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

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 demonstrated that the adjuvant CpG or Nanoemulsion could significantly enhance the immune effect of inactivated virus vaccine. Compared with CpG, Nanoemulsion induces stronger cellular immunity and humoral immunity, which is more suitable for virus-inactivated vaccines research.