An Ebola Virus-Like Particle-Based Reporter System Enables Evaluation of Antiviral Drugs In Vivo under Non-Biosafety Level 4 Conditions

Abstract

Ebola virus (EBOV) is a highly contagious lethal pathogen. As a biosafety level 4 (BSL-4) agent, however, EBOV is restricted to costly BSL-4 laboratories for experimentation, thus significantly impeding the evaluation of EBOV vaccines and drugs. Here, we report an EBOV-like particle (EBOVLP)-based luciferase reporter system that enables the evaluation of anti-EBOV agents in vitro and in vivo outside BSL-4 facilities. Cotransfection of HEK293T cells with four plasmids encoding the proteins VP40, NP, and GP of EBOV and firefly luciferase (Fluc) resulted in the production of Fluc-containing filamentous particles that morphologically resemble authentic EBOV. The reporter EBOVLP was capable of delivering Fluc into various cultured cells in a GP-dependent manner and was recognized by a conformation-dependent anti-EBOV monoclonal antibody (MAb). Significantly, inoculation of mice with the reporter EBOVLP led to the delivery of Fluc protein into target cells and rapid generation of intense bioluminescence signals that could be blocked by the administration of EBOV neutralizing MAbs. This BSL-4-free reporter system should facilitate high-throughput screening for anti-EBOV drugs targeting viral entry and efficacy testing of candidate vaccines. IMPORTANCE Ebola virus (EBOV) researches have been limited to costly biosafety level 4 (BSL-4) facilities due to the lack of animal models independent of BSL-4 laboratories. In this study, we reveal that a firefly luciferase-bearing EBOV-like particle (EBOVLP) with typical filamentous EBOV morphology is capable of delivering the reporter protein into murine target cells both in vitro and in vivo.Moreover, we demonstrate that the reporter delivery can be inhibited both in vitro and in vivo by a known anti-EBOV protective monoclonal antibody, 13C6. Our work provides a BSL-4-free system that can facilitate the in vivo evaluation of anti-EBOV antibodies, drugs, and vaccines. The system may also be useful for mechanistic study of the viral entry process. E bola virus (EBOV) is one of the most virulent and lethal human pathogens known. It is responsible for the 2013-2015 Ebola epidemic in West Africa, the greatest outbreak in history (1, 2), in which more than 28,000 suspected cases had been reported and over 11,000 deaths had been recorded as of September 2015. Due to the high transmissibility and mortality associated with the virus and the growing globalization that may facilitate the rapid spread of the virus around the world, EBOV is now recognized as a major threat to global public health and social stability. Therefore, the development of vaccines and therapeutics against EBOV is urgently needed (3, 4). However, EBOV is a biosafety level 4 (BSL-4) pathogen (5). Handling of various infectious forms of EBOV, including clinical isolates (6, 7); mouse/guinea pigadapted strains (7-9); and recombinant EBOVs expressing reporter proteins, such as green fluorescent protein (10) or firefly luciferase (Fluc) (11), is highly restricted and can be performed only in BSL-4 facilities, grea...

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection

“…Disparities in previous antibody-mediated neutralization results could be due to differing experimental conditions and model systems (Davidson et al, 2015; Li et al, 2016; Wilkinson et al, 2017; Wilson et al, 2000). Thus, we characterized the neutralization of each mAb in three assays: i) replication-competent vesicular stomatitis virus bearing EBOV GP (rVSV); ii) biologically contained EBOV (ΔVP30) (Halfmann et al, 2008); and iii) authentic EBOV (Figure 2) performed under BSL-2+, BSL-3 and BSL-4 containment, respectively, as previously described, to ensure consistency with previous studies.…”

Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood

“…We reasoned against chemically synthesized RNA as a template because blood nucleases rapidly degrade such biomaterials at ambient temperatures, 20 which could prove problematic with minimal assay formulations. On the other hand, de novo construction of in vitro produced, non-infectious, and/or replication-deficient EBOV virions, 21 an approach common for many well-studied species of mammalian viruses, was actively discouraged outside high containment for reasons of health, safety, and biosecurity. We thus elected to use commercially available recombinant MS2 coliphage as our primary surrogate template (AR14), despite the robustness of this virion compared to the easily fragmentable nature of filovirus particles, 22 and pseudotyped lentivirus (PV) based on HIV manufactured in-house as a fallback template (see ESI, Fig.…”

Antibody-dependent-cellular-cytotoxicity-inducing antibodies significantly affect the post-exposure treatment of Ebola virus infection

“…Recently, some pseudoviral-particle neutralization assays based on the luciferase reporter system have been reported that allow the evaluation of anti-EBOV agents in laboratories with a lower biosafety level of containment (BSL-2)1920. The EBOV glycoprotein (GP) is solely responsible for viral attachment to, fusion with, and entry into new host cells, and it is therefore a major target of vaccine and drug design efforts2122.…”