Potent Single-Domain Antibodies that Arrest Respiratory Syncytial Virus Fusion Protein in its Prefusion State

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Continuous flexibility analysis of SARS-CoV-2 spike prefusion structures

Using a new consensus-based image-processing approach together with principal component analysis, the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state have been analysed. These studies revealed concerted motions involving the receptor-binding domain (RBD), N-terminal domain, and subdomains 1 and 2 around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. It is shown that in this data set there are not well defined, stable spike conformations, but virtually a continuum of states. An ensemble map was obtained with minimum bias, from which the extremes of the change along the direction of maximal variance were modeled by flexible fitting. The results provide a warning of the potential image-processing classification instability of these complicated data sets, which has a direct impact on the interpretability of the results.The authors would like to acknowledge financial support from CSIC (PIE/COVID-19 No. 202020E079), the Comunidad de Madrid through grant CAM (S2017/BMD-3817), the Spanish Ministry of Science and Innovation through projects SEV 2017-0712, FPU-2015/264 and PID2019-104757RB-I00/AEI/ FEDER, the Instituto de Salud Carlos III [PT17/0009/0010 (ISCIII-SGEFI/ERDF)], and the European Union and Horizon 2020 through grants INSTRUCT–ULTRA (INFRADEV-03-2016-2017, Proposal 731005), EOSC Life (INFRAEOSC-04-2018, Proposal 824087), HighResCells (ERC-2018-SyG, Proposal 810057), IMpaCT (WIDESPREAD- 03-2018, Proposal 857203), CORBEL (INFRADEV-1-2014-1, Proposal 654248) and EOSC–Synergy (EINFRA-EOSC-5, Proposal 857647). HDT and BF were supported by NIH grant GM125769 and JSM was supported by NIH grant R01-AI12752

Broad neutralization of SARS-related viruses by human monoclonal antibodies

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses

Structural Insights into Coronavirus Spike Proteins and Implications for Therapeutic Interventions

The family Coronaviridae contains over forty distinct viruses, which cause a wide variety of disease in diverse hosts, ranging from severe enteric disease in pigs, to asymptomatic infection in bats. Although human coronaviruses were first identified over fifty years ago, there are currently no FDA approved therapeutics for coronavirus infection. Coronavirus disease outbreaks such as SARS, MERS, and the current COVID-19 pandemic illustrate the urgent need for both prophylactic and therapeutic treatments against these pathogens. Coronaviruses make use of spike (S), the largest known class I viral fusion protein, to both attach to host cells and to mediate the process of membrane fusion. Because of the critical role that S plays in the earliest stages of viral infection, it represents a clear target for the development of vaccines and immunotherapeutics. On the viral surface, S exists in a metastable prefusion conformation. Host-cell receptor binding gradually destabilizes this prefusion conformation, causing S to undergo dramatic conformational rearrangements that result in viral membrane fusion and the formation of the highly stable postfusion conformation. The first several chapters of this thesis describe the determination of structures of the S protein in the prefusion state and detail how these observations have been leveraged to design mutations to stabilize this conformation. These findings have important implications for vaccine development, and some of the preliminary results of their implementation are also described here. The second portion of this thesis describes potential immunotherapeutics, isolated using these stabilized coronavirus spikes. Several of these antibodies are capable of cross-neutralizing multiple coronaviruses and by determining their structures in complex with their viral targets, it was possible to elucidate their mechanisms of neutralization. Some of these antibodies and their derivatives are currently being investigated as potential therapeutics to combat the current COVID-19 pandemic and to help prevent future coronavirus outbreaks

Site-specific glycan analysis of the SARS-CoV-2 spike

The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.</p

Local computational methods to improve the interpretability and analysis of cryo-EM maps

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, ‘over-sharpening’ due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.N

Structural rearrangements allow nucleic acid discrimination by type I-D Cascade

CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Ã, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3′ binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems