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Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.
Gilchuk P, Murin CD, Milligan JC, Cross RW, Mire CE, Ilinykh PA, Huang K, Kuzmina N, Altman PX, Hui S, Gunn BM, Bryan AL, Davidson E, Doranz BJ, Turner HL, Alkutkar T, Flinko R, Orlandi C, Carnahan R, Nargi R, Bombardi RG, Vodzak ME, Li S, Okoli A, Ibeawuchi M, Ohiaeri B, Lewis GK, Alter G, Bukreyev A, Saphire EO, Geisbert TW, Ward AB, Crowe JE
(2020) Immunity 52: 388-403.e12
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Cell Line, Disease Models, Animal, Drug Therapy, Combination, Ebolavirus, Epitopes, Female, Glycoproteins, Hemorrhagic Fever, Ebola, Humans, Immunoglobulin Fab Fragments, Macaca mulatta, Male, Mice, Mice, Inbred BALB C, Molecular Mimicry, Protein Conformation
Show Abstract · Added March 31, 2020
Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.
Copyright © 2020 Elsevier Inc. All rights reserved.
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Early Human B Cell Response to Ebola Virus in Four U.S. Survivors of Infection.
Williamson LE, Flyak AI, Kose N, Bombardi R, Branchizio A, Reddy S, Davidson E, Doranz BJ, Fusco ML, Saphire EO, Halfmann PJ, Kawaoka Y, Piper AE, Glass PJ, Crowe JE
(2019) J Virol 93:
MeSH Terms: Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, B-Lymphocytes, Ebolavirus, Female, Hemorrhagic Fever, Ebola, Humans, Immunologic Memory, Male, Survivors, United States, Viral Envelope Proteins
Show Abstract · Added March 31, 2019
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. 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. 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.
Copyright © 2019 American Society for Microbiology.
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Antibody-Mediated Protective Mechanisms Induced by a Trivalent Parainfluenza Virus-Vectored Ebolavirus Vaccine.
Kimble JB, Malherbe DC, Meyer M, Gunn BM, Karim MM, Ilinykh PA, Iampietro M, Mohamed KS, Negi S, Gilchuk P, Huang K, Wolf YI, Braun W, Crowe JE, Alter G, Bukreyev A
(2019) J Virol 93:
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Cell Line, Drug Combinations, Ebola Vaccines, Ebolavirus, Epitopes, Female, Ferrets, Genetic Vectors, Glycoproteins, Guinea Pigs, Hemorrhagic Fever, Ebola, Parainfluenza Virus 3, Human, Viral Envelope Proteins, Viral Vaccines
Show Abstract · Added March 31, 2019
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. 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.
Copyright © 2019 American Society for Microbiology.
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In Vivo Delivery of Synthetic Human DNA-Encoded Monoclonal Antibodies Protect against Ebolavirus Infection in a Mouse Model.
Patel A, Park DH, Davis CW, Smith TRF, Leung A, Tierney K, Bryan A, Davidson E, Yu X, Racine T, Reed C, Gorman ME, Wise MC, Elliott STC, Esquivel R, Yan J, Chen J, Muthumani K, Doranz BJ, Saphire EO, Crowe JE, Broderick KE, Kobinger GP, He S, Qiu X, Kobasa D, Humeau L, Sardesai NY, Ahmed R, Weiner DB
(2018) Cell Rep 25: 1982-1993.e4
MeSH Terms: Animals, Antibodies, Monoclonal, DNA, Disease Models, Animal, Ebolavirus, Epitope Mapping, Epitopes, Female, Glycoproteins, HEK293 Cells, Hemorrhagic Fever, Ebola, Humans, Mice, Inbred BALB C, Muscles, Mutagenesis, Recombinant Proteins
Show Abstract · Added March 31, 2019
Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
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Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.
Gilchuk P, Kuzmina N, Ilinykh PA, Huang K, Gunn BM, Bryan A, Davidson E, Doranz BJ, Turner HL, Fusco ML, Bramble MS, Hoff NA, Binshtein E, Kose N, Flyak AI, Flinko R, Orlandi C, Carnahan R, Parrish EH, Sevy AM, Bombardi RG, Singh PK, Mukadi P, Muyembe-Tamfum JJ, Ohi MD, Saphire EO, Lewis GK, Alter G, Ward AB, Rimoin AW, Bukreyev A, Crowe JE
(2018) Immunity 49: 363-374.e10
MeSH Terms: 3T3 Cells, Adult, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, CHO Cells, Cell Line, Chlorocebus aethiops, Cricetulus, Disease Models, Animal, Drosophila, Ebolavirus, Female, Ferrets, Glycoproteins, Guinea Pigs, Hemorrhagic Fever, Ebola, Humans, Immunoglobulin G, Jurkat Cells, Male, Mice, Mice, Inbred BALB C, Mice, Knockout, THP-1 Cells, Vero Cells
Show Abstract · Added March 3, 2020
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.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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A "Trojan horse" bispecific-antibody strategy for broad protection against ebolaviruses.
Wec AZ, Nyakatura EK, Herbert AS, Howell KA, Holtsberg FW, Bakken RR, Mittler E, Christin JR, Shulenin S, Jangra RK, Bharrhan S, Kuehne AI, Bornholdt ZA, Flyak AI, Saphire EO, Crowe JE, Aman MJ, Dye JM, Lai JR, Chandran K
(2016) Science 354: 350-354
MeSH Terms: Animals, Antibodies, Bispecific, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Binding Sites, Carrier Proteins, Cell Line, Tumor, Ebolavirus, Endosomes, Hemorrhagic Fever, Ebola, Humans, Immunotherapy, Intracellular Signaling Peptides and Proteins, Membrane Glycoproteins, Mice, Mice, Inbred BALB C, Receptors, Virus, Viral Envelope Proteins, Virus Internalization
Show Abstract · Added April 13, 2017
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.
Copyright © 2016, American Association for the Advancement of Science.
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Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.
Pallesen J, Murin CD, de Val N, Cottrell CA, Hastie KM, Turner HL, Fusco ML, Flyak AI, Zeitlin L, Crowe JE, Andersen KG, Saphire EO, Ward AB
(2016) Nat Microbiol 1: 16128
MeSH Terms: Amino Acid Sequence, Antibodies, Monoclonal, Antibody Formation, Cross Reactions, Cryoelectron Microscopy, Ebolavirus, Epitopes, Glycoproteins, Hemorrhagic Fever, Ebola, Humans, Membrane Glycoproteins, Models, Structural, Mutation, Protein Multimerization, Sequence Alignment, Viral Proteins
Show Abstract · Added April 13, 2017
The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.
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Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.
Bornholdt ZA, Ndungo E, Fusco ML, Bale S, Flyak AI, Crowe JE, Chandran K, Saphire EO
(2016) mBio 7: e02154-15
MeSH Terms: Antibodies, Neutralizing, Antibodies, Viral, Carrier Proteins, Cell Line, Crystallography, X-Ray, Ebolavirus, Hemorrhagic Fever, Ebola, Humans, Hydrophobic and Hydrophilic Interactions, Intracellular Signaling Peptides and Proteins, Membrane Glycoproteins, Mutagenesis, Mutation, Protein Binding, Receptors, Virus, Viral Envelope Proteins, Virus Internalization
Show Abstract · Added May 4, 2016
UNLABELLED - The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.
IMPORTANCE - Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs.
Copyright © 2016 Bornholdt et al.
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Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection.
Flyak AI, Shen X, Murin CD, Turner HL, David JA, Fusco ML, Lampley R, Kose N, Ilinykh PA, Kuzmina N, Branchizio A, King H, Brown L, Bryan C, Davidson E, Doranz BJ, Slaughter JC, Sapparapu G, Klages C, Ksiazek TG, Saphire EO, Ward AB, Bukreyev A, Crowe JE
(2016) Cell 164: 392-405
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Cross Reactions, Disease Models, Animal, Ebolavirus, Epitope Mapping, Guinea Pigs, Hemorrhagic Fever, Ebola, Humans, Mice, Mice, Inbred BALB C, Microscopy, Electron, Models, Molecular, Mutagenesis, Survivors, Uganda
Show Abstract · Added January 26, 2016
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.
Copyright © 2016 Elsevier Inc. All rights reserved.
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Structural basis for Marburg virus neutralization by a cross-reactive human antibody.
Hashiguchi T, Fusco ML, Bornholdt ZA, Lee JE, Flyak AI, Matsuoka R, Kohda D, Yanagi Y, Hammel M, Crowe JE, Saphire EO
(2015) Cell 160: 904-912
MeSH Terms: Amino Acid Sequence, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Antigen-Antibody Complex, Cell Line, Cross Reactions, Crystallography, X-Ray, Drosophila, Ebolavirus, Humans, Immunoglobulin Fab Fragments, Marburg Virus Disease, Marburgvirus, Models, Molecular, Molecular Sequence Data, Mucins, Sequence Alignment, Viral Envelope Proteins
Show Abstract · Added January 26, 2016
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.
Copyright © 2015 Elsevier Inc. All rights reserved.
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20 MeSH Terms