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

Abstract

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...

Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity

“…The hinge has been shown to play a critical role in the conformational change that E protein undergoes at low pH to fuse with cellular endosomes, allowing viral uncoating and the release of viral RNA into the cellular cytoplasm (7). We hypothesize that hinge-targeting NAbs act through mechanisms that block this structural transition, consistent with what has been reported for West Nile virus (20,21). Our data are consistent with models where the EDI/EDII hinge of each serotype contains a single or multiple overlapping epitopes targeted by primate NAbs.…”

“…Our data are consistent with models where the EDI/EDII hinge of each serotype contains a single or multiple overlapping epitopes targeted by primate NAbs. Indeed, structure studies with human mAbs that bind to the EDI/EDII hinge region of flaviviruses indicate that this region contains overlapping quaternary epitopes (9,20). Although primary cross-reactive T-cell responses and antibodies do not confer long-term protection against heterologous challenge in primates, it is possible that the EDI/EDII hinge region contains peptides that can be recognized by DENV-specific T cells that also contribute to protection against DENV reinfection.…”

Recognition determinants of broadly neutralizing human antibodies against dengue viruses

“…The EDE is totally circumscribed to the E dimer and therefore it does not depend on the higher order arrangement of dimers, as recently suggested for other quaternary epitopes on the DENV particle 25 based on studies on a different flavivirus, the West Nile virus 26 . Recent studies on DENV--2 detected a particle expansion at physiological temperatures of humans, causing the E dimers to reorient with respect to each other and presenting a different surface pattern as in mosquito grown viruses 27,28 .…”

“…This process effectively displaces the equilibrium toward dimers, similar to the bnAb--induced shift toward dimers of the sE monomer--dimer equilibrium in solution (data not shown). Because the degree of prM cleavage is variable and depends on the particular cell in which the virus was replicated, the fact that these bnAbs bind efficiently partially mature particles is expected to be important for protection in humans.The EDE is totally circumscribed to the E dimer and therefore it does not depend on the higher order arrangement of dimers, as recently suggested for other quaternary epitopes on the DENV particle 25 based on studies on a different flavivirus, the West Nile virus 26 . Recent studies on DENV--2 detected a particle expansion at physiological temperatures of humans, causing the E dimers to reorient with respect to each other and presenting a different surface pattern as in mosquito grown viruses 27,28 .…”

Human antibodies stop dengue virus by jamming its mechanics

“…The footprint of the Fab fragment is on DI, the DI-DII hinge of one E protein, and DIII of a neighboring E protein. HMAb CR4354 is a highly neutralizing West Nile virus-specific antibody (15); although it binds to nonoverlapping residues as 14c10, the footprint lies in a similar region. Another potent antibody from chimpanzee (5H12) complexed with dengue rE protein was solved by X-ray crystallography (16).…”