Abstract：
Spring viremia of carp (SVC) remains as a vaccine orphan disease mostly affecting juvenile specimens. Young fish are especially difficult to vaccinate and oral administration of vaccine combined with food would be the election system to minimise stress and the vaccination costs associated to injection. However, administration of prophylactics with food pellets faces off several drawbacks mainly related with vaccine degradation and weak protection correlates of oral vaccines. Here we present a platform based on recombinant proteins (subunit vaccines) manufactured as highly resistant nanostructured materials, and providing excellent levels of protection against SVC virus in a preliminary i.p injection challenge. The G3 domain of SVCV glycoprotein G was overexpressed in E. coli together with IFNγ and the modular protein was purified from bacterial aggregates (inclusion bodies) as highly organised nanostructured biomaterial (nanopellets, NP). These SVCV-IFNNP were taken up by zebrafish cells leading to the enhanced expression of different antiviral and IFN markers (e.g vig1, mx, lmp2 or ifngr1 among others) in zebrafish liver cells (ZFL). To monitor if SVCVNP and SVCV-IFNNP can be taken up by intestinal epithelia and can induce antiviral response we performed experiments with SVCVNP and SVCV-IFNNP in 3 days post fertilization (dpf) zebrafish larvae. Both, SVCVNP and SVCV-IFNNP were taken up and accumulated in the intestine without signs of toxicity. The antiviral response in larvae showed a different induction pattern: SVCV-IFNNP did not induce an antiviral response while SVCVNP showed a good antiviral induction. Interestingly ZF4, an embryonic derived cell line, showed an antiviral response like ZFL cells, although the lmp2 and ifngr1 (markers of the IFNγ response) were not overexpressed. Experiments with adult zebrafish indicated an excellent level of protection against a SVCV model infection where SVCV-IFNNP vaccinated fish reached 20% cumulative mortality while control fish reached over 80% cumulative mortality.
摘要：
鲤鱼春季病毒血症（SVC）仍然是一种疫苗孤儿病，主要影响青少年标本。幼鱼特别难以接种疫苗，口服疫苗与食物相结合将是最大程度地减少压力和与注射相关的疫苗接种成本的选择系统。然而,用食物颗粒施用预防剂克服了几个主要与疫苗降解和口服疫苗弱保护相关的缺点。在这里，我们提出了一个基于重组蛋白（亚单位疫苗）的平台，制造为高抗性纳米结构材料，并在初步i.p.注射挑战中提供针对SVC病毒的出色保护水平。SVCV糖蛋白G的G3结构域在大肠杆菌中与IFNγ一起过表达，并且模块蛋白从细菌聚集体（包涵体）中纯化为高度组织化的纳米结构生物材料（纳米小片，NP).这些SVCV-IFNNP被斑马鱼细胞吸收，导致不同抗病毒和IFN标记物的表达增强（例如gvig1,mx,lmp2或ifngr1等）在斑马鱼肝细胞（ZFL）中。为了监测SVCVNP和SVCV-IFNNP是否可以被肠上皮吸收并且可以诱导抗病毒反应,我们在受精后3天(dpf)斑马鱼幼虫中进行了SVCVNP和SVCV-IFNNP的实验。两者,SVCVNP和SVCV-IFNNP被摄取并在肠中积累,没有毒性迹象。幼虫的抗病毒反应显示出不同的诱导模式：SVCV-IFNNP不诱导抗病毒反应，而SVCVNP显示出良好的抗病毒诱导。有趣的是ZF4，一种胚胎来源的细胞系，像ZFL细胞一样表现出抗病毒反应，尽管lmp2和ifngr1（IFNγ反应的标记）没有过表达。用成年斑马鱼进行的实验表明对SVCV模型感染具有优异的保护水平,其中SVCV-IFNNP接种的鱼达到20%累积死亡率,而对照鱼达到超过80%累积死亡率。
