Spike Conformation Research Articles

Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy.

2343. SARS-CoV-2 Spike Conformation Essentially Influences Vaccine Immunogenicity – Results from Two Clinical Phase I Trials of the Vector Vaccine Candidates MVA-SARS-2-S and MVA-SARS-2-ST

Abstract Background Recombinant Modified Vaccinia virus Ankara (MVA) is a replication-deficient viral vector platform with a favorable safety profile that is currently developed for multiple indications, including vaccines against emerging infections. We assessed the safety and immunogenicity of two MVA-based vaccine candidates against COVID-19 encoding the native or pre-fusion stabilized SARS-CoV-2 spike antigen, namely MVA-SARS-2-S and MVA-SARS-2-ST, in two clinical Phase I trials. Methods We conducted a first-in-human trial of the vaccine candidate MVA-SARS-2-S (NCT04569383) and a Phase Ib trial of the successor vaccine MVA-SARS-2-ST (NCT05226390) in healthy, SARS-CoV-2 immune-naïve adults aged between 18-55 (MVA-SARS-2-S) and 18-64 (MVA-SARS-2-ST) years. MVA-SARS-2-S expresses the native, full-length spike glycoprotein of SARS-CoV-2 (S), while MVA-SARS-2-ST delivers a pre-fusion stabilized spike antigen (ST). Both trials were designed as open-label dose-escalation trials and vaccinations were administered as a prime-boost regimen on days 0 and 28. Detailed safety and immunogenicity assessments were conducted at multiple timepoints throughout the study until day 168. Results In NCT04569383, 30 individuals received either 1 x 107 ± 0.5 log IU (IU=International Units, n=15, lower dose) or 1 x 108 ± 0.5 log IU (n=15, higher dose) of MVA-SARS-2-S. In NCT05226390, 8 participants received a lower (≥ 1 x 107 IU) and 7 participants a higher (≥ 5 x 107 IU) dose of MVA-SARS-2-ST. Both vaccines were well-tolerated and no vaccine-related severe adverse events (SAEs) occurred in either trial. However, seroconversion rates, measured as binding anti-SARS-CoV-2 S1 antibodies, could only be observed in 3/15 participants in the high dose cohort and none in the low dose cohort after vaccination with MVA-SARS-2-S. In contrast, all participants who received MVA-SARS-2-ST (n=15) achieved seroconversion independent of administered dose. Conclusion Both vaccine candidates were safe and well-tolerated. While MVA-SARS-2-S did not induce adequate immunogenicity, vaccination with the pre-fusion stabilized successor vaccine MVA-SARS-2-ST induced seroconversion in all vaccinees, demonstrating the significance of SARS-CoV-2 spike antigen conformation for vaccine immunogenicity. Disclosures Anahita Fathi, MD, Biotest: Honoraria Sibylle C. Mellinghoff, MD, Gilead: Grant|Octapharma: Advisor/Consultant

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

Exploring Conformational Landscapes and Cryptic Binding Pockets in Distinct Functional States of the SARS-CoV-2 Omicron BA.1 and BA.2 Trimers: Mutation-Induced Modulation of Protein Dynamics and Network-Guided Prediction of Variant-Specific Allosteric Binding Sites.

A significant body of experimental structures of SARS-CoV-2 spike trimers for the BA.1 and BA.2 variants revealed a considerable plasticity of the spike protein and the emergence of druggable binding pockets. Understanding the interplay of conformational dynamics changes induced by the Omicron variants and the identification of cryptic dynamic binding pockets in the S protein is of paramount importance as exploring broad-spectrum antiviral agents to combat the emerging variants is imperative. In the current study, we explore conformational landscapes and characterize the universe of binding pockets in multiple open and closed functional spike states of the BA.1 and BA.2 Omicron variants. By using a combination of atomistic simulations, a dynamics network analysis, and an allostery-guided network screening of binding pockets in the conformational ensembles of the BA.1 and BA.2 spike conformations, we identified all experimentally known allosteric sites and discovered significant variant-specific differences in the distribution of binding sites in the BA.1 and BA.2 trimers. This study provided a structural characterization of the predicted cryptic pockets and captured the experimentally known allosteric sites, revealing the critical role of conformational plasticity in modulating the distribution and cross-talk between functional binding sites. We found that mutational and dynamic changes in the BA.1 variant can induce the remodeling and stabilization of a known druggable pocket in the N-terminal domain, while this pocket is drastically altered and may no longer be available for ligand binding in the BA.2 variant. Our results predicted the experimentally known allosteric site in the receptor-binding domain that remains stable and ranks as the most favorable site in the conformational ensembles of the BA.2 variant but could become fragmented and less probable in BA.1 conformations. We also uncovered several cryptic pockets formed at the inter-domain and inter-protomer interface, including functional regions of the S2 subunit and stem helix region, which are consistent with the known role of pocket residues in modulating conformational transitions and antibody recognition. The results of this study are particularly significant for understanding the dynamic and network features of the universe of available binding pockets in spike proteins, as well as the effects of the Omicron-variant-specific modulation of preferential druggable pockets. The exploration of predicted druggable sites can present a new and previously underappreciated opportunity for therapeutic interventions for Omicron variants through the conformation-selective and variant-specific targeting of functional sites involved in allosteric changes.

Secondary Structures of MERS-CoV, SARS-CoV, and SARS-CoV-2 Spike Proteins Revealed by Infrared Vibrational Spectroscopy.

All coronaviruses are characterized by spike glycoproteins whose S1 subunits contain the receptor binding domain (RBD). The RBD anchors the virus to the host cellular membrane to regulate the virus transmissibility and infectious process. Although the protein/receptor interaction mainly depends on the spike's conformation, particularly on its S1 unit, their secondary structures are poorly known. In this paper, the S1 conformation was investigated for MERS-CoV, SARS-CoV, and SARS-CoV-2 at serological pH by measuring their Amide I infrared absorption bands. The SARS-CoV-2 S1 secondary structure revealed a strong difference compared to those of MERS-CoV and SARS-CoV, with a significant presence of extended β-sheets. Furthermore, the conformation of the SARS-CoV-2 S1 showed a significant change by moving from serological pH to mild acidic and alkaline pH conditions. Both results suggest the capability of infrared spectroscopy to follow the secondary structure adaptation of the SARS-CoV-2 S1 to different environments.

Detergent modulates the conformational equilibrium of SARS-CoV-2 Spike during cryo-EM structural determination

The Spike glycoprotein of SARS-CoV-2 mediates viral entry into the host cell via the interaction between its receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2). Spike RBD has been reported to adopt two primary conformations, a closed conformation in which the binding site is shielded and unable to interact with ACE2, and an open conformation that is capable of binding ACE2. Many structural studies have probed the conformational space of the homotrimeric Spike from SARS-CoV-2. However, how sample buffer conditions used during structural determination influence the Spike conformation is currently unclear. Here, we systematically explored the impact of commonly used detergents on the conformational space of Spike. We show that in the presence of detergent, the Spike glycoprotein predominantly occupies a closed conformational state during cryo-EM structural determination. However, in the absence of detergent, such conformational compaction was neither observed by cryo-EM, nor by single-molecule FRET designed to visualize the movement of RBD in solution in real-time. Our results highlight the highly sensitive nature of the Spike conformational space to buffer composition during cryo-EM structural determination, and emphasize the importance of orthogonal biophysical approaches to validate the structural models obtained.

Novel bispecific human antibody platform specifically targeting a fully open spike conformation potently neutralizes multiple SARS-CoV-2 variants

Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines.

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.

SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Vaccine-elicited murine antibody WS6 neutralizes diverse beta-coronaviruses by recognizing a helical stem supersite of vulnerability

Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike elicits diverse antibodies, but it is unclear if any of the antibodies can neutralize broadly against other beta-coronaviruses. Here, we report antibody WS6 from a mouse immunized with mRNA encoding the SARS-CoV-2 spike. WS6 bound diverse beta-coronavirus spikes and neutralized SARS-CoV-2 variants, SARS-CoV, and related sarbecoviruses. Epitope mapping revealed WS6 to target a region in the S2 subunit, which was conserved among SARS-CoV-2, Middle East respiratory syndrome (MERS)-CoV, and hCoV-OC43. The crystal structure at 2 Å resolution of WS6 revealed recognition to center on a conserved S2 helix, which was occluded in both pre- and post-fusion spike conformations. Structural and neutralization analyses indicated WS6 to neutralize by inhibiting fusion and post-viral attachment. Comparison of WS6 with other recently identified antibodies that broadly neutralize beta-coronaviruses indicated a stem-helical supersite—centered on hydrophobic residues Phe1148, Leu1152, Tyr1155, and Phe1156—to be a promising target for vaccine design.

Structural diversity of the SARS-CoV-2 Omicron spike

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

SARS-CoV-2 Variants Increase Kinetic Stability of Open Spike Conformations as an Evolutionary Strategy.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) harbor mutations in the spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity toward conformations accessible to the human angiotensin-converting enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Förster resonance energy transfer (smFRET) studies of critical mutations observed in VOCs, including D614G and E484K, in the context of virus particles. Investigated variants predominately occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited slower transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

Energy landscape of the SARS-CoV-2 reveals extensive conformational heterogeneity.

Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As with any protein, some of the spike's conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the glycosylated spike protein, and identify the diversity of low-energy conformations in the vicinity of its open (so called 1RBD-up) state. The resulting atomic refinement reveal global and local molecular rearrangements that cannot be inferred from an average 1RBD-up cryo-EM model. Here we report varied degrees of “openness” in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these low-energy conformations. Five out of six binding sites we analyzed, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This apo-to-holo switching is reminiscent of a conformational preequilibrium. We found only one binding site, namely that of AB-C135 remains in apo state within all the sampled free energy-minimizing models, suggesting an induced fit mechanism for the docking of this antibody to the spike.

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo-electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)-up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer.

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.

Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS-CoV-2 Spike Protein*.

We investigate binding of linoleate and other potential ligands to the recently discovered fatty acid binding site in the SARS‐CoV‐2 spike protein, using docking and molecular dynamics simulations. Simulations suggest that linoleate and dexamethasone stabilize the locked spike conformation, thus reducing the opportunity for ACE2 interaction. In contrast, cholesterol may expose the receptor‐binding domain by destabilizing the closed structure, preferentially binding to a different site in the hinge region of the open structure. We docked a library of FDA‐approved drugs to the fatty acid site using an approach that reproduces the structure of the linoleate complex. Docking identifies steroids (including dexamethasone and vitamin D); retinoids (some known to be active in vitro, and vitamin A); and vitamin K as potential ligands that may stabilize the closed conformation. The SARS‐CoV‐2 spike fatty acid site may bind a diverse array of ligands, including dietary components, and therefore provides a promising target for therapeutics or prophylaxis.

Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS‐CoV‐2 Spike Protein**

AbstractWe investigate binding of linoleate and other potential ligands to the recently discovered fatty acid binding site in the SARS‐CoV‐2 spike protein, using docking and molecular dynamics simulations. Simulations suggest that linoleate and dexamethasone stabilize the locked spike conformation, thus reducing the opportunity for ACE2 interaction. In contrast, cholesterol may expose the receptor‐binding domain by destabilizing the closed structure, preferentially binding to a different site in the hinge region of the open structure. We docked a library of FDA‐approved drugs to the fatty acid site using an approach that reproduces the structure of the linoleate complex. Docking identifies steroids (including dexamethasone and vitamin D); retinoids (some known to be active in vitro, and vitamin A); and vitamin K as potential ligands that may stabilize the closed conformation. The SARS‐CoV‐2 spike fatty acid site may bind a diverse array of ligands, including dietary components, and therefore provides a promising target for therapeutics or prophylaxis.

PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen’s kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.