通过双调控体系条件性敲除红内期必需基因构建疟原虫减毒双期活疫苗的研究

WHO proposed malaria vaccine routine in 2006, however the current vaccine strategs have not achieved the objective.The previous studies have indicated that attenuated vaccines were the most potential design. We propose to design attenuated vaccine by combinating Tet-on and DD conditional technology to knockout erythrocytic essential gene. There are thred advatages over the current attenuated vaccines. First, this vaccine not only induce immune protection for the liver stage of plasmodium, but also for the erythrocytic stage by very low dose parasitemia.Second, plasmodium infection can not cause clinical symptoms since there are not the inducers in the body, thus the safety is gauranted.Third, plasmoidum can proliferation in vitro when the inducers are added to culture medium, and the knocked gene do not affect mosquito and liver stage development, thus the sporozoites could produce for immunization.We will use CRISP/Cas9 gene editing technology in the plasmodium to realize the proposal. And we expect that the protection potential are superior to the existing stratedgy.

现有进入临床试验的疟疾疫苗未达到WHO提出的保护目标，初期研究显示减毒活疫苗最具潜力。但现有的减毒活疫苗要么分别诱导针对疟原虫肝期或红内期抗原的免疫保护，要么现场推广的可行性差。我们提出利用双调控体系Tet-on/DD条件性敲除疟原虫红内期必需基因制备减毒、双期、全虫活疫苗的设想。该策略综合了现有的减毒疫苗的优点：（1）诱导双期免疫：既能诱导针对肝期的免疫保护，也能通过极低剂量的红内期疟原虫感染诱导针对红内期的免疫保护；（2）安全性有保证：体内没有诱导剂，疟原虫感染无法导致临床症状；（3）可进行生产：培养环境中加入诱导剂，可进行Pf体外培养，敲除基因不影响蚊期和肝期发育，可以获得用于接种免疫的子孢子。疟原虫基因信息的揭示和基因编辑技术CRISP/Cas9在疟原虫中的应用为实现该设想提供了技术保证。预期这种疫苗策略的保护力优于现有的疫苗，为解决疟疾疫苗设计的难题提供一种研发新思路。

疟疾是一种危险的全球性传染病，由于疟原虫的生活史复杂，抗原的成分多变，疫苗研究的实验模型不完善等原因，疫苗研发的进展不大，不能满足疟疾保护的需求。现有的减毒活疫苗主要是通过辐照以及药物减毒，限制较大，保护力较低。我们通过利用DDD调控系统调控疟原虫必须基因EF1γ，实现了在添加调控药物TMP时疟原虫表达必须基因，从而高效的激活机体针对疟疾的免疫，不添加调控药物后必须基因不表达，疟原虫全部死亡，避免存活的疟原虫引起疟疾传播，由此在动物模型中开发了疟原虫减毒疫苗，并在小鼠中成功验证其保护力，在小鼠中有100%保护效果。在验证疟原虫必须基因的过程中，开发了在伯氏疟原虫中连续基因编辑的技术和在恶性疟原虫中的自杀-拯救基因编辑技术，相关成果已发表SCI论文3篇，申请专利1项。为了在不同的疟原虫中得到更低的调控背景，获得更好的减毒效果，后续仍在开展Tet-On/DDD联合调控系统的工作。