3种TLR配体对猪圆环病毒2型病毒样颗粒疫苗佐剂活性的比较

Comparison of adjuvant activities of three molecular adjuvants for virus-like particle vaccine of porcine circovirus type 2

为比较3种TLR配体对猪圆环病毒2型(PCV2)病毒样颗粒(VLP)疫苗的佐剂活性,本研究利用重组大肠杆菌表达脑膜炎奈瑟菌Ag473脂蛋白、创伤弧菌FlaB鞭毛蛋白和结核分枝杆菌热应激蛋白70(Hsp70),采用镍亲和柱纯化获得该3种重组蛋白;分别将相同剂量(10μg)3种重组蛋白、ISA206佐剂(100μL)和不完全弗氏佐剂(FIA)(100μL)与PCV2 VLP疫苗(50μg)混合,肌肉注射免疫小鼠,初免后14 d加强免疫1次;初免后每周采血分离血清,采用ELISA检测抗原特异抗体;加强免疫后14 d利用PCV2攻毒,攻毒后7 d和14 d采血分离血清,采用荧光定量PCR检测病毒DNA拷贝数;攻毒后14 d迫杀小鼠,制备并培养其脾细胞,经PCV2 VLP疫苗刺激后,采用试剂盒检测细胞因子的表达。SDS-PAGE分析结果显示,3种TLR配体在重组大肠杆菌中均获得正确表达,纯化重组蛋白的纯度大于90%。从初免14 d起,3个分子佐剂组中Ag473佐剂组的PCV2抗体水平最高,但低于ISA206佐剂组却高于FIA组,差异不显著(p>0.05)。在PCV2攻毒前,3个分子佐剂组中Ag473佐剂组的小鼠脾细胞TNF-α和IFN-γ表达水平最高,但低于ISA206佐剂组却高于FIA组,差异显著(p<0.05);Hsp70佐剂组的IL-4表达水平最高,Ag473佐剂组次之,但均高于ISA206和FIA组。在攻毒后第7 d,5个免疫组小鼠的病毒拷贝数均较对照组显著下降(p<0.05),但免疫组间差异不显著;在攻毒后第14 d,5个免疫组小鼠的病毒拷贝数进一步下降,免疫组间差异不显著。研究结果表明在测试的3种分子佐剂中,Ag473脂蛋白具有较强的总体免疫佐剂活性,有望进一步开发为PCV2等病毒的亚单位疫苗佐剂。

To compare the adjuvant activities of three TLR agonists for the virus-like particle(VLP)vaccine of porcine circovirus type 2(PCV2),in this study the Ag473 lipoprotein from Neisseria meningitides,the FlaB flagellin from Vibrio vulnificus and the heat shock protein 70(Hsp70)from Mycobacterium tuberculosis were expressed in E.coli as His-tagged proteins,and purified using nickel affinity chromatography.Each of three molecular adjuvants at the dose of 10μg,ISA206(100μL)and Freunds incomplete adjuvant(FIA)(100μL)were mixed individually with PCV2 VLP vaccine(50μg)and injected intramuscularly into mice.At day 14 post immunization,the immunized mice were boosted with the same vaccine formulations.From the primary immunization,the serum samples were collected weekly and tested for PCV2-specific antibody by ELISA.At day 14 post boosting immunization,the immunized mice was challenged with PCV2.At days 7 and 14 after challenge,the serum samples were collected and the number of viral DNA copies was detected by quantitative PCR.At day 14 after challenge,three mice from each group were sacrificed for splenocyte culture preparation.After stimulation with PCV2 VLP vaccine,the expression of three cytokines were measured using commercial ELISA kits.SDS-PAGE analysis showed that the three molecular adjuvants were correctly expressed in E.coli,and the purity of the purified recombinant proteins reached up to more than 90%.From day 14 post primary immunization,the antibody level of PCV2 in the Ag473 adjuvant group was the highest among the three molecular adjuvant groups,which was lower than that in the ISA206 adjuvant group but higher than that in the FIA group,with no significant difference.Before PCV2 challenge,the expression levels of TNF-αand IFN-γin the splenocyte cultures of the Ag473 adjuvant group were the highest among the three molecular adjuvant groups,which were lower than that in the ISA206 adjuvant group but higher than that in the FIA group,with significant difference.The expression level of IL-4 in the Ag473 adjuvant group was lower than that in the Hsp70 adjuvant group but higher than that in the ISA206 and FIA adjuvant groups.From days 7 to 14 post challenge,the viremic titers in the three molecular adjuvant groups were similar to that in the two commercial adjuvant groups.These data suggest the overall strong immune adjuvant activity of Ag473 lipoprotein,which could be futher developed as a molecular adjuvant for PCV2 and other viral subunit vaccines.