不同佐剂对病毒灭活疫苗免疫效果的影响  

Impacts of different adjuvants on immune responses of inactivated virus vaccine

作　　者：时小光 李秀丽[2] 张闯年 黄平升 张建华[1] 王伟伟 鄢明华[2] SHI Xiao-guang;LI Xiu-li;ZHANG Chuang-nian;HUANG Ping-sheng;ZHANG Jian-hua;WANG Wei-wei;YAN Ming-hua(School of Chemical Engineering and Technology,Tianjin University,Tianjin 300072,China;Tianjin Animal Science and Veterinary Research Institute,Tianjin 300381,China;Institute of Biomedical Engineering,Chinese Academy of Medical Sciences and Peking Union Medical College,Tianjin 300192,China)

机构地区：[1]天津大学化工学院,天津300072 [2]天津市畜牧兽医研究所,天津300381 [3]中国医学科学院·北京协和医学院生物医学工程研究所,天津300192

出　　处：《生物医学工程与临床》2020年第5期604-610,共7页Biomedical Engineering and Clinical Medicine

基　　金：天津市高端兽药先进制造科技重大专项(17ZXGSNC00080)。

摘　　要：目的探讨两种不同佐剂CpG和纳米乳(Nanoemulsion)对病毒灭活疫苗免疫效果的影响。方法以灭活猪德尔塔冠状病毒(PDCoV)培养液为例,将骨髓来源树突状细胞(BMDC)与含有不同免疫佐剂的PDCoV灭活疫苗共孵育。抗体染色后使用流式细胞仪检测BMDC成熟、活化情况。成年健康C57BL/6J雌性小鼠20只,鼠龄6~8周;随机分成4组,分别接种磷酸盐缓冲溶液(PBS)、灭活PDCoV液、CpG/PDCoV疫苗和Nanoemulsion/PDCoV疫苗,皮下免疫3次,时间间隔为1周;第21天杀死小鼠获取脾脏和血液,检测不同PDCoV灭活疫苗接种小鼠免疫反应情况。结果 Nanoemulsion可负载灭活PDCoV的抗原成分,形成平均粒径为(157.6±0.4) nm的纳米粒,而CpG与灭活PDCoV则是无规则混合。Cp G/PDCoV组或Nanoemulsion/PDCoV组促BMDC成熟程度分别为45.57%和53.32%(CD86+MHCⅡ+BMDC),明显高于PDCoV组(38.49%),差异有统计学意义(P <0.01、P <0.05)。第21天,CpG/PDCoV组小鼠脾脏中Th1(P <0.05)和Tc2(P <0.05)细胞比例分别为5.81%和0.24%,显著高于PDCoV组(3.56%和0.14%);Nanoemulsion/PDCoV组小鼠脾脏中Tc1(P <0.05)细胞比例为6.46%,高于PDCoV组(9.28%);CpG/PDCoV组小鼠血清中总免疫球蛋白(IgG)、IgG2a和干扰素-γ(IFN-γ)水平明显高于PDCoV组,差异有统计学意义(P <0.05);Nanoemulsion/PDCoV组小鼠血清中总IgG、IgG2a、IFN-γ、肿瘤坏死因子-α(TNF-α)和白细胞介素(IL-4)水平明显高于PDCoV组,差异有统计学意义(P <0.05);另外,与CpG相比,Nanoemulsion能够诱导更高水平的IgG2a、IFN-γ、TNF-α和IL-4。结论佐剂CpG或者Nanoemulsion的添加能够显著地增强病毒灭活疫苗免疫效果。相较于CpG,Nanoemulsion可以诱导更强的细胞免疫和体液免疫,更适用于病毒灭活疫苗的研发。Objective To investigate the impacts of CpG and Nanoemulsion on immune responses of inactivated virus vaccine.Methods The inactivated porcine deltacoronavirus(PDCoV) was taken as an example, bone marrow-derived dendritic cells(BMDC) were co-incubated with inactivated PDCoV vaccines comprising of CpG or Nanoemulsion. The maturation and activation of BMDC were detected by flow cytometry after antibody staining. Twenty healthy C57BL/6J mice(aged 6-8 weeks) were randomly divided into 4 groups, and administrated with phosphate buffered saline(PBS), inactivated PDCoV solution, CpG/PDCoV or Nanoemulsion/PDCoV vaccines once a week for 3 times. On day 21, mice were sacrificed and spleens and blood were harvested to detect immune response. Results Nanoemulsion loaded inactivated PDCoV antigen components and formed into nanoparticle with mean diameter of(157.6 ± 0.4) nm, while CpG and inactivated PDCoV were irregularly mixed. The maturations of BMDC incubated with Cp G/PDCoV group and Nanoemulsion/PDCoV group were 45.57 % and 53.32 %, respectively, as indicated by percentage of CD86+MHCⅡ+BMDC, which were distinctly higher than that of PDCoV group(38.49 %)(P < 0.01, P <0.05). On day 21, Th1(P < 0.05) and Tc2(P < 0.05) in spleens of CpG/PDCoV group were 5.81 % and 0.24 %, which were significantly higher than those of PDCoV group(3.56 % and 0.14 %). Tc1 in spleens of Nanoemulsion/PDCoV group(6.46 %)was higher than that of PDCoV group(9.28 %). The levels of total immunoglobulin G(IgG), IgG2a and interferon gamma(IFN-γ) in serum of CpG/PDCoV group were apparently higher that those in PDCoV group(P < 0.05), and the difference was statistically significant(P < 0.05). The levels of total IgG, IgG2a, IFN-γ, tumor necrosis factor α(TNF-α) and interleukin 4(IL-4)in serum of Nanoemulsion/PDCoV group were significantly higher than those of PDCoV group, and the difference was statistically significant(P < 0.05). Compared with CpG, Nanoemulsion was induced higher levels of IgG2a, IFN-γ, TNF-α and IL-4.Conclusion It is demonstrate

关 键 词：病毒灭活疫苗 免疫佐剂 纳米疫苗 细胞免疫 体液免疫 CPG 纳米乳 

分 类 号：TK448.21[动力工程及工程热物理—动力机械及工程]