多阶段抗原的结核分枝杆菌亚单位疫苗SHP／TMD的免疫原性及保护性研究

Immunogenicity and protective efficacy of a subunit vaccine SHP/TMD consisting of multi - stage antigens against Mycobacterlum tuberculosis infection

目的：设计构建包含结核分枝杆菌（M．tb）感染多阶段抗原的融合蛋白亚单位疫苗 SHP，在中国 M．tb 感染者和小鼠模型中评价其免疫原性和抗感染保护性。方法选择 M．tb 复制期分泌抗原 Rv0577、休眠期抗原 Rv2623和 PE ／PPE 家族抗原 Rv3478，构建并原核表达融合蛋白 SHP；以基于 TB-IGRA 的全血IFN-γ分析试验（WBIA）检测 SHP 及其亚组分蛋白诱导中国 M．tb 感染者和健康对照者外周血淋巴细胞产生的特异性 IFN-γ水平及其差异；以 SHP 联合佐剂 TMD 皮下免疫 C57BL／6小鼠，9周后处死并检测特异性抗体滴度、脾淋巴细胞培养上清中特异性 Th1型细胞因子水平以及肺脏 IFN-γ、TNF-α、IL-10和 iNOS 的表达水平；以 M．tb H37Rv 毒株攻毒4周后评价 SHP／TMD 的抗感染保护性。结果成功构建、表达融合蛋白 SHP，SHP及其亚组分蛋白刺激 M．tb 感染者淋巴细胞产生的 IFN-γ浓度均显著性高于健康对照者[如 SHP 刺激值分别为（1420．0±253．9）pg／mL 和（236．1±95．1）pg／mL，t ＝2．063，P ＜0．01]。SHP／TMD 诱导产生显著高水平的特异性 IgG 抗体及其亚类，IgG2a／IgG1趋向 Th1型应答；SHP／TMD 免疫小鼠的脾淋巴细胞诱导显著高水平的特异性 IL-2、IFN-γ和 TNF-α；SHP／TMD 免疫小鼠肺脏组织 IFN-γ、TNF-α及 iNOS 表达水平显著上升。此外， SHP／TMD 显著降低 M．tb 急性感染小鼠肺、脾脏荷菌量（如 SHP／TMD 组肺脏荷菌量相对 TMD 组减少了1．03 log， F ＝1518，P ＜0．05），同时肺部病理病变减轻。结论以中国 M．tb 感染者 T 细胞识别的多阶段优势表达抗原构建亚单位疫苗 SHP／TMD，免疫小鼠可提供显著的抗 M．tb 急性感染的保护性，主要与 SHP 抗原特异性CD ＋4 T h1型应答有关。

Objective To construct a fusion protein SHP of subunit vaccine containing multi -stage anti-gens,and to confirm the immunogenicity and protective effect of SHP through M.tb infected Chinese population and C57BL/6 mice model.Methods Rv0577 （secreted by replicating bacilli）,Rv2623 （expressed in dormant bacilli） and Rv3478（from PE /PPE family）were selected and to construct fusion protein SHP,WBIA（based on TB -IGRA） was performed to detect the specific concentration of IFN -γstimulated from peripheral -blood lymphocyte of Chinese M.tb infections and non -infections.C57BL/6 mice were immunized（s.c.）with SHP emulsified in the adjuvant TMD.Nine weeks after immunized,SHP -specific antibody titers（IgG,IgG1,IgG2a）,as well as the levels of Th1 cytokines by splenic lymphocytes stimulated with SHP and the mRNA expression levels of molecules in the lung of vaccinated mice were detected.Four weeks after challenge by M.tb H37Rv,the protective efficacy of SHP/TMD was＆amp;nbsp;evaluated.Results Recombinant plasmid pET30b -SHP and subgroups were constructed and expressed successfully, which stimulated statistically higher levels of IFN -γin M.tb infected Chinese subjects than in healthy donors[SHP stimulated IFN -γlevels in infected and healthy groups were （1 420.0 ±253.9）pg/mL and （236.1 ±95.1）pg/mL, t =2.063,P 〈0.01].SHP/TMD induced higher levels of specific IgG,IgG2a and IgG1 antibodies,and the ratio of IgG2a/IgG1 indicated that SHP/TMD mainly induced the Th1 -type cell -mediated immune response.Meanwhile, SHP/TMD induced statistically higher levels of specific IFN -γ,TNF -αand IL -2 from splenic lymphocytes,also the mRNA expression levels of IFN -γ,TNF -αand iNOS from the lung.Moreover,SHP/TMD group resulted in the significant reduction of bacterial load in the lung and spleen organ（the reduction of bacterial load of SHP/TMD in comparison of TMD group was 1.03 log,F =1518,P 〈0.05）,as well as lessened inflammation in alveolar tissue. Conclusion Based on the predominant antigens expressed during multi stages which can be recognized by T cells of M.tb infections from China,a subunit vaccine SHP/TMD was constructed.SHP/TMD can provide significant protective efficacy against acute infection of M.tb,which is mainly attributed to the SHP -specific CD ＋4 Th1 -type cell immune response.