SARS-CoV-2 Omicron (B.1.1.529) shows minimal neurotropism in a double-humanized mouse model.

Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) initially infects the respiratory tract, it also directly or indirectly affects other organs, including the brain. However, little is known about the relative neurotropism of SARS-CoV-2 variants of concern (VOCs), including Omicron (B.1.1.529), which emerged in November 2021 and has remained the dominant pathogenic lineage since then. To address this gap, we examined the relative ability of Omicron, Beta (B.1.351), and Delta (B.1.617.2) to infect the brain in the context of a functional human immune system by using human angiotensin-converting enzyme 2 (hACE2) knock-in triple-immunodeficient NGC mice with or without reconstitution with human CD34+ stem cells. Intranasal inoculation of huCD34+-hACE2-NCG mice with Beta and Delta resulted in productive infection of the nasal cavity, lungs, and brain on day 3 post-infection, but Omicron was surprisingly unique in its failure to infect either the nasal tissue or brain. Moreover, the same infection pattern was observed in hACE2-NCG mice, indicating that antiviral immunity was not responsible for the lack of Omicron neurotropism. In independent experiments, we demonstrate that nasal inoculation with Beta or with D614G, an ancestral SARS-CoV-2 with undetectable replication in huCD34+-hACE2-NCG mice, resulted in a robust response by human innate immune cells, T cells, and B cells, confirming that exposure to SARS-CoV-2, even without detectable infection, is sufficient to induce an antiviral immune response. Collectively, these results suggest that modeling of the neurologic and immunologic sequelae of SARS-CoV-2 infection requires careful selection of the appropriate SARS-CoV-2 strain in the context of a specific mouse model.

Thermodynamic analysis of the interactions between human ACE2 and Spike RBD of Betacoronaviruses (SARS-CoV-1 and SARS-CoV-2).

There are many scientific reports on the interaction of the SARS-CoV-2 virus S protein (and its RBD) with the human ACE2 receptor protein. However, there are no reliable data on how this interaction differs from the interaction of the receptor binding domain of SARS-CoV-1 with ACE2, in terms of binding strength and changes in reaction enthalpy and entropy. Our studies have revealed these differences as well as the impact of zinc ions on this interaction. Intriguingly, the binding affinity of both RBDs (of SARS-CoV-1 and of SARS-CoV-2) to the ACE2 receptor protein is almost identical; however, there are some differences in the entropic and enthalpic contributions to these interactions.

The Development of Pharmacophore Models for the Search of New Natural Inhibitors of SARS-CoV-2 Spike RBD-ACE2 Binding Interface.

To date, some succeeding variants of SARS-CoV-2 have become more contagious. This virus is known to enter human cells by binding the receptor-binding domain (RBD) of spike protein with the angiotensin-converting enzyme 2 (ACE2), the latter being a membrane protein that regulates the renin-angiotensin system. Since the host cell receptor plays a critical role in viral entry, inhibition of the RBD-ACE2 complex is a promising strategy for preventing COVID-19 infection. In the present communication, we propose and utilize an approach based on the generation of a complex of pharmacophore models and subsequent Induced Fit Docking (IFD) to identify potential inhibitors of the main binding sites of the Omicron SARS-CoV-2 RBD(S1)-ACE2 complex (PDB ID: 7T9L) among a number of natural products of various types and origins. Several natural compounds have been found to provide a high affinity for the receptor of interest. It is expected that the present results will stimulate further research aimed at the development of specialized drugs against this virus.

Atomistic insights into the binding of SARS-CoV-2 spike receptor binding domain with the human ACE2 receptor: The importance of residue 493.

SARS-CoV-2 is a coronavirus that has created a global pandemic. The virus contains a spike protein which has been shown to bind to the ACE2 receptor on the surface of human cells. Vaccines have been developed that recognize elements of the SARS-CoV-2 spike protein and they have been successful in preventing infection. Recently, the Omicron variant of the SARS-CoV-2 virus was reported and quickly became a variant of concern due to its transmissibility. This variant contained an unusually large number (32) of point mutations, of which 15 of those mutations are in the receptor binding domain of the spike protein. While several computational and experimental investigations comparing the binding of the Omicron and wild type RBD to the human ACE2 receptor have been conducted, many of these report contradictory findings. In order to assess the differential binding ability, we conducted 2 µs of classical molecular dynamics (cMD) simulation to estimate the binding affinities and behaviors. Based upon MM-GBSA binding affinity, per-residue energy decomposition analysis, center of mass distance measurements, ensemble clustering, pairwise residue decomposition and hydrogen bonding analysis, our results suggest that a single point mutation is responsible for the enhanced binding of the Omicron mutant relative to the WT. While the 15-point mutations in the receptor binding domain contribute positively and negatively to the affinity of the spike protein for the human ACE2 receptor, it is the point mutation Q493R that confers enhanced binding while the Q493K mutation results in similar binding. The MM-GBSA binding estimations over a 2 µs trajectory, suggest that the wild type binds to ACE2 with a value of -29.69 kcal/mol while the Q493K and Q493R Omicron mutants bind with energy values of -26.67 and -34.56 kcal/mol, respectively. These values are significantly different, given the error estimates associated with the MM-GBSA method. In general, while some mutations increase binding, more mutations diminish binding, leading to an overall similar picture of binding for Q493K and enhanced binding for Q493R.

Human ACE2 Polymorphisms from Different Human Populations Modulate SARS-CoV-2 Infection.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 6 million deaths worldwide. The high variability in COVID-19 symptoms remains one of the most interesting mysteries of the pandemic. Genetic and environmental factors are likely to be key determinants of COVID-19 symptomatology. Here, we explored ACE2 as a genetic determinant for SARS-CoV-2 infection and COVID-19 symptomatology. Each human genome encodes two alleles of ACE2, which encodes the cell entry receptor for SARS-CoV-2. Here, we determined whether naturally occurring human ACE2 (hACE2) polymorphisms in the human population affect SARS-CoV-2 infection and the severity of COVID-19 symptoms. ACE2 variants S19P, I21V, E23K, K26R, K31R, N33I, H34R, E35K, and T92I showed increased virus infection compared to wild-type ACE2; thus, these variants could increase the risk for COVID-19. In contrast, variants D38V, Y83H, I468V, and N638S showed reduced infection, indicating a potential protective effect. hACE2 variants K26R and T92I increased infection by three-fold without changing the levels of ACE2 on the surface of the cells, suggesting that these variants may increase the risk of severe COVID-19. On the contrary, hACE2 variants D38V and Y83H decreased SARS-CoV-2 infection by four- and ten-fold, respectively, without changing surface expression, suggesting that these variants may protect against severe COVID-19. Remarkably, all protective hACE2 Polymorphisms were found almost exclusively in Asian populations, which may provide a partial explanation for the low COVID-19 mortality rates in Asian countries. Thus, hACE2 polymorphisms may modulate susceptibility to SARS-CoV-2 in the host and partially account for the differences in severity of COVID-19 among different ethnic groups.

Comparative Structural Analysis of Human ACE2 Receptor with Spike Protein of SARS-CoV-2 Variants: Implications to Understand Infectivity of the Virus.

Purpose: Spike protein on SARS-CoV-2 virus plays an integral part during infection as cell entry depends on binding of this protein to human ACE2 receptor. Understanding of infectivity by these variants necessitates a comparative structural analysis of complexes of spike protein-receptor binding domain (RBD) of these variants to receptor. Methodology: Wild type SARS-CoV-2 spike protein sequence was retrieved from the UniProt database, and mutations of five variants at receptor binding domain were manually incorporated and aligned using Clustal Omega. Crystal structure complexes of human ACE2 receptor with spike protein RBD domain of SARS-CoV-2 variants of wild type, α, ß, and δ were extracted from the RCSB database. Wild type SARS-CoV-2 complex with receptor was used as template to generate model complexes of receptor with spike protein RBD of γ and omicron variants through WinCoot program. These were energy minimized and validated and molecular dynamic simulation was performed using Desmond simulation program. Results: Mutations are distributed across the entire length of RBD, but the maximum number of mutations are seen at 11 positions within binding interface motifs of six variant sequences. Interface of spike protein RBDs with human ACE2-receptor shows different mix of hydrogen bonded and ionic interactions. Alpha and ß variants have few interactions, while γ and δ variants have higher number of interactions compared to wild type variant. Omicron variant, with 10 polar interactions including two ionic bonds, has the highest binding energy. Conclusion: Different mutations on RBD of spike protein results in varying quantity and quality of interactions, thereby affecting potency of each variant. Variations in binding are due to interactions of mutant residues and induced conformational changes on loops of RBDs. Variants α and ß have a low potency, while, γ, δ, and omicron have a higher potency. These results correlate with viral infectivity and place clinical observations in the right perspective.

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.

Molecular Aspects of Spike–ACE2 Interaction

DefinitionA new betacoronavirus (CoV-2) is responsible for the pandemic of severe acute respiratory syndrome (SARS) that began in China at the end of 2019, today known as COronaVIrus Disease 2019 (COVID-19). Subsequent studies confirmed the human angiotensin-converting enzyme 2 (hACE2) as the main cell receptor of spike trimeric glycoprotein, located on the viral envelope, mediating the CoV-2 invasion into the host cells through the receptor-binding domain (RBD) of the spike. Computational analysis of the known experimental 3D structures of spike–ACE2 complexes evidenced distinguishing features in the molecular interactions at the RBD-cell receptor binding interface between CoV-2 and previous CoV-1. The spike represents a key target for drug design as well as an optimal antigen for RNA/viral vector vaccines and monoclonal antibodies in order to maximize prevention and therapy of COVID-19.

An engineered nano-liposome-human ACE2 decoy neutralizes SARS-CoV-2 Spike protein-induced inflammation in both murine and human macrophages.

Rationale: Macrophages are the frontline immune cells in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) serves as the binding receptor to SARS-CoV-2 Spike glycoprotein for fusion and internalization into the human host cells. However, the mechanisms underlying SARS-CoV-2-elicited macrophage inflammatory responses remain elusive. Neutralizing SARS-CoV-2 by human ACE2 (hACE2) decoys has been proposed as a therapeutic approach to ameliorate SARS-CoV-2-stimulated inflammation. This study aims to investigate whether an engineered decoy receptor can abrogate SARS-CoV-2-induced macrophage inflammation. Methods: hACE2 was biotinylated to the surface of nano-liposomes (d = 100 nm) to generate Liposome-human ACE2 complex (Lipo-hACE2). Lentivirus expressing Spike protein (D614G) was also created as a pseudo-SARS-CoV-2 (Lenti-Spike). Liposome-hACE2 was used as a decoy receptor or competitive inhibitor to inhibit SARS-CoV-2 or Lenti-Spike-induced macrophage inflammation in vitro and in vivo. Results: Both SARS-CoV-2 and Lenti-Spike stimulated strong inflammatory responses by inducing the expression of key cytokine and chemokines, including IL-1ß, IL-6, TNFα, CCL-2, and CXCL-10, in murine and human macrophages in vitro, whereas Lipo-hACE2 decoy abolished these effects in macrophages. Furthermore, intravenous injection of Lenti-Spike led to increased macrophage and tissue inflammation in wild type mice, which was also abolished by Lipo-hACE2 treatment. Mechanistically, Spike protein stimulated macrophage inflammation by activating canonical NF-κB signaling. RNA sequencing analysis revealed that Lenti-Spike induced over 2,000 differentially expressed genes (DEGs) in murine macrophages, but deficiency of IκB kinase ß (IKKß), a key regulator for NF-κB activation, abrogated Lenti-Spike-elicited macrophage inflammatory responses. Conclusions: We demonstrated that the engineered Lipo-hACE2 acts as a molecular decoy to neutralize SARS-CoV-2 or Spike protein-induced inflammation in both murine and human macrophages, and activation of the canonical IKKß/NF-κB signaling is essential for SARS-CoV-2-elicited macrophage inflammatory responses.

Analyzing the interaction of human ACE2 and RBD of spike protein of SARS-CoV-2 in perspective of Omicron variant.

The newly identified Omicron (B.1.1.529) variant of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has steered concerns across the world due to the possession of a large number of mutations leading to high infectivity and vaccine escape potential. The Omicron variant houses 32 mutations in spike (S) protein alone. The viral infectivity is determined mainly by the ability of S protein Receptor Binding Domain (RBD) to bind to the human Angiotensin I Converting Enzyme 2 (hACE2) receptor. In this paper, the interaction of the RBDs of SARS-CoV-2 variants with hACE2 was analyzed by using protein-protein docking and compared with the novel Omicron variant. Our findings reveal that the Omicron RBD interacts strongly with hACE2 receptor via unique amino acid residues as compared to the Wuhan and many other variants. However, the interacting residues of RBD are found to be the same in Lamda (C.37) variant. This unique binding of Omicron RBD with hACE2 suggests an increased potential of infectivity and vaccine evasion potential of the new variant. The evolutionary drive of the SARS-CoV-2 may not be exclusively driven by RBD variants but surely provides for the platform for emergence of new variants.

Structural modeling of Omicron spike protein and its complex with human ACE-2 receptor: Molecular basis for high transmissibility of the virus.

Omicron is a new variant of SARS-CoV-2, which is currently infecting people around the world. Spike glycoprotein, an important molecule in pathogenesis of infection has been modeled and the interaction of its Receptor Binding Domain with human ACE-receptor has been analysed by simulation studies. Structural analysis of Omicron spike glycoprotein shows the 30 mutations to be distributed over all domains of the trimeric protein, wherein the mutant residues are seen to be participating in higher number of intra-molecular interactions including two salt bridges emanating from mutant residues thereby stabilizing their conformation, as compared to wild type. Complex of Receptor Binding Domain (RBD) with human ACE-2 receptor shows seven mutations at interacting interface comprising of two ionic interactions, eight hydrogen bonds and seven Van der Waals interactions. The number and quality of these interactions along with other binding biophysical parameters suggests more potency of RBD domain to the receptor as compared to the wild type counterpart. Results of this study explains the high transmissibility of Omicron variant of SARS-CoV-2 that is currently observed across the world.

Biparatopic nanobodies protect mice from lethal challenge with SARS-CoV-2 variants of concern.

The ongoing COVID-19 pandemic and the emergence of new SARS-CoV-2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nbs) specific for the receptor-binding domain (RBD) of SARS-CoV-2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS-CoV-2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb-treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS-CoV-2 VOCs and represent easily applicable drug candidates.

Rapid Assessment of Binding Affinity of SARS-COV-2 Spike Protein to the Human Angiotensin-Converting Enzyme 2 Receptor and to Neutralizing Biomolecules Based on Computer Simulations.

SARS-CoV-2 infects humans and causes Coronavirus disease 2019 (COVID-19). The S1 domain of the spike glycoprotein of SARS-CoV-2 binds to human angiotensin-converting enzyme 2 (hACE2) via its receptor-binding domain, while the S2 domain facilitates fusion between the virus and the host cell membrane for entry. The spike glycoprotein of circulating SARS-CoV-2 genomes is a mutation hotspot. Some mutations may affect the binding affinity for hACE2, while others may modulate S-glycoprotein expression, or they could result in a virus that can escape from antibodies generated by infection with the original variant or by vaccination. Since a large number of variants are emerging, it is of vital importance to be able to rapidly assess their characteristics: while changes of binding affinity alone do not always cause direct advantages for the virus, they still can provide important insights on where the evolutionary pressure is directed. Here, we propose a simple and cost-effective computational protocol based on Molecular Dynamics simulations to rapidly screen the ability of mutated spike protein to bind to the hACE2 receptor and selected neutralizing biomolecules. Our results show that it is possible to achieve rapid and reliable predictions of binding affinities. A similar approach can be used to perform preliminary screenings of the potential effects of S-RBD mutations, helping to prioritize the more time-consuming and expensive experimental work.

Computational prediction of the effect of amino acid changes on the binding affinity between SARS-CoV-2 spike RBD and human ACE2.

The association of the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with human angiotensin-converting enzyme 2 (hACE2) represents the first required step for cellular entry. SARS-CoV-2 has continued to evolve with the emergence of several novel variants, and amino acid changes in the RBD have been implicated with increased fitness and potential for immune evasion. Reliably predicting the effect of amino acid changes on the ability of the RBD to interact more strongly with the hACE2 can help assess the implications for public health and the potential for spillover and adaptation into other animals. Here, we introduce a two-step framework that first relies on 48 independent 4-ns molecular dynamics (MD) trajectories of RBD-hACE2 variants to collect binding energy terms decomposed into Coulombic, covalent, van der Waals, lipophilic, generalized Born solvation, hydrogen bonding, π-π packing, and self-contact correction terms. The second step implements a neural network to classify and quantitatively predict binding affinity changes using the decomposed energy terms as descriptors. The computational base achieves a validation accuracy of 82.8% for classifying single-amino acid substitution variants of the RBD as worsening or improving binding affinity for hACE2 and a correlation coefficient of 0.73 between predicted and experimentally calculated changes in binding affinities. Both metrics are calculated using a fivefold cross-validation test. Our method thus sets up a framework for screening binding affinity changes caused by unknown single- and multiple-amino acid changes offering a valuable tool to predict host adaptation of SARS-CoV-2 variants toward tighter hACE2 binding.

Recent advances in SARS-CoV-2 Spike protein and RBD mutations comparison between new variants Alpha (B.1.1.7, United Kingdom), Beta (B.1.351, South Africa), Gamma (P.1, Brazil) and Delta (B.1.617.2, India).

New variants of SARS-CoV-2 Alpha (B.1.1.7); Beta (B.1.351) Gamma (P.1) and Delta (B.1.617.2) quickly spread in the UK, South Africa, Brazil and India, respectively. To address whether mutations in SARS-CoV-2 RBD spike protein could affect virus infectivity, peptides containing RBD amino acids mutations have been constructed and interacted with human ACE2 by computational methods. Our results suggest that mutations in RBD amino acids K417, E484, L452, T478 and N501 are expressively increasing the affinity of this protein with human angiotensin-converting enzyme 2 (ACE2), consequently, variants Alpha (B.1.1.7), Beta (B1.351), Gamma (P.1) and Delta (B.1.617.2) could be more infective in human cells compared with SARS-CoV-2 isolated in Wuhan-2019 and the Gamma and Delta variants could be the most infective among them.

Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ∼30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions, the RNA is protected by viral capsid proteins, membrane proteins, and nucleocapsid proteins. To utilize RNases as antiviral strategy, we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We expressed six proteins in E. coli cells: (1) MBP-ACE2NTD, (2) ACE2NTD-GFP, (3) RNase I (6×His), (4) RNase III (6×His), (5) RNase I-ACE2NTD (6×His), and (6) human RNase A-ACE2NTD (6×His). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6×His) and hRNase A-ACE2NTD (6×His) fusions are active in cleaving SARS-CoV-2 RNA fragment in vitro. The recombinant RNase I (6×His) and RNase III (6×His) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human receptor and nuclease that may be used to degrade viral nucleic acids.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Structural Dissection of Viral Spike-Protein Binding of SARS-CoV-2 and SARS-CoV-1 to the Human Angiotensin-Converting Enzyme 2 (ACE2) as Cellular Receptor.

An outbreak by a new severe acute respiratory syndrome betacoronavirus (SARS-CoV-2) has spread CoronaVirus Disease 2019 (COVID-19) all over the world. Immediately, following studies have confirmed the human Angiotensin-Converting Enzyme 2 (ACE2) as a cellular receptor of viral Spike-Protein (Sp) that mediates the CoV-2 invasion into the pulmonary host cells. Here, we compared the molecular interactions of the viral Sp from previous SARS-CoV-1 of 2002 and SARS-CoV-2 with the host ACE2 protein by in silico analysis of the available experimental structures of Sp-ACE2 complexes. The K417 amino acid residue, located in the region of Sp Receptor-Binding Domain (RBD) of the new coronavirus SARS-CoV-2, showed to have a key role for the binding to the ACE2 N-terminal region. The R426 residue of SARS-CoV-1 Sp-RBD also plays a key role, although by interacting with the central region of the ACE2 sequence. Therefore, our study evidenced peculiarities in the interactions of the two Sp-ACE2 complexes. Our outcomes were consistent with previously reported mutagenesis studies on SARS-CoV-1 and support the idea that a new and different RBD was acquired by SARS-CoV-2. These results have interesting implications and suggest further investigations.

HD5 and LL-37 Inhibit SARS-CoV and SARS-CoV-2 Binding to Human ACE2 by Molecular Simulation.

The coronavirus (COVID-19) pandemic is still spreading all over the world. As reported, angiotensin-converting enzyme-2 (ACE2) is a receptor of SARS-CoV-2 spike protein that initializes viral entry into host cells. Previously, the human defensin 5 (HD5) has been experimentally confirmed to be functional against the SARS-CoV-2. The present study proposes a human cathelicidin known as LL37 that strongly binds to the carboxypeptidase domain of human ACE2 compared to HD5. Therefore, LL37 bears a great potential to be tested as an anti-SARS-CoVD-2 peptide. We investigated the molecular interactions formed between the LL37 and ACE2 as well as HD5 and ACE2 tailed by their thermodynamic stability. The MM-PBSA and free energy landscape analysis outcomes confirmed its possible inhibitory effect against the SARS-CoV-2. The results obtained here could help propose a promising therapeutic strategy against the havoc caused by SARS-CoV-2 infections.

Implementation of Adenovirus-Mediated Pulmonary Expression of Human ACE2 in HLA Transgenic Mice Enables Establishment of a COVID-19 Murine Model for Assessment of Immune Responses to SARS-CoV-2 Infection.

HLA transgenic mice are instrumental for evaluation of human-specific immune responses to viral infection. Mice do not develop COVID-19 upon infection with SARS-CoV-2 due to the strict tropism of the virus to the human ACE2 receptor. The aim of the current study was the implementation of an adenovirus-mediated infection protocol for human ACE2 expression in HLA transgenic mice. Transient pulmonary expression of the human ACE2 receptor in these mice results in their sensitisation to SARS-CoV-2 infection, consequently providing a valuable animal model for COVID-19. Infection results in a transient loss in body weight starting 3 days post-infection, reaching 20-30% loss of weight at day 7 and full recovery at days 11-13 post-infection. The evolution of the disease revealed high reproducibility and very low variability among individual mice. The method was implemented in two different strains of HLA immunized mice. Infected animals developed strong protective humoral and cellular immune responses specific to the viral spike-protein, strictly depending on the adenovirus-mediated human ACE2 expression. Convalescent animals were protected against a subsequent re-infection with SARS-CoV-2, demonstrating that the model may be applied for assessment of efficacy of anti-viral immune responses.