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Zika virus (ZIKV) is a major human pathogen and member of the genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.
West Nile virus (WNV), a member of the Flavivirus genus, is a leading cause of viral encephalitis in the United States. The development of neutralizing antibodies against the flavivirus envelope (E) protein is critical for immunity and vaccine protection. Previously identified candidate therapeutic mouse and human neutralizing monoclonal antibodies (mAbs) target epitopes within the E domain III lateral ridge and the domain I-II hinge region, respectively. To explore the neutralizing antibody repertoire elicited by WNV infection for potential therapeutic application, we isolated ten mAbs from WNV-infected individuals. mAb WNV-86 neutralized WNV with a 50% inhibitory concentration of 2 ng ml, one of the most potently neutralizing flavivirus-specific antibodies ever isolated. WNV-86 targets an epitope in E domain II, and preferentially recognizes mature virions lacking an uncleaved form of the chaperone protein prM, unlike most flavivirus-specific antibodies. In vitro selection experiments revealed a neutralization escape mechanism involving a glycan addition to E domain II. Finally, a single dose of WNV-86 administered two days post-infection protected mice from lethal WNV challenge. This study identifies a highly potent human neutralizing mAb with therapeutic potential that targets an epitope preferentially displayed on mature virions.
Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have 'elite' activity with 50 and 99% effective inhibitory concentrations (EC and EC) of less than 10 and 100 ng ml, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.
The role that broadly neutralizing antibodies (bNAbs) play in natural clearance of human hepatitis C virus (HCV) infection and the underlying mechanisms remain unknown. Here, we investigate the mechanism by which bNAbs, isolated from two humans who spontaneously cleared HCV infection, contribute to HCV control. Using viral gene sequences amplified from longitudinal plasma of the two subjects, we found that these bNAbs, which target the front layer of the HCV envelope protein E2, neutralized most autologous HCV strains. Acquisition of resistance to bNAbs by some autologous strains was accompanied by progressive loss of E2 protein function, and temporally associated with HCV clearance. These data demonstrate that bNAbs can mediate clearance of human HCV infection by neutralizing infecting strains and driving escaped viruses to an unfit state. These immunopathologic events distinguish HCV from HIV-1 and suggest that development of an HCV vaccine may be achievable.
Copyright © 2018 Elsevier Inc. All rights reserved.
Hepatitis C virus (HCV) vaccine efforts are hampered by the extensive genetic diversity of HCV envelope glycoproteins E1 and E2. Structures of broadly neutralizing antibodies (bNAbs) (e.g., HEPC3, HEPC74) isolated from individuals who spontaneously cleared HCV infection facilitate immunogen design to elicit antibodies against multiple HCV variants. However, challenges in expressing HCV glycoproteins previously limited bNAb-HCV structures to complexes with truncated E2 cores. Here we describe crystal structures of full-length E2 ectodomain complexes with HEPC3 and HEPC74, revealing lock-and-key antibody-antigen interactions, E2 regions (including a target of immunogen design) that were truncated or disordered in E2 cores, and an antibody CDRH3 disulfide motif that exhibits common interactions with a conserved epitope despite different bNAb-E2 binding orientations. The structures display unusual features relevant to common genetic signatures of HCV bNAbs and demonstrate extraordinary plasticity in antibody-antigen interactions. In addition, E2 variants that bind HEPC3/HEPC74-like germline precursors may represent candidate vaccine immunogens.
Copyright © 2018 Elsevier Inc. All rights reserved.
Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%-90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CXCC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific "wing" antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure.
Copyright © 2017 Elsevier Inc. All rights reserved.
An atomic-detail model of the Marburg virus glycoprotein in complex with a neutralizing human monoclonal antibody designated MR78 was constructed using Phenix.Rosetta starting from a 3.6Å crystallographic density map. The Asp at T6 in the HCDR3's bulged torso cannot form the canonical salt bridge as position T2 lacks an Arg or Lys residue. It instead engages in a hydrogen bond interaction with a Tyr contributed by the HCDR1 loop. This inter-CDR loop interaction stabilizes the bulged conformation needed for binding to the viral glycoprotein: a Tyr to Phe mutant displays a binding affinity reduced by a factor of at least 10. We found that 5% of a database of 465 million human antibody sequences has the same residues at T2 and T6 positions in HCDR3 and Tyr in HCDR1 that could potentially form this Asp-Tyr interaction, and that this interaction might contribute to a non-canonical bulged torso conformation.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, and VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138- and VACV-304-binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Uncovering mechanisms of antibody-mediated neutralization for viral infections requires epitope and paratope mapping in the context of whole viral particle interactions with the antibody in solution. In this study, we use amide hydrogen/deuterium exchange mass spectrometry to describe the interface of a dengue virus-neutralizing antibody, 2D22, with its target epitope. 2D22 binds specifically to DENV2, a serotype showing strain-specific structural expansion at human host physiological temperatures of 37°C. Our results identify the heavy chain of 2D22 to be the primary determinant for binding DENV2. Temperature-mediated expansion alters the mode of interaction of 2D22 binding. Importantly, 2D22 interferes with the viral expansion process and offers a basis for its neutralization mechanism. The relative magnitude of deuterium exchange protection upon antibody binding across the various epitope loci allows a deconstruction of the antibody-viral interface in host-specific environments and offers a robust approach for targeted antibody engineering.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Antibodies are the principal immune effectors that mediate protection against reinfection following viral infection or vaccination. Robust techniques for human mAb isolation have been developed in the last decade. The study of human mAbs isolated from subjects with prior immunity has become a mainstay for rational structure-based, next-generation vaccine development. The plethora of detailed molecular and genetic studies coupling the structure of antigen-antibody complexes with their antiviral function has begun to reveal common principles of critical interactions on which we can build better vaccines and therapeutic antibodies. This review outlines the approaches to isolating and studying human antiviral mAbs and discusses the common principles underlying the basis for their activity. This review also examines progress toward the goal of achieving a comprehensive understanding of the chemical and physical basis for molecular recognition of viral surface proteins in order to build predictive molecular models that can be used for vaccine design.
Copyright © 2017. Published by Elsevier Inc.