新信号分子c-di-AMP为佐剂的rBCG疫苗的构建及其免疫学特性研究

Bacillus Calmette-Guérin (BCG) is the solely vaccine available against Tuberculosis (TB). However, the protective efficacy of BCG against highly contagious pulmonary infection is controversial. In addition, as an attenuated vaccine, BCG is still not safe enough to be administered to immunosuppressed population. Although a number of TB vaccine candidates are now in clinical trials, novel vaccine candidates are still urgently needed. Cyclic diadenosine monophosphate (c-di-AMP) is a newly recognized signaling molecules following cyclic diguanosine monophosphate (c-di-GMP), which have been reported to affect the pathogenicity of a number of bacteria. Several studies have now convincingly demonstrated that c-di-AMP has strong immunostimulatory function, especially in promoting mucosal immunity. Enhanced antigen-specific systemic humoral and Th1 type cellular immune responses were induced after co-administration with c-di-AMP as compared to immunization of antigen alone. Vaccination of mice with c-di-AMP as an adjuvant also reduced the bacterial burdens in the spleens and livers organs post challenge with some bacteria. The genes associated with c-di-AMP in Mycobacterium tuberculosis (Mtb) have been reported. We have recently characterized the diadenylate cyclase (Dac) and a phosphodiesterase (CnpB) of c-di-AMP in Mtb. Then we found that CnpB mutant BCG can induce stronger INF-γ response in mice. In this proposal, we will take advantage of our understanding of these nucleotides to construct recombinant BCG (rBCG) strains with deletion of phosphodiesterase and overexpress diadenylate, further combine with immundominant antigen Ag85B and ESAT-6 fusion protein to enhance the immunogenicity of BCG. The immunological characteristics of all the rBCG mutants will be determined after vaccination of mice. The efficacy of vaccine will be evaluated with a mouse model challenged with Mtb. Our goal is to construct a more effective and/or safer vaccine candidate against TB.

卡介苗（BCG）作为目前预防结核病（TB）的唯一疫苗，尚存在保护效率不完善、不能用于免疫低下人群接种等不足。目前尚未获得免疫保护力超过BCG并可推广应用的新型TB疫苗，表明仍需探索新的疫苗策略。c-di-AMP是新近发现的细菌信号分子，参与细菌致病力在内的多种生理过程，与抗原联合免疫可诱导更强的免疫应答，具有强的免疫佐剂作用。申请人前期首次报告了结核分枝杆菌的c-di-AMP合成酶（DacA），并鉴定出一种分解酶（CnpB）；发现CnpB突变的rBCG生长迟缓，更可诱导小鼠更强的免疫应答。本项目拟通过敲除CnpB和过表达DAC酶构建c-di-AMP水平升高的rBCG，联合免疫优势抗原Ag85B和ESAT-6的表达,通过动物实验明确其诱导的免疫应答水平，评价其作为新型抗TB疫苗的潜力。本研究有可能获得免疫原性更强、更安全的新型TB疫苗，并为c-di-AMP用于其他细菌性疾病研究提供依据。

卡介苗（BCG）是目前预防结核病（TB）的惟一疫苗，但存在保护效率不完善、不能用于免疫低下人群接种等不足。虽然有很多新疫苗在研究中，但目前尚未获得免疫保护力超过BCG并可推广应用的新型TB疫苗，表明仍需探索新的疫苗策略。c-di-AMP是新近发现的细菌信号分子，参与细菌致病力在内的多种生理过程，与抗原联合免疫可诱导更强的免疫应答，具有强的免疫佐剂作用。本课题通过同源重组技术构建了c-di-AMP合成酶和分解酶缺陷的重组耻垢分枝杆菌rMS，获得c-di-AMP内源性水平显著升高的rMS；以内源性c-di-AMP水平升高的rMS为载体，过表达相应的Mtb抗原；进一步以该rMS免疫小鼠，明确了c-di-AMP为内源性佐剂的rMS的免疫学特性。研究表明，内源性c-di-AMP升高的rMS菌株感染小鼠和人巨噬细胞，可诱导IL-1β、IFN-γ升高为特征的固有免疫应答，其机制可能与自噬和炎症小体的激活有关；以内源性c-di-AMP升高的rMS菌株免疫小鼠，可诱导特异性体液免疫应答和细胞免疫应答；以内源性c-di-AMP升高的rMS免疫的小鼠感染Mtb后，诱导的Th1免疫应答明显升高，而Th2免疫应答并不提高，并对Mtb感染具有一定的免疫保护力。因此，本研究获得的rMS有可能用于TB新型疫苗或免疫策略的研制。