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1.
Methods Mol Biol ; 1877: 351-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536015

RESUMO

The BCL-2 protein family plays central roles in the mitochondrial pathway of cell apoptosis. The BCL-2-Associated X protein (BAX), along with other proapoptotic proteins, induces cell death in response to a variety of stress stimuli. Upon receipt of killing signals, cytosolic BAX is activated and translocates to mitochondria where it causes mitochondrial outer membrane permeabilization (MOMP) and initials a series of cellular events that eventually lead to cell destruction. Despite recent progress toward understanding the structure, function, and activation mechanism of BAX, detailed information about how cytosolic BAX can be inhibited is still limited. Here we describe a method of selecting synthetic antibody fragments (Fabs) against BAX using phage display. Synthetic antibodies discovered from the selection have been used as structural probes to gain novel mechanistic details on BAX inhibition. This synthetic antibody selection method could be potentially applied to other BCL-2 proteins.


Assuntos
Anticorpos/química , Anticorpos/metabolismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo , Apoptose/fisiologia , Citosol/metabolismo , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
2.
Nature ; 563(7732): 559-563, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30464266

RESUMO

The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.

3.
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.

4.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976679

RESUMO

Dengue virus is the most globally prevalent mosquito-transmitted virus. Primary infection with one of four cocirculating serotypes (DENV-1 to -4) causes a febrile illness, but secondary infection with a heterologous serotype can result in severe disease, due in part to antibody-dependent enhancement of infection (ADE). In ADE, cross-reactive but nonneutralizing antibodies, or subprotective levels of neutralizing antibodies, promote uptake of antibody-opsonized virus in Fc-γ receptor-positive cells. Thus, elicitation of broadly neutralizing antibodies (bNAbs), but not nonneutralizing antibodies, is desirable for dengue vaccine development. Domain III of the envelope glycoprotein (EDIII) is targeted by bNAbs and thus is an attractive immunogen. However, immunization with EDIII results in sera with limited neutralization breadth. We developed "resurfaced" EDIII immunogens (rsDIIIs) in which the A/G strand epitope that is targeted by bNAb 4E11 is maintained but less desirable epitopes are masked. RsDIIIs bound 4E11, but not serotype-specific or nonneutralizing antibodies. One rsDIII and, unexpectedly, wild-type (WT) DENV-2 EDIII elicited cross-neutralizing antibody responses against DENV-1 to -3 in mice. While these sera were cross-neutralizing, they were not sufficiently potent to protect AG129 immunocompromised mice at a dose of 200 µl (50% focus reduction neutralization titer [FRNT50], ∼1:60 to 1:130) against mouse-adapted DENV-2. Our results provide insight into immunogen design strategies based on EDIII.IMPORTANCE Dengue virus causes approximately 390 million infections per year. Primary infection by one serotype causes a self-limiting febrile illness, but secondary infection by a heterologous serotype can result in severe dengue syndrome, which is characterized by hemorrhagic fever and shock syndrome. This severe disease is thought to arise because of cross-reactive, non- or poorly neutralizing antibodies from the primary infection that are present in serum at the time of secondary infection. These cross-reactive antibodies enhance the infection rather than controlling it. Therefore, induction of a broadly and potently neutralizing antibody response is desirable for dengue vaccine development. Here, we explore a novel strategy for developing immunogens based on domain III of the E glycoprotein, where undesirable epitopes (nonneutralizing or nonconserved) are masked by mutation. This work provides fundamental insight into the immune response to domain III that can be leveraged for future immunogen design.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/imunologia , Vírus da Dengue/genética , Domínios Proteicos/genética , Proteínas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/efeitos adversos , Anticorpos Facilitadores , Técnicas de Visualização da Superfície Celular , Reações Cruzadas , Dengue/virologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/química , Vírus da Dengue/imunologia , Epitopos/imunologia , Camundongos , Domínios Proteicos/imunologia , Engenharia de Proteínas/métodos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Virais/química , Proteínas Virais/genética
5.
J Biol Chem ; 293(16): 6201-6211, 2018 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-29500195

RESUMO

Filoviruses (family Filoviridae) include five ebolaviruses and Marburg virus. These pathogens cause a rapidly progressing and severe viral disease with high mortality rates (generally 30-90%). Outbreaks of filovirus disease are sporadic and, until recently, were limited to less than 500 cases. However, the 2013-2016 epidemic in western Africa, caused by Ebola virus (EBOV), illustrated the potential of filovirus outbreaks to escalate to a much larger scale (over 28,000 suspected cases). mAbs against the envelope glycoprotein represent a promising therapeutic platform for managing filovirus infections. However, mAbs that exhibit neutralization or protective properties against multiple filoviruses are rare. Here we examined a panel of engineered bi- and trispecific antibodies, in which variable domains of mAbs that target epitopes from multiple filoviruses were combined, for their capacity to neutralize viral infection across filovirus species. We found that bispecific combinations targeting EBOV and Sudan virus (another ebolavirus), provide potent cross-neutralization and protection in mice. Furthermore, trispecific combinations, targeting EBOV, Sudan virus, and Marburg virus, exhibited strong neutralization potential against all three viruses. These results provide important insights into multispecific antibody engineering against filoviruses and will inform future immunotherapeutic discoveries.

6.
Bioorg Med Chem ; 25(20): 5790-5798, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28947103

RESUMO

Monoclonal antibodies (mAbs) are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, developing anticarbohydrate antibodies to target glycans for those purposes has been less successful because the molecular basis for glycan-mAb interactions is poorly understood relative to protein- or peptide-binding mAbs. Here, we report our investigation on glycan-mAb interactions by using the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120, as the template for engineering highly specific mAbs to target glycans. We first analyzed 24 different X-ray structures of antiglycan mAbs from the Protein Data Bank to determine side chain amino acid distributions in of glycan-mAb interactions. We identified Tyr, Arg, Asn, Ser, Asp, and His as the six most prevalent residues in the glycan-mAb contacts. We then utilized this information to construct two phage display libraries ("Lib1" and "Lib2") in which positions on the heavy chain variable domains of 2G12 were allowed to vary in restricted manner among Tyr, Asp, Ser, His, Asn, Thr, Ala and Pro to interrogate the minimal physicochemical requirements for oligomannose recognition. We analyzed the sequences of 39 variants from Lib1 and 14 variants from Lib2 following selection against gp120, the results showed that there is a high degree of malleability within the 2G12 for glycan recognitions. We further characterized five unique phage clones from both libraries that exhibited a gp120-specific binding profile. Expression of two of these variants as soluble mAbs indicated that, while specificity of gp120-binding was retained, the affinity of these mutants was significantly reduced relative to WT 2G12. Nonetheless, the results indicate these is some malleability in the identity of contact residues and provide a novel insight into the nature of glycan-antibody interactions and how they may differ from protein-antibody binding interactions.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Técnicas de Visualização da Superfície Celular , Biblioteca Gênica , Mutação , Polissacarídeos/química , Polissacarídeos/metabolismo
7.
Immunol Lett ; 190: 289-295, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28890093

RESUMO

The Sudan virus (SUDV), an ebolavirus, causes severe hemorrhagic fever with human case fatality rates of ∼50%. Previous work from our lab demonstrated the synthetic antibody F4 potently inhibits viral entry and protects against lethal virus challenge in mice [Chen et al., ACS Chem. Biol., 2014, 9, 2263-2273]. Here, we explore mechanistic requirements as well as contribution of the Fc region and function on neutralization and in vivo protection. Live cell imaging demonstrates that the antibody colocalizes with vesicular stomatitis virus particles containing the Sudan virus glycoprotein (VSV-GPSUDV) and that the antibody is rapidly degraded within cellular endosomes. A viral escape mutant contained substitutions on the N-heptad repeat (NHR) segment of GP2, the fusion subunit. Truncation studies indicated that the size of the Fc impacts virus neutralization potential. Finally, we examined the protective efficacy of Fc-null mutants in mice, and found that Fc function was not required for high levels of protection. Altogether, these results indicate that neutralization of SUDV GP-mediated cell entry likely involves blockade of viral membrane fusion within endosomes, and that inhibition of viral entry is the likely mechanism of in vivo protection.


Assuntos
Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Células HEK293 , Humanos , Fusão de Membrana , Camundongos , Camundongos Knockout , Mutação/genética , Proteínas do Envelope Viral/imunologia , Internalização do Vírus
8.
Cell ; 169(5): 878-890.e15, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28525755

RESUMO

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, we mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Our findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Vacinas contra Ebola/imunologia , Doença pelo Vírus Ebola/imunologia , Sobreviventes , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Cercopithecus aethiops , Reações Cruzadas , Ebolavirus/classificação , Ebolavirus/imunologia , Feminino , Furões , Doença pelo Vírus Ebola/virologia , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Alinhamento de Sequência , Células Vero
9.
Clin Vaccine Immunol ; 24(5)2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28356257

RESUMO

Analysis of monoclonal antibodies (MAbs) derived from single B cell cloning has been highly beneficial for antimicrobial immunotherapy, vaccine design, and advancing our understanding of pathogen-triggered effects on the human immunoglobulin repertoire. Sequencing of variable domains of single B cells, and characterization of binding and functional activities of MAbs derived from those sequences, provides in-depth insight not only into sites of susceptibility for antibody-mediated neutralization or opsonization of the pathogen but also into the dynamics of protective antibody evolution during infection. This information can be utilized to rapidly develop novel immunotherapies of completely human origin and provides a roadmap for structure-based vaccine design that aims to elicit similar protective antibody responses. Here, we summarize recent aspects of the single B cell cloning approach.


Assuntos
Anti-Infecciosos/imunologia , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Anticorpos Neutralizantes/imunologia , Células Clonais , Humanos
10.
Hum Vaccin Immunother ; 13(4): 836-842, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-27786606

RESUMO

Bispecific antibody engineering, in which binding specificities toward 2 distinct epitopes are combined into a single molecule, can greatly enhance immunotherapeutic properties of monoclonal antibodies. While the bispecific antibody approach has been applied widely to targets for indications such as cancer and inflammation, the development of such agents for viral immunotherapy is only now emerging. Here, we review recent advances in the development of bispecific antibodies for viral immunotherapy, highlighting promising in vitro and in vivo results.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Fatores Imunológicos/imunologia , Imunoterapia/métodos , Viroses/terapia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Descoberta de Drogas/tendências , Humanos , Fatores Imunológicos/administração & dosagem , Camundongos
11.
Science ; 354(6310): 350-354, 2016 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-27608667

RESUMO

There is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other filoviruses. The conserved, essential interaction between the filovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs but is shielded by multiple mechanisms, including physical sequestration in late endosomes. Here, we describe a bispecific-antibody strategy to target this interaction, in which mAbs specific for NPC1 or the GP receptor-binding site are coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viral particles themselves for endosomal delivery and conferred postexposure protection against multiple ebolaviruses in mice. Such "Trojan horse" bispecific antibodies have potential as broad antifilovirus immunotherapeutics.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas de Transporte/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Glicoproteínas de Membrana/imunologia , Receptores Virais/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Endossomos/virologia , Doença pelo Vírus Ebola/terapia , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Internalização do Vírus
12.
Cell Rep ; 15(7): 1514-1526, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27160900

RESUMO

Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Ebolavirus/fisiologia , Epitopos/imunologia , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/imunologia , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes , Anticorpos Antivirais/química , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/ultraestrutura , Cobaias , Células HEK293 , Humanos , Cinética , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação/genética , Coloração Negativa , Testes de Neutralização , Resultado do Tratamento
13.
Sci Rep ; 6: 19193, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26758505

RESUMO

Filoviruses (Ebola and Marburg) cause severe hemorrhagic fever. There are five species of ebolavirus; among these, the Ebola (Zaire) and Sudan viruses (EBOV and SUDV, respectively) are highly pathogenic and have both caused recurring, large outbreaks. However, the EBOV and SUDV glycoprotein (GP) sequences are 45% divergent and thus antigenically distinct. Few antibodies with cross-neutralizing properties have been described to date. We used antibody engineering to develop novel bispecific antibodies (Bis-mAbs) that are cross-reactive toward base epitopes on GP from EBOV and SUDV. These Bis-mAbs exhibit potent neutralization against EBOV and SUDV GP pseudotyped viruses as well as authentic pathogens, and confer a high degree (in one case 100%) post-exposure protection of mice from both viruses. Our studies show that a single agent that targets the GP base epitopes is sufficient for protection in mice; such agents could be included in panfilovirus therapeutic antibody cocktails.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Profilaxia Pós-Exposição , Animais , Anticorpos Biespecíficos/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Afinidade de Anticorpos/imunologia , Linhagem Celular , Reações Cruzadas , Modelos Animais de Doenças , Ebolavirus/classificação , Humanos , Camundongos , Testes de Neutralização , Ligação Proteica , Engenharia de Proteínas
14.
J Biol Chem ; 291(1): 89-102, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26565029

RESUMO

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.


Assuntos
Anticorpos/farmacologia , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/química , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Sítios de Ligação , Citocromos c/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Permeabilidade/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
15.
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
16.
Virology ; 485: 371-82, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26339794

RESUMO

Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Domínios e Motivos de Interação entre Proteínas/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Técnicas de Visualização da Superfície Celular , Vírus da Dengue/classificação , Mapeamento de Epitopos/métodos , Epitopos/química , Epitopos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Biblioteca de Peptídeos , Ligação Proteica/imunologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Reprodutibilidade dos Testes , Alinhamento de Sequência , Sorogrupo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
17.
ACS Infect Dis ; 1(1): 42-52, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25984565

RESUMO

The Ebolaviruses are members of the family Filoviridae ("filoviruses") and cause severe hemhorragic fever with human case fatality rates as high as 90%. Infection requires attachment of the viral particle to cells and triggering of membrane fusion between the host and viral membranes, a process that occurs in the host endosome and is facilitated by the envelope glycoprotein (GP). One potential strategy for therapeutic intervention is the development of agents (antibodies, peptides, and small molecules) that can interfere with viral entry aspects such as attachment, uptake, priming, or membrane fusion. This paper highlights recent developments in the discovery and evaluation of therapeutic entry inhibitors and identifies opportunities moving forward.

18.
J Infect Dis ; 212 Suppl 2: S146-53, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25786917

RESUMO

Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion. Here, we describe the first structural and functional studies of a protein corresponding to the MARV GP2 FL. We found that this protein undergoes a pH-dependent conformational change, as monitored by circular dichroism and nuclear magnetic resonance. Furthermore, we report that, under low pH conditions, the MARV GP2 FL can induce content leakage from liposomes. The general aspects of this pH-dependent structure and lipid-perturbing behavior are consistent with previous reports on Ebola virus GP2 FL. However, nuclear magnetic resonance studies in lipid bicelles and mutational analysis indicate differences in structure exist between MARV and Ebola virus GP2 FL. These results provide new insight into the mechanism of MARV GP2-mediated cell entry.


Assuntos
Marburgvirus/química , Marburgvirus/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Animais , Escherichia coli/virologia , Concentração de Íons de Hidrogênio , Lipídeos/química , Lipossomos/metabolismo , Doença do Vírus de Marburg/virologia , Conformação Proteica , Internalização do Vírus
19.
Biopolymers ; 104(3): 178-85, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25753192

RESUMO

The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms a well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. These novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.


Assuntos
Peptídeos/química , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Selenometionina/química
20.
Biochemistry ; 54(8): 1589-99, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25658332

RESUMO

Fusion of host and viral membranes is a critical step during infection by membrane-bound viruses. The HIV-1 glycoproteins gp120 (surface subunit) and gp41 (fusion subunit) represent the prototypic system for studying this process; in the prevailing model, the gp41 ectodomain forms a trimeric six-helix bundle that constitutes a critical intermediate and provides the energetic driving force for overcoming barriers associated with membrane fusion. However, most structural studies of gp41 variants have been performed either on ectodomain constructs lacking one or more of the membrane-associated segments (the fusion peptide, FP, the membrane-proximal external region, MPER, and the transmembrane domain, TM) or on variants consisting of these isolated segments alone without the ectodomain. Several recent reports have suggested that the HIV-1 ectodomain, as well as larger construct containing the membrane-bound segments, dissociates from a trimer to a monomer in detergent micelles. Here we compare the properties of a series of gp41 variants to delineate the roles of the ectodomain, FP, and MPER and TM, all in membrane-mimicking environments. We find that these proteins are prone to formation of a monomer in detergent micelles. In one case, we observed exclusive monomer formation at pH 4 but conditional trimerization at pH 7 even at low micromolar (∼5 µM) protein concentrations. Liposome release assays demonstrate that these gp41-related proteins have the capacity to induce content leakage but that this activity is also strongly modulated by pH with much higher activity at pH 4. Circular dichroism, nuclear magnetic resonance, and binding assays with antibodies specific to the MPER provide insight into the structural and functional roles of the FP, MPER, and TM and their effect on structure within the larger context of the fusion subunit.


Assuntos
Proteína gp41 do Envelope de HIV/química , HIV-1/química , Multimerização Proteica , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Concentração de Íons de Hidrogênio , Micelas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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