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1.
Int J Mol Sci ; 25(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38732142

RESUMO

The high mortality rate among patients with acute myocardial infarction (AMI) is one of the main problems of modern cardiology. It is quite obvious that there is an urgent need to create more effective drugs for the treatment of AMI than those currently used in the clinic. Such drugs could be enzyme-resistant peptide analogs of glucagon-like peptide-1 (GLP-1). GLP-1 receptor (GLP1R) agonists can prevent ischemia/reperfusion (I/R) cardiac injury. In addition, chronic administration of GLP1R agonists can alleviate the development of adverse cardiac remodeling in myocardial infarction, hypertension, and diabetes mellitus. GLP1R agonists can protect the heart against oxidative stress and reduce proinflammatory cytokine (IL-1ß, TNF-α, IL-6, and MCP-1) expression in the myocardium. GLP1R stimulation inhibits apoptosis, necroptosis, pyroptosis, and ferroptosis of cardiomyocytes. The activation of the GLP1R augments autophagy and mitophagy in the myocardium. GLP1R agonists downregulate reactive species generation through the activation of Epac and the GLP1R/PI3K/Akt/survivin pathway. The GLP1R, kinases (PKCε, PKA, Akt, AMPK, PI3K, ERK1/2, mTOR, GSK-3ß, PKG, MEK1/2, and MKK3), enzymes (HO-1 and eNOS), transcription factors (STAT3, CREB, Nrf2, and FoxO3), KATP channel opening, and MPT pore closing are involved in the cardioprotective effect of GLP1R agonists.


Assuntos
Cardiotônicos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Transdução de Sinais , Humanos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Animais , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/patologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Agonistas do Receptor do Peptídeo 1 Semelhante ao Glucagon
2.
Cell Rep ; 42(10): 113254, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37858466

RESUMO

Ebola virus (EBOV) and Bundibugyo virus (BDBV) belong to the family Filoviridae and cause a severe disease in humans. We previously isolated a large panel of monoclonal antibodies from B cells of human survivors from the 2007 Uganda BDBV outbreak, 16 survivors from the 2014 EBOV outbreak in the Democratic Republic of the Congo, and one survivor from the West African 2013-2016 EBOV epidemic. Here, we demonstrate that EBOV and BDBV are capable of spreading to neighboring cells through intercellular connections in a process that depends upon actin and T cell immunoglobulin and mucin 1 protein. We quantify spread through intercellular connections by immunofluorescence microscopy and flow cytometry. One of the antibodies, BDBV223, specific to the membrane-proximal external region, induces virus accumulation at the plasma membrane. The inhibiting activity of BDBV223 depends on BST2/tetherin.


Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Antígeno 2 do Estroma da Médula Óssea , Ebolavirus , Doença pelo Vírus Ebola , Humanos , Antígenos CD , Antígeno 2 do Estroma da Médula Óssea/imunologia , Ebolavirus/imunologia , Proteínas Ligadas por GPI , Doença pelo Vírus Ebola/virologia
3.
Cell Rep ; 35(2): 108984, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33852862

RESUMO

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form ß-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos , Sítios de Ligação , Microscopia Crioeletrônica , Ebolavirus/crescimento & desenvolvimento , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Epitopos/química , Epitopos/imunologia , Feminino , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Humanos , Células Jurkat , Camundongos , Modelos Moleculares , Polissacarídeos/química , Polissacarídeos/imunologia , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
4.
Cell Host Microbe ; 27(6): 976-991.e11, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32320678

RESUMO

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here, we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP, termed the wing region. One mAb targeting the GP2 wing, MR228, showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb, MR235, to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs, resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B , Chlorocebus aethiops , Modelos Animais de Doenças , Ebolavirus/imunologia , Epitopos/imunologia , Feminino , Glicoproteínas/imunologia , Cobaias , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sobreviventes , Células THP-1 , Células Vero , Proteínas do Envelope Viral/imunologia
5.
Cell Mol Life Sci ; 77(13): 2579-2603, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31562565

RESUMO

Ebola virus (EBOV) causes severe human disease with a high case fatality rate. The balance of evidence implies that the virus circulates in bats. The molecular basis for host-viral interactions, including the role for phosphorylation during infections, is largely undescribed. To address this, and to better understand the biology of EBOV, the phosphorylation of EBOV proteins was analyzed in virions purified from infected monkey Vero-E6 cells and bat EpoNi/22.1 cells using high-resolution mass spectrometry. All EBOV structural proteins were detected with high coverage, along with phosphopeptides. Phosphorylation sites were identified in all viral structural proteins. Comparison of EBOV protein phosphorylation in monkey and bat cells showed only partial overlap of phosphorylation sites, with shared sites found in NP, VP35, and VP24 proteins, and no common sites in the other proteins. Three-dimensional structural models were built for NP, VP35, VP40, GP, VP30 and VP24 proteins using available crystal structures or by de novo structure prediction to elucidate the potential role of the phosphorylation sites. Phosphorylation of one of the identified sites in VP35, Thr-210, was demonstrated to govern the transcriptional activity of the EBOV polymerase complex. Thr-210 phosphorylation was also shown to be important for VP35 interaction with NP. This is the first study to compare phosphorylation of all EBOV virion proteins produced in primate versus bat cells, and to demonstrate the role of VP35 phosphorylation in the viral life cycle. The results uncover a novel mechanism of EBOV transcription and identify novel targets for antiviral drug development.


Assuntos
Ebolavirus/genética , Ebolavirus/metabolismo , Regulação Viral da Expressão Gênica , Nucleoproteínas/metabolismo , Transcrição Gênica , Proteínas do Core Viral/metabolismo , Animais , Quirópteros , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas do Nucleocapsídeo , Nucleoproteínas/química , Fosforilação , Proteômica , Ribonucleoproteínas/metabolismo , Células Vero , Proteínas do Core Viral/química , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Vírion/genética , Vírion/metabolismo
6.
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
7.
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
8.
J Virol ; 93(4)2019 02 15.
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 broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent vaccine 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 sera from guinea pigs receiving the monovalent vaccines, sera from guinea pigs receiving the trivalent vaccine bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only a slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389 to 403, 397 to 415, and 477 to 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 infection survivors demonstrated that the immune sera block the binding of antibodies specific for the GP glycan cap, the GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, administration of a cocktail of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus.IMPORTANCE The symptoms of the disease caused by the ebolaviruses Ebola, Bundibugyo, and Sudan are similar, and their areas of endemicity overlap. However, because of the limited antigenic relatedness of the ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous and not against heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, and this 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, an in-depth analysis of the antibody responses to administration of a cocktail of human parainfluenza virus 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.


Assuntos
Vacinas contra Ebola/genética , Ebolavirus/genética , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Combinação de Medicamentos , Epitopos/imunologia , Feminino , Furões , Vetores Genéticos , Glicoproteínas/imunologia , Cobaias , Doença pelo Vírus Ebola/virologia , Vírus da Parainfluenza 3 Humana/genética , Vacinas Virais/genética
9.
Cell Rep ; 24(7): 1802-1815.e5, 2018 08 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.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Anticorpos Facilitadores , Doença pelo Vírus Ebola/virologia , Doença do Vírus de Marburg/virologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Ebolavirus/efeitos dos fármacos , Ebolavirus/genética , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/terapia , Humanos , Soros Imunes/química , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/mortalidade , Doença do Vírus de Marburg/terapia , Marburgvirus/efeitos dos fármacos , Marburgvirus/genética , Marburgvirus/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/virologia , Cultura Primária de Células , Receptores de IgG/genética , Receptores de IgG/imunologia , Análise de Sobrevida , Sobreviventes , Células THP-1 , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
10.
PLoS Pathog ; 14(8): e1007204, 2018 08.
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.


Assuntos
Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Proteínas do Envelope Viral/antagonistas & inibidores , Anticorpos Monoclonais/imunologia , Humanos
11.
J Infect Dis ; 218(suppl_5): S418-S422, 2018 11 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.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Doença pelo Vírus Ebola/imunologia , Camundongos , Camundongos Knockout , Células Vero , Proteínas do Envelope Viral/imunologia
12.
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
13.
J Virol ; 90(8): 3890-3901, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26819310

RESUMO

UNLABELLED: Recent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection. IMPORTANCE: The study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Filoviridae/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Chlorocebus aethiops , Filoviridae/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Genética Reversa , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
14.
J Virol ; 89(15): 7567-83, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25972536

RESUMO

UNLABELLED: Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC. IMPORTANCE: This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal effects of mutations disrupting the two IRADs differ, and the lists of affected genes only partially overlap such that VP35 and VP24 IRADs each have profound effects on antigen presentation by exposed DC. The global modulation of DC gene expression and the resulting lack of their maturation represent a major mechanism by which EBOV disables the T cell response and suggests that these suppressive pathways are a therapeutic target that may unleash the T cell responses during EBOV infection.


Assuntos
Células Dendríticas/metabolismo , Ebolavirus/metabolismo , Expressão Gênica , Doença pelo Vírus Ebola/genética , Interferons/genética , Nucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Virais/metabolismo , Células Cultivadas , Células Dendríticas/virologia , Ebolavirus/química , Ebolavirus/genética , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Humanos , Interferons/metabolismo , Proteínas do Nucleocapsídeo , Nucleoproteínas/química , Nucleoproteínas/genética , Estrutura Terciária de Proteína , Transdução de Sinais , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas Virais/química , Proteínas Virais/genética
15.
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
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