Research ArticleEbola virus

Successful Treatment of Ebola Virus–Infected Cynomolgus Macaques with Monoclonal Antibodies

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Science Translational Medicine  13 Jun 2012:
Vol. 4, Issue 138, pp. 138ra81
DOI: 10.1126/scitranslmed.3003876

Figures

  • Fig. 1

    ZMAb improves survival of EBOV-challenged NHPs. (A) Nine cynomolgus macaques were challenged with 1000 PFU of EBOV (red arrow). Two groups (n = 4) were treated with ZMAb beginning either at 24 hours (days 1, 4, and 7; blue arrows) or at 48 hours (days 2, 5, and 8; pink arrows). A control animal (n = 1) received PBS (days 1, 4, and 7). The animals were sampled on treatment days plus days 14 and 21 (black arrows) and examined for (B) serum murine mAb levels by an Ebola virus–like particle (eVLP) ELISA, (C) survival, and (E) viremia measured by real-time RT-PCR (GEQ, genome equivalents) or (F) TCID50. Only PCR-positive samples were assayed for TCID50. (D) Historical cynomolgus macaque controls over the last 2 years, using the same challenge virus stock and dose, are presented with their time to death after challenge. All macaques have consistent symbols across panels.

  • Fig. 2

    Clinical overview. During the EBOV experiment, various clinical parameters were assessed for the 24-hour, 48-hour, and control animals. (A) The percent change in weight was expressed as a change from baseline values taken on day 1 (control and 24 hours) or day 2 (48 hours). (B) Temperature for each animal on each exam date. (C) The animals were scored daily for signs of illness, and the daily clinical score reached over 19 days is presented. (D to F) Variations in blood biochemistry and hematological levels were noted for (D) alanine aminotransferase (ALT), (E) alkaline phosphatase (ALP), and (F) platelets (PLT). All macaques have consistent symbols across panels.

  • Fig. 3

    EBOV-GP–specific antibody response. (A and B) During the EBOV challenge experiment, the NHP serum was quantified by eVLP ELISA to determine the levels of EBOV-GP–specific NHP (A) IgM and (B) IgG. (C) A fluorescence neutralization assay using EBOV-eGFP was used to determine the EBOV-GP–specific NHP neutralizing antibody levels. All macaques have consistent symbols across panels.

  • Fig. 4

    Cellular immune response in surviving animals 21 days after challenge. On 21 dpi, the surviving animals’ PBMCs were isolated by Ficoll gradient and exposed in vitro to four EBOV-GP peptide pools spanning GP. (A and B) The frequency of PBMCs secreting IFN-γ was measured by (A) ELISpot as represented by the number of spot-forming cells (SFC) per million PBMCs for each animal and by (B) flow cytometry to demonstrate which subpopulations of PBMCs produced IFN-γ. Flow cytometry density plots gated on lymphocytes represent IFN-γ versus CD8 (left panel) and CD4 (right panel) expression in PBMCs stimulated in vitro with the EBOV-GP peptide pool 3. (C and D) A graphical representation of the number of (C) CD8+ or (D) CD4+ IFN-γ–producing cells per million lymphocytes was determined by flow cytometry for survivors.

  • Fig. 5

    Identification of IL-2 and IFN-γ–producing cells upon in vitro stimulation by EBOV-GP peptide pools using flow cytometry. (A) Sample plot demonstrating the gating using NHP A3 PBMCs stimulated by EBOV-GP peptide pool 3. (B and C) Relative distribution of (B) IL-2+ and (C) IFN-γ PBMCs from day 21, summed for all four peptide pools in the CD4+, CD8+, and double-negative (CD4CD8) cell populations. In (C), the light blue part of the small circles (right panel) represents the IL-2+ population of IFN-γ+ cells, whereas the darker shade represents cells that only produce IFN-γ+.

  • Fig. 6

    EBOV-GP sequences isolated from nonsurvivors. EBOV RNA was isolated on 8 and 11 dpi from the blood of the two nonsurvivors B2 and B3, respectively, and compared to the challenge EBOV (CHALLENGE), as well as other EBOV-GP sequences found in GenBank. Reference sequences for the EBOV-GP were obtained from GenBank by searching for [(Ebolavirus glycoprotein) AND “Zaire ebolavirus”[porgn,__txid186538]”]. Every sequence of at least 2000 base pairs that was not a full genome was used for the alignment. The alignment was performed with DNASTAR Lasergene 9 MegAlign software using the Clustal W algorithm. The aligned sequences were prepared using the GeneDoc software. For the amino acid sequence associated with the amino acid 508 mutation, an additional A was added at position 885 to account for the transcriptional insertion that is required for full-length GP. (A) Alignment of the sequences around amino acid 275 showing no previous isolates having the W275L mutation. (B) Alignment of the sequences around amino acid 508 showing no previous isolates having the Q508R mutation.

Tables

  • Table 1

    Viral shedding detected by real-time RT-PCR. Values are expressed as GEQ per swab. A1 to A4 animals were part of the 24-hour treatment group; the samples for these animals and the control animal (C1) were taken at 1, 4, 7, and 14 dpi. B1 to B4 animals were part of the 48-hour treatment group; the samples for these animals were taken at 2, 5, 8, and 14 dpi. ND, not done; —, not detected.

    dpiA1A2A3A4B1B2B3B4C1
    1OralNDNDNDND
    NasalNDNDNDND
    RectalNDNDNDND
    2OralNDNDNDNDND
    NasalNDNDNDNDND
    RectalNDNDNDNDND
    4OralNDNDNDND
    NasalNDNDNDND
    RectalNDNDNDND31.8
    5OralNDNDNDND5.77 × 103
    NasalNDNDNDND5.38 × 103
    RectalNDNDNDND35.31.48 × 104
    7OralNDNDNDNDND
    NasalNDNDNDNDND
    RectalNDNDNDNDND
    8OralNDNDNDND12.0ND
    NasalNDNDNDND4.39 × 10342.0ND
    RectalNDNDNDND2.06 × 102ND
    11OralNDNDNDNDNDND13.4NDND
    NasalNDNDNDNDNDND6.94 × 102NDND
    RectalNDNDNDNDNDND4.85 × 102NDND
    14OralNDNDND
    NasalNDNDND
    RectalNDNDND
  • Table 2

    Clinical findings. Fever was defined as a temperature ≥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 white blood cells (WBCs) and platelets, respectively. Leukocytosis and thrombocytosis were defined as a twofold or greater increase in numbers of platelets and WBCs over baseline and also a WBC count >11,000. ↑, two- to threefold increase; ↑↑, four- to fivefold increase; ↑↑↑, greater than fivefold increase. ALP, alkaline phosphatase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; GLOB, globulin; GLU, glucose; TBIL, total bilirubin; ND, not done; —, no change.

    Animal ID sex
    group
    Day 4 or 5Day 7 or 8Day 11Day 14Day of death
    A1LeukocytosisNDThrombocytopenia,
    leukocytosis
    Survived
    A2Fever, thrombocytosisThrombocytosis, GLU↑NDThrombocytosisSurvived
    A3Fever, TBIL↑, GLOB↑,
    leukocytosis
    NDThrombocytosis,
    GLOB↑
    Survived
    A4LeukocytosisFever, ALP↑NDThrombocytosis,
    ALP↑, ALT↑
    Survived
    B1FeverThrombocytopenia,
    TBIL↑, GLOB↑,
    leukocytosis
    NDThrombocytosis,
    GLOB↑
    Survived
    B2FeverFever, leukocytopenia,
    mild rash, anorexia,
    ALT↑, ALP↑, GLOB↑
    ND8
    B3LeukocytosisFever,
    thrombocytopenia,
    anorexia, ALT↑,ALP↑,
    TBIL↑, BUN↑, GLOB↑,
    leukocytosis
    Fever,
    thrombocytopenia,
    anorexia, ALT↑↑↑,
    ALP↑↑↑, TBIL↑,
    BUN↑, GLOB↑,
    GLU↑, leukocytosis
    11
    B4Thrombocytopenia, ALP↑NDThrombocytosis,
    leukocytosis
    Survived
    C1Fever, anorexia,
    leukocytosis, ALT↑,
    ALP↑, BUN↑
    ND5