Surface enhanced Raman spectroscopy based analysis of SARS-CoV-2 spike protein binding to ACE2 receptor

The SARS-CoV-2 virus is still a challenge because of its diversity and mutations. The binding interactions of the angiotensin converting enzyme 2 (ACE2) receptor and the spike protein are relevant for the SARS-CoV-2 virus to enter the cell. Consequently, it is important and helpful to analyze binding activities and the changes in the structure of the ACE2 receptor and the spike protein. Surface enhanced Raman spectroscopy is able to analyze small concentrations of the proteins without contact, non-invasively and label-free. In this work, we present a SERS based approach in the visible wavelength range to analyze and study the binding interactions of the ACE2 receptor and the spike protein. SERS measurements of the ACE2 receptor, the spike protein and the ACE2-spike complex were performed. Additionally, an inhibitor was used to prevent the spike protein from binding to ACE2 and to compare the results. The analysis of the measured SERS spectra reveals structural differences and changes due to binding activities. Thus, we show that the performed SERS based approach can help for rapid and non-invasive analysis of binding interactions of the ACE2-spike complex and also of protein binding in general. © 2023 SPIE.

Antibody- and aptamer-free SERS substrate for ultrasensitive and anti-interference detection of SARS-CoV-2 spike protein in untreated saliva.

Sensitive and anti-interference detection of targeted signal(s) in body fluids is one of the paramount tasks in biosensing. Overcoming the complication and high cost of antibody/aptamer-modification, surface-enhanced Raman spectroscopy (SERS) based on antibody/aptamer-free (AAF) substrates has shown great promise, yet with rather limited detection sensitivity. Herein, we report ultrasensitive and anti-interference detection of SARS-CoV-2 spike protein in untreated saliva by an AAF SERS substrate, applying the evanescent field induced by the high-order waveguide modes of well-defined nanorods for SERS for the first time. A detection limit of 3.6 × 10-17 M and 1.6 × 10-16 M are obtained in phosphate buffered saline and untreated saliva, respectively; the detection limits are three orders of magnitude improved than the best records from AAF substrates. This work unlocks an exciting path to design AAF SERS substrates for ultrasensitive biosensing, not limited to detection of viral antigens.

Dihydroisocoumarins of Hydrangea macrophylla var. thunbergia inhibit binding of the SARS-CoV-2 spike protein to ACE2.

Binding of the spike protein of severe acute respiratory syndrome coronavirus 2 to the cognate angiotensin-converting enzyme 2 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 angiotensin-converting enzyme 2 receptor and inhibit angiotensin-converting enzyme 2 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 severe acute respiratory syndrome coronavirus 2 infection.

Balancing Functional Tradeoffs between Protein Stability and ACE2 Binding in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB Lineages: Dynamics-Based Network Models Reveal Epistatic Effects Modulating Compensatory Dynamic and Energetic Changes.

Evolutionary and functional studies suggested that the emergence of the Omicron variants can be determined by multiple fitness trade-offs including the immune escape, binding affinity for ACE2, conformational plasticity, protein stability and allosteric modulation. In this study, we systematically characterize conformational dynamics, structural stability and binding affinities of the SARS-CoV-2 Spike Omicron complexes with the host receptor ACE2 for BA.2, BA.2.75, XBB.1 and XBB.1.5 variants. We combined multiscale molecular simulations and dynamic analysis of allosteric interactions together with the ensemble-based mutational scanning of the protein residues and network modeling of epistatic interactions. This multifaceted computational study characterized molecular mechanisms and identified energetic hotspots that can mediate the predicted increased stability and the enhanced binding affinity of the BA.2.75 and XBB.1.5 complexes. The results suggested a mechanism driven by the stability hotspots and a spatially localized group of the Omicron binding affinity centers, while allowing for functionally beneficial neutral Omicron mutations in other binding interface positions. A network-based community model for the analysis of epistatic contributions in the Omicron complexes is proposed revealing the key role of the binding hotspots R498 and Y501 in mediating community-based epistatic couplings with other Omicron sites and allowing for compensatory dynamics and binding energetic changes. The results also showed that mutations in the convergent evolutionary hotspot F486 can modulate not only local interactions but also rewire the global network of local communities in this region allowing the F486P mutation to restore both the stability and binding affinity of the XBB.1.5 variant which may explain the growth advantages over the XBB.1 variant. The results of this study are consistent with a broad range of functional studies rationalizing functional roles of the Omicron mutation sites that form a coordinated network of hotspots enabling a balance of multiple fitness tradeoffs and shaping up a complex functional landscape of virus transmissibility.

SARS-Cov-2 Spike-S1 Antigen Test Strip with High Sensitivity Endowed by High-Affinity Antibodies and Brightly Fluorescent QDs/Silica Nanospheres.

The extensive research into developing novel strategies for detecting respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens, especially the sensitive point-of-care testing method, is still urgently needed to reach rapid screening of viral infections. Herein, a new lateral flow immunoassay (LFIA) platform was reported for the detection of SARS-CoV-2 spike-S1 protein antigens, in which four sensitive and specific SARS-CoV-2 mouse monoclonal antibodies (MmAbs) were tailored by using quantum dot (QD)-loaded dendritic mesoporous silica nanoparticles modified further for achieving the -COOH group surface coating (named Q/S-COOH nanospheres). Importantly, compact QD adsorption was achieved in mesoporous channels of silica nanoparticles on account of highly accessible central-radial pores and electrostatic interactions, leading to significant signal amplification. As such, a limit of detection for SARS-CoV-2 spike-S1 testing was found to be 0.03 ng/mL, which is lower compared with those of AuNPs-LFIA (traditional colloidal gold nanoparticles, Au NPs) and enzyme-linked immunosorbent assay methods. These results show that optimizing the affinity of antibody and the intensity of fluorescent nanospheres simultaneously is of great significance to improve the sensitivity of LFIA.

SARS-CoV-2 Spike Protein S1 Exposure Increases Susceptibility to Angiotensin II-Induced Hypertension in Rats by Promoting Central Neuroinflammation and Oxidative Stress.

The SARS-CoV-2 spike S1 subunit (S1) can cross the blood-brain barrier and elicit neuroinflammatory response independent of viral infection. Here we examined whether S1 influences blood pressure (BP) and sensitizes the hypertensive response to angiotensin (ANG) II by enhancing neuroinflammation and oxidative stress in hypothalamic paraventricular nucleus (PVN), a key brain cardiovascular regulatory center. Rats received central S1 or vehicle (VEH) injection for 5 days. One week after injection, ANG II or saline (control) was subcutaneously delivered for 2 weeks. S1 injection induced greater increases in BP, PVN neuronal excitation and sympathetic drive in ANG II rats but had no effects in control rats. One week after S1 injection, mRNA for proinflammatory cytokines and oxidative stress marker were higher but mRNA of Nrf2, the master regulator of inducible antioxidant and anti-inflammatory responses, was lower in the PVN in S1-injected rats than in VEH-injected rats. Three weeks after S1 injection, mRNA for proinflammatory cytokines and oxidative stress marker, microglia activation and reactive oxygen species in the PVN were comparable between S1 and VEH treated control rats but were elevated in two groups of ANG II rats. Notably, ANG II-induced elevations in these parameters were exaggerated by S1. Interestingly, ANG II increased PVN Nrf2 mRNA in VEH-treated rats but not in S1-treated rats. These data suggest that S1 exposure has no effect on BP, but post-S1 exposure increases susceptibility to ANG II-induced hypertension by downregulating PVN Nrf2 to promote neuroinflammation and oxidative stress and augment sympathetic excitation.

Age, Sex and BMI Relations with Anti-SARS-CoV-2-Spike IgG Antibodies after BNT162b2 COVID-19 Vaccine in Health Care Workers in Northern Greece.

The aim of this work was to study age, sex, and BMI (Body Mass Index)-related differences in the development of anti-SARS-CoV-2-Spike IgG antibodies, after vaccination with the BNT162b2 COVID-19 vaccine, in health care workers of a General Hospital in a city in Northern Greece. Blood sampling was drawn two to four weeks following the second dose of the vaccine, and six months after the first blood sample collection. Measurement of serum IgG antibodies against the spike domain of SARS-CoV-2 was performed using the SARS-CoV-2 IgG II Quant assay. All participants had sufficient serum IgG titers in the first measurement. Women developed higher IgG titers than men. The IgG titers were inversely related to age in both sexes; there was also a small, insignificant tendency to be inversely related to BMI. Six months after the first measurement, the IgG titers decreased dramatically to values less than 5% of the initial. This decrease was observed in both men and women and was inversely related to age. Multivariate regression analysis showed that age and sex explained with statistical significance 9% of the variance in SARS-CoV-2 IgG titers in our study population; the role of BMI was limited and insignificant.

Sensitive Detection of SARS-CoV-2 Spike Protein based on Electrochemical Impedance Spectroscopy of Fe3O4@SiO2-Au/GCE Biosensor

Highly contagious COVID-19 disease is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which poses a serious threat to global public health. Therefore, the development of a fast and reliable method for the detection of SARS-CoV-2 is an urgent research need. The Fe3O4@SiO2-Au is enriched with a variety of functional groups, which can be used to fabricate a sensitive electrochemical biosensor by biofunctionalization with angiotensin-converting enzyme 2 (ACE2). Accordingly, we developed a novel electrochemical sensor by chemically modifying a glassy carbon electrode (GCE) with Fe3O4@SiO2-Au nanocomposites (hereafter Fe3O4@SiO2-Au/GCE) for the rapid detection of S-protein spiked SARS-CoV-2 by electrochemical impedance spectroscopy (EIS). The new electrochemical sensor has a low limit detection (viz., 4.78 pg/mL) and a wide linear dynamic range (viz., 0.1 ng/mL to 10 μg/mL) for detecting the EIS response signal of S-protein. The robust Fe3O4@SiO2-Au/GCE biosensor has high selectivity, stability, and reproducibility for the detection of S-protein with good recovery of saliva samples.

High-Density Lipoprotein function is modulated by the SARS-CoV-2 spike protein in a lipid-type dependent manner.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.

A surface molecularly imprinted electrochemical biosensor for the detection of SARS-CoV-2 spike protein by using Cu7S4-Au as built-in probe.

Sensitive detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein (S protein) is of significant clinical importance in the diagnosis of COVID-19 pandemic. In this work, a surface molecularly imprinted (SMI) electrochemical biosensor is fabricated for the detection of SARS-CoV-2 S protein. Cu7S4-Au is used as the built-in probe and modified on the surface of a screen-printed carbon electrode (SPCE). 4-Mercaptophenylboric acid (4-MPBA) is anchored to the surface of the Cu7S4-Au through Au-SH bonds, which can be used for the immobilization of the SARS-CoV-2 S protein template through boronate ester bonds. After that, 3-aminophenylboronic acid (3-APBA) is electropolymerized on the electrode surface and used as the molecularly imprinted polymers (MIPs). The SMI electrochemical biosensor is obtained after the elution of the SARS-CoV-2 S protein template with an acidic solution by the dissociation of the boronate ester bonds, which can be utilized for sensitive detection of the SARS-CoV-2 S protein. The developed SMI electrochemical biosensor displays high specificity, reproducibility and stability, which might be a potential and promising candidate for the clinical diagnosis of COVID-19.

Immune responses related to the immunogenicity and reactogenicity of COVID-19 mRNA vaccines.

Vaccination for the prevention of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is considered the most promising approach to control the pandemic of coronavirus disease 2019 (COVID-19). Although various COVID-19 vaccines have been developed worldwide using several modalities, the vaccines that have shown the highest efficacy to date are mRNA vaccines. Despite their extensive usage, the mechanisms that stimulate the immune responses associated with their immunogenicity and reactogenicity remain largely unknown. In this review, we summarize and discuss current knowledge on immune responses to COVID-19 mRNA vaccines, including potential immune responses and correlating factors underlying the immunogenicity and reactogenicity of mRNA vaccines. We also describe recent trends in the optimization of lipid nanoparticles and vaccination routes. Further understanding of vaccine-elicited immune responses will guide the development of more effective and safe vaccines.

Exposure to SARS-CoV-2 and Infantile Diseases.

Background and Aim Immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in newborns and children after prophylactic immunization is currently a relevant research topic. The present study analyzes the issue by examining the possibility that the anti-SARS-CoV-2 immune responses are not uniquely directed against the virus but can-via molecular mimicry and the consequent cross-reactivity-also hit human proteins involved in infantile diseases. Methods Human proteins that-if altered-associate with infantile disorders were searched for minimal immune pentapeptide determinants shared with SARS-CoV-2 spike glycoprotein (gp). Then, the shared pentapeptides were analyzed for immunologic potential and immunologic imprinting phenomena. Results Comparative sequence analysis shows that: (1) numerous pentapeptides (namely, 54) are common to SARS-CoV-2 spike gp and human proteins that, when altered, are linked to infantile diseases; (2) all the shared peptides have an immunologic potential since they are present in experimentally validated SARS-CoV-2 spike gp-derived epitopes; and (3) many of the shared peptides are also hosted in infectious pathogens to which children can have already been exposed, thus making immunologic imprint phenomena feasible. Conclusion Molecular mimicry and the consequent cross-reactivity can represent the mechanism that connects exposure to SARS-CoV-2 and various pediatric diseases, with a fundamental role of the immunologic memory and the history of the child's infections in determining and specifying the immune response and the pathologic autoimmune sequela.

Synergistically functionalized molybdenum disulfide-reduced graphene oxide nanohybrid based ultrasensitive electrochemical immunosensor for real sample analysis of COVID-19.

Herein, we have proposed a straightforward and label-free electrochemical immunosensing strategy supported on a glassy carbon electrode (GCE) modified with a biocompatible and conducting biopolymer functionalized molybdenum disulfide-reduced graphene oxide (CS-MoS2/rGO) nanohybrid to investigate the SARS-CoV-2 virus. CS-MoS2/rGO nanohybrid-based immunosensor employs recombinant SARS-CoV-2 Spike RBD protein (rSP) that specifically identifies antibodies against the SARS-CoV-2 virus via differential pulse voltammetry (DPV). The antigen-antibody interaction diminishes the current responses of the immunosensor. The obtained results indicate that the fabricated immunosensor is extraordinarily capable of highly sensitive and specific detection of the corresponding SARS-CoV-2 antibodies with a LOD of 2.38 zg mL-1 in phosphate buffer saline (PBS) samples over a broad linear range between 10 zg mL-1-100 ng mL-1. In addition, the proposed immunosensor can detect attomolar concentrations in spiked human serum samples. The performance of this immunosensor is assessed using actual serum samples from COVID-19-infected patients. The proposed immunosensor can accurately and substantially differentiate between (+) positive and (-) negative samples. As a result, the nanohybrid can provide insight into the conception of Point-of-Care Testing (POCT) platforms for cutting-edge infectious disease diagnostic methods.

SARS-CoV-2 spike host cell surface exposure promoted by a COPI sorting inhibitor.

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.

In Silico Identification of Propolis Compounds Potential as COVID-19 Drug Candidates Against SARS-CoV-2 Spike Protein

Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a global health issue resulting in mortality and morbidity across the world. There is an urgent need to find treatments to inhibit virus infections and their consequences. Propolis compounds are predicted to have interactions with the SARS-CoV-2 protein since it has various phytochemicals that have been used in medicine. Here, we conducted in silico study to analyze the interaction between propolis compounds and SARS-CoV-2 spike protein by performing molecular docking. The target protein of this research is the crystal structure of the SARS-CoV-2 spike receptor-binding domain (RBD) bound with ACE2 (PDB ID: 6M0J). The ligand of this study is the bioactive compounds from Propolis of Tetragonula sapiens. The docking analysis revealed that Broussoflavonol F and Glyasperin A were the most promising propolis compounds that potentially block the binding of the SARS-CoV-2 spike protein to the host ACE2 receptor, with the binding affinity of -7.6 kcal/mol and -7.3 kcal/mol and the geometric score of 4582 and 4382, respectively. Based on this finding, those compounds are the potential to be developed as COVID-19 drug candidates. © (2023). All Rights Reserved.

Post COVID-19 AA amyloidosis of the kidneys with rapidly progressive renal failure.

Coronavirus disease 2019 (COVID-19) pandemic has taken the world by a storm, posing a gruelling challenge to the medical fraternity globally. Besides its very high infectivityinfectivity, significant organ dysfunction occurs in critically ill COVID-19 patients, leading to severe morbidity and mortality. Pulmonary involvement is the leading cause of death in these patients to be followed by the cardiovascular involvement. Kidney involvement due to COVID-19 is becoming more discernible with AKI adversely affecting the outcome. Besides AKI, a few cases of collapsing FSGS in genetically vulnerable patients and thrombotic microangiopathies have been reported as well. We report a case of AA amyloidosis of the kidney with a rapidly progressive renal failure and congestive heart failure with preserved left ventricular functions, which complicated a moderate COVID-19 pneumonia providing some clues to a possible association of this novel virus disease with this complication, which needs to be confirmed in future studies.

SARS-CoV-2 Enters Human Leydig Cells and Affects Testosterone Production In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a SARS-like coronavirus, continues to produce mounting infections and fatalities all over the world. Recent data point to SARS-CoV-2 viral infections in the human testis. As low testosterone levels are associated with SARS-CoV-2 viral infections in males and human Leydig cells are the main source of testosterone, we hypothesized that SARS-CoV-2 could infect human Leydig cells and impair their function. We successfully detected SARS-CoV-2 nucleocapsid in testicular Leydig cells of SARS-CoV-2-infected hamsters, providing evidence that Leydig cells can be infected with SARS-CoV-2. We then employed human Leydig-like cells (hLLCs) to show that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 is highly expressed in hLLCs. Using a cell binding assay and a SARS-CoV-2 spike-pseudotyped viral vector (SARS-CoV-2 spike pseudovector), we showed that SARS-CoV-2 could enter hLLCs and increase testosterone production by hLLCs. We further combined the SARS-CoV-2 spike pseudovector system with pseudovector-based inhibition assays to show that SARS-CoV-2 enters hLLCs through pathways distinct from those of monkey kidney Vero E6 cells, a typical model used to study SARS-CoV-2 entry mechanisms. We finally revealed that neuropilin-1 and cathepsin B/L are expressed in hLLCs and human testes, raising the possibility that SARS-CoV-2 may enter hLLCs through these receptors or proteases. In conclusion, our study shows that SARS-CoV-2 can enter hLLCs through a distinct pathway and alter testosterone production.

SARS-CoV-2: The Self-Nonself Issue and Diagnostic Tests.

Objective At present, false negatives/positives have been reported in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. Searching for the molecular basis of such tests' unreliability, this study aimed at defining how specific are the sequences used in serological and polymerase chain reaction (PCR) tests to detect SARS-CoV-2. Materials and Methods Analyses were performed on the leading SARS-CoV-2 biomarker spike glycoprotein (gp). Sharing of peptide sequences between the spike antigen and the human host was analyzed using the Peptide Search program from Uniprot database. Sharing of oligonucleotide sequences was investigated using the nucleotide Basic Local Alignment Search Tool (BLASTn) from National Center for Biotechnology Information (NCBI). Results Two main points stand out: (1) a massive pentapeptide sharing exists between the spike gp and the human proteome, and only a limited number of pentapeptides (namely 107) identify SARS-CoV-2 spike gp as nonself when compared with the human proteome, and (2) the small phenetic difference practically disappears at the genetic level. Indeed, almost all of the 107 pentadecameric nucleotide sequences coding for the pentapeptides unique to SARS-CoV-2 spike gp are present in human nucleic acids too. Conclusion The data are of immunological significance for defining the issue of the viral versus human specificity and likely explain the fact that false positives can occur in serological and PCR tests for SARS-CoV-2 detection.

A COVID-19 vaccine candidate based on SARS-CoV-2 spike protein and immune-stimulating complexes.

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.

Coarse-Grained Molecular Simulations and Ensemble-Based Mutational Profiling of Protein Stability in the Different Functional Forms of the SARS-CoV-2 Spike Trimers: Balancing Stability and Adaptability in BA.1, BA.2 and BA.2.75 Variants.

Evolutionary and functional studies have suggested that the emergence of Omicron variants can be determined by multiple fitness tradeoffs including immune escape, binding affinity, conformational plasticity, protein stability, and allosteric modulation. In this study, we embarked on a systematic comparative analysis of the conformational dynamics, electrostatics, protein stability, and allostery in the different functional states of spike trimers for BA.1, BA.2, and BA.2.75 variants. Using efficient and accurate coarse-grained simulations and atomistic reconstruction of the ensembles, we examined the conformational dynamics of the spike trimers that agree with the recent functional studies, suggesting that BA.2.75 trimers are the most stable among these variants. A systematic mutational scanning of the inter-protomer interfaces in the spike trimers revealed a group of conserved structural stability hotspots that play a key role in the modulation of functional dynamics and are also involved in the inter-protomer couplings through local contacts and interaction networks with the Omicron mutational sites. The results of mutational scanning provided evidence that BA.2.75 trimers are more stable than BA.2 and comparable in stability to the BA.1 variant. Using dynamic network modeling of the S Omicron BA.1, BA.2, and BA.2.75 trimers, we showed that the key network mediators of allosteric interactions are associated with the major stability hotspots that are interconnected along potential communication pathways. The network analysis of the BA.1, BA.2, and BA.2.75 trimers suggested that the increased thermodynamic stability of the BA.2.75 variant may be linked with the organization and modularity of the residue interaction network that allows for allosteric communications between structural stability hotspots and Omicron mutational sites. This study provided a plausible rationale for a mechanism in which Omicron mutations may evolve by targeting vulnerable sites of conformational adaptability to elicit immune escape while maintaining their control on balancing protein stability and functional fitness through robust allosteric communications with the stability hotspots.