Lau Alice, Singh Vijender, Soualhine Hafid, Hmama Zakaria
Division of Infectious Diseases, Department of Medicine and Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, BC V6H 3Z6, Canada.
Division of Infectious Diseases, Department of Medicine and Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, BC V6H 3Z6, Canada.
Vaccine. 2017 Apr 11;35(16):2060-2068. doi: 10.1016/j.vaccine.2017.02.065. Epub 2017 Mar 17.
BCG vaccine, introduced almost 100years ago, is the only option to prevent TB disease. It effectively protects newborns from meningeal TB but fails to prevent adult pulmonary TB. TB kills 1.3million people annually in areas where BCG vaccination is widely practiced. Thus, more effective TB vaccines are urgently needed. Others and we have shown that BCG mimics features of virulent M. tuberculosis, in particular attenuation of essential macrophage functions such as phagosome maturation and antigen presentation. One of these studies revealed that defect in antigen presentation is largely due to down-regulation of the cysteine protease Cathepsin S (CatS), which prevents MHC II molecule maturation and proper antigen peptide loading. Recent studies also suggested a potential role for cysteine proteases in the regulation of apoptosis, a key cellular process used by the macrophage to (i) contain and process ingested bacteria and (ii) facilitate cross-talk antigen presentation between the macrophage and dendritic cells.
To reverse the phenotype of vaccine-mediated macrophage attenuation, we engineered a novel BCG strain that expresses and secretes active CatS (rBCG-CatS) to examine its pro-apoptotic properties in vitro, and subsequently, immunogenicity in mice.
Transcriptomic profiling of macrophages infected with rBCG-CatS, but not BCG, revealed upregulation of key pro-apoptotic genes and downregulation of anti-apoptotic genes, which were further confirmed by RT-qPCR analyses of expression of selected genes. Macrophages infected with rBCG-CatS undergo apoptosis as indicated by increased levels of annexin V staining and intracellular caspase-3 cleavage. Consistent with these findings, mice vaccinated with rBCG-CatS showed increased antigen-specific CD4 T-cell responses, as well as enhanced cytokine production and proliferation in CD4 upon ex vivo re-stimulation.
Collectively, this study shows that a pro-apoptotic BCG strain alleviates adverse traits of the wild-type strain, resulting in a highly immunogenic TB vaccine.
卡介苗于近100年前问世，是预防结核病的唯一选择。它能有效保护新生儿免受结核性脑膜炎侵害，但无法预防成人肺结核。在广泛接种卡介苗的地区，结核病每年导致130万人死亡。因此，迫切需要更有效的结核病疫苗。我们和其他人已经表明，卡介苗模拟了毒力结核分枝杆菌的特征，特别是对诸如吞噬体成熟和抗原呈递等关键巨噬细胞功能的减弱。其中一项研究表明，抗原呈递缺陷主要是由于半胱氨酸蛋白酶组织蛋白酶S（CatS）的下调，这阻止了MHC II分子的成熟和适当的抗原肽加载。最近的研究还表明，半胱氨酸蛋白酶在细胞凋亡调节中可能发挥作用，细胞凋亡是巨噬细胞用于（i）容纳和处理摄入的细菌以及（ii）促进巨噬细胞与树突状细胞之间的串扰抗原呈递的关键细胞过程。
为了逆转疫苗介导的巨噬细胞减弱的表型，我们构建了一种新型卡介苗菌株，该菌株表达并分泌活性CatS（rBCG-CatS），以在体外检查其促凋亡特性，随后在小鼠中检查其免疫原性。
感染rBCG-CatS而非卡介苗的巨噬细胞的转录组分析显示，关键促凋亡基因上调，抗凋亡基因下调，这通过对选定基因表达的RT-qPCR分析得到进一步证实。感染rBCG-CatS的巨噬细胞发生凋亡，这通过膜联蛋白V染色水平增加和细胞内半胱天冬酶-3裂解来表明。与这些发现一致，接种rBCG-CatS的小鼠显示出抗原特异性CD4 T细胞反应增加，以及在体外重新刺激后CD4中细胞因子产生和增殖增强。
总体而言，这项研究表明，一种促凋亡的卡介苗菌株减轻了野生型菌株的不良特征，从而产生了一种高度免疫原性的结核病疫苗。
