Molecular Dynamics Analysis of Fast-Spreading Severe Acute Respiratory Syndrome Coronavirus 2 Variants and Their Effects on the Interaction with Human Angiotensin-Converting Enzyme 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving with mutations in the spike protein, especially in the receptor-binding domain (RBD). The failure of public health measures in some countries to contain the spread of the disease has given rise to novel viral variants with increased transmissibility. However, key questions about how quickly the variants can spread remain unclear. Herein, we performed a structural investigation using molecular dynamics simulations and determined dissociation constant (K D) values using surface plasmon resonance assays of three fast-spreading SARS-CoV-2 variants, alpha, beta, and gamma, as well as genetic factors in host cells that may be related to the viral infection. Our results suggest that the SARS-CoV-2 variants facilitate their entry into the host cell by moderately increased binding affinities to the human ACE2 receptor, different torsions in hACE2 mediated by RBD variants, and an increased spike exposure time to proteolytic enzymes. We also found that other host cell aspects, such as gene and isoform expression of key genes for the infection (ACE2, FURIN, and TMPRSS2), may have few contributions to the SARS-CoV-2 variant infectivity. In summary, we concluded that a combination of viral and host cell factors allows SARS-CoV-2 variants to increase their abilities to spread faster than the wild type.

Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants.

Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.

Cross-neutralization of RBD mutant strains of SARS-CoV-2 by convalescent patient derived antibodies.

BACKGROUND: The emergence of COVID-19 pandemic resulted in an urgent need for the development of therapeutic interventions. Of which, neutralizing antibodies play a crucial role in the prevention and resolution of viral infection. METHODS: We generated antibody libraries from 18 different COVID-19 recovered patients and screened neutralizing antibodies to SARS-CoV-2 and its mutants. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Clones were then reconstituted into whole human IgG for epitope binning assay and all 19 IgG were classified into 6 different epitope groups or Bins. RESULTS: Although all antibodies were found to bind RBD, the antibodies in Bin2 had superior inhibitory ability of the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 showed stronger binding affinity or ability to mutant RBDs (N501Y, W463R, R408I, N354D, V367F, and N354D/D364Y) derived from different SARS-CoV-2 strains as well, suggesting the great potential of these antibodies in preventing infection of SARS-CoV-2 and its mutations. Furthermore, such neutralizing antibodies strongly restricted the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized wildtype and more transmissible mutant pseudovirus entry into hACE2 overexpressed 293T cells. In Vero-E6 cells, one of these antibodies can even block the entry of live SARS-CoV-2 into cells at 12.5 nM. CONCLUSIONS: These results indicate that the neutralizing human antibodies from the patient-derived antibody libraries have the potential to fight SARS-CoV-2 and its mutants in this global pandemic.

Chicken Egg Yolk Antibodies (IgYs) block the binding of multiple SARS-CoV-2 spike protein variants to human ACE2.

The SARS-CoV-2 virus is still spreading worldwide, and there is an urgent need to effectively prevent and control this pandemic. This study evaluated the potential efficacy of Egg Yolk Antibodies (IgY) as a neutralizing agent against the SARS-CoV-2. We investigated the neutralizing effect of anti-spike-S1 IgYs on the SARS-CoV-2 pseudovirus, as well as its inhibitory effect on the binding of the coronavirus spike protein mutants to human ACE2. Our results show that the anti-Spike-S1 IgYs showed significant neutralizing potency against SARS-CoV-2 pseudovirus, various spike protein mutants, and even SARS-CoV in vitro. It might be a feasible tool for the prevention and control of ongoing COVID-19.

Molecular Dynamics Reveals Complex Compensatory Effects of Ionic Strength on the Severe Acute Respiratory Syndrome Coronavirus 2 Spike/Human Angiotensin-Converting Enzyme 2 Interaction.

The SARS-CoV-2 pandemic has already killed more than one million people worldwide. To gain entry, the virus uses its Spike protein to bind to host hACE-2 receptors on the host cell surface and mediate fusion between viral and cell membranes. As initial steps leading to virus entry involve significant changes in protein conformation as well as in the electrostatic environment in the vicinity of the Spike/hACE-2 complex, we explored the sensitivity of the interaction to changes in ionic strength through computational simulations and surface plasmon resonance. We identified two regions in the receptor-binding domain (RBD), E1 and E2, which interact differently with hACE-2. At high salt concentration, E2-mediated interactions are weakened but are compensated by strengthening E1-mediated hydrophobic interactions. These results provide a detailed molecular understanding of Spike RBD/hACE-2 complex formation and stability under a wide range of ionic strengths.

Self-assemblies of 5'-cholesteryl-ethyl-phosphoryl zidovudine.

Anti-HIV prodrugs are recently focused on due to their ability of self-assembly, macrophage targeting, and enhanced antiviral effects. Here, an amphiphilic prodrug of zidovudine, an anti-HIV nucleoside analogue, 5'-cholesteryl-ethyl-phosphoryl zidovudine (CEPZ) was synthesized. CEPZ showed some unique physicochemical properties. The solubility of CEPZ in the noncompetitive solvents chloroform and tetrahydrofuran (THF) was very high based on the hydrogen bonds between zidovudine groups, though CEPZ was sparing soluble in alcohols and almost insoluble in water. The typical amphiphilic property of CEPZ was demonstrated according to the Langmuir monolayers at the air/water interface. The LogP of CEPZ was high to 13.78, indicating the high hydrophobicity of amphiphilic CEPZ similar to phospholipids. Homogenous and stable self-assemblies were formed with the mean size of 128.7nm and the zeta potential of -35.4mV after injecting the CEPZ-in-THF solution into water. Hydrophobic interaction between the cholesteryl moieties of CEPZ could drive molecular self-assembly and lead to the formation of spherical vesicles. CEPZ self-assemblies showed strong stability even under high temperature and gravity probably due to the high surface charge. CEPZ was very slowly degraded in neutral solutions (e.g., pH 7.4), but fast in acid solutions (e.g., pH 5.0) and some tissue homogenates. CEPZ was quickly eliminated from the circulation and distributed into the mononuclear phagocyte system (MPS) including the liver, spleen and lung after bolus intravenous administration of CEPZ self-assemblies to mice. The MPS targeting effect of CEPZ self-assemblies makes them become a promising self-assembled drug delivery system to eradicate the HIV hidden in the macrophages.

Magnetic resonance imaging of osteosarcoma using a bis(alendronate)-based bone-targeted contrast agent.

Magnetic resonance (MR) is currently used for diagnosis of osteosarcoma but not well even though contrast agents are administered. Here, we report a novel bone-targeted MR imaging contrast agent, Gd2-diethylenetriaminepentaacetate-bis(alendronate) (Gd2-DTPA-BA) for the diagnosis of osteosarcoma. It is the conjugate of a bone cell-seeking molecule (i.e., alendronate) and an MR imaging contrast agent (i.e., Gd-DTPA). Its physicochemical parameters were measured, including pKa, complex constant, and T1 relaxivity. Its bone cell-seeking ability was evaluated by measuring its adsorption on hydroxyapatite. Hemolysis was investigated. MR imaging and biodistribution of Gd2-DTPA-BA and Gd-DTPA were studied on healthy and osteosarcoma-bearing nude mice. Gd2-DTPA-BA showed high adsorption on hydroxyapatite, the high MR relaxivity (r1) of 7.613mM-1s-1 (2.6 folds of Gd-DTPA), and no hemolysis. The MR contrast effect of Gd2-DTPA-BA was much higher than that of Gd-DTPA after intravenous injection to the mice. More importantly, the MR imaging of osteosarcoma was significantly improved by Gd2-DTPA-BA. The signal intensity of Gd2-DTPA-BA reached 120.3% at 50min, equal to three folds of Gd-DTPA. The bone targeting index (bone/blood) of Gd2-DTPA-BA in the osteosarcoma-bearing mice was very high to 130 at 180min. Furthermore, the contrast enhancement could also be found in the lung due to metastasis of osteosarcoma. Gd2-DTPA-BA plays a promising role in the diagnoses of osteosacomas, including the primary bone tumors and metastases.

Nasal delivery of analgesic ketorolac tromethamine thermo- and ion-sensitive in situ hydrogels.

Ketorolac tromethamine (KT) was potent to treat moderate to moderately severe pains. However, KT solutions for nasal delivery lost quickly from the nasal route. Thermo- and ion-sensitive in-situ hydrogels (ISGs) are appropriate for nasal drug delivery because the intranasal temperature maintains ∼37 °C and nasal fluids consist of plentiful cations. In this study, a novel nasal thermo- and ion-sensitive ISG of KT was prepared with thermo-sensitive poloxamer 407 (P407) and ion-sensitive deacetylated gellan gum (DGG). The optimal formulation of the KT ISG consisted of 3% (w/v) DGG and 18% (w/v) P407 and its viscosity was up to 7.63 Pas at 37 °C. Furthermore, penetration enhancers and bacterial inhibitors were added and their fractions in the ISG were optimized based on transmucosal efficiencies and toxicity on toad pili. Sulfobutyl ether-ß-cyclodextrin of 2.5% (w/v) and chlorobutanol of 0.5% (w/v) were chosen as the penetration enhancer and the bacterial inhibitor, respectively. The Fick's diffusion and dissolution of KT could drive it continuous release from the dually sensitive ISG according to the in vitro investigation. Two methods, writhing frequencies induced by acetic acid and latency time of tails retracting from hot water, were used to evaluate the pharmacodynamics of the KT ISG on the mouse models. The writhing frequencies significantly decreased and the latency time of tail retracting was obviously prolonged (p<0.05) for the KT ISG compared to the control. The thermo- and ion-sensitive KT ISG had appropriate gelation temperature, sustained drug release, improved intranasal absorption, obvious pharmacodynamic effect, and negligible nasal ciliotoxicity. It is a promising intranasal analgesic formulation.