Ebola virus-induced eye sequelae: a murine model for evaluating glycoprotein-targeting therapeutics.

BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.

Effects of Toll-like receptor 1 and 2 agonist Pam3CSK4 on uveal melanocytes and relevant experimental mouse model.

Pam3CSK4 activates Toll-like receptors 2 and 1 (TLR1/2), which recognize mainly molecules from gram-positive pathogens. The effect of Pam3CSK4 on various cytokine and chemokine expression in cultured human uveal melanocytes (UM) has not been studied systematically. The purpose of this study was to investigate the mechanistic expressions of seven cytokines and chemokines of interleukin- (IL-) 6, IL-10, MCP-1 (CCL-2), CXCL-1 (GRO-α), CXCL-8 (IL-8), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in UM. These cytokines are reported to be increased in intraocular fluids or tissues of the patients with endophthalmitis and non-infectious uveitis, as well as in various experimental animal uveitic models in the literature. Flow cytometry was used to measure the effects of Pam3CSK4 on the expression of TLR1/2 in UM. ELISA and Real-time PCR analysis were used to estimate the ability of Pam3CSK4 to elevate these cytokines and chemokines levels in conditioned media and cell lysates of UM, respectively. Flow cytometry measured and compared the phosphorylated MAPK pathway and activated NF-κB signals pathway in UM, treated with and without Pam3CSK4. ELISA analysis tested the effect of various signal inhibitors (ERK1/2, JNK1/2, p38 and NF-κB) on Pam3CSK4-induced IL-6 levels in cultured UM. The role of TLR2 in Pam3CSK4-induced acute anterior uveitis in experimental mouse model was tested in TLR2 knockout (TLR2 KO) mice and their wild-type C57Bl/6 controls. Pam3CSK4 increased the expression of TLR1/2 proteins in cultured UM. Pam3CSK4 significantly elevated the IL-6, MCP-1, CXCL-1, CXCL-8 protein, and mRNA levels in cultured UM, but not IL-10, TNF-α, or IFN-γ. Pam3CSK4 activated NF-κB, ERK, JNK, and p38 expression. Pam3CSK4-induced expression of IL-6 was decreased by NF-κB, ERK, INK, and p38 inhibitors; especially the NF-κB inhibitor, which can completely block the IL-6 stimulation. Intravitreal injection of Pam3CSK4 induced acute anterior uveitis in C57Bl/6 mice, this effect was significantly reduced in TLR2 KO mice. TLR1/2 plays an important role against invading pathogens, especially gram-positive bacteria; but an excessive reaction to molecules from gram-positive bacteria may promote non-infectious uveitis. UM can produce IL-6, MCP-1, CXCL-1, and CXCL-8, and are one of the target cells of TNF-α and IFN-γ. TLR-2 inhibitors might have a beneficial effect in the treatment of certain types of uveitis and other ocular inflammatory-related diseases and warrant further investigation.