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1.  Immune Memory to Sudan Virus: Comparison between Two Separate Disease Outbreaks 
Viruses  2015;7(1):37-51.
Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda—Gulu 2000–2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1–649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1–649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.
doi:10.3390/v7010037
PMCID: PMC4306827  PMID: 25569078
ebolavirus; human survivors; memory immunity; cross-reactivity
2.  Lassa virus entry requires a trigger-induced receptor switch 
Science (New York, N.Y.)  2014;344(6191):1506-1510.
Lassa virus spreads from rodents to humans and can lead to lethal hemorrhagic fever. Despite its broad tropism, chicken cells were reported to resist infection thirty years ago. We show that Lassa virus readily engaged its cell surface receptor α-dystroglycan in avian cells, but virus entry in susceptible species involved a pH-dependent switch to an intracellular receptor, the lysosome-resident protein LAMP1. Iterative haploid screens revealed that the sialyltransferase ST3GAL4 was required for the interaction of the virus glycoprotein with LAMP1. A single glycosylated residue in LAMP1, present in susceptible species but absent in birds, was essential for interaction with the Lassa virus envelope protein and subsequent infection. The resistance of Lamp1-deficient mice to Lassa virus highlights the relevance of this receptor switch in vivo.
doi:10.1126/science.1252480
PMCID: PMC4239993  PMID: 24970085
3.  Euthanasia Assessment in Ebola Virus Infected Nonhuman Primates 
Viruses  2014;6(11):4666-4682.
Multiple products are being developed for use against filoviral infections. Efficacy for these products will likely be demonstrated in nonhuman primate models of filoviral disease to satisfy licensure requirements under the Animal Rule, or to supplement human data. Typically, the endpoint for efficacy assessment will be survival following challenge; however, there exists no standardized approach for assessing the health or euthanasia criteria for filovirus-exposed nonhuman primates. Consideration of objective criteria is important to (a) ensure test subjects are euthanized without unnecessary distress; (b) enhance the likelihood that animals exhibiting mild or moderate signs of disease are not prematurely euthanized; (c) minimize the occurrence of spontaneous deaths and loss of end-stage samples; (d) enhance the reproducibility of experiments between different researchers; and (e) provide a defensible rationale for euthanasia decisions that withstands regulatory scrutiny. Historic records were compiled for 58 surviving and non-surviving monkeys exposed to Ebola virus at the US Army Medical Research Institute of Infectious Diseases. Clinical pathology parameters were statistically analyzed and those exhibiting predicative value for survival are reported. These findings may be useful for standardization of objective euthanasia assessments in rhesus monkeys exposed to Ebola virus and may serve as a useful approach for other standardization efforts.
doi:10.3390/v6114666
PMCID: PMC4246243  PMID: 25421892
filovirus; nonhuman primate; viral hemorrhagic fever; euthanasia; clinical pathology; Ebola virus
4.  Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names 
Kuhn, Jens H. | Andersen, Kristian G. | Bào, Yīmíng | Bavari, Sina | Becker, Stephan | Bennett, Richard S. | Bergman, Nicholas H. | Blinkova, Olga | Bradfute, Steven | Brister, J. Rodney | Bukreyev, Alexander | Chandran, Kartik | Chepurnov, Alexander A. | Davey, Robert A. | Dietzgen, Ralf G. | Doggett, Norman A. | Dolnik, Olga | Dye, John M. | Enterlein, Sven | Fenimore, Paul W. | Formenty, Pierre | Freiberg, Alexander N. | Garry, Robert F. | Garza, Nicole L. | Gire, Stephen K. | Gonzalez, Jean-Paul | Griffiths, Anthony | Happi, Christian T. | Hensley, Lisa E. | Herbert, Andrew S. | Hevey, Michael C. | Hoenen, Thomas | Honko, Anna N. | Ignatyev, Georgy M. | Jahrling, Peter B. | Johnson, Joshua C. | Johnson, Karl M. | Kindrachuk, Jason | Klenk, Hans-Dieter | Kobinger, Gary | Kochel, Tadeusz J. | Lackemeyer, Matthew G. | Lackner, Daniel F. | Leroy, Eric M. | Lever, Mark S. | Mühlberger, Elke | Netesov, Sergey V. | Olinger, Gene G. | Omilabu, Sunday A. | Palacios, Gustavo | Panchal, Rekha G. | Park, Daniel J. | Patterson, Jean L. | Paweska, Janusz T. | Peters, Clarence J. | Pettitt, James | Pitt, Louise | Radoshitzky, Sheli R. | Ryabchikova, Elena I. | Saphire, Erica Ollmann | Sabeti, Pardis C. | Sealfon, Rachel | Shestopalov, Aleksandr M. | Smither, Sophie J. | Sullivan, Nancy J. | Swanepoel, Robert | Takada, Ayato | Towner, Jonathan S. | van der Groen, Guido | Volchkov, Viktor E. | Volchkova, Valentina A. | Wahl-Jensen, Victoria | Warren, Travis K. | Warfield, Kelly L. | Weidmann, Manfred | Nichol, Stuart T.
Viruses  2014;6(9):3663-3682.
Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences.
doi:10.3390/v6093663
PMCID: PMC4189044  PMID: 25256396
Bundibugyo virus; cDNA clone; cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; Ravn virus; RefSeq; Reston virus; reverse genetics; Sudan virus; Taï Forest virus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
5.  Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus 
Journal of Virology  2013;87(9):4952-4964.
There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.
doi:10.1128/JVI.03361-12
PMCID: PMC3624300  PMID: 23408633
6.  VIRUS NOMENCLATURE BELOW THE SPECIES LEVEL: A STANDARDIZED NOMENCLATURE FOR LABORATORY ANIMAL-ADAPTED STRAINS AND VARIANTS OF VIRUSES ASSIGNED TO THE FAMILY FILOVIRIDAE 
Archives of virology  2013;158(6):1425-1432.
The International Committee on Taxonomy of Viruses (ICTV) organizes the classification of viruses into taxa, but is not responsible for the nomenclature for taxa members. International experts groups, such as the ICTV Study Groups, recommend the classification and naming of viruses and their strains, variants, and isolates. The ICTV Filoviridae Study Group has recently introduced an updated classification and nomenclature for filoviruses. Subsequently, and together with numerous other filovirus experts, a consistent nomenclature for their natural genetic variants and isolates was developed that aims at simplifying the retrieval of sequence data from electronic databases. This is a first important step toward a viral genome annotation standard as sought by the US National Center for Biotechnology Information (NCBI). Here, this work is extended to include filoviruses obtained in the laboratory by artificial selection through passage in laboratory hosts. The previously developed template for natural filovirus genetic variant naming ( ///-) is retained, but it is proposed to adapt the type of information added to each field for laboratory animal-adapted variants. For instance, the full-length designation of an Ebola virus Mayinga variant adapted at the State Research Center for Virology and Biotechnology “Vector” to cause disease in guinea pigs after seven passages would be akin to “Ebola virus VECTOR/C.porcellus-lab/COD/1976/Mayinga-GPA-P7”. As was proposed for the names of natural filovirus variants, we suggest using the full-length designation in databases, as well as in the method section of publications. Shortened designations (such as “EBOV VECTOR/C.por/COD/76/May-GPA-P7”) and abbreviations (such as “EBOV/May-GPA-P7”) could be used in the remainder of the text depending on how critical it is to convey information contained in the full-length name. “EBOV” would suffice if only one EBOV strain/variant/isolate is addressed.
doi:10.1007/s00705-012-1594-2
PMCID: PMC3669655  PMID: 23358612
cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
7.  Toll-Like Receptor Agonist Augments Virus-Like Particle-Mediated Protection from Ebola Virus with Transient Immune Activation 
PLoS ONE  2014;9(2):e89735.
Identifying safe and effective adjuvants is critical for the advanced development of protein-based vaccines. Pattern recognition receptor (PRR) agonists are increasingly being explored as potential adjuvants, but there is concern that the efficacy of these molecules may be dependent on potentially dangerous levels of non-specific immune activation. The filovirus virus-like particle (VLP) vaccine protects mice, guinea pigs, and nonhuman primates from viral challenge. In this study, we explored the impact of a stabilized dsRNA mimic, polyICLC, on VLP vaccination of C57BL/6 mice and Hartley guinea pigs. We show that at dose levels as low as 100 ng, the adjuvant increased the efficacy of the vaccine in mice. Antigen-specific, polyfunctional CD4 and CD8 T cell responses and antibody responses increased significantly upon inclusion of adjuvant. To determine whether the efficacy of polyICLC correlated with systemic immune activation, we examined serum cytokine levels and cellular activation in the draining lymph node. PolyICLC administration was associated with increases in TNFα, IL6, MCP1, MIP1α, KC, and MIP1β levels in the periphery and with the activation of dendritic cells (DCs), NK cells, and B cells. However, this activation resolved within 24 to 72 hours at efficacious adjuvant dose levels. These studies are the first to examine the polyICLC-induced enhancement of antigen-specific immune responses in the context of non-specific immune activation, and they provide a framework from which to consider adjuvant dose levels.
doi:10.1371/journal.pone.0089735
PMCID: PMC3933660  PMID: 24586996
9.  Virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae 
Archives of virology  2012;158(1):301-311.
The task of international expert groups is to recommend the classification and naming of viruses. The ICTV Filoviridae Study Group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. Here, further guidelines are suggested to include their natural genetic variants. First, this term is defined. Second, a template for full-length virus names (such as “Ebola virus H.sapiens-tc/COD/1995/Kikwit-9510621”) is proposed. These names contain information on the identity of the virus (e.g., Ebola virus), isolation host (e.g., members of the species Homo sapiens), sampling location (e.g., Democratic Republic of the Congo (COD)), sampling year, genetic variant (e.g., Kikwit), and isolate (e.g., 9510621). Suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). We suggest that these comprehensive names are to be used specifically in the methods section of publications. Suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. The proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. If applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the National Center for Biotechnology Information (NCBI). Furthermore, adoption of this nomenclature would increase the general understanding of filovirus-related publications and presentations and improve figures such as phylograms, alignments, and diagrams. Most importantly, it would counter the increasing confusion in genetic variant naming due to the identification of ever more sequences through technological breakthroughs in high-throughput sequencing and environmental sampling.
doi:10.1007/s00705-012-1454-0
PMCID: PMC3535543  PMID: 23001720
cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
10.  Niemann-Pick C1 (NPC1)/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor 
Viruses  2012;4(11):2471-2484.
We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C) of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor.
doi:10.3390/v4112471
PMCID: PMC3509659  PMID: 23202491
Ebola virus; Marburg virus; filovirus; viral entry; Niemann-Pick C1; NPC1; Niemann-Pick C1-like1; NPC1L1; host factor; viral receptor
11.  A Shared Structural Solution for Neutralizing Ebolaviruses 
Nature structural & molecular biology  2011;18(12):1424-1427.
Sudan virus (genus ebolavirus) is lethal, yet no monoclonal antibody is known to neutralize it. Here we describe antibody 16F6 that neutralizes Sudan virus and present its structure bound to the trimeric viral glycoprotein. Unexpectedly, the 16F6 epitope overlaps that of KZ52, the only other antibody against the GP1,2 core to be visualized. Further, both antibodies against this key GP1–GP2-bridging epitope neutralize at a post-internalization step, likely fusion.
doi:10.1038/nsmb.2150
PMCID: PMC3230659  PMID: 22101933
12.  Structural Basis for Differential Neutralization of Ebolaviruses 
Viruses  2012;4(4):447-470.
There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed GP1,2. Curiously, some antibodies against them are known to neutralize in vitro but not protect in vivo, whereas other antibodies are known to protect animal models in vivo, but not neutralize in vitro. A detailed understanding of what constitutes a neutralizing and/or protective antibody response is critical for development of novel therapeutic strategies. Here, we show that paradoxically, a lower affinity antibody with restricted access to its epitope confers better neutralization than a higher affinity antibody against a similar epitope, suggesting that either subtle differences in epitope, or different characteristics of the GP1,2 molecules themselves, confer differential neutralization susceptibility. Here, we also report the crystal structure of trimeric, prefusion GP1,2 from the original 1976 Boniface variant of Sudan virus complexed with 16F6, the first antibody known to neutralize Sudan virus, and compare the structure to that of Sudan virus, variant Gulu. We discuss new structural details of the GP1-GP2 clamp, thermal motion of various regions in GP1,2 across the two viruses visualized, details of differential interaction of the crystallized neutralizing antibodies, and their relevance for virus neutralization.
doi:10.3390/v4040447
PMCID: PMC3347318  PMID: 22590681
Filovirus; Ebola; ebolavirus; Sudan virus; neutralization: glycoprotein; antibodies; structure
13.  Profiling the Native Specific Human Humoral Immune Response to Sudan Ebola Virus Strain Gulu by Chemiluminescence Enzyme-Linked Immunosorbent Assay 
Clinical and Vaccine Immunology : CVI  2012;19(11):1844-1852.
Ebolavirus, a member of the family Filoviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP1-294 and GP1-649) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP1-649, followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP1-649, and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future.
doi:10.1128/CVI.00363-12
PMCID: PMC3491549  PMID: 22993411
14.  Ebola Virus Genome Plasticity as a Marker of Its Passaging History: A Comparison of In Vitro Passaging to Non-Human Primate Infection 
PLoS ONE  2012;7(11):e50316.
To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.
doi:10.1371/journal.pone.0050316
PMCID: PMC3509072  PMID: 23209706
15.  Designing and Testing Broadly-Protective Filoviral Vaccines Optimized for Cytotoxic T-Lymphocyte Epitope Coverage 
PLoS ONE  2012;7(10):e44769.
We report the rational design and in vivo testing of mosaic proteins for a polyvalent pan-filoviral vaccine using a computational strategy designed for the Human Immunodeficiency Virus type 1 (HIV-1) but also appropriate for Hepatitis C virus (HCV) and potentially other diverse viruses. Mosaics are sets of artificial recombinant proteins that are based on natural proteins. The recombinants are computationally selected using a genetic algorithm to optimize the coverage of potential cytotoxic T lymphocyte (CTL) epitopes. Because evolutionary history differs markedly between HIV-1 and filoviruses, we devised an adapted computational technique that is effective for sparsely sampled taxa; our first significant result is that the mosaic technique is effective in creating high-quality mosaic filovirus proteins. The resulting coverage of potential epitopes across filovirus species is superior to coverage by any natural variants, including current vaccine strains with demonstrated cross-reactivity. The mosaic cocktails are also robust: mosaics substantially outperformed natural strains when computationally tested against poorly sampled species and more variable genes. Furthermore, in a computational comparison of cross-reactive potential a design constructed prior to the Bundibugyo outbreak performed nearly as well against all species as an updated design that included Bundibugyo. These points suggest that the mosaic designs would be more resilient than natural-variant vaccines against future Ebola outbreaks dominated by novel viral variants. We demonstrate in vivo immunogenicity and protection against a heterologous challenge in a mouse model. This design work delineates the likely requirements and limitations on broadly-protective filoviral CTL vaccines.
doi:10.1371/journal.pone.0044769
PMCID: PMC3463593  PMID: 23056184
16.  A Characterization of Aerosolized Sudan Virus Infection in African Green Monkeys, Cynomolgus Macaques, and Rhesus Macaques 
Viruses  2012;4(10):2115-2136.
Filoviruses are members of the genera Ebolavirus, Marburgvirus, and “Cuevavirus”. Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable in aerosols, retain virulence after lyophilization, and can be present on contaminated surfaces for extended periods of time. This study explores the characteristics of aerosolized Sudan virus (SUDV) Boniface in non-human primates (NHP) belonging to three different species. Groups of cynomolgus macaques (cyno), rhesus macaques (rhesus), and African green monkeys (AGM) were challenged with target doses of 50 or 500 plaque-forming units (pfu) of aerosolized SUDV. Exposure to either viral dose resulted in increased body temperatures in all three NHP species beginning on days 4–5 post-exposure. Other clinical findings for all three NHP species included leukocytosis, thrombocytopenia, anorexia, dehydration, and lymphadenopathy. Disease in all of the NHPs was severe beginning on day 6 post-exposure, and all animals except one surviving rhesus macaque were euthanized by day 14. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were elevated during the course of disease in all three species; however, AGMs had significantly higher ALT and AST concentrations than cynos and rhesus. While all three species had detectable viral load by days 3-4 post exposure, Rhesus had lower average peak viral load than cynos or AGMs. Overall, the results indicate that the disease course after exposure to aerosolized SUDV is similar for all three species of NHP.
doi:10.3390/v4102115
PMCID: PMC3497044  PMID: 23202456
filovirus; ebolavirus; aerosol; primate; animal model
17.  Structure of an Antibody in Complex with Its Mucin Domain Linear Epitope That Is Protective against Ebola Virus 
Journal of Virology  2012;86(5):2809-2816.
Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.
doi:10.1128/JVI.05549-11
PMCID: PMC3302272  PMID: 22171276
18.  Filovirus vaccines 
Human Vaccines  2011;7(6):701-711.
Filoviruses can cause severe and often fatal hemorrhagic fever in humans and non-human primates (NHPs). Although there are currently no clinically proven treatments for filovirus disease, much progress has been made in recent years in the discovery of therapeutics and vaccines against these viruses. A variety of vaccine platforms have been shown to be effective against filovirus infection. This review summarizes the literature in this field, focusing on vaccines that have been shown to protect NHPs from infection. Furthermore, the uses of rodent models in vaccine development, as well as correlates of immunity, are discussed.
doi:10.4161/hv.7.6.15398
PMCID: PMC3219077  PMID: 21519188
ebola; ebola virus; ebolavirus; filovirid; filoviridae; filovirus; marburg virus; marburgvirus; mononegavirad; mononegavirales; mononegavirus; vaccine
19.  Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector▿  
Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.
doi:10.1128/CVI.00467-09
PMCID: PMC2849326  PMID: 20181765
20.  Synthetic Antibodies with a Human Framework That Protect Mice from Lethal Sudan Ebolavirus Challenge 
ACS Chemical Biology  2014;9(10):2263-2273.
The ebolaviruses cause severe and rapidly progressing hemorrhagic fever. There are five ebolavirus species; although much is known about Zaire ebolavirus (EBOV) and its neutralization by antibodies, little is known about Sudan ebolavirus (SUDV), which is emerging with increasing frequency. Here we describe monoclonal antibodies containing a human framework that potently inhibit infection by SUDV and protect mice from lethal challenge. The murine antibody 16F6, which binds the SUDV envelope glycoprotein (GP), served as the starting point for design. Sequence and structural alignment revealed similarities between 16F6 and YADS1, a synthetic antibody with a humanized scaffold. A focused phage library was constructed and screened to impart 16F6-like recognition properties onto the YADS1 scaffold. A panel of 17 antibodies were characterized and found to have a range of neutralization potentials against a pseudotype virus infection model. Neutralization correlated with GP binding as determined by ELISA. Two of these clones, E10 and F4, potently inhibited authentic SUDV and conferred protection and memory immunity in mice from lethal SUDV challenge. E10 and F4 were further shown to bind to the same epitope on GP as 16F6 with comparable affinities. These antibodies represent strong immunotherapeutic candidates for treatment of SUDV infection.
doi:10.1021/cb5006454
PMCID: PMC4201348  PMID: 25140871
21.  Protective Cytotoxic T-Cell Responses Induced by Venezuelan Equine Encephalitis Virus Replicons Expressing Ebola Virus Proteins 
Journal of Virology  2005;79(22):14189-14196.
Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8+ T-cell responses to six Ebola virus proteins were examined. CD8+ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8+ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8+ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.
doi:10.1128/JVI.79.22.14189-14196.2005
PMCID: PMC1280180  PMID: 16254354

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