不同黏膜佐剂对猪丁型冠状病毒诱导小鼠免疫应答的影响  

Effects of different mucosal adjuvants on the immune responses induced by porcine deltacoronavirus in mice

作　　者：李厚伟 张先锋 王彦辉 姬星宇 王蕾 张真真 李倩倩 张云飞[3] 胡永浩[1] LI Houwei;ZHANG Xianfeng;WANG Yanhui;JI Xingyu;WANG Lei;ZHANG Zhenzhen;LI Qianqian;ZHANG Yunfei;HU Yonghao(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,China;Henan Puhua Gene Technology Co.,Ltd.,Zhengzhou 450045,Henan,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,Henan,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]河南普华基因科技有限公司,河南郑州450045 [3]河南农业大学动物医学院,河南郑州450046

出　　处：《微生物学报》2022年第7期2686-2701,共16页Acta Microbiologica Sinica

基　　金：国家自然科学基金面上项目(31972678);河南省重大科技专项(181100110500)。

摘　　要：【目的】旨在为猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)灭活疫苗黏膜免疫筛选理想佐剂,降低疫苗副作用。利用小鼠模型评价不同佐剂制备的PDCoV灭活疫苗对体液免疫、细胞免疫和黏膜免疫应答的影响。【方法】将甘露聚糖肽(PA)、CpGODN2395、单磷酰脂质A(MPLA)佐剂分别与IMS 1313、GEL02佐剂联合制备PDCoV灭活疫苗,经鼻腔免疫BALB/c小鼠;将ISA201佐剂制备的PDCoV灭活疫苗经皮下免疫BALB/c小鼠,将PDCoV灭活抗原经鼻腔免疫BALB/c小鼠作为对照,间隔14 d加强免疫一次。用ELISA方法检测小鼠血清、支气管肺泡灌洗液(BALF)中的IgG、IgG1、IgG2a、IL-4、IFN-γ及粪便和BALF中sIgA表达水平;用MTT方法检测疫苗免疫后对小鼠脾淋巴细胞增殖的影响;观察并记录小鼠免疫后的临床表现,HE染色方法观察免疫小鼠主要器官组织的病理学变化,评价疫苗的安全性。【结果】ISA201组小鼠BALF和血清中的抗体(IgG、IgG1)及IL-4表达水平相对较高,但IgG2a、IFN-γ和粪便中sIgA表达水平较低;相较于IMS 1313组、IMS1313+2395组、IMS1313+PA组、IMS1313+MPLA组,GEL02组、GEL02+2395组、GEL02+PA组、GEL02+MPLA组小鼠BALF和血清中的抗体(IgG、IgG1、IgG2a)、IL-4、IFN-γ及粪便中sIgA表达水平均较高;其中GEL02+2395组小鼠BALF和血清中IgG2a抗体、IFN-γ及BALF和粪便中sIgA表达水平显著高于其他组;GEL02+2395疫苗、IMS1313+2395疫苗可促进T淋巴细胞增殖;小鼠主要器官组织未见明显病理变化,GEL02疫苗、GEL02+2395疫苗免疫小鼠的副反应最轻微【。结论】GEL02与CpG ODN2395佐剂联合制备PDCoV灭活疫苗经鼻腔免疫小鼠,不仅可增强小鼠体液免疫水平,也可有效提高细胞免疫和黏膜免疫水平,为黏膜疫苗的研发提供数据支持。[Objective]The aim of this study is to screen an ideal adjuvant for an inactivated porcine deltacoronavirus(PDCoV)vaccine to induce mucosal immunity and reduce the side effect of the vaccine.We used different mucosal adjuvants to prepare the inactivated PDCoV vaccines.We then used mouse model to evaluate the humoral,cellular and mucosal immune responses induced by the inactivated vaccines via different immunization routes.[Methods]The adjuvants IMS1313 and GEL02 were respectively combined with polyactin A(PA),CpG ODN2395,and monophosphoryl lipid A(MPLA)to prepare the inactivated PDCoV vaccines,which were then used to immunize BALB/c mice intranasally.The inactivated PDCoV vaccine prepared with ISA201 adjuvant was used to immunize BALB/c mice subcutaneously.The inactivated PDCoV vaccine without adjuvant was used as a control to immunize BALB/c mice intranasally.The mice were immunized once again at the same doses in the same ways 14 days post the primary immunization.Enzyme-linked immunosorbent assay(ELISA)was employed to determine the expression levels of IgG,IgG1,IgG2a,IL-4,and IFN-γin serum and bronchial lavage fluid(BALF)samples,as well as the levels of sIgA in feces and BALF samples.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the proliferation of spleen lymphocytes.We observed and recorded the clinical manifestations of immunized mice.Meanwhile,we observed the pathological changes of major organs and tissues of immunized mice via hematoxylin-eosin(HE)staining to evaluate the safety of the vaccines.[Results]The ISA201 group had high expression levels of antibodies(IgG and IgG1)and IL-4 in BALF and serum and low expression levels of IgG2a,IFN-γ,and sIgA in feces.GEL02,GEL02+2395,GEL02+PA,and GEL02+MPLA groups showed higher expression levels of antibodies(IgG,IgG1,and IgG2a),IL-4,and IFN-γin BALF and serum and higher expression level of sIgA in feces than IMS1313,IMS1313+2395,IMS1313+PA,and IMS1313+MPLA groups.In particularly,the expression levels of IgG2a in BALF and serum samples and IFN-γand

关 键 词：猪丁型冠状病毒 佐剂 黏膜免疫 

分 类 号：S852.4[农业科学—基础兽医学]