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Developing potent therapeutics and effective vaccines are the ultimate goals in controlling infectious diseases. Lassa virus (LASV), the causative pathogen of Lassa fever (LF), infects hundreds of thousands annually, but effective antivirals or vaccines against LASV infection are still lacking. Furthermore, neutralizing antibodies against LASV are rare. Here, we describe biochemical analyses and high-resolution cryo-electron microscopy structures of a therapeutic cocktail of three broadly protective antibodies that target the LASV glycoprotein complex (GPC), previously identified from survivors of multiple LASV infections. Structural and mechanistic analyses reveal compatible neutralizing epitopes and complementary neutralization mechanisms that offer high potency, broad range, and resistance to escape. These antibodies either circumvent or exploit specific glycans comprising the extensive glycan shield of GPC. Further, they require mammalian glycosylation, native GPC cleavage, and proper GPC trimerization. These findings guided engineering of a next-generation GPC antigen suitable for future neutralizing antibody and vaccine discovery. Together, these results explain protective mechanisms of rare, broad, and potent antibodies and identify a strategy for the rational design of therapeutic modalities against LF and related infectious diseases.

SummaryThe mammarenavirus Lymphocytic choriomeningitis virus (LCMV) is a globally distributed zoonotic pathogen that can be lethal in immunocompromised patients and cause severe birth defects if acquired during pregnancy. Despite the fundamental importance of LCMV for studying immunobiology, the structure of the trimeric surface glycoprotein, essential for entry, vaccine design and antibody neutralization, remains unknown. In this study, we present the cryoEM structure of the LCMV surface glycoprotein (GP) in its trimeric prefusion assembly both alone and in complex with a rationally engineered monoclonal neutralizing antibody termed 18.5C-M28 (M28). Additionally, we show that passive administration of M28 protects mice from LCMV clone 13 (LCMVcl13) challenge when administered as either a prophylactic or therapeutic. Our study illuminates not only the overall structural organization of LCMV GP and the mechanism for its inhibition by M28, but also presents a promising therapeutic candidate to prevent severe or fatal disease in individuals who are at risk of infection by a virus that poses a threat worldwide.HighlightsRationally-engineered antibody M28 neutralizes lymphocytic choriomeningitis virus in vitro.First high-resolution cryoEM structure of the pre-fusion trimeric lymphocytic choriomeningitis virus glycoprotein alone and in complex with M28.M28 neutralizes by bridging adjacent glycoprotein protomers and locking it in the pre-fusion state.Prophylactic and therapeutic administration of M28 protects mice from chronic lymphocytic choriomeningitis virus infection.

Significance Lymphocytic choriomeningitis virus (LCMV) is the prototypic arenavirus and has been utilized for decades as a model to understand the host immune response against viral infection. LCMV infection can lead to fatal meningitis in immunocompromised people and can lead to congenital birth defects and spontaneous abortion if acquired during pregnancy. Using a genetic screen, we uncover host factors involved in LCMV entry that were previously unknown and are candidate therapeutic targets to combat LCMV infection. This study expands our understanding of the entry pathway of LCMV, revealing that its glycoprotein switches from utilizing the known receptor α-DG and heparan sulfate at the plasma membrane to binding the lysosomal mucin CD164 at pH levels found in endolysosomal compartments, facilitating membrane fusion.

Lassa virus (LASV) is the etiologic agent of Lassa Fever, a hemorrhagic disease that is endemic to West Africa. During LASV infection, LASV glycoprotein (GP) engages with multiple host receptors for cell entry. Neutralizing antibodies against GP are rare and principally target quaternary epitopes displayed only on the metastable, pre-fusion conformation of GP. Currently, the structural features of the neutralizing GPC-A antibody competition group are understudied. Structures of two GPC-A antibodies presented here demonstrate that they bind the side of the pre-fusion GP trimer, bridging the GP1 and GP2 subunits. Complementary biochemical analyses indicate that antibody 25.10C, which is broadly specific, neutralizes by inhibiting binding of the endosomal receptor LAMP1 and also by blocking membrane fusion. The other GPC-A antibody, 36.1F, which is lineage-specific, prevents LAMP1 association only. These data illuminate a site of vulnerability on LASV GP and will guide efforts to elicit broadly reactive therapeutics and vaccines.

Summary A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. Dynamic tracking of variant frequencies revealed a recurrent pattern of G614 increase at multiple geographic levels: national, regional and municipal. The shift occurred even in local epidemics where the original D614 form was well established prior to the introduction of the G614 variant. The consistency of this pattern was highly statistically significant, suggesting that the G614 variant may have a fitness advantage. We found that the G614 variant grows to higher titer as pseudotyped virions. In infected individuals G614 is associated with lower RT-PCR cycle thresholds, suggestive of higher upper respiratory tract viral loads, although not with increased disease severity. These findings illuminate changes important for a mechanistic understanding of the virus, and support continuing surveillance of Spike mutations to aid in the development of immunological interventions., Highlights: - A SARS-CoV-2 variant with Spike G614 has replaced D614 as the dominant pandemic form - The consistent increase of G614 at regional levels may indicate a fitness advantage - G614 is associated with lower RT PCR Ct’s, suggestive of higher viral loads in patients - The G614 variant grows to higher titers as pseudotyped virions

Bacterial global post-transcriptional regulators execute hundreds of interactions with targets that display varying molecular features while retaining specificity. Herein, we develop, validate, and apply a biophysical, statistical thermodynamic model of canonical target mRNA interactions with the CsrA global post-transcriptional regulator to understand the molecular features that contribute to target regulation. Altogether, we model interactions of CsrA with a pool of 236 mRNA: 107 are experimentally regulated by CsrA and 129 are suspected interaction partners. Guided by current understanding of CsrA-mRNA interactions, we incorporate (i) mRNA nucleotide sequence, (ii) cooperativity of CsrA-mRNA binding, and (iii) minimization of mRNA structural changes to identify an ensemble of likely binding sites and their free energies. The regulatory impact of bound CsrA on mRNA translation is determined with the RBS calculator. Predicted regulation of 66 experimentally regulated mRNAs adheres to the principles of canonical CsrA-mRNA interactions; the remainder implies that other, diverse mechanisms may underlie CsrA-mRNA interaction and regulation. Importantly, results suggest that this global regulator may bind targets in multiple conformations, via flexible stretches of overlapping predicted binding sites. This novel observation expands the notion that CsrA always binds to its targets at specific consensus sequences.