27 results on '"Murin CD"'
Search Results
2. Spatial localization of CD16a at the human NK cell ADCC lytic synapse.
- Author
-
Ross P, Fatima H, Leaman DP, Matthias J, Spencer K, Zwick MB, Henderson SC, Mace EM, and Murin CD
- Abstract
Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc γ RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells., Competing Interests: Conflict of Interest JM is an employee of the company Abberior Instruments America, which commercializes super-resolution microscopy systems, including MINFLUX. The other authors declare no conflict of interest.
- Published
- 2024
- Full Text
- View/download PDF
3. Editorial: Antibody Therapeutics for the Treatment of Filoviral Infection.
- Author
-
Murin CD, Gunn BM, Parren PWHI, and Kobinger GP
- Subjects
- Ebolavirus immunology, Filoviridae, Humans, Hemorrhagic Fever, Ebola immunology, Hemorrhagic Fever, Ebola therapy
- Abstract
Competing Interests: PP is an employee of Lava Therapeutics, a publicly traded biotechnology company that develops therapeutic antibodies including bispecific antibodies and bispecific antibody technology. He obtains stock options as part of his employment.
- Published
- 2022
- Full Text
- View/download PDF
4. Structural Biology Illuminates Molecular Determinants of Broad Ebolavirus Neutralization by Human Antibodies for Pan-Ebolavirus Therapeutic Development.
- Author
-
Murin CD, Gilchuk P, Crowe JE Jr, and Ward AB
- Subjects
- Antibodies, Monoclonal therapeutic use, Antibodies, Monoclonal, Humanized therapeutic use, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Antiviral Agents immunology, Antiviral Agents therapeutic use, Drug Combinations, Ebolavirus drug effects, Ebolavirus physiology, Epitopes chemistry, Epitopes immunology, Glycoproteins chemistry, Glycoproteins immunology, Hemorrhagic Fever, Ebola drug therapy, Hemorrhagic Fever, Ebola virology, Humans, Models, Molecular, Protein Domains immunology, Viral Proteins chemistry, Viral Proteins immunology, Antibodies, Monoclonal immunology, Antibodies, Monoclonal, Humanized immunology, Ebolavirus immunology, Hemorrhagic Fever, Ebola immunology
- Abstract
Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses., Competing Interests: JC has served as a consultant for Luna Biologics and Merck Sharp & Dohme Corp., is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics and AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Murin, Gilchuk, Crowe and Ward.)
- Published
- 2022
- Full Text
- View/download PDF
5. Pan-ebolavirus protective therapy by two multifunctional human antibodies.
- Author
-
Gilchuk P, Murin CD, Cross RW, Ilinykh PA, Huang K, Kuzmina N, Borisevich V, Agans KN, Geisbert JB, Zost SJ, Nargi RS, Sutton RE, Suryadevara N, Bombardi RG, Carnahan RH, Bukreyev A, Geisbert TW, Ward AB, and Crowe JE Jr
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal immunology, Antibodies, Neutralizing immunology, Binding Sites, Cell Line, Cryoelectron Microscopy, Ebolavirus ultrastructure, Epitopes immunology, Female, Glycoproteins chemistry, Glycoproteins immunology, Hemorrhagic Fever, Ebola virology, Humans, Hydrogen-Ion Concentration, Mice, Inbred BALB C, Models, Molecular, Primates, Receptors, Fc metabolism, Recombinant Proteins immunology, Viremia immunology, Mice, Antibodies, Viral immunology, Ebolavirus immunology, Hemorrhagic Fever, Ebola immunology, Hemorrhagic Fever, Ebola prevention & control
- Abstract
Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here, we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies, rEBOV-515 and rEBOV-442, that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity, resisted viral escape, and possessed differing requirements for their Fc-regions for optimal in vivo activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy., Competing Interests: Declaration of interests J.E.C. has served as a consultant for Eli Lilly, GlaxoSmithKline and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from Takeda Vaccines, IDBiologics, and AstraZeneca. Vanderbilt University has applied for patents concerning ebolavirus antibodies that are related to this work. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
6. Convergence of a common solution for broad ebolavirus neutralization by glycan cap-directed human antibodies.
- Author
-
Murin CD, Gilchuk P, Ilinykh PA, Huang K, Kuzmina N, Shen X, Bruhn JF, Bryan AL, Davidson E, Doranz BJ, Williamson LE, Copps J, Alkutkar T, Flyak AI, Bukreyev A, Crowe JE Jr, and Ward AB
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal metabolism, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing metabolism, Antibodies, Viral chemistry, Antibodies, Viral metabolism, Antibody Specificity, Binding Sites, Cryoelectron Microscopy, Ebolavirus growth & development, Ebolavirus immunology, Ebolavirus pathogenicity, Epitopes chemistry, Epitopes immunology, Female, HEK293 Cells, HeLa Cells, Hemorrhagic Fever, Ebola immunology, Hemorrhagic Fever, Ebola pathology, Hemorrhagic Fever, Ebola virology, Humans, Jurkat Cells, Mice, Models, Molecular, Polysaccharides chemistry, Polysaccharides immunology, Protein Binding, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Sequence Alignment, Sequence Homology, Amino Acid, Viral Envelope Proteins antagonists & inhibitors, Viral Envelope Proteins genetics, Viral Envelope Proteins metabolism, Antibodies, Monoclonal pharmacology, Antibodies, Neutralizing pharmacology, Antibodies, Viral pharmacology, Ebolavirus drug effects, Hemorrhagic Fever, Ebola drug therapy, Viral Envelope Proteins chemistry
- Abstract
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 β-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., Competing Interests: Declaration of interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is the founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from and IDBiologics and AstraZeneca. Vanderbilt University has applied for a patent that is related to antibodies discussed in this work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
7. Considerations of Antibody Geometric Constraints on NK Cell Antibody Dependent Cellular Cytotoxicity.
- Author
-
Murin CD
- Subjects
- Animals, Antibodies chemistry, Humans, Immunomodulation, Killer Cells, Natural metabolism, Ligands, Lymphocyte Activation immunology, Organ Specificity immunology, Protein Binding, Receptors, Immunologic chemistry, Receptors, Immunologic metabolism, Structure-Activity Relationship, Antibodies immunology, Antibody-Dependent Cell Cytotoxicity immunology, Killer Cells, Natural immunology
- Abstract
It has been well-established that antibody isotype, glycosylation, and epitope all play roles in the process of antibody dependent cellular cytotoxicity (ADCC). For natural killer (NK) cells, these phenotypes are linked to cellular activation through interaction with the IgG receptor FcγRIIIa, a single pass transmembrane receptor that participates in cytoplasmic signaling complexes. Therefore, it has been hypothesized that there may be underlying spatial and geometric principles that guide proper assembly of an activation complex within the NK cell immune synapse. Further, synergy of antibody phenotypic properties as well as allosteric changes upon antigen binding may also play an as-of-yet unknown role in ADCC. Understanding these facets, however, remains hampered by difficulties associated with studying immune synapse dynamics using classical approaches. In this review, I will discuss relevant NK cell biology related to ADCC, including the structural biology of Fc gamma receptors, and how the dynamics of the NK cell immune synapse are being studied using innovative microscopy techniques. I will provide examples from the literature demonstrating the effects of spatial and geometric constraints on the T cell receptor complex and how this relates to intracellular signaling and the molecular nature of lymphocyte activation complexes, including those of NK cells. Finally, I will examine how the integration of high-throughput and "omics" technologies will influence basic NK cell biology research moving forward. Overall, the goal of this review is to lay a basis for understanding the development of drugs and therapeutic antibodies aimed at augmenting appropriate NK cell ADCC activity in patients being treated for a wide range of illnesses., (Copyright © 2020 Murin.)
- Published
- 2020
- Full Text
- View/download PDF
8. Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.
- Author
-
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, and Crowe JE Jr
- Subjects
- Animals, Antibodies, Monoclonal therapeutic use, Antibodies, Neutralizing therapeutic use, Antibodies, Viral therapeutic use, Cell Line, Disease Models, Animal, Drug Therapy, Combination, Epitopes, Female, Glycoproteins chemistry, Hemorrhagic Fever, Ebola prevention & control, Humans, Immunoglobulin Fab Fragments immunology, Macaca mulatta, Male, Mice, Mice, Inbred BALB C, Molecular Mimicry, Protein Conformation, Antibodies, Monoclonal immunology, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Ebolavirus immunology, Glycoproteins immunology, Hemorrhagic Fever, Ebola immunology
- Abstract
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., Competing Interests: Declaration of Interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Sanofi and is on the Scientific Advisory Boards of CompuVax and Meissa Vaccines, is a recipient of previous unrelated research grants from Moderna and Sanofi, and is founder of IDBiologics. Vanderbilt University has applied for a patent that is related to this work. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
9. Antibody responses to viral infections: a structural perspective across three different enveloped viruses.
- Author
-
Murin CD, Wilson IA, and Ward AB
- Subjects
- Animals, Antibody Formation, Humans, Virus Diseases virology, Viruses genetics, Antibodies, Viral immunology, Virus Diseases immunology, Viruses immunology
- Abstract
Antibodies serve as critical barriers to viral infection. Humoral immunity to a virus is achieved through the dual role of antibodies in communicating the presence of invading pathogens in infected cells to effector cells, and in interfering with processes essential to the viral life cycle (chiefly entry into the host cell). For individuals that successfully control infection, virus-elicited antibodies can provide lifelong surveillance and protection from future insults. One approach to understand the nature of a successful immune response has been to utilize structural biology to uncover the molecular details of antibodies derived from vaccines or natural infection and how they interact with their cognate microbial antigens. The ability to isolate antigen-specific B-cells and rapidly solve structures of functional, monoclonal antibodies in complex with viral glycoprotein surface antigens has greatly expanded our knowledge of the sites of vulnerability on viruses. In this Review, we compare the adaptive humoral immune responses to human immunodeficiency virus (HIV), influenza and filoviruses, with a particular focus on neutralizing antibodies. The pathogenesis of each of these viruses is quite different, providing an opportunity for comparison of immune responses: HIV causes a persistent, chronic infection; influenza, an acute infection with multiple exposures during a lifetime and annual vaccination; filoviruses, a virulent, acute infection. Neutralizing antibodies that develop under these different constraints are therefore sentinels that can provide insight into the underlying humoral immune responses, as well as important lessons to guide future development of vaccines and immunotherapeutics.
- Published
- 2019
- Full Text
- View/download PDF
10. Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates.
- Author
-
Pascal KE, Dudgeon D, Trefry JC, Anantpadma M, Sakurai Y, Murin CD, Turner HL, Fairhurst J, Torres M, Rafique A, Yan Y, Badithe A, Yu K, Potocky T, Bixler SL, Chance TB, Pratt WD, Rossi FD, Shamblin JD, Wollen SE, Zelko JM, Carrion R Jr, Worwa G, Staples HM, Burakov D, Babb R, Chen G, Martin J, Huang TT, Erlandson K, Willis MS, Armstrong K, Dreier TM, Ward AB, Davey RA, Pitt MLM, Lipsich L, Mason P, Olson W, Stahl N, and Kyratsous CA
- Subjects
- Animals, Antibodies, Monoclonal isolation & purification, Glycoproteins immunology, Guinea Pigs, HEK293 Cells, Humans, Macaca mulatta, Male, Mice, Antibodies, Monoclonal therapeutic use, Antibodies, Neutralizing therapeutic use, Antibodies, Viral therapeutic use, Hemorrhagic Fever, Ebola drug therapy
- Abstract
Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases., Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses., Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent., Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.
- Published
- 2018
- Full Text
- View/download PDF
11. Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop.
- Author
-
Murin CD, Bruhn JF, Bornholdt ZA, Copps J, Stanfield R, and Ward AB
- Subjects
- Antibodies, Neutralizing genetics, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Antibodies, Viral metabolism, Cryoelectron Microscopy, Crystallography, Ebolavirus genetics, Filoviridae genetics, Glycoproteins genetics, Promoter Regions, Genetic genetics, Protein Structure, Secondary, Antibodies, Neutralizing metabolism, Ebolavirus metabolism, Filoviridae metabolism, Glycoproteins immunology, Glycoproteins metabolism
- Abstract
Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The antibody ADI-15878 was previously identified from a human survivor of Ebola virus Makona variant (EBOV/Mak) infection. This mAb demonstrated potent neutralizing activity against all known ebolaviruses and provided protection in rodent and ferret models against three ebolavirus species. Here, we describe the unliganded crystal structure of ADI-15878 as well as the cryo-EM structures of ADI-15878 in complex with the EBOV/Mak and Bundibugyo virus (BDBV) glycoproteins (GPs). ADI-15878 binds through an induced-fit mechanism by targeting highly conserved residues in the internal fusion loop (IFL), bridging across GP protomers via the heptad repeat 1 (HR1) region. Our structures provide a more complete description of the ebolavirus immunogenic landscape, as well as a molecular basis for how rare but potent antibodies target conserved filoviral fusion machinery., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
12. Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.
- Author
-
Saphire EO, Schendel SL, Fusco ML, Gangavarapu K, Gunn BM, Wec AZ, Halfmann PJ, Brannan JM, Herbert AS, Qiu X, Wagh K, He S, Giorgi EE, Theiler J, Pommert KBJ, Krause TB, Turner HL, Murin CD, Pallesen J, Davidson E, Ahmed R, Aman MJ, Bukreyev A, Burton DR, Crowe JE Jr, Davis CW, Georgiou G, Krammer F, Kyratsous CA, Lai JR, Nykiforuk C, Pauly MH, Rijal P, Takada A, Townsend AR, Volchkov V, Walker LM, Wang CI, Zeitlin L, Doranz BJ, Ward AB, Korber B, Kobinger GP, Andersen KG, Kawaoka Y, Alter G, Chandran K, and Dye JM
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, Female, Hemorrhagic Fever, Ebola immunology, Hemorrhagic Fever, Ebola virology, Immunization, Mice, Mice, Inbred BALB C, Treatment Outcome, Antibodies, Monoclonal immunology, Antibodies, Monoclonal isolation & purification, Ebolavirus immunology, Epitopes immunology, Hemorrhagic Fever, Ebola prevention & control, Membrane Glycoproteins immunology
- Abstract
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., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
13. Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.
- Author
-
Flyak AI, Kuzmina N, Murin CD, Bryan C, Davidson E, Gilchuk P, Gulka CP, Ilinykh PA, Shen X, Huang K, Ramanathan P, Turner H, Fusco ML, Lampley R, Kose N, King H, Sapparapu G, Doranz BJ, Ksiazek TG, Wright DW, Saphire EO, Ward AB, Bukreyev A, and Crowe JE Jr
- Subjects
- Animals, Antibodies, Neutralizing metabolism, Antibodies, Viral metabolism, Antibodies, Viral pharmacology, Binding Sites drug effects, Chlorocebus aethiops, Cross Reactions, Ferrets, Guinea Pigs, Hemorrhagic Fever, Ebola metabolism, Humans, Membrane Glycoproteins immunology, Membrane Glycoproteins metabolism, Mice, Models, Molecular, Protein Binding, Rabbits, Vero Cells, Viral Proteins chemistry, Viral Proteins metabolism, Antibodies, Neutralizing pharmacology, Ebolavirus immunology, Epitopes immunology, Hemorrhagic Fever, Ebola immunology, Membrane Glycoproteins chemistry
- Abstract
Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection
1 and has been used under compassionate-treatment protocols in humans2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs)3-6 that target EBOV-specific epitopes on the surface glycoprotein7,8 . However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.- Published
- 2018
- Full Text
- View/download PDF
14. Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.
- Author
-
Wec AZ, Herbert AS, Murin CD, Nyakatura EK, Abelson DM, Fels JM, He S, James RM, de La Vega MA, Zhu W, Bakken RR, Goodwin E, Turner HL, Jangra RK, Zeitlin L, Qiu X, Lai JR, Walker LM, Ward AB, Dye JM, Chandran K, and Bornholdt ZA
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing immunology, Antibodies, Viral chemistry, Antibodies, Viral immunology, Chlorocebus aethiops, Cross Reactions, Ebolavirus classification, Ebolavirus immunology, Female, Ferrets, Hemorrhagic Fever, Ebola virology, Humans, Kinetics, Mice, Mice, Inbred BALB C, Models, Molecular, Sequence Alignment, Vero Cells, Antibodies, Neutralizing isolation & purification, Antibodies, Viral isolation & purification, Ebola Vaccines immunology, Hemorrhagic Fever, Ebola immunology, Survivors
- Abstract
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 (GP
CL ) 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
15. Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.
- Author
-
Pallesen J, Murin CD, de Val N, Cottrell CA, Hastie KM, Turner HL, Fusco ML, Flyak AI, Zeitlin L, Crowe JE Jr, Andersen KG, Saphire EO, and Ward AB
- Subjects
- Amino Acid Sequence, Antibodies, Monoclonal immunology, Antibody Formation, Cross Reactions, Cryoelectron Microscopy, Ebolavirus pathogenicity, Epitopes immunology, Glycoproteins genetics, Glycoproteins immunology, Hemorrhagic Fever, Ebola virology, Humans, Membrane Glycoproteins genetics, Membrane Glycoproteins immunology, Mutation, Protein Multimerization, Sequence Alignment, Viral Proteins chemistry, Viral Proteins immunology, Antibodies, Monoclonal chemistry, Ebolavirus immunology, Glycoproteins chemistry, Hemorrhagic Fever, Ebola immunology, Membrane Glycoproteins chemistry, Models, Structural
- Abstract
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.
- Published
- 2016
- Full Text
- View/download PDF
16. Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site.
- Author
-
Howell KA, Qiu X, Brannan JM, Bryan C, Davidson E, Holtsberg FW, Wec AZ, Shulenin S, Biggins JE, Douglas R, Enterlein SG, Turner HL, Pallesen J, Murin CD, He S, Kroeker A, Vu H, Herbert AS, Fusco ML, Nyakatura EK, Lai JR, Keck ZY, Foung SKH, Saphire EO, Zeitlin L, Ward AB, Chandran K, Doranz BJ, Kobinger GP, Dye JM, and Aman MJ
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal ultrastructure, Antibodies, Neutralizing, Antibodies, Viral chemistry, Binding Sites, Disease Models, Animal, Female, Glycoproteins chemistry, Glycoproteins genetics, Glycoproteins ultrastructure, Guinea Pigs, HEK293 Cells, Humans, Kinetics, Mice, Inbred BALB C, Models, Molecular, Mutation genetics, Negative Staining, Neutralization Tests, Treatment Outcome, Antibodies, Monoclonal therapeutic use, Ebolavirus physiology, Epitopes immunology, Glycoproteins metabolism, Hemorrhagic Fever, Ebola immunology, Receptors, Virus metabolism
- Abstract
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., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
17. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak.
- Author
-
Bornholdt ZA, Turner HL, Murin CD, Li W, Sok D, Souders CA, Piper AE, Goff A, Shamblin JD, Wollen SE, Sprague TR, Fusco ML, Pommert KB, Cavacini LA, Smith HL, Klempner M, Reimann KA, Krauland E, Gerngross TU, Wittrup KD, Saphire EO, Burton DR, Glass PJ, Ward AB, and Walker LM
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal therapeutic use, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing therapeutic use, Antibodies, Viral chemistry, Antibodies, Viral therapeutic use, Antibody Formation, Antigen-Antibody Complex chemistry, Democratic Republic of the Congo epidemiology, Disease Outbreaks, Ebola Vaccines immunology, Ebola Vaccines therapeutic use, Hemorrhagic Fever, Ebola epidemiology, Hemorrhagic Fever, Ebola therapy, Humans, Immunization, Passive, Mice, Survivors, Tissue Donors, Viral Envelope Proteins chemistry, Virion immunology, Antibodies, Monoclonal isolation & purification, Antibodies, Neutralizing isolation & purification, Antibodies, Viral isolation & purification, Ebolavirus immunology, Hemorrhagic Fever, Ebola immunology, Viral Envelope Proteins immunology
- Abstract
Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies., (Copyright © 2016, American Association for the Advancement of Science.)
- Published
- 2016
- Full Text
- View/download PDF
18. Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection.
- Author
-
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, and Crowe JE Jr
- Subjects
- Animals, Cross Reactions, Disease Models, Animal, Epitope Mapping, Guinea Pigs, Humans, Mice, Mice, Inbred BALB C, Microscopy, Electron, Models, Molecular, Mutagenesis, Uganda, Antibodies, Monoclonal immunology, Antibodies, Neutralizing immunology, Ebolavirus immunology, Hemorrhagic Fever, Ebola immunology, Survivors
- Abstract
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.)
- Published
- 2016
- Full Text
- View/download PDF
19. Correction: Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs.
- Author
-
Fusco ML, Hashiguchi T, Cassan R, Biggins JE, Murin CD, Warfield KL, Li S, Holtsberg FW, Shulenin S, Vu H, Olinger GG, Kim DH, Whaley KJ, Zeitlin L, Ward AB, Nykiforuk C, Aman MJ, Berry JD, and Saphire EO
- Published
- 2015
- Full Text
- View/download PDF
20. Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs.
- Author
-
Fusco ML, Hashiguchi T, Cassan R, Biggins JE, Murin CD, Warfield KL, Li S, Holtsberg FW, Shulenin S, Vu H, Olinger GG, Kim DH, Whaley KJ, Zeitlin L, Ward AB, Nykiforuk C, Aman MJ, Berry JD, and Saphire EO
- Subjects
- Animals, Antibodies, Viral immunology, Cross Reactions immunology, Ebolavirus immunology, Female, Male, Marburg Virus Disease immunology, Mice, Inbred BALB C, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacology, Hemorrhagic Fever, Ebola immunology, Immunization, Marburg Virus Disease prevention & control, Marburgvirus immunology
- Abstract
The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.
- Published
- 2015
- Full Text
- View/download PDF
21. Mechanism of human antibody-mediated neutralization of Marburg virus.
- Author
-
Flyak AI, Ilinykh PA, Murin CD, Garron T, Shen X, Fusco ML, Hashiguchi T, Bornholdt ZA, Slaughter JC, Sapparapu G, Klages C, Ksiazek TG, Ward AB, Saphire EO, Bukreyev A, and Crowe JE Jr
- Subjects
- Adult, Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal metabolism, Antibodies, Neutralizing isolation & purification, Antibodies, Neutralizing metabolism, Antibodies, Viral chemistry, Antibodies, Viral immunology, Antibodies, Viral metabolism, B-Lymphocytes immunology, Female, Humans, Immunoglobulin Fab Fragments chemistry, Immunoglobulin Fab Fragments metabolism, Marburgvirus genetics, Marburgvirus immunology, Models, Molecular, Mutation, Protein Structure, Tertiary, Viral Envelope Proteins metabolism, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing immunology, Antigen-Antibody Complex ultrastructure, Marburg Virus Disease immunology, Marburgvirus chemistry, Viral Envelope Proteins chemistry
- Abstract
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., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
22. Structures of protective antibodies reveal sites of vulnerability on Ebola virus.
- Author
-
Murin CD, Fusco ML, Bornholdt ZA, Qiu X, Olinger GG, Zeitlin L, Kobinger GP, Ward AB, and Saphire EO
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Antibodies, Viral chemistry, Antibody Affinity immunology, Antibody Specificity immunology, Binding Sites, Antibody immunology, Cell Line, Ebolavirus metabolism, Epitopes chemistry, Glycoproteins immunology, Hemorrhagic Fever, Ebola immunology, Hemorrhagic Fever, Ebola virology, Immunoglobulin Fab Fragments immunology, Microscopy, Electron, Models, Molecular, Protein Structure, Tertiary, Viral Proteins immunology, Antibodies, Monoclonal immunology, Antibodies, Viral immunology, Ebolavirus immunology, Epitopes immunology
- Abstract
Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.
- Published
- 2014
- Full Text
- View/download PDF
23. Structure of 2G12 Fab2 in complex with soluble and fully glycosylated HIV-1 Env by negative-stain single-particle electron microscopy.
- Author
-
Murin CD, Julien JP, Sok D, Stanfield RL, Khayat R, Cupo A, Moore JP, Burton DR, Wilson IA, and Ward AB
- Subjects
- Antibodies, Neutralizing chemistry, Antibodies, Neutralizing metabolism, Image Processing, Computer-Assisted, Microscopy, Electron, Protein Binding, Protein Interaction Domains and Motifs, Protein Multimerization, Staining and Labeling methods, HIV Antibodies chemistry, HIV Antibodies metabolism, Immunoglobulin Fab Fragments chemistry, Immunoglobulin Fab Fragments metabolism, Macromolecular Substances ultrastructure, env Gene Products, Human Immunodeficiency Virus chemistry, env Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Unlabelled: The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody that binds exclusively to conserved N-linked glycans that form the high-mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high-mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here, we present a negative-stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ∼17-Å resolution that reveals the antibody's interaction with its native and fully glycosylated epitope. We also mapped relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knockouts. In addition, a reconstruction at ∼26 Å of the ternary complex formed by 2G12 Fab2, soluble CD4, and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env's interaction with coreceptor(s). These structures provide a basis for understanding 2G12 binding and neutralization, and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope., Importance: HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here, we present structural data that reveal the viral binding site of one of the first HIV-1-neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
- Full Text
- View/download PDF
24. Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120.
- Author
-
Kong L, Lee JH, Doores KJ, Murin CD, Julien JP, McBride R, Liu Y, Marozsan A, Cupo A, Klasse PJ, Hoffenberg S, Caulfield M, King CR, Hua Y, Le KM, Khayat R, Deller MC, Clayton T, Tien H, Feizi T, Sanders RW, Paulson JC, Moore JP, Stanfield RL, Burton DR, Ward AB, and Wilson IA
- Subjects
- 1-Deoxynojirimycin analogs & derivatives, 1-Deoxynojirimycin pharmacology, Alkaloids pharmacology, Amino Acid Motifs, Amino Acid Sequence, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing metabolism, Antigen-Antibody Reactions, Binding Sites, Antibody, Biopolymers, CD4 Antigens immunology, CD4 Antigens metabolism, Carbohydrate Sequence, Crystallography, X-Ray, Epitopes chemistry, Epitopes immunology, Glycosylation drug effects, HEK293 Cells, HIV Antibodies chemistry, HIV Antibodies metabolism, HIV Envelope Protein gp120 chemistry, HIV Envelope Protein gp120 metabolism, HIV Envelope Protein gp120 ultrastructure, Humans, Immunoglobulin Fab Fragments immunology, Immunoglobulin Fab Fragments metabolism, Microscopy, Electron, Models, Molecular, Molecular Docking Simulation, Molecular Sequence Data, Polysaccharides physiology, Protein Conformation, Protein Processing, Post-Translational drug effects, Structure-Activity Relationship, env Gene Products, Human Immunodeficiency Virus immunology, Antibodies, Neutralizing immunology, HIV Antibodies immunology, HIV Envelope Protein gp120 immunology
- Abstract
A substantial proportion of the broadly neutralizing antibodies (bnAbs) identified in certain HIV-infected donors recognize glycan-dependent epitopes on HIV-1 gp120. Here we elucidate how the bnAb PGT 135 binds its Asn332 glycan-dependent epitope from its 3.1-Å crystal structure with gp120, CD4 and Fab 17b. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield and access the gp120 protein surface. EM reveals that PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. Combined structural studies of PGT 135, PGT 128 and 2G12 show that this Asn332-dependent antigenic region is highly accessible and much more extensive than initially appreciated, which allows for multiple binding modes and varied angles of approach; thereby it represents a supersite of vulnerability for antibody neutralization.
- Published
- 2013
- Full Text
- View/download PDF
25. Expression vectors for Acinetobacter baylyi ADP1.
- Author
-
Murin CD, Segal K, Bryksin A, and Matsumura I
- Subjects
- Acinetobacter metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, Cloning, Molecular, DNA, Bacterial analysis, DNA, Bacterial genetics, Glucuronidase genetics, Glucuronidase metabolism, Molecular Sequence Data, Polymerase Chain Reaction, Acinetobacter genetics, Genetic Engineering methods, Genetic Vectors, Plasmids genetics
- Abstract
Acinetobacter baylyi ADP1 is naturally competent and proficient at homologous recombination, so it can be transformed without restriction digests or ligation reactions. Expression vectors for this system, however, are not yet widely available. Here we describe the construction and characterization of inducible expression vectors that replicate as plasmids in A. baylyi or integrate into a nonessential part of its chromosome. These tools will facilitate the engineering of genes and genomes in this promising model organism.
- Published
- 2012
- Full Text
- View/download PDF
26. CO2 fixation kinetics of Halothiobacillus neapolitanus mutant carboxysomes lacking carbonic anhydrase suggest the shell acts as a diffusional barrier for CO2.
- Author
-
Dou Z, Heinhorst S, Williams EB, Murin CD, Shively JM, and Cannon GC
- Subjects
- Carbon chemistry, Catalysis, Diffusion, Dimerization, Genetic Complementation Test, Hydrogen-Ion Concentration, Kinetics, Models, Biological, Plasmids metabolism, Ribulose-Bisphosphate Carboxylase chemistry, Time Factors, Carbon Dioxide chemistry, Carbonic Anhydrases chemistry, Halothiobacillus genetics, Halothiobacillus physiology, Mutation
- Abstract
The widely accepted models for the role of carboxysomes in the carbon-concentrating mechanism of autotrophic bacteria predict the carboxysomal carbonic anhydrase to be a crucial component. The enzyme is thought to dehydrate abundant cytosolic bicarbonate and provide ribulose 1.5-bisphosphate carboxylase/oxygenase (RubisCO) sequestered within the carboxysome with sufficiently high concentrations of its substrate, CO(2), to permit its efficient fixation onto ribulose 1,5-bisphosphate. In this study, structure and function of carboxysomes purified from wild type Halothiobacillus neapolitanus and from a high CO(2)-requiring mutant that is devoid of carboxysomal carbonic anhydrase were compared. The kinetic constants for the carbon fixation reaction confirmed the importance of a functional carboxysomal carbonic anhydrase for efficient catalysis by RubisCO. Furthermore, comparisons of the reaction in intact and broken microcompartments and by purified carboxysomal RubisCO implicated the protein shell of the microcompartment as impeding diffusion of CO(2) into and out of the carboxysome interior.
- Published
- 2008
- Full Text
- View/download PDF
27. Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus.
- Author
-
Heinhorst S, Williams EB, Cai F, Murin CD, Shively JM, and Cannon GC
- Subjects
- Carbon Dioxide metabolism, Cell Compartmentation physiology, Halothiobacillus physiology, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism, Bacterial Proteins metabolism, Carbonic Anhydrases metabolism, Halothiobacillus enzymology
- Abstract
In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.
- Published
- 2006
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.