Factors Affecting Confirmed COVID-19 Patient's Recovery Time at King Abdulaziz Medical City, Jeddah.

INTRODUCTION: The COVID-19 pandemic has become a threat to the public's health, especially to the elderly and those with chronic conditions. It is capable of spreading from carriers who are both asymptomatic and symptomatic. Associated factors such as age, sex, severe symptoms of COVID-19 disease, and chronic disease have a significant impact on the recovery time of patients. AIM: The study aimed to determine associated factors on recovery time in COVID-19 patients hospitalized at King Abdulaziz Medical city. METHODS: A single-center retrospective study was utilized to recruit 1776 confirmed COVID-19 patients from 13 September to 24 October 2020 at King Abdulaziz Medical City (KAMC) in Jeddah. RESULTS: The patients were categorized into three age groups: below 5 years, 5 to 65 years, and above 65 years. The number of male patients in each group was 49, 764, and 73, and the number of female patients in each group was 54, 754, and 82, respectively. Impact recovery time on female patients was 11.75 days; with male patients was 10.95 days. Symptoms such as sore throat, diarrhea, and fever in female patients declined the recovery time. On the other hand, symptoms such as runny nose, diarrhea, fever, and headache in male patients declined the recovery time. DISCUSSION AND CONCLUSION: It was revealed that older aged COVID-19 patients, male sex, and some symptoms decline recovery time. The study findings show an independent predictor of particular symptoms and sign which delay the time of recovery in the COVID-19 patients enrolled in the study differently, for male and female patients. Thus, patients who are infected with COVID-19 should be monitored keenly to prevent a prolonged rate of recovery and should be eligible for priority management to enhance a good clinical outcome.

Containing the spread of COVID-19 virus facing to its high mutation rate: approach to intervention using a nonspecific way of blocking its entry into the cells.

Viruses have multiple mutation rates that are higher than any other member of the kingdom of life. This gives them the ability to evolve, even within the course of a single infection, and to evade multiple host defenses, thereby impacting pathogenesis. Additionally, there are also interplays between mutation and recombination and the high multiplicity of infection (MOI) that enhance viral adaptability and increase levels of recombination leading to complex and conflicting effects on genome selection, and the net results is difficult to predict. Recently, the outbreak of COVID-19 virus represents a pandemic threat that has been declared a public health emergency of international concern. Up to present, however, due to the high mutation rate of COVID-19 virus, there are no effective procedures to contain the spread of this virus across the globe. For such a purpose, there is then an urgent need to explore new approaches. As an opinion, the present approach emphasizes on (a) the use of a nonspecific way of blocking the entry of COVID-19 virus as well as its variants into the cells via a therapeutic biocompatible compound (ideally, "in a pill") targeting its spike (S) glycoprotein; and (b) the construction of expression vectors via the glycosyl-phosphatidylinositol, GPI, anchor for studying intermolecular interactions between the spike S of COVID-19 virus as well as its variants and the angiotensin-converting enzyme 2 (ACE2) of its host receptor for checking the efficacy of any therapeutic biocompatible compound of the nonspecific way of blocking. Such antiviral drug would be safer than the ACE1 and ACE2 inhibitors/angiotensin receptor blockers, and recombinant human ACE2 as well as nucleoside analogs or protease inhibitors used for fighting the spread of the virus inside the cells, and it would also be used as a universal one for any eventual future pandemic related to viruses, especially the RNA viruses with high mutation rates.

Sustained Antibody-Dependent NK Cell Functions in Mild COVID-19 Outpatients During Convalescence.

The coronavirus disease 2019 (COVID19) pandemic has left researchers scrambling to identify the humoral immune correlates of protection from COVID-19. To date, the antibody mediated correlates of virus neutralization have been extensively studied. However, the extent that non-neutralizing functions contribute to anti-viral responses are ill defined. In this study, we profiled the anti-spike antibody subtype/subclass responses, along with neutralization and antibody-dependent natural killer cell functions in 83 blood samples collected between 4 and 201 days post-symptoms onset from a cohort of COVID-19 outpatients. We observed heterogeneous humoral responses against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Overall, anti-spike profiles were characterized by a rapid rise of IgA and sustained IgG titers. In addition, strong antibody-mediated natural killer effector responses correlated with milder disease and being female. While higher neutralization profiles were observed in males along with increased severity. These results give an insight into the underlying function of antibodies beyond neutralization and suggest that antibody-mediated natural killer cell activity is a key function of the humoral response against the SARS-CoV-2 spike protein.

Functional Trimeric SARS-CoV-2 Envelope Protein Expressed in Stable CHO Cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. One of the three membrane-bound envelope proteins is the spike protein (S), the one responsible for docking to the cellular surface protein ACE2 enabling infection with SARS-CoV-2. Although the structure of the S-protein has distinct similarities to other viral envelope proteins, robust and straightforward protocols for recombinant expression and purification are not described in the literature. Therefore, most studies are done with truncated versions of the protein, like the receptor-binding domain. To learn more about the interaction of the virus with the ACE2 and other cell surface proteins, it is mandatory to provide recombinant spike protein in high structural quality and adequate quantity. Additional mutant variants will give new insights on virus assembly, infection mechanism, and therapeutic drug development. Here, we describe the development of a recombinant CHO cell line stably expressing the extracellular domain of a trimeric variant of the SARS CoV-2 spike protein and discuss significant parameters to be considered during the expression and purification process.

Stabilized coronavirus spike stem elicits a broadly protective antibody.

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive ß-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies.

Synthetic SARS-CoV-2 Spike-Based DNA Vaccine Elicits Robust and Long-Lasting Th1 Humoral and Cellular Immunity in Mice.

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. The spike (S) glycoprotein of severe acute respiratory syndrome-coronavirus (SARS-CoV-2) is a major immunogenic and protective protein and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2, denoted VIU-1005. The design was based on a codon-optimized coding sequence of a consensus full-length S glycoprotein. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models 4 weeks after three injections with 100 µg of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Such immunization induced long-lasting IgG and memory T cell responses in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower or fewer doses were able to elicit significantly high levels of Th1-biased systemic S-specific immune responses, as demonstrated by the significant levels of binding IgG antibodies, nAbs and IFN-γ, TNF and IL-2 cytokine production from memory CD8+ and CD4+ T cells in BALB/c mice. Furthermore, compared to intradermal needle injection, which failed to induce any significant immune response, intradermal needle-free immunization elicited a robust Th1-biased humoral response similar to that observed with intramuscular immunization. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting Th1-skewed humoral and cellular immunity in mice. Furthermore, we show that the use of a needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.

Computational Studies on T2Rs Agonist-Based Anti-COVID-19 Drug Design.

The expeditious and world pandemic viral disease of new coronavirus (SARS-CoV-2) has formed a prompt urgency to discover auspicious target-based ligand for the treatment of COVID-19. Symptoms of novel coronavirus disease (COVID-19) typically include dry cough, fever, and shortness of breath. Recent studies on many COVID-19 patients in Italy and the United Kingdom found increasing anosmia and ageusia among the COVID-19-infected patients. SARS-CoV-2 possibly infects neurons in the nasal passage and disrupts the senses of smell and taste, like other coronaviruses, such as SARS-CoV and MERS-CoV that could target the central nervous system. Developing a drug based on the T2Rs might be of better understanding and worth finding better molecules to act against COVID-19. In this research, we have taken a taste receptor agonist molecule to find a better core molecule that may act as the best resource to design a drug or corresponding derivatives. Based on the computational docking studies, the antibiotic tobramycin showed the best interaction against 6LU7 COVID-19 main protease. Aromatic carbonyl functional groups of the molecule established intermolecular hydrogen bonding interaction with GLN189 amino acid and it showed the two strongest carbonyl interactions with receptor protein resulting in a glide score of -11.159. To conclude, depending on the molecular recognition of the GPCR proteins, the agonist molecule can be recognized to represent the cell secondary mechanism; thus, it provides enough confidence to design a suitable molecule based on the tobramycin drug.

Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Based Novel Epitopes Induce Potent Immune Responses in vivo and Inhibit Viral Replication in vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiates infection by attachment of the surface-exposed spike glycoprotein to the host cell receptors. The spike glycoprotein (S) is a promising target for inducing immune responses and providing protection; thus the ongoing efforts for the SARS-CoV-2 vaccine and therapeutic developments are mostly spiraling around S glycoprotein. The matured functional spike glycoprotein is presented on the virion surface as trimers, which contain two subunits, such as S1 (virus attachment) and S2 (virus fusion). The S1 subunit harbors the N-terminal domain (NTD) and the receptor-binding domain (RBD). The RBD is responsible for binding to host-cellular receptor angiotensin-converting enzyme 2 (ACE2). The NTD and RBD of S1, and the S2 of S glycoprotein are the major structural moieties to design and develop spike-based vaccine candidates and therapeutics. Here, we have identified three novel epitopes (20-amino acid peptides) in the regions NTD, RBD, and S2 domains, respectively, by structural and immunoinformatic analysis. We have shown as a proof of principle in the murine model, the potential role of these novel epitopes in-inducing humoral and cellular immune responses. Further analysis has shown that RBD and S2 directed epitopes were able to efficiently inhibit the replication of SARS-CoV-2 wild-type virus in vitro suggesting their role as virus entry inhibitors. Structural analysis revealed that S2-epitope is a part of the heptad repeat 2 (HR2) domain which might have plausible inhibitory effects on virus fusion. Taken together, this study discovered novel epitopes that might have important implications in the development of potential SARS-CoV-2 spike-based vaccine and therapeutics.

An Overview of Spike Surface Glycoprotein in Severe Acute Respiratory Syndrome-Coronavirus.

The novel coronavirus originated in December 2019 in Hubei, China. This contagious disease named as COVID-19 resulted in a massive expansion within 6 months by spreading to more than 213 countries. Despite the availability of antiviral drugs for the treatment of various viral infections, it was concluded by the WHO that there is no medicine to treat novel CoV, SARS-CoV-2. It has been confirmed that SARS-COV-2 is the most highly virulent human coronavirus and occupies the third position following SARS and MERS with the highest mortality rate. The genetic assembly of SARS-CoV-2 is segmented into structural and non-structural proteins, of which two-thirds of the viral genome encodes non-structural proteins and the remaining genome encodes structural proteins. The most predominant structural proteins that make up SARS-CoV-2 include spike surface glycoproteins (S), membrane proteins (M), envelope proteins (E), and nucleocapsid proteins (N). This review will focus on one of the four major structural proteins in the CoV assembly, the spike, which is involved in host cell recognition and the fusion process. The monomer disintegrates into S1 and S2 subunits with the S1 domain necessitating binding of the virus to its host cell receptor and the S2 domain mediating the viral fusion. On viral infection by the host, the S protein is further cleaved by the protease enzyme to two major subdomains S1/S2. Spike is proven to be an interesting target for developing vaccines and in particular, the RBD-single chain dimer has shown initial success. The availability of small molecules and peptidic inhibitors for host cell receptors is briefly discussed. The development of new molecules and therapeutic druggable targets for SARS-CoV-2 is of global importance. Attacking the virus employing multiple targets and strategies is the best way to inhibit the virus. This article will appeal to researchers in understanding the structural and biological aspects of the S protein in the field of drug design and discovery.

Molecular Mechanisms Behind Anti SARS-CoV-2 Action of Lactoferrin.

Despite the huge effort to contain the infection, the novel SARS-CoV-2 coronavirus has rapidly become pandemic, mainly due to its extremely high human-to-human transmission capability, and a surprisingly high viral charge of symptom-less people. While the seek for a vaccine is still ongoing, promising results have been obtained with antiviral compounds. In particular, lactoferrin is regarded to have beneficial effects both in preventing and soothing the infection. Here, we explore the possible molecular mechanisms with which lactoferrin interferes with SARS-CoV-2 cell invasion, preventing attachment and/or entry of the virus. To this aim, we search for possible interactions lactoferrin may have with virus structural proteins and host receptors. Representing the molecular iso-electron surface of proteins in terms of 2D-Zernike descriptors, we 1) identified putative regions on the lactoferrin surface able to bind sialic acid present on the host cell membrane, sheltering the cell from the virus attachment; 2) showed that no significant shape complementarity is present between lactoferrin and the ACE2 receptor, while 3) two high complementarity regions are found on the N- and C-terminal domains of the SARS-CoV-2 spike protein, hinting at a possible competition between lactoferrin and ACE2 for the binding to the spike protein.

Repurposing drugs and identification of inhibitors of integral proteins (spike protein and main protease) of SARS-CoV-2.

The outbreak of Coronavirus infection (COVID-19) has prompted the World Health Organisation (WHO) to declare the outbreak, a Public Health Emergency of International concern. As part of the efforts to discover lead compounds for clinical use, 53 molecules were screened using molecular docking and dynamic simulations (MDS) techniques to identify potential inhibitors of SARS-CoV-2 spike protein (COVID-19 Sgp) and main protease (COVID-19 Mpro) or both. Lopinavir (LPV), nelfinavir (NEF), hydroxychloroquine (HCQ), remdesivir (RDV) and an irreversible inhibitor of SARS-CoV (N3) were used as standard drugs for COVID-19 Mpro, while zafirlukast (ZFK) and cefoperazone (CSP)) as standard drugs for COVID-19 Sgp. After 100 ns of MDS, with reference to standard drugs (N3, -52.463 Kcal/mol, NEF, -51.618 Kcal/mol, RDV, -48.780 Kcal/mol, LPV, -46.788 Kcal/mol, DRV, -33.655 Kcal/mol and HCQ, -21.065 Kcal/mol), five molecules, HCR, GRN, C3G, EGCG, and K7G were predicted to be promising inhibitors of COVID-19 Mpro with binding energies of -53.877 kcal/mol, -50.653 Kcal/mol, -48.600 kcal/mol, -47.798 kcal/mol and -46.902 kcal/mol, respectively. These lead molecules were then docked at receptor-binding domain (RBD) of COVID-19 Sgp to examine their inhibitory effects. C3G, GRN and K7G exhibited higher binding energies of -42.310 kcal/mol, -32.210 kcal/mol, -26.922 kcal/mol than the recorded values for the reference drugs (CSP, -35.509 kcal/mol, ZFK, -24.242 kcal/mol), respectively. The results of the binding energy and structural analyses from this study revealed that C3G, GRN and K7G could serve as potential dual inhibitors of COVID-19 Sgp and COVID-19 Mpro, while HCR and EGCG would be inhibitors of COVID-19 Mpro.Communicated by Ramaswamy H. Sarma.

In silico Screening of Natural Compounds as Potential Inhibitors of SARS-CoV-2 Main Protease and Spike RBD: Targets for COVID-19.

Historically, plants have been sought after as bio-factories for the production of diverse chemical compounds that offer a multitude of possibilities to cure diseases. To combat the current pandemic coronavirus disease 2019 (COVID-19), plant-based natural compounds are explored for their potential to inhibit the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the cause of COVID-19. The present study is aimed at the investigation of antiviral action of several groups of phytoconstituents against SARS-CoV-2 using a molecular docking approach to inhibit Main Protease (Mpro) (PDB code: 6LU7) and spike (S) glycoprotein receptor binding domain (RBD) to ACE2 (PDB code: 6M0J) of SARS-CoV-2. For binding affinity evaluation, the docking scores were calculated using the Extra Precision (XP) protocol of the Glide docking module of Maestro. CovDock was also used to investigate covalent docking. The OPLS3e force field was used in simulations. The docking score was calculated by preferring the conformation of the ligand that has the lowest binding free energy (best pose). The results are indicative of better potential of solanine, acetoside, and rutin, as Mpro and spike glycoprotein RBD dual inhibitors. Acetoside and curcumin were found to inhibit Mpro covalently. Curcumin also possessed all the physicochemical and pharmacokinetic parameters in the range. Thus, phytochemicals like solanine, acetoside, rutin, and curcumin hold potential to be developed as treatment options against COVID-19.

Exploring the efficacy of naturally occurring biflavone based antioxidants towards the inhibition of the SARS-CoV-2 spike glycoprotein mediated membrane fusion.

Molecular docking studies were done to show the inhibitory effect of two naturally occurring biflavone based anti-HIV agents, hinokiflavone and robustaflavone against the SARS-CoV-2 spike (S) protein mediated attack on the human ACE2 receptors via membrane fusion mechanism. Nefamostat, a FDA approved drug, well-known as a serine protease inhibitor for MERS-CoV infection, was used as the reference compound. Both the biflavones, showed potential as inhibitors for SARS-CoV-2 S protein-mediated viral entry. The binding affinities of these naturally occurring biflavones for RBD-S2 subunit protein of SARS-CoV-2 were explored for the first time. Such binding affinities play a critical role in the virus-cell membrane fusion process. These biflavones are able to interact more strongly with the residues of heptad repeat 1 and 2 (HR1 and HR2) regions of S2 protein of SARS-CoV-2 compared to nefamostat, and thus, these biflavones can effectively block the formation of six-helix bundle core fusion structure (6-HB) leading to the inhibition of virus-target cell-membrane fusion.