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The action of mRNA-based vaccines requires the expression of the antigen in cells targeted by lipid nanoparticle–mRNA complexes. When the vaccine antigen is not fully retained by the producer cells, its local and systemic diffusion can have consequences depending on both the levels of antigen expression and its biological activity. A peculiarity of mRNA-based COVID-19 vaccines is the extraordinarily high amounts of the Spike antigen expressed by the target cells. In addition, vaccine Spike can be shed and bind to ACE-2 cell receptors, thereby inducing responses of pathogenetic significance including the release of soluble factors which, in turn, can dysregulate key immunologic processes. Moreover, the circulatory immune responses triggered by the vaccine Spike is quite powerful, and can lead to effective anti-Spike antibody cross-binding, as well as to the emergence of both auto- and anti-idiotype antibodies. In this paper, the immunologic downsides of the strong efficiency of the translation of the mRNA associated with COVID-19 vaccines are discussed together with the arguments supporting the idea that most of them can be avoided with the advent of next-generation, mucosal COVID-19 vaccines. [ABSTRACT FROM AUTHOR]

Chemical prototypes with broad-spectrum antiviral activity are important toward developing new therapies that can act on both existing and emerging viruses. Binding of the SARS-CoV-2 spike protein to the host angiotensin-converting enzyme 2 (ACE2) receptor is required for cellular entry of SARS-CoV-2. Toward identifying new chemical leads that can disrupt this interaction, including in the presence of SARS-CoV-2 adaptive mutations found in variants like omicron that can circumvent vaccine, immune, and therapeutic antibody responses, we synthesized 5-chloro-3-(2-(2,4-dinitrophenyl)hydrazono)indolin-2-one (H2L) from the condensation reaction of 5-chloroisatin and 2,4-dinitrophenylhydrazine in good yield. H2L was characterised by elemental and spectral (IR, electronic, Mass) analyses. The NMR spectrum of H2L indicated a keto–enol tautomerism, with the keto form being more abundant in solution. H2L was found to selectively interfere with binding of the SARS-CoV-2 spike receptor-binding domain (RBD) to the host angiotensin-converting enzyme 2 receptor with a 50% inhibitory concentration (IC50) of 0.26 μM, compared to an unrelated PD-1/PD-L1 ligand–receptor-binding pair with an IC50 of 2.06 μM in vitro (Selectivity index = 7.9). Molecular docking studies revealed that the synthesized ligand preferentially binds within the ACE2 receptor-binding site in a region distinct from where spike mutations in SARS-CoV-2 variants occur. Consistent with these models, H2L was able to disrupt ACE2 interactions with the RBDs from beta, delta, lambda, and omicron variants with similar activities. These studies indicate that H2L-derived compounds are potential inhibitors of multiple SARS-CoV-2 variants, including those capable of circumventing vaccine and immune responses. [ABSTRACT FROM AUTHOR]

The receptor‐binding domain (RBD) of the spike glycoprotein of SARS‐CoV‐2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor‐binding domain soon became the major target for COVID‐19 research and the development of diagnostic tools and new‐generation vaccines. Here, we provide a detailed protocol for high‐yield expression and one‐step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum‐free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L−1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA‐based vaccination of mice.

The action of mRNA-based vaccines requires the expression of the antigen in cells targeted by lipid nanoparticle–mRNA complexes. When the vaccine antigen is not fully retained by the producer cells, its local and systemic diffusion can have consequences depending on both the levels of antigen expression and its biological activity. A peculiarity of mRNA-based COVID-19 vaccines is the extraordinarily high amounts of the Spike antigen expressed by the target cells. In addition, vaccine Spike can be shed and bind to ACE-2 cell receptors, thereby inducing responses of pathogenetic significance including the release of soluble factors which, in turn, can dysregulate key immunologic processes. Moreover, the circulatory immune responses triggered by the vaccine Spike is quite powerful, and can lead to effective anti-Spike antibody cross-binding, as well as to the emergence of both auto- and anti-idiotype antibodies. In this paper, the immunologic downsides of the strong efficiency of the translation of the mRNA associated with COVID-19 vaccines are discussed together with the arguments supporting the idea that most of them can be avoided with the advent of next-generation, mucosal COVID-19 vaccines.

ABSTRACTSARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33°C and 37°C, respectively). In addition, fever is a common COVID-19 symptom. SARS-CoV-2 has been shown to replicate more efficiently at low temperatures, but the effect of temperature on different viral proteins remains poorly understood. Here, we investigate how temperature affects the SARS-CoV-2 spike function and evolution. We first observed that increasing temperature from 33°C to 37°C or 39°C increased spike-mediated cell–cell fusion. We then experimentally evolved a recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike at these different temperatures. We found that spike-mediated cell–cell fusion was maintained during evolution at 39°C but was lost in a high proportion of viruses that evolved at 33°C or 37°C. Consistently, sequencing of the spikes evolved at 33°C or 37°C revealed the accumulation of mutations around the furin cleavage site, a region that determines cell–cell fusion, whereas this did not occur in spikes evolved at 39°C. Finally, using site-directed mutagenesis, we found that disruption of the furin cleavage site had a temperature-dependent effect on spike-induced cell–cell fusion and viral fitness. Our results suggest that variations in body temperature may affect the activity and diversification of the SARS-CoV-2 spike.IMPORTANCEWhen it infects humans, SARS-CoV-2 is exposed to different temperatures (e.g., replication site and fever). Temperature has been shown to strongly impact SARS-CoV-2 replication, but how it affects the activity and evolution of the spike protein remains poorly understood. Here, we first show that high temperatures increase the SARS-CoV-2 spike fusogenicity. Then, we demonstrate that the evolution of the spike activity and variants depends on temperature. Finally, we show that the functional effect of specific spike mutations is temperature-dependent. Overall, our results suggest that temperature may be a factor influencing the activity and adaptation of the SARS-CoV-2 spike in vivo, which will help understanding viral tropism, pathogenesis, and evolution.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The receptor‐binding domain (RBD) of the spike glycoprotein of SARS‐CoV‐2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor‐binding domain soon became the major target for COVID‐19 research and the development of diagnostic tools and new‐generation vaccines. Here, we provide a detailed protocol for high‐yield expression and one‐step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum‐free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L−1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA‐based vaccination of mice. [ABSTRACT FROM AUTHOR]

• Production of Gag-Spike VLPs using suspension serum-free HEK 293 cells. • Purification of VLPs by ultracentrifugation, tangential flow filtration and affinity chromatography. • Incorporation of SARS-CoV-2 components into Gag-Spike VLPs. • Evaluation of humoral and cellular responses to Gag-Spike VLPs through mouse studies. The virus-like particle (VLP) platform is a robust inducer of humoral and cellular immune responses; hence, it has been used in vaccine development for several infectious diseases. In the current work, VLPs carrying SARS-CoV-2 Spike (S) protein (Wuhan strain) with an HIV-1 Gag core were produced using suspension HEK 293SF-3F6 cells by transient transfection. The Gag was fused with green fluorescent protein (GFP) for rapid quantification of the VLPs. Five different versions of Gag-Spike VLPs (Gag-S-VLPs) consisting of Gag-S alone or combined with other SARS-CoV-2 components, namely Gag-S-Nucleocapsid (N), Gag-S-Matrix (M), Gag-S-Envelope (E), Gag-S-MEN, along with Gag alone were produced and processed by clarification, nuclease treatment, concentration by tangential flow filtration (TFF) and diafiltration. A pilot mouse study was performed to evaluate the immunogenicity of the Gag-S-VLPs through the measurement of the humoral and/or cellular responses against all the mentioned SARS-CoV-2 components. Antibody response to Spike was observed in all variants. The highest number of Spike-specific IFN-γ + T cells was detected with Gag-S-VLPs. No induction of antigen-specific cellular responses to M, N or E proteins were detected with any of the Gag-S, M, E/or N VLPs tested. Therefore, the Gag-S-VLP, by reason of consistently eliciting strong antigen-specific cellular and antibody responses, was selected for further evaluation. The purification process was improved by replacing the conventional centrifugation by serial microfiltration in the clarification step, followed by Spike-affinity chromatography to get concentrated VLPs with higher purity. Three different doses of Gag-S-VLP in conjunction with two adjuvants (Quil-A or AddaVax) were used to assess the dose-dependent antigen-specific cellular and antibody responses in mice. The Gag-S-VLP adjuvanted with Quil-A resulted in a stronger Spike-specific cellular response compared to that adjuvanted with AddaVax. A strong spike neutralisation activity was observed for all doses, independent of the adjuvant combination. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can infect various human tissues and cell types, principally via interaction with its cognate receptor angiotensin-converting enzyme-2 (ACE2). However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After twenty passages of a spike-expressing recombinant vesicular stomatitis virus (VSV), cell-type-specific phenotypic changes were observed and sequencing allowed the identification of sixteen adaptive spike mutations. We used VSV pseudotyping to measure the entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, and entry pathway usage of all the mutants, alone or in combination. The fusogenicity of the mutant spikes was assessed with a cell–cell fusion assay. Finally, mutant recombinant VSVs were used to measure the fitness advantage associated with selected mutations. We found that the effects of these mutations varied across cell types, both in terms of viral entry and replicative fitness. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution. [ABSTRACT FROM AUTHOR]

Via an insufficient coat protein complex I (COPI) retrieval signal, the majority of SARS-CoV-2 spike (S) is resident in host early secretory organelles and a tiny amount is leaked out in cell surface. Only surface-exposed S can be recognized by B cell receptor (BCR) or anti-S therapeutic monoclonal antibodies (mAbs) that is the trigger step for B cell activation after S mRNA vaccination or infected cell clearance by S mAbs. Now, a drug strategy to promote S host surface exposure is absent. Here, we first combined structural and biochemical analysis to characterize S COPI sorting signals. A potent S COPI sorting inhibitor was then invented, evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity (ADCC). Importantly, with the inhibitor as a probe, we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations, possibly corresponding to its ER chaperone association. Our findings not only suggest COPI is a druggable target against COVID-19, but also highlight SARS-CoV-2 evolution mechanism driven by S folding and trafficking mutations.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) enters the host cell by binding to angiotensin‐converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS‐CoV‐2 viruses impact species specificity. Reverse zoonoses led to SARS‐CoV‐2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink‐associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio‐layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink‐derived S glycoproteins of SARS‐CoV‐2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo‐electron microscopy (cryo‐EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor‐binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S‐ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS‐CoV‐2 entry. [ABSTRACT FROM AUTHOR]

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches.

Binding of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the cognate angiotensin-converting enzyme 2 (ACE2) receptor is the initial step in the viral infection process. In this study, we screened an in-house extract library to identify food materials with inhibitory activity against this binding using enzyme-linked immunosorbent assays and attempted to ascertain their active constituents. Hydrangea macrophylla var. thunbergia leaves were identified as candidate materials. Its active compounds were purified using conventional chromatographic methods and identified as naringenin, dihydroisocoumarins, hydrangenol, and phyllodulcin, which have affinities for the ACE2 receptor and inhibit ACE2 receptor–spike S1 binding. Given that boiled water extracts of H. macrophylla leaves are commonly consumed as sweet tea in Japan, we speculated that this tea could be used as a potential natural resource to reduce the risk of SARS-CoV-2 infect Hydrangenol and phyllodulcin from H. macrophylla inhibit the binding between the spike protein of SARS-CoV-2 and ACE2. [ABSTRACT FROM AUTHOR]

New vaccine delivery technologies, such as mRNA, have played a critical role in the rapid and efficient control of SARS-CoV-2, helping to end the COVID-19 pandemic. Enveloped virus-like particles (eVLPs) are often more immunogenic than protein subunit immunogens and could be an effective vaccine platform. Here, we investigated whether the genetic delivery of eVLPs could achieve strong immune responses in mice as previously reported with the immunization of in vitro purified eVLPs. We utilized Newcastle disease virus-like particles (NDVLPs) to display SARS-CoV-2 prefusion-stabilized spikes from the WA-1 or Beta variant (S-2P or S-2Pᵦ, respectively) and evaluated neutralizing murine immune responses achieved by a single-gene-transcript DNA construct for the WA-1 or Beta variant (which we named S-2P-NDVLP-1T and S-2Pᵦ-NDVLP-1T, respectively), by multiple-gene-transcript DNA constructs for the Beta variant (S-2Pᵦ-NDVLP-3T), and by a protein subunit–DNA construct for the WA-1 or Beta variant (S-2P-TM or S-2Pᵦ-TM, respectively). The genetic delivery of S-2P-NDVLP-1T or S-2Pᵦ-NDVLP-1T yielded modest neutralizing responses after a single immunization and high neutralizing responses after a second immunization, comparable to previously reported results in mice immunized with in vitro purified S-2P-NDVLPs. Notably, genetic delivery of S-2Pᵦ-NDVLP-3T yielded significantly higher neutralizing responses in mice after a second immunization than S-2Pᵦ-NDVLP-1T or S-2Pᵦ-TM. Genetic delivery also elicited high spike-specific T-cell responses. Collectively, these results indicate that genetic delivery can provide an effective means to immunize eVLPs and that a multiple-gene transcript eVLP platform may be especially efficacious and inform the design of improved vaccines. [ABSTRACT FROM AUTHOR]

Via an insufficient coat protein complex I (COPI) retrieval signal, the majority of SARS-CoV-2 spike (S) is resident in host early secretory organelles and a tiny amount is leaked out in cell surface. Only surface-exposed S can be recognized by B cell receptor (BCR) or anti-S therapeutic monoclonal antibodies (mAbs) that is the trigger step for B cell activation after S mRNA vaccination or infected cell clearance by S mAbs. Now, a drug strategy to promote S host surface exposure is absent. Here, we first combined structural and biochemical analysis to characterize S COPI sorting signals. A potent S COPI sorting inhibitor was then invented, evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity (ADCC). Importantly, with the inhibitor as a probe, we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations, possibly corresponding to its ER chaperone association. Our findings not only suggest COPI is a druggable target against COVID-19, but also highlight SARS-CoV-2 evolution mechanism driven by S folding and trafficking mutations. SARS-CoV-2 spike is largely hidden in cell organelles, resulting in compromised host immune surveillance. We invented a COPI inhibitor capable of promoting spike surface exposure to enhance host immune response. [Display omitted] [ABSTRACT FROM AUTHOR]

Spike protein from SARS-CoV-2, the etiologic agent of the COVID-19 pandemic disease, constitutes a structural protein that proved to be the main responsible for neutralizing antibody production. Thus, its sequence is highly considered for the design of candidate vaccines. Animal cell culture represents the best option for the production of subunit vaccines based on recombinant proteins since they introduce post-translational modifications that are important to mimic the natural antigenic epitopes. Particularly, the human cell line HEK293T has been explored and used for the production of biotherapeutics since the products derived from them present human-like post-translational modifications that are important for the protein's activity and immunogenicity. The aim of this study was to produce and characterize a potential vaccine for COVID-19 based on the spike ectodomain (S-ED) of SARS-CoV-2 and two different adjuvants: aluminum hydroxide (AH) and immune-stimulating complexes (ISCOMs). The S-ED was produced in sHEK293T cells using a 1-L stirred tank bioreactor operated in perfusion mode and purified. S-ED characterization revealed the expected size and morphology. High N-glycan content was confirmed. S-ED-specific binding with the hACE2 (human angiotensin-converting enzyme 2) receptor was verified. The immunogenicity of S-ED was evaluated using AH and ISCOMs. Both formulations demonstrated the presence of anti-RBD antibodies in the plasma of immunized mice, being significantly higher for the latter adjuvant. Also, higher levels of IFN-γ and IL-4 were detected after the ex vivo immune stimulation of spleen-derived MNCs from ISCOMs immunized mice. Further analysis confirmed that S-ED/ISCOMs elicit neutralizing antibodies against SARS-CoV-2. Key points: Trimeric SARS-CoV-2 S-ED was produced in stable recombinant sHEK cells in serum-free medium. A novel S-ED vaccine formulation induced potent humoral and cellular immunity. S-ED formulated with ISCOMs adjuvant elicited a highly neutralizing antibody titer. [ABSTRACT FROM AUTHOR]

The attachment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike to angiotensin-converting enzyme 2 (ACE-2) leads the cell fusion process, so spike blockade may be a promising therapy combating COVID-19. Bee pollen bioflavonoids with intrinsic bioactivities are of outmost importance to block SARS-CoV-2-ACE-2 interaction. Herein, we conducted a molecular docking assessment through natural phenolics/non-phenolics of pollen to investigate their affinity against SARS-CoV-2 spike. Finally, kaempferol 3-neohesperidoside 7-O-rhamnoside (compound a), quercetin 7-rhamnoside (compound b), delphinidin-3-O-(6-p-coumaroyl) glucoside (compound c), and luteolin-7-O-6″-malonylglucoside (compound d) showed the lowest binding affinity of −8.1, −7.7, −7.3 and −6.7 kcal/mol. The docking procedure was validated using protein-protein interactions between ACE-2 and SARS-CoV-2 RBD via HADDOCK webserver. MD simulations were fulfilled to investigate different ligands' effects on protein movements. Collectively, compound a may possess the potency to disturb the binding of SARS-CoV-2 spike-ACE-2, which can be on the call for further in vitro and in vivo study to investigate its antiviral potential against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 spike protein contains a functionally important fatty acid (FA) binding site, which is also found in some other coronaviruses, e.g. SARS-CoV and MERS-CoV. The occupancy of the FA site by linoleic acid (LA) reduces infectivity by 'locking' the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the allosteric responses of spike variants to LA removal. D-NEMD simulations show that the FA site is coupled to other functional regions of the protein, e.g. the receptor-binding motif (RBM), N-terminal domain (NTD), furin cleavage site, and regions surrounding the fusion peptide. D-NEMD simulations also identify the allosteric networks connecting the FA site to these functional regions. The comparison between the wild-type spike and four variants (Alpha, Delta, Delta plus, and Omicron BA.1) shows that the variants differ significantly in their responses to LA removal. The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein, with the exception of the RBM and the S71–R78 region, which show a weaker link to the FA site. In contrast, Omicron is the most different variant, exhibiting significant differences in the RBM, NTD, V622–L629, and furin cleavage site. These differences in the allosteric modulation may be of functional relevance, potentially affecting transmissibility and virulence. Experimental comparison of the effects of LA on SARS-CoV-2 variants, including emerging variants, is warranted. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 has evolved as several variants. Immunization to boost the Ab response to Spike antigens is effective, but similar vaccines could not enhance Ab efficacy enough. Effective Ab responses against the human ACE2 (hACE2)-mediated infection of the emerging SARS-CoV-2 variants are needed. We identified closed linear epitopes of the SARS-CoV-2 Spike molecule that induced neutralizing Abs (nAbs) against both S1-RBD, responsible for attachment to hACE2, and S2-HR1/2, in convalescents and vaccine recipients. They inhibited a pseudo-virus infection mediated by the hACE2 pathway. The epitope sequences included epitopes #7 (aa411-432), #11 (aa459-480) and #111 (aa1144-1161), in S1-RBD and S2-HR2. Epitope #111 was conserved in Wuhan and variant strains, whereas #7 and #11 were conserved in Wuhan carried mutations K417N and S477N/T478K in Omicron BA.4/5. These mutations were recognized by the original epitope-specific Abs. These epitopes in RBD and HR2 neither contained, nor overlapped with, those responsible for the antibody-dependent enhancement of the SARS-CoV-2 infection. The sublingual administration of multiple epitope-conjugated antigens increased the IgG and IgA Abs specific to the neutralizing epitopes in mice pre-immunized subcutaneously. The findings indicated that S1-RBD and S2-HR2 epitopes were responsible for pseudo-virus SARS-CoV-2 infections and that sublingual boosts with multiple epitope-conjugated antigens could enhance the protection by nAbs of IgG and IgA against infection by a wide range of variants. [ABSTRACT FROM AUTHOR]

Under ER stress conditions, the ER form of transmembrane proteins can reach the plasma membrane via a Golgi‐independent unconventional protein secretion (UPS) pathway. However, the targeting mechanisms of membrane proteins for UPS are unknown. Here, this study reports that TMED proteins play a critical role in the ER stress‐associated UPS of transmembrane proteins. The gene silencing results reveal that TMED2, TMED3, TMED9 and TMED10 are involved in the UPS of transmembrane proteins, such as CFTR, pendrin and SARS‐CoV‐2 Spike. Subsequent mechanistic analyses indicate that TMED3 recognizes the ER core‐glycosylated protein cargos and that the heteromeric TMED2/3/9/10 complex mediates their UPS. Co‐expression of all four TMEDs improves, while each single expression reduces, the UPS and ion transport function of trafficking‐deficient ΔF508‐CFTR and p.H723R‐pendrin, which cause cystic fibrosis and Pendred syndrome, respectively. In contrast, TMED2/3/9/10 silencing reduces SARS‐CoV‐2 viral release. These results provide evidence for a common role of TMED3 and related TMEDs in the ER stress‐associated, Golgi‐independent secretion of transmembrane proteins. [ABSTRACT FROM AUTHOR]

Multisystem inflammatory syndrome in children (MIS-C) is a rare hyperinflammatory disease occurring several weeks after SARS-CoV-2 infection. The clinical similarities between MIS-C and the toxic shock syndrome, together with the preferential expansion of T cells with a T-cell receptor variable β chain (TCRVβ) skewing, suggested a superantigen theory of MIS-C. For instance, recent in silico modelling evidenced the presence of a highly conserved motif within SARS-CoV-2 spike protein similar in structure to the superantigenic fragment of staphylococcal enterotoxin B (SEB). However, experimental data on the superantigenic activity of the SARS-CoV-2 spike have not yet been provided. Here, we assessed the superantigenic activity of the SARS-CoV-2 spike by analysing inflammatory cytokine production in both Jurkat cells and the peripheral blood CD4+ T cells stimulated with the SARS-CoV-2 spike or SEB as a control. We found that, unlike SEB, the SARS-CoV-2 spike does not exhibit an intrinsic superantigen-like activity. [ABSTRACT FROM AUTHOR]

We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. Given the cell-entry scenario dominated by the endosomal “late pathway”, the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell–cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell–cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections. [ABSTRACT FROM AUTHOR]

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design. [ABSTRACT FROM AUTHOR]

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.

Among numerous point mutation differences between the SARS-CoV-2 and the bat RaTG13 coronavirus, only the 12-nucleotide furin cleavage site (FCS) exceeds 3 nucleotides. A BLAST search revealed that a 19 nucleotide portion of the SARS-CoV-2 genome encompassing the furin cleavage site is a 100% complementary match to a codon-optimized proprietary sequence that is the reverse complement of the human mutS homolog (MSH3). The reverse complement sequence present in SARS-CoV-2 may occur randomly but other possibilities must be considered. Recombination in an intermediate host is an unlikely explanation. Single stranded RNA viruses such as SARS-CoV-2 utilize negative strand RNA templates in infected cells, which might lead through copy choice recombination with a negative sense SARS-CoV-2 RNA to the integration of the MSH3 negative strand, including the FCS, into the viral genome. In any case, the presence of the 19-nucleotide long RNA sequence including the FCS with 100% identity to the reverse complement of the MSH3 mRNA is highly unusual and requires further investigations.

Among numerous point mutation differences between the SARS-CoV-2 and the bat RaTG13 coronavirus, only the 12-nucleotide furin cleavage site (FCS) exceeds 3 nucleotides. A BLAST search revealed that a 19 nucleotide portion of the SARS-CoV- 2 genome encompassing the furin cleavage site is a 100% complementary match to a codon-optimized proprietary sequence that is the reverse complement of the human mutS homolog (MSH3). The reverse complement sequence present in SARS-CoV-2 may occur randomly but other possibilities must be considered. Recombination in an intermediate host is an unlikely explanation. Single stranded RNA viruses such as SARS- CoV-2 utilize negative strand RNA templates in infected cells, which might lead through copy choice recombination with a negative sense SARS-CoV-2 RNA to the integration of the MSH3 negative strand, including the FCS, into the viral genome. In any case, the presence of the 19-nucleotide long RNA sequence including the FCS with 100% identity to the reverse complement of the MSH3 mRNA is highly unusual and requires further investigations. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell-cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S20 fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S20 cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S20 cleavage at the R815 site was also broadly shared by other SARSCoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 has evolved as several variants. Immunization to boost the Ab response to Spike antigens is effective, but similar vaccines could not enhance Ab efficacy enough. Effective Ab responses against the human ACE2 (hACE2)-mediated infection of the emerging SARS-CoV-2 variants are needed. We identified closed linear epitopes of the SARS-CoV-2 Spike molecule that induced neutralizing Abs (nAbs) against both S1-RBD, responsible for attachment to hACE2, and S2-HR1/2, in convalescents and vaccine recipients. They inhibited a pseudo-virus infection mediated by the hACE2 pathway. The epitope sequences included epitopes #7 (aa411-432), #11 (aa459-480) and #111 (aa1144-1161), in S1-RBD and S2-HR2. Epitope #111 was conserved in Wuhan and variant strains, whereas #7 and #11 were conserved in Wuhan carried mutations K417N and S477N/T478K in Omicron BA.4/5. These mutations were recognized by the original epitope-specific Abs. These epitopes in RBD and HR2 neither contained, nor overlapped with, those responsible for the antibody-dependent enhancement of the SARS-CoV-2 infection. The sublingual administration of multiple epitope-conjugated antigens increased the IgG and IgA Abs specific to the neutralizing epitopes in mice pre-immunized subcutaneously. The findings indicated that S1-RBD and S2-HR2 epitopes were responsible for pseudo-virus SARS-CoV-2 infections and that sublingual boosts with multiple epitope-conjugated antigens could enhance the protection by nAbs of IgG and IgA against infection by a wide range of variants.

Abstract SARS‐CoV‐2 spike protein (S) binds to human angiotensin‐converting enzyme 2 (hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we use high‐speed atomic force microscopy (HS‐AFM) for real‐time visualization of S, and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show conformational heterogeneity of S, flexibility of S stalk and receptor‐binding domain (RBD), and pH/temperature‐induced conformational change of S. S in an S‐ACE2 complex appears as an all‐RBD up conformation. The complex acquires a distinct topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S‐hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2‐expressing sEVs for viral neutralization. In contrary, S2 subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking the viral entry scenario. Altogether, our study provides a platform that is suitable for real‐time visualization of various entry inhibitors, neutralizing antibodies, and sEV‐based decoy in blocking viral entry. Teaser: Comprehensive observation of SARS‐CoV‐2 spike and its interaction with receptor ACE2 and sEV‐based decoy in real time using HS‐AFM.

The SARS-CoV-2 spike has been regarded as the main target of antibody design against COVID-19. Two single-site mutations, R190K and N121Q, were deemed to weaken the binding affinity of biliverdin although the underlying molecular mechanism is still unknown. Meanwhile, the effect of the two mutations on the conformational changes of “lip” and “gate” loops was also elusive. Thus, molecular dynamics simulation and molecular mechanics/generalized Born surface area (MM/GBSA) free energy calculation were conducted on the wild-type and two other SARS-CoV-2 spike mutants. Our simulations indicated that the R190K mutation causes Lys190 to form six hydrogen bonds, guided by Asn99 and Ile101, which brings Lys190 closer to Arg102 and Asn121, thereby weakening the interaction energy between biliverdin and Ile101 as well as Lys190. For the N121Q mutation, Gln121 still maintained a hydrogen bond with biliverdin; nevertheless, the overall binding mode deviated significantly under the reversal of the side chain of Phe175. Moreover, the two mutants would stabilize the lip loop, which would restrain the meaningful upward movement of the lip. In addition, N121Q significantly promoted the gate loop deviating to the biliverdin binding site and compressed the site. This work would be useful in understanding the dynamics binding biliverdin to the SARS-CoV-2 spike.

SARS‐CoV‐2 spike protein (S) binds to human angiotensin‐converting enzyme 2 (hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we use high‐speed atomic force microscopy (HS‐AFM) for real‐time visualization of S, and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show conformational heterogeneity of S, flexibility of S stalk and receptor‐binding domain (RBD), and pH/temperature‐induced conformational change of S. S in an S‐ACE2 complex appears as an all‐RBD up conformation. The complex acquires a distinct topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S‐hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2‐expressing sEVs for viral neutralization. In contrary, S2 subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking the viral entry scenario. Altogether, our study provides a platform that is suitable for real‐time visualization of various entry inhibitors, neutralizing antibodies, and sEV‐based decoy in blocking viral entry. Teaser: Comprehensive observation of SARS‐CoV‐2 spike and its interaction with receptor ACE2 and sEV‐based decoy in real time using HS‐AFM. [ABSTRACT FROM AUTHOR]

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches. [ABSTRACT FROM AUTHOR]

Many of the approved SARS-CoV-2 vaccines are based on a stabilized variant of the spike protein. This raises the question of whether the immune response against the stabilized spike is identical to the immune response that is elicited by the native spike in the case of a SARS-CoV-2 infection. Using a peptide array-based approach, we analysed the binding of antibodies from Comirnaty-elicited, convalescent, and control sera to the peptides covering the spike protein. A total of 37 linear epitopes were identified. A total of 26 of these epitopes were almost exclusively recognized by the convalescent sera. Mapping these epitopes to the spike structures revealed that most of these 26 epitopes are masked in the pre-fusion structure. In particular, in the conserved central helix, three epitopes that are only exposed in the post-fusion conformation were identified. This indicates a higher spike-specific antibody diversity in convalescent sera. These differences could be relevant for the breadth of spike-specific immune response.

We observe that a residue R of the spike glycoprotein of SARS-CoV-2 that has mutated in one or more of the current variants of concern or interest, or under monitoring, rarely participates in a backbone hydrogen bond if R lies in the S1 subunit and usually participates in one if R lies in the S2 subunit. A partial explanation for this based upon free energy is explored as a potentially general principle in the mutagenesis of viral glycoproteins. This observation could help target future vaccine cargos for the evolving coronavirus as well as more generally. A related study of the Delta and Omicron variants suggests that Delta was an energetically necessary intermediary in the evolution from Wuhan-Hu-1 to Omicron.

Here, we describe a protocol for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA). The protocol was developed with a keen focus on optimizing several key parameters, including antigen coating concentration, antibody and sample dilutions, and assay development time. The final protocol features the following characteristics:•The capability to detect SARS-CoV-2 spike-specific IgG in both plasma and serum samples.•A streamlined procedure that requires only 1 hour and 20 minutes of hands-on time.•Reliable assay performance, with a remarkable sensitivity of 98.1 % and specificity of 99.5 %., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The generation of antigen-specific memory B cells is crucial to the long-term effectiveness of vaccines. When the protective antibodies circulating in the blood wane, memory B cells (MBC) can be rapidly reactivated and differentiated into antibody-secreting cells during a new infection. Such MBC responses are considered to be key in providing long-term protection after infection or vaccination. Here, we describe the optimization and qualification of a FluoroSpot assay to measure MBCs directed against the SARS-CoV-2 spike protein in the peripheral blood, for use in COVID-19 vaccine trials. We developed a FluoroSpot assay enabling simultaneous enumeration of B cells secreting IgA or IgG spike-specific antibodies after polyclonal stimulation of peripheral blood mononuclear cells (PBMCs) with interleukin-2 and the toll-like receptor agonist R848 for 5 days. The antigen coating was optimized using a capture antibody directed against the spike subunit-2 glycoprotein of SARS-CoV-2 to immobilize recombinant trimeric spike protein onto the membrane. Compared to a direct spike protein coating, the addition of a capture antibody increased the number and the quality of detected spots for both spike-specific IgA and IgG secreting cells in PBMCs from COVID-19 convalescents. The qualification showed good sensitivity of the dual-color IgA-IgG FluoroSpot assay, with lower limits of quantitation of 18 background-subtracted (BS) antibody-secreting cells (ASCs)/well for spike-specific IgA and IgG responses. Linearity was demonstrated at values ranging from 18 to 73 and from 18 to 607 BS ASCs/well for spike-specific IgA and IgG, respectively, as was precision, with intermediate precision (percentage geometric coefficients of variation) of 12% and 26% for the proportion of spike-specific IgA and IgG MBCs (ratio specific/total IgA or Ig). The assay was specific, since no spike-specific MBCs were detected in PBMCs from pre-pandemic samples; the results were below the limit of detection of 17 BS ASCs/well. These results show that the dual-color IgA-IgG FluoroSpot provides a sensitive, specific, linear, and precise tool to detect spike-specific MBC responses. This MBC FluoroSpot assay is a method of choice for monitoring spike-specific IgA and IgG MBC responses induced by COVID-19 candidate vaccines in clinical trials. [Display omitted] • We developed a dual-color SARS-CoV-2 spike-specific IgA-IgG memory B cell (MBC) FluoroSpot assay. • We selected a high-affinity capture antibody to optimize the antigen coating. • Qualification of the assay confirmed specificity, sensitivity, linearity and precision. • This assay is suitable to evaluate MBC responses in COVID-19 vaccine clinical trials. [ABSTRACT FROM AUTHOR]