv-Fc 融合体成功地在小鼠疾病模型中灭活了 VEEV，因此它可能具有治疗用途。当在挑战后 6 小时给予重组抗体时，80%至 100%的小鼠能够从致死性 VEEV IA/B 或 IE 感染中存活。当在 VEEV 亚型 II 和前 IIIA 挑战后 6 小时应用 scFv-Fc ToR67-3B4 时，40%至 60%的小鼠存活。与 E2 中和抗体联合使用，该 NHP 抗体可以显著提高 VEE 的被动保护和通用治疗效果。
