CpG-ODN及PolyICLC在结核亚单位疫苗中的佐剂效应

The adjuvant effects of CpG-ODN and PolyICLC in tuberculosis subunit vaccine

目的观察TLR识别配体不同组合佐剂对结核分枝杆菌融合蛋白免疫原性的影响及DDA对TLR识别配体的辅助效应。方法 CpG-ODN(CpG)和/或PolyICLC联合/不联合DDA,分别与融合蛋白Mtb10.4-HspX(MH)混合,制备亚单位疫苗,于第1、4、7周皮下免疫C57BL/6小鼠。以PBS和BCG(仅免疫1次)作为对照。末次免疫后6周,采血检测血清抗体水平,并分离脾淋巴细胞,检测分泌IFN-γ的淋巴细胞水平。结果经MH及HspX抗原刺激后,MH+CpG+PolyICLC+DDA组小鼠分泌IFN-γ的脾淋巴细胞数高于其它各组(P<0.05),MH+CpG+PolyICLC组其次,显著高于MH+CpG及MH+PolyICLC组(P<0.05);联合DDA佐剂组均分别高于对应的未联合DDA佐剂组(P<0.05)。各亚单位疫苗组诱导产生的抗MH及HspX的IgG1、IgG2b、IgG2c水平均明显高于BCG组(P<0.05),其中MH+CpG+PolyICLC+DDA组3种抗体水平最高;各亚单位疫苗组IgG2c/IgG1均高于BCG组(P<0.05)。结论 CpG+PolyICLC+DDA佐剂增强了结核分枝杆菌融合蛋白的免疫原性;CpG和PolyICLC具有佐剂协同作用;DDA有助于CpG和/或PolyICLC诱导特异性细胞免疫应答。

This study is aimed to observe whether the TLR ligands combined as adjuvant can enhance the immunogenicity of Mycobacterium tuberculosis fusion protein and to analyze synergetic effects of DDA on TLR ligands. Mycobacterium tuberculosis fusion protein Mtbl0.4-HspX （MH） subunit vaccine was prepared by mixing CpG-ODN （CpG） and/or PolyICLC with/without DDA, and then C57BI./6 mice were immunized subcutaneously with the vaccine at week 1, 4 and 7. PBS and BCG immunized groups received one time inoculation were used as controls. The serum samples of the mice were collected at 6 weeks after the last immunization for antibodies detection. Meanwhile, the splenic lymphocytes of mice were isolated for the detection of IFN-γproduction. After the stimulation of MH and HspX antigens, the mice immunized with MH＋CpG＋PolyICLC＋DDA vaccine generated the highest level of IFN-γ（P〈 0.05）, followed by MH＋CpG＋PolylCLC vaccine immunized mice with significantly higher production of IFN-γ（P 〈 0.05）, as compared with MH＋CpG and MH ＋PolyICLC. Furthermore, the levels of IFN-γ induced by vaccines combined with DDA were respectively higher than these without DDA （P〈 0.05）. MH and HspX-specific IgG1, IgG2b and IgG2c induced by all subunit vaccines were significantly higher than BCG （P〈0.05）. Notably, these antibodies were the highest in the mice immunized with MH＋CpG＋ PolyICLC＋DDA vaccine. In addition, IgG2c/IgG1 from every subunit vaccine group was higher than that of BCG group （P〈0.05）. All the result suggested that CpG＋ PolyICLC ＋DDA could enhance the immunogenicity of Myeobacterium tuberculosis fusion protein. There is a synergism between CpG and PolyICLC as adjuvant, and DDA is contributed to the specific cellular immune response induced by CpG and/or PolyICLC.