Conditional male lethal Anopheles stephensi line for the efficient manufacture of malaria vaccines

项目摘要 2020年，疟疾病例（2.41亿）和死亡人数（62.7万）达到2012年以来的最高水平，强调迫切需要 需要新的工具来预防、控制和消除这种疾病。萨纳里亚氏恶性疟原虫 (Pf)子孢子（SPZ）疫苗，由通过静脉注射施用的放射减毒PfSPZ组成， 在美国、欧洲和非洲6个国家的1740例年龄为5个月至61岁的受试者中进行了评估， 在非洲，PfSPZ-CVac（化学减毒）可提供100%的保护， 异源控制人疟疾感染至少12周。PfSPZ采用无菌方法生产 饲养雌性斯氏按蚊。只有雌性才能吸血，所以在目前的生产中 在这个过程中，无菌雄性蚊子是多余的，消耗生长培养基，占据空间， 否则用于生产更多的女性在没有额外的成本或努力。在这个项目中，我们计划 无菌蚊子饲养过程效率提高2倍（疫苗生产成本降低10-15%） 通过在胚胎阶段将雄蚊从系统中移除。这将通过创建一个 一种蚊子品系，其中雄性蚊子有条件地表达一种致命的昆虫特异性杂交毒素，最初 由蜘蛛产生。使用四环素控制的基因， 表达系统在我们的具体目标，我们将：1.建立驱动系：转基因A.斯蒂芬西线 携带Tet-On反式激活因子（rtTA）。将通过将rtTA插入到转基因株系的下游来产生转基因株系。 严格控制vasa启动子，其在产卵后的最初几个小时内表达。该构建体将 使用基于piggyBac的种系修饰插入。在不存在多西环素的情况下，rtTA不应结合至 四环素反应元件（TRE）和因此致死基因将是无活性的，当表达不被激活时， 想要的2.建立一个Y型连接的对接A。斯蒂芬西线我们已经确定了特定的Y染色体 可用于靶向X（lox）对接位点的基因座的整合的基因组基因座。3xP 3下的RFP 使用CRISPR-Cas9将侧翼为两个lox位点的启动子引入鉴定的Y序列 介导的同源性修复。3.建立效应线：A。带Y染色体蜘蛛 四环素反应元件（TRE）控制下的杂交毒素。Y形连接的对接线将 用于通过lox位点整合含有TRE、最小启动子和杂合毒素的基因盒， cre/lox重组酶介导的盒交换。在这个转基因品系中，致命的杂交毒素将被 当多西环素和rtTA都可用时，仅在雄性蚊子中表达;在缺乏 多西环素和rtTA预期无致死性。4.生成转基因条件性雄性致死性别鉴定 菌株A.史蒂芬西来自驱动器线的雌性将与来自效应器线的雄性杂交。在 在雄性卵祖细胞中，rtTA将在多西环素的存在下结合TRE，驱动TRE的表达。 毒素并诱导雄性死亡，而在没有强力霉素的情况下，两性都将存活。

Project Abstract In 2020 malaria cases (241M) and deaths (627,000) reached the highest since 2012 emphasizing the urgent need for new tools for prevention, control, and elimination of this disease. Sanaria’s Plasmodium falciparum (Pf) sporozoite (SPZ) Vaccine, composed of radiation attenuated PfSPZ administered by intravenous injection, assessed in 1740 subjects aged 5 months to 61 years in 6 countries in US, Europe and Africa, is safe and protective for 18 months in Africa, while PfSPZ-CVac (chemo-attenuated) conferred 100% protection against heterologous controlled human malaria infection for at least 12 weeks. PfSPZ are produced using aseptically reared female Anopheles stephensi mosquitoes. Only females ingest blood, so in the current manufacturing process, aseptic male mosquitoes are superfluous, consuming growth medium and occupy space that could otherwise be used for production of more females at no additional cost or effort. In this project we plan to make the aseptic mosquito rearing process 2-fold more efficient (reducing costs of vaccine manufacture by 10-15%) by removing male mosquitoes from the system at the embryonic stage. This will be achieved by creating a mosquito line in which males are conditionally expressing a lethal insect-specific hybrid toxin, originally produced by spiders. The lethality will be induced on a switch, using the Tetracycline-controlled gene expression system. In our specific aims we will: 1. Establish a driver line: a transgenic A. stephensi line carrying the Tet-On transactivator (rtTA). A transgenic line will be created by inserting the rtTA, under the tight control of the vasa promoter, which is expressed in the first few hours after egg laying. The construct will be inserted using piggyBac-based germline modification. In the absence of doxycycline, rtTA should not bind to the Tetracycline responsive element (TRE) and thus the lethal gene will be inactive when expression is not wanted. 2. Establish a Y-linked docking A. stephensi line. We have identified specific Y-chromosome genomic loci which can be used to target integration of the locus of X (lox) docking sites. RFP under the 3xP3 promoter, flanked by two lox sites will be introduced to the identified Y sequences using CRISPR-Cas9 mediated homology-derived repair. 3. Establish an effector line: A. stephensi carrying Y-linked spider hybrid toxin under the control of the Tetracycline Response Element (TRE). The Y-linked docking line will be used to integrate a gene cassette containing TRE, minimal promoter and the hybrid toxin via lox sites using the cre/lox recombinase-mediated cassette exchange. In this transgenic line the lethal hybrid toxin will be expressed only in male mosquitoes when both doxycycline and rtTA are available; in the absence of doxycycline and rtTA no lethality is anticipated. 4. Generate a transgenic conditional male-lethal sexing strain of A. stephensi. Females from the driver line will be crossed with males from the effector line. In the progenitor male eggs, the rtTA will bind to TRE in the presence of doxycycline, driving the expression of the toxin and induce male lethality, while in the absence of doxycycline both sexes will survive.