新型Trop2-CD40L嵌合VLPs疫苗增强老龄小鼠免疫应答及体内抑瘤作用研究

Lung cancer is the first leading cause of malignant cancer and cancer related death, and more than 50% cases happened among aged group (＞65 years old). Immunosenescence, influences the ability of aged subject to react against exogenous antigens and in particular, reduce the capacity of anti-tumoral immune defences in the elderly. To provide better protection for the increased aging population，new and improved vaccines are needed. Oncogene Trop2 is a cell surface glycoprotein, which can induce both humoral and cellular immune response; CD40L is a type II transmembrane protein, which has the adjuvant activity in immune response; virus like particles(VLPs) is a novel vaccine strategy which has multiple advantages, such as high immnunogenecity, mimic the conformation of native virus but lack the viral genome, can induce both humoral and cellular immune responses effectively. In the project, we will develop a Trop2-CD40L chimeric VLPs as novel vaccine targeting aged mice. We will first make the chimeric VLPs using baculovirus expression system, characterize the VLPs by means of Western Blot, EM, and stimulation of DC. After immunization,we will use ELISA, Cytokine ELISA,ELISPOT,FACS to detect the protect efficiency of Trop2-CD40L chimeric VLPs against mouse lewis(Trop2+)lung cancer cell challenge; on the other hand, we will first inoculate the aged mice with lewis(Trop2+) cancer cells and then immunize mice with the vaccine, and then detect the cellular and humoral immune response, the tumor formation and survival rate. From both the preventive and therapeutic efficiency, our project will detect the immune genecity of our novel lung cancer vaccine in aged mice model, and provide laboratory foundation for other age related vaccine development.

肺癌位居我国恶性肿瘤发病和致死率首位，且老龄群体为主要罹患对象，免疫应答低下是共性问题且直接影响治疗性疫苗效果，针对老龄群体的新型疫苗亟待研发。癌基因Trop2表达于胞膜表面，可诱发体液和细胞免疫反应而备受关注；而CD40L 为具有免疫佐剂作用的II类跨膜蛋白；病毒样颗粒（VLPs）因抗原呈现好、免疫原性强、不含病毒核酸成分等优势，成为疫苗研发热点 。本课题拟用杆状病毒表达系统制备Trop2-CD40L嵌合VLPs，WB、电镜、刺激DC检测VLPs组装效率及活性；VLPs分别免疫成年或老龄鼠，ELISA、ELISPOT、FACS 等检测体液和细胞免疫应答；用鼠源lewis(Trop2+)肺癌细胞接种免疫后小鼠，监测肿瘤生长及生存率；同时预先制备荷瘤鼠再行免疫，分析上述指标。探讨嵌合VLPs对老龄荷瘤鼠作用，为研发对老龄群体有效的新型肿瘤疫苗提供依据，并为其它老年性疾病疫苗的研发提供借鉴。

肺癌位居我国恶性肿瘤发病率和致死率首位，且老龄群体为主要罹患对象，免疫应答低下是共性问题且直接影响治疗性疫苗效果，针对老龄群体的新型疫苗亟待研发。癌基因Trop2表达于胞膜表面，可诱发体液和细胞免疫反应而备受关注；而CD40L 为具有免疫佐剂作用的II类跨膜蛋白；病毒样颗粒（VLPs）成功用于宫颈癌疫苗研发并投放市场，对新型肿瘤疫苗研发推波助澜。本课题成功制备Trop2、CD40L的真核表达载体和杆状病毒表达载体，证实Trop2能够成功表达并表达在细胞膜上，进而利用杆状病毒表达系统成功制备Trop2 VLPs和Trop2-CD40L嵌合VLPs，WB、电镜检测VLPs组装效率，结果显示VLPs制备成功，嵌合VLPs直径大小和gag VLPs一致，约为110nm；VLPs分别免疫老龄鼠，ELISA、FACS 等检测小鼠体液和细胞免疫应答，结果显示嵌合VLPs免疫效果较好；用鼠源lewis(Trop2+)肺癌细胞接种免疫后小鼠，监测肿瘤生长及生存率，抑瘤实验结果显示，Trop2-CD40L嵌合 VLPs 注射组小鼠成瘤体积最小，小于TROP2 VLPs组。本研究的实验结果有望为研发对老龄群体有效的新型VLPs疫苗提供依据，并为其它老年性疾病疫苗的研发提供借鉴依据。