Research ArticleMARBURG VIRUS

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

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Science Translational Medicine  20 Aug 2014:
Vol. 6, Issue 250, pp. 250ra116
DOI: 10.1126/scitranslmed.3009706
  • 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.

  • Table 1. Clinical description and outcome of MARV-Angola–challenged NHPs.

    Days after MARV challenge are in parentheses. Fever is defined as a temperature more than 2.5°C over baseline or at least 1.5°C over baseline and ≥39.7°C. Mild rash: focal areas of petechiae covering less than 10% of the skin; moderate rash: areas of petechiae covering between 10 and 40% of the skin; severe rash: areas of petechiae and/or ecchymosis covering more than 40% of the skin. Lymphopenia and thrombocytopenia are defined as a ≥35% drop in the number of lymphocytes and platelets, respectively. Leukocytosis and granulocytosis are defined as a ≥35% increase in the number of white blood cells. Hypoalbuminemia is defined as a ≥35% decrease in the level of albumin. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; GGT, γ-glutamyltransferase; CRE, creatine; CRP, C-reactive protein.

    Subject no.SexGroupClinical illnessClinical and gross pathology
    0308904FControlDepression (d7); lethargy (d7);
    loss of appetite (d6–7);
    moderate rash (d6–7);
    animal expired in p.m. on d7
    ALT >10-fold ↑ (d6);
    AST >10-fold ↑ (d6);
    ALP >10-fold ↑ (d6);
    GGT >4-fold ↑ (d6);
    CRP >4-fold ↑ (d6)
    0904099MControlFever (d6); depression (d7–8);
    lethargy (d7–8); loss of appetite (d7–8);
    mild rash (d8); rectorrhagia (d8);
    animal euthanized on d8
    Leukocytosis (d8); lymphopenia (d6);
    ALT >10-fold ↑ (d8); AST >6-fold ↑ (d6);
    AST >10-fold ↑ (d8); ALP >2-fold ↑ (d8);
    GGT >3-fold ↑ (d8); BUN >4-fold ↑ (d8);
    CRE >5-fold ↑ (d8); CRP >10-fold ↑ (d8)
    0707061MControlFever (d6); depression (d8–9);
    lethargy (d8–9); loss of appetite (d8–9);
    moderate rash (d9); rectorrhagia (d9);
    animal euthanized on d9
    Leukocytosis (d9); lymphopenia (d6);
    granulocytosis (d9); ALT >10-fold ↑ (d9);
    AST >10-fold ↑ (d9); BUN >3-fold ↑ (d9);
    CRE >3-fold ↑ (d9)
    0907042FControlFever (d6–7); depression (d6–8);
    lethargy (d6–8); loss of appetite (d5–8);
    dehydration (d5–8); mild rash (d7);
    moderate rash (d8); rectorrhagia (d8);
    animal euthanized on d8
    Thrombocytopenia (d6); granulocytosis (d6, d8);
    hypoalbuminemia (d8); ALT >10-fold ↑ (d8);
    AST >10-fold ↑ (d8); ALP >10-fold ↑ (d8);
    GGT >5-fold ↑ (d8); CRP >10-fold ↑ (d6, d8)
    0903269MControlFever (d4,6); depression (d6–8);
    lethargy (d6–8); loss of appetite (d7–8);
    mild rash (d7); moderate rash (d8);
    animal euthanized on d8
    Leukocytosis (d8); lymphopenia (d3, d6);
    hypoalbuminemia (d8); ALT >6-fold ↑ (d6);
    ALT >10-fold ↑ (d10); AST >10-fold ↑ (d6, d8);
    ALP >4-fold ↑ (d6); ALP >10-fold ↑ (d8);
    GGT >4-fold ↑ (d8); CRE >3-fold ↑ (d8);
    CRP >10-fold ↑ (d8)
    0908097M30–45 minFever (d2, d4, d10); loss
    of appetite (d3–5)
    Thrombocytopenia (d3); lymphopenia (d10);
    CRP >10-fold ↑ (d3, d6, d14);
    CRP >4-fold ↑ (d10)
    08083572F30–45 minNoneThrombocytopenia (d3);
    lymphopenia (d3, d6)
    0906057M30–45 minNoneLeukocytosis (d10); granulocytosis (d10);
    CRP >5-fold ↑ (d3); CRP >10-fold ↑ (d6);
    CRP >8-fold (d10)
    R080062F30–45 minLoss of appetite (d2)Thrombocytopenia (d3); AST >4-fold ↑ (d10);
    CRP >10-fold ↑ (d3, d6); CRP >2-fold ↑ (d10)
    0806012F24 hoursFever (d4, d7)ALT >2-fold ↑ (d6); ALT >4-fold ↑ (d10);
    AST >4-fold ↑ (d6); AST >3-fold ↑ (d10);
    ALP >2-fold ↑ (d10); GGT >2-fold ↑ (d14);
    CRP >5-fold ↑ (d6); CRP >2-fold ↑ (d10)
    0704019M24 hoursNoneLeukocytosis (d3); granulocytosis (d3);
    CRP >10-fold ↑ (d3); CRP >3-fold ↑ (d6)
    0908021M24 hoursNoneLymphopenia (d3)
    08R0002F24 hoursNoneLeukocytosis (d6, d10); granulocytosis
    (d6, d10); CRP >10-fold ↑ (d6)
    0904047M48 hoursNoneLeukocytosis (d3, d6); granulocytosis (d3, d6);
    ALT >5-fold ↑ (d10); AST >3-fold ↑ (d10);
    CRP >10-fold ↑ (d3, d6); CRP >3-fold ↑ (d10)
    1002065M48 hoursFever (d5)Granulocytosis (d6); ALT >2-fold ↑ (d10);
    AST >2-fold ↑ (d6, d10); CRP >10-fold ↑ (d6)
    0705010F48 hoursFever (d4)Leukocytosis (d6); granulocytosis (d6);
    AST >10-fold ↑ (d10); CRP >10-fold ↑ (d6)
    0904088F48 hoursNoneLeukocytosis (d21); granulocytosis (d21);
    lymphopenia (d10); ALT >10-fold ↑ (d10);
    AST >10-fold ↑ (d10)
    0906220F72 hoursNoneLeukocytosis (d6); granulocytosis (d6);
    thrombocytopenia (d10); ALT >6-fold ↑ (d10);
    AST >7-fold ↑ (d10); ALP >2-fold ↑ (d10, d14);
    GGT >2-fold ↑ (d10, d14, d21);
    CRP >10-fold ↑ (d6, d10)
    0810254F72 hoursNoneGranulocytosis (d10); thrombocytopenia (d10);
    ALT >10-fold ↑ (d10); AST >10-fold ↑ (d10);
    CRP >10-fold ↑ (d10)
    0906145M72 hoursFever (d4)Leukocytosis (d10); granulocytosis (d10);
    lymphopenia (d6); thrombocytopenia
    (d6, d10, d14); ALT >10-fold ↑ (d10);
    ALT >4-fold ↑ (d14); AST >10-fold ↑ (d10);
    ALP >3-fold ↑ (d10); ALP >2-fold ↑ (d14);
    GGT >2-fold ↑ (d10); CRP >10-fold ↑ (d6, d10)
    0807175M72 hoursFever (d7–9); loss of appetite (d5–11);
    mild rash (d9–11)
    Leukocytosis (d10); granulocytosis
    (d10); thrombocytopenia (d10);
    hypoalbuminemia (d6, d10, d14);
    ALT >8-fold ↑ (d10); ALT >3-fold ↑ (d14);
    AST >10-fold ↑ (d10); ALP >2-fold ↑ (d10);
    GGT >4-fold ↑ (d10); CRP >10-fold ↑ (d6, d10)

Supplementary Materials

  • Supplementary Material for:

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

    Emily P. Thi, Chad E. Mire, Raul Ursic-Bedoya, Joan B. Geisbert, Amy C. H. Lee, Krystle N. Agans, Marjorie Robbins, Daniel J. Deer, Karla A. Fenton, Ian MacLachlan,* Thomas W. Geisbert*

    *Corresponding author. E-mail: twgeisbe@utmb.edu (T.W.G.); IMacLachlan@tekmirapharm.com (I.M.)

    Published 20 August 2014, Sci. Transl. Med. 6, 250ra116 (2014)
    DOI: 10.1126/scitranslmed.3009706

    This PDF file includes:

    • Fig. S1. NP-718m–LNP RNAi mechanism of action confirmation.
    • Fig. S2. Clinical pathology parameters of MARV HF in NP-718m–LNP–treated, untreated, and Luc LNP–administered animals.
    • Table S1. Hematology results.
    • Table S2. Clinical chemistry results.

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