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1.
Cell ; 174(4): 938-952.e13, 2018 08 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.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Ebolavirus/imunologia , Epitopos/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Glicoproteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Feminino , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Resultado do Tratamento
2.
Cell ; 164(3): 392-405, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26806128

RESUMO

Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Sobreviventes , Animais , Reações Cruzadas , Modelos Animais de Doenças , Mapeamento de Epitopos , Cobaias , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Modelos Moleculares , Mutagênese , Uganda
3.
Cell ; 160(5): 904-912, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25723165

RESUMO

The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Marburgvirus/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Complexo Antígeno-Anticorpo/química , Linhagem Celular , Reações Cruzadas , Cristalografia por Raios X , Drosophila , Ebolavirus/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Doença do Vírus de Marburg/imunologia , Marburgvirus/genética , Marburgvirus/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Mucinas/química , Alinhamento de Sequência , Proteínas do Envelope Viral/metabolismo
4.
Cell ; 160(5): 893-903, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25723164

RESUMO

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Complexo Antígeno-Anticorpo/ultraestrutura , Doença do Vírus de Marburg/imunologia , Marburgvirus/química , Proteínas do Envelope Viral/química , Adulto , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Linfócitos B/imunologia , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Marburgvirus/genética , Marburgvirus/imunologia , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/metabolismo
5.
Immunity ; 49(2): 363-374.e10, 2018 08 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.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Ebolavirus/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Células 3T3 , Adulto , Animais , Células CHO , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Modelos Animais de Doenças , Drosophila , Feminino , Furões , Cobaias , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/imunologia , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células THP-1 , Células Vero
6.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728263

RESUMO

The human B cell response to natural filovirus infections early after recovery is poorly understood. Previous serologic studies suggest that some Ebola virus survivors exhibit delayed antibody responses with low magnitude and quality. Here, we sought to study the population of individual memory B cells induced early in convalescence. We isolated monoclonal antibodies (MAbs) from memory B cells from four survivors treated for Ebola virus disease (EVD) 1 or 3 months after discharge from the hospital. At the early time points postrecovery, the frequency of Ebola-specific B cells was low and dominated by clones that were cross-reactive with both Ebola glycoprotein (GP) and with the secreted GP (sGP) form. Of 25 MAbs isolated from four donors, only one exhibited neutralization activity. This neutralizing MAb, designated MAb EBOV237, recognizes an epitope in the glycan cap of the surface glycoprotein. In vivo murine lethal challenge studies showed that EBOV237 conferred protection when given prophylactically at a level similar to that of the ZMapp component MAb 13C6. The results suggest that the human B cell response to EVD 1 to 3 months postdischarge is characterized by a paucity of broad or potent neutralizing clones. However, the neutralizing epitope in the glycan cap recognized by EBOV237 may play a role in the early human antibody response to EVD and should be considered in rational design strategies for new Ebola virus vaccine candidates.IMPORTANCE The pathogenesis of Ebola virus disease (EVD) in humans is complex, and the mechanisms contributing to immunity are poorly understood. In particular, it appears that the quality and magnitude of the human B cell response early after recovery from EVD may be reduced compared to most viral infections. Here, we isolated human monoclonal antibodies from B cells of four survivors of EVD at 1 or 3 months after hospital discharge. Ebola-specific memory B cells early in convalescence were low in frequency, and the antibodies they encoded demonstrated poor neutralizing potencies. One neutralizing antibody that protected mice from lethal infection, EBOV237, was identified in the panel of 25 human antibodies isolated. Recognition of the glycan cap epitope recognized by EBOV237 suggests that this antigenic site should be considered in vaccine design and treatment strategies for EVD.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Memória Imunológica , Sobreviventes , Proteínas do Envelope Viral/imunologia , Feminino , Humanos , Masculino , Estados Unidos
7.
J Virol ; 90(1): 266-78, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26468533

RESUMO

UNLABELLED: The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE: Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Filoviridae/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Imunização Passiva , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/administração & dosagem , Proteção Cruzada , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Camundongos Endogâmicos BALB C , Resultado do Tratamento
8.
PLoS Pathog ; 11(6): e1005016, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26115029

RESUMO

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Doença pelo Vírus Ebola/imunologia , Imunização , Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Ebolavirus/imunologia , Feminino , Masculino , Doença do Vírus de Marburg/imunologia , Camundongos Endogâmicos BALB C
9.
Proc Natl Acad Sci U S A ; 111(48): 17182-7, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25404321

RESUMO

Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Epitopos/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular , Ebolavirus/metabolismo , Epitopos/química , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Fragmentos Fab das Imunoglobulinas/imunologia , Microscopia Eletrônica , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas Virais/imunologia
10.
J Infect Dis ; 214(suppl 3): S210-S217, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27587634

RESUMO

BACKGROUND: Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. METHODS: Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. RESULTS: The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 105-9.0 × 108 genomes/mL. CONCLUSIONS: The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015.


Assuntos
Antígenos Virais/sangue , Surtos de Doenças , Ebolavirus/imunologia , Doença pelo Vírus Ebola/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Proteínas da Matriz Viral/sangue , África Ocidental/epidemiologia , Animais , Ebolavirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Doença pelo Vírus Ebola/virologia , Humanos , Imunoensaio , Limite de Detecção , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
11.
J Infect Dis ; 212 Suppl 2: S359-67, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26232440

RESUMO

BACKGROUND: Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. METHODS: Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. RESULTS: Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. CONCLUSIONS: The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins.


Assuntos
Antígenos Virais/imunologia , Filoviridae/imunologia , Imunoensaio/métodos , África Ocidental , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Ebolavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Doença do Vírus de Marburg/sangue , Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/virologia , Marburgvirus/imunologia , Serra Leoa
12.
Nature ; 454(7201): 177-82, 2008 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-18615077

RESUMO

Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and fusion of viral and host membranes. Here we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the amino terminus of GP1. This structure provides a template for unravelling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.


Assuntos
Anticorpos Antivirais/imunologia , Ebolavirus/química , Glicoproteínas/química , Glicoproteínas/imunologia , Sobreviventes , Animais , Anticorpos Antivirais/genética , Sítios de Ligação de Anticorpos , Catepsinas/metabolismo , Linhagem Celular , Cricetinae , Cricetulus , Cristalografia por Raios X , República Democrática do Congo , Ebolavirus/imunologia , Glicoproteínas/metabolismo , Glicosilação , Humanos , Fusão de Membrana , Modelos Moleculares , Conformação Proteica , Receptores Virais/química , Receptores Virais/metabolismo
13.
J Virol ; 86(1): 364-72, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22031933

RESUMO

Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ∼10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.


Assuntos
Catepsina L/metabolismo , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/enzimologia , Fusão de Membrana , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Ebolavirus/química , Ebolavirus/genética , Endossomos/enzimologia , Doença pelo Vírus Ebola/virologia , Humanos , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/genética
14.
J Virol ; 86(5): 2809-16, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22171276

RESUMO

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 ß-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.


Assuntos
Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Animais , Sítios de Ligação de Anticorpos , Ebolavirus/química , Ebolavirus/genética , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Sequências Repetidas Invertidas , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/genética
16.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 11): 1162-80, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19923712

RESUMO

The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature (London), 454, 177-182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions.


Assuntos
Ebolavirus/química , Glicoproteínas/química , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Linhagem Celular , Sequência Conservada , Cristalografia por Raios X , Ebolavirus/genética , Ebolavirus/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/isolamento & purificação , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/isolamento & purificação
17.
Nat Struct Mol Biol ; 26(3): 204-212, 2019 03.
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.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Proteínas Virais de Fusão/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Cristalografia por Raios X , Humanos , Estrutura Terciária de Proteína , Sobreviventes
18.
mBio ; 9(5)2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206174

RESUMO

Only one naturally occurring human antibody has been described thus far that is capable of potently neutralizing all five ebolaviruses. Here we present two crystal structures of this rare, pan-ebolavirus neutralizing human antibody in complex with Ebola virus and Bundibugyo virus glycoproteins (GPs), respectively. The structures delineate the key protein and glycan contacts for binding that are conserved across the ebolaviruses, explain the antibody's unique broad specificity and neutralization activity, and reveal the likely mechanism behind a known escape mutation in the fusion loop region of GP2. We found that the epitope of this antibody, ADI-15878, extends along the hydrophobic paddle of the fusion loop and then dips down into a highly conserved pocket beneath the N-terminal tail of GP2, a mode of recognition unlike any other antibody elicited against Ebola virus, and likely critical for its broad activity. The fold of Bundibugyo virus glycoprotein, not previously visualized, is similar to the fold of Ebola virus GP, and ADI-15878 binds to each virus's GP with a similar strategy and angle of attack. These findings will be useful in deployment of this antibody as a broad-spectrum therapeutic and in the design of immunogens that elicit the desired broadly neutralizing immune response against all members of the ebolavirus genus and filovirus family.IMPORTANCE There are five different members of the Ebolavirus genus. Provision of vaccines and treatments able to protect against any of the five ebolaviruses is an important goal of public health. Antibodies are a desired result of vaccines and can be delivered directly as therapeutics. Most antibodies, however, are effective against only one or two, not all, of these pathogens. Only one human antibody has been thus far described to neutralize all five ebolaviruses, antibody ADI-15878. Here we describe the molecular structure of ADI-15878 bound to the relevant target proteins of Ebola virus and Bundibugyo virus. We explain how it achieves its rare breadth of activity and propose strategies to design improved vaccines capable of eliciting more antibodies like ADI-15878.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Glicoproteínas/imunologia , Epitopos/imunologia , Humanos , Conformação Proteica , Proteínas do Envelope Viral/imunologia
19.
Cell Host Microbe ; 23(1): 101-109.e4, 2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29324225

RESUMO

Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%-90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific "wing" antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Marburgvirus/imunologia , Receptores Virais/imunologia , Receptores Virais/ultraestrutura , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/ultraestrutura , Agrobacterium tumefaciens , Animais , Anticorpos Monoclonais/ultraestrutura , Sítios de Ligação/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Chlorocebus aethiops , Cristalografia por Raios X , Drosophila melanogaster , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Marburgvirus/metabolismo , Glicoproteínas de Membrana/imunologia , Proteína C1 de Niemann-Pick , Nicotiana , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Ligação Viral
20.
Nat Microbiol ; 3(6): 670-677, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29736037

RESUMO

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs)3-6 that target EBOV-specific epitopes on the surface glycoprotein7,8. However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.


Assuntos
Anticorpos Neutralizantes/farmacologia , Ebolavirus/imunologia , Epitopos/imunologia , Doença pelo Vírus Ebola/imunologia , Glicoproteínas de Membrana/química , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Sítios de Ligação/efeitos dos fármacos , Chlorocebus aethiops , Reações Cruzadas , Furões , Cobaias , Doença pelo Vírus Ebola/metabolismo , Humanos , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Coelhos , Células Vero , Proteínas Virais/química , Proteínas Virais/metabolismo
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