以疟原虫遗传减毒子孢子为载体的MAGE3疫苗的抗肺癌作用及免疫机制的研究

Although MAGE3-engineered DC-base and MAGE3-expressing nonreplicating adenoviral vector could induce MAGE3-specific Th1 response, their inhibitory effect on the growth of lung carcinoma need to be largely improved. As a tolerogenic organ, lung is supposed to potentially suppress the immune response induced by vaccines, which might be a main cause resulted in the failure of the current vaccines against lung carcinoma. Genetically attenuated sporozoites, which could successfully induce robust CSP-specific CD4+ and CD8+ T responses, was thought to be able to overcome the tolerogenic mechanisms of lung. Our preliminary data has shown that MAGE3-expressing genetically attenuated sporozoites could effectively resist to the challenge of LLC, but the underlined menchanisms are still unknown. In this project, we would like to investigate whether the MAGE3-expressing genetically attenuated sporozoite could also inhibit the proliferation of the pre-inoculated LLC and the CD4+/CD8+T response was responsible for this effect. As our previous study showed that TLR2 was involved in the recognition of the attenuated sporozoite lysate and essential for the protective immunity induced by the attenuated sporozoite, we would also like to investigate whether TLR2 is also critical for the anti-tumor protective immunity induced by the MAGE3-expressing genetically attenuated sporozoite acting through DC and Treg. This study would help us to understand the mechanism of anti-tumor protective immunity induced by the MAGE3-expressing genetically attenuated sporozoite, and provide us with novel clues to design effective subunited vaccines against lung carcinoma.

；选择能诱导强烈CD4+/CD8+T反应的载体是设计肿瘤疫苗的关键。现有疫苗大多采用病毒为载体，但效果并不是很理想。遗传减毒子孢子是一种定向敲除疟原虫肝期发育相关基因的子孢子。减毒子孢子能克服机体的免疫耐受，诱导强烈的疟原虫特异性CD4/CD8+T细胞反应。能否选择遗传减毒子孢子作为构建肺癌疫苗的理想载体诱导强烈的肺癌特异性CD4/CD8+T细胞免疫应答？课题组预实验证实，构建的表达MAGE3的遗传减毒子孢子疫苗确实能有效抵御小鼠肺癌株LLC的攻击。鉴此，本项目拟首先在前期研究基础上，一方面观察该疫苗能否诱导MAGE3特异的抗体和CD4/CD8+T 细胞反应，以抑制肺癌细胞的增殖；另一方面利用B、CD4+和CD8+T细胞敲除小鼠探讨其抗肿瘤的效应机制。同时根据TLR2识别减毒子孢子的前期发现探讨其抗肺癌过程中TLR2信号的作用以及对DC和Treg活化的影响，明确遗传减毒子孢子的载体效应机制

肿瘤疫苗不但能预防肿瘤的发生，而且还可以用于肿瘤的治疗，是重要的肿瘤免疫治疗策略之一。大量的研究证实，CD4/CD8+T细胞免疫应答是机体的免疫系统监视和抑制肿瘤的最主要效应机制，因此是设计有效肿瘤疫苗的重要依据。然而目前并无较理想的肿瘤疫苗。究其原因，可能与机体的免疫耐受抑制肿瘤疫苗诱导强烈的CD4/CD8+T细胞免疫应答有关。因此，寻找能够有效诱导机体CD4/CD8+T应答的肿瘤疫苗载体是目前研究的焦点。. 鉴于疟原虫遗传减毒子孢子（GAS）可以克服机体免疫耐受，并高效诱导疟原虫特异性CD8+/CD4+T 细胞反应，本项目以遗传减毒子孢子作为载体，构建表达主要肺癌相关抗原MAGE-A3的重组遗传减毒子孢子疫苗，观察能否活化MAGE-A3特异性CD4/CD8+T细胞，以达到抗肺癌的作用，并进一步探讨相关机制，这可为设计有效的肺癌等肿瘤疫苗提供新思路。. 为了探讨表达肺癌相关抗原MAGE-A3的重组遗传减毒子孢子是否具有抗肺癌的作用，本研究拟利用CRIPSR-cas9技术对伯氏疟原虫ANKA株（P.b ANKA）子孢子UIS3基因序列替换成肺癌相关抗原MAGE-A3，从而获得能够成功入侵肝细胞但不能在体内继续增殖发育，而且能够表达MAGE-A3的重组遗传减毒子孢子（GAS/MAGE-A3）。. 为了探讨表达肺癌相关抗原MAGE-A3的重组遗传减毒子孢子能否诱导小鼠CD4+/CD8+T细胞应答，以达到抗肺癌作用。本实验用PBS、GAS、GAS/MAGE-A3分别多次尾静脉注射免疫HLA-A2小鼠，末次免疫后7天分别检测各组小鼠外周血及脾脏CD4+/CD8+T细胞反应，然后采用Elispot检测分泌IFN-γ的MAGE-A3特异性CD8+T 细胞。然后将分离收集的免疫小鼠脾脏的CD8+T细胞过继到皮下接种A549的裸鼠体内，观察其抗肺癌效果。