利用 RNAi 瞬时敲低斯氏按蚊 LRIM1 可增加疟原虫子孢子唾液腺感染。
Transient knockdown of Anopheles stephensi LRIM1 using RNAi increases Plasmodium falciparum sporozoite salivary gland infections.
机构信息
Sanaria Inc, Suite A209, 9800 Medical Center Drive, Rockville, MD, 20850, USA.
Institute for Bioscience and Biotechnology Research and Department of Entomology, University of Maryland, Gudelsky Drive, Rockville, MD, 20850, USA.
出版信息
Malar J. 2021 Jun 26;20(1):284. doi: 10.1186/s12936-021-03818-8.
BACKGROUND
Plasmodium falciparum (Pf) sporozoites (PfSPZ) can be administered as a highly protective vaccine conferring the highest protection seen to date. Sanaria® PfSPZ vaccines are produced using aseptically reared Anopheles stephensi mosquitoes. The bionomics of sporogonic development of P. falciparum in A. stephensi to fully mature salivary gland PfSPZ is thought to be modulated by several components of the mosquito innate immune system. In order to increase salivary gland PfSPZ infections in A. stephensi and thereby increase vaccine production efficiency, a gene knock down approach was used to investigate the activity of the immune deficiency (IMD) signaling pathway downstream effector leucine-rich repeat immune molecule 1 (LRIM1), an antagonist to Plasmodium development.
METHODS
Expression of LRIM1 in A. stephensi was reduced following injection of double stranded (ds) RNA into mosquitoes. By combining the Gal4/UAS bipartite system with in vivo expression of short hairpin (sh) RNA coding for LRIM1 reduced expression of LRIM1 was targeted in the midgut, fat body, and salivary glands. RT-qPCR was used to demonstrate fold-changes in gene expression in three transgenic crosses and the effects on P. falciparum infections determined in mosquitoes showing the greatest reduction in LRIM1 expression.
RESULTS
LRIM1 expression could be reduced, but not completely silenced, by expression of LRIM1 dsRNA. Infections of P. falciparum oocysts and PfSPZ were consistently and significantly higher in transgenic mosquitoes than wild type controls, with increases in PfSPZ ranging from 2.5- to tenfold.
CONCLUSIONS
Plasmodium falciparum infections in A. stephensi can be increased following reduced expression of LRIM1. These data provide the springboard for more precise knockout of LRIM1 for the eventual incorporation of immune-compromised A. stephensi into manufacturing of Sanaria's PfSPZ products.
背景
恶性疟原虫（Pf）孢子（PfSPZ）可作为一种高度保护性疫苗进行给药，提供迄今为止所见的最高保护。Sanaria® PfSPZ 疫苗是使用无菌饲养的斯氏按蚊生产的。疟原虫在斯氏按蚊中进行孢子生殖发育并完全成熟的唾液腺 PfSPZ 被认为是由蚊子先天免疫系统的几个成分调节的。为了增加斯氏按蚊中的唾液腺 PfSPZ 感染，从而提高疫苗生产效率，采用基因敲低方法研究了免疫缺陷（IMD）信号通路下游效应物富含亮氨酸重复免疫分子 1（LRIM1）的活性，LRIM1 是疟原虫发育的拮抗剂。
方法
通过向蚊子注射双链（ds）RNA，降低斯氏按蚊中 LRIM1 的表达。通过结合 Gal4/UAS 二部分系统和体内表达编码 LRIM1 的短发夹（sh）RNA，靶向降低中肠、脂肪体和唾液腺中的 LRIM1 表达。RT-qPCR 用于证明三个转基因品系中的基因表达倍数变化，并在显示 LRIM1 表达降低最大的蚊子中确定对疟原虫感染的影响。
结果
LRIM1 表达可以通过 LRIM1 dsRNA 的表达降低，但不能完全沉默。与野生型对照相比，转基因蚊子中 Pf 孢子和 PfSPZ 的感染始终显著增加，PfSPZ 的增加范围从 2.5 倍到 10 倍。
结论
在降低 LRIM1 表达后，斯氏按蚊中的疟原虫感染可以增加。这些数据为更精确地敲除 LRIM1 提供了基础，以便最终将免疫功能低下的斯氏按蚊纳入 Sanaria 的 PfSPZ 产品的制造。
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