不同硫代修饰CpG ODN佐剂活性的比较

目的评价相同序列不同硫代修饰CpG ODN佐剂联合重组乙型肝炎疫苗表面抗原(rHBsAg)在BALB/c小鼠中的免疫原性,及在HEK-Blue^TM hTLR9细胞上的活性。方法将4种(全硫代修饰的QCX1、部分硫代修饰的HS1和HS2及非硫代修饰的NS)CpG ODN及其阳性对照(全硫代修饰的CpG7909)按照相同配比分别与rHBsAg混合,使CpG ODN和rHBsAg终浓度均为20μg/mL,同时设等剂量抗原对照组(rHBsAg)及空白对照组(生理盐水)。分别肌肉免疫BALB/c小鼠,每只100μL,于免疫后1、2、4周摘眼球采血后处死小鼠,分离血清,应用化学发光微粒子免疫分析技术检测乙型肝炎病毒表面抗体(antibody to hepatitis B virus surface antigen,Anti-HBs)水平;取小鼠脾脏,制备脾细胞悬液,分离脾单个核细胞(mononuclear cell,MNCs),应用ELISPOT技术检测抗原特异性IFNγ、IL-2的分泌水平。体外应用HEK-Blue^TM hTLR9细胞,检测4种CpG ...

Comparison of adjuvant activities of different phosphorothioate backbone modified CpG ODN

Objective To evaluate the immunogenicity of different phosphorothioate modified CpG ODN adjuvant combined with recombinant hepatitis B surface antigen(rHBsAg)in BALB/c mice and their activities in HEK-BlueTM hTLR9 cells.Methods Four kinds of CpG ODNs(completely phosphorothioate modified QCX1,partially phosphorothioate modified HS1,partially phosphorothioate modified HS2 and NS without phosphorothioate modification)and positive control(completely phosphorothioate modified CpG7909)were mixed with rHBsAg at the same proportions,in which the final concentrations of CpG ODN and rHBsAg were both 20μg/mL,while rHBsAg at the same dosage was set up as antigen control group,and physiological saline as blank control,with which BALB/c mice were immunized intramuscularly,100μL for each.Serum samples were collected 1,2 and 4 weeks after immunization and determined for antibody to HBsAg(Anti-HBs)by chemiluminesent microparticle immunoassay kit.Meanwhile,the spleen cell suspension of mice was prepared,from which spleen mononuclear cells(MNCs)were isolated and determined for the secretion levels of antigen-specific IFNγand IL-2 by enzyme linked immunospot assay(ELISPOT).The activities of four kinds of CpG ODN and positive control CpG7909 in HEK-BlueTM hTLR9 cells,as well as the activities after storage in sera for various days,were determined in vitro.Results The anti-HBs levels in sera of mice in various groups increased gradually 1,2 and 4 weeks after immunization,while those in QCX1 group 2 and 4 weeks after immunization were higher than those in rHBsAg control,HS1,HS2 and NS groups.The ability of MNCs stimulated by rHBsAg in QCX1 group in secreting IFNγand IL-2 was significantly higher than those in HS1,HS2,NS and rHBsAg control groups(each P<0.01).The half effective concentrations(EC50)for stimulation of HEK-BlueTM hTLR9 cell activity in QCX1 and HS2 groups were 5.94 and 6.52μg/mL respectively,which were significantly lower than those in HS1(46.54μg/mL)and NS(49.10μg/mL)groups.With the increasing time for storage of CpG ODN in sera,only the A655 values of cell cultures in QCX1 and CpG7909 groups showed little change,while those in other groups decreased at different degrees.Conclusion The completely phosphorothioate modified QCX1 adjuvant was equivalent to CpG 7909 in enhancing the cellular and humoral immunities of mice,which showed high activity in HEK-BlueTM hTLR9 and high stability in sera.