Spike Glycoprotein Research Articles

Development of Long-Term Stability of Enveloped rVSV Viral Vector Expressing SARS-CoV-2 Antigen Using a DOE-Guided Approach

Liquid formulations have been successfully used in many viral vector vaccines including influenza (Flu), hepatitis B, polio (IPV), Ebola, and COVID-19 vaccines. The main advantage of liquid formulations over lyophilized formulations is that they are cost-effective, as well as easier to manufacture and distribute. However, studies have shown that the liquid formulations of enveloped viral vector vaccines are not stable over extended periods of time. In this study, we explored the development of the liquid formulations of an enveloped recombinant Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. To do so, we used a design of experiments (DOE) method, which allowed us to assess the stability dynamics of the viral vector in an effective manner. An initial stability study showed that trehalose, gelatin, and histidine were effective at maintaining functional viral titers during freeze–thaw stress and at different temperatures (−20, 4, 20, and 37 °C). These preliminary data helped to identify critical factors for the subsequent implementation of the DOE method that incorporated a stress condition at 37 °C. We used the DOE results to identify the optimal liquid formulations under the selected accelerated stress conditions, which then guided the identification of long-term storage conditions for further evaluation. In the long-term stability study, we identified several liquid formulations made of sugar (sucrose, trehalose, and sorbitol), gelatin, and a histidine buffer that resulted in the improved stability of rVSV-SARS-CoV-2 at 4 °C for six months. This study highlights an effective approach for the development of liquid formulations for viral vector vaccines, contributing significantly to the existing knowledge on enveloped viral vector thermostability.

Delineating the functional activity of antibodies with cross-reactivity to SARS-CoV-2, SARS-CoV-1 and related sarbecoviruses.

The recurring spillover of pathogenic coronaviruses and demonstrated capacity of sarbecoviruses, such SARS-CoV-2, to rapidly evolve in humans underscores the need to better understand immune responses to this virus family. For this purpose, we characterized the functional breadth and potency of antibodies targeting the receptor binding domain (RBD) of the spike glycoprotein that exhibited cross-reactivity against SARS-CoV-2 variants, SARS-CoV-1 and sarbecoviruses from diverse clades and animal origins with spillover potential. One neutralizing antibody, C68.61, showed remarkable neutralization breadth against both SARS-CoV-2 variants and viruses from different sarbecovirus clades. C68.61, which targets a conserved RBD class 5 epitope, did not select for escape variants of SARS-CoV-2 or SARS-CoV-1 in culture nor have predicted escape variants among circulating SARS-CoV-2 strains, suggesting this epitope is functionally constrained. We identified 11 additional SARS-CoV-2/SARS-CoV-1 cross-reactive antibodies that target the more sequence conserved class 4 and class 5 epitopes within RBD that show activity against a subset of diverse sarbecoviruses with one antibody binding every single sarbecovirus RBD tested. A subset of these antibodies exhibited Fc-mediated effector functions as potent as antibodies that impact infection outcome in animal models. Thus, our study identified antibodies targeting conserved regions across SARS-CoV-2 variants and sarbecoviruses that may serve as therapeutics for pandemic preparedness as well as blueprints for the design of immunogens capable of eliciting cross-neutralizing responses.

S-RBD-modified and miR-486-5p-engineered exosomes derived from mesenchymal stem cells suppress ferroptosis and alleviate radiation-induced lung injury and long-term pulmonary fibrosis

BackgroundRadiation-induced lung injury (RILI) is associated with alveolar epithelial cell death and secondary fibrosis in injured lung. Mesenchymal stem cell (MSC)-derived exosomes have regenerative effect against lung injury and the potential to intervene of RILI. However, their intervention efficacy is limited because they lack lung targeting characters and do not carry sufficient specific effectors. SARS-CoV-2 spike glycoprotein (SARS-CoV-2-S-RBD) binds angiotensin-converting enzyme 2 (ACE2) receptor and mediates interaction with host cells. MiR-486-5p is a multifunctional miRNA with angiogenic and antifibrotic potential and acts as an effector in MSC-derived exosomes. Ferroptosis is a form of cell death associated with radiation injury, its roles and mechanisms in RILI remain unclear. In this study, we developed an engineered MSC-derived exosomes with SARS-CoV-2-S-RBD- and miR-486-5p- modification and investigated their intervention effects on RIPF and action mechanisms via suppression of epithelial cell ferroptosis.ResultsAdenovirus-mediated gene modification led to miR-486-5p overexpression in human umbilical cord MSC exosomes (p < 0.05), thereby constructing miR-486-5p engineered MSC exosomes (miR-486-MSC-Exo). MiR-486-MSC-Exo promoted the proliferation and migration of irradiated mouse lung epithelial (MLE-12) cells in vitro and inhibited RILI in vivo (all p < 0.05). MiR-486-MSC-Exo suppressed ferroptosis in MLE-12 cells, and an in vitro assay revealed that the expression of fibrosis-related genes is up-regulated following ferroptosis (both p < 0.05). MiR-486-MSC-Exo reversed the up-regulated expression of fibrosis-related genes induced by TGF-β1 in vitro and improved pathological fibrosis in RIPF mice in vivo (all p < 0.05). SARS-CoV-2-S-RBD-modified and miR-486-5p-engineered MSC exosomes (miR-486-RBD-MSC-Exo) were also constructed, and the distribution of DiR dye-labeled miR-486-RBD-MSC-Exo in hACE2CKI/CKI Sftpc-Cre+ mice demonstrated long-term retention in the lung (p < 0.05). MiR-486-RBD-MSC-Exo significantly improved the survival rate and pathological changes in hACE2CKI/CKI Sftpc-Cre+ RIPF mice (all p < 0.05). Furthermore, miR-486-MSC-Exo exerted anti-fibrotic effects via targeted SMAD2 inhibition and Akt phosphorylation activation (p < 0.05).ConclusionsEngineered MSC exosomes with SARS-CoV-2-S-RBD- and miR-486-5p-modification were developed. MiR-486-RBD-MSC-Exo suppressed ferroptosis and fibrosis of MLE-12 cells in vitro, and alleviated RILI and long-term RIPF in ACE2 humanized mice in vivo. MiR-486-MSC-Exo exerted anti-fibrotic effects via SMAD2 inhibition and Akt activation. This study provides a potential approach for RIPF intervention.Graphical

Cross-Reactive Immune Response of Bovine Coronavirus Spike Glycoprotein to SARS-CoV-2 Variants of Concern

The high variability observed in the clinical symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has been attributed to the presence, in a proportion of infection-naive subjects, of pre-existing cross-reactive immune responses. Here, we demonstrate that the bovine coronavirus spike protein (BoS) may represent a source of protective immunity to SARS-CoV-2. Indeed, vaccination of BALB/c mice with a Bovine herpesvirus 4 (BoHV-4)-based vector expressing BoS induced both cell-mediated and humoral immune responses that cross-react with SARS-CoV-2 spike protein. Although the spike-specific antibodies induced by BoS did not neutralize SARS-CoV-2, the T lymphocytes activated by BoS were able to induce cytotoxicity of cells expressing spike proteins derived from several SARS-CoV-2 variants. These results demonstrate that immunization with BoS may represent a source of cross-reactive immunity to SARS-CoV-2, and that these cross-reactive immune responses may exert protective functions. These results contribute to deciphering the mechanisms responsible for lack or mildness of symptoms observed in many individuals upon SARS-CoV-2 infection and may open new ways for the development of new vaccines for coronaviruses.

Potential Beneficial Role of Nitric Oxide in SARS-CoV-2 Infection: Beyond Spike-Binding Inhibition

SARS-CoV-2, the causative virus for the COVID-19 disease, uses its spike glycoprotein to bind to human ACE2 as a first step for viral entry into the cell. For this reason, great efforts have been made to find mechanisms that disrupt this interaction, avoiding the infection. Nitric oxide (NO) is a soluble endogenous gas with known antiviral and immunomodulatory properties. In this study, we aimed to test whether NO could inhibit the binding of the viral spike to ACE2 in human cells and its effects on ACE2 enzymatic activity. Our results show that ACE2 activity was decreased by the NO donors DETA-NONOate and GSNO and by the NO byproduct peroxynitrite. Furthermore, we found that DETA-NONOate could break the spike–ACE2 interaction using the spike from two different variants (Alpha and Gamma) and in two different human cell types. Moreover, the same result was obtained when using NO-producing murine macrophages, while no significant changes were observed in ACE2 expression or distribution within the cell. These results support that it is worth considering NO as a therapeutic agent for COVID-19, as previous reports have suggested.

Antibodies as Promising Molecules to Block Spike Glycoprotein from SARS-CoV2

Abstract: COVID-19 is caused by a new SARS-CoV-2 virus from the Coronaviridae family, responsible for developing severe acute respiratory syndrome (SARS-CoV). Its emergence in 2019 presented several challenges to global health. The virus can infect host cells using the ACE2 receptor, and after infection, several clinical manifestations appear, ranging from mild symptoms to death. One approach that has shown promising results is convalescent plasma since convalescent plasma (CP) consists of a concentrate of immunoglobulins in the blood of patients recently infected with the coronavirus for those currently infected. Therefore, this therapy uses antibodies in the plasma to neutralize the virus. This literature review aims to evaluate the efficacy of CP therapy in symptomatic COVID-19 patients by analyzing clinical trials and studies published between 2020 and 2023. Therefore, the search identified studies in which the majority reported the efficacy of using convalescent plasma to treat and alleviate the clinical condition of patients diagnosed with COVID-19. Important factors influence the efficacy of CP therapy, such as the timing of administration and the severity of the disease. Early administration in mild and moderate cases has shown promise. Furthermore, molecular docking analysis suggests that antibodies can cause conformational changes in the SARS-CoV-2 spike protein, raising the hypothesis that using CP may interfere with viral entry into host cells.

Inhibitory Effect of Luteolin on Spike S1 Glycoprotein-Induced Inflammation in THP-1 Cells via the ER Stress-Inducing Calcium/CHOP/MAPK Pathway.

The global SARS-CoV-2 outbreak has escalated into a critical public health emergency, with the spike glycoprotein S1 subunit of SARS-CoV-2 (spike-S1) linked to inflammation in lung tissue and immune cells. Luteolin, a flavone with anti-inflammatory properties, shows promise, but research on its effectiveness against long-COVID-related inflammation and spike protein-induced responses remains limited. This study aims to elucidate the underlying mechanisms of inflammation in THP-1 cells induced by the spike-S1. Additionally, it seeks to assess the potential of luteolin in mitigating inflammatory responses induced by the spike-S1 in a THP-1 macrophage model. The gene expression profiles of spike-S1 in THP-1 cells were analyzed by transcriptome sequencing. The inhibitory effect of luteolin on ER stress and inflammation in spike-S1-induced THP-1 cells was investigated using Western blotting, RT-PCR, and ELISA. The candidate genes (CAMK2A, SIGLEC7, PPARGC1B, SEC22B, USP28, IER2, and TIRAP) were upregulated in the spike-S1-induced THP-1 group compared to the control group. Among these, calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) was identified as the most promising molecule in spike-S1-induced THP-1 cells. Our results indicate that the spike S1 significantly increased the expression of ER-stress markers at both gene and protein levels. Luteolin significantly reduced ER stress by decreasing the expression of ER-stress marker genes and ER-stress marker proteins (p < 0.01). Additionally, luteolin exhibited anti-inflammatory properties upon spike S1-induction in THP-1 cells by significantly suppressing IL-6, IL-8, and IL-1β cytokine secretion in a dose-dependent manner (p < 0.05). Furthermore, our results revealed that luteolin exhibited the downregulation of the MAPK pathway, as evidenced by modulating the phosphorylation of p-ERK1/2, p-JNK and p-p38 proteins (p < 0.05). The results from this study elucidate the mechanisms by which the spike S1 induces inflammation in THP-1 cells and supports the use of naturally occurring bioactive compounds, like luteolin, against inflammation-related SARS-CoV-2 infection.

Modelling of SARS-CoV-2 spike protein structures at varying pH values

ABSTRACT Recognising the fact that the SARS-CoV-2 spike glycoprotein is the most crucial protein for the virus's attachment to surfaces and subsequent infection, we carried out massive all-atom molecular dynamics studies of the conformational states of S-protein in solvents of various pH values, ranging from pH = 1 through pH = 11. Our studies found that: (1) The S-protein’s structural deviation measured in the backbone at pH = 1 is 185% higher than that of pH = 7, and most of this overall deviation occurs at the receptor-binding domain and N-terminal domain. (2) Structural changes are also observed at both pH = 3 and pH = 11, albeit to a lesser extent than at pH = 1. (3) Changes, as measured by various metrics at pH = 5, pH = 7 and pH = 9, are insignificant. Such a high degree of structural tolerance of the S-protein at these pH values corroborates indirectly with in vitro observations that the virus retains significant infectivity over a wide range of pH. The multiscale structural analyses pinpoint the critical regions that are responsible for the S-protein’s structural deviations.

Prevalent and persistent new-onset autoantibodies in mild to severe COVID-19

Autoantibodies have been shown to be implied in COVID-19 but the emerging autoantibody repertoire remains largely unexplored. We investigated the new-onset autoantibody repertoire in 525 healthcare workers and hospitalized COVID-19 patients at five time points over a 16-month period in 2020 and 2021 using proteome-wide and targeted protein and peptide arrays. Our results show that prevalent new-onset autoantibodies against a wide range of antigens emerged following SARS-CoV-2 infection in relation to pre-infectious baseline samples and remained elevated for at least 12 months. We found an increased prevalence of new-onset autoantibodies after severe COVID-19 and demonstrated associations between distinct new-onset autoantibodies and neuropsychiatric symptoms post-COVID-19. Using epitope mapping, we determined the main epitopes of selected new-onset autoantibodies, validated them in independent cohorts of neuro-COVID and pre-pandemic healthy controls, and identified sequence similarities suggestive of molecular mimicry between main epitopes and the conserved fusion peptide of the SARS-CoV-2 Spike glycoprotein. Our work describes the complexity and dynamics of the autoantibody repertoire emerging with COVID-19 and supports the need for continued analysis of the new-onset autoantibody repertoire to elucidate the mechanisms of the post-COVID-19 condition.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Sarbecovirus RBD indels and specific residues dictating multi-species ACE2 adaptiveness

Our comprehensive understanding of the multi-species ACE2 adaptiveness of sarbecoviruses remains elusive, particularly for those with various receptor binding motif (RBM) insertions/deletions (indels). Here, we analyzed RBM sequences from 268 sarbecoviruses categorized into four RBM indel types. We examined the ability of 20 representative sarbecovirus Spike glycoproteins (S) and derivatives in utilizing ACE2 from various bats and several other mammalian species. We reveal that sarbecoviruses with long RBMs (type-I) can achieve broad ACE2 tropism, whereas viruses with single deletions in Region 1 (type-II) or Region 2 (type-III) exhibit narrower ACE2 tropism. Sarbecoviruses with double region deletions (type-IV) completely lost ACE2 usage, which is restricted by clade-specific residues within and outside RBM. Lastly, we propose the evolution of sarbecovirus RBM indels and illustrate how loop lengths, disulfide, and residue determinants shape multi-species ACE2 adaptiveness. This study provides profound insights into the mechanisms governing ACE2 usage and spillover risks of sarbecoviruses.

The HCoV-HKU1 N-Terminal Domain Binds a Wide Range of 9-O-Acetylated Sialic Acids Presented on Different Glycan Cores.

Coronaviruses (CoVs) recognize a wide array of protein and glycan receptors by using the S1 subunit of the spike (S) glycoprotein. The S1 subunit contains two functional domains: the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD). The S1-NTD of SARS-CoV-2, MERS-CoV, and HCoV-HKU1 possesses an evolutionarily conserved glycan binding cleft that facilitates weak interactions with sialic acids on cell surfaces. HCoV-HKU1 employs 9-O-acetylated α2-8-linked disialylated structures for initial binding, followed by TMPRSS2 receptor binding and virus-cell fusion. Here, we demonstrate that the HCoV-HKU1 NTD has a broader receptor binding repertoire than previously recognized. We presented HCoV-HKU1 NTD Fc chimeras on a nanoparticle system to mimic the densely decorated surface of HCoV-HKU1. These proteins were expressed by HEK293S GnTI- cells, generating species carrying Man-5 structures, often observed near the receptor binding site of CoVs. This multivalent presentation of high mannose-containing NTD proteins revealed a much broader receptor binding profile compared to that of its fully glycosylated counterpart. Using glycan microarrays, we observed that 9-O-acetylated α2-3-linked sialylated LacNAc structures are also bound, comparable to OC43 NTD, suggesting an evolutionarily conserved glycan-binding modality. Further characterization of receptor specificity indicated promiscuous binding toward 9-O-acetylated sialoglycans, independent of the glycan core (glycolipids, N- or O-glycans). We demonstrate that HCoV-HKU1 may employ additional sialoglycan receptors to trigger conformational changes in the spike glycoprotein to expose the S1-CTD for proteinaceous receptor binding.

Insilco vaccine design of spike and hemagglutinin esterase proteins of bovine coronavirus

BackgroundBovine coronavirus (BCoV) is a widespread, fatal illness in cattle that has a large economic impact in particularly disease-prone hosts. BCoV does not have an effective vaccine. As a result, the objective of this study was to use immunoinformatics and computational tools to design a multi-epitope vaccine against Spike and haemagglutinin proteins of Bovine coronavirus. BCoV proteins were also subjected to protein analysis.MethodsA variety of tools of bioinformatics were used for data analysis. Conserved B and T cell epitopes against BCoV target proteins were predicted using the Immune Epitope Database (IEDB). Proteins were characterized utilizing a variety of servers, including Protparam, PSIPRED and the GOR IV servers. The antigenicity, allergenicity, and toxicity of the anticipated epitopes were assessed as well.ResultsSeveral MHCI epitopes were predicted from S and HE proteins. As top epitopes, the peptides 77NMALKGTLL85 and 56SYMDLNPAL65 were proposed from Spike and hemagglutinin proteins, respectively. These epitopes exhibited high scores of antigenicity, no allergenicity, no toxicity, and a strong connection to Bola alleles. Moreover, three epitopes (1204YYYPE1208, 379TCQPQ384, and 720QLQPINY726) from Spike glycoprotein were selected as surface, linear, and antigenic epitopes using B cell scales. The methods dropped to anticipate effective and safe epitope(s) to cover all B cell scales from HE protein.ConclusionThree B cell epitopes (1204YYYPE1208, 379TCQPQ384, and 720QLQPINY726) were predicted from Spike protein (S) of BCoV only. MHC I epitopes of S and HE proteins of BCoV predicted two epitopes (77NMALKGTLL85 and 56SYMDLNPAL65 respectively) to have a strong link to Bola alleles, as well as high antigenicity and safety. The predicted epitopes’ activity should be tested experimentally as a multi-epitope vaccine against BCoV using in vitro and in vivo trials.

The Impact of United Kingdom SARS-CoV-2 Spike Mutations on Spike-ACE2 Interactions Investigated Through Molecular Dynamic Simulation

Background: A newly emergent betacoronavirus, SARS-CoV-2, poses a tremendous global threat and causes severe acute respiratory syndrome coronavirus-2, a major pandemic that began worldwide in 2019. The trimeric spike glycoprotein (S) is located on the envelope of the virus and facilitates the fusion of viral and host cell membranes by interacting with a cellular receptor called angiotensin-converting enzyme 2 (ACE2). In the United Kingdom (UK), a variant of SARS-CoV-2 has been detected using sequencing technology. There has been a significant surge in the number of COVID-19 cases in South East England. These lineages are significantly more transmissible than previously circulating variants. Objectives: The aim of this study is to investigate the effects of UK SARS-CoV-2 spike mutations on its interactions with ACE2. Methods: In this study, the structure of the UK spike protein with multiple mutations, including deletion 69 - 70, deletion 144, N501Y, A570D, T716I, D614G, P681H, S982A, and D1118H, was investigated. The research focuses on studying the impact of these mutations on spike function and its interactions with ACE2 through molecular dynamic simulation. Results: The results indicated that hydrophobic interactions, hydrogen bonds, and double/triple bonds are more prevalent in the mutated spike-ACE2 complex. Additionally, the UK spike-ACE2 complex maintained a consistent conformation throughout the simulation, experiencing minimal changes in structure. The mutant spike protein structure is less stable compared to the wild-type spike structure. Conclusions: Multiple mutations, including deletion 69 - 70, deletion 144, N501Y, A570D, T716I, D614G, P681H, S982A, and D1118H in the spike protein of UK SARS-CoV-2, can affect its interaction with the ACE2 receptor and the transmissibility of this variant of SARS-CoV-2.

Neutralisation Resistance of SARS-CoV-2 Spike-Variants is Primarily Mediated by Synergistic Receptor Binding Domain Substitutions

The evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence.

Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold.

Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by glycoprotein (G)spikes, which also comprise the primary target for the humoral immune response. RABV glycoprotein (RABV-G) displays several antigenic sites that are targeted by neutralizing monoclonal antibodies (mAbs). In this study, we determined the epitope of a potently neutralizing human mAb, CR57, which we engineered into a diabody format to facilitate crystallization. We report the crystal structure of the CR57 diabody alone at 2.38Å resolution, and in complex with RABV-G domain III at 2.70Å resolution. The CR57-RABV-G structure reveals critical interactions at the antigen interface, which target the conserved "KLCGVL" peptide and residues proximal to it on RABV-G. Structural analysis combined with a cell-cell fusion assay demonstrates that CR57 effectively inhibits RABV-G-mediated fusion by obstructing the fusogenic transitions of the spike protein. Altogether, this investigation provides a structural perspective on RABV inhibition by a potently neutralizing human antibody.

Protein mimics of fusion core from SARS-CoV-1 can inhibit SARS-CoV-2 entry

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the genus Betacoronavirus (subgenus Sarbecovirus) and shares significant genomic and phylogenetic similarities with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). SARS-CoV-2 infection occurs through membrane fusion between the virus and host cell membranes, which is facilitated by the spike glycoprotein subunit 2 (S2). The folding of three heptad-repeat regions 1 (HR1) into a central trimeric core structure, along with the binding of three heptad-repeat regions 2 (HR2) in an antiparallel manner within the groove formed between the HR1 regions, which provides the driving force for membrane fusion. In this study, trimeric and monomeric six-helix bundles (6HB) were created by combining various truncations of the sequences from SARS-CoV-2 HR1 and HR2. In addition, monomeric five-helix bundles (5HB) were constructed using a similar method. Finally, we demonstrated a protein mimic, 5HB_V1 (from SARS-CoV-1), that exhibits activity in inhibiting SARS-CoV-2. These findings suggest a strategy to design monomeric 6HB and 5HB based on the SARS-CoV-2 fusion core: maintain the flanking sequences outside the α-helix region in HR2 and introduce point mutations to enhance hydrogen bonding between the helix bundles. The 5HB could be a target for designing new inhibitors against SARS-CoV-1 and SARS-CoV-2.

Synthesis of SARS-CoV-2 Spike Glycoprotein Peptide Fragments and Study of Their Binding to Human Blood Cells

Synthesis of SARS-CoV-2 Spike Glycoprotein Peptide Fragments and Study of Their Binding to Human Blood Cells

SARS-CoV-2 Delta and Omicron variants resist spike cleavage by human airway trypsin-like protease.

Soluble host factors in the upper respiratory tract can serve as the first line of defense against SARS-CoV-2 infection. In this study, we described the identification and function of a human airway trypsin-like protease (HAT), capable of reducing the infectivity of ancestral SARS-CoV-2. Further, in mouse models, HAT analogue expression was upregulated by SARS-CoV-2 infection. The antiviral activity of HAT functioned through the cleavage of the SARS-CoV-2 spike glycoprotein at R682. This cleavage resulted in inhibition of the attachment of ancestral spike proteins to host cells, which inhibited the cell-cell membrane fusion process. Importantly, exogenous addition of HAT notably reduced the infectivity of ancestral SARS-CoV-2 in vivo. However, HAT was ineffective against the Delta variant and most circulating Omicron variants, including the BQ.1.1 and XBB.1.5 subvariants. We demonstrate that the P681R mutation in Delta and P681H mutation in the Omicron variants, adjacent to the R682 cleavage site, contributed to HAT resistance. Our study reports what we believe to be a novel soluble defense factor against SARS-CoV-2 and resistance of its actions in the Delta and Omicron variants.

Structure-guided assembly of an influenza spike nanobicelle vaccine provides pan H1 intranasal protection.

Development of intranasal vaccines for respiratory viruses has gained popularity. However, currently only a live-attenuated influenza vaccine is FDA-approved for intranasal administration. Here, we focused on influenza virus as it circulates seasonally, has pandemic potential, and has vaccine formulations that present hemagglutinin (HA) in different structural arrangements. These display differences have not been correlated with induction of pan-H1 antibodies or shown to provide intranasal protection. Using electron microscopy, biochemistry and animal studies, we identified HA complexes arranged as lipid discs with multiple trimeric HAs displayed along the perimeter, termed spike nanobicelles (SNB). We utilized a structure-guided approach to synthesize in vitro assembled spiked nanobicelles (IA-SNB) from a classical 1934 H1N1 influenza virus. IA-SNBs elicited pan-H1 antibodies and provided protection against antigenically divergent H1N1 viruses via intranasal immunizations. Viral glycoprotein spikes displayed as SNBs could aid in combating antigenic variation and provide innovative intranasal vaccines to aid universal influenza vaccine development.