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2.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
3.
J Gen Virol ; 100(6): 911-912, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31021739

RESUMO

Members of the family Filoviridae produce variously shaped, often filamentous, enveloped virions containing linear non-segmented, negative-sense RNA genomes of 15-19 kb. Several filoviruses (e.g., Ebola virus) are pathogenic for humans and are highly virulent. Several filoviruses infect bats (e.g., Marburg virus), whereas the hosts of most other filoviruses are unknown. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on Filoviridae, which is available at www.ictv.global/report/filoviridae.

4.
Nat Struct Mol Biol ; 26(3): 204-212, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30833785

RESUMO

The structural features that govern broad-spectrum activity of broadly neutralizing anti-ebolavirus antibodies (Abs) outside of the internal fusion loop epitope are currently unknown. Here we describe the structure of a broadly neutralizing human monoclonal Ab (mAb), ADI-15946, which was identified in a human survivor of the 2013-2016 outbreak. The crystal structure of ADI-15946 in complex with cleaved Ebola virus glycoprotein (EBOV GPCL) reveals that binding of the mAb structurally mimics the conserved interaction between the EBOV GP core and its glycan cap ß17-ß18 loop to inhibit infection. Both endosomal proteolysis of EBOV GP and binding of mAb FVM09 displace this loop, thereby increasing exposure of ADI-15946's conserved epitope and enhancing neutralization. Our work also mapped the paratope of ADI-15946, thereby explaining reduced activity against Sudan virus, which enabled rational, structure-guided engineering to enhance binding and neutralization of Sudan virus while retaining the parental activity against EBOV and Bundibugyo virus.

5.
Arch Virol ; 164(4): 1233-1244, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30663023

RESUMO

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).


Assuntos
Mononegavirais/classificação , Mononegavirais/genética , Mononegavirais/isolamento & purificação , Filogenia , Virologia/organização & administração
6.
J Virol ; 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518655

RESUMO

Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV) and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce a broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV and BDBV. As immunization with a cocktail of antigenically-related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to the monovalent vaccines, sera from trivalent vaccinated guinea pigs bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389-403, 397-415 and 477-493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus survivors demonstrated that the immune sera block binding of antibodies specific for the GP glycan cap, GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, cocktail administration of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus.IMPORTANCE Symptoms of the disease caused by ebolaviruses Ebola, Bundibugyo and Sudan are similar, and their endemic areas overlap. However, because of the limited antigenic relatedness of ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous but not heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, which might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here in-depth analysis of the antibody responses to cocktail administration of human parainfluenza type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses, compared to that induced by individual vaccines.

7.
J Infect Dis ; 218(suppl_5): S475-S485, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30289506

RESUMO

The outer leaflet of the viral membrane of Ebola virus (EBOV) virions is enriched with phosphatidylserine (PtdSer), which is thought to play a central role in viral tropism, entry, and virus-associated immune evasion. We investigated the effects of inhibiting synthesis and/or export of PtdSer to the cell surface of infected cells on viral infectivity. Knockdown of both PtdSer synthase enzymes, PTDSS1 and PTDSS2, effectively decreased viral production. Decreased PtdSer expression resulted in an accumulation of virions at the plasma membrane and adjacent of intracellular organelles, suggesting that virion budding is impaired. The addition of inhibitors that block normal cellular trafficking of PtdSer to the plasma membrane resulted in a similar accumulation of virions and reduced viral replication. These findings demonstrate that plasma membrane-associated PtdSer is required for efficient EBOV budding, increasing EBOV infectivity, and could constitute a potential therapeutic target for the development of future countermeasures against EBOV.

8.
J Infect Dis ; 218(suppl_5): S335-S345, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30289531

RESUMO

Viral apoptotic mimicry, which is defined by exposure of phosphatidylserine (PtdSer) into the outer leaflet of budding enveloped viruses, increases viral tropism, infectivity and promotes immune evasion. Here, we report that the calcium (Ca2+)-dependent scramblase, transmembrane protein 16F (TMEM16F), is responsible for the incorporation of PtdSer into virion membranes during Ebola virus infection. Infection of Huh7 cells with Ebola virus resulted in a pronounced increase in plasma membrane-associated PtdSer, which was demonstrated to be dependent on TMEM16F function. Analysis of virions using imaging flow cytometry revealed that short hairpin RNA-mediated down-regulation of TMEM16F function directly reduced virion-associated PtdSer. Taken together, these studies demonstrate that TMEM16F is a central cellular factor in the exposure of PtdSer in the outer leaflet of viral membranes.

9.
MBio ; 9(5)2018 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-30301857

RESUMO

Ebola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3' and 5' extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.IMPORTANCE Ebola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.

10.
Trends Microbiol ; 2018 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-30201511

RESUMO

Testing vaccine efficacy against the highly lethal Ebola virus (EBOV) in humans is almost impossible due to obvious ethical reasons and the sporadic nature of outbreaks. For such situations, the 'animal rule' was established, requiring the product be tested in animal models, expected to predict the response observed in humans. For vaccines, this testing aims to identify immune correlates of protection, such as antibody or cell-mediated responses. In the wake of the 2013-2016 EBOV epidemic, and despite advancement of promising candidates into clinical trials, protective correlates remain ambiguous. In the hope of identifying a reliable correlate by comparing preclinical and clinical trial data on immune responses to vaccination, we conclude that correlates are not universal for all EBOV vaccines.

11.
J Infect Dis ; 218(suppl_5): S627-S635, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30169869

RESUMO

Background: Ebola virus (EBOV) infection causes severe hemorrhagic fever. EBOV transcription is controlled by host protein phosphatase 1 (PP1), which dephosphorylates VP30 protein. We previously developed 1E7-03, a compound targeting a noncatalytic site of PP1 that induced VP30 phosphorylation and inhibited EBOV transcription. Here, we attempted to further improve 1E7-03, which was not stable in murine serum. Results: High-throughput screening with EBOV-green fluorescent protein was conducted on 72 1E7-03 analogs and identified 6 best inhibitory and the least toxic compounds. A parallel in silico screening of compounds from the ZINC database by docking to PP1 identified the best-binding compound C31, which was also present among the top 6 compounds found in the viral screen. C31 showed the best EBOV inhibitory activity among the top 6 compounds and also inhibited EBOV minigenome. C31 bound to the PP1 C-terminal groove in vitro and increased VP30 phosphorylation in cultured cells. C31 demonstrated improved stability in mouse plasma and cell permeability, compared with 1E7-03. It was also detected for 24 hours after injection in mice. Conclusion: C31 represents a novel PP1-targeting EBOV inhibitor with improved pharmacological properties that can be further evaluated for future antifiloviral therapy.

12.
Cell Rep ; 24(7): 1802-1815.e5, 2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110637

RESUMO

Some monoclonal antibodies (mAbs) recovered from survivors of filovirus infections can protect against infection. It is currently unknown whether natural infection also induces some antibodies with the capacity for antibody-dependent enhancement (ADE). A panel of mAbs obtained from human survivors of filovirus infection caused by Ebola, Bundibugyo, or Marburg viruses was evaluated for their ability to facilitate ADE. ADE was observed readily with all mAbs examined at sub-neutralizing concentrations, and this effect was not restricted to mAbs with a particular epitope specificity, neutralizing capacity, or subclass. Blocking of specific Fcγ receptors reduced but did not abolish ADE that was associated with high-affinity binding antibodies, suggesting that lower-affinity interactions still cause ADE. Mutations of Fc fragments of an mAb that altered its interaction with Fc receptors rendered the antibody partially protective in vivo at a low dose, suggesting that ADE counteracts antibody-mediated protection and facilitates dissemination of filovirus infections.

13.
Cell Rep ; 24(7): 1816-1829, 2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110638

RESUMO

Comparative immune response profiling is important for selecting next-generation vaccines. We comprehensively evaluated the antibody responses from a panel of nine respiratory vaccines against Ebola virus (EBOV) derived from human and avian paramyxoviruses expressing EBOV glycoprotein (GP). Most vaccines were protective in guinea pigs but yielded antibody repertoires that differed in proportion targeting key antigenic regions, avidity, neutralizing antibody specificities, and linear epitope preferences. Competition studies with monoclonal antibodies from human survivors revealed that some epitopes in GP targeted for neutralization were vector dependent, while EBOV-neutralizing titers correlated with the response magnitude toward the receptor-binding domain and GP1/GP2 interface epitopes. While an immunogen determines the breadth of antibody response, distinct vaccine vectors can induce qualitatively different responses, affecting protective efficacy. These data suggest that immune correlates of vaccine protection cannot be generalized for all vaccines against the same pathogen, even if they use the exact same immunogen.

14.
J Infect Dis ; 218(suppl_5): S327-S334, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30085081

RESUMO

A better understanding of the mechanisms used by Ebola virus to disable the host immune system and spread the infection are of great importance for development of new therapeutic strategies. We demonstrate that treatment of monocytic cells with Ebola virus shed glycoprotein (GP) promotes their differentiation resulting in increased infection and cell death. The effects were inhibited by blocking Toll-like receptor 4 pathway. In addition, high levels of shed GP were detected in supernatants of cells treated with Ebola vaccines. This study highlights the role of shed GP in Ebola pathogenesis and also in adverse effects associated with Ebola vaccines.

15.
J Virol ; 2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135121

RESUMO

The filoviruses Marburg (MARV) and Ebola (EBOV) cause hemorrhagic fever in humans and non-human primates with case high fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation at amino acid residues 46-53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, co-immunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule 1E7-03 targeting a non-catalytic site of the enzyme, which previously showed to increase EBOV VP30 phosphorylation, was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at the cluster of phosphorylated amino acids Ser-46-Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35 and dramatically reduced replication of infectious MARV particles. Thus MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds.IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004-2005 and caused 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of MARV genome demonstrated differences compared to EBOV. The identified phosphorylation sites appeared to inhibit transcription, and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility of development of antivirals based on compounds targeting PP1.

16.
PLoS Pathog ; 14(8): e1007204, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30138408

RESUMO

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach.

17.
Cell ; 174(4): 938-952.e13, 2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096313

RESUMO

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

18.
Antiviral Res ; 158: 288-302, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30144461

RESUMO

Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/ß and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.

19.
Immunity ; 49(2): 363-374.e10, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30029854

RESUMO

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

20.
J Infect Dis ; 218(suppl_5): S418-S422, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30060231

RESUMO

Screening of monoclonal antibodies against ebolaviruses requires small-animal models. Wild-type mice require adaptation of ebolaviruses, whereas immunodeficient mice are still resistant to nonadapted Bundibugyo ebolavirus. Swapping of Ebola virus glycoprotein with that from Bundibugyo virus resulted in a replication-competent chimeric virus, which caused 100% lethal infection in STAT1 knockout mice. Monoclonal antibody BDBV223 isolated from a human survivor of Bundibugyo virus infection protected mice from challenge with the chimeric virus. These data demonstrate the suitability of the approach for in vivo screening of antibodies and suggest the greater contribution of internal Ebola proteins in pathogenesis compared to Bundibugyo virus proteins.

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