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目的探讨以含有多拷贝CpG寡脱氧核苷酸(CpG ODN)的质粒作为治疗性乙肝疫苗佐剂的可行性。方法构建含有6个拷贝D型CpG ODN的质粒pKO-CG6,将该质粒以及载体pKO分别刺激健康人及HBV感染者的外周血单核细胞(PBMC),检测PBMC的增殖及分泌的细胞因子IFN-γ、IL-12,进一步将重组HBsAg分别联合这两种质粒免疫小鼠,检测小鼠的细胞和体液免疫应答。结果质粒pKO-CG6与载体pKO在体外均能有效激活健康人及HBV感染者PBMC增殖反应,并促进IFN-γ、IL-12的产生,其中pKO-CG6的免疫刺激活性强于载体pKO。小鼠体内试验表明,虽然载体pKO也具有免疫佐剂作用,但pKO-CG6更能显著增强HBsAg诱导的免疫应答,尤其是细胞免疫应答。结论含有多拷贝D型CpG ODN的质粒能有效激活HBV感染者的PBMC,并增强HBsAg在小鼠体内的免疫原性。
Objective To investigate the feasibility of using a plasmid containing multiple copies of CpG ODN as therapeutic adjuvant for hepatitis B vaccine. Methods Plasmid pKO-CG6 containing 6 copies of D-type CpG ODN was constructed. The plasmid and pKO were used to stimulate peripheral blood mononuclear cells (PBMCs) of healthy individuals and HBV infected individuals respectively. The proliferation and secretion of cytokines IFN -γ, IL-12. The recombinant HBsAg was further immunized with the two plasmids respectively to detect the cellular and humoral immune responses in mice. Results The plasmid pKO-CG6 and pKO could effectively activate the PBMC proliferation and promote the production of IFN-γ and IL-12 in healthy and HBV infected patients. The immunostimulatory activity of pKO-CG6 was stronger than that of pKO. In vivo experiments in mice showed that pKO-CG6 significantly enhanced the HBsAg-induced immune response, especially the cellular immune response, in spite of the immune adjuvant effect. CONCLUSION: Plasmids containing multiple copies of CpG ODN can effectively activate PBMC in HBV infected patients and enhance the immunogenicity of HBsAg in mice.