Transient knockdown of Anopheles stephensi LRIM1 using RNAi increases Plasmodium falciparum sporozoite salivary gland infections

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.

Keywords: Anopheles stephensi; Gene silencing; Immune system; Mosquito; Oocyst; PfSPZ vaccine; Plasmodium falciparum; Sporozoite.

MeSH terms

  • Animals
  • Anopheles / genetics
  • Anopheles / parasitology*
  • Female
  • Gene Knockdown Techniques
  • Insect Proteins / genetics*
  • Insect Proteins / metabolism
  • Plasmodium falciparum / physiology*
  • RNA Interference*
  • Salivary Glands / parasitology
  • Sporozoites / physiology


  • Insect Proteins
  • LRIM1 protein, Anopheles gambiae