Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)– and ex–US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.

Currently, no approved therapeutics exist to treat or prevent infections induced by Ebola viruses, and recent events have demonstrated an urgent need for rapid discovery of new treatments. Repurposing approved drugs for emerging infections remains a critical resource for potential antiviral therapies. We tested ~2600 approved drugs and molecular probes in an in vitro infection assay using the type species, Zaire ebolavirus. Selective antiviral activity was found for 80 U.S. Food and Drug Administration–approved drugs spanning multiple mechanistic classes, including selective estrogen receptor modulators, antihistamines, calcium channel blockers, and antidepressants. Results using an in vivo murine Ebola virus infection model confirmed the protective ability of several drugs, such as bepridil and sertraline. Viral entry assays indicated that most of these antiviral drugs block a late stage of viral entry. By nature of their approved status, these drugs have the potential to be rapidly advanced to clinical settings and used as therapeutic countermeasures for Ebola virus infections.

Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC₅₀ 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann–Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.

Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1 ⁺ LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. IMPORTANCE Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1 ⁺ LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1 ⁺ LE/Lys, as a therapeutic target for SARS and EBOV.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus capable of causing a severe hemorrhagic fever disease in humans. There are currently no licensed vaccines to prevent CCHFV-associated disease. We developed a DNA vaccine expressing the M-segment glycoprotein precursor gene of CCHFV and assessed its immunogenicity and protective efficacy in two lethal mouse models of disease: type I interferon receptor knockout (IFNAR^-/-) mice; and a novel transiently immune suppressed (IS) mouse model. Vaccination of mice by muscle electroporation of the M-segment DNA vaccine elicited strong antigen-specific humoral immune responses with neutralizing titers after three vaccinations in both IFNAR^-/- and IS mouse models. To compare the protective efficacy of the vaccine in the two models, groups of vaccinated mice (7–10 per group) were intraperitoneally (IP) challenged with a lethal dose of CCHFV strain IbAr 10200. Weight loss was markedly reduced in CCHFV DNA-vaccinated mice as compared to controls. Furthermore, whereas all vector-control vaccinated mice succumbed to disease by day 5, the DNA vaccine protected >60% of the animals from lethal disease. Mice from both models developed comparable levels of antibodies, but the IS mice had a more balanced Th1/Th2 response to vaccination. There were no statistical differences in the protective efficacies of the vaccine in the two models. Our results provide the first comparison of these two mouse models for assessing a vaccine against CCHFV and offer supportive data indicating that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV. Crimean-Congo hemorrhagic Fever Virus (CCHFV) is a tick-borne virus capable of causing lethal human disease against which there are currently no approved vaccines. In this study, we compared the immunogenicity and protective efficacy of a candidate DNA vaccine expressing the glycoprotein precursor gene of CCHFV in two mouse models. In addition to the recently established IFNAR^-/- mouse pathogenesis model, we also tested the vaccine in a novel murine system in which the interferon (IFN) α/β signaling response of immunocompetent mice is transiently suppressed. We found that the DNA vaccine elicited high humoral immune responses and provided significant protection against challenge with CCHFV in both mouse models. These findings further our understanding of the requirements for a CCHFV vaccine and provide a new mouse model for the development of CCHFV countermeasures.

An antibody targeting the CCHFV GP38 molecule protects adult mice against lethal infection in a severe disease model.