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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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      Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp™

      Without an approved vaccine or treatment, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. Here we show that a combination of monoclonal antibodies (ZMapp™), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viremia, and abnormalities in blood count and chemistry were evident in many animals before ZMapp™ intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal hemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp™ is cross-reactive with the Guinean variant of Ebola. ZMapp™ currently exceeds all previous descriptions of efficacy with other therapeutics, and results warrant further development of this cocktail for clinical use.
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        Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!)

        Summary Background rVSV-ZEBOV is a recombinant, replication competent vesicular stomatitis virus-based candidate vaccine expressing a surface glycoprotein of Zaire Ebolavirus. We tested the effect of rVSV-ZEBOV in preventing Ebola virus disease in contacts and contacts of contacts of recently confirmed cases in Guinea, west Africa. Methods We did an open-label, cluster-randomised ring vaccination trial (Ebola ça Suffit!) in the communities of Conakry and eight surrounding prefectures in the Basse-Guinée region of Guinea, and in Tomkolili and Bombali in Sierra Leone. We assessed the efficacy of a single intramuscular dose of rVSV-ZEBOV (2×107 plaque-forming units administered in the deltoid muscle) in the prevention of laboratory confirmed Ebola virus disease. After confirmation of a case of Ebola virus disease, we definitively enumerated on a list a ring (cluster) of all their contacts and contacts of contacts including named contacts and contacts of contacts who were absent at the time of the trial team visit. The list was archived, then we randomly assigned clusters (1:1) to either immediate vaccination or delayed vaccination (21 days later) of all eligible individuals (eg, those aged ≥18 years and not pregnant, breastfeeding, or severely ill). An independent statistician generated the assignment sequence using block randomisation with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 individuals vs >20 individuals). Ebola response teams and laboratory workers were unaware of assignments. After a recommendation by an independent data and safety monitoring board, randomisation was stopped and immediate vaccination was also offered to children aged 6–17 years and all identified rings. The prespecified primary outcome was a laboratory confirmed case of Ebola virus disease with onset 10 days or more from randomisation. The primary analysis compared the incidence of Ebola virus disease in eligible and vaccinated individuals assigned to immediate vaccination versus eligible contacts and contacts of contacts assigned to delayed vaccination. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201503001057193. Findings In the randomised part of the trial we identified 4539 contacts and contacts of contacts in 51 clusters randomly assigned to immediate vaccination (of whom 3232 were eligible, 2151 consented, and 2119 were immediately vaccinated) and 4557 contacts and contacts of contacts in 47 clusters randomly assigned to delayed vaccination (of whom 3096 were eligible, 2539 consented, and 2041 were vaccinated 21 days after randomisation). No cases of Ebola virus disease occurred 10 days or more after randomisation among randomly assigned contacts and contacts of contacts vaccinated in immediate clusters versus 16 cases (7 clusters affected) among all eligible individuals in delayed clusters. Vaccine efficacy was 100% (95% CI 68·9–100·0, p=0·0045), and the calculated intraclass correlation coefficient was 0·035. Additionally, we defined 19 non-randomised clusters in which we enumerated 2745 contacts and contacts of contacts, 2006 of whom were eligible and 1677 were immediately vaccinated, including 194 children. The evidence from all 117 clusters showed that no cases of Ebola virus disease occurred 10 days or more after randomisation among all immediately vaccinated contacts and contacts of contacts versus 23 cases (11 clusters affected) among all eligible contacts and contacts of contacts in delayed plus all eligible contacts and contacts of contacts never vaccinated in immediate clusters. The estimated vaccine efficacy here was 100% (95% CI 79·3–100·0, p=0·0033). 52% of contacts and contacts of contacts assigned to immediate vaccination and in non-randomised clusters received the vaccine immediately; vaccination protected both vaccinated and unvaccinated people in those clusters. 5837 individuals in total received the vaccine (5643 adults and 194 children), and all vaccinees were followed up for 84 days. 3149 (53·9%) of 5837 individuals reported at least one adverse event in the 14 days after vaccination; these were typically mild (87·5% of all 7211 adverse events). Headache (1832 [25·4%]), fatigue (1361 [18·9%]), and muscle pain (942 [13·1%]) were the most commonly reported adverse events in this period across all age groups. 80 serious adverse events were identified, of which two were judged to be related to vaccination (one febrile reaction and one anaphylaxis) and one possibly related (influenza-like illness); all three recovered without sequelae. Interpretation The results add weight to the interim assessment that rVSV-ZEBOV offers substantial protection against Ebola virus disease, with no cases among vaccinated individuals from day 10 after vaccination in both randomised and non-randomised clusters. Funding WHO, UK Wellcome Trust, the UK Government through the Department of International Development, Médecins Sans Frontières, Norwegian Ministry of Foreign Affairs (through the Research Council of Norway's GLOBVAC programme), and the Canadian Government (through the Public Health Agency of Canada, Canadian Institutes of Health Research, International Development Research Centre and Department of Foreign Affairs, Trade and Development).
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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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            Author and article information

            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

            Contributors
            Role: Writing – original draft, Role: Writing – review & editing
            Role: Data curation, Role: Formal analysis, Role: Investigation, Role: Project administration, Role: Validation, Role: Visualization, Role: Writing – original draft, Role: Writing – review & editing
            Role: Data curation, Role: Formal analysis, Role: Investigation, Role: Project administration, Role: Visualization, Role: Writing – original draft, Role: Writing – review & editing
            Role: Investigation, Role: Writing – review & editing
            Role: Investigation, Role: Writing – review & editing
            Role: Resources, Role: Writing – review & editing
            Role: Data curation, Role: Formal analysis, Role: Investigation, Role: Validation, Role: Visualization, Role: Writing – original draft, Role: Writing – review & editing
            Role: Formal analysis, Role: Investigation
            Role: Investigation
            Role: Investigation
            Role: Investigation
            Role: Investigation
            Role: Investigation
            Role: Resources
            Role: Resources
            Role: Supervision, Role: Writing – review & editing
            Role: Supervision, Role: Writing – review & editing
            Role: Conceptualization, Role: Supervision, Role: Writing – review & editing
            ORCID: http://orcid.org/0000-0002-0532-996X, Role: Conceptualization, Role: Data curation, Role: Formal analysis, Role: Funding acquisition, Role: Project administration, Role: Supervision, Role: Writing – original draft, Role: Writing – review & editing
            Role: Conceptualization, Role: Funding acquisition, Role: Project administration, Role: Supervision, Role: 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
            31356611
            6687191
            10.1371/journal.pntd.0007595
            PNTD-D-18-02019
            (Editor)

            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.

            Counts
            Figures: 7, Tables: 0, Pages: 20
            Product
            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 : ORCID: http://orcid.org/0000-0002-0532-996X
            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.
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            All relevant data are within the manuscript and its Supporting Information files.

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