Development and optimization of biologically contained Marburg virus for high-throughput antiviral screening.

Comparable to the related Ebola virus, Marburg virus is an emerging zoonotic pathogen that causes hemorrhagic fever with a high mortality rate. Therefore, handling of Ebola virus and Marburg virus is limited to biosafety level 4 facilities, of which only a limited number exists worldwide. However, researchers have developed several virus alternatives that are safe to handle in lower biosafety settings. One particularly interesting approach is the engineering of biologically contained Ebola virus by removing an essential gene from the virus genome and providing this missing gene in trans in a specific cell line. Because the virus is confined to this specific cell line, this results in a system that is safe to handle. So far, Ebola virus is the only virus for which biological containment has been reported. Here, we describe the first successful rescue of biologically contained Marburg virus and demonstrate that biological containment is also feasible for other filoviruses. Specifically, we describe the development of containment cell lines for Marburg virus through lentiviral transduction and show the growth and safety characteristics of eGFP-expressing, biologically contained Marburg virus in these cell lines. Additionally, we exploited this newly established Marburg virus system to screen over 500 compounds from available libraries. Lastly, we also validated the applicability of our biologically contained Marburg virus system in a 384-well format, to further illustrate the usefulness of this novel system as an alternative for high-throughput MARV screening of compound libraries.

Combination therapy protects macaques against advanced Marburg virus disease.

Monoclonal antibodies (mAbs) and remdesivir, a small-molecule antiviral, are promising monotherapies for many viruses, including members of the genera Marburgvirus and Ebolavirus (family Filoviridae), and more recently, SARS-CoV-2. One of the major challenges of acute viral infections is the treatment of advanced disease. Thus, extending the window of therapeutic intervention is critical. Here, we explore the benefit of combination therapy with a mAb and remdesivir in a non-human primate model of Marburg virus (MARV) disease. While rhesus monkeys are protected against lethal infection when treatment with either a human mAb (MR186-YTE; 100%), or remdesivir (80%), is initiated 5 days post-inoculation (dpi) with MARV, no animals survive when either treatment is initiated alone beginning 6 dpi. However, by combining MR186-YTE with remdesivir beginning 6 dpi, significant protection (80%) is achieved, thereby extending the therapeutic window. These results suggest value in exploring combination therapy in patients presenting with advanced filovirus disease.

Discovery of Marburg virus neutralizing antibodies from virus-naïve human antibody repertoires using large-scale structural predictions.

Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. We computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a full-length native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.

A biaryl sulfonamide derivative as a novel inhibitor of filovirus infection.

Ebolaviruses and marburgviruses, members of the family Filoviridae, are known to cause fatal diseases often associated with hemorrhagic fever. Recent outbreaks of Ebola virus disease in West African countries and the Democratic Republic of the Congo have made clear the urgent need for the development of therapeutics and vaccines against filoviruses. Using replication-incompetent vesicular stomatitis virus (VSV) pseudotyped with the Ebola virus (EBOV) envelope glycoprotein (GP), we screened a chemical compound library to obtain new drug candidates that inhibit filoviral entry into target cells. We discovered a biaryl sulfonamide derivative that suppressed in vitro infection mediated by GPs derived from all known human-pathogenic filoviruses. To determine the inhibitory mechanism of the compound, we monitored each entry step (attachment, internalization, and membrane fusion) using lipophilic tracer-labeled ebolavirus-like particles and found that the compound efficiently blocked fusion between the viral envelope and the endosomal membrane during cellular entry. However, the compound did not block the interaction of GP with the Niemann-Pick C1 protein, which is believed to be the receptor of filoviruses. Using replication-competent VSVs pseudotyped with EBOV GP, we selected escape mutants and identified two EBOV GP amino acid residues (positions 47 and 66) important for the interaction with this compound. Interestingly, these amino acid residues were located at the base region of the GP trimer, suggesting that the compound might interfere with the GP conformational change required for membrane fusion. These results suggest that this biaryl sulfonamide derivative is a novel fusion inhibitor and a possible drug candidate for the development of a pan-filovirus therapeutic.

Monoterpenoid-based inhibitors of filoviruses targeting the glycoprotein-mediated entry process.

In this study, we screened a large library of (+)-camphor and (-)-borneol derivatives to assess their filovirus entry inhibition activities using pseudotype systems. Structure-activity relationship studies revealed several compounds exhibiting submicromolar IC50 values. These compounds were evaluated for their effect against natural Ebola virus (EBOV) and Marburg virus. Compound 3b (As-358) exhibited the good antiviral potency (IC50 = 3.7 µM, SI = 118) against Marburg virus, while the hydrochloride salt of this compound 3b·HCl had a strong inhibitory effect against Ebola virus (IC50 = 9.1 µM, SI = 31) and good in vivo safety (LD50 > 1000 mg/kg). The results of molecular docking and in vitro mutagenesis analyses suggest that the synthesized compounds bind to the active binding site of EBOV glycoprotein similar to the known inhibitor toremifene.

Remdesivir (GS-5734) Is Efficacious in Cynomolgus Macaques Infected With Marburg Virus.

Marburg virus (MARV) is a filovirus with documented human case-fatality rates of up to 90%. Here, we evaluated the therapeutic efficacy of remdesivir (GS-5734) in nonhuman primates experimentally infected with MARV. Beginning 4 or 5 days post inoculation, cynomolgus macaques were treated once daily for 12 days with vehicle, 5 mg/kg remdesivir, or a 10-mg/kg loading dose followed by 5 mg/kg remdesivir. All vehicle-control animals died, whereas 83% of animals receiving a 10-mg/kg loading dose of remdesivir survived, as did 50% of animals receiving a 5-mg/kg remdesivir regimen. Remdesivir-treated animals exhibited improved clinical scores, lower plasma viral RNA, and improved markers of kidney function, liver function, and coagulopathy versus vehicle-control animals. The small molecule remdesivir showed therapeutic efficacy in this Marburg virus disease model with treatment initiation 5 days post inoculation, supporting further assessment of remdesivir for the treatment of Marburg virus disease in humans.

T-705 induces lethal mutagenesis in Ebola and Marburg populations in macaques.

Nucleoside analogues (NA) disrupt RNA viral RNA-dependent RNA polymerase (RdRP) function and fidelity for multiple viral families. The mechanism of action (MOA) of T-705 has been attributed alternatively or concurrently to chain termination and lethal mutagenesis depending on the viral species during in vitro studies. In this study, we evaluated the effect of T-705 on the viral population in non-human primates (NHPs) after challenge with Ebola virus (EBOV) or Marburg virus (MARV) to identify the predominant in vivo MOA. We used common virological assays in conjunction with deep sequencing to characterize T-705 effects. T-705 exhibited antiviral activity that was associated with a reduction in specific infectivity and an accumulation of low frequency nucleotide variants in plasma samples collected day 7 post infection. Stranded analysis of deep sequencing data to identify chain termination demonstrated no change in the transcriptional gradient in negative stranded viral reads and minimal changes in positive stranded viral reads in T-705 treated animals, questioning as a MOA in vivo. These findings indicate that lethal mutagenesis is a MOA of T-705 that may serve as an indication of therapeutic activity of NAs for evaluation in clinical settings. This study expands our understanding of MOAs of these compounds for the Filovirus family and provides further evidence that lethal mutagenesis could be a preponderant MOA for this class of therapeutic compounds.

Vaccinomics strategy for developing a unique multi-epitope monovalent vaccine against Marburg marburgvirus.

Marburg virus is known to cause a severe hemorrhagic fever (MHF) in both humans and non-human primates with high degree of infectivity and lethality. To date no approved treatment is available for Marburg virus infection. A study was employed to design a novel chimeric subunit vaccine against Marburg virus by adopting reverse vaccinology approach. The entire viral proteome was retrieved from UniprotKB and assessed to design highly antigenic epitopes by antigenicity screening, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach. Envelope glycoprotein (GP) and matrix protein (VP40) were identified as most antigenic viral proteins which generated a plethora of epitopes. The final vaccine was constructed by the combination of highly immunogenic epitopes along with suitable adjuvant and linkers. Physicochemical and secondary structure of the designed vaccine was assessed to ensure its thermostability, hydrophilicity, theoretical PI and structural behaviors. Disulfide engineering, molecular dynamic simulation and codon adaptation were further employed to develop a unique multi-epitope monovalent vaccine. Docking analysis of the refined vaccine structure with different MHC molecules and human immune TLR8 receptor present on lymphocyte cells demonstrated higher interaction. Moreover, disulfide engineering served to lessen the high mobility region of the designed vaccine in order to extend its stability. Complexed structure of the modeled vaccine and TLR8 showed minimal deformability at molecular level. Finally, translational potency and microbial expression of the modeled vaccine was analyzed with pET28a(+) vector for E. coli strain K12. However, further in vitro and in vivo investigation could be implemented for the acceptance and validation of the predicted vaccine against Marburg virus.

Filovirus Virulence in Interferon α/ß and γ Double Knockout Mice, and Treatment with Favipiravir.

The 2014 Ebolavirus outbreak in West Africa highlighted the need for vaccines and therapeutics to prevent and treat filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would facilitate the screening of anti-filovirus agents. To that end, we characterized knockout mice lacking α/ß and γ interferon receptors (IFNAGR KO) as a model for wild-type filovirus infection. Intraperitoneal challenge of IFNAGR KO mice with several known human pathogenic species from the genus Ebolavirus and Marburgvirus, except Bundibugyo ebolavirus and Taï Forest ebolavirus, caused variable mortality rate. Further characterization of the prototype Ebola virus Kikwit isolate infection in this KO mouse model showed 100% lethality down to a dilution equivalent to 1.0 × 10-1 pfu with all deaths occurring between 7 and 9 days post-challenge. Viral RNA was detectable in serum after challenge with 1.0 × 10² pfu as early as one day after infection. Changes in hematology and serum chemistry became pronounced as the disease progressed and mirrored the histological changes in the spleen and liver that were also consistent with those described for patients with Ebola virus disease. In a proof-of-principle study, treatment of Ebola virus infected IFNAGR KO mice with favipiravir resulted in 83% protection. Taken together, the data suggest that IFNAGR KO mice may be a useful model for early screening of anti-filovirus medical countermeasures.

The Calcium Channel Blocker Bepridil Demonstrates Efficacy in the Murine Model of Marburg Virus Disease.

No therapeutics are approved for the treatment of filovirus infections. Bepridil, a calcium channel blocker developed for treating angina, was identified as a potent inhibitor of filoviruses in vitro, including Ebola and Marburg viruses, and Ebola virus in vivo. We evaluated the efficacy of bepridil in a lethal mouse model of Marburg virus disease. A dose of 12 mg/kg bepridil once or twice daily resulted in 80% or 90% survival, respectively. These data confirm bepridil's broad-spectrum anti-filovirus activity warranting further investigation of bepridil, or improved compounds with a similar mechanism, as a pan-filovirus therapeutic agent.

Monoclonal Antibody Cocktail Protects Hamsters From Lethal Marburg Virus Infection.

Marburg virus (MARV), family Filoviridae, causes Marburg hemorrhagic fever (MHF) in humans and nonhuman primates with case fatality rates of up to 90%. There is no approved therapeutic for MHF, yet several experimental approaches have been evaluated in preclinical studies including small interfering RNA and monoclonal antibody (mAb) treatment. In this study we attempted to improve the therapeutic efficacy of the neutralizing mAb M4 by combining treatment with 1 or 2 of blocking but nonneutralizing mAbs 126-15 and 127-8. We found that single-dose treatment early after infection with the neutralizing mAb M4 or any of the mAb combinations resulted in similar protection in the MARV hamster model. However, a single-dose treatment with the cocktail of all 3 mAbs provided the best protection in delayed treatment, with 67%-100% of the animals surviving a lethal challenge depending on the time of treatment. This study identified a new promising mAb cocktail as a therapeutic option for MHF.

Small Animal Models for Evaluating Filovirus Countermeasures.

The development of novel therapeutics and vaccines to treat or prevent disease caused by filoviruses, such as Ebola and Marburg viruses, depends on the availability of animal models that faithfully recapitulate clinical hallmarks of disease as it is observed in humans. In particular, small animal models (such as mice and guinea pigs) are historically and frequently used for the primary evaluation of antiviral countermeasures, prior to testing in nonhuman primates, which represent the gold-standard filovirus animal model. In the past several years, however, the filovirus field has witnessed the continued refinement of the mouse and guinea pig models of disease, as well as the introduction of the hamster and ferret models. We now have small animal models for most human-pathogenic filoviruses, many of which are susceptible to wild type virus and demonstrate key features of disease, including robust virus replication, coagulopathy, and immune system dysfunction. Although none of these small animal model systems perfectly recapitulates Ebola virus disease or Marburg virus disease on its own, collectively they offer a nearly complete set of tools in which to carry out the preclinical development of novel antiviral drugs.

Successful treatment of Marburg virus with orally administrated T-705 (Favipiravir) in a mouse model.

Filoviruses, such as Marburg and Ebola viruses, cause severe disease in humans with high case fatality rates and are therefore considered biological threat agents. To date, no licensed vaccine or therapeutic exists for their treatment. T-705 (favipiravir) is a pyrazinecarboxamide derivative that has shown broad antiviral activity against a number of viruses and is clinically licenced in Japan to treat influenza. Here we report the efficacy of T-705 against Marburg virus infection in vitro and in vivo. Notably, oral administration of T-705 beginning one or two days post-infection and continuing for eight days resulted in complete survival of mice that had been intraperitoneally infected with mouse-adapted Marburg virus (variant Angola). Moreover, lower doses of T-705 and higher doses administered later during infection (day 3 or 4 post-infection) showed partial efficacy, with at least half the infected mice surviving. Accordingly, we observed reductions in infectious virus particles and virus RNA levels following drug treatment that appeared to correlate with survival. Our findings suggest that T-705 may be an effective therapeutic against Marburg virus and might be especially promising for use in the event of an outbreak, where it could be orally administered quickly and safely even after exposure.

Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus.

Favipiravir is a broad-spectrum antiviral agent that has demonstrated efficacy against Ebola virus (EBOV) in rodents. However, there are no published reports of favipiravir efficacy for filovirus infection of nonhuman primates (NHPs). Here we evaluated the pharmacokinetic profile of favipiravir in NHPs, as well as in vivo efficacy against two filoviruses, EBOV and Marburg virus (MARV). While no survival benefit was observed in two studies employing once- or twice-daily oral dosing of favipiravir during EBOV infection of NHPs, an antiviral effect was observed in terms of extended time-to-death and reduced levels of viral RNA. However, oral dosing in biosafety level-4 (BSL-4) presents logistical and technical challenges, and repeated anesthesia events may potentially worsen survival outcome in animals. For the third study of treatment of MARV infection, we therefore made use of catheters, jackets, and tethers for intravenous (IV) dosing and blood collection, which minimized the requirement for repeated anesthesia events. When MARV infection was treated with IV favipiravir, five of six animals (83%) survived infection, while all untreated NHPs succumbed. An accompanying report presents the results of favipiravir treatment of EBOV infection in mice.

siRNA rescues nonhuman primates from advanced Marburg and Ravn virus disease.

Ebolaviruses and marburgviruses belong to the family Filoviridae and cause high lethality in infected patients. There are currently no licensed filovirus vaccines or antiviral therapies. The development of broad-spectrum therapies against members of the Marburgvirus genus, including Marburg virus (MARV) and Ravn virus (RAVV), is difficult because of substantial sequence variability. RNAi therapeutics offer a potential solution, as identification of conserved target nucleotide sequences may confer activity across marburgvirus variants. Here, we assessed the therapeutic efficacy of lipid nanoparticle (LNP) delivery of a single nucleoprotein-targeting (NP-targeting) siRNA in nonhuman primates at advanced stages of MARV or RAVV disease to mimic cases in which patients begin treatment for fulminant disease. Sixteen rhesus monkeys were lethally infected with MARV or RAVV and treated with NP siRNA-LNP, with MARV-infected animals beginning treatment four or five days after infection and RAVV-infected animals starting treatment three or six days after infection. While all untreated animals succumbed to disease, NP siRNA-LNP treatment conferred 100% survival of RAVV-infected macaques, even when treatment began just 1 day prior to the death of the control animals. In MARV-infected animals, day-4 treatment initiation resulted in 100% survival, and day-5 treatment resulted in 50% survival. These results identify a single siRNA therapeutic that provides broad-spectrum protection against both MARV and RAVV.

Identification of a coumarin-based antihistamine-like small molecule as an anti-filoviral entry inhibitor.

Filoviruses, consisting of Ebola virus, Marburg virus and Cuevavirus, cause severe hemorrhagic fevers in humans with high mortality rates up to 90%. Currently, there is no approved vaccine or therapy available for the prevention and treatment of filovirus infection in humans. The recent 2013-2015 West African Ebola epidemic underscores the urgency to develop antiviral therapeutics against these infectious diseases. Our previous study showed that GPCR antagonists, particularly histamine receptor antagonists (antihistamines) inhibit Ebola and Marburg virus entry. In this study, we screened a library of 1220 small molecules with predicted antihistamine activity, identified multiple compounds with potent inhibitory activity against entry of both Ebola and Marburg viruses in human cancer cell lines, and confirmed their anti-Ebola activity in human primary cells. These small molecules target a late-stage of Ebola virus entry. Further structure-activity relationship studies around one compound (cp19) reveal the importance of the coumarin fused ring structure, especially the hydrophobic substituents at positions 3 and/or 4, for its antiviral activity, and this identified scaffold represents a favorable starting point for the rapid development of anti-filovirus therapeutic agents.

The Toll-Like Receptor 4 Antagonist Eritoran Protects Mice from Lethal Filovirus Challenge.

The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa, which has seen intermittent reemergence since it was officially declared over in February of 2016, has demonstrated the need for the rapid development of therapeutic intervention strategies. Indirect evidence has suggested that the EBOV infection shares several commonalities associated with the onset of bacterial sepsis, including the development of a "cytokine storm." Eritoran, a Toll-like receptor 4 (TLR4) antagonist, was previously shown to result in protection of mice against lethal influenza virus infection. Here, we report that eritoran protects against the lethality caused by EBOV and the closely related Marburg virus (MARV) in mice. Daily administration of eritoran reduced clinical signs of the disease and, unexpectedly, resulted in reduced viral titers. Analysis of peripheral blood indicated that eritoran reduced granulocytosis despite an apparent increase in the percentage of activated neutrophils. Surprisingly, the increased survival rate and reduced viremia were not accompanied by increased CD3+ T lymphocytes, as lymphopenia was more pronounced in eritoran-treated mice. Overall, a global reduction in the levels of multiple cytokines, chemokines, and free radicals was detected in serum, suggesting that eritoran treatment may alleviate the severity of the "cytokine storm." Last, we provide compelling preliminary evidence suggesting that eritoran treatment may alter the kinetics of cytokine responses. Hence, these studies are the first to demonstrate the role of TLR4 in the pathogenesis of EBOV disease and indicate that eritoran is a prime candidate for further evaluation as a clinically viable therapeutic intervention strategy for EBOV and MARV infections.IMPORTANCE A hallmark of bacterial sepsis is the uncontrolled activation of the TLR4 pathway, which is the primary cause of the pathological features associated with this disease. Considering the importance of TLR4 signaling in bacterial sepsis and the remarkable pathological similarities associated with infections caused by filoviruses Ebola virus (EBOV) and Marburg virus (MARV), we assessed the ability of eritoran, a TLR4 antagonist, to protect mice against these viruses. Here, we show that eritoran effectively promotes survival of mice of filovirus infection, as 70% and 90% of mice receiving daily eritoran treatment survived lethal EBOV and MARV infections, respectively. Eritoran treatment resulted in a remarkable global reduction of inflammatory mediators, which is suggestive of the mechanism of action of this therapeutic treatment. These studies are the first to show the critical importance of the TLR4 pathway in the pathogenesis of filovirus infection and may provide a new avenue for therapeutic interventions.

Therapeutic treatment of Marburg and Ravn virus infection in nonhuman primates with a human monoclonal antibody.

As observed during the 2013-2016 Ebola virus disease epidemic, containment of filovirus outbreaks is challenging and made more difficult by the lack of approved vaccine or therapeutic options. Marburg and Ravn viruses are highly virulent and cause severe and frequently lethal disease in humans. Monoclonal antibodies (mAbs) are a platform technology in wide use for autoimmune and oncology indications. Previously, we described human mAbs that can protect mice from lethal challenge with Marburg virus. We demonstrate that one of these mAbs, MR191-N, can confer a survival benefit of up to 100% to Marburg or Ravn virus-infected rhesus macaques when treatment is initiated up to 5 days post-inoculation. These findings extend the small but growing body of evidence that mAbs can impart therapeutic benefit during advanced stages of disease with highly virulent viruses and could be useful in epidemic settings.