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

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.


Introduction
As of December 13, 2015, the current outbreak of Ebola virus disease (EVD) in West Africa has resulted in 28,633 cumulative cases and 11,314 deaths [1]. Two potential vaccine candidates, rVSVΔG-ZEBOV and ChAd3-EBO Z, have shown durable protection from lethal Ebola challenge in mice [2] and macaques [3] respectively, and are part of the phase II/III PREVAIL trial in Liberia and Guinea (https://clinicaltrials.gov/ct2/show/NCT02344407). Other potential therapeutics, such as convalescent plasma and the antibody cocktail ZMapp [4] have been approved for an emergency phase II/III trial in Guinea (https://clinicaltrials.gov/ct2/show/ NCT02342171) and a phase I trial in Liberia (https://clinicaltrials.gov/ct2/show/ NCT02363322), respectively. However, to date there is no licensed vaccine or treatment for EVD, although improvements in supportive care are increasing survival rates [5].
Repurposing antivirals used for other viral infections, based on knowledge of mechanisms of action, has prompted accumulating interest in the application of different nucleoside/nucleotide analogs and type I interferons (IFNs) for the treatment of Ebola virus disease (EVD). Experimental nucleoside analogs may have therapeutic efficacy for EVD, given the evidence of protection in primate and rodent disease models, 2-6 days after lethal Ebola or the related hemorrhagic Marburg virus challenges [6,7]. Favipiravir, a viral polymerase inhibitor, provides 100% protection when administered 6 days after challenge with a lethal dose of Ebola virus [6] and has been evaluated in the phase II/III JIKI trial in Guinea (https://clinicaltrials.gov/ct2/ show/NCT02329054). TKM-100802, a cocktail of siRNAs targeting VP35 and L polymerase and brincidofovir (BCV), a viral polymerase inhibitor that has activity against dsDNA viruses such as adenovirus and cytomegalovirus [8], were also considered for treatment against EVD. The brincidofovir trial was halted, ostensibly because of projections of low recruitment.
Despite infecting different target cells, Ebola and HIV-1 share many similar features early in their replication cycle. Both are RNA viruses that package a viral polymerase (L for Ebola, RT for HIV-1) required for early replication in the cytosol of the host cell [9]. Homology-based structural prediction of the RNA-dependant RNA polymerase of Ebola indicates the polymerase contains conserved structural motifs in the catalytic palm subdomain similar to viral DNA polymerases [10], supportive of nucleoside analogs potentially inhibiting Ebola replication. Inhibiting HIV-1 reverse transcription with nucleoside analogs such as lamivudine (3TC, cytidine analog), zidovudine (AZT, thymidine analog) or tenofovir (TFV, adenosine monophosphate analog) is the basis for highly active antiretroviral treatment (HAART) [11,12]. Nucleoside analogs are on the WHO list of essential medicines and can be deployed in limited resource settings [13]. Moreover, AZT binds RNA through G-C and A-U bases [14], prompting us to evaluate whether these nucleoside analogs might also inhibit Ebola replication.
Type I IFNs mediate diverse biological effects, including cell type-independent antiviral responses and cell type-restricted responses of immunological relevance. IFNs inhibit viral infection by preventing viral entry into target cells and by blocking different stages of the viral replication cycle for different viruses. Moreover, type I IFNs have a critical role in linking the innate and adaptive immune responses to viral challenge. IFN-α/β expression occurs as the earliest non-specific response to viral infection. Indeed, viruses have evolved immune evasion strategies specifically targeted against an IFN response, confirming the importance of IFNs as antivirals. This immune evasion strategy is relevant when one considers the IFN response to Ebola infection [15]. Ebola proteins VP24 and VP35 inhibit host cell systems that lead to IFN production and also inhibit events associated with an IFN response [16][17][18]. VP24 blocks the binding of importins to phosphorylated STAT1, preventing STAT1 nuclear translocation required for transcription of interferon simulated genes [16]. VP35 binds viral dsRNA, preventing dsRNA degradation [17] and inhibits the phosphorylation of IRF-3 and the SUMOylation of IRF-3 and IRF-7, thereby limiting IFN production [18]. Despite these virally-encoded mechanisms to limit an IFN response to infection, different rodent and non-human primate studies provide evidence for IFN-induced partial protection: the effects of IFN-α/β treatment in lethal Ebola virus infection reduced viremia and prolonged survival [19][20][21]. Thus, a potential therapeutic effect for IFNs as monotherapy in EVD, or in combination with other anti-Ebola therapies, has not been resolved.

Results
We employed an established mini-genome system to rapidly evaluate candidate drugs that could inhibit Ebola Zaire replication under BSL 2 conditions [22][23][24]. At the outset we established the experimental conditions for infection with replication and transcription-competent virus like particles (trVLPs), by examining luciferase activity under various transfection and drug treatment conditions, which included transfection with viral support protein plasmids (S1 Fig). We included treatment with maraviroc, a CCR5 inhibitor, that would have no effect on trVLP entry and infection, thereby serving as a negative control for subsequent treatment regimens.
In a first series of experiments, we examined the inhibitory effects of IFN-α (0.5μM/10,000 U/mL), IFN-ß (0.2μM/1,000 U/ml), TOR (5μM), CDF (100μM), FPV (100μM), and a combination of 3TC, AZT and TFV (5μM each) on trVLP infection of 293T cells (Fig 1). Specifically, the 293 T cells were treated with the different drugs at four different times relative to infection with trVLP, as indicated. We provide evidence that for each of the individual drugs and for the triple drug combination, at the doses indicated, trVLP infection of 293 T cells is inhibited when treatment is initiated at +2, +6 or +24 hours post-infection. Interestingly, TOR, an estrogen receptor modulator discovered in a high throughput screen as a potent inhibitor of Ebola [25], significantly reduced viral luciferase activity at all time-points tested. For IFN-α, IFN-ß, TOR and FPV treatments, maximal inhibition of trVLP infection was achieved when the cells were treated prior to challenge with trVLP. By contrast, pre-treatment with CDF at 100μM, 24 hours prior to infection with trVLP, resulted in enhanced infection.
In subsequent dose-response studies, we compared the inhibitory effects of IFN-α, IFN-ß, TOR, CDF, FPV, 3TC, AZT or TFV when administered 24 hours post trVLP infection (Fig 2). The data in Fig 2I summarize the IC 50 dose for each drug. The IFNs exhibited the lowest IC 50 values at 0.016μM for IFN-ß and 0.038μM for IFN-α. The data show a log-fold difference in IC 50 values for IFN-α and IFN-ß when compared in terms of U/ml, the norm for antiviral activity measurements (Fig 2A and 2B). TOR had the next lowest IC 50 (0.36μM) and completely inhibited infection at doses > 5μM ( Fig 2C). TFV had an IC 50 at 0.98μM. CDF, 3TC and AZT all exhibited similar IC 50 values in the dose range 4.2-7.8μM, while FPV had the highest IC 50 of the nucleoside analogs at 36.8μM. At their IC 50 concentration, none of these drugs directly inhibited luciferase reporter activity (S2 Fig). We observed a relatively small antiviral dose range for CDF (1.5-25μM) (Fig 2D), beyond which the drug appeared to enhance viral infection (S3 Fig). In cell viability assays we observe that at doses >10μM CDF affect cell viability, confounding the interpretation of the effects of CDF on viral replication.
In an orthogonal assay to confirm these findings, we next measured viral replication and transcription by qRT-PCR, following trVLP infection. trVLP-infected cells were either left untreated, or treated with the different drugs 24 hours post-infection, then viral replication and  transcription evaluated 24 hours later (Fig 3). All treatments, with the exception of TOR, significantly reduced the amount of genomic vRNA detected within cells ( Fig 3A) and all treatments significantly reduced the synthesis of cRNA and mRNA isolated from infected cells ( Fig 3B). Notably, IFN-ß treatment of trVLP-infected cells resulted in the greatest reduction in viral replication and transcription.  Next we examined the effectiveness of two and three drug combinations on trVLP infection. We first examined 28 two-drug combinations, using each drug at its IC 50 value, and used the median-effect equation and combination index theorem [26] to determine drug synergy, additive or sub-additive effects (Fig 4A). Synergy is defined as greater than additive effect when drugs were combined (CI<1), additive as the effect expected when combining each drug (CI = 1) and sub-additive as a smaller than expected additive effect (CI>1). When administered 24 hours post-infection, many of the two-drug combinations showed strong synergism in inhibiting trVLP replication (Fig 4J), with IFN-β + 3TC demonstrating the greatest synergism (97.3% inhibition, CI = 0.028). 3TC was synergistic with all seven other drugs tested. Notably, when CDF was used in combination with FPV, AZT, TFV or IFN-α, it produced a sub-additive effect.
Next we tested all possible 56 three-drug combinations, using each drug at its IC 50 value, to assess whether adding a third drug enhanced efficacy compared with two-drug combinations (Fig 4B-4I). This series of experiments served to validate our two-drug findings, as synergistic two-drug combinations such as IFN-β + 3TC and IFN-β + AZT, predicted strong synergy for the triple drug combination of IFN-ß + 3TC + AZT. As anticipated from the two-drug polygonogram, CDF was sub-additive when combined in three-drug combinations ( Fig 4E). This was most evident even when CDF was administered in conjunction with two-drug combinations that had shown strong synergy, such as IFN-β + 3TC or FPV + TFV, further indicating that CDF diminishes the antiviral effects of other drugs. IFN-ß, 3TC, AZT and TFV all promoted strong synergism when included in triple drug combinations, with IFN-β + AZT specifically providing strong synergism when combined in three unique triple therapies.
From these two-drug and three-drug screens, we calculated the combination index (CI) and fractional inhibition (Fi) (Fig 4J and 4K). Many of the synergistic drug combinations (i.e. low CI) included one nucleoside analog and an IFN, while those drug combinations that were subadditive all included CDF. IFN-β was predominant in the most efficacious two-and three-drug combinations. In particular, IFN-β + 3TC and IFN-β + 3TC + AZT consistently exhibited the strongest synergism and highest Fi when administered 24 hours post-infection. Refer also to S1 and S2 Tables.
In a final series of experiments, in order to validate our findings from the trVLP infection studies, we examined the antiviral effectiveness of IFN-ß, IFN-α, TOR, FVP, AZT, 3TC and TFV in 293T cells infected with ZEBOV (ZEBOV contained an eGFP reporter). CDF was excluded from these experiments. Initial dose-response studies were conducted at doses reflective of those used in the trVLP experiments in Fig 2. A higher dose of each drug was required to inhibit ZEBOV infection compared with trVLP infection (S4 Fig). Using the IC 25 of each drug, we next evaluated 2 and 3 drug combinations for additive or synergistic effects against ZEBOV infection. All seven 2 drug combinations were synergistic (low CI) (Fig 5A), similar to the most synergistic combinations against trVLP in Fig 4J. IFN-β + 3TC proved to be the most synergistic 2 drug combination, analogous to trVLP infection. Of the most synergistic 3 drug combinations identified in the trVLP infection system, all seven exhibited synergy against ZEBOV infection, with IFN-β + 3TC + AZT and IFN-β + TOR + AZT exhibiting the strongest synergy (Fig 5B). The CIs determined from trVLP infection correlated well with those determined using ZEBOV infection; specifically, the correlation coefficients (R 2 values) confirm this (Fig 5C and 5D).

Discussion
In September 2014, the WHO hosted a conference to facilitate development of a global action plan to deal with the Ebola outbreak in West Africa. Delegates from affected West African countries, ethicists, scientists, health care providers, logisticians and representatives from different funding agencies were in attendance. A committee had been struck to evaluate the different vaccine candidates and therapeutic interventions being developed, which subsequently received an overwhelming number of submissions for consideration, and was hampered by an inability to compare antiviral effectiveness, since in vitro and pre-clinical in vivo model systems  vary, treatment regimens vary from prophylaxis to post-exposure administration, and direct readouts of antiviral efficacy differ. Moreover, given the virulence and high mortality associated with EVD, all of these studies have been conducted under BSL 4 conditions, limiting the number of laboratories that can engage in these antiviral studies. Cognizant of these limitations, we employed the trVLP model system to compare the antiviral effectiveness of eight antiviral candidates from three drug classes. We evaluated their antiviral activities in the context of inhibition of Ebola replication, using this mini-genome model that allows for rapid comparisons among compounds under BSL 2 conditions. The tetracistronic minigenome represents the most sophisticated in vitro replication model of Ebola virus to date. trVLPs proceed through every replication step as wild-type Ebola virus, and have been tested in multiple cell lines. Using TOR, there has been some validation of the trVLP assay. Specifically, TOR has been evaluated in limiting Ebola virus infection of VeroE6 and HepG2 cells, and exhibited IC 50 values of 0.2 μM and 0.03 μM, respectively [27], in line with the IC 50 dose for TOR (0.36μM) observed with trVLP infection. Likewise, the IC 50 identified in the trVLP system for FPV (36.8μM), is consistent with that of 67μM recorded using Ebola virus infection [6], suggesting that this Ebola mini-genome system has relevance for screening potential antiviral compounds. Indeed, our validation studies using ZEBOV (ZEBOV-eGFP) suggest that the trVLP infection model has utility as an in vitro screening assay when comparing different drugs as monotherapies or in 2 and 3 drug combinations.
As mentioned, the Ebola virus encodes in its genome factors that limit a type I IFN response to infection [16][17][18]. Yet, both rodent and non-human primate studies suggest that IFN-α and IFN-ß treatment can confer partial protection from infection, reducing viremia and prolonging survival [19][20][21], suggesting that it may be possible to override the inhibitory effects of the virus by treatment with IFN. At the outset, we conducted a series of experiments to compare the antiviral activities of IFN-α and IFN-ß in the trVLP infection system, and our findings suggest that whether treatment is administered prior to or post-infection, both IFN-α and IFN-ß exhibit antiviral activity. These findings only have relevance for the direct antiviral activities of these IFNs, since the effects of IFN-α or IFN-ß on immune modulation for viral clearance cannot be determined using this system. Nevertheless, these data contributed to the decision to conduct a clinical trial of IFN-ß treatment for EVD in Guinea.
We provide evidence that the nucleoside/nucleotide analogs 3TC, AZT, TFV, FPV and CDF inhibit Ebola trVLP replication in vitro. The results with 3TC are in contrast to published data that show no evidence for 3TC inhibiting Ebola virus infection in vitro [27]. These studies examined the antiviral effectiveness of 3TC when administered one hour prior to infection, in contrast to our studies that have focused on post-exposure protection. In cells, the kinetics of 3TC phosphorylation are such that a minimum of four hours are required for optimal activity, perhaps distinguishing why our 24 hour pre-treatment, specifically a combination treatment, offered protection. Post-exposure treatment with 3TC and the other nucleoside/nucleotide analogs we examined, would more likely reveal activity against viral RNA synthesis than pretreatment. When comparing the IC 50 values of each of the nucleoside analogs that we tested, TFV exhibited the lowest IC 50 at~1μM. Whether this reflects the fact that this adenosine monophosphate analog only requires two phosphorylation events to become an active drug versus three for the other nucleoside analogs, remains undetermined. Extensive published data reveal both the safety profiles [11,28,29] and the biodistribution of 3TC, AZT and TFV in the circulation and liver [30,31], the same compartments where Ebola infects monocytes, macrophages, dendritic cells, endothelial cells and hepatocytes. Moreover, drug interactions with other nucleoside analogs have been well studied: e.g. tenofovir disoproxil fumarate, when used alone or in combination with emtricitabine effectively prevents HIV-1 infection in antiretroviral pre-exposure prophylaxis (PrEP) [29].
Our studies also revealed that the active metabolite of brincidofovir, CDF, has a narrow therapeutic window of efficacy (6.25-25μM) when assessed in the trVLP assay, enhancing viral replication at higher doses when added either prior to or post-infection. In cell viability assays, CDF exhibits cytotoxicity at doses >10μM. These findings suggest that caution is required if CDF is to be considered further for the treatment of EVD, specifically that phase I/II trials define the safety profile of this drug for EVD.
Another advantage of this in vitro system is that it allowed us to evaluate various 2 and 3 drug combinations and demonstrates that combination treatments limit viral replication up to 97.3%. A benefit of combination treatment is the potential to limit/avoid the emergence of drug resistance. Interestingly, IFN-ß was predominant among all the 8 antivirals considered in terms of contributing very strong synergism in combination treatments: e.g. IFN-ß + 3TC; IFN-ß + 3TC + AZT. Using this system, we observe that FPV, when administered 24 hours post-infection, has an IC 50 of~37μM. To date, the phase II/III JIKI trial examining the efficacy of FPV against EVD has reported only modestly encouraging results. In our 2 drug combination treatment studies we show that, with the exception of CDF, whenever FPV is included, synergy occurs, effectively reducing the CI. It may transpire that for treating EVD, FPV is most effective in a drug combination regimen.
Viewed altogether, we present an in vitro Ebola trVLP screening system, that requires only level 2 biocontainment, which allowed us to compare the antiviral activities of 8 compounds, either alone or in combination. We provide evidence that IFNs are effective inhibitors of Ebola replication, with IFN-ß exhibiting greater efficacy over IFN-α, or when used in combination with nucleoside analogs. We infer from our data that whether IFN-ß treatment is administered 24 hours prior to, or up to 24 hours post-infection, reduced Ebola replication is achieved. As additional antiviral therapeutic candidates become available, we now have the capability to measure and compare their direct antiviral activities with the existing panel. This allows for rapid in vitro evaluation and the opportunity to prioritize antiviral candidates for further preclinical and clinical trial studies.

Cell culture and trVLP infection
We employed an established mini-genome system to rapidly evaluate candidate drugs that could inhibit Ebola Zaire replication under BSL 2 conditions [22]. The mini-genome encodes 3 of the 7 Ebola proteins (VP24, VP40 and GP 1,2 ) and a luciferase reporter gene. Expression plasmids for the remaining four Ebola nucleocapsid proteins (L, NP, VP30 and VP35) were also included during transfection. Cell culture conditions and virus infections were performed as previously described [22]. Briefly, 80,000 producer 293 T cells (American Type Culture Collection; ATCC, Rockville, USA) were seeded in individual wells of 24-well plates in 400μL Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, 1% penicillin and 1% streptomycin, and grown in 5% C0 2 atmosphere at 37°C. Cells were transfected with the viral replication protein plasmids (L, NP, VP30, VP35), a tetracistronic Ebola mini-genome and the T7 polymerase, using the CalPhos Mammalian Transfection Kit (Clontech Laboratories). 24 hours later, medium was replaced with 800μL DMEM with 5% FBS. The replication and transcription-competent virus like particles (trVLPs) were harvested 3 days later. Virus stock was frozen at -80°C.
For infection, 293 T target cells were seeded at 80,000 cells in 400μL of DMEM supplemented with 10% FBS. Target cells were then transfected with the four viral replication protein plasmids, as well as Tim-1, to allow efficient virus binding and entry. 24hr post-transfection, 25μL of trVLP stock was diluted in 600μL of DMEM with 5% FBS, warmed to 37°C for 30 min, then added to target cells. Medium was removed the following day and replaced with 800μL DMEM with 5% FBS. Four days post-infection, the medium was aspirated and cells were resuspended in 200μL of 1x Renila Luciferase Assay Lysis Buffer (Renilla Luciferase Assay System, Promega). Lysates were assayed for luciferase activity.

ZEBOV-eGFP infection
We generated recombinant ZEBOV expressing enhanced green fluorescent protein (eGFP) from cDNA clones of full-length infectious ZEBOV, as previously described [32]. The eGFP reporter protein was expressed as an eighth gene, and the virus exhibited an in vitro phenotype similar to wild-type ZEBOV. Notably, in vivo, incorporation of GFP into wild-type ZEBOV results in some attenuation of disease [32].

Mini-genome RNA extraction and qRT-PCR quantification of viral RNA
Forty-eight hours after trVLP infection, medium was aspirated from 293 T cells that had either been left untreated or treated with the various drugs and total RNA extracted from cell lysates with 500μL of TRIzol (Thermo Fisher Scientific). cDNA synthesis was performed on 5 μg of total RNA, using the First-Strand cDNA Synthesis Kit (GE Healthcare Life Sciences), according to the manufacturer's instructions. A 20 μl reaction also contained bulk first-strand cDNA reaction mix, DTT solution and 40 pmol of one of two trVLP specific primers [33]: vRNA forward (5'-GGC CTC TTC TTA TTT ATG GCG A -3'), or cRNA/mRNA reverse (5'-AGA ACC ATT ACC AGA TTT GCC TGA-3'). Both primers were synthesized by the Center for Applied Genomics (The Hospital for Sick Children, Toronto, Canada). Real-time qPCR reactions (25 μl) were conducted in duplicate, using the Rotor-Gene RG-3000 thermocycler (Corbett Research, Montreal, Canada). Each reaction contained 100 ng template cDNA, 12.5 μL 2 x SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), 300 nM of both the forward (vRNA) and reverse (cRNA/mRNA) primers, and PCR grade H 2 O (Roche Diagnostics, Indianapolis, USA). Samples lacking reverse transcriptase (No RT) during first-strand cDNA synthesis served as negative controls. Cycling parameters were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of amplification with 95°C for 15 seconds, 56°C for 30 seconds, and 60°C for 30 seconds. Biological triplicates in the drug-treated groups were normalized to the average Ct of infected cells given DMSO solvent alone, by the 2 -ΔCT comparative C T method.

Cell viability assay
Dose-response cytotoxicity/viability assays were conducted in 293 T cells 4 days post-infection for each of the drugs examined, either alone or in the various combinations indicated, using the MTT assay as previously described [34].

Statistics
Means were compared using a two-tailed, unpaired Student's t test and corrected for multiple comparisons. For all figures, ( Ã ) denotes a p value <0.05, ( ÃÃ ) denotes a p value <0.01 and ( ÃÃÃ ) denotes a p value <0.001. Error bars shown are the standard error around the mean (SEM). Synergy between two and three-drug combinations, combination index (CI) and dose-reduction index (DRI) were calculated with CompuSyn Version 1.0 [26]. The coefficient of determination (R 2 ) was determined for simple linear regressions.