霍乱毒素B亚基展示HIV-1 gp41 MPER表位的研究

Study on the cholera toxin B subunit displaying HIV-1 gp41 membrane-proximal external region epitope

目的 评价霍乱毒素B亚基融合HIV-1 gp41 MPER表位的免疫效果.方法 利用霍乱毒素的B亚基（CTB）作为载体展示靶向于MPER的广谱中和抗体2F5和4E10的表位.表达纯化后的融合蛋白与弗氏佐剂混合乳化后免疫小鼠,免疫4次后对小鼠的血清进行综合评价.ELISA法评价融合蛋白的免疫原性;检测免疫血清中IgA类抗体分析小鼠黏膜免疫应答;对小鼠血清中的不同类型的抗体量进行分析,通过流式细胞仪检测PBMC细胞因子IFN-γ和IL-4用以分析小鼠免疫激活途径;病毒中和实验检测免疫血清对病毒感染细胞的抑制能力.结果 CTB作为载体蛋白具有很好的免疫原性,能够诱导免疫系统产生针对MPER表位的抗体,刺激小鼠产生黏膜免疫应答,诱导针对MPER表位的IgA抗体.同时,融合蛋白可以刺激机体的IgG1,IgG2a和IgG2b类抗体的产生和刺激小鼠体内的Th2类免疫途径.中和实验结果显示,CTB-4E10免疫后的血清可以抑制NL4-3病毒对细胞的感染.结论 CTB融合蛋白具有强免疫原性,可以刺激体液免疫反应,黏膜免疫反应和Th2类免疫途径,并且CTB-4E10蛋白免疫血清能够抑制NL4-3病毒感染细胞.本研究为HIV-1 gp41 MPER作为候选疫苗的探索奠定基础.

Objective To evaluate the immune effect of fusion protein of CTB and HIV-1 gp41 MPER epitope.MethodsThe cholera toxin B subunit was used as a carrier to display the epitopes of bNAbs 2F5 and 4E10 targeting the MPER.The purified fusion proteins with Freund adjuvant were immunized in mice and the immunized sera were analyzed after the four times immunization.The immunogenicity of the fusion proteins was analyzed by ELISA.And the IgA antibodies were detected to evaluate the mucosal immune response in mice.The different types of antibodies and the cytokines INF-γ and IL-4 of PBMC were used to evaluate the immune response pathway.Besides, neutralization assay was carried out to detected the antiviral ability of immunized serum.ResultsCTB as a carrier has good immunogenicity and fusion proteins can induced specific antibodies against MPER epitopes.In addition, the fusion proteins stimulated mucosal immunity, induced IgA, IgG1, IgG2a and IgG2b antibodies and the Th2 immune response pathway in mice.Importantly, CTB-4E10 immune serum had the neutralizing activity against the NL4-3 virus.Conclusion CTB fusion proteins had good immunogenicity and could induce humoral immunity, mucosal immunity and Th2 immune response pathway in mice.The serum from the CTB-4E10 immunized mice could inhibit virus infecting cells.This study laid the foundation for the exploration of HIV-1 gp41 MPER as candidate vaccine.