6种新型CpG分子增强新型冠状病毒重组亚单位疫苗的免疫效果比较  

Comparative study on 6 novel CpG molecules in enhancing the immune effect of SARS-CoV-2 recombinant subunit vaccine

作　　者：李思奇 高孟 安彤 张柯欣 马培凯 沈钱通 朱赟 庄昉成 陈刚 Li Siqi;Gao Meng;An Tong;Zhang Kexin;Ma Peikai;Shen Qiantong;Zhu Yun;Zhuang Fangcheng;Chen Gang(Zhejiang Key Laboratory of Bio-medical Vaccine Research and Development,School of Basic Medical Sciences and Forensic Medicine,Hangzhou Medical College,Hangzhou 310053,China)

机构地区：[1]杭州医学院基础医学与法医学院、浙江省医学生物工程疫苗研发重点实验室,杭州310053

出　　处：《国际流行病学传染病学杂志》2023年第2期98-103,共6页International Journal of Epidemiology and Infectious Disease

基　　金：浙江省基础公益研究计划(LGD22C080002);浙江省重点研发计划应急攻关项目(2021C03202);浙江省教育厅一般科研项目(Y202045358);浙江省医学生物工程疫苗研发重点实验室(2008F3022)。

摘　　要：目的了解新设计的6组CpG分子在动物体内对新型冠状病毒(SARS-CoV-2)重组亚单位疫苗免疫效果的提升作用,以初步筛选出有潜力的新型CpG佐剂。方法通过序列检索比对,确定人鼠同源的CpG基序,以不同的组合方式设计出6条不同的CpG寡聚脱氧核苷酸(CpG-ODN)序列,并人工合成相应6种CpG,分别与SARS-CoV-2重组亚单位疫苗联用,已上市的佐剂CpG1018作为阳性对照。以间隔2周的方式免疫BALB/c小鼠2次,免疫后每周取眼眶血检测IgG1和IgG2a指标。2针免疫后21 d处死小鼠,取眼球血检测结合抗体抑制率;取脾脏淋巴细胞进行酶联免疫斑点试验检测。结果初次免疫后14 d,CpG6组小鼠血清IgG2a和IgG1水平分别为3.63±0.27和3.44±1.03,显著高于CpG1018组的2.06±1.30和2.06±0.39。加强免疫后14 d,CpG6组小鼠血清IgG2a水平为6.52±0.21,高于CpG1018组的6.33±0.40。在针对原型株的结合抗体抑制率指标上,初次免疫14 d、加强免疫21 d后,各组间差异存在统计学意义(F=13.66、12.58、19.38和19.38,P均<0.001)。初次免疫14 d后,稀释10倍血清中CpG6组小鼠血清抑制率为(64.67±20.41)%,高于CpG1018组[(27.84±20.23)%],差异有统计学意义(t=2.70,P=0.031);加强免疫21 d后,稀释1000倍的血清中CpG6组小鼠血清抑制率针对原型株[(89.71±4.83)%]和Delta株[(79.04±6.32)%]的抑制率水平均显著高于CpG1018组[(70.84±17.20)%和(52.61±14.22)%],差异有统计学意义(t=2.36,P=0.046;t=3.80,P=0.005)。酶联免疫斑点实验结果显示,CpG1018组针对SARS-CoV-2原性株敏感和Delta突变株敏感的特异性IFN-γ分泌细胞数与CpG6组相比,差异均无统计学意义(t=0.76,P=0.469;t=0.78,P=0.459);在特异性IL-2分泌细胞数上,两组差异亦无统计学意义(t=0.70,P=0.503;t=0.90,P=0.395)。结论CpG6与SARS-CoV-2重组亚单位疫苗联用,能在疫苗初次免疫后更快速地在小鼠体内产生体液免疫应答,且加强免疫后体液免疫和细胞免疫效果均不弱于已上市的CpGObjective To understand the boosting effect of 6 newly designed CpG molecules on the immune efficacy of SARS-CoV-2 recombinant subunit vaccine in animals,so as to initially screen out the potential CpG adjuvants.Methods The human-mouse homologous CpG motifs were identified by sequence search and comparison,and 6 different CpG-oligodeoxynucleotide(ODN)molecules were designed and synthesized.These CpG molecules were used in combination with SARS-CoV-2 recombinant subunit vaccine,with the existing adjuvant CpG1018 as a positive control.BALB/c mice were immunized twice at two-week intervals,with weekly orbital blood sampling for IgG1 and IgG2a indicators.The mice were sacrificed at day 21 after the booster immunization,and the eye blood was collected to detect binding antibody inhibition rate,and the lymphocytes were collected for enzyme-linked immunospot assay.Results At day 14 after the initial immunization,the serum levels of IgG2a and IgG1 in CpG6 group were 3.63±0.27 and 3.44±1.03,which were both higher than those of 2.06±1.30 and 2.06±0.39 in CpG1018 group.At day 14 after the booster immunization,the serum level of IgG2a in CpG6 group was 6.52±0.21,which was higher than that of 6.33±0.40 in CpG1018 group.The inhibition rates of conjugated antibodies against the prototype strain were significantly different in all groups after 14 days of initial immunization and 21 days of booster immunization(F=13.66,12.58,19.38 and 19.38,P all<0.001).In CpG6 group,the serum inhibition rate in a 10-fold dilution was(64.67±20.41)%at day 14 after the initial immunization,which was significantly higher than that of(27.84±20.23)%in CpG1018 group(t=2.70,P=0.031).At day 21 after the booster immunization,the serum inhibition rates of conjugated antibodies against the prototype strain and Delta strain in a 1000-fold dilution were(89.71±4.83)%and(79.04±6.32)%in CpG6 group,respectively,which were significantly higher than those of(70.84±17.20)%and(52.61±14.22)%in CpG1018 group(t=2.36,P=0.046;t=3.80,P=0.005).The results of enz

关 键 词：佐剂 免疫 亚单位疫苗 CPG寡核苷酸 增强免疫 体液免疫 细胞免疫 

分 类 号：R392[医药卫生—免疫学]