Prokaryotic expression and immunogenicity in rabbit of Ebola virus VP40 matrix protein

2017 ◽  
Author(s):  
Yinbiao Wang ◽  
Xiao Liu ◽  
Xingdong Yang ◽  
Ling Tao ◽  
Pengwei Xu ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Large Scale ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Polyclonal Antibodies ◽  
Hemorrhagic Fever ◽  
Water Soluble ◽  
Health Concern ◽  
Water Soluble Protein ◽  
Antibody Titers

Ebola hemorrhagic fever is among severe and fatal viral hemorrhagic fevers and a global health concern. Here, we constructed a prokaryotic expression plasmid pET32a-VP40 for the large-scale expression of VP40 in E. coli Rosetta (DE3) cells. Water-soluble protein was obtained through optimization of the expression temperature, time and IPTG concentrations. After purification through Ni-NTA affinity chromatography, VP40 protein was applied to immunize rabbit for the analysis of immunogenicity and the production of polyclonal antibodies. Western blot showed that recombinant VP40 protein could be recognized by anti-His6-tag MAb and rabbit polyclonal antibodies against VP40 protein. Polyclonal antibody titers in ELISA reached 1:51200. Moreover, the produced rabbit polyclonal antibodies could react specifically with VP40 protein expressed in BHK-21 cells by transfection of pcDNA-eGFP-VP40 in IPMA. The preparation of VP40 protein and its polyclonal antibodies laid the basis for developing immunoassays for EBOV detection.

scholarly journals Equine-Origin Immunoglobulin Fragments Protect Nonhuman Primates from Ebola Virus Disease

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Hualei Wang ◽  
Gary Wong ◽  
Wenjun Zhu ◽  
Shihua He ◽  
Yongkun Zhao ◽  
...  
Keyword(s):  
Large Scale ◽  
Nonhuman Primates ◽  
Ebola Virus ◽  
Virus Disease ◽  
Hemorrhagic Fever ◽  
West African ◽  
Clinical Testing ◽  
The Past ◽  
Over 40 Years

ABSTRACT Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab′)2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab′)2 was found to potently neutralize West African and Central African EBOV in vitro. Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab′)2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab′)2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab′)2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics. IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab′)2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab′)2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab′)2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.

scholarly journals Exploiting the Reverse Vaccinology Approach to Design Novel Subunit Vaccine against Ebola Virus

2020 ◽  
Author(s):  
Md. Asad Ullah ◽  
Bishajit Sarkar ◽  
Syed Sajidul Islam
Keyword(s):  
Molecular Docking ◽  
Mhc Class Ii ◽  
Large Scale ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Rna Virus ◽  
Docking Study ◽  
Dynamics Simulation ◽  
Molecular Docking Study ◽  
Class I Mhc

AbstractEbola virus is a highly pathogenic RNA virus that causes haemorrhagic fever in human. With very high mortality rate, Ebola virus is considered as one of the dangerous viruses in the world. Although, the Ebola outbreaks claimed many lives in the past, no satisfactory treatment or vaccine have been discovered yet to fight against Ebola. For this reason, in this study, various tools of bioinformatics and immunoinformatics were used to design possible vaccines against Zaire Ebola virus strain Mayinga-76. To construct the vaccine, three potential antigenic proteins of the virus, matrix protein VP40, envelope glycoprotein and nucleoprotein were selected against which the vaccines would be designed. The MHC class-I, MHC class-II and B-cell epitopes were determined and after robust analysis through various tools and molecular docking analysis, three vaccine candidates, designated as EV-1, EV-2 and EV-3, were constructed. Since the highly conserved epitopes were used for vaccine construction, these vaccine constructs are also expected to be effective on other strains of Ebola virus like strain Gabon-94 and Kikwit-95. Next, the molecular docking study on these vaccine constructs were analyzed by molecular docking study and EV-1 emerged as the best vaccine construct. Later, molecular dynamics simulation study revealed the good performances as well as good stability of the vaccine protein. Finally, codon adaptation and in silico cloning were conducted to design a possible plasmid (pET-19b plasmid vector was used) for large scale, industrial production of the EV-1 vaccine.

scholarly journals Ebola from emergence to epidemic: the virus and the disease, global preparedness and perspectives

2015 ◽  
Vol 9 (05) ◽  
pp. 441-455 ◽  
Author(s):  
Kuldeep Dhama ◽  
Yashpal Singh Malik ◽  
Satya Veer Singh Malik ◽  
Raj Kumar Singh
Keyword(s):  
Ebola Virus ◽  
Control Strategies ◽  
International Health ◽  
Hemorrhagic Fever ◽  
Viral Disease ◽  
Health Concern ◽  
Swine Flu ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hygienic Measures ◽  
And Control

Humans constantly encounter threats from many infectious, zoonotic, and devastating pathogens. Outbreaks of severe acute respiratory syndrome (SARS), bird flu, and swine flu posing pandemic threats have compelled health agencies to follow global preparedness for combating the emerging deadly pathogens. The outbreak in West Africa of highly contagious Ebola viral disease (EVD) that started in Guinea in December 2013, assumed global proportions to become the largest outbreak of EVD and the most prominent international health concern. With fatality rates of nearly 50%–90%, it has claimed, as of 11 April 2015, 10,619 human lives out of a total of 25,626 cases reported worldwide. Ebola virus (EBOV), a member of Filoviridae family, is associated with severe, often lethal, hemorrhagic fever disease in humans and animals. The animal hosts, including non-human primates and reservoir hosts (fruit bats), play a significant role in transmission and maintenance of EBOV in nature. Although no approved vaccine for the prevention of EVD currently exists, disease control can be greatly enhanced by timely laboratory confirmation through blood tests using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Adherence to strict sanitary and hygienic measures, monitoring and surveillance of EBOV, as well as quarantine checks on international trade, transport, and visitors from affected countries are mandatory to prevent and control the spread of EVD. This review describes the salient properties of EBOV and the development of novel diagnostics, vaccines, and control strategies for this emerging disease of high public health concern and international emergency.

scholarly journals A THEORETICAL STUDY ON FRACTIONAL EBOLA HEMORRHAGIC FEVER MODEL

Fractals ◽  
2021 ◽  
Author(s):  
SHAHER MOMANI ◽  
R. P. CHAUHAN ◽  
SUNIL KUMAR ◽  
SAMIR HADID
Keyword(s):  
Virus Infection ◽  
Ebola Virus ◽  
Virus Disease ◽  
Numerical Technique ◽  
Disease Model ◽  
Hemorrhagic Fever ◽  
Health Concern ◽  
Ebola Hemorrhagic Fever ◽  
Conformable Derivatives ◽  
Ebola Virus Infection

The Ebola virus infection (EVI), generally known as Ebola hemorrhagic fever, is a major health concern. The occasional outbreaks of virus occur primarily in certain parts of Africa. Many researches have been devoted to the study of the Ebola virus disease. In this paper, we have taken susceptible-infected-recovered-deceased-environment (SIRDP) system to investigate the dynamics of Ebola virus infection. We adopted fractional operators for a better illustration of model dynamics and memory effects. Initially, the Ebola disease model is modified with Caputo–Fabrizio arbitrary operator in Caputo sense (CFC) and we employed the fixed-point results for the existence and uniqueness of the solution of the fractional system. Further, we adopted the arbitrary fractional conformable and [Formula: see text]-conformable derivatives to the alternative representation of the model. For the numerical approximation of the system, we show a numerical technique based on the fundamental theorem of fractional calculus for CFC derivative and a numerical scheme called the Adams–Moulton for conformable derivatives. Finally, for the validation of theoretical results, the numerical simulations are displayed.

scholarly journals Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Ziying Han ◽  
Cari A. Sagum ◽  
Fumio Takizawa ◽  
Gordon Ruthel ◽  
Corbett T. Berry ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Ebola Virus ◽  
E3 Ubiquitin Ligase ◽  
Hemorrhagic Fever ◽  
Efficient Production ◽  
Ww Domain ◽  
Ww Domains ◽  
E3 Ligases

ABSTRACT Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress. IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.

scholarly journals Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression

2015 ◽  
Vol 90 (4) ◽  
pp. 1839-1848 ◽  
Author(s):  
Shun-ichiro Oda ◽  
Takeshi Noda ◽  
Kaveesha J. Wijesinghe ◽  
Peter Halfmann ◽  
Zachary A. Bornholdt ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Virus Assembly ◽  
Hemorrhagic Fever ◽  
Marburg Virus ◽  
Molecular Structures ◽  
Matrix Assembly ◽  
Terminal Domain ◽  
Terminal Domains

ABSTRACTMarburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly.IMPORTANCEMarburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function.

scholarly journals A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 52
Author(s):  
Ryan P. Bennett ◽  
Courtney L. Finch ◽  
Elena N. Postnikova ◽  
Ryan A. Stewart ◽  
Yingyun Cai ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Virus Disease ◽  
Screening Method ◽  
Marburg Virus ◽  
Health Concern ◽  
Screening Assay ◽  
Particle Assembly ◽  
High Throughput Screening Assay

Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.

scholarly journals Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Marc Schweneker ◽  
Andrea S. Laimbacher ◽  
Gert Zimmer ◽  
Susanne Wagner ◽  
Elisabeth M. Schraner ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Viral Vectors ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Virus Disease ◽  
Hemorrhagic Fever ◽  
Human Cells ◽  
Transient Expression Assay ◽  
Virus Like Particles ◽  
Modified Vaccinia Virus Ankara

ABSTRACT There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant. IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines.

scholarly journals PI(4,5)P2 Binding Sites in the Ebola Virus Matrix Protein Modulate Assembly and Budding

2018 ◽  
Author(s):  
Kristen A. Johnson ◽  
Melissa R. Budicini ◽  
Sarah Urata ◽  
Nisha Bhattarai ◽  
Bernard S. Gerstman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Treatment Options ◽  
Treatment Strategies ◽  
Hemorrhagic Fever ◽  
Lipid Binding ◽  
Cellular Assays ◽  
Cell Plasma

AbstractEbola virus (EBOV) causes sever hemorrhagic fever in humans, can cause death in a large percentage of those infected, and still lacks FDA approved treatment options. In this study, we investigated how the essential EBOV protein, VP40, forms stable oligomers to mediate budding and assembly from the host cell plasma membrane. An array of in vitro and cellular assays identified and characterized two lysine rich regions that bind to PI(4,5)P2 and serve distinct functions through the lipid binding and assembly of the viral matrix layer. We found that when VP40 binds PI(4,5)P2, VP40 oligomers become extremely stable and long lived. Together, this work characterizes the molecular basis of PI(4,5)P2 binding by VP40, which stabilizes formation of VP40 oligomers necessary for viral assembly and budding. Quercetin, a natural product that lowers PI(4,5)P2 in the plasma membrane, inhibited budding of VP40 VLPs and may inform future treatment strategies against EBOV.

scholarly journals A proteomic survey of Junín virus interactions with human proteins reveals host factors required for arenavirus replication

2017 ◽  
pp. JVI.01565-17 ◽  
Author(s):  
Christopher M. Ziegler ◽  
Philip Eisenhauer ◽  
Jamie A. Kelly ◽  
Loan N. Dang ◽  
Vedran Beganovic ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Large Scale ◽  
Matrix Protein ◽  
Hemorrhagic Fever ◽  
Etiologic Agent ◽  
Human Pathogens ◽  
Host Proteins ◽  
Protein Z ◽  
Argentine Hemorrhagic Fever ◽  
Human Proteins

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenvirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Further, there is a little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside of cells or within virus-like particles (VLPs) and/or (ii) are incorporated intobona fideJUNV strain Candid #1 particles. Bioinformatic analyses revealed that multiple classes of human proteins were overrepresented in the datasets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ARF1, ATP6V0D1 and PRDX3), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP5B, IMPDH2). Further, we show that release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This dataset provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCEArenaviruses are deadly human pathogens for which there are no United States Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a dataset that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

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