免疫增强剂CVC1302诱导高亲和力抗体机制分析

Analysis of the mechanism of immunopotentiator CVC1302 inducing high affinity antibody

为探究免疫增强剂CVC1302诱导机体产生高亲和力抗体的免疫机制,本研究利用4-羟基-3-硝基苯乙酰基耦联鸡卵白蛋白(NP-OVA)与免疫增强剂CVC1302配伍后,与ISA206佐剂乳化制备疫苗。将33只6周龄BALB/c雌性小鼠随机分为3组,分别后腿肌肉注射NP、NP-ISA206和NP-CVC1302-ISA206,每只100μL疫苗,50μg NP-OVA/只,利用ELISA检测高亲和力NP特异性抗体水平;利用流式细胞仪分析生发中心B细胞增殖水平、生发中心B细胞诱导活化的胞苷脱氨酶(AID)表达水平;利用荧光定量PCR检测AID基因和HoxC4基因转录水平。结果表明:1)免疫增强剂CVC1302可诱导小鼠产生高亲和力的NP特异性抗体。2)免疫增强剂CVC1302可显著提升抗原特异性生发中心B细胞数量占总B细胞数量比例。3)免疫增强剂CVC1302可显著增加生发中心B细胞HoxC4基因转录水平。4)免疫增强剂CVC1302可显著增强生发中心B细胞AID蛋白表达水平。生发中心B细胞表达的AID可针对B细胞免疫球蛋白序列可变区进行点突变,发生体细胞高频突变,提升B细胞BCR针对抗原的亲和力,进而在辅助性T细胞的阳性选择下分化为长寿浆细胞,持续性分泌高亲和力抗体,因此本研究推断免疫增强剂CVC1302依赖于正向调控AID诱导机体产生高亲和力抗体,为机体提供免疫保护力,以抵抗病原体的感染,为后续新型免疫增强剂的研制提供思路和理论基础。

In order to elucidate the immune-mechanism of immunopotentiator CVC1302inducing high affinity antibody,NP-OVA,as model antigen,mixed with CVC1302,then emulsified with ISA206,was prepared for vaccine.33Six week-old BALB/c female mice were divided into 3groups and immunized with NP-CVC1302-ISA206,NP-ISA206or NP,each mouse immunized with 100μL vaccine,as well as 50μg NP-OVA.The levels of high-affinity NP-specific antibody were detected by ELISA,the percentages of NP-specific germinal center(GC)B cells in B cells and expression levels of AID in GC B cells were analyzed by flow cytometry.GC B cells were harvested by FACS sorting.The transcriptional levels of AID and HoxC4 in GC B cells were analyzed by real-time PCR.The results showed that:1)Immunopotentiator CVC1302induced high affinity NP-specific antibodies.2)CVC1302induced higher percentage of NP-specific GC B cells in B cells.3)CVC1302improved transcriptional levels of HoxC4 in GC B cells.4)CVC1302enhanced the expression levels of AID in GC B cells.Considering AID in GC B cells inducing high affinity antibodies through point mutation,then GC B cells differentiated into long-term plasma cells under the positive selection of follicular helper T cells,we concluded that CVC1302induced high affinity antibody dependent on positive control of AID,which shed light on the development of new immunopotentiators.