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      A modified anthrax toxin-based enzyme-linked immunospot assay reveals robust T cell responses in symptomatic and asymptomatic Ebola virus exposed individuals

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          Abstract

          Background

          Ebola virus (EBOV) caused more than 11,000 deaths during the 2013–2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses.

          Methodology/Principle findings

          Between August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors.

          Conclusion/Significance

          Our data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development.

          Author summary

          The 2013–2016 West African Ebola virus (EBOV) outbreak is the largest on record with over 28,000 reported symptomatic cases and more than 11,000 deaths. We developed a simple and inexpensive modified anthrax toxin-based ELISPOT assay to detect and characterize the T cell responses elicited by prior exposure to EBOV. Our data show robust T cell responses to several EBOV proteins in individuals who experienced both severe and asymptomatic EBOV infections. These results provide further evidence that EBOV transmission events can go undetected. We also show that the seropositive asymptomatic individuals have stronger T cell responses compared to survivors, which has important implications for vaccine development.

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          Most cited references17

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          Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations.

          The taxonomy of the family Filoviridae (marburgviruses and ebolaviruses) has changed several times since the discovery of its members, resulting in a plethora of species and virus names and abbreviations. The current taxonomy has only been partially accepted by most laboratory virologists. Confusion likely arose for several reasons: species names that consist of several words or which (should) contain diacritical marks, the current orthographic identity of species and virus names, and the similar pronunciation of several virus abbreviations in the absence of guidance for the correct use of vernacular names. To rectify this problem, we suggest (1) to retain the current species names Reston ebolavirus, Sudan ebolavirus, and Zaire ebolavirus, but to replace the name Cote d'Ivoire ebolavirus [sic] with Taï Forest ebolavirus and Lake Victoria marburgvirus with Marburg marburgvirus; (2) to revert the virus names of the type marburgviruses and ebolaviruses to those used for decades in the field (Marburg virus instead of Lake Victoria marburgvirus and Ebola virus instead of Zaire ebolavirus); (3) to introduce names for the remaining viruses reminiscent of jargon used by laboratory virologists but nevertheless different from species names (Reston virus, Sudan virus, Taï Forest virus), and (4) to introduce distinct abbreviations for the individual viruses (RESTV for Reston virus, SUDV for Sudan virus, and TAFV for Taï Forest virus), while retaining that for Marburg virus (MARV) and reintroducing that used over decades for Ebola virus (EBOV). Paying tribute to developments in the field, we propose (a) to create a new ebolavirus species (Bundibugyo ebolavirus) for one member virus (Bundibugyo virus, BDBV); (b) to assign a second virus to the species Marburg marburgvirus (Ravn virus, RAVV) for better reflection of now available high-resolution phylogeny; and (c) to create a new tentative genus (Cuevavirus) with one tentative species (Lloviu cuevavirus) for the recently discovered Lloviu virus (LLOV). Furthermore, we explain the etymological derivation of individual names, their pronunciation, and their correct use, and we elaborate on demarcation criteria for each taxon and virus.
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            Ebola haemorrhagic fever in Sudan, 1976. Report of a WHO/International Study Team.

            A large outbreak of haemorrhagic fever (subsequently named Ebola haemorrhagic fever) occurred in southern Sudan between June and November 1976. There was a total of 284 cases; 67 in the source town of Nzara, 213 in Maridi, 3 in Tembura, and 1 in Juba. The outbreak in Nzara appears to have originated in the workers of a cotton factory. The disease in Maridi was amplified by transmission in a large, active hospital. Transmission of the disease required close contact with an acute case and was usually associated with the act of nursing a patient. The incubation period was between 7 and 14 days. Although the link was not well established, it appears that Nzara could have been the source of infection for a similar outbreak in the Bumba Zone of Zaire.In this outbreak Ebola haemorrhagic fever was a unique clinical disease with a high mortality rate (53% overall) and a prolonged recovery period in those who survived. Beginning with an influenza-like syndrome, including fever, headache, and joint and muscle pains, the disease soon caused diarrhoea (81%), vomiting (59%), chest pain (83%), pain and dryness of the throat (63%), and rash (52%). Haemorrhagic manifestations were common (71%), being present in half of the recovered cases and in almost all the fatal cases.Two post mortems were carried out on patients in November 1976. The histopathological findings resembled those of an acute viral infection and although the features were characteristic they were not exclusively diagnostic. They closely resembled the features described in Marburg virus infection, with focal eosinophilic necrosis in the liver and destruction of lymphocytes and their replacement by plasma cells. One case had evidence of renal tubular necrosis.Two strains of Ebola virus were isolated from acute phase sera collected from acutely ill patients in Maridi hospital during the investigation in November 1976. Antibodies to Ebola virus were detected by immunofluorescence in 42 of 48 patients in Maridi who had been diagnosed clinically, but in only 6 of 31 patients in Nzara. The possibility of the indirect immunofluorescent test not being sufficiently sensitive is discussed.Of Maridi case contacts, in hospital and in the local community, 19% had antibodies. Very few of them gave any history of illness, indicating that Ebola virus can cause mild or even subclinical infections. Of the cloth room workers in the Nzara cotton factory, 37% appeared to have been infected, suggesting that the factory may have been the prime source of infection.
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              Successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies.

              Ebola virus (EBOV) is considered one of the most aggressive infectious agents and is capable of causing death in humans and nonhuman primates (NHPs) within days of exposure. Recent strategies have succeeded in preventing acquisition of infection in NHPs after treatment; however, these strategies are only successful when administered before or minutes after infection. The present work shows that a combination of three neutralizing monoclonal antibodies (mAbs) directed against the Ebola envelope glycoprotein (GP) resulted in complete survival (four of four cynomolgus macaques) with no apparent side effects when three doses were administered 3 days apart beginning at 24 hours after a lethal challenge with EBOV. The same treatment initiated 48 hours after lethal challenge with EBOV resulted in two of four cynomolgus macaques fully recovering. The survivors demonstrated an EBOV-GP-specific humoral and cell-mediated immune response. These data highlight the important role of antibodies to control EBOV replication in vivo, and support the use of mAbs against a severe filovirus infection.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draft
                Role: InvestigationRole: MethodologyRole: ValidationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: Project administrationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ValidationRole: Writing – review & editing
                Role: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: 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
                24 May 2018
                May 2018
                : 12
                : 5
                : e0006530
                Affiliations
                [1 ] Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, United States of America
                [2 ] College of Medicine, University of Lagos, Akoka, Lagos, Nigeria
                [3 ] Lagos University Teaching Hospital, Idi Oro, Lagos, Nigeria
                [4 ] African Centre of Excellence for Genomics of Infectious Diseases (ACEGID), Redeemer’s University, Ede, Osun State, Nigeria
                [5 ] Department of Biological Sciences, College of Natural Sciences, Redeemer’s University, Ede, Osun State, Nigeria
                [6 ] Haikou VTI Biological Institute, Haikou, Hainan, China
                University of Geneva Hospitals, SWITZERLAND
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-7424-6060
                http://orcid.org/0000-0003-4249-9850
                http://orcid.org/0000-0003-4739-4572
                Article
                PNTD-D-18-00472
                10.1371/journal.pntd.0006530
                5991402
                29795572
                d663beba-a7be-4d49-9ac8-789e501cad65
                © 2018 Herrera et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 March 2018
                : 15 May 2018
                Page count
                Figures: 5, Tables: 3, Pages: 14
                Funding
                Funded by: McLennan Family Fund - Harvard T.H. Chan School of Public Health
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000051, National Human Genome Research Institute;
                Award ID: U01HG007480
                Award Recipient : x
                Funded by: funder-id http://dx.doi.org/10.13039/100000051, National Human Genome Research Institute;
                Award ID: U54HG007480
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004421, World Bank Group;
                Award ID: ACE019
                Award Recipient :
                This study was supported by the McLennan Family Fund - Harvard T.H. Chan School of Public Health Dean’s Challenge grant. Additional support from NIH-National Human Genome Research Institute U01HG007480 and U54HG007480 and the World Bank Project ACE019. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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