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

A Novel Humanized Chi3l1 Blocking Antibody Attenuates Acetaminophen-Induced Liver Injury in Mice

Acetaminophen (APAP) overdose is a leading cause of acute liver injury in the USA. The chitinase 3-like-1 (Chi3l1) protein contributes to APAP-induced liver injury (AILI) by promoting hepatic platelet recruitment. Here, we report the development of a Chi3l1-targeting antibody as a potential therapy for AILI. By immunizing a rabbit successively with the human and mouse Chi3l1 proteins, we isolated cross-reactive monoclonal antibodies (mAbs) from single memory B cells. One of the human and mouse Chi3l1 cross-reactive mAbs was humanized and characterized in bot

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

Immunogenicity of a DNA vaccine candidate for COVID-19

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study

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

The 3.1-Angstrom Cryo-electron Microscopy Structure of the Porcine Epidemic Diarrhea Virus Spike Protein in the Prefusion Conformation

Coronavirus spike proteins are large, densely glycosylated macromolecular machines that mediate receptor binding and membrane fusion to facilitate entry into host cells. This report describes the atomic-resolution structure of the spike protein from porcine epidemic diarrhea virus, a pathogenic alphacoronavirus that causes severe agricultural damage. The structure reveals a novel position for the sialic acid-binding attachment domain in the intact spike. We also observed shed fusion-suppressive capping subunits that displayed the putative receptor-binding domain in an accessible conformation. These observations provide a basis for understanding the molecular mechanisms that drive the earliest stages of alphacoronavirus infection and will inform future efforts to rationally design vaccines.</jats: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