Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates

Abstract

The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.

Extended Data Figure 1. Antiviral activity of siEbola-3 in cells infected with EBOV Makona

For comparison, siEbola-3 activity was also assessed against the Central African EBOV Kikwit strain and siEbola-2 activity was evaluated against both EBOV strains. Data is viral RNA copies/mL of each sample normalized to untreated infected cells. Results are mean ± SD from one biological replicate, conducted in technical triplicate.

Extended Data Figure 2. siEbola-3 LNP treatment provides partial protection against EBOV Makona clinical pathologies, and infection with EBOV Makona infection induces a lesser degree of liver dysfunction compared to EBOV Kikwit infection

a. No differences in viremia levels were observed in untreated animals infected with EBOV Makona or Kikwit. b–e. Liver dysfunction markers. Normal values for uninfected NHPs ranges are GGT (40–115 U/L), AST (20–45 U/L), ALT (20–165 U/L), ALP (130–500 U/L). f, g. Protection against EBOV Makona-induced CRE and BUN elevation was observed. Normal values for uninfected NHPs range from BUN (10–25 mg/dL) and CRE (0.8–1.2 mg/dL).

Extended Data Figure 3. Comparison of coagulation and hematology characteristics between untreated control animals infected with EBOV Makona or Kikwit

a, b. Coagulopathies are not as marked in EBOV Makona infection when compared to historical EBOV Kikwit data. c. Lymphopenia is observed in all infected animals. d. Thrombocytopenia levels are similar between EBOV Makona and EBOV Kikwit infected control animals.

Figure 1. siEbola-3 is active against EBOV Makona target sequences

a. The TKM-Ebola siRNA cocktail of siVP35-2 and siLpol-2 targets gene regions in Central African EBOV sequences but West African outbreak sequences contain mutations at these locations. siEbola-3 has these target site mismatches corrected. b. siEbola-3 and its individual components, siVP35-3 and siLpol-3, are active against EBOV Makona sequences. Activity was assessed by dual luciferase reporter assay (see Methods). Shown is RLuc/FLuc of each sample normalized to untreated cells. Results are mean ± SEM from one (negative control) or two biological replicates (other data), conducted in technical triplicate.

Figure 2. siEbola-3 LNP treatment confers survival and reduces viral load

a. NHPs lethally challenged with EBOV Makona survive when treated with siEbola-3 LNP starting 72 h post-infection. b. Clinical signs were improved in treated animals. Treatment reduces c. infectious virus load (*p=0.0450, one-sided t-test, day 6) and d. viral RNA in blood (**p=0.0023, one-sided t-test, day 6); e. tissues. Lower limit of detection is 5 pfu/mL. d, e. qRT-PCR data shown are means ± SD of two technical replicates. ND = Not detected. LLOQ = Lower limit of quantitation, 4.8 log₁₀ copies/g or 5.1 log₁₀ copies/mL. N = 3/group.

Figure 3. EBOV Makona tissue pathology and antigen in NHPs untreated or treated with siEbola-3 LNP

a. Immmunolabeling of sinusoidal lining and Kupffer cells in untreated animal. b, c, d. No immunolabeling of treated animals. e. Immunolabeling of dendriform mononuclear cells in red and white pulp of untreated animal. f, g, h. No immunolabeling of treated animals. i. Immunolabeling of cortical and interstitial cells in untreated animal. j, k, l. No immunolabeling of treated animals. m. Immunolabeling, dendriform mononuclear cells within subcapsular and medullary sinuses in untreated animal. n, o, p. No immunolabeling of treated animals.