Fulber Julia Puppin Chaves, Farnós Omar, Kiesslich Sascha, Yang Zeyu, Dash Shantoshini, Susta Leonardo, Wootton Sarah K, Kamen Amine A
Viral Vectors and Vaccines Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H3A 0G4, Canada.
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.
Vaccines (Basel). 2021 Nov 16;9(11):1335. doi: 10.3390/vaccines9111335.
The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms-technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 10 TCID/mL for NDV-GFP and 1.33 × 10 TCID/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 10 TCID/mL for NDV-GFP and 3.16 × 10 TCID/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.
持续的新冠疫情使全球关注传染病，吸引了大量资源用于制定大流行防范计划以及开发具有强大生产流程、可迅速转向针对新出现疾病的疫苗平台技术。新城疫病毒（NDV）已被作为人类和兽用疫苗的病毒载体进行研究，但其生产严重依赖鸡胚，在细胞培养中生产NDV的研究很少。在此，NDV在悬浮的Vero细胞中生产，并开发了分析检测方法（TCID和ddPCR）来量化感染性病毒滴度和总病毒滴度。NDV-GFP和NDV-FLS（严重急性呼吸综合征冠状病毒2全长刺突蛋白）构建体通过使用无血清培养基适应在Vero和HEK293悬浮培养物中复制，同时微调诸如感染复数、温度和胰蛋白酶浓度等参数。用Vero细胞进行摇瓶生产时，NDV-GFP的感染性滴度为1.07×10 TCID/mL，NDV-FLS为1.33×10 TCID/mL。在1L批次生物反应器中生产也使培养上清液中的滴度很高，NDV-GFP达到2.37×10 TCID/mL，NDV-FLS达到3.16×10 TCID/mL。这表明在细胞培养中能有效生产NDV，为可扩展的载体疫苗生产工艺奠定了基础，该工艺可应用于不同靶点。
