含免疫增强剂CVC1302口蹄疫灭活疫苗缩短免疫应答窗口期机制的研究

The mechanism of killed foot-and-mouth disease virus vaccine adjuvanted with immunopotentiator CVC1302 in shortening the immune response period in micetitis

为探究免疫增强剂CVC1302缩短口蹄疫灭活疫苗(KV)免疫应答窗口期的机制,本研究将灭活的O型口蹄疫病毒(FMDV)与CVC1302按照1.50配比后,与ISA206佐剂乳化制备疫苗(KV-CVC1302);将灭活的FMDV与ISA206佐剂乳化制备疫苗(KV),将上述疫苗分别免疫6周龄BALB/c雌性小鼠。免疫后7 d、28 d、56 d、90 d、120 d、150 d和180 d采血分离血清,利用O型FMD液相阻断ELISA(LBP ELISA)试剂盒检测各组小鼠血清FMDV特异性抗体水平。免疫后1 d,取注射位点肌肉,分别利用qPCR和ELISA检测其中细胞趋化因子的转录及表达水平。免疫后1 d、3 d、5 d和7 d分别采集注射位点肌肉及腹股沟淋巴结,制备单个淋巴细胞,利用流式细胞仪检测注射位点肌肉和腹股沟淋巴结的抗原递呈细胞(APC)[树突状细胞(DC)、巨噬细胞(Mph)、单核细胞(Mo)]的数量;免疫后1 d,利用流式细胞仪检测注射位点肌肉中DC的活化情况。结果显示,KV-CVC1302组小鼠FMDV特异性抗体水平、注射位点趋化因子转录及表达水平均显著高于KV组(p<0.01、p<0.001);且注射位点APC的数量及DC的活化水平也显著提高(p<0.001),该组小鼠腹股沟淋巴结APC的数量也明显高于KV组(p<0.05、p<0.01或p<0.001)。综上结果表明,CVC1302通过增强KV于注射位点诱导产生趋化因子的能力,募集大量的APC,进而对FMDV进行有效的捕获加工和递呈,转运至腹股沟淋巴结,诱导B细胞的形成,并分泌高水平抗体,从而缩短免疫应答窗口期。本研究首次证实CVC1302是在疫苗诱导免疫应答的启动阶段发挥作用,其通过募集活化的APC捕获更为有效的抗原以激活免疫系统,缩短了免疫应答窗口期,为后续研制新型免疫增强剂提供了新思路。

In order to decipher the mechanisms of CVC1302 in reducing the immune response period induced by serotype O killed foot-and-mouth disease virus(FMDV)vaccine,inactivated FMDV were mixed with CVC1302 in a ratio of 1􀏑50 followed by emulsified with ISA206,named as KV-CVC1302,inactivated FMDV were emulsified with ISA206,named as KV.Six-week-old female BABL/c mice were immunized with KV-CVC1302,KV or ISA206,and FMDV-specific antibody titers were detected at 7,28,56,90,120,150 and 180 days post-immunization(dpi)by using FMD liquid-phase blocking(LBP)ELISA kits.The relative transcription and expression levels of chemokine(CCL2,CCL3 and CCL4)induced at injection muscle were measured at 1 dpi by using qPCR and ELISA.The injection muscle and draining lymph node were sampled to detect the numbers of APCs at 1,3,5 and 7 dpi.At 1 dpi,the levels of activation of DC were also analyzed by detecting the expression of several molecular markers(CD40,CD80,CD86 and MHCII).Through the detection,we found that FMDV-specific antibody titers were significantly higher in KV CVC1302 group when compared with KV group(p<0.01).Significantly higher levels of chemokine at injection muscle were induced in KV-CVC1302 group compared with KV group(p<0.01 or p<0.001).Moreover,significantly higher numbers of DC,Mo and Mph were recruited into the injection muscle of mice received with KV-CVC1302 when compared with those received KV,as well as the levels of activation of DCs(p<0.001).Significantly higher numbers of DC,Mo and Mph were recruited into the draining lymph nodes of mice received with KV-CVC1302 when compared with those received KV(p<0.05,p<0.01 or p<0.001).Results of the study revealed that CVC1302 improved the ability of KV to produce the chemokines at injection sites,then recruited APCs to efficiently capture,process and present FMDV,finally induced B cells to express higher levels of antibodies,shorten the immune response period.Through the study,CVC1302 were demonstrated to activate APCs at injection sites,initate immune response,then shorten immune response period,which provides the blueprint for designing new generations of immunopotentiators.