口服Ag85A DNA疫苗肠黏膜细胞内传感器及传导途径与适应性免疫应答诱导

DNA vaccines have not been applied clinically for prophylaxis and treatment, mainly due to the weak immunogenicity. So far there are very limited studies on molecular mechanisms of DNA vaccine and moreover, the immune mechanisms through which, oral DNA vaccine would be processed and presented to initiate an immune response in intestine mucous system remain unclear. The aim of this research is: via oral delivery of artificial Ag85A DNA vaccine, followed by, from different aspects of antigen presentation, to investigate sensor molecules, which will recognize dsDNA vaccine inside the APC or non APC in intestine mucous system and the way of transduction, as well as induced cellular and humeral immune responses in this way, so as to understand in depth, key factors which will affect immunogenicity of Ag85 DNA vaccine. This investigation will provide a meaningful mode of action and data for design of DNA vaccine with strong immunogenicity.

DNA疫苗至今未能用于人类疾病的预防和治疗，其主要原因是免疫原性低。有关DNA疫苗在体内作用的细胞和分子机制知道较少，经口接种DNA疫苗在肠道黏膜局部如何进行抗原处理提呈和诱导产生适应性免疫应答的机制更不清楚。为此，本项目拟通过口服自行构建的Ag85A DNA疫苗，从与传统蛋白质抗原提呈方式不同的角度，对肠道黏膜局部APC和非APC细胞内识别双股DNA（dsDNA）疫苗的传感器分子及其信号传导途径，经此方式诱导细胞和体液免疫应答的机制进行研究，进一步深入了解影响口服 Ag85A DNA疫苗免疫原性的关键因素，为提高口服DNA疫苗的免疫原性及疫苗设计提供实验和理论依据。

本项研究是通过口服接种Ag85A DNA疫苗，对肠道黏膜局部抗原提呈细胞识别双股DNA（dsDNA）疫苗的传感器分子及其信号传导途径，探讨新的抗原提呈机制和深入了解影响口服 Ag85A DNA免疫原性的关键因素。自行设计构建了具有转录mRNA活性，无转译Ag85A蛋白分子活性的突变型Ag85A 基因DNA质粒疫苗。将其与无突变（野生型）Ag85A 基因DNA质粒疫苗同样转染至抗原提呈细胞CD2.4细胞系，并采用CRISPR/Cas9技术将突变型和野生型Ag85A基因分别敲进小鼠。经基因瞬转和稳转CD2.4细胞系检测结果显示，DNA传感器DAI、IFI16、DDX41、TLR9和 MyD88分子在突变型和野生型Ag85A基因质粒转染组与正常细胞对照组未见明显差异；细胞因子IL-1、IFN-、IL-6和IL12-p40分子表达水平也未见明显差异；RNA传感器Mda5和RIG-I在突变型和野生型Ag85A基因质粒转染组与正常细胞对照组可见明显差异；Adaptors IFNβ、IRF3、IRF7和MAVS也见明显差异。结果提示，双股DNA疫苗可能通过RNA传感器分子及其传导通路，将免疫原信息从抗原提呈细胞提呈给Th细胞。而不是通过DNA传感器及其传导途径。目前正应用突变型和野生型Ag85A基因敲进小鼠做进一步体内研究。可为研发RNA传感器的刺激剂，提高核酸疫苗的免疫原性提供科学依据。