Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

Lok, Shee-Mei; Kostyuchenko, V.A.; Ge, Nybakken; Ha, Holdaway; Aj, Battisti; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Ms, Diamond; Rj, Kuhn; Mg, Rossmann

doi:10.1038/nsmb.1382

Cited by 318 publications

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“…2). Thus, CR4354 Fab binding does not induce disturbance in the outer E protein shell, as was observed for the interaction of dengue virus with Fab fragments of 1A1D-2, a neutralizing mouse MAb that binds an epitope in DIII of the E protein (22).…”

Section: Resultsmentioning

confidence: 96%

“…The E monomer conformation in immature WNV is virtually identical to recombinant, soluble E protein (28), and thus it is improbable that CR4354 can bind to completely immature virus, which has been demonstrated to be noninfectious (29). However, although CR4354 may not bind the static cryoEM image of the immature virion, it remains possible (and perhaps even likely) that it recognizes conformational subsets of immature virions that occur during particle "breathing" (22).…”

Section: Discussionmentioning

confidence: 99%

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Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

Kaufmann

Vogt²,

Goudsmit³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

123

121

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Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.antibody | cryoelectron microscopy | flavivirus W est Nile virus (WNV) is a human pathogen that causes a febrile illness, which can progress to encephalitis, paralysis, and death. The virus is endemic in parts of Africa, Asia, and Europe, and in the past decade has spread throughout North America and into Central and South America (1). WNV is closely related to other arthropod-transmitted, medically relevant flaviviruses, such as dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses. These lipid-enveloped viruses enter host cells by receptor-mediated endocytosis. The singlestranded, positive-sense RNA genome is released into the cytoplasm after low pH induces the fusion of the viral lipid envelope with the endosomal membrane.Mature WNV virions are roughly spherical with a diameter of about 500 Å. The outer viral surface is composed of an icosahedral scaffold of 180 closely packed copies of the membrane-anchored envelope (E) glycoprotein. Sets of three, nearly parallel E homodimers are associated into rafts that form a herringbone pattern on the surface of mature virions. The ectodomain of E has three structural domains, DI, DII, and DIII (2-5), with domain DI positioned structurally between DII and DIII. DII contains a fusion loop at its distal end that is indispensable for virus-cell membrane fusion. The Ig-like C-terminal domain DIII undergoes a major, pH-triggered positional rearrangement essential for fusion, and may also be involved in receptor binding (2, 6-12). During cell entry of flaviviruses, low endosomal pH triggers a proposed E protein rearrangement cascade, including the dissociation of E dimers and the outward rotation of DII during the repositioning of E monomers into fusion-active trimers (6, 9, 13).The humoral immune response is essential for protection against flavivirus infection and disease (14,15). The E glycoprotein is the principal antigen that elicits neutralizing antibodies agai...

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Section: Resultsmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

Kaufmann

Vogt²,

Goudsmit³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

123

121

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“…This is relevant because a previous structure of DENV--4 sE in complex with an antibody that binds away from the EDE also had the 150 loop disordered 13 , highlighting an intrinsic mobility in this area depending on the E amino acid sequence. Displacement of the 150 loop allows the EDE1 light chain to come closer to sE and interact with domain III (compare panel e with f and g in Fig.1) in the region of the "A strand" epitope, which has been structurally characterized previously for murine DENV cross--reactive antibodies 16,17 . These domain III contacts are centered on the conserved E residue K310, the side chain of which makes a lid covering the indole ring of W101 of the fusion loop (ED Fig.…”

mentioning

confidence: 89%

Recognition determinants of broadly neutralizing human antibodies against dengue viruses

Rouvinski

Guardado-Calvo

Barba-Spaeth

et al. 2015

Nature

303

353

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2Dengue disease is caused by four different flavivirus 1 serotypes, which infect 390 million people yearly with 25% symptomatic cases 2 and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (DENV--2) 3,4 . Structural studies so far have characterized only epitopes recognized by serotype specific human antibodies 5,6 . We recently isolated human antibodies potently neutralizing all four DENV serotypes 7 . Here we describe the X--ray structures of four of these broadly neutralizing antibodies (bnAbs) in complex with the envelope glycoprotein E from DENV--2, revealing that the recognition determinants are at a serotype conserved site at the E dimer interface, including the exposed main chain of the E fusion loop 8 and the two conserved glycan chains.This "E--dimer dependent epitope" (EDE) is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell 9 , explaining its conservation across serotypes and highlighting an Achilles heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.Exposed at the surface of infectious mature DENV particles, protein E is the sole target of neutralizing antibodies. It displays an icosahedral arrangement in which 90 E dimers completely coat the viral surface 10,11 and which is sensitive to the environmental pH. Upon entry of DENV into cells via receptor--mediated endocytosis, the acidic 3 endosomal environment triggers an irreversible fusogenic conformational change in E that leads to fusion of viral and endosomal membranes 1 . The structure of the isolated E dimer has been determined by X--ray crystallography using the soluble ectodomain (sE) 8,12 . Protein E is relatively conserved, displaying about 65% amino acid sequence identity when comparing the most distant DENV serotypes. In particular, there are two conserved N--linked glycosylation sites at positions N67 and N153. To examine its interaction with the antibodies, we selected four highly potent bnAbs identified in the accompanying work: 747(4) A11 and 747 B7 (EDE2 group, requiring glycosylation at position N153 for efficient binding) and 752--2 C8 and 753(3) C10 (EDE1 group, binding regardless of the glycosylation at N153) 7 -referred to as A11, B7, C8 and C10 from hereon. The EDE2 bnAbs were isolated from the same patient (who had a secondary infection with DENV--2), and are somatic variants of the same IgG clone, derived from the IGHV3--74 and IGLV2--23 germ lines. The heavy chain has a very long (26 amino acids, IMGT convention) complementarity--determining region 3 (CDR H3). The EDE1 bnAbs were isolated from different patients and derive from (EDE1 C8, the patient appeared to have a primary infection of undetermined serotype) and IGHV1--3* and IGLV2--14 (EDE1 C10, from a patient with secondary DENV--1 infecti...

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“…In contrast, antibodies that recognize the "A" β-strand of E protein domain III (EDIII) have been shown to potently neutralize some-but rarely all four-serotypes (SI Appendix, Fig. S1) (16). We asked whether we could, through computational chemistry, redesign an A-strand-specific antibody, namely 4E11 (17, 18) (SI Appendix, Fig.…”

mentioning

confidence: 99%

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

Tharakaraman¹,

Robinson²,

Hatas³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Affinity improvement of proteins, including antibodies, by computational chemistry broadly relies on physics-based energy functions coupled with refinement. However, achieving significant enhancement of binding affinity (>10-fold) remains a challenging exercise, particularly for cross-reactive antibodies. We describe here an empirical approach that captures key physicochemical features common to antigen-antibody interfaces to predict proteinprotein interaction and mutations that confer increased affinity. We apply this approach to the design of affinity-enhancing mutations in 4E11, a potent cross-reactive neutralizing antibody to dengue virus (DV), without a crystal structure. Combination of predicted mutations led to a 450-fold improvement in affinity to serotype 4 of DV while preserving, or modestly increasing, affinity to serotypes 1-3 of DV. We show that increased affinity resulted in strong in vitro neutralizing activity to all four serotypes, and that the redesigned antibody has potent antiviral activity in a mouse model of DV challenge. Our findings demonstrate an empirical computational chemistry approach for improving protein-protein docking and engineering antibody affinity, which will help accelerate the development of clinically relevant antibodies. (1). Engineering improved affinity and specificity of these compounds can augment their potency and safety while decreasing required dosages. Production of antibodies with binding properties of interest typically relies on methods involving screening large numbers of clones generated by the immune system or by mutant libraries (2, 3). Alternatively, computer-based design offers the potential to rationally mutate available antibodies for improved properties, including enhanced affinity and specificity to target antigens. Recently, several successful examples of antibody affinity improvement by computational methods using physical modeling with energy minimization have been described (4-6). However, such approaches require a 3D structure of the antibody-antigen complex and rarely result in affinity gains greater than 10-fold. Further, these approaches are sensitive to precise atomic coordinates, rendering them inapplicable to computer-generated models. More significantly, enhancement of affinity in the context of an antibody that recognizes multiple antigens (i.e., crossreactive) remains a particular challenge.Dengue is the most medically relevant arboviral disease in humans, with an estimated 3.6 billion people at risk for infection. More than 200 million infections of dengue virus (DV) are estimated to occur globally each year (7). The incidence, geographical outreach, and number of severe disease cases of dengue are increasing (8, 9), making DV of increasing concern as a human pathogen. The complex of DVs is composed of four distinct serotypes (designated DV1-4) (10), which vary from one another at the amino acid level by 25-40%. The sequence and antigenic variability of DVs have challenged efforts to develop an effective vaccine or therapeutic against a...

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Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

Cited by 318 publications

References 44 publications

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

Recognition determinants of broadly neutralizing human antibodies against dengue viruses

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

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