Immune Deficient Mosquitoes for Improved Malaria Sporozoite Vaccine Manufacture

免疫缺陷蚊子用于改进疟疾子孢子疫苗的生产

• 批准号: 9407543
• 负责人: Peter F. Billingsley
• 金额: $ 99.88万
• 依托单位: SANARIA, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-02 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9407543
• 关键词: Alleles; Anopheles Genus; Antimalarials; Attenuated; Biological Sciences; Biotechnology; Blood; C10; CRISPR/Cas technology; Cessation of life; Characteristics; Chloroquine; Culicidae; Data; Down-Regulation; Engineering; Gene Expression; Gene Targeting; Genes; Geographic Locations; Goals; Guide RNA; Hemolymph; Hepatocyte; Immune; Immune signaling; Immune system; Individual; Infection; Innate Immune Response; Institutes; Invaded; MAPK8 gene; Malaria; Maryland; Measures; Midgut; Mutagenesis; Mutate; Mutation; Oocysts; Parasites; Pathway interactions; Pharmaceutical Preparations; Phase; Phase II Clinical Trials; Phenotype; Plasmodium falciparum; Plasmodium falciparum vaccine; Population; Prevalence; Production; RNA Interference; Research; Running; Salivary Glands; Signal Pathway; Small Business Innovation Research Grant; Sporozoite vaccine; Sporozoites; TEP1 gene; Technology; Testing; TimeLine; Transgenic Organisms; Underserved Population; Universities; Vaccination; Vaccines; Whole Organism; base; commercialization; cost; disease transmission; genome editing; improved; manufacturing process; mutant; nuclease; potency testing; prevent; success; tool; transcription factor; volunteer

Abstract An ideal tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, would be a highly effective vaccine that prevents blood stage infection and thereby prevents both disease and transmission. Sanaria has developed two effective vaccines that are phase 2 clinical trials; Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in >80% of recipients and PfSPZ Challenge (infectious PfSPZ) used in PfSPZ-CVac, a vaccination approach utilizing PfSPZ Challenge administered in the presence of an anti-malarial drug such as chloroquine which prevents blood stage infection in 100% of the volunteers. Both these vaccines are on an aggressive timeline to commercialization. PfSPZ used in these products are extracted from aseptically reared mosquitoes. Increasing the number of PfSPZ/mosquito will reduce the cost of goods. This project will develop a line of immune deficient Anopheles stephensi mosquitoes highly susceptible to PfSPZ infections. Sanaria and the Univ. of Maryland Institute for Bioscience and Biotechnology Research used transgenic technologies to introduce and express genes that resulted in the down-regulation of REL2 immune pathway in A. stephensi, including Caspar (negative regulator of Rel2), Rel2 (transcription factor) and LRIM1 (effector). Reduction of LRIM1 expression using transgenic RNA interference resulted in increased Pf infections of Pf oocysts and PfSPZ (3.6-fold increase) in non-aseptic A. stephensi. These results are encouraging because transgenic RNA interference results in hypomorphic phenotypes. Null alleles are expected to have stronger phenotypes with even more intense infections. We will use CRISPR/Cas9 technologies to create in A. stephensi null alleles through mutagenesis of LRIM1 and three additional immune genes (TEP1, LL3 and JNK) that are implicated in limiting Pf infections in Anopheles mosquitoes. These immune deficient A. stephensi lines, will provide a stable and much more efficient PfSPZ-production platform for Sanaria's manufacturing process. We will: 1) Generate mosquito lines null for LRIM1, TEP1, LL3 and JNK using CRISPR/Cas9 driven gene specific mutagenesis; 2) Test the mutant lines and select at least one line that produce ≥2- fold higher PfSPZ infection intensities compared to wild type, adapt this line to aseptic PfSPZ manufacturing process and test the potency of PfSPZ manufactured in this selected line; 3) test the prevalence and intensity of Pf infection in A. stephensi mutant lines when infected with other strains of Pf; 5) Submit a Biologics Master File to the FDA describing the strain and its incorporation into Sanaria's manufacturing process.

抽象的 消除恶性疟原虫（PF）的理想工具，该疟疾是所有疟疾死亡的99％的病因， 将是一种高效的疫苗，可防止血液阶段感染，从而防止疾病和 传播。 Sanaria开发了两种有效的疫苗，分别是第二阶段临床试验。 PF Sporozoite（SPZ） 疫苗可防止> 80％的受体和PFSPZ挑战中PF血液阶段感染（感染性PFSPZ） 用于PFSPZ-CVAC，一种利用PFSPZ挑战的疫苗接种方法 抗疟疾药物，例如氯喹，可防止100％志愿者的血液阶段感染。两个都 这些疫苗是商业化的积极时间表。这些产品中使用的PFSPZ是 从美学饲养的蚊子中提取。增加PFSPZ/蚊子的数量将降低成本 货物。该项目将高度发展一系列免疫缺陷的肛门蚊子蚊子 容易受到PFSPZ感染的影响。 Sanaria和Univ。马里兰州生物科学与生物技术学院的 研究使用转基因技术引入和表达基因，导致下调 A. stephensi中的RER2免疫条道，包括Caspar（REL2的负调节剂），REL2（转录） 因子）和LRIM1（效应子）。使用转基因RNA干扰降低LRIM1表达导致 在非无动物A. stephensi中，PF卵囊和PFSPZ的PF感染增加了。这些结果 令人鼓舞的是，转基因RNA干扰会导致肌型表型。无等位基因是 预计将具有更强的表型，并具有更强烈的感染。我们将使用CRISPR/CAS9 通过LRIM1的诱变和三个其他免疫 在末端蚊子中限制PF感染的基因（TEP1，LL3和JNK）。这些 免疫缺陷的A. Stephensi线将提供稳定且更有效的PFSPZ生产平台 用于Sanaria的制造过程。我们将：1）为LRIM1，TEP1，LL3和JNK生成蚊子无效 使用CRISPR/CAS9驱动基因特异性诱变； 2）测试突变线并至少选择一条线 与野生型相比，产生≥2-倍的PFSPZ感染实例，将此线适应无菌PFSPZ 制造过程并测试该行中生产的PFSPZ的效力； 3）测试 当受到其他PF菌株感染时，A. stephensi突变系中PF感染的患病率和强度； 5） 向FDA提交生物制品大师文件，该文件描述了这种压力及其就业 制造过程。