Multifunctional human monoclonal antibody combination mediates protection against Rift Valley fever virus at low doses.

The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic.

Mechanistic basis for potent neutralization of Sin Nombre hantavirus by a human monoclonal antibody.

Rodent-borne hantaviruses are prevalent worldwide and upon spillover to human populations, cause severe disease for which no specific treatment is available. A potent antibody response is key for recovery from hantavirus infection. Here we study a highly neutralizing human monoclonal antibody, termed SNV-42, which was derived from a memory B cell isolated from an individual with previous Sin Nombre virus (SNV) infection. Crystallographic analysis demonstrates that SNV-42 targets the Gn subcomponent of the tetrameric (Gn-Gc)4 glycoprotein assembly that is relevant for viral entry. Integration of our 1.8 Å structure with the (Gn-Gc)4 ultrastructure arrangement indicates that SNV-42 targets the membrane-distal region of the virus envelope. Comparison of the SNV-42 paratope encoding variable genes with inferred germline gene segments reveals high sequence conservation, suggesting that germline-encoded antibodies inhibit SNV. Furthermore, mechanistic assays reveal that SNV-42 interferes with both receptor recognition and fusion during host-cell entry. This work provides a molecular-level blueprint for understanding the human neutralizing antibody response to hantavirus infection.

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen.

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nnHTN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nnHTN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.

Molecular rationale for antibody-mediated targeting of the hantavirus fusion glycoprotein.

The intricate lattice of Gn and Gc glycoprotein spike complexes on the hantavirus envelope facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Through study of a neutralizing monoclonal antibody termed mAb P-4G2, which neutralizes the zoonotic pathogen Puumala virus (PUUV), we provide a molecular-level basis for antibody-mediated targeting of the hantaviral glycoprotein lattice. Crystallographic analysis demonstrates that P-4G2 binds to a multi-domain site on PUUV Gc and may preclude fusogenic rearrangements of the glycoprotein that are required for host-cell entry. Furthermore, cryo-electron microscopy of PUUV-like particles in the presence of P-4G2 reveals a lattice-independent configuration of the Gc, demonstrating that P-4G2 perturbs the (Gn-Gc)4 lattice. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism.

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-Å-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.

Structures of enveloped virions determined by cryogenic electron microscopy and tomography.

Enveloped viruses enclose their genomes inside a lipid bilayer which is decorated by membrane proteins that mediate virus entry. These viruses display a wide range of sizes, morphologies and symmetries. Spherical viruses are often isometric and their envelope proteins follow icosahedral symmetry. Filamentous and pleomorphic viruses lack such global symmetry but their surface proteins may display locally ordered assemblies. Determining the structures of enveloped viruses, including the envelope proteins and their protein-protein interactions on the viral surface, is of paramount importance. These structures can reveal how the virions are assembled and released by budding from the infected host cell, how the progeny virions infect new cells by membrane fusion, and how antibodies bind surface epitopes to block infection. In this chapter, we discuss the uses of cryogenic electron microscopy (cryo-EM) in elucidating structures of enveloped virions. Starting from a detailed outline of data collection and processing strategies, we highlight how cryo-EM has been successfully utilized to provide unique insights into enveloped virus entry, assembly, and neutralization.

Beyond structures of highly symmetric purified viral capsids by cryo-EM.

Cryogenic transmission electron microscopy (cryo-EM) is widely used to determine high-resolution structures of symmetric virus capsids. The method holds promise for extending studies beyond purified capsids and their symmetric protein shells. The non-symmetric genome component has been addressed in dsRNA cypoviruses and ssRNA bacteriophages Qß and MS2. The structure of human herpes simplex virus type 1 capsids has been determined within intact virions to resolve capsid-tegument interactions. Electron tomography under cryogenic conditions (cryo-ET), has allowed resolving an early membrane fusion intermediate of Rift Valley fever virus. Antibody-affinity based sample grids allow capturing of virions directly from cell cultures or even clinical samples. These and other emerging methods will support studies to address viral entry, assembly and neutralization processes at increasingly high resolutions and native conditions.

Structural Transitions of the Conserved and Metastable Hantaviral Glycoprotein Envelope.

Hantaviruses are zoonotic pathogens that cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in northeastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface.IMPORTANCE Although outbreaks of Korean hemorrhagic fever were first recognized during the Korean War (1950 to 1953), it was not until 1978 that they were found to be caused by Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Here, we describe the crystal structure of HTNV envelope glycoprotein Gn, an integral component of the Gn-Gc glycoprotein spike complex responsible for host cell entry. HTNV Gn is structurally conserved with the Gn of a genetically and geographically distal hantavirus, Puumala virus, indicating that the observed α/ß fold is well preserved across the Hantaviridae family. The combination of our crystal structure with solution state analysis of recombinant protein and electron cryo-microscopy of acidified hantavirus allows us to propose a model for endosome-induced reorganization of the hantaviral glycoprotein lattice. This provides a molecular-level rationale for the exposure of the hydrophobic fusion loops on the Gc, a process required for fusion of viral and cellular membranes.

Expression and purification of tau protein and its frontotemporal dementia variants using a cleavable histidine tag.

Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.