Department of Pathobiology, University of Guelph.
Department of Pathobiology, University of Guelph;
J Vis Exp. 2022 May 25(183). doi: 10.3791/63817.
Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.
新城疫病毒（NDV），也称为禽正黏病毒血清型 1，是一种负义、单链 RNA 病毒，已被开发为溶瘤病毒和病毒载体疫苗。由于其成熟的反向遗传学系统、强大的免疫刺激特性和极好的安全性，NDV 是一种有吸引力的治疗和预防药物。当作为溶瘤病毒或病毒载体疫苗给药时，NDV 会引发强大的抗肿瘤或抗原特异性免疫反应，激活免疫系统的先天和适应性 arms。鉴于这些理想的特性，NDV 已在众多临床试验中进行了评估，是研究最多的溶瘤病毒之一。目前，有两项涉及 NDV 的注册临床试验：一项评估用于 SARS-CoV-2 的重组 NDV 载体疫苗（NCT04871737），另一项评估编码白细胞介素 12 的重组 NDV 与抗 PD-L1 抗体 Durvalumab 联合用于治疗癌症（NCT04613492）。为了促进这种极具前景的病毒载体的进一步发展，需要简化产生高滴度、体内级、重组 NDV（rNDV）的方法。本文描述了在特定病原体无（SPF）鸡胚中扩增 rNDV 和从羊水纯化 rNDV 的详细程序，并改进了纯化过程以减少损失。还描述了推荐的质量控制检测，应进行这些检测以确认无污染物和病毒完整性。总之，该详细程序使高滴度、体内级、重组、减毒和中等毒力的 lentogenic 和 mesogenic NDV 的合成、纯化和储存成为可能，可用于临床前研究。
