Liu Wei, Xu Ying, Shen Hongbo, Yan Jingran, Yang Enzhuo, Wang Honghai
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, P.R. China.
Exp Ther Med. 2017 May;13(5):2339-2347. doi: 10.3892/etm.2017.4273. Epub 2017 Mar 28.
The only available vaccine against pulmonary tuberculosis is (BCG). As the efficacy reported of the vaccine is not up to the mark, there is an urgent need to develop improved anti-tuberculosis vaccines. Antigen 85B (Ag85B) is a very promising vaccine candidate molecule of and interferon (IFN)-γ and has been considered the most attractive correlate of protective immunity. The aim of this study was to construct a novel recombinant BCG (rBCG) to secrete Ag85B and mouse IFN-γ under control of the Mycobacterial heat shock protein 60 () promoter and the antigen signal sequence. Second aim of the present study is to evaluate the immune response in C57BL/6 elicted by the new rBCG. Expression of the fusion protein was readily detectable by western blotting and IFN-γ bioactivity was detected indirectly by enzyme-linked immunosorbent assay (ELISA). Compared with BCG, rBCG::Ag85B-IFN-γ was substantially more active in inducing the production of IFN-γ and tumor necrosis factor (TNF)-α from mouse splenocytes. ELISA analysis for IgG, IgG1 and IgG2c showed that rBCG::Ag85B-IFN-γ induced higher titer of Ag85B and facilitated Th1 type immune response. rBCG::Ag85B-IFN-γ also improved nitric oxide production levels and enhanced antigen-specific splenocyte proliferation. Moreover, rBCG::Ag85B-IFN-γ induced human monocytes such as THP-1 cells to enhance expression of CD80, CD86, CD40 and HLA-DR. Flow cytometry analysis confirmed that rBCG::Ag85B-IFN-γ significantly activated CD4 T cells. Assessing combinations of IFN-γ, TNF-α and interleukin-2 at the single-cell level by multiparameter flow cytometry, we found that rBCG::Ag85B-IFN-γ improved the multifunctional T cells level in comparison to BCG. In conclusion, the present study indicates that rBCG::Ag85B-IFN-γ increases cell mediated immune response and is a potential candidate vaccine for immunotherapeutic protocols against pulmonary tuberculosis.
唯一可用于预防肺结核的疫苗是卡介苗（BCG）。由于报道的该疫苗效力未达标准，因此迫切需要研发改良的抗结核疫苗。抗原85B（Ag85B）是一种非常有前景的结核疫苗候选分子，与干扰素（IFN）-γ相关，并且被认为是保护性免疫最具吸引力的关联因素。本研究的目的是构建一种新型重组卡介苗（rBCG），使其在分枝杆菌热休克蛋白60（hsp60）启动子和抗原信号序列的控制下分泌Ag85B和小鼠IFN-γ。本研究的第二个目的是评估新型rBCG在C57BL/6小鼠中引发的免疫反应。通过蛋白质免疫印迹法很容易检测到融合蛋白的表达，并且通过酶联免疫吸附测定（ELISA）间接检测IFN-γ的生物活性。与卡介苗相比，rBCG::Ag85B-IFN-γ在诱导小鼠脾细胞产生IFN-γ和肿瘤坏死因子（TNF）-α方面活性显著更高。针对IgG、IgG1和IgG2c的ELISA分析表明，rBCG::Ag85B-IFN-γ诱导产生更高滴度的Ag85B，并促进Th1型免疫反应。rBCG::Ag85B-IFN-γ还提高了一氧化氮的产生水平，并增强了抗原特异性脾细胞增殖。此外，rBCG::Ag85B-IFN-γ诱导人单核细胞如THP-1细胞增强CD80、CD86、CD40和HLA-DR的表达。流式细胞术分析证实，rBCG::Ag85B-IFN-γ显著激活CD4 T细胞。通过多参数流式细胞术在单细胞水平评估IFN-γ、TNF-α和白细胞介素-2的组合，我们发现与卡介苗相比，rBCG::Ag85B-IFN-γ提高了多功能T细胞水平。总之，本研究表明rBCG::Ag85B-IFN-γ增强了细胞介导的免疫反应，是用于肺结核免疫治疗方案的潜在候选疫苗。
