Transgenic Mosquitoes for Improved Malaria Sporozoite Vaccine Manufacture

描述（由申请人提供）：消灭恶性疟原虫（Pf）的理想工具是一种非常有效的疫苗，它可以防止血液阶段感染，从而防止疾病和传播。恶性疟原虫是导致99%疟疾死亡的病原体。Sanaria的目标是开发并商业化一种Pf孢子子（SPZ）疫苗，该疫苗可在90%的接种者中预防Pf血期感染。Sanaria(R) PfSPZ疫苗由减毒、纯化、无菌、低温保存的PfSPZ组成。为生产疫苗而开发的平台技术还允许生产用于攻击感染的PfSPZ，以测试疫苗和药物（PfSPZ challenge）和用于氯喹保护下的攻击疫苗接种（PfSPZ- cvac）。这些产品组合正处于商业化的积极时间表上。这些产品中使用的PfSPZ是从无菌饲养的蚊子中提取的。增加每只蚊子的SPZ数量直接降低了商品的成本。该项目将培育一种对PfSPZ感染高度敏感的转基因斯氏按蚊。通过沉默蚊子免疫效应基因LRIM1 （LRR基因家族的一员），我们可重复地增加斯氏伊蚊的SPZ负荷。我们还证实了一种表达SRPN6双链RNA （dsRNA）序列的转基因史蒂芬氏杆菌株卵囊负荷增加的表型，该序列在发夹结构中完全整合到基因组中。这些主要结果证明了该二期项目的可行性，该项目将创建减少或消除LRIM1或APL1表达的GM a . stephensi细胞系，为Sanaria的制造过程提供稳定高效的spz生产平台。LRIM1的沉默有两种策略：1)通过表达基因特异性dsRNA直接沉默基因，引发针对LRIM1或APL1转录物的内源性RNA干扰反应；2)通过过表达负调控蛋白Caspar或转录因子Rel2的显性负变异体，破坏相关调控信号通路，间接使基因沉默。我们成功的I期策略是通过驱动基因特异性dsRNA的表达来引发内源性RNA干扰，从而直接沉默基因；这种方法将被改进，以创造转基因黄芪。我们将通过结合GAL4/UAS二元表达系统和“双靶点”基因沉默技术来创建基因沉默转基因。这一策略将使我们能够限制昂贵和耗时的感染表型评估，因为它能够在感染性喂养之前有效地确定基因沉默。这种方法将增强我们生产具有最佳基因型和表型的菌株的能力。我们将对转基因菌株进行筛选，以确定那些与当前生产菌株相比，持续产生2倍以上PfSPZ的菌株。选择的菌株将在标准昆虫条件下进行研究，并在GMP合规生产的无菌条件下进行研究。我们将把最佳的转基因菌株纳入Sanaria的生产过程中。

DESCRIPTION (provided by applicant): 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's goal is to develop and commercialize a Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in > 90% of recipients. The Sanaria(R) PfSPZ Vaccine is composed of attenuated, purified, aseptic, cryopreserved PfSPZ. The platform technology developed to manufacture the vaccine has also allowed the manufacture of PfSPZ for challenge infections to test vaccines and drugs (PfSPZ Challenge) and for vaccination by challenge under chloroquine protection (PfSPZ-CVac). These comprise a portfolio of products that are on an aggressive timeline to commercialization. The PfSPZ used in these products are extracted from aseptically reared mosquitoes. Increasing the number of SPZ per mosquito directly reduces the cost of goods. This project will develop a strain of genetically modified (GM) Anopheles stephensi mosquitoes highly susceptible to PfSPZ infections. By silencing a mosquito immune effector gene, LRIM1 (a member of the LRR gene family), we have reproducibly increased SPZ loads in A. stephensi. We have also demonstrated a phenotype of increased oocyst burdens in a transgenic A. stephensi strain expressing a SRPN6 double- stranded RNA (dsRNA) sequence in a hairpin construct that was fully integrated into the genome. These major results justify this Phase II project which will create GM A. stephensi lines in which expression of LRIM1 or APL1 is reduced or eliminated, providing a stable and efficient SPZ-production platform for Sanaria's manufacturing process. Two strategies are planned for silencing LRIM1: 1) directly silencing the genes through the expression of gene-specific dsRNA to elicit an endogenous RNA interference response against LRIM1 or APL1 transcripts; 2) indirectly silencing the genes by disrupting the relevant regulatory signaling pathway through either the over-expression of the negative-regulatory protein Caspar or a dominant-negative variant of the transcription factor Rel2. Our successful Phase I strategy was to directly silence genes by driving the expression of gene-specific dsRNA to elicit endogenous RNA interference; this approach will be improved upon for the creation of GM A. stephensi. We will create gene-silencing transgenes by incorporating the GAL4/UAS binary expression system and a 'two-target' gene silencing technology. This strategy will enable us to limit the costly and time consuming assessments of infection phenotype as it enables efficient determination of gene silencing prior to infectious feeding. This approach will enhance our capacity to produce strains with the optimal genotypes and phenotypes. We will screen GM lines to identify those that consistently produce e 2 fold more PfSPZ compared to the current production strain of A. stephensi. Selected strains will be studied under standard insectary conditions, and under the aseptic conditions used for GMP compliant manufacturing. We will incorporate the optimal GM strain of A. stephensi into Sanaria's manufacturing process. PUBLIC HEALTH RELEVANCE: To protect individuals against malaria and to aid in eradicating this disease, a highly effective vaccine is needed. Sanaria has developed a platform technology, manufacture of live Plasmodium falciparum sporozoites (PfSPZ) to make such a vaccine. Two are in clinical development. The first is a live attenuated whole organism vaccine called the PfSPZ Vaccine. The second is a live infectious vaccine administered with an antimalarial drug, a vaccine called PfSPZ-CVac. In addition Sanaria has developed live, infectious PfSPZ, a product called PfSPZ Challenge, for use in testing malaria drugs and vaccines. All the products require the growth of PfSPZ in aseptic Anopheles stephensi mosquitoes. This proposal aims to increase the yields of sporozoites by genetically modifying the mosquito immune system. Success in this project will greatly reduce cost of vaccine manufacture, thereby making the vaccine available more cheaply for travelers and developing world populations.