Search

• This review includes the most relevant methodologies for assessing both immune response and demographic implication against SARS-CoV-2 virus. • Cellular response can be measured mainly by flow cytometry, ELISpot and cytokine-detection methods. • Semi-quantitative techniques (like ELISA) are the most common and reliable methodologies to study humoral response. • Epidemiological studies are relevant for estimating SARS-CoV-2 infectivity and response to the general population. • Combination of different techniques enable to achieve more extensive information in SARS-CoV-2 response studies. The SARS-CoV-2 pandemic has a huge impact on public health and global economy, meaning an enormous scientific, political, and social challenge. Studying how infection or vaccination triggers both cellular and humoral responses is essential to know the grade and length of protection generated in the population. Nowadays, scientists and authorities around the world are increasingly concerned about the arrival of new variants, which have a greater spread, due to the high mutation rate of this virus. The aim of this review is to summarize the different techniques available for the study of the immune responses after exposure or vaccination against SARS-CoV-2, showing their advantages and limitations, and proposing suitable combinations of different techniques to achieve extensive information in these studies. We wish that the information provided here will helps other scientists in their studies of the immune response against SARS-CoV-2 after vaccination with new vaccine candidates or infection with upcoming variants. [ABSTRACT FROM AUTHOR]

The many carbohydrate chains on Covid-19 coronavirus SARS-CoV-2 and its S-protein form a glycan-shield that masks antigenic peptides and decreases uptake of inactivated virus or S-protein vaccines by APC. Studies on inactivated influenza virus and recombinant gp120 of HIV vaccines indicate that glycoengineering of glycan-shields to present α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) enables harnessing of the natural anti-Gal antibody for amplifying vaccine efficacy, as evaluated in mice producing anti-Gal. The α-gal epitope is the ligand for the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. Upon administration of vaccines presenting α-gal epitopes, anti-Gal binds to these epitopes at the vaccination site and forms immune complexes with the vaccines. These immune complexes are targeted for extensive uptake by APC as a result of binding of the Fc portion of immunocomplexed anti-Gal to Fc receptors on APC. This anti-Gal mediated effective uptake of vaccines by APC results in 10–200-fold higher anti-viral immune response and in 8-fold higher survival rate following challenge with a lethal dose of live influenza virus, than same vaccines lacking α-gal epitopes. It is suggested that glycoengineering of carbohydrate chains on the glycan-shield of inactivated SARS-CoV-2 or on S-protein vaccines, for presenting α-gal epitopes, will have similar amplifying effects on vaccine efficacy. α-Gal epitope synthesis on coronavirus vaccines can be achieved with recombinant α1,3galactosyltransferase, replication of the virus in cells with high α1,3galactosyltransferase activity as a result of stable transfection of cells with several copies of the α1,3galactosyltransferase gene (GGTA1), or by transduction of host cells with replication defective adenovirus containing this gene. In addition, recombinant S-protein presenting multiple α-gal epitopes on the glycan-shield may be produced in glycoengineered yeast or bacteria expression systems containing the corresponding glycosyltransferases. Prospective Covid-19 vaccines presenting α-gal epitopes may provide better protection than vaccines lacking this epitope because of increased uptake by APC. [ABSTRACT FROM AUTHOR]

Abstract: Currently, there is an increasing interest in the use of probiotics as an alternative strategy to antimicrobial compounds. In this study, two high adhesive strains Lactobacillus crispatus K313 adhering to HT-29 cells as well as Lb. crispatus K243 adhering to collagen type IV were isolated from chicken intestines. SDS-PAGE analysis revealed the presence of the potential S-proteins SlpA and SlpB in Lb. crispatus K243 and K313. SlpA and SlpB, rich in hydrophobic amino acids, were proved to be involved in adhering to collagen type IV and HT-29 cells, respectively, based on the LiCl treatment assay. After removal of S-proteins, the viability and tolerance of the two Lb. crispatus strains to simulated gastric and small intestinal juice were reduced, indicating the protective role of S-proteins against the hostile environments. Lb. crispatus K313 exhibited the stronger autoaggregation ability and inhibitive activity against Salmonella braenderup H9812 adhesion to HT-29 cells than the strain K243. To elucidate the inhibitive mechanism, cultured epithelial cells were exposed with Lb. crispatus strains, and followed by a challenge with S. braenderup H9812. The pro-inflammatory signaling factors (IL-8, CXCL1 and CCL20) from HT-29 were detected by real-time PCR technology. The results showed that both of Lb. crispatus strains down-regulated the transcription level of those pro-inflammatory genes induced by S. braenderup H9812 by 36.2–58.8%. ELISA analysis was further confirmed that Lb. crispatus K243 and K313 inhibited the IL-8 secretion triggered by S. braenderup H9812 by 32.8% and 47.0%, indicating that the two isolates could attenuate the pro-inflammatory signaling induced by S. braenderup H9812, and have the potential application in clinical practice to prevent diarrhea. [Copyright &y& Elsevier]

The increasing number of deaths due to the COVID-19 pandemic has raised serious global concerns. Increased testing capacity and ample intensive care availability could explain lower mortality in some countries compared to others. Nevertheless, it is also plausible that the SARS-CoV-2 mutations giving rise to different phylogenetic clades are responsible for the apparent death rate disparities around the world. Current research literature linking the genetic make-up of SARS-CoV-2 with fatalities is lacking. Here, we suggest that this disparity in fatality rates may be attributed to SARS-CoV-2 evolving mutations and urge the international community to begin addressing the phylogenetic clade classification of SARS-CoV-2 in relation to clinical outcomes. [ABSTRACT FROM AUTHOR]

Rapid, mass diagnosis of the coronavirus disease 2019 (COVID-19) is critical to stop the ongoing infection spread. The two standard screening methods to confirm the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are polymerase chain reaction (PCR), through the RNA of the virus, and serology by detecting antibodies produced as a response to the viral infection. However, given the detection complexity, cost and relatively long analysis times of these techniques, novel technologies are urgently needed. Here, we report an aptamer-based biosensor developed on a screen-printed carbon electrode platform for rapid, sensitive, and user-friendly detection of SARS-CoV-2. The aptasensor relies on an aptamer targeting the receptor-binding domain (RBD) in the spike protein (S-protein) of the SARS-CoV-2. The aptamer immobilization on gold nanoparticles, and the presence of S-protein in the aptamer-target complex, investigated for the first time by photo-induced force microscopy mapping between 770 and 1910 cm−1 of the electromagnetic spectrum, revealed abundant S-protein homogeneously distributed on the sensing probe. The detection of SARS-CoV-2 S-protein was achieved by electrochemical impedance spectroscopy after 40 min incubation with several analyte concentrations, yielding a limit of detection of 1.30 pM (66 pg/mL). Moreover, the aptasensor was successfully applied for the detection of a SARS-CoV-2 pseudovirus, thus suggesting it is a promising tool for the diagnosis of COVID-19. • Aptamer targeting the SARS-CoV-2 S-protein was immobilized on Au nanoparticles. • Aptamer-target complex on the probe was studied by photo-induce force microscopy. • PiFM revealed abundant S-protein present on the sensing probe. • LOD of aptasensor is lower than LOD of ELISA for SARS-CoV-2 S-protein. • The aptasensor can detect a SARS-CoV-2 pseudovirus. [ABSTRACT FROM AUTHOR]

The present study attempts to decipher the site-specific amino acid alterations at certain positions experiencing preferential selectivity and their effect on proteins' stability and flexibility. The study examines the selection preferences by considering pair-wise non-bonded interaction energies of adjacent and interacting amino acids present at the interacting site, along with their evolutionary history. For the study, variations in the interacting residues of spike protein (S-Protein) receptor-binding domain (RBD) of different coronaviruses were examined. The MD simulation trajectory analysis revealed that, though all the variants studied were structurally stable at their native and bound confirmations, the RBD of 2019-nCoV/SARS-CoV-2 was found to be more flexible and more dynamic. Furthermore, a noticeable change observed in the non-bonded interaction energies of the amino acids interacting with the receptor corroborated their selection at respective positions. The conformational changes exerted by the altered amino acids could be the reason for a broader range of interacting receptors among the selected proteins. The results envisage a strong indication that the residue selection at certain positions is governed by a well-orchestrated feedback mechanism, which follows increased stability and flexibility in the folded structure compared to its evolutionary predecessor. [Display omitted] [ABSTRACT FROM AUTHOR]

The D614G variant of SARS-CoV-2 S-protein emerged in early 2020 and quickly became the dominant circulating strain in Europe and its environs. The variant was characterized by the higher viral load, which is not associated with disease severity, higher incorporation into the virion, and high cell entry via ACE-2 and TMPRSS2. Previous strains of the coronavirus and the current SARS-CoV-2 have demonstrated the selection of mutations as a mechanism of escaping immune responses. In this study, we used molecular dynamics simulation and MM-PBSA binding energy analysis to provide insights into the behaviour of the D614G S-protein at the molecular level and describe the neutralization mechanism of this variant. Our results show that the D614G S-protein adopts distinct conformational dynamics which is skewed towards the open-state conformation more than the closed-state conformation of the wild-type S-protein. Residue-specific variation of amino acid flexibility and domain-specific RMSD suggest that the mutation causes an allosteric conformational change in the RBD. Evaluation of the interaction energies between the S-protein and neutralizing antibodies show that the mutation may enhance, reduce or not affect the neutralizing interactions depending on the neutralizing antibody, especially if it targets the RBD. The results of this study have shed insights into the behaviour of the D614G S-protein at the molecular level and provided a glimpse of the neutralization mechanism of this variant. • The D614G S-protein adopts distinct conformation dynamics compared to the wild-type. • Structural dynamics of the D614G S-protein is skewed towards the open-state conformation of wild-type S-protein. • The mutation induces an allosteric conformation change in the receptor-binding domain of the S-protein. • The potency of neutralizing the D614G variant may depend on the type of neutralizing antibody. [ABSTRACT FROM AUTHOR]

In recent years, a total of seven human pathogenic coronaviruses (HCoVs) strains were identified, i.e., SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1. Here, we performed an analysis of the protease recognition sites and antigenic variation of the S-protein of these HCoVs. We showed tissue-specific expression pattern, functions, and a number of recognition sites of proteases in S-proteins from seven strains of HCoVs. In the case of SARS-CoV-2, we found two new protease recognition sites, each of calpain-2, pepsin-A, and caspase-8, and one new protease recognition site each of caspase-6, caspase-3, and furin. Our antigenic mapping study of the S-protein of these HCoVs showed that the SARS-CoV-2 virus strain has the most potent antigenic epitopes (highest antigenicity score with maximum numbers of epitope regions). Additionally, the other six strains of HCoVs show common antigenic epitopes (both B-cell and T-cell), with low antigenicity scores compared to SARS-CoV-2. We suggest that the molecular evolution of structural proteins of human CoV can be classified, such as (i) HCoV-NL63 and HCoV-229E, (ii) SARS-CoV-2, and SARS-CoV and (iii) HCoV-OC43 and HCoV-HKU1. In conclusion, we can presume that our study might help to prepare the interventions for the possible HCoVs outbreaks in the future. • Analysis of the protease recognition sites and antigenic variation of S-protein of seven HCoVs. • Tissue specific- expression pattern analysis of the proteases of seven HCoVs. • Grouping of seven HCoVs strains through molecular phylogenetics using of S, M, E, N proteins. • Immunogenicity and conservancy analysis of common B-cell and T-cell epitopes of seven HCoVs. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel member of the betacoronaviruses family affecting the lower respiratory tract mainly through binding to angiotensin converting enzyme 2 (ACE2) via its S-protein. Genetic analysis of (ACE2) gene revealed several variants that have been suggested to regulate the interaction with S protein. This study investigates the N720D variant, positioned in the collectrin-like domain (CLD) at proximity to type II transmembrane serine protease (TMPRSS2) cleavage site. The effect of N720D variant on ACE2 structure and thermodynamic stability was studied by DynaMut. HDOCK was utilised to model TMPRSS2 protease binding to ACE2 WT and D720 variant cleavage site. PRODIGY was used to calculate binding affinities and MD simulation tools calculated the at 100 ns for ACE2 apo structure and the ACE2-TMPRSS2 complex. The N720D variant is a more dynamic structure with a free energy change (ΔΔG): −0.470 kcal/mol. As such, introducing a tighter binding affinity of K d = 3.2 × 10−10 M between TMPRSS2 and N720D variant. RMSD, RMSF calculations showed the N720D variant is less stable, however, RMSF values of the D720-TMPRSS2 complex reflected a slower dynamic motion. The hotspot N720D variant in the CLD of ACE2 affected the stability and flexibility of ACE2 by increasing the level of motion in the loop region, resulting in a more favourable site for TMPRSS2 binding and cleavage. Consequently, this would facilitate S-protein binding and can potentially increase viral entry highlighting the importance of variants affecting the ACE2-TMPRSS2 complex. [ABSTRACT FROM AUTHOR]

• Network pharmacology, protein docking and molecular docking computation was adopted. • Glu329/Gln325 and Gln42/Asp38 in ACE2 play an important role in the binding process. • Phillyrin and chlorogenic acid could block the combination of S-protein and ACE2. This article intends to use molecular docking technology to find potential inhibitors that can respond to COVID-19 from active compounds in Mongolian medicine. Mongolian medicine with anti-inflammatory and antiviral effects is selected from Mongolian medicine prescription preparations. TCMSP, ETCM database and document mining methods were used to collect active compounds. Swiss TargetPrediction and SuperPred server were used to find targets of compounds with smiles number. Drugbank and Genecard database were used to collect antiviral drug targets. Then the above targets were compared and analyzed to screen out antiviral targets of Mongolia medicine. Metascape database platform was used to enrich and analyze the GO (Gene ontology) annotation and KEGG pathway of the targets. In view of the high homology of gene sequences between SARS-CoV-2 S-protein RBD domain and SARS virus, as well as their similarities in pathogenesis and clinical manifestations, we established SARS-CoV-2 S-protein model using Swiss-Model. The ZDOCK protein docking software was applied to dock the S-protein with the human angiotensin ACE2 protein to find out the key amino acids of the binding site. Taking ACE2 as the receptor, the molecular docking between the active ingredients and the target protein was studied by AutoDock molecular docking software. The interaction between ligand and receptor is applied to provide a choice for screening anti-COVID-19 drugs. A total of 253 active components were predicted. Metascape analysis showed that key candidate targets were significantly enriched in multiple pathways related to different toxins. These key candidate targets were mainly derived from phillyrin and chlorogenic acid. Through the protein docking between S-protein and ACE2, it is found that Glu329/Gln325 and Gln42/Asp38 in ACE2 play an important role in the binding process of the two. The results of molecular docking virtual calculation showed that phillyrin and chlorogenic acid could stably combine with Gln325 and Gln42/Asp38 in ACE2, respectively, which hindered the combination between S- protein and ACE2. Phillyrin and chlorogenic acid can effectively prevent the combination of SARS-CoV-2 S-protein and ACE2 at the molecular level. Phillyrin and chlorogenic acid can be used as potential inhibitors of COVID-19 for further research and development. [ABSTRACT FROM AUTHOR]