A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus

Abstract

To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038μM and 0.016μM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50–62%) or in combination (87%). They exhibited lower IC50 (0.98–6.2μM) compared with FPV (36.8μM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25–25μM). When dosed >50μM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.

Evaluation of the Activity of Lamivudine and Zidovudine against Ebola Virus

“…A recent publication reported that HIV-1 drugs inhibit EBOV virus-like particles (trVLPs) that undergo a single cycle of transcription and replication [16]. The drugs showed activity alone or in combination against the transcription-competent trVLPs, but limited efficacy against EBOV-eGFP [16].…”

“…The drugs showed activity alone or in combination against the transcription-competent trVLPs, but limited efficacy against EBOV-eGFP [16]. To investigate further these findings, we repeated the studies using the same cell lines, compound source, time of drug addition, and length of infection time to compare with the conditions we used to perform the drug screen study on EBOV/Mak (S2 Fig).…”

“…We previously reported lamivudine had no antiviral activity against EBOV in Vero E6, Hep G2, or monocyte-derived macrophages (MDMs) infected with the 1995 EBOV Kikwit variant [15]. Given the continued interest of using lamivudine to treat EVD and the results of a recent article showing in vitro antiviral activity against EBOV infection [16], we further evaluated the efficacy of lamivudine and a second HIV-1 drug, zidovudine, in a broad range of infected cell lines. Tested cell lines included Vero E6, Huh 7, HeLa, Hep G2, and 293T cells, and primary MDMs, using varying multiplicities of infections (MOIs), drugs from different sources, and different time courses of drug treatment and assay endpoints.…”

Antiviral Screening of Multiple Compounds against Ebola Virus

“…In this screen, the drug Tasigna was not retested due to compound unavailability, but additionally, the drug Gleevec was included since it was reported to operate via the same mechanism and demonstrated better in vitro efficacy. The in vitro screens used viral RNA levels as a readout based on previous antiviral testing work [33] and infectious doses of MOI 0.01–0.1, similar to levels used in other anti-EBOV testing studies [6,34,35]. Given that the compounds tested in our study were chosen due to their perceived potential effectiveness for use against EBOV, the negative results demonstrate the importance of testing therapies using the actual live pathogen to determine effects.…”

Clinical Evaluation of Ebola Virus Disease Therapeutics

“…In another study, six doses of human IFN-β (10.5 ug/kg) administered subcutaneously (SC) extended the survival time of NHPs challenged with EBOV or Marburg virus , but did not improve the survival rate [53], suggesting that IFN treatments might be beneficial, but likely not fully protective by themselves. Based on the in vitro observation that IFN-β could inhibit the replication of recombinant EBOV in HEK293 cells more strongly than IFN-α could [55], a historically controlled clinical trial tested the efficacy of IFN-β-1a in nine EVD patients in Guinea [56]. Within 2 days following qRT-PCR-mediated confirmation of EVD, IFN-β-1a (30 ug/day) was administered SC to patients daily, until patients were tested negative for EBOV, or perished [56].…”