Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

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.