Zaitseva Marina, Kapnick Senta, Golding Hana
Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA.
Methods Mol Biol. 2012;890:161-76. doi: 10.1007/978-1-61779-876-4_10.
Preclinical evaluation of novel anti-smallpox vaccines and antiviral treatments often rely on mouse -challenge models using pathogenic vaccinia virus, such as Western Reserve (WR) strain or other orthopoxviruses. Traditionally, efficacy of treatment is evaluated using various readouts, such as lethality (rare), measurements of body weight loss, pox lesion scoring, and determination of viral loads in internal organs by enumerating plaques in sensitive cell lines. These methodologies provide valuable information about the contribution of the treatment to protection from infection, yet all have similar limitations: they do not evaluate dissemination of the virus within the same animal and require large numbers of animals. These two problems prompted us to turn to a recently developed whole body imaging technology, where replication of recombinant vaccinia virus expressing luciferase enzyme (WRvFire) is sensed by detecting light emitted by the enzyme in the presence of D: -luciferin substrate administered to infected animal. Bioluminescence signals from infected organs in live animals are registered by the charge-coupled device camera in IVIS instrument developed by Caliper, and are converted into numerical values. This chapter describes whole body bioimaging methodology used to determine viral loads in normal live BALB/c mice infected with recombinant WRvFire vaccinia virus. Using Dryvax vaccination as a model, we show how bioluminescence data can be used to determine efficacy of treatment. In addition, we illustrate how bioluminescence and survival outcome can be combined in Receiver Operating Characteristic curve -analysis to develop predictive models of lethality that can be applied for testing of new therapeutics and second-generation vaccines.
新型抗天花疫苗和抗病毒治疗的临床前评估通常依赖于使用致病性痘苗病毒（如西储（WR）株或其他正痘病毒）的小鼠攻毒模型。传统上，治疗效果通过各种指标进行评估，如致死率（罕见）、体重减轻测量、痘疹病变评分以及通过在敏感细胞系中计数噬斑来测定内脏器官中的病毒载量。这些方法提供了关于治疗对预防感染的贡献的有价值信息，但都有类似的局限性：它们无法评估同一动物体内病毒的传播情况，并且需要大量动物。这两个问题促使我们转向最近开发的全身成像技术，在该技术中，通过检测在给感染动物施用D - 荧光素底物时由表达荧光素酶的重组痘苗病毒（WRvFire）发出的光来感知病毒的复制。活体动物感染器官发出的生物发光信号由Caliper公司开发的IVIS仪器中的电荷耦合器件相机记录，并转换为数值。本章描述了用于确定感染重组WRvFire痘苗病毒的正常活体BALB / c小鼠体内病毒载量的全身生物成像方法。以Dryvax疫苗接种为模型，我们展示了如何利用生物发光数据来确定治疗效果。此外，我们还说明了如何在受试者工作特征曲线分析中将生物发光和生存结果结合起来，以开发可用于测试新疗法和第二代疫苗的致死率预测模型。
