Viral Spike Research Articles

What Role Does Microthrombosis Play in Long COVID?

Soon after the outbreak of coronavirus disease 2019 (COVID-19), unexplained sustained fatigue, cognitive disturbance, and muscle ache/weakness were reported in patients who had recovered from acute COVID-19 infection. This abnormal condition has been recognized as "long COVID (postacute sequelae of COVID-19 [PASC])" with a prevalence estimated to be from 10 to 20% of convalescent patients. Although the pathophysiology of PASC has been studied, the exact mechanism remains obscure. Microclots in circulation can represent one of the possible causes of PASC. Although hypercoagulability and thrombosis are critical mechanisms of acute COVID-19, recent studies have reported that thromboinflammation continues in some patients, even after the virus has cleared. Viral spike proteins and RNA can be detected months after patients have recovered, findings that may be responsible for persistent thromboinflammation and the development of microclots. Despite this theory, long-term results of anticoagulation, antiplatelet therapy, and vascular endothelial protection are inconsistent, and could not always show beneficial treatment effects. In summary, PASC reflects a heterogeneous condition, and microclots cannot explain all the presenting symptoms. After clarification of the pathomechanisms of each symptom, a symptom- or biomarker-based stratified approach should be considered for future studies.

Distinguishing SARS-CoV-2 infection and vaccine responses up to 18 months post-infection using nucleocapsid protein and receptor-binding domain antibodies.

The prediction of the durability of immunity against COVID-19 is relevant, and longitudinal studies are essential for unraveling the details regarding protective SARS-CoV-2 antibody responses. It has become challenging to discriminate between COVID-19 vaccine- and infection-induced immune responses since all approved vaccines in Europe and the USA are based on the viral spike (S) protein, which is also the most commonly used antigen in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a nucleocapsid (N) protein-based sandwich ELISA for detecting pan anti-SARS-CoV-2 Ig with a sensitivity and specificity of 97%. Generalized mixed models were used to determine the degree of long-term humoral immunity against the N protein and the receptor-binding domain (RBD) of the S protein in a cohort of infected individuals to distinguish between COVID-19 vaccine- and infection-induced immunity. N-specific waning could be observed in individuals who did not experience reinfection, while individuals who experienced reinfection had a new significant increase in N-specific Ig levels. In individuals that seroconverted without a reinfection, 70.1% remained anti-N seropositive after 550 days. The anti-RBD Ig dynamics were unaffected by reinfection but exhibited a clear increase in RBD-specific Ig when vaccination was initiated. In conclusion, a clear difference in the dynamics of the antibody response against N protein and RBD was observed over time. Anti-N protein-specific Igs can be detected up to 18 months after SARS-CoV-2 infection allowing long-term discrimination of infectious and vaccine antibody responses.IMPORTANCELongitudinal studies are essential to unravel details regarding the protective antibody responses after COVID-19 infection and vaccination. It has become challenging to distinguish long-term immune responses to SARS-CoV-2 infection and vaccination since most approved vaccines are based on the viral spike (S) protein, which is also mostly used in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a novel nucleocapsid (N) protein-based sandwich ELISA for detecting pan-anti-SARS-CoV-2 Ig, exhibiting high sensitivity and specificity. Generalized mixed models were used to determine long-term humoral immunity in a cohort of infected individuals from the Faroe Islands, distinguishing between COVID-19 vaccine- and infection-induced immunity. A clear difference in the dynamics of the antibody response against N protein and S protein was observed over time, and the anti-N protein-specific Igs could be detected up to 18 months after SARS-CoV-2 infection. This enables long-term discrimination between natural infection and vaccine-dependent antibody responses.

Designing and Expression of Recombinant Chimeric Spike Protein from SARS-CoV-2 in Escherichia coli and Its Immunogenicity Assessment.

Since December 2019, the world has been grappling with an ongoing global COVID-19 pandemic. Various virus variants have emerged over the past two years, each posing a greater threat than its predecessors. The recent appearance of the omicron variant (B.1.1.529) has raised significant alarm within the field of epidemiology due to its highly contagious nature and rapid transmission rate. The omicron variant possessed mutations in the key receptor-binding domain (RBD) region, the S region, and these modifications have shown a notable impact on the strain's susceptibility to neutralizing antibodies. Developing safe and efficient vaccines to prevent a future severe acute respiratory outbreak of coronavirus syndrome 2 (SARS-CoV-2) is significant. Viral surface spike proteins are ideal targets for vaccines. This study aimed to find a multi-subunit chimeric vaccine. After conducting bioinformatics analysis, the recombinant spike (RS) protein of SARS-CoV-2 was deliberately designed and subsequently produced using E. coli expression systems. The immunogenicity of RS and neutralizing antibody responses were evaluated on immunized BALB/c mice. There was a significant difference in antibody titers between RS-immunized mice and control groups. The endpoint of the serum antibody titer of mice immunized with our chimeric protein was 2.5 times higher than that of the negative control. The chimeric construct could present multiple antigens simultaneously, influentially affecting immunization. Sera from mice vaccinated by RS could recognize the SARS-CoV-2 virus and neutralize antibodies. Our chimeric peptide could bind to antibodies in the serum of patients infected with different serotypes of the SARS-CoV-2 virus, such as alpha, delta, and omicron variants. The results indicated that the RS protein would be a potential novel antigenic candidate for subunit vaccine development and could be used as a useful alternative to generate diagnostic serological tests for SARS-CoV-2 infection.

Affinity Maturated Transferrin Receptor Apical Domain Blocks Machupo Virus Glycoprotein Binding

Transferrin receptor 1 (TfR) delivers iron across cellular membranes by shuttling the ion carrier protein transferrin. This ability to deliver large protein ligands inside cells is taken advantage of by pathogens to infiltrate human cells. Notably, the receptor’s outermost ectodomain, the apical domain, is used as a point of attachment for several viruses including hemorrhagic arenaviruses. To better understand interactions with the receptor it would be advantageous to probe sequence determinants in the apical domain with viral spike proteins. Here, we carried out affinity maturation of our computationally designed apical domain from human TfR to identify underlying driving forces that lead to better binding. The improved variants were confirmed by in vitro surface plasmon resonance measurements with dissociation constants obtained in the lower nanomolar range. It was found that the strong binding affinities for the optimized variants matched the strength of interactions with the native receptor. The structure of the best variant was determined experimentally indicating that the conformational change in the hairpin binding motif at the protein–protein interface plays a crucial role. The experimental methodology can be straightforwardly applied to other arenavirus or pathogens that use the apical domain. It can further be useful to probe host–virus compatibility or therapeutic strategies based on the transferrin receptor decoys.

Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

Mannose-Binding Lectins as Potent Antivirals against SARS-CoV-2.

The SARS-CoV-2 entry into host cells is mainly mediated by the interactions between the viral spike protein (S) and the ACE-2 cell receptor, which are highly glycosylated. Therefore, carbohydrate binding agents may represent potential candidates to abrogate virus infection. Here, we evaluated the in vitro anti-SARS-CoV-2 activity of two mannose-binding lectins isolated from the Brazilian plants Canavalia brasiliensis and Dioclea violacea (ConBR and DVL). These lectins inhibited SARS-CoV-2 Wuhan-Hu-1 strain and variants Gamma and Omicron infections, with selectivity indexes (SI) of 7, 1.7, and 6.5, respectively for ConBR; and 25, 16.8, and 22.3, for DVL. ConBR and DVL inhibited over 95% of the early stages of the viral infection, with strong virucidal effect, and also protected cells from infection and presented post-entry inhibition. The presence of mannose resulted in the complete lack of anti-SARS-CoV-2 activity by ConBR and DVL, recovering virus titers. ATR-FTIR, molecular docking, and dynamic simulation between SARS-CoV-2 S and either lectins indicated molecular interactions with predicted binding energies of -85.4 and -72.0 Kcal/Mol, respectively. Our findings show that ConBR and DVL lectins possess strong activities against SARS-CoV-2, potentially by interacting with glycans and blocking virus entry into cells, representing potential candidates for the development of novel antiviral drugs.

Severe acute respiratory syndrome coronavirus 2 infection leads to Tau pathological signature in neurons.

COVID-19 has represented an issue for global health since its outbreak in March 2020. It is now evident that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in a wide range of long-term neurological symptoms and is worryingly associated with the aggravation of Alzheimer's disease. Little is known about the molecular basis of these manifestations. Here, several strain variants were used to infect SH-SY5Y neuroblastoma cells and K18-hACE C57BL/6J mice. The Tau phosphorylation profile and aggregation propensity upon infection were investigated on cellular extracts, subcellular fractions, and brain tissue. The viral proteins spike, nucleocapsid, and membrane were overexpressed in SH-SY5Y cells, and the direct interaction and effect on Tau phosphorylation were checked using immunoblot experiments. Upon infection, Tau is phosphorylated at several pathological epitopes associated with Alzheimer's disease and other tauopathies. Moreover, this event increases Tau's propensity to form insoluble aggregates and alters its subcellular localization. Our data support the hypothesis that SARS-CoV-2 infection in the central nervous system triggers downstream effects altering Tau function, eventually leading to the impairment of neuronal function.

Abstract P431: SARS-CoV-2-induced Inflammation In Human Endothelial Cells Involves ACE2

COVID-19 is associated with hypertension, where endotheliitis and endothelial dysfunction are risk factors. Considering the relationship between the viral spike protein and ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether contributes to vascular injury induced by SARS-CoV-2. Here we tested whether ACE2 acts as a receptor for SARS-CoV-2 pro-inflammatory responses, independently of ACE2 enzymatic function, in human endothelial cells (ECs). ECs and ACE2-expressing HEK cells were exposed to recombinant SARS-CoV-2 spike protein [(rS1p) 0.66 μg/mL] for 24h. Gene expression was assessed by RT-PCR and cytokine release was assessed by ELISA. Protein expression was assessed by immunoblotting. ACE2 was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Label-free quantitation of full proteome of ECs after rS1p exposure or ACE2 co-immunoprecipitation was performed. Co-localization studies were performed by confocal microscopy. Proteomics identified 1150 proteins associated with NFκB signalling and inflammation. 21 proteins were found to be differentially expressed vs vehicle-treated cells (fold change >2, p<0.05), including upregulation of ICAM-1. rS1p increased levels of IL6 (1221.2±18.3 vs. C:22.77±3.2 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU); PAI1 (5.6±0.7 vs. C: 2.9±0.2) and NFκB activation (0.13±0.004 vs. C:0.06±0.01 AU); p<0.05. MLN4760 and ACE2 siRNA blocked rS1P effects. rS1p did not alter ACE2 enzymatic activity. HEK-ACE2 cells had increased IL-8 (78.7±8 vs. C:56.2±4 pg/mL), while rS1p induced IL-8 production only in HEK expressing ACE2 (ACE2:112.8±5.3 vs. C:60.3±3.3 pg/mL); p<0.05. ACE2 CO-IP proteomics identified 216 proteins (filtered with ≥1 unique peptide, 1% FDR) along with ACE-2 (11 peptides). Potential candidate interacting proteins include caveolin 1 (Cav 1). Co-localization studies confirmed interactions between ACE2/Cav-1 in ECs, which was lost after rS1p stimulation. SARS-CoV2 spike protein induces pro-inflammatory responses in ECs via receptor-like function of ACE2. These molecular processes may contribute to vascular damage after COVID-19.

Frequency of SARS-CoV-2 Antibodies in PCR-Positive COVID-19 Patients and High-Risk Exposed Subjects at Multan


 
 Objective: To investigate the degree of association between Reverse Transcription Polymerase Chain Reaction positivity and seroconversion after natural COVID-19 infection in Multan, City of Pakistan.
 Study Design: Cross-sectional study.
 Place and Duration of Study: Combined Military Hospital, Multan Pakistan from Apr 2021 to Sep 2021.
 Methodology: In this study, 219 Healthcare Workers with suspected SARS-CoV-2 infection were screened via Reverse Transcription Polymerase Chain Reaction for viral genome, followed by detection of corresponding antibody response in serum samples within ten weeks of their first exposure against spike protein via Chemiluminescence immunoassay. 
 Results: There was a significant association between positive RT-PCR and detectable corresponding antibodies (p=0.001). However, we found no evidence of an association between age and RT-PCR positivity and between age and detectable antibodies (p=0.874 and 0.842, respectively). Furthermore, results indicated no association between gender and RT-PCR positivity and between gender and detectable antibodies (p=0.536 and 0.285, respectively).
 Conclusions: It is concluded that antibody detection against SARS-CoV-2 virus spike protein is a useful laboratory tool for screening for COVID-19 infection.Keywords: Antibody test, Asymptomatic cases, COVID-19, Healthcare workers, PCR, SARS-CoV-2.
 

The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein.

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 μg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Endothelial Glycocalyx Injury in SARS-CoV-2 Infection: Molecular Mechanisms and Potential Targeted Therapy.

This review aims at summarizing state-of-the-art knowledge on glycocalyx and SARS-CoV-2. The endothelial glycocalyx is a dynamic grid overlying the surface of the endothelial cell (EC) lumen and consists of membrane-bound proteoglycans and glycoproteins. The role of glycocalyx has been determined in the regulation of EC permeability, adhesion, and coagulation. SARS-CoV-2 is an enveloped, single-stranded RNA virus belonging to β-coronavirus that causes the outbreak and the pandemic of COVID-19. Through the respiratory tract, SARS-CoV-2 enters blood circulation and interacts with ECs possessing angiotensin-converting enzyme 2 (ACE2). Intact glycolyx prevents SARS-CoV-2 invasion of ECs. When the glycocalyx is incomplete, virus spike protein of SARS-CoV-2 binds with ACE2 and enters ECs for replication. In addition, cytokine storm targets glycocalyx, leading to subsequent coagulation disorder. Therefore, it is intriguing to develop a novel treatment for SARS-CoV-2 infection through the maintenance of the integrity of glycocalyx. This review aims to summarize state-of-the-art knowledge of glycocalyx and its potential function in SARS-CoV-2 infection.

Venom Peptides of Crotalus atrox Against SARS-Cov-2 Spike Protein and Human ACE2 Receptor by Molecular Docking Analysis

Venoms are composed of about 100 to 500 pharmacologically active compounds. Less than 0.01% of these compounds have been identified and a significant majority of them act on unknown receptors. Here, the potential Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) activities of selected Crotalus atrox venom peptides (CVPs) including Atrolysin D (AD), vascular apoptosis-inducing protein-1 (VAIP-1), Catrocollastatin (CC), and Calcium-Free Phospholipase A2 (CFP) were investigated via molecular docking analysis. CVPs were docked against human angiotensin-converting enzyme-2 (ACE-2) and 3-chymotrypsin-like protease (3CLpro) viral spike protein. All CVPs had low binding energies to both 3CLpro and ACE2, suggesting that they interacted strongly with the active sites of enzymes, compared to the reference drugs lopinavir and ritonavir. The binding energy of 3CLpro was -139.517 kcal/mol, -96.239 kcal/mol, -121.590 kcal/mol, -259.424 kcal/mol with AD, VAIP-1, CC, and CFP, respectively. CFP showed a very strong binding activity with 3CLpro, suggesting that it could be a very effective compound in inhibiting the SARS-CoV-2 virus. The binding energy of ACE2 was -101.165 kcal/mol, -73.064 kcal/mol, -106.918 kcal/mol, -82.830 kcal/mol with AD, VAIP-1, CC, and CFP, respectively. AD made a much stronger bond with ACE2 than reference drugs, showing that it could be used as a virus-protective component in humans. The results suggest a potential drug candidate for the development of therapeutics against Coronavirus disease 2019 (COVID-19). In vitro and in vivo experiments are needed to confirm these compounds' potential preventive and therapeutic effects.

The Possible Mechanisms of Cu and Zn in the Treatment and Prevention of HIV and COVID-19 Viral Infection.

Due to their unique properties and their potential therapeutic and prophylactic applications, heavy metals have attracted the interest of many researchers, especially during the outbreak of COVID-19. Indeed, zinc (Zn) and copper (Cu) have been widely used during viral infections. Zn has been reported to prevent excessive inflammatory response and cytokine storm, improve the response of the virus to Type I interferon (IFN-1), and enhance the production of IFN-a to counteract the antagonistic effect of SARS-CoV-2 virus protein on IFN. Additionally, Zn has been found to promote the proliferation and differentiation of T and B lymphocytes, thereby improving immune function, inhibiting RNA-dependent RNA polymerase (RdRp) in SARS- CoV-2 reducing the viral replication and stabilizing the cell membrane by preventing the proteolytic processing of viral polyprotein and proteases enzymes. Interestingly, Zn deficiency has been correlated with enhanced SARS-CoV-2 viral entry through interaction between the ACE2 receptor and viral spike protein. Along with zinc, Cu possesses strong virucidal capabilities and is known to be effective at neutralizing a variety of infectious viruses, including the poliovirus, influenza virus, HIV type 1, and other enveloped or nonenveloped, single- or double-stranded DNA and RNA viruses. Cu-related antiviral action has been linked to different pathways. First, it may result in permanent damage to the viral membrane, envelopes, and genetic material of viruses. Second, Cu produces reactive oxygen species to take advantage of the redox signaling mechanism to eradicate the virus. The present review focused on Zn and Cu in the treatment and prevention of viral infection. Moreover, the application of metals such as Cu and gold in nanotechnology for the development of antiviral therapies and vaccines has been also discussed.

Peptide foldamer-based inhibitors of the SARS-CoV-2 S protein–human ACE2 interaction

The entry of the SARS-CoV-2 virus into a human host cell begins with the interaction between the viral spike protein (S protein) and human angiotensin-converting enzyme 2 (hACE2). Therefore, a possible strategy for the treatment of this infection is based on inhibiting the interaction of the two abovementioned proteins. Compounds that bind to the SARS-CoV-2 S protein at the interface with the alpha-1/alpha-2 helices of ACE2 PD Subdomain I are of particular interest. We present a stepwise optimisation of helical peptide foldamers containing trans-2-aminocylopentanecarboxylic acid residues as the folding-inducing unit. Four rounds of optimisation led to the discovery of an 18-amino-acid peptide with high affinity for the SARS-CoV-2 S protein (Kd = 650 nM) that inhibits this protein–protein interaction with IC50 = 1.3 µM. Circular dichroism and nuclear magnetic resonance studies indicated the helical conformation of this peptide in solution.

Electrochemical capacitance spectroscopy based determination of antibodies against SARS-CoV-2 virus spike protein

In this study, we are reporting a novel electrochemical capacitance spectroscopy (ECS) platform designed for the sensitive and label-free detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus spike protein (anti-rS) in diluted blood serum. The determination of anti-rS is crucial for identification individuals who have been infected by SARS-CoV-2 virus and may have acquired immunity. The rS protein was immobilized on a screen-printed carbon electrode, which was incubated in diluted blood serum containing anti-rS antibodies. Label-free ECS was applied for the determination of interaction between immobilized rS and free-standing anti-rS. Here reported bioanalytical platform demonstrated high sensitivity and specificity in detecting anti-rS, achieving a limit of detection of 4.38 nM. This versatile platform could be further enhanced by applying various electrode materials and adapting this platform to detect antibodies against some other proteins. Our findings have significant implications for the development of affordable, scalable biosensing platforms capable to provide rapid and accurate public health screening and monitoring, particularly in the context of the coronavirus disease 2019 (COVID-19) pandemic.

Inhibitory Potential of Chitosan Derivatives against Severe Acute Respiratory Syndrome Coronavirus 2: An In Silico Prospective

The present work was designed to investigate the antiviral potential of novel monomeric and oligomeric chitosan derivatives through in silico approaches. The goal was to identify potent broad-spectrum antiviral compounds as promising drug candidates against severe acute respiratory syndrome coronavirus 2 and understand their mode of action. Chitosan biopolymer and its derivatives were virtually screened against the spike glycoprotein and human angiotensin-converting enzyme 2 (ACE2) receptor of novel coronavirus-19. Hydroxypropyl trimethyl ammonium chloride chitosan (HTCC), a polymeric chitosan, has been reported to interact with the corona viral spike (S) protein and blocks its interaction with the ACE2 receptor. The enhancement of antiviral activity relies on better biocompatibility, structural correlations, variation in the degree of deacetylation, and molecular weight of modified chitosan derivatives. The chitosan derivatives constructively interact with viral S protein. Among the chitosan derivatives, N-carboxymethyl chitosan (NCMC) displayed efficient binding affinity. Comparing NCMC to mHTCC, monomeric chitosan, for their interaction with the S protein, receptor binding domain site, and ACE2 receptor, NCMC displayed better binding affinity of −7.9, −6.3, and −7.4 with binding energies of −6.2, −4.8, and −5.5 kcal/mol, respectively. Furthermore, through flexible docking, the interactions of the S protein with ACE2 receptor and ligand mHTCC-S protein complex and NCMC-S protein complex with ACE2 receptor were calculated, showing an efficient reduction of binding energy from −901.2 kJ/mol to −765.06 kJ/mol and −814.72 kJ/mol, respectively. This points to the decrease binding affinity of the viral S protein for the ACE2 receptor in the presence of NCMC/mHTCC. For the first time, the computational study envisages the antiviral efficiency of NCMC, mHTCC, and biocompatible chitosan derivatives as a preventive intervention against COVID-19.

Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides.

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.

Molecular Docking-based Screening of Natural Heterocyclic Compounds as a Potential Drug for COVID-19

Introduction: The coronavirus pandemic poses significant challenges for the pharmaceutical industry. Methods: Coronavirus enters host cells via the angiotensin-converting enzyme 2 receptors (ACE2). The SARS-CoV-2 spike glycoprotein is a potential target for medicinal chemists in the development of specific drugs. The current study investigates molecular modeling studies to identify potential drug candidates. Molecular docking simulations were run on 11 natural heterocyclic compounds/flavonoids. Results: When tested against the viral spike protein receptor, isoquercetin had a docking binding energy of -6.74kcal/mol (PDBID:6LU7). Conclusion: A docking study revealed the interaction of the receptor-binding domain with various flavonoid compounds.

Impact of mAb-induced A475V substitution on viral fitness and antibody neutralization of SARS-CoV-2 omicron variants in the presence of monoclonal antibodies and human convalescent sera.

The emergence and rapid evolution of SARS-CoV-2 variants have posed a major challenge to the global efforts to control the COVID -19 pandemic. In this study, we investigated the potential of two SARS-CoV-2 variants, BA.2 and BA.5, to evade neutralization by a human monoclonal antibody targeting the virus's spike RBD (mAb 1D1). By subjecting the viruses to serial propagation in the presence of the antibody, we found that BA.2 exhibited poor growth, whereas BA.5 regained robust growth with significantly higher kinetics than the parental virus. Genetic analysis identified a single mutation, A475V, in the spike protein of BA.5 that substantially reduced the neutralizing activities of monoclonal antibodies and convalescent sera. In addition, the A475V mutation alone in BA.2 moderately reduced the neutralizing activity but completely abolished the neutralizing effect of mAb 1D1 when F486V or L452R were also present. Our results shed light on the possible evolutionary development of SARS-CoV-2 variants under selection pressure by monoclonal antibodies and have implications for the development of effective antibody therapies and vaccines against the virus.

Evaluation of DPP4/CD26 Potential Role for the Management of Inflammation in COVID-19 Patients

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is re-sponsible for Coronavirus Disease 2019 (COVID-19) pandemic. Cell entry of the virus can be me-diated by several enzymes, such as ACE-2, DPP4/CD26, and TMPRSS2, upon viral membrane fu-sion through SARS-CoV-2 envelope spike glycoproteins. In docked molecular complexes, DPP4/CD26 functional receptors and viral spike proteins have a large interface, potentially leading to inflammation in severe COVID-19. Objective: The aim of this study is to review the role of DPP4/CD26 in the immune system and its effects on the production of cytokine storms in COVID-19. Furthermore, we hypothesize that tar-geting DPP4/CD26 as a therapeutic strategy could reduce the inflammatory complications of SARS-CoV-2 infection. Methods: The current review was conducted using keywords such as COVID-19, SARS-CoV-2, dipeptidyl peptidase-4, CD26, cytokine storm, and treatment to search for articles in Google Scholar and PubMed databases that were specifically oriented towards our objectives. Results: The regulation or inhibition of DPP4/CD26 might affect one or more stages in COVID-19 immuno- pathogenesis due to its associations with many immunological functions, such as modulat-ing the NF-kB pathway, upregulating CD86 expression, activating proliferation of T cells, and in-fluencing the antiviral response and cytokine storm in COVID-19. In this regard, the applications of DPP4/CD26 inhibitors, DPP4/CD26 siRNAs, and CD26 antibodies have been demonstrated to pre-vent cytokine storms and airway inflammation. Conclusion: It is suggested to utilize novel technologies such as CRISPR/Cas and chimeric antigen receptor T cells, based on their many advantages, to increase the sensitivity and specificity of future treatment methods.