Fig 1.
Human mAb provides limited protection from Ebola virus disease in ferrets.
Ferrets were inoculated with EBOV variant Makona-C07 and treated with a human mAb cocktail in two independent experiments. In both the first (A, B) and second (C, D) experiment, ferrets were inoculated with a target dose of 1000 TCID50 EBOV via the intranasal (IN; n = 10/experiment) or intramuscular (IM; n = 10/experiment) routes. In the first experiment, 8 ferrets from each inoculation group were treated with 30 mg/kg each antibody delivered via the intraperitoneal (IP) route “early”, on 2 and 5 days post-infection (DPI), or “late”, on 3 and 6 DPI. Two ferrets from each inoculation group served as control animals and were treated with PBS only on 2 and 5 DPI. Animals were monitored for 29 days for survival (A) and weight change (B). In the second experiment, 8 ferrets from each inoculation group were treated with 30 mg/kg each antibody delivered via the IP route “very early”, on 2 and 4 DPI, or “mid”, on 3 and 5 DPI. One ferret from the IN group served as a control animal and was treated with PBS only on 2 and 4 DPI. Animals were monitored for 36 days for survival (C) and weight change (D). On all graphs, the vertical area shaded grey, from 12 to 18 DPI, represents the window in which atypical disease was observed.
Table 1.
Outcomes in mAb-treated ferrets.
Fig 2.
Levels of viral RNA and infectious virus differ among acute, atypical, and surviving ferrets.
Viral RNA loads (genome equivalents [GEQ] per millilitre) in the blood were determined over time for the acute, atypical, and surviving ferrets (A). The vertical area shaded grey, from 12 to 18 DPI, represents the window in which atypical disease was observed. Viral RNA loads (in GEQ/ml or GEQ/g) at the terminal time points are depicted separately for the blood (B), liver (D), spleen (F), and brain (H) samples, with means and standard deviations indicated. Mean levels were compared using a one-way ANOVA with Tukey’s multiple comparison test. Infectious virus loads at the terminal time points are depicted in median tissue culture infectious dose (TCID50) per ml or g, with means and standard deviations indicated, for the blood (C), liver (E), spleen (G), and brain (I) samples. Mean levels were compared using a t test. ns, not significant; *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001.
Fig 3.
Brain histopathology in animals that died of atypical EVD.
Ferret brain tissue sections stained by RNA in situ hybridization (red; A–C) or immunohistochemistry (brown; D) from an acute ferret (A, B) and an atypical ferret (C, D). EBOV genomic RNA (red) was only detected in endothelial vessels in the brain (A, B) of the acute ferret. Both EBOV genomic RNA (red; C) and antigen (brown; D) were detected in the inflammatory cells of meninges and ventricular system of the atypical animal. Quantitative analysis of EBOV genomic RNA distribution was performed by scoring the severity of RNA ISH signal in the meninges and ventricular systems in the acute and atypical ferrets (E). Representative brain tissue sections stained with hematoxylin and eosin (H&E) from an acute ferret (F, G) and an atypical ferret (H–K), revealing meningoencephalitis (H, I), ventriculitis (J), and choroid plexitis (K) in the brains of atypical ferrets (H–K) in comparison to unnoticeable changes in the brains of acute animals (F, G). Quantitative analysis of histopathological lesions detected in the brain regions of atypical and acute ferrets, including the ventricular system, meninges, and cerebral cortex (L). Nuclei were counterstained blue with hematoxylin (A-B, F-K). ISH (E) and histopathology (L) severity scores are indicated for each analyzed animal (represented by a single dot), with means and standard deviations indicated. Mean levels were compared using unpaired t-tests (Holm-Sidak). ns, not significant; *, p ≤ 0.05.
Fig 4.
Transcriptomic and proteomic analyses of ferret brain tissue.
Brain tissue samples from a subset of ferrets (all from the second challenge experiment) were subjected to transcriptomic and proteomic analyses. A heatmap from the transcriptomic analysis shows the differential expression of the fifty named genes with the most variable expression, with log2 fold change centered for each gene (A). A heatmap from the proteomic analysis shows the differential abundance of the fifty proteins with the highest variance among all animals, with log2 fold change centered for each protein (B).
Fig 5.
Blood biochemistry parameters for acute, atypical, and surviving ferrets.
Levels of alanine aminotransferase (ALT) (A, B), alkaline phosphatase (ALP) (C,D), total bilirubin (E, F), albumin (G, H), globulin (I, J), glucose (K, L), and total protein (M, N) were quantified in all blood samples from all animals. Levels of each of these analytes are depicted over time (A, C, E, G, I, K, M). The vertical area shaded grey, from 12 to 18 DPI, represents the window in which atypical disease was observed, while the horizontal area shaded grey represents the normal range for each parameter. The levels of each analyte at the terminal time points are also depicted separately (B, D, F, H, J, L, N), with means and standard deviations indicated. Mean levels were compared using a one-way ANOVA with Tukey’s multiple comparison test. ns, not significant; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
Fig 6.
Blood cell counts for acute, atypical, and surviving ferrets.
Lymphocytes (A, B), neutrophils (C, D), monocytes (E, F), white blood cells (G, H), and platelets (I, J) were enumerated in all blood samples from all animals. Cell counts are depicted over time (A, C, E, G, I). The vertical area shaded grey, from 12 to 18 DPI, represents the window in which atypical disease was observed, while the horizontal area shaded grey represents the normal range for each cell type. Cell counts at the terminal time points are also depicted separately (B, D, F, H, J), with means and standard deviations indicated. Mean levels were compared using a one-way ANOVA with Tukey’s multiple comparison test. ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
Fig 7.
The cytokine response in acute, atypical, and surviving ferrets.
Levels of IP-10 (A, B), MCP-1 (C, D), MIP-1β (E, F), IL-6 (G, H), IL-12p40 (I, J), IL-8 (K, L), and IL-4 (M, N) were quantified in all serum samples from all animals via Luminex assay. Levels of each cytokine are depicted over time (A, C, E, G, I, K, M). The vertical area shaded grey, from 12 to 18 DPI, represents the window in which atypical disease was observed. The levels of each cytokine at the terminal time points are also depicted separately (B, D, F, H J, L, N), with means and standard deviations indicated. Mean levels were compared using a one-way ANOVA with Tukey’s multiple comparison test. ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
Fig 8.
Antibody quantification in acute, atypical, and surviving ferrets.
Levels of EBOV GP-specific IgG were determined via ELISA (A, B) in all serum samples from the atypical and surviving animals, starting with samples collected on 9 DPI. Levels of antibody are depicted over time (A), with each line in the graph off-set slightly relative to the x-axis to ensure that data from individual animals could be discriminated. EBOV GP-specific IgG levels at the terminal time points, as well as at 14 DPI for the surviving animals, are also depicted separately (B), with means and standard deviations indicated. The red dotted line indicates the lower limit of quantification for this assay. Total levels of human IgG were determined via ELISA (C, D) in all serum samples from all animals. Levels of antibody are depicted over time (C). Human IgG levels at the terminal time points are also depicted separately (D), with means and standard deviations indicated. The vertical area shaded grey, from 12 DPI to 18 DPI, in (A) and (C) represents the window in which atypical disease was observed. Mean levels in (B) and (C) were compared using a one-way ANOVA with Tukey’s multiple comparison test. ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.