Immune Deficient Mosquitoes for Improved Malaria Sporozoite Vaccine Manufacture

摘要 一种消除恶性疟原虫（Pf）的理想工具，恶性疟原虫是99%疟疾死亡的病原体， 将是一种非常有效的疫苗，可以预防血液阶段感染，从而预防疾病和 传输Sanaria已经开发了两种有效的疫苗，正在进行2期临床试验; Pf子孢子（SPZ） 在>80%的接受者中预防Pf血液期感染的疫苗和PfSPZ攻毒（传染性PfSPZ） PfSPZ-CVac中使用，这是一种在存在疫苗的情况下使用PfSPZ攻毒的疫苗接种方法。 抗疟疾药物，如氯喹，可100%预防志愿者的血液期感染。两 这些疫苗正处于积极的商业化时间轴上。这些产品中使用的PfSPZ是 从无菌饲养的蚊子中提取的。增加PfSPZ/蚊子的数量将降低成本 的物品.本项目将培育出免疫缺陷型斯氏按蚊品系， 易受PfSPZ感染。Sanaria和马里兰州大学生物科学与生物技术研究所 研究使用转基因技术引入和表达基因，导致基因表达下调。 A. stephensi，包括Caspar（Rel 2的负调节子）、Rel 2（转录 因子）和LRIM 1（效应子）。使用转基因RNA干扰降低LRIM 1表达， 在非无菌A中，Pf卵囊和PfSPZ的Pf感染增加（3.6倍增加）。史蒂芬西这些结果 是令人鼓舞的，因为转基因RNA干扰的结果在亚型表型。等位基因是 预计具有更强的表型，感染更严重。我们将使用CRISPR/Cas9 在A.通过诱变LRIM 1和三种另外免疫 基因（TEP 1，LL 3和JNK）参与限制按蚊Pf感染。这些 免疫缺陷型A. stephensi生产线，将提供一个稳定和更有效的PfSPZ生产平台 生产过程中的一个重要环节我们将：1）为LRIM 1、TEP 1、LL 3和JNK生成空蚊线 使用CRISPR/Cas9驱动的基因特异性诱变; 2）测试突变系并选择至少一个系 与野生型相比，产生≥2倍高PfSPZ感染强度，使该品系适应无菌PfSPZ 生产工艺，并检测在该选择的生产线中生产的PfSPZ的效价; 3）检测 A.当感染其他Pf菌株时，斯氏突变株系; 5） 向FDA提交生物制品主文件，描述菌株及其并入Sanaria的 制造过程

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.