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The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here, we use two-component protein nanoparticles consisting of trimeric and pentameric subunits to stabilize GPC in a trimeric conformation. These GPC nanoparticles present twenty prefusion GPC trimers on the surface of an icosahedral particle. Cryo-EM studies of GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against the lethal Lassa virus challenge in guinea pigs. Additionally, we isolated a neutralizing antibody that mapped to the putative receptor-binding site, revealing a previously undefined site of vulnerability. Collectively, these findings offer potential approaches to vaccine and therapeutic design for the Lassa virus.

SummaryLassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences amongst LASV lineages. Despite the sequence diversity of GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VI, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of GPC in complex with GP1-A antibodies reveal their neutralization mechanisms. Finally, we present the isolation and characterization of a novel trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.HighlightsStructural characterization of soluble glycoproteins from four Lassa virus lineages.MAb 12.1F, belonging to the GP1-A cluster, inhibits matriglycan and LAMP-1 binding.GP1-A mAbs show glycan-dependence with 19.7E demonstrating lineage-dependent binding.A novel trimer-preferring NAb S370.7 targets the GPC-B epitope.

Lassa virus is endemic in large parts of West Africa and causes a hemorrhagic fever. Recent years have seen several serious outbreaks of Lassa fever with high mortality rates. A vaccine to curtail infection is urgently needed. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here we use two-component protein nanoparticles to stabilize GPC in a trimeric conformation and present twenty prefusion GPC trimers on the surface of an icosahedral nanoparticle. Cryo-EM studies of assembled GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against a lethal Lassa virus challenge in guinea pigs. We isolated a neutralizing antibody which was mapped to the putative receptor-binding site, revealing a novel site of vulnerability on GPC.

Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic., Here, the authors present cryoEMPEM, a method for high-resolution structural analysis of vaccine-elicited polyclonal antibody responses. They apply cryoEMPEM in combination with standard serology experiments to characterize the polyclonal antibody (pAb) responses elicited in rhesus macaques by HIV Env trimer immunogens and were able to determine up to 8 different polyclonal antibody structures in complex with their respective antigen from a single cryoEM dataset.

Bacteriophages are abundant in the environment, yet the vast majority have not been discovered or described. Many characterized bacteriophages infect a small subset of Enterobacteriaceae hosts. Despite its similarity to Escherichia coli, the pathogenic Shigella flexneri has relatively few known phages, which exhibit significant differences from many E. coli phages. This suggests that isolating additional Shigella phages is necessary to further explore these differences. To address questions of novelty and prevalence, high school students isolated bacteriophages on non-pathogenic strains of enteric bacteria. Results indicate that Shigella phages are abundant in the environment and continue to differ significantly from E. coli phages. Our findings suggest that Shigella-infecting members of the Ounavirinae subfamily continue to be over-represented and show surprisingly low diversity within and between sampling sites. Additionally, a podophage with distinct genomic and structural properties suggests that continued isolation on non-model species of bacteria is necessary to truly understand bacteriophage diversity.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities., Graphical Abstract, Highlights • SARS-CoV-2 spike protein interacts with heparan sulfate and ACE2 through the RBD • Heparan sulfate promotes Spike-ACE2 interaction • SARS-CoV-2 infection is co-dependent on heparan sulfate and ACE2 • Heparin and non-anticoagulant derivatives block SARS-CoV-2 binding and infection, Clausen et al. provide evidence that heparan sulfate is a necessary co-factor for SARS-CoV-2 infection. They show that heparan sulfate interacts with the receptor-binding domain of the SARS-CoV-2 spike glycoprotein, adjacent to ACE2, shifting the spike structure to an open conformation to facilitate ACE2 binding.

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities. Funding: This work was supported by RAPID grant 2031989 from the National Science Foundation and Project 3 of NIH P01 HL131474 to J.D.E.; The Alfred Benzon Foundation to T.M.C; NIH R01 AI146779 and a Massachusetts Consortium on Pathogenesis Readiness MassCPR grant to A.G.S.; DOD grant W81XWH-20-1-0270 and Fluomics/NOSI U19 AI135972 to S.K.C; a Career Award for Medical Scientists from the Burroughs Wellcome Fund to A.F.C.; Bill and Melinda Gates Foundation grant OPP1170236 to A.B.W.; COVID19 seed funding from the Huck Institutes of the Life Sciences and Penn State start-up funds to J.J.; and T32 training grants GM007753 for B.M.H. and T.C and AI007245 for J.F.; J.P. received funding from the Innovation Fund Denmark and VAR2 Pharmaceuticals. Conflict of Interest: J.D.E. is a co-founder of TEGA Therapeutics. J.D.E. and The Regents of the University of California have licensed a University invention to and have an equity interest in TEGA Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. C.A.G and B.E.T are employees of TEGA Therapeutics. Ethical Approval: The collection of human tissue in this study abided by the Helsinki Principles. This work included postmortem human tissue, collected at the University Hospital, at the University of Copenhagen in Denmark. The patient provided informed consent for the tissue to be used for research purposes. All samples were completely deidentified before transfer to the researchers and this did not need specific IRB approval.