A Rapid Screening Assay Identifies Monotherapy with Interferon-？ 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-？, 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-？ 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-？ 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.
References
[1]
World Health Organization, Ebola Situation Report – 16 December 2015. .
[2]
Wong G, Audet J, Fernando L, Fausther-Bovendo H, Alimonti JB, Kobinger GP, et al. Immunization with vesicular stomatitis virus vaccine expressing the Ebola glycoprotein provides sustained long-term protection in rodents. Vaccine. 2014;32: 5722–9. doi: 10.1016/j.vaccine.2014.08.028. pmid:25173474
[3]
Stanley DA, Honko AN, Asiedu C, Trefry JC, Lau-Kilby AW, Johnson JC, et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge. Nat Med. 2014;20: 1126–9. doi: 10.1038/nm.3702. pmid:25194571
[4]
Qiu X, Wong G, Audet J, Bello A, Fernando L, Alimonti JB, et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 2014;514: 47–53. doi: 10.1038/nature13777. pmid:25171469
[5]
Wong KK, Perdue CL, Malia J, Kenney JL, Peng S, Gwathney JK, et al. Supportive Care of the First 2 Ebola Virus Disease Patients at the Monrovia Medical Unit. Clin Infect Dis. 2015;61:e47–51. doi: 10.1093/cid/civ420. pmid:26021993
[6]
Oestereich L, Ludtke A, Wurr S, Rieger T, Munoz-Fontela C, Gunther S. Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model. Antiviral Res. 2014;105:17–21. doi: 10.1016/j.antiviral.2014.02.014. pmid:24583123
[7]
Warren TK, Wells J, Panchal RG, Stuthman KS, Garza NL, Van Tongeren SA, et al. Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430. Nature. 2014;508: 402–5. doi: 10.1038/nature13027. pmid:24590073
[8]
Florescu DF, Keck MA. Development of CMX001 (Brincidofovir) for the treatment of serious diseases or conditions caused by dsDNA viruses. Expert Rev Anti Infect Ther. 2014;12: 1171–8. doi: 10.1586/14787210.2014.948847. pmid:25120093
[9]
Muhlberger E, Weik M, Volchkov VE, Klenk HD, Becker S. Comparison of the transcription and replication strategies of marburg virus and Ebola virus by using artificial replication systems. J Virol. 1999;73: 2333–42. pmid:9971816
[10]
Jácome R, Becerra A, de León SP, Lazcano A. Structural Analysis of Monomeric RNA- Dependent Polymerases: Evolutionary and Therapeutic Implications. PLoS One. 2015;10:e0139001. doi: 10.1371/journal.pone.0139001. pmid:26397100
[11]
Cassetti I, Madruga JV, Suleiman JM, Etzel A, Zhong L, Cheng AK, et al. The safety and efficacy of tenofovir DF in combination with lamivudine and efavirenz through 6 years in antiretroviral-naive HIV-1-infected patients. HIV Clin Trials. 2007;8: 164–72. pmid:17621463 doi: 10.1310/hct0803-164
[12]
Phanuphak N, Ananworanich J, Teeratakulpisarn N, Jadwattanakul T, Kerr SJ, Chomchey N, et al. A 72-week randomized study of the safety and efficacy of a stavudine to zidovudine switch at 24 weeks compared to zidovudine or tenofovir disoproxil fumarate when given with lamivudine and nevirapine. Antivir Ther. 2012;17: 1521–31. doi: 10.3851/IMP2497. pmid:23220732
[13]
World Health Organization, WHO Model List of Essential Medicines: 18th list October 2013: .
[14]
Ahmed Ouameur A, Marty R, Neault JF, Tajmir-Riahi HA. AZT binds RNA at multiple sites. DNA Cell Biol. 2004;23: 783–8. pmid:15585137 doi: 10.1089/dna.2004.23.783
[15]
Cardenas WB. Evasion of the interferon-mediated antiviral response by filoviruses. Viruses. 2010;2: 262–82. doi: 10.3390/v2010262. pmid:21994610
[16]
Xu W, Edwards MR, Borek DM, Feagins AR, Mittal A, Alinger JB, et al. Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1. Cell Host Microbe. 2014;16: 187–200. doi: 10.1016/j.chom.2014.07.008. pmid:25121748
[17]
Fabozzi G, Nabel CS, Dolan MA, Sullivan NJ. Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. J Virol. 2011;85: 2512–23. doi: 10.1128/JVI.01160-10. pmid:21228243
[18]
Chang TH, Kubota T, Matsuoka M, Jones S, Bradfute SB, Bray M, et al. Ebola Zaire virus blocks type I interferon production by exploiting the host SUMO modification machinery. PLoS Pathog. 2009;5: e1000493. doi: 10.1371/journal.ppat.1000493. pmid:19557165
[19]
Smith LM, Hensley LE, Geisbert TW, Johnson J, Stossel A, Honko A, et al. Interferon-beta therapy prolongs survival in rhesus macaque models of Ebola and Marburg hemorrhagic fever. J Infect Dis. 2013;208: 310–8. doi: 10.1093/infdis/jis921. pmid:23255566
[20]
Qiu X, Wong G, Fernando L, Audet J, Bello A, Strong J, et al. mAbs and Ad-vectored IFN-alpha therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms. Sci Transl Med. 2013;5: 207ra143. doi: 10.1126/scitranslmed.3006605. pmid:24132638
[21]
Qiu X, Wong G, Fernando L, Ennis J, Turner JD, Alimonti JB, et al. Monoclonal antibodies combined with adenovirus-vectored interferon significantly extend the treatment window in Ebola virus-infected guinea pigs. J Virol. 2013;87: 7754–7. doi: 10.1128/JVI.00173-13. pmid:23616649
[22]
Hoenen T, Watt A, Mora A, Feldmann H. Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes. J Vis Exp. 2014;91: 52381. doi: 10.3791/52381. pmid:25285674
[23]
Watt A, Moukambi F, Banadyga L, Groseth A, Callison J, Herwig A, et al. A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity. J Virol. 2014;88: 10511–10524. doi: 10.1128/JVI.01272-14. pmid:24965473
[24]
Hoenen T, Feldmann H. Reverse genetics systems as tools for the development of novel therapies against filoviruses. Expert Rev Anti Infect Ther. 2014;12: 1253–1263. doi: 10.1586/14787210.2014.948848. pmid:25169588
[25]
Johansen LM, Brannan JM, Delos SE, Shoemaker CJ, Stossel A, Lear C, et al. FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. Sci Transl Med. 2013;5:190ra79. doi: 10.1126/scitranslmed.3005471. pmid:23785035
[26]
Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006;58: 621–81. pmid:16968952 doi: 10.1124/pr.58.3.10
[27]
Johansen LM, DeWald LE, Shoemaker CJ, Hoffstrom BG, Lear-Rooney CM, Stossel A et al. A screen of approved drugs and molecular probes identifies therapeutics with anti-Ebola virus activity. Science Transl Med. 2015;7:290ra89. doi: 10.1126/scitranslmed.aaa5597
[28]
Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology. 2003;125: 1714–22. pmid:14724824 doi: 10.1053/j.gastro.2003.09.033
[29]
Baeten JM, Donnell D, Mugo NR, Ndase P, Thomas KK, Campbell JD, et al. Single-agent tenofovir versus combination emtricitabine plus tenofovir for pre-exposure prophylaxis for HIV-1 acquisition: an update of data from a randomised, double-blind, phase 3 trial. Lancet Infect Dis. 2014;14: 1055–64. doi: 10.1016/S1473-3099(14)70937-5. pmid:25300863
[30]
Di Mascio M, Srinivasula S, Bhattacharjee A, Cheng L, Martiniova L, Herscovitch P, et al. Antiretroviral tissue kinetics: in vivo imaging using positron emission tomography. Antimicrob Agents Chemother. 2009;53: 4086–95. doi: 10.1128/AAC.00419-09. pmid:19667288
[31]
Parang K, Wiebe LI, Knaus EE. Pharmacokinetics and tissue distribution of (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine, a prodrug of 3'-azido-2',3'-dideoxythymidine (AZT) in mice. J Pharm Pharmacol. 1998;50: 989–96. pmid:9811159 doi: 10.1111/j.2042-7158.1998.tb06913.x
[32]
Ebihara H, Theriault S, Neumann G, Alimonti J, Geisbert J, Hensley L, et al. In vitro and in vivo characterization of recombinant Ebola viruses expressing enhanced green fluorescent protein. J Infect Dis. 2007;196 Suppl 2:S313–22. pmid:17940966 doi: 10.1086/520590
[33]
Hoenen T, Jung S, Herwig A, Groseth A, Becker S. Both matrix proteins of Ebola virus contribute to the regulation of viral genome replication and transcription. J Virol. 2010;403: 56–66. doi: 10.1016/j.virol.2010.04.002
[34]
Mehrotra S, Sharma B, Joshi S, Kroczynska B, Majchrzak B, Stein BL, et al. Essential role for the Mnk pathway in the inhibitory effects of type I interferons on myeloproliferative neoplasm (MPN) precursors. J Biol Chem. 2013;288: 23814–22. doi: 10.1074/jbc.M113.476192. pmid:23814052
