Primers used for synthetic construct design.

Infectious bronchitis (IB) is a highly contagious, acute respiratory disease in chickens, with a severe economic impact on poultry production globally. The rapid emergence of regional variants of this Gammacoronavirus warrants new vaccine approaches that are more humane and rapid to produce than the current embryonated chicken egg-based method used for IB variant vaccine propagation (chemically-inactivated whole viruses). The production of virus-like particles (VLPs) expressing the Spike (S) glycoprotein, the major antigen which induces neutralizing antibodies, has not been achieved in planta up until now. In this study, using the Agrobacterium-mediated Nicotiana benthamiana (tobacco plant) transient expression system, the highest levels of VLPs displaying a modified S protein of a QX-like IB variant were obtained when the native transmembrane (TM) domain and cytoplasmic tail were substituted with that of the Newcastle disease virus (NDV) fusion glycoprotein, co-infiltrated with the NDV Matrix protein. In comparison, the native IB modified S co-infiltrated with IB virus membrane, envelope and nucleocapsid proteins, or substituted with the TM and CT of an H6-subtype influenza A virus hemagglutinin glycoprotein yielded lower VLP expression levels. Strong immunogenicity was confirmed in specific pathogen free chickens immunized intramuscularly with VLPs adjuvanted with Emulsigen®-P, where birds that received doses of 5 μg or 20 μg (S protein content) seroconverted after two weeks with mean hemaggluttination inhibition titres of 9.1 and 10 log2, respectively. Plant-produced IB VLP variant vaccines are safer, more rapid and cost effective to produce than VLPs produced in insect cell expression systems or the traditional egg-produced inactivated whole virus oil emulsion vaccines currently in use, with great potential for improved IB disease control in future.

传染性支气管炎（Infectious bronchitis, IB）是一种危害鸡只的高度传染性急性呼吸道疾病，对全球家禽养殖业造成严重经济损失。这种γ冠状病毒（Gammacoronavirus）的区域性变异株快速出现，亟需研发比当前用于IB变异株疫苗增殖（化学灭活全病毒法）的鸡胚接种生产方式更具人道性且生产更快捷的新型疫苗方案。此前始终无法在植物宿主中成功制备表达刺突糖蛋白（Spike glycoprotein, S）的病毒样颗粒（virus-like particles, VLPs）——该糖蛋白是诱导中和抗体的主要抗原。本研究利用农杆菌介导的本氏烟（Nicotiana benthamiana，烟草植株）瞬时表达系统，当将天然跨膜（transmembrane, TM）结构域与胞质尾替换为新城疫病毒（Newcastle disease virus, NDV）融合糖蛋白的对应序列，并与NDV基质蛋白共浸润时，可获得最高水平的、展示QX样IB变异株修饰S蛋白的VLPs。与之相比，将天然IB修饰S蛋白与IB病毒膜蛋白、包膜蛋白及核衣壳蛋白共浸润，或是将其替换为H6亚型甲型流感病毒血凝素糖蛋白的TM与胞质尾（cytoplasmic tail, CT），VLPs的表达水平均较低。使用Emulsigen®-P作为佐剂的VLPs经肌肉免疫无特定病原体（specific pathogen free, SPF）鸡只后，其较强的免疫原性得到验证：接种5μg或20μg（以S蛋白含量计）剂量的鸡只在两周后均发生血清阳转，平均血凝抑制滴度分别为9.1和10 log₂。与昆虫细胞表达系统制备的VLPs以及当前广泛使用的传统鸡胚灭活全病毒油乳剂疫苗相比，植物源IB VLP变异株疫苗的生产更安全、更快捷且成本效益更优，未来在IB疾病防控领域具有巨大应用潜力。