Research ArticleEbola

mAbs and Ad-Vectored IFN-α Therapy Rescue Ebola-Infected Nonhuman Primates When Administered After the Detection of Viremia and Symptoms

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Science Translational Medicine  16 Oct 2013:
Vol. 5, Issue 207, pp. 207ra143
DOI: 10.1126/scitranslmed.3006605
  • Fig. 1. Survival and clinical overview of cynomolgus macaques treated with mAbs and Ad-IFN starting at 3 dpi.

    (A) Animals (Cyno72h) were challenged with EBOV (red arrow) and administered intravenously with ZMAb and intramuscularly with Ad-IFN (blue arrow) at 3 dpi and then two more intravenous ZMAb treatments at 6 and 9 dpi (green arrows). Control animals received either PBS (B1) or mouse IgG (B2). (B to E) Animals were sampled on treatment and postmortem days in addition to 14, 21, and 28 dpi and examined for (B) survival [*P = 0.0253, log-rank test Cyno72h versus two controls (B1 and B2)], (C) viremia, (D) clinical score, and (E) rectal temperature. (F to I) Variations in blood biochemistry and hematological levels were recorded for (F) alanine aminotransferase (ALT), (G) alkaline phosphatase (ALP), (H) platelets (PLT), and (I) white blood cells (WBCs). Animals A1 to A4 received the treatment; animals B1 and B2 were the controls.

  • Fig. 2. Survival and clinical overview of cynomolgus macaques treated with Ad-IFN at 1 dpi, and then mAbs starting at 4 dpi.

    (A) Animals (Cyno96h) were challenged with EBOV (red arrow) and administered intramuscularly with Ad-IFN (purple arrow) at 1 dpi and then three intravenous ZMAb treatments at 4, 7, and 10 dpi (green arrows). Control animals received either PBS (D1) or PBS at 1 dpi followed by mouse IgG at 4 dpi (D2). (B to E) Animals were sampled on treatment and postmortem days in addition to 14, 21, and 28 dpi and examined for (B) survival [P = 0.0624, log-rank test for survival comparing Cyno96h to two controls (D1 and D2)], (C) viremia, (D) clinical score, and (E) rectal temperature. (F to I) Variations in blood biochemistry and hematological levels were recorded for (F) ALT, (G) ALP, (H) platelets, and (I) WBCs. Animals C1 to C4 received the treatment; animals D1 and D2 were the controls.

  • Fig. 3. Survival and clinical overview of rhesus macaques treated with mAbs and Ad-IFN starting at 3 dpi.

    (A) Animals (Rhesus72h) were challenged with EBOV (red arrow) and administered intravenously with ZMAb and intramuscularly with Ad-IFN (blue arrow) at 3 dpi and then two more intravenous ZMAb treatments at 6 and 9 dpi (green arrows). One control animal received mouse IgG (F1), and the other received Ad-IFN alone (F2). (B to E) Animals were sampled on treatment and postmortem days in addition to 14, 21, and 28 dpi and examined for (B) survival [*P = 0.0177, log-rank test for survival comparing Rhesus72h to two controls (F1 and F2)], (C) viremia, (D) clinical score, and (E) rectal temperature. (F to I) Variations in blood biochemistry and hematological levels were recorded for (F) ALT, (G) ALP, (H) platelets, and (I) WBCs. Animals E1 to E4 received the treatment; animals F1 and F2 were the controls.

  • Fig. 4. Ebola-specific antibody responses.

    (A to F) Sera from the Cyno72h (A1 to A4), Cyno96h (C1 to C4), and Rhesus72h (E1 to E4) group were quantified by recombinant EBOV-GP ELISA to determine levels of EBOV-GP–specific IgM (A to C) and IgG (D to F), respectively.

  • Fig. 5. Ebola-specific T cell responses.

    PBMCs from each animal still alive at 21 dpi (22 dpi for group A) were stimulated with peptide pools spanning the EBOV surface GP. (A to C) The production of IFN-γ was assessed by ELISpot for groups A (A), C (B), and E (C). The background number of spots (from a medium-only stimulation) was subtracted from each pool’s response. (D and E) The CD4+ (D) and CD8+ (E) T cell responses were analyzed by flow cytometry. The first larger graphs show the frequencies of cells that are positive for individual cytokines. The four smaller graphs represent the frequencies of cells positive for one marker that are also positive for a second one (for example, the frequency of CD107a+ cells that are also IFN-γ+). The positivity threshold was defined as two times the background (medium-only stimulation) for the specified gate, in which case the background was subtracted. The responses shown are based on the sum of positive cells for all three pools; the horizontal bar shows the median of each group. Individual animals that died at later time points (A2 and C4) are designated by colored symbols.

  • Table 1. Clinical findings on days 1 to 28 after EBOV challenge.

    Hypothermia was defined as below 35°C. Fever was defined as >1.0°C higher than baseline. Mild rash was defined as focal areas of petechiae covering <10% of the skin, moderate rash as areas of petechiae covering 10 to 40% of the skin, and severe rash as areas of petechiae and/or ecchymosis covering >40% of the skin. Leukocytopenia and thrombocytopenia were defined as a >30% decrease in numbers of WBCs and platelets, respectively. Leukocytosis and thrombocytosis were defined as a twofold or greater increase in numbers of WBCs and platelets over baseline, where WBC count >11.000. ↑, two- to threefold increase; ↑↑, four- to fivefold increase; ↑↑↑, greater than fivefold increase; ↓, two- to threefold decrease; ↓↓, four- to fivefold decrease; ↓↓↓, greater than fivefold decrease. ALB, albumin; AMY, amylase; TBIL, total bilirubin; BUN, blood urea nitrogen; PHOS, phosphate; CRE, creatinine; GLU, glucose; GLOB, globulin; —, no change.

    Animal IDTreatmentsFindingsStatus
    Cyno72h
      A13 dpi Ad-IFN and
    50 mg/kg mAb
    Leukocytopenia (9 dpi), thrombocytopenia (6, 9 dpi)Survived
      A23 dpi Ad-IFN and
    50 mg/kg mAb
    Fever (22 dpi), leukocytopenia (6, 9, 25 dpi), leukocytosis (22 dpi),
    thrombocytopenia (9, 16 dpi), thrombocytosis (3 dpi)
    Died at 25 dpi
    ALT↑ (3 dpi), ALP↑↑↑ (25 dpi), AMY↑ (3, 6, 16 dpi), BUN↓ (9 dpi),
    CRE↓ (25 dpi), GLU↑↑↑ (25 dpi)
      A33 dpi Ad-IFN and
    50 mg/kg mAb
    Fever (22 dpi), leukocytopenia (9 dpi), leukocytosis (16, 22 dpi),
    thrombocytopenia (6 dpi)
    Survived
    ALB↓ (16 dpi), ALT↑ (6, 9, 16 dpi)
      A43 dpi Ad-IFN and
    50 mg/kg mAb
    Leukocytosis (6, 16, 22 dpi), thrombocytopenia (3, 6, 8, 28 dpi)Survived
    TBIL↑ (6, 8 dpi)
      B12 dpi mAb controlSevere rash (5 dpi), leukocytopenia (5 dpi), thrombocytopenia (5 dpi)Died at 5 dpi
    TBIL↑ (5 dpi), BUN↑ (5 dpi), CRE↑↑ (5 dpi)
      B22 dpi PBS controlSevere rash (5 dpi), thrombocytopenia (5 dpi)Died at 5 dpi
    ALP↑ (5 dpi), ALT↑↑↑ (5 dpi), BUN↑ (5 dpi), CRE↑↑ (5 dpi)
    Cyno96h
      C11 dpi Ad-IFN and
    4 dpi 50 mg/kg mAb
    Fever (4 dpi), severe rash (6 dpi),
    leukocytopenia (6 dpi), thrombocytopenia (4, 6 dpi)
    Died at 6 dpi
    ALT↑↑↑ (6 dpi), AMY↑↑↑ (6 dpi), TBIL↑↑ (6 dpi), BUN↑↑ (6 dpi),
    PHOS↑↑↑ (6 dpi), CRE↑↑↑ (6 dpi), GLU↑↑↑ (6 dpi)
      C21 dpi Ad-IFN and
    4 dpi 50 mg/kg mAb
    Fever (21 dpi), mild rash (14 dpi), leukocytosis (10, 21, 28 dpi),
    thrombocytopenia (7, 10, 14 dpi)
    Survived
    ALP↑ (14, 21, 28 dpi), GLU↑ (4, 7, 10 dpi)
      C31 dpi Ad-IFN and
    4 dpi 50 mg/kg mAb
    Hypothermia (14 dpi), leukocytopenia (7, 14 dpi), thrombocytopenia (4, 7, 10, 14, 21 dpi)Survived
    CRE↑ (7 dpi)
      C41 dpi Ad-IFN and
    4 dpi 50 mg/kg mAb
    Hypothermia (10, 23 dpi), leukocytopenia (7, 14 dpi), leukocytosis (21 dpi),
    thrombocytopenia (7, 10, 14 dpi)
    Died at 23 dpi
    BUN↑ (23 dpi), GLU↑ (23 dpi), GLOB↑ (21, 23 dpi)
      D11 dpi PBS and
    4 dpi mAb control
    Severe rash (6 dpi), leukocytopenia (6 dpi), thrombocytopenia (4, 6 dpi)Died at 6 dpi
    ALP↑ (6 dpi), TBIL↑ (6 dpi), GLU↑ (6 dpi)
      D21 dpi PBS and
    4 dpi PBS control
    Severe rash (6 dpi), leukocytopenia (5 dpi), leukocytosis (2 dpi),
    thrombocytopenia (4, 5 dpi)
    Died at 5 dpi
    ALB↓↓↓ (5 dpi), ALP↑↑↑ (5 dpi), ALT↑↑↑ (5 dpi), AMY↑↑ (5 dpi),
    BUN↑↑ (5 dpi), PHOS↑↑↑ (5 dpi), CRE↑↑↑ (5 dpi), GLU↓ (5 dpi), GLOB↓↓↓ (5 dpi)
    Rhesus72h
      E13 dpi Ad-IFN and
    50 mg/kg mAb
    Survived
      E23 dpi Ad-IFN and
    50 mg/kg mAb
    Thrombocytopenia (6 dpi)Survived
      E33 dpi Ad-IFN and 50
    mg/kg mAb
    Survived
      E43 dpi Ad-IFN and
    50 mg/kg mAb
    Leukocytosis (3, 9 dpi)Survived
      F13 dpi mAb controlModerate rash (6 dpi), leukocytopenia (7 dpi), thrombocytopenia
    (6, 7 dpi), ALB↑ (7 dpi), ALT↑↑↑ (6, 7 dpi), TBIL↑ (6, 7 dpi), BUN↑ (6, 7 dpi),
    PHOS↑↑ (7 dpi), CRE↑↑ (6 dpi) ↑↑↑ (7 dpi), GLU↓ (7 dpi), GLOB↑↑↑ (7 dpi)
    Died at 7 dpi
      F23 dpi Ad-IFN only controlMild rash (6 dpi), moderate rash (9 dpi), leukocytosis (3 dpi),
    thrombocytopenia (6, 9 dpi)
    Died at 9 dpi
    ALT↑↑ (6, 7 dpi), PHOS↓ (9 dpi)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/5/207/207ra143/DC1

    Fig. S1. EBOV/May-eGFP–neutralizing antibody assays.

    Fig. S2. Survival curves and time to death of historical control rhesus and cynomolgus macaques.

    Table S1. Ebola viremia values at various times post-challenge as measured by qRT-PCR.

    Table S2. Viremia by TCID50.

    Table S3. Clinical score.

    Table S4. Temperature.

    Table S5. ALT.

    Table S6. ALP.

    Table S7. PLT.

    Table S8. WBCs.

    Table S9. PCR results for animal C4 for EBOV and 16S DNA.

    Table S10. IgM titers.

    Table S11. IgG titers.

    Table S12. nAb titers.

    Table S13. ELISpot counts.

    Table S14. Cytokine positivity of CD8+ cells.

    Table S15. Cytokine positivity of CD8+ CD107a+ cells.

    Table S16. Cytokine positivity of CD8+ IL-2+ cells.

    Table S17. Cytokine positivity of CD8+ IFN-γ+ cells.

    Table S18. Cytokine positivity of CD8+ IL-4+ cells.

    Table S19. Cytokine positivity of CD4+ cells.

    Table S20. Cytokine positivity of CD4+ CD107a+ cells.

    Table S21. Cytokine positivity of CD4+ IL-2+ cells.

    Table S22. Cytokine positivity of CD4+ IFN-γ+ cells.

    Table S23. Cytokine positivity of CD4+ IL-4+ cells.

  • Supplementary Material for:

    mAbs and Ad-Vectored IFN-α Therapy Rescue Ebola-Infected Nonhuman Primates When Administered After the Detection of Viremia and Symptoms

    Xiangguo Qiu, Gary Wong, Lisa Fernando, Jonathan Audet, Alexander Bello, Jim Strong, Judie B. Alimonti, Gary P. Kobinger*

    *Corresponding author. E-mail: Gary.Kobinger@phac-aspc.gc.ca

    Published 16 October 2013, Sci. Transl. Med. 5, 207ra143 (2013)
    DOI: 10.1126/scitranslmed.3006605

    This PDF file includes:

    • Fig. S1. EBOV/May-eGFP–neutralizing antibody assays.
    • Fig. S2. Survival curves and time to death of historical control rhesus and cynomolgus macaques.
    • Table S1. Ebola viremia values at various times post-challenge as measured by qRT-PCR.
    • Table S2. Viremia by TCID50.
    • Table S3. Clinical score.
    • Table S4. Temperature.
    • Table S5. ALT.
    • Table S6. ALP.
    • Table S7. PLT.
    • Table S8. WBCs.
    • Table S9. PCR results for animal C4 for EBOV and 16S DNA.
    • Table S10. IgM titers.
    • Table S11. IgG titers.
    • Table S12. nAb titers.
    • Table S13. ELISpot counts.
    • Table S14. Cytokine positivity of CD8+ cells.
    • Table S15. Cytokine positivity of CD8+ CD107a+ cells.
    • Table S16. Cytokine positivity of CD8+ IL-2+ cells.
    • Table S17. Cytokine positivity of CD8+ IFN-γ+ cells.
    • Table S18. Cytokine positivity of CD8+ IL-4+ cells.
    • Table S19. Cytokine positivity of CD4+ cells.
    • Table S20. Cytokine positivity of CD4+ CD107a+ cells.
    • Table S21. Cytokine positivity of CD4+ IL-2+ cells.
    • Table S22. Cytokine positivity of CD4+ IFN-γ+ cells.
    • Table S23. Cytokine positivity of CD4+ IL-4+ cells.

    [Download PDF]

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