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Malaria Journal
Published in: Malaria Journal 1/2021

Open Access 01-12-2021 | Plasmodium Falciparum | Research

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

Authors: Peter F. Billingsley, Kasim I. George, Abraham G. Eappen, Robert A. Harrell II, Robert Alford, Tao Li, Sumana Chakravarty, B. Kim Lee Sim, Stephen L. Hoffman, David A. O’Brochta

Published in: Malaria Journal | Issue 1/2021

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Abstract

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.
Appendix
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Literature
1.
WHO. World malaria report 2020. Geneva: World Health Organization; 2020.
2.
Billingsley PF, Maas CD, Olotu A, Schwabe C, García GA, Rivas MR, et al. The Equatoguinean malaria vaccine initiative: from the launching of a clinical research platform to malaria elimination planning in central west Africa. Am J Trop Med Hyg. 2020;103:947–54.PubMedPubMedCentralCrossRef
3.
Weiss DJ, Bertozzi-Villa A, Rumisha SF, Amratia P, Arambepola R, Battle KE, et al. Indirect effects of the Covid-19 pandemic on malaria intervention coverage, morbidity, and mortality in Africa: a geospatial modelling analysis. Lancet Infect Dis. 2020;21:59–69.PubMedCrossRef
4.
5.
Sissoko MS, Healy SA, Katile A, Omaswa F, Zaidi I, Gabriel EE, et al. Safety and efficacy of PfSPZ vaccine against Plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in Mali: a randomised, double-blind phase 1 trial. Lancet Infect Dis. 2017;17:498–509.PubMedPubMedCentralCrossRef
6.
Jongo SA, Shekalage SA, Church LWP, Ruben AJ, Schindler T, Zenklusen I, et al. Safety, immunogenicity, and protective efficacy against controlled human malaria infection of Plasmodium falciparum sporozoite vaccine in Tanzanian adults. Am J Trop Med Hyg. 2018;99:338–49.PubMedPubMedCentralCrossRef
7.
Mordmüller B, Surat G, Lagler H, Chakravarty S, Ishizuka AS, Lalremruata A, et al. Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature. 2017;542:445–9.PubMedCrossRef
8.
Jongo SA, Church LWP, Mtoro AT, Chakravarty S, Ruben AJ, Swanson Ii PA, et al. Increase of dose associated with decrease in protection against controlled human malaria infection by PfSPZ vaccine in Tanzanian adults. Clin Infect Dis. 2020;71:2849–57.PubMedCrossRef
9.
Lyke KE, Ishizuka AS, Berry AA, Chakravarty S, Dezure A, Enama ME, et al. Attenuated PfSPZ vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection. Proc Natl Acad Sci USA. 2017;114:2711–6.PubMedPubMedCentralCrossRef
10.
Ishizuka AS, Lyke KE, Dezure A, Berry AA, Richie TL, Mendoza FH, et al. Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. Nat Med. 2016;22:614–23.PubMedCrossRef
11.
Hoffman SL, Billingsley PF, James E, Richman A, Loyevsky M, Li T, et al. Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria. Hum Vaccin. 2010;6:97–106.PubMedCrossRef
12.
Richie TL, Billingsley PF, Sim BK, Epstein JE, Lyke KE, Mordmuller B, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines. Vaccine. 2015;33:7452–61.PubMedPubMedCentralCrossRef
13.
Epstein JE, Paolino KM, Richie TL, Sedegah M, Singer A, Ruben AJ, et al. Protection against Plasmodium falciparum malaria by PfSPZ vaccine. JCI Insight. 2017;2:e89154.PubMedPubMedCentralCrossRef
14.
Epstein JE, Tewari K, Lyke KE, Sim BK, Billingsley PF, Laurens MB, et al. Live attenuated malaria vaccine designed to protect through hepatic CD8+T cell immunity. Science. 2011;334:475–80.PubMedCrossRef
15.
Olotu A, Urbano V, Hamad A, Eka M, Chemba M, Nyakarungu E, et al. Advancing global health through development and clinical trials partnerships: A randomized, placebo-controlled, double-blind assessment of safety, tolerability, and immunogenicity of Plasmodium falciparum sporozoite vaccine for malaria in healthy Equatoguinean men. Am J Trop Med Hyg. 2018;98:308–18.PubMedCrossRef
16.
Lyke KE, Laurens MB, Strauss K, Adams M, Billingsley PF, James E, et al. Optimizing intradermal administration of cryopreserved Plasmodium falciparum sporozoites in controlled human malaria infection. Am J Trop Med Hyg. 2015;93:1274–84.PubMedPubMedCentralCrossRef
17.
Lell B, Mordmuller B, Dejon Agobe JC, Honkpehedji J, Zinsou J, Mengue JB, et al. Impact of sickle cell trait and naturally acquired immunity on uncomplicated malaria after controlled human malaria infection in adults in Gabon. Am J Trop Med Hyg. 2018;98:508–15.PubMedCrossRef
18.
Obiero JM, Shekalaghe S, Hermsen CC, Mpina M, Bijker EM, Roestenberg M, et al. Impact of malaria pre-exposure on anti-parasite cellular and humoral immune responses after controlled human malaria infection. Infect Immun. 2015;83:2185–96.PubMedPubMedCentralCrossRef
19.
Hodgson SH, Juma E, Salim A, Magiri C, Njenga D, Molyneux S, et al. Lessons learnt from the first controlled human malaria infection study conducted in Nairobi, Kenya. Malar J. 2015;14:182.PubMedPubMedCentralCrossRef
20.
Mordmüller B, Supan C, Sim KL, Gómez-Pérez GP, Ospina Salazar CL, Held J, et al. Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres. Malar J. 2015;14:117.PubMedPubMedCentralCrossRef
21.
Hodgson SH, Juma EA, Salim A, Magiri C, Kimani D, Njenga D, et al. Evaluating controlled human malaria infection in Kenyan adults with varying degrees of prior exposure to Plasmodium falciparum using sporozoites administered by intramuscular injection. Front Microbiol. 2014;5:686.PubMedPubMedCentralCrossRef
22.
Roestenberg M, Bijker EM, Sim BK, Billingsley PF, James ER, Bastiaens GJ, et al. Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites. Am J Trop Med Hyg. 2013;88:5–13.PubMedPubMedCentralCrossRef
23.
Sheehy SH, Spencer AJ, Douglas AD, Sim BK, Longley RJ, Edwards NJ, et al. Optimising controlled human malaria infection studies using cryopreserved parasites administered by needle and syringe. PLoS ONE. 2013;8:e65960.PubMedPubMedCentralCrossRef
24.
Shekalaghe S, Rutaihwa M, Billingsley PF, Chemba M, Daubenberger CA, James ER, et al. Controlled human malaria infection of Tanzanians by intradermal injection of aseptic, purified, cryopreserved Plasmodium falciparum sporozoites. Am J Trop Med Hyg. 2014;91:471–80.PubMedPubMedCentralCrossRef
25.
Gomez-Perez GP, Legarda A, Munoz J, Sim BK, Ballester MR, Dobano C, et al. Controlled human malaria infection by intramuscular and direct venous inoculation of cryopreserved Plasmodium falciparum sporozoites in malaria-naive volunteers: effect of injection volume and dose on infectivity rates. Malar J. 2015;14:306.PubMedPubMedCentralCrossRef
26.
Bastiaens GJ, Van Meer MP, Scholzen A, Obiero JM, Vatanshenassan M, Van Grinsven T, et al. Safety, immunogenicity, and protective efficacy of intradermal immunization with aseptic, purified, cryopreserved Plasmodium falciparum sporozoites in volunteers under chloroquine prophylaxis: a randomized controlled trial. Am J Trop Med Hyg. 2016;94:663–73.PubMedPubMedCentralCrossRef
27.
28.
Roestenberg M, Walk J, Van Der Boor SC, Langenberg MCC, Hoogerwerf MA, Janse JJ, et al. A double-blind, placebo-controlled phase 1/2a trial of the genetically attenuated malaria vaccine PfSPZ-GA1. Sci Transl Med. 2020;12:eaaz5629.PubMed
29.
Fraiture M, Baxter RH, Steinert S, Chelliah Y, Frolet C, Quispe-Tintaya W, et al. Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium. Cell Host Microbe. 2009;5:273–84.PubMedCrossRef
30.
Waterhouse RM, Povelones M, Christophides GK. Sequence-structure-function relations of the mosquito leucine-rich repeat immune proteins. BMC Genomics. 2010;11:531.PubMedPubMedCentralCrossRef
31.
Garver LS, Bahia AC, Das S, Souza-Neto JA, Shiao J, Dong Y, et al. Anopheles IMD pathway factors and effectors in infection intensity-dependent anti-Plasmodium action. PLoS Pathog. 2012;8:e1002737.PubMedPubMedCentralCrossRef
32.
Warr E, Lambrechts L, Koella JC, Bourgouin C, Dimopoulos G. Anopheles gambiae immune responses to sephadex beads: involvement of anti-Plasmodium factors in regulating melanization. Insect Biochem Mol Biol. 2006;36:769–78.PubMedCrossRef
33.
Jaramillo-Gutierrez G, Rodrigues J, Ndikuyeze G, Povelones M, Molina-Cruz A, Barillas-Mury C. Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes. BMC Microbiol. 2009;9:154.PubMedPubMedCentralCrossRef
34.
Habtewold T, Povelones M, Blagborough AM, Christophides GK. Transmission blocking immunity in the malaria non-vector mosquito Anopheles quadriannulatus species A. PLoS Pathog. 2008;4:e1000070.PubMedPubMedCentralCrossRef
35.
Povelones M, Waterhouse RM, Kafatos FC, Christophides GK. Leucine-rich repeat protein complex activates mosquito complement in defense against Plasmodium parasites. Science. 2009;324:258–61.PubMedPubMedCentralCrossRef
36.
Povelones M, Upton LM, Sala KA, Christophides GK. Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1. PLoS Pathog. 2011;7:e1002023.PubMedPubMedCentralCrossRef
37.
Baxter RH, Steinert S, Chelliah Y, Volohonsky G, Levashina EA, Deisenhofer J. A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae. Proc Natl Acad Sci USA. 2010;107:16817–22.PubMedPubMedCentralCrossRef
38.
Han YS, Thompson J, Kafatos FC, Barillas-Mury C. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J. 2000;19:6030–40.PubMedPubMedCentralCrossRef
39.
Osta MA, Christophides GK, Kafatos FC. Effects of mosquito genes on Plasmodium development. Science. 2004;303:2030–2.PubMedCrossRef
40.
Marinotti O, Nguyen QK, Calvo E, James AA, Ribeiro JM. Microarray analysis of genes showing variable expression following a blood meal in Anopheles gambiae. Insect Mol Biol. 2005;14:365–73.PubMedCrossRef
41.
Feldmann AM, Ponnudurai T. Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum. Med Vet Entomol. 1989;3:41–52.PubMedCrossRef
42.
Feldmann AM, Billingsley PF, Savelkoul E. Bloodmeal digestion by strains of Anopheles stephensi Liston (Diptera: Culicidae) of differing susceptibility to Plasmodium falciparum. Parasitology. 1990;101:193–200.PubMedCrossRef
43.
44.
Billingsley PF, Rudin W. The role of the mosquito peritrophic membrane in bloodmeal digestion and infectivity of Plasmodium species. J Parasitol. 1992;78:430–40.PubMedCrossRef
45.
Li T, Eappen AG, Richman AM, Billingsley PF, Abebe Y, Li M, et al. Robust, reproducible, industrialized, standard membrane feeding assay for assessing the transmission blocking activity of vaccines and drugs against Plasmodium falciparum. Malar J. 2015;14:150.PubMedPubMedCentralCrossRef
46.
Handler AM, Harrell RA 2nd. Germline transformation of Drosophila melanogaster with the piggybac transposon vector. Insect Mol Biol. 1999;8:449–57.PubMedCrossRef
47.
48.
49.
Jiang X, Peery A, Hall AB, Sharma A, Chen X-G, Waterhouse RM, et al. Genome analysis of a major urban malaria vector mosquito Anopheles stephensi. Genome Biology. 2014;15:459.PubMedPubMedCentralCrossRef
50.
O’brochta DA, Alford RT, Pilitt KL, Aluvihare CU, Harrell RA II. Piggybac transposon remobilization and enhancer detection in Anopheles mosquitoes. Proc Natl Acad Sci USA. 2011;108:16339–44.PubMedPubMedCentralCrossRef
51.
Osta MA, Christophides GK, Vlachou D, Kafatos FC. Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics. J Exp Biol. 2004;207:2551–63.PubMedCrossRef
52.
Mendes AM, Awono-Ambene PH, Nsango SE, Cohuet A, Fontenille D, Kafatos FC, et al. Infection intensity-dependent responses of Anopheles gambiae to the African malaria parasite Plasmodium falciparum. Infect Immun. 2011;79:4708–15.PubMedPubMedCentralCrossRef
53.
Blandin S, Moita LF, Kocher T, Wilm M, Kafatos FC, Levashina EA. Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the defensin gene. EMBO Rep. 2002;3:852–6.PubMedPubMedCentralCrossRef
54.
Catteruccia F, Levashina EA. RNAi in the malaria vector, Anopheles gambiae. Methods Mol Biol. 2009;555:63–75.PubMedCrossRef
55.
Kennerdell JR, Carthew RW. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell. 1998;95:1017–26.PubMedCrossRef
56.
Misquitta L, Paterson BM. Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA. 1999;96:1451–6.PubMedPubMedCentralCrossRef
57.
Brown AE, Bugeon L, Crisanti A, Catteruccia F. Stable and heritable gene silencing in the malaria vector Anopheles stephensi. Nucleic Acids Res. 2003;31:e85.PubMedPubMedCentralCrossRef
58.
Dimopoulos G, Richman A, Muller HM, Kafatos FC. Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc Natl Acad Sci USA. 1997;94:11508–13.PubMedPubMedCentralCrossRef
59.
Dimopoulos G, Christophides GK, Meister S, Schultz J, White KP, Barillas-Mury C, et al. Genome expression analysis of Anopheles gambiae: responses to injury, bacterial challenge, and malaria infection. Proc Nat Acad Sci USA. 2002;99:8814–9.PubMedPubMedCentralCrossRef
60.
Yassine H, Kamareddine L, Chamat S, Christophides GK, Osta MA. A serine protease homolog negatively regulates TEP1 consumption in systemic infections of the malaria vector Anopheles gambiae. J Innate Immun. 2014;6:806–18.PubMedPubMedCentralCrossRef
61.
Dekmak AS, Yang X, Dohna HZ, Buchon N, Osta MA. The route of infection influences the contribution of key immunity genes to antibacterial defense in Anopheles gambiae. J Inn Immun. 2020;13:107–26.CrossRef
62.
Cohuet A, Osta MA, Morlais I, Awono-Ambene PH, Michel K, Simard F, et al. Anopheles and Plasmodium: from laboratory models to natural systems in the field. EMBO Rep. 2006;7:1285–9.PubMedPubMedCentralCrossRef
Metadata
Title
Transient knockdown of Anopheles stephensi LRIM1 using RNAi increases Plasmodium falciparum sporozoite salivary gland infections
Authors
Peter F. Billingsley
Kasim I. George
Abraham G. Eappen
Robert A. Harrell II
Robert Alford
Tao Li
Sumana Chakravarty
B. Kim Lee Sim
Stephen L. Hoffman
David A. O’Brochta
Publication date
01-12-2021
Publisher
BioMed Central
Published in
Malaria Journal / Issue 1/2021
Electronic ISSN: 1475-2875
DOI
https://doi.org/10.1186/s12936-021-03818-8

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