嵌合HAdV六邻体蛋白抗原表位的rFLU/HAdV/Hexon疫苗候选株构建及免疫效果研究

Recombinant influenza virus carrying human adenovirus epitopes induce protective antibodies in mice

目的构建、拯救以PR8流感病毒为载体嵌合HAdV六邻体蛋白抗原表位的疫苗候选株,对其免疫原性及免疫保护性进行评价。方法应用反向遗传学技术,以流感病毒PR8为载体,将HAdV六邻体蛋白抗原表位的基因插入流感病毒非结构蛋白NS基因,构建重组质粒NS1-Hexon,将重组质粒与PR8流感病毒其他7个基因组质粒共转染COS-1/MDCK共培养细胞,成功拯救嵌合HAdV六邻体抗原表位重组疫苗候选株,命名为rFLU/HAdV/Hexon,通过HA、RT-PCR、IFA、Western blotting和电镜等方法对rFLU/HAdV/Hexon进行鉴定。rFLU/HAdV/Hexon经鸡胚培养、浓缩纯化后滴鼻免疫Balb/c小鼠,评价小鼠机体产生的体液免疫、细胞免疫和黏膜免疫应答,进一步采用PR8流感病毒和HAdV-3病毒攻击小鼠评价其免疫保护性。结果成功拯救出基于PR8流感病毒为载体嵌合HAdV六邻体蛋白抗原表位的rFLU/HAdV/Hexon疫苗候选株。rFLU/HAdV/Hexon疫苗株形态符合流感病毒典型特征,能够诱导小鼠机体产生高效价的针对PR8和HAdV-3的特异性抗体,肺鼻灌洗液可检测到高效价的sIgA抗体;攻毒实验结果显示,rFLU/HAdV/Hexon疫苗株免疫小鼠可明显减轻感染症状、有效降低肺鼻的病毒载量、显著改善肺组织的病理病变情况。结论研制的嵌合HAdV六邻体蛋白抗原表位的rFLU/HAdV/Hexon疫苗株是有发展前景的腺病毒候选疫苗株,为腺病毒疫苗的研究提供了新思路。

It has been reported that human adenoviruses （HAdVs） can cause a broad spectrum of diseases in pediatric and adult patient. As yet, there is still no registered vaccine available for this major pathogen. In this study, by applying reverse genetics （RG） we have successfully rescued a recombinant PR8 influenza virus vector expressing the HAdV hexon protein antigenic epitopes inserted into the influenza nonstructural （NS1） protein gene. Sequence analysis of transfectant viral RNA has verified that the transfectant virus contained the chimeric RNA, and the expression of NS1-Hexon protein was detected by Western blotting, rFLU/HAdV/Hexon morphological characteristic was observed with electron microscope. Balb/c mice vaccinated intranasally with rFLU/HAdV/Hexon could produce neutralizing anti-Ad3 antibodies, in addition to antibodies against influenza. We detected specificimmune responses （IgG, IgG1, IgG2a and slgA） against HAdV-3 viruses, which demonstrated that immunization with rFLU/HAdV/Hexon elicited systemic immunity in serum and mucosal immunty in murine mucosal secretions. These specific neutralizing antibodies could provid protection against subsequent HAdV-3 and PR8 influenza viruses challenge, showing a significantreduction o f viral load, noticeable alleviation of histopathological changes in the challenged mouse lung. The findings manifested that the recombinant rFLU/HAdV/Hexon merit further investigation as a promising candidate vaccine and the immunoprotection should be certified exactly in monkey model.