Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA

Abstract

Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)-targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti-MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease.

Fig. 1. Treatment with LNP-encapsulated NP-718m siRNA protects NHPs against MARV-Angola up to 72 hours after infection

(A) Survival of animals infected with 1000 PFU of MARV-Angola and then treated with NP-718m–LNP at 30 to 45 min (n = 4), 24 hours (n = 4), 48 hours (n = 4), or 72 hours (n = 4) after infection. Untreated and Luc LNP–treated animals were used as negative controls (n = 5). NP-718m–LNP significantly protected NHPs against MARV-Angola infection (100% survival, four of four animals surviving for each group), whereas untreated animals and animals administered Luc LNP succumbed (*P = 0.0286, Fisher's exact test). Numbers in parentheses beside each group in the legend indicate the number of surviving animals/total number of animals in that group. (B) NP-718m–LNP treatment effectively reduces serum viremia. Plasma samples taken from untreated, negative control Luc LNP–administered, and NP-718m–LNP–treated animals were assessed by plaque assay for infectious viral particles at various time points after infection. NP-718m–LNP treatment significantly reduced infectious peak viral load in infected animals by 5 to 7 log₁₀ PFU/ml compared to control animals when administered at 30 to 45 min, 24 hours, 48 hours, and 72 hours after infection [***P < 0.0001, two-way analysis of variance (ANOVA), with Bonferroni correction for pairwise comparisons]. Data are group means ± SD. The lower limit of detection is 15 PFU/ml. (C) NP-718m–LNP treatment significantly abrogates serum viral RNA. Viral RNA was quantified using quantitative RT-PCR (qRT-PCR) in serum samples taken from untreated, negative control Luc LNP–administered, and NP-718m–LNP–treated animals at various time points after infection. Animals treated with NP-718m–LNP at 30 to 45 min, 24 hours, 48 hours, and 72 hours after infection with MARV-Angola had lower peak levels [5 to 11 log₁₀ genome equivalents (GEq)/ml] of viral RNA compared to control animals (*P = 0.0497 (day 3), ***P < 0.0001 (day 6), two-way ANOVA, with Bonferroni correction for pairwise comparisons). Data are group means ± SD. The lower limit of quantitation is 1 × 10⁵ GEq/ml. (D) Viral RNA levels in tissues from NP-718m–LNP–treated animals are reduced. RNA was extracted from various tissues taken at necropsy from untreated, control Luc LNP–administered, and NP-718m–LNP–treated animals infected with MARV-Angola, and quantified for viral RNA using qRT-PCR. Viral RNA levels in all assessed tissues from NP-718m–LNP–treated animals were 4 to 10 log₁₀ GEq/g lower than peak viral RNA loads in infected tissues taken from untreated and negative control Luc LNP–administered animals [*P = 0.0457, two-way ANOVA, with Bonferroni correction for pairwise comparisons]. ND, not detected; Ax LN, axillary lymph nodes; Ig LN, inguinal lymph nodes. Data are group means ± SEM. The lower limit of quantitation (LLOQ) is 1 × 10⁵ GEq/g. Tissues were taken on day 7 to day 9 for untreated animals, on day 8 for Luc LNP groups, and on day 28, the scheduled study end date for animals in treated groups, which all survived to study end.

Fig. 2. Clinical pathology and clinical scoring in MARV-infected cynomolgus monkeys

NP-718m–LNP treatment effectively abrogates clinical signs of MARV-Angola infection when administered 30 to 45 min, 24 hours, 48 hours, or 72 hours after infection. MARV-mediated liver dysfunction either is completely prevented in NP-718m–LNP–treated animals (n = 4 per treatment group) or manifests transiently with delayed onset and reduced severity. (A) ALT activity. (B) AST activity. Data are group means ± SD. (C and D) Retention of (C) prothrombin time (PT) [*P = 0.0011 (30–45 min Tx delay), *P = 0.0011 (24 h Tx delay), *P = 0.0004 (48 h Tx delay), *P < 0.0001 (72 h Tx delay), two-way ANOVA, with Bonferroni correction for pairwise comparisons] and (D) activated partial thromboplastin time (APTT) [*P = 0.0193 (1 h Tx delay), *P = 0.0034 (24 h Tx delay), *P = 0.0226 (48 h Tx delay), *P = 0.0176 (72 h Tx delay), two-way ANOVA, with Bonferroni correction for pairwise comparisons] indicates protection against HF coagulopathy. Data are group means ± SEM. (E) Clinical scores for each individual within each group after MARV challenge.

Fig. 3. Comparison of MARV pathology and antigen in representative tissues of rhesus monkeys either treated or not treated with NP-718m–LNP

(A) Liver, multifocal necrotizing hepatitis, sinusoidal leukocytosis, and eosinophilic cytoplasmic inclusion bodies in a MARV-infected control animal. (B) Liver, diffuse cytoplasmic immunolabeling (brown) of sinusoidal lining cells, Kupffer cells, and hepatocytes. (C and D) No overt lesions (C) or immunolabeling (D) in the liver of an NP-718m–LNP–treated animal. (E) Spleen, diffuse lymphoid depletion of the white pulp and fibrin deposition in the red pulp in a MARV-infected control animal. (F) Spleen, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells in the red and white pulp of a MARV-infected control animal. (G and H) No overt lesions (G) or immunolabeling (H) in the spleen of an NP-718m–LNP–treated animal. All representative images taken at ×40 from treated animal 0807175 or control animal 0904099. Scale bars, 50 μm.