Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study

Abstract

Background: We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever.

Methods: A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV.

Findings: Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection.

Interpretation: This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections.

Funding: Defense Threat Reduction Agency.

Figure 1

Effect of small interfering RNAs (siRNAs) targeting virion protein genes VP24 (A) and VP35 (B) of Zaire Ebola virus (ZEBOV) expressed in a non-viral plasmid-based system in HepG2 cells Error bars represent SD of triplicate tissue culture wells.

Figure 2

Effect of small interfering RNAs (siRNAs) on synthesis of interleukin 6 (A) and interferon α (B), induction of interferon-induced protein with tetratricopeptide repeats (IFIT1) mRNA in mice (C), and induction of interferon α in human peripheral blood mononuclear cell cultures (D) Data are mean (SD). Error bars represent SD. Luc=luciferase. PBS=phosphate-buffered saline. Luc mod=modified luciferase. GAPDH=glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

Rapid amplification of cDNA ends (RACE)-PCR of small interfering RNA (siRNA)-mediated cleavage of Zaire Ebola virus (ZEBOV) L polymerase, virion protein (VP) 24 (VP24), and VP35 mRNAs in ZEBOV-infected Vero E6 cells Vero E6 cells were treated with stable nucleic acid-lipid particles (SNALPs) containing ZEBOV siRNA cocktail, modified luciferase (Luc mod), or phosphate-buffered saline (PBS). (B) Vero E6 cells were treated with SNALPs containing EK-1-mod, VP24-1160-mod, VP35-855-mod, ZEBOV siRNA cocktail, Luc mod, or PBS. The order of samples for each RACE PCR shown in the gel is SNALPs containing PBS, single gene-specific siRNAs (EK-1-mod, VP24-1160-mod, or VP35-855-mod), ZEBOV cocktail, and Luc mod. Lanes 1 and 17 are the 100 base pair (bp) ladder, lanes 2–5 are EK-1 RACE PCR, lanes 7–10 are VP24-1160 RACE, and lanes 12–15 are VP35-855 RACE. Lanes 6, 11, and 16 are empty.

Figure 4

Proportion of differentiated cells with uptake of stable nucleic acid-lipid particles (SNALPs) containing fluorescein-isothiocyanate-labelled modified luciferase small interfering RNAs

Figure 5

Effect of daily administration of stable nucleic acid-lipid particles (SNALPs) containing Zaire Ebola virus (ZEBOV) small interfering RNAs on activities (siRNAs) of alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase (A), and blood cell counts (B) in mice PBS=phosphate-buffered saline.

Figure 6

Survival curves for Zaire Ebola virus (ZEBOV)-infected rhesus macaques treated after challenge with stable nucleic acid-lipid particles containing ZEBOV small interfering RNAs Study 1: four postexposure treatments. Study 2: seven post exposure treatments.