Natural history of nonhuman primates after conjunctival exposure to Ebola virus

Abstract

Transmission of Ebola virus (EBOV) primarily occurs via contact exposure of mucosal surfaces with infected body fluids. Historically, nonhuman primate (NHP) challenge studies have employed intramuscular (i.m.) or small particle aerosol exposure, which are largely lethal routes of infection, but mimic worst-case scenarios such as a needlestick or intentional release, respectively. When exposed by more likely routes of natural infection, limited NHP studies have shown delayed onset of disease and reduced mortality. Here, we performed a series of systematic natural history studies in cynomolgus macaques with a range of conjunctival exposure doses. Challenge with 10,000 plaque forming units (PFU) of EBOV was uniformly lethal, whereas 5/6 subjects survived lower dose challenges (100 or 500 PFU). Conjunctival challenge resulted in a protracted time-to death compared to i.m. Asymptomatic infection was observed in survivors with limited detection of EBOV replication. Inconsistent seropositivity in survivors may suggest physical or natural immunological barriers are sufficient to prevent widespread viral dissemination.

Figure 1

Survival analysis and determination of viral load in EBOV-challenged macaques. (A) Kaplan–Meier survival curves of cynomolgus macaques challenged with low (100–500 PFU) and high (10,000 PFU) doses of EBOV variant Makona. Arrows below x-axis denote scheduled sampling days. Colored asterisks denote statistical significance to the same colored group. **p ≤ 0.01. Viral load was determined by plaque titration of plasma (B–D) and RT-qPCR detection of EBOV vRNA (E–G) from whole blood.

Figure 2

Representative gross pathology in cynomolgus macaques infected with EBOV Makona variant via conjunctival route. (A) Lack of significant hepatic lesions in a 100PFU survivor (100-5), (B) marked necrotizing hepatitis (500-4), (C) marked diffuse necrotizing hepatitis (100-4), (D) marked axillary petechial rash (10K-6), (E) multifocal hemorrhagic interstitial pneumonia (black arrows) (10K-6).

Figure 3

Representative histologic lesions in cynomolgus macaques infected with EBOV Makona variant via conjunctival route. Representative tissues of cynomolgus macaques from 100 PFU, survivor (100-6) (A–F) and succumbed 11 dpi (100-4) (G–L), 500 PFU, succumbed 11 dpi (500-4) (M-R) and 10,000 PFU, succumbed 9 dpi (10K-6) (S–X). All images captured at 20x, insets captured at 40x. Hematoxylin and eosin (H&E) staining (A,G,M,S,C,I,O,U,E,K,Q,W) and immunohistochemistry (IHC) for anti-EBOV antigen (B,H,N,T,D,J,J,Inset,P,P inset,V,V inset, F,L,R,X). No significant lesions (NSL) and no significant immunolabeling (NSI) for spleen (A,B), lung (C,D) and liver (E,F) of 100 PFU survivor. Splenitis with lymphoid depletion and fibrin deposition in NHPs that succumb at 100 PFU (G), 500 PFU (M) and 10,000 PFU (S). Diffuse cytoplasmic immunolabeling of mononuclear cells (brown) in red and white pulp of the spleen in those that succumb (H,N,T). Diffuse interstitial pneumonia (I,O,U) with alveolar hemorrhage (U, black arrow). Diffuse cytoplasmic immunolabeling (brown) of mononuclear cells within the lung (J,P,V) and alveolar spaces (insets J,P,V, black arrows). Necrotizing hepatitis (K,Q,W). Diffuse cytoplasmic immunolabeling (brown) of Kupffer cells, sinusoidal lining cells and rarely hepatocytes (L,R,X).

Figure 4

Representative histologic lesions in the brain of cynomolgus macaques infected with EBOV Makona variant via conjunctival route. Representative tissues of cynomolgus macaques from 100 PFU, survivor (100-6) (A–C) and succumbed 11 dpi (100-4) (D–F), 500 PFU, succumbed 11 dpi (500-4) (G–I) and 10,000PFU, succumbed 9 dpi (10K-6) (J–L). All images captured at 40×. Hematoxylin and eosin (H&E) staining (A,D,G,J), immunohistochemistry (IHC) for anti-EBOV antigen (B,E,H,K), in situ hybridization (C,F,I,L). Mild gliosis (A,D,G,J). Diffuse cytoplasmic immunolabeling for anti-EBOV antigen in neuronal cells (B, brown), endothelium of small caliber vessels (E,H,K). Punctate labeling (red) in a single neuronal cell of 100 PFU survivor (100-6) (c, black arrow) and endothelium of small caliber vessels (F,I,L).

Figure 5

Circulating levels of inflammatory markers in EBOV-challenged macaques. Absolute values of each analyte measured for each subject at the indicated timepoints, and normalized to baseline (day 0/challenge) values. Shown is the average value for all animals in the indicated group. Data is shown as a proportion of baseline (set to 1, indicated by dashed horizontal line in each panel) to facilitate plotting on a log scale. Data from historical control animals inoculated i.m. with the homologous virus were included for statistical purposes. (A) IFN-γ; (B) IL-1β; (C) TNF-α; (D) IL-18; (E) IL-6; (F) IL-1RA; (G) MIP-1α; (H) MCP-1; (I) IL-4; (J) IL-13. All assays were run in duplicate reactions.