West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

Kaufmann, Bärbel; Nybakken, Grant E.; Chipman, Paul R.; Zhang, Wei; Diamond, Michael S.; Fremont, Daved H.; Kühn, Richard; Rossmann, Michael G.

doi:10.1073/pnas.0603488103

Cited by 120 publications

(132 citation statements)

References 43 publications

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“…The density related to the variable domains of the Fab fragments was almost as strong as the density of the glycoprotein shell, indicating ∼90% occupancy of the 120 binding sites (87% for X1, 93% for X2). The density related to the constant regions of Fab CR4354 is only about 0.7-fold as strong as the variable region, suggesting flexibility of the elbow angle between variable and constant domains, as often observed with antibody structures (23)(24)(25)(26)(27).…”

Section: Resultsmentioning

confidence: 55%

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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.

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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: 55%

“…Initial manual placement of the E protein structure of mature WNV (27) into the WNV-CR4354 complex density suggested that the general arrangement of the E monomers in the complex was essentially the same as in mature flavivirions, indicating that Fab binding did not significantly change the conformation of the E proteins.…”

Section: Methodsmentioning

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.

Self Cite

124

121

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“…Fig. 4 A shows a reconstructed cryo-EM model using the electron density map for E16 (38) along with a representative binding configuration from the simulation for E16. In the simulation, E16 shows 100% occupancy at A and B epitopes and 20% occupancy for epitope C, as a result of steric exclusion by neighboring bound Abs.…”

Section: Partial Occupancy and Heterogeneitymentioning

confidence: 99%

“…For example, Kaufmann et al (38) and Fibriansah et al (17) attribute the lack of binding of E16 to epitope C and of 1F4 to epitope B, respectively, to possible steric hindrance of neighboring E-proteins in those epitopes to Ab binding. Dejnirattisai et al (22) suggest that differences in epitope presentation between epitope B and epitopes A and C are responsible for the weaker Ab binding to epitope B in EDE2-A11.…”

Section: Partial Occupancy and Heterogeneitymentioning

confidence: 99%

Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses

Ripoll

Khavrutskii

Wallqvist

et al. 2016

Biophysical Journal

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Cryo-electron-microscopy (cryo-EM) structures of flaviviruses reveal significant variation in epitope occupancy across different monoclonal antibodies that have largely been attributed to epitope-level differences in conformation or accessibility that affect antibody binding. The consequences of these variations for macroscopic properties such as antibody binding and neutralization are the results of the law of mass action-a stochastic process of innumerable binding and unbinding events between antibodies and the multiple binding sites on the flavivirus in equilibrium-that cannot be directly imputed from structure alone. We carried out coarse-grained spatial stochastic binding simulations for nine flavivirus antibodies with epitopes defined by cryo-EM or x-ray crystallography to assess the role of epitope spatial arrangement on antibody-binding stoichiometry, occupancy, and neutralization. In our simulations, all epitopes were equally competent for binding, representing the upper limit of binding stoichiometry that results from epitope spatial arrangement alone. Surprisingly, our simulations closely reproduced the relative occupancy and binding stoichiometry observed in cryo-EM, without having to account for differences in epitope accessibility or conformation, suggesting that epitope spatial arrangement alone may be sufficient to explain differences in binding occupancy and stoichiometry between antibodies. Furthermore, we found that there was significant heterogeneity in binding configurations even at saturating antibody concentrations, and that bivalent antibody binding may be more common than previously thought. Finally, we propose a structure-based explanation for the stoichiometric threshold model of neutralization.

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“…Many studies have shown that mAb binding to ED3 is most efficient at blocking virus attachment to cells (7)(8)(9)(10), and recently it has been suggested that some anti-ED3 mAbs may also impair virus membrane fusion (11,12). We have previously determined using virological techniques that residue Thr-332 in ED3 is a major neutralization site of WNV (13,14) (Fig.…”

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confidence: 99%

Long Range Communication in the Envelope Protein Domain III and Its Effect on the Resistance of West Nile Virus to Antibody-mediated Neutralization

Maillard

Jordan

Barrett

et al. 2008

Journal of Biological Chemistry

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The envelope protein domain III (ED3) of West Nile virus is the major virus-specific neutralization domain and harbors most of the critical mutations that induce resistance against antibody-mediated neutralization. We investigated the molecular mechanisms of neutralization resistance by studying the biophysical perturbations of monoclonal antibody (mAb)-resistant mutations on ED3 wild type. Our results showed that although the solution structure between ED3 wild type and mutants was preserved, the mutations that confer the highest degree of resistance to mAbs showed low protein stability and high local dynamic motions. Interestingly, the latter was observed in regions outside the mutation sites, indicating long range communications within ED3. Thus, we hypothesized that the mechanisms involved in resistance to mAb neutralization may include, in addition to mutations in the epitope, long range effects among distant structural elements. This hypothesis is consistent with reported mutations in other flaviviruses whose surfaces are not exposed for the interaction with other macromolecules, yet they confer mAb neutralization resistance.The genus Flavivirus of the family Flaviviridae consists of a number of pathogens of major public health importance that cause diseases, including yellow fever, Japanese encephalitis, tick-borne encephalitis, dengue, and, most recently, in the Americas, West Nile virus (WNV) 2 (1). Since it was first isolated in 1999, the number of human cases reporting West Nile viral encephalitis has increased dramatically. Currently there is no human vaccine to prevent WNV infections. All studies to date indicate that neutralizing antibodies are the major mechanism of protective immunity against flaviviruses (2), as demonstrated by the effectiveness of vaccines to prevent other flavivirus diseases (yellow fever, Japanese encephalitis, and tickborne encephalitis). The primary target of virus-neutralizing antibodies is the envelope (E) protein (3). The E protein is the major structural protein on the surface of flaviviruses and consists of three distinct structural domains (ED1-3). ED1 and ED2 are the central and dimerization domains, respectively, and ED3 (amino acids 295-395 in the E protein) is the putative receptor-binding domain (4, 5). Consistent with the functional role of ED3 is its orientation in the context of the intact virion. The ED3 of WNV clearly projects above the rest of the E protein facilitating the potential interaction with other macromolecules, such as antibodies or cell receptors (6).Although all three domains in the E protein contain immunogenic sites, suggesting that there is not a single defined antigenic site, most of the experimental evidence strongly indicates that ED3 is the major immunogenic domain for virus typespecific neutralizing monoclonal antibodies (mAbs) (3). Many studies have shown that mAb binding to ED3 is most efficient at blocking virus attachment to cells (7-10), and recently it has been suggested that some anti-ED3 mAbs may also impair virus membrane fusio...

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West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

Cited by 120 publications

References 43 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

Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses

Long Range Communication in the Envelope Protein Domain III and Its Effect on the Resistance of West Nile Virus to Antibody-mediated Neutralization

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