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      Inhibition of Ebola Virus by a Molecularly Engineered Banana Lectin

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          Abstract

          Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013–2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50–80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.

          Author summary

          There are no approved vaccines or treatments to combat infections with ebolaviruses, which cause Ebola virus disease (EVD), an often rapidly fatal disease characterized by fever and bleeding that results in death in up to ~90% of cases. Ebolaviruses are among the most pathogenic viruses that cause human disease and represent a threat to global public health. Outbreaks of EVD occur periodically in African countries and can be exported elsewhere, with recent outbreaks including one ongoing in the Democratic Republic of the Congo and the largest ever in Western Africa in 2013–2016. There is therefore a great need to develop new vaccines and treatments that target ebolaviruses. We examined whether a lectin (carbohydrate-binding protein), predicted to bind to carbohydrates present on the surface of many viruses and thereby interfere with infection, could block ebolavirus infection and be used for prevention and/or treatment of EVD. We found that the protein blocked ebolavirus infection in cell cultures and, moreover, protected a significant proportion of ebolavirus-infected mice from death, even when administered only once before exposure to virus as a preventive. The protein hence shows promise as a potential agent to prevent and/or treat EVD.

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          Experimental Treatment with Favipiravir for Ebola Virus Disease (the JIKI Trial): A Historically Controlled, Single-Arm Proof-of-Concept Trial in Guinea

          Background Ebola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies. Methods and Findings Inclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in “cycle threshold” [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A “target value” of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log10 genome copies/ml. Mortality was 20% (95% CI 11.6%–32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%–91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 μmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 μmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 μmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 μmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log10 copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within <72 h of symptoms compared to others. Favipiravir was well tolerated. Conclusions In the context of an outbreak at its peak, with crowded care centers, randomizing patients to receive either standard care or standard care plus an experimental drug was not felt to be appropriate. We did a non-randomized trial. This trial reaches nuanced conclusions. On the one hand, we do not conclude on the efficacy of the drug, and our conclusions on tolerance, although encouraging, are not as firm as they could have been if we had used randomization. On the other hand, we learned about how to quickly set up and run an Ebola trial, in close relationship with the community and non-governmental organizations; we integrated research into care so that it improved care; and we generated knowledge on EVD that is useful to further research. Our data illustrate the frequency of renal dysfunction and the powerful prognostic value of low Ct values. They suggest that drug trials in EVD should systematically stratify analyses by baseline Ct value, as a surrogate of viral load. They also suggest that favipiravir monotherapy merits further study in patients with medium to high viremia, but not in those with very high viremia. Trial registration ClinicalTrials.gov NCT02329054
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            FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection.

            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.
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              Playing Hide and Seek: How Glycosylation of the Influenza Virus Hemagglutinin Can Modulate the Immune Response to Infection

              Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA) glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity.
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                Author and article information

                Contributors
                Role: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Project administrationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Project administrationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Resources
                Role: Resources
                Role: SupervisionRole: Writing – review & editing
                Role: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                29 July 2019
                July 2019
                : 13
                : 7
                : e0007595
                Affiliations
                [1 ] Medical Scientist Training Program, University of Michigan, Ann Arbor, Michigan, United States of America
                [2 ] Department of Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan, United States of America
                [3 ] Integrated Research Facility, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, United States of America
                [4 ] Division of Infectious Diseases, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America
                [5 ] Department of Cell Biology, University of Virginia, Charlottesville, Virginia, United States of America
                [6 ] Department of Pathology, University of Virginia, Charlottesville, Virginia, United States of America
                [7 ] Antibody Engineering and Technology Core, University of Virginia, Charlottesville, Virginia, United States of America
                [8 ] Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, United States of America
                [9 ] Department of Microbiology, University of Virginia, Charlottesville, Virginia, United States of America
                [10 ] Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, Michigan, United States of America
                [11 ] Graduate Program in Immunology, University of Michigan, Ann Arbor, Michigan, United States of America
                [12 ] Cancer Biology Program, University of Michigan, Ann Arbor, Michigan, United States of America
                Institute of Tropical Medicine, BELGIUM
                Author notes

                I have read the journal's policy and the authors of this manuscript have the following competing interests: DMM is an inventor on a patent for H84T BanLec. He is also founder of Virule, a company that aims to commercialize H84T.

                [¤a]

                Current address: Emergent BioSolutions, Gaithersburg, Maryland, United States of America

                [¤b]

                Current address: University of Saint Joseph, West Hartford, Connecticut, United States of America

                [¤c]

                Current address: Bioqual, Inc., Rockville, Maryland, United States of America

                [¤d]

                Current address: Indiana Biosciences Research Institute, Indianapolis, Indiana, United States of America

                [¤e]

                Current address: Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America

                [¤f]

                Current address: MRIGlobal-Global Health Surveillance and Diagnostics, Gaithersburg, MD, United States of America

                Author information
                http://orcid.org/0000-0002-0532-996X
                Article
                PNTD-D-18-02019
                10.1371/journal.pntd.0007595
                6687191
                31356611
                128c8b1b-d9ba-402e-9a6e-13d4c06211ec

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 21 December 2018
                : 2 July 2019
                Page count
                Figures: 7, Tables: 0, Pages: 20
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000774, Defense Threat Reduction Agency;
                Award ID: HDTRA1-15-0067
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: 1RO1AI114776
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: 1F31AI136615-01
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: T32 GM07863
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: T32 AI007528
                Award Recipient :
                This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID), Integrated Research Facility (NIAID, Division of Clinical Research), and Battelle Memorial Institute’s prime contract with the NIAID (Contract # HHSN272200700016I). RG, DMG, JYL, and LED performed this work as employees of Battelle Memorial Institute (BMI). Subcontractors to BMI who performed this work are JD and EP as employees of Tunnell Consulting, Inc., LT as an employee of Charles River, and GGO Jr as an employee of MRIGlobal. This work was also supported by grants to DMM from the Defense Threat Reduction Agency (HDTRA1-15-0067; http://www.dtra.mil) and to JMW from the NIAID (1RO1AI114776; https://www.niaid.nih.gov). EMC-D was supported by National Institutes of Health training grants to the University of Michigan Medical Scientist Training Program (T32 GM07863) and Molecular Mechanisms of Microbial Pathogenesis (T32 AI007528), as well as by a National Research Service Award (1F31AI136615-01) from the NIAID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                All relevant data are within the manuscript and its Supporting Information files.

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