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

用于高效生产疟疾疫苗的条件性雄性致死史氏按蚊品系

• 批准号: 10602811
• 负责人: Peter F. Billingsley
• 金额: $ 30万
• 依托单位: SANARIA, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-14 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10602811
• 关键词: Adult; Africa; African; Anopheles Genus; Antimalarials; Attenuated; Attenuated Vaccines; Automobile Driving; Binding; Bioreactors; Blood; CCL20 gene; CRISPR/Cas technology; Cessation of life; Consumption; Country; Cryopreservation; Culicidae; DNA cassette; Disease; Docking; Dose; Doxycycline; Elements; Embryo; Europe; European; Female; Gene Deletion; Gene Expression; Genes; Genomics; Geographic Locations; Grant; Growth; Hour; Human; Hybrids; Immunization; Impairment; Individual; Infection; Ingestion; Insecta; Lethal Genes; Link; Maintenance; Malaria; Malaria Vaccines; Mediating; Modification; Nutrient; Partner in relationship; Physiologic pulse; Plasmodium falciparum; Plasmodium falciparum vaccine; Prevalence; Prevention; Process; Production; Radiation; Regimen; Reproducibility; Response Elements; Site; Small Business Innovation Research Grant; Spiders; Sporozoite vaccine; Sporozoites; Sterility; System; Testing; Tetracycline Control; Tetracyclines; Toxin; Trans-Activators; Transgenic Organisms; Vaccines; Vial device; Whole Organism; Y Chromosome; aged; cost; egg; feeding; genomic locus; in vivo; intravenous administration; intravenous injection; malaria infection; male; manufacture; manufacturing process; pathogenic fungus; progenitor; promoter; prophylactic; recombinase-mediated cassette exchange; red fluorescent protein; repaired; sex; technology platform; tool; vaccine access

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.

项目摘要 2020 年，疟疾病例（2.41 亿）和死亡人数（627,000 例）达到 2012 年以来的最高水平，这凸显了紧迫性 需要新的工具来预防、控制和消除这种疾病。 (Pf)子孢子(SPZ)疫苗，由通过静脉注射施用的辐射减毒PfSPZ组成， 该研究对美国、欧洲和非洲 6 个国家的 1740 名 5 个月至 61 岁的受试者进行了评估，是安全且可靠的 在非洲具有 18 个月的保护作用，而 PfSPZ-CVac（化疗减毒）可提供 100% 的保护 异源受控人类疟疾感染至少 12 周是使用无菌方法生产的。 饲养的雌性史氏按蚊只有雌性吸食血液，因此在目前的制造中。 在此过程中，无菌雄性蚊子是多余的，消耗生长介质并占据可能的空间 否则可用于生产更多雌性，无需额外成本或精力。在这个项目中，我们计划进行。 无菌蚊子饲养过程效率提高 2 倍（疫苗制造成本降低 10-15%） 通过在胚胎阶段将雄性蚊子从系统中去除，这将通过创建一个来实现。 雄性蚊子有条件地表达致命的昆虫特异性混合毒素，最初 蜘蛛产生的致死性将通过四环素控制的基因在开关上诱导。 在我们的具体目标中，我们将： 1. 建立驱动系：转基因 A.stephensi 系。 携带 Tet-On 反式激活因子 (rtTA) 的转基因系将通过在 rtTA 下插入来创建。 vasa启动子的严格控制，该启动子在产卵后的最初几个小时内表达。 使用基于 PiggyBac 的种系修饰插入 在没有多西环素的情况下，rtTA 不应结合。 四环素反应元件（TRE），因此当表达不活跃时致死基因将失活 2.建立Y连锁对接A.stephensi系。我们已鉴定出特定的Y染色体。 基因组基因座，可用于靶向整合 3xP3 下的 X (lox) RFP 基因座。 使用 CRISPR-Cas9 将侧翼有两个 lox 位点的启动子引入到已识别的 Y 序列中 介导的同源修复 3. 建立效应系：携带 Y 联蜘蛛的 A. stephensi。 四环素反应元件 (TRE) 控制下的混合毒素 Y 连接对接线将。 用于通过 lox 位点整合含有 TRE、最小启动子和混合毒素的基因盒 在该转基因系中，cre/lox重组酶介导的盒交换将是致命的杂种毒素。 当多西环素和 rtTA 都存在且缺乏时，仅在雄性蚊子中表达； 强力霉素和 rtTA 预计不会产生致死性。 4. 产生转基因条件性雄性致死性性别鉴定。 来自驱动系的雌性菌株将与来自效应系的雄性杂交。 在雄性祖细胞中，在多西环素存在的情况下，rtTA 将与 TRE 结合，驱动 毒素并导致雄性死亡，而在没有强力霉素的情况下，两性都可以生存。