Structural genetics of circulating variants affecting the SARS-CoV-2 spike/human ACE2 complex

<h3>Abstract</h3> SARS-CoV-2 entry in human cells is mediated by the interaction between the viral Spike protein and the human ACE2 receptor. This mechanism evolved from the ancestor bat coronavirus and is currently one of the main targets for antiviral strategies. However, there currently exist several Spike protein variants in the SARS-CoV-2 population as the result of mutations, and it is unclear if these variants may exert a specific effect on the affinity with ACE2 which, in turn, is also characterized by multiple alleles in the human population. In the current study, the GBPM analysis, originally developed for highlighting host-guest interaction features, has been applied to define the key amino acids responsible for the Spike/ACE2 molecular recognition, using four different crystallographic structures. Then, we intersected these structural results with the current mutational status, based on more than 295,000 sequenced cases, in the SARS-CoV-2 population. We identified several Spike mutations interacting with ACE2 and mutated in at least 20 distinct patients: S477N, N439K, N501Y, Y453F, E484K, K417N, S477I and G476S. Among these, mutation N501Y in particular is one of the events characterizing SARS-CoV-2 lineage B.1.1.7, which has recently risen in frequency in Europe. We also identified five ACE2 rare variants that may affect interaction with Spike and susceptibility to infection: S19P, E37K, M82I, E329G and G352V. <h3>Significance Statement</h3> We developed a method to identify key amino acids responsible for the initial interaction between SARS-CoV-2 (the COVID-19 virus) and human cells, through the analysis of Spike/ACE2 complexes. We further identified which of these amino acids show variants in the viral and human populations. Our results will facilitate scientists and clinicians alike in identifying the possible role of present and future Spike and ACE2 sequence variants in cell entry and general susceptibility to infection.

The worse outcome of COVID-19 in people with diabetes mellitus could be related to the non-enzymatic glycation of human ACE2, leading to a more susceptible interaction with virus Spike protein. We aimed to evaluate, through a computational approach, the interaction between human ACE2 receptor and SARS-CoV-2 Spike protein under different conditions of hyperglycemic environment. A computational analysis was performed, based on the X-ray crystallographic structure of the Spike Receptor-Binding Domain (RBD)-ACE2 system. The possible scenarios of lysine aminoacid residues on surface transformed by glycation were considered: (1) on ACE2 receptor; (2) on Spike protein; (3) on both ACE2 receptor and Spike protein. In comparison to the native condition, the number of polar bonds (comprising both hydrogen bonds and salt bridges) in the poses considered are 10, 6, 6, and 4 for the states ACE2/Spike both native, ACE2 native/Spike glycated, ACE2 glycated/Spike native, ACE2/Spike both glycated, respectively. The analysis highlighted also how the number of non-polar contacts (in this case, van der Waals and aromatic interactions) significantly decreases when the lysine aminoacid residues undergo glycation. Following non-enzymatic glycation, the number of interactions between human ACE2 receptor and SARS-CoV-2 Spike protein is decreased in comparison to the unmodified model. The reduced affinity of the Spike protein for ACE2 receptor in case of non-enzymatic glycation may shift the virus to multiple alternative entry routes.

COVID-19 is an infectious disease caused by the virus SARS-CoV-2. To access the internal machinery necessary for its replication, the virus needs to latch onto and then enter host cells. Such processes rely on specific ‘glycoproteins’ that carry complex sugar molecules (or glycans), and can be found at the surface of both viruses and host cells. In particular, the viral ‘Spike’ glycoprotein can attach to human proteins called ACE2, which coat the cells that line the inside of the lungs, heart, kidney and brain. Yet the roles played by glycans in these processes remains unclear. To investigate the role of Spike and ACE-2 glycans, Yang et al. designed a form of SARS-CoV-2 that could be handled safely in the laboratory. How these viruses infect human kidney cells that carry ACE2 was then examined, upon modifying the structures of the sugars on the viral Spike protein as well as the host ACE2 receptor. In particular, the sugar structures displayed by the virus were modified either genetically or chemically, using a small molecule that disrupts the formation of the glycans. Similar methods were also applied to modify the glycans of ACE2. Together, these experiments showed that the sugars present on the Spike protein play a minor role in helping the virus stick to human cells.However, they were critical for the virus to fuse and enter the host cells. These findings highlight the important role of Spike protein sugars in SARS-CoV-2 infection, potentially offering new paths to treat COVID-19 and other coronavirus-related illnesses. In particular, molecules designed to interfere with Spike-proteins and the viral entrance into cells could be less specific to SARS-CoV-2 compared to vaccines, allowing treatments to be efficient even if the virus changes.

The increasing number of SARS-CoV-2 variants associated with highly transmissible phenotypes is a health-public concern in the current pandemic scenario. Herein, we developed a comprehensive in silico analysis of the changes occurring upon mutations in the viral spike. We focused on mutants located in the receptor-binding domain of the viral spike protein and analyzed whether these mutants modulate the interaction with the human host receptor angiotensin-converting enzyme II (ACE2). Thirty-two highly prevalent mutants were retrieved from the GISAID database, and their structural models were built using the SWISS-Model server. The stabilization effect for each mutation was assessed by the DUET and DeepDGG software. By applying molecular docking using both Z-Dock and Haddock software we found that multiple mutations, including A475V, V455E, V445L, and V445I, resulted in the higher binding free energy as compared to the wild type (WT) spike protein, thus had a destabilizing effect on the binding to ACE2. On the other hand, several mutants, including the most prevalent N501Y and B.1.1.7 variants, as well as the K444R, L455F, Q493R, and Y505W variants exhibited lower binding free energy as compared to the WT spike. These mutants showed an increased number of electrostatic interactions with ACE2 than the WT spike protein, and they changed the interaction pattern of the neighboring residues. Together, the results presented in this study contribute to a better understanding of the changes in the interaction between SARS-CoV-2 and the human host ACE2 receptor associated with point mutations in the viral spike protein.

COVID-19 patients experience, in 10-20% of the cases, a prolonged long-COVID syndrome, defined as the persistence of symptoms for at least two months after the infection. The underlying biological mechanisms of this syndrome remain poorly understood. Several hypotheses have been proposed, among which are the potential autoimmunity resulting from molecular mimicry between viral spike protein and human proteins, the reservoir and viral reproduction hypothesis, and the viral integration hypothesis. Although official data state that vaccinal spike protein is harmless and remains at the site of infection, several studies proposed spike protein toxicity and found it in blood circulation several months after the vaccination. To search for the presence of viral and vaccine spike protein in a cohort of long-COVID patients. In this study, we employed a proteomic-based approach utilizing mass spectrometry to analyze the serum of 81 patients with long-COVID syndrome. Moreover, viral integration in patients' leukocytes was assessed with a preliminary study, without further investigation. We identified the presence of the viral spike protein in one patient after infection clearance and negativity of COVID-19 test and the vaccine spike protein in two patients two months after the vaccination. This study, in agreement with other published investigations, demonstrates that both natural and vaccine spike protein may still be present in long-COVID patients, thus supporting the existence of a possible mechanism that causes the persistence of spike protein in the human body for much longer than predicted by early studies. According to these results, all patients with long-COVID syndrome should be analyzed for the presence of vaccinal and viral spike protein.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes. eLife digest The new coronavirus, SARS-CoV-2, which causes the disease COVID-19, has had a serious worldwide impact on human health. The virus was virtually unknown at the beginning of 2020. Since then, intense research efforts have resulted in sequencing the coronavirus genome, identifying the structures of its proteins, and creating a wide range of tools to search for effective vaccines and therapies. Antibodies, which are immune molecules produced by the body that target specific segments of viral proteins can neutralize virus particles and trigger the immune system to kill cells infected with the virus. Several technologies are currently under development to exploit antibodies, including vaccines, blood plasma from patients who were previously infected, manufactured antibodies and more. The spike proteins on the surface of SARS-CoV-2 are considered to be prime antibody targets as they are accessible and have an essential role in allowing the virus to attach to and infect host cells. Antibodies bind to spike proteins and some can block the virus' ability to infect new cells. But some viruses, such as HIV and influenza, are able to mutate their equivalent of the spike protein to evade antibodies. It is unknown whether SARS-CoV-2 is able to efficiently evolve to evade antibodies in the same way. Weisblum, Schmidt et al. addressed this question using an artificial system that mimics natural infection in human populations. Human cells grown in the laboratory were infected with a hybrid virus created by modifying an innocuous animal virus to contain the SARS-CoV-2 spike protein, and treated with either manufactured antibodies or antibodies present in the blood of recovered COVID-19 patients. In this situation, only viruses that had mutated in a way that allowed them to escape the antibodies were able to survive. Several of the virus mutants that emerged had evolved spike proteins in which the segments targeted by the antibodies had changed, allowing these mutant viruses to remain undetected. An analysis of more than 50,000 real-life SARS-CoV-2 genomes isolated from patient samples further showed that most of these virus mutations were already circulating, albeit at very low levels in the infected human populations. These results show that SARS-CoV-2 can mutate its spike proteins to evade antibodies, and that these mutations are already present in some virus mutants circulating in the human population. This suggests that any vaccines that are deployed on a large scale should be designed to activate the strongest possible immune response against more than one target region on the spike protein. Additionally, antibody-based therapies that use two antibodies in combination should prevent the rise of viruses that are resistant to the antibodies and maintain the long-term effectiveness of vaccines and therapies. Introduction Neutralizing antibodies are a key component of adaptive immunity against many viruses that can be elicited by natural infection or vaccination (Plotkin, 2010). Antibodies can also be administered as recombinantly produced proteins or as convalescent plasma to confer a state of passive immunity in prophylactic or therapeutic settings. These paradigms are of particular importance given the emergence of SARS-CoV-2, and the devastating COVID19 pandemic that has ensued. Indeed, interventions to interrupt SARS-CoV-2 replication and spread are urgently sought, and passively administered antibodies are currently among the most promising therapeutic and prophylactic antiviral agents. Moreover, an understanding of the neutralizing antibody response to SARS-CoV-2 is critical for the elicitation of effective and durable immunity by vaccination (Kellam and Barclay, 2020). Recent studies have shown that related, potently neutralizing monoclonal antibodies that recognize the SARS-CoV-2 receptor-binding domain (RBD) are often elicited in SARS-CoV-2 infection (Robbiani et al., 2020; Brouwer et al., 2020; Cao et al., 2020; Chen et al., 2020; Chi et al., 2020; Rogers et al., 2020; Shi et al., 2020; Wu et al., 2020a; Wec et al., 2020; Kreer et al., 2020; Hansen et al., 2020; Ju et al., 2020; Seydoux et al., 2020; Liu et al., 2020; Zost et al., 2020). These antibodies have great potential to be clinically impactful in the treatment and prevention of SARS-CoV-2 infection. The low levels of somatic hypermutation and repetitive manner in which similar antibodies (e.g. those based on IGHV3-53 Robbiani et al., 2020; Barnes et al., 2020; Yuan et al., 2020) have been isolated from COVID19 convalescents suggests that potently neutralizing responses should be readily elicited. Paradoxically, a significant fraction of COVID19 convalescents, including some from whom potent neutralizing antibodies have been cloned, exhibit low levels of plasma neutralizing activity (Robbiani et al., 2020; Wu et al., 2020b; Luchsinger et al., 2020). Together, these findings suggest that natural SARS-CoV-2 infection may often fail to induce sufficient B-cell expansion and maturation to generate high-titer neutralizing antibodies. The degree to, and pace at which SARS-CoV-2 might evolve to escape neutralizing antibodies is unclear. The aforementioned considerations raise the possibility that SARS-CoV-2 evolution might be influenced by frequent encounters with sub-optimal concentrations of potently neutralizing antibodies during natural infection. Moreover, the widespread use of convalescent plasma containing unknown, and often suboptimal, levels of neutralizing antibodies might also increase the acquisition of neutralizing antibody resistance by circulating SARS-CoV-2 populations (Bloch et al., 2020; Al‐Riyami et al., 2020). Reinfection of previously infected individuals who have incomplete or waning serological immunity might similarly drive emergence of antibody escape variants. As human neutralizing antibodies are discovered and move into clinical development as therapeutics and prophylactics, and immunogens based on prototype SARS-CoV-2 spike protein sequences are deployed as vaccines, it is important to anticipate patterns of antibody resistance that might arise. Here, we describe a recombinant chimeric virus approach that can rapidly generate and evaluate SARS-CoV-2 S mutants that escape antibody neutralization. We show that mutations conferring resistance to convalescent plasma or RBD-specific monoclonal antibodies can be readily generated in vitro. Notably, these antibody resistance mutations are present at low frequency in natural populations. Importantly, the use of candidate monoclonal antibody combinations that target distinct epitopes on the RBD (and therefore have non-overlapping resistance mutations) can suppress the emergence of antibody resistance. Results Selection of SARS-CoV-2 S variants using a replication-competent VSV/SARS-CoV-2 chimeric virus To select SARS-CoV-2 S variants that escape neutralization by antibodies, we used a recently described replication-competent chimeric virus based on vesicular stomatitis virus that encodes the SARS-CoV-2 spike (S) protein and green fluorescent protein (rVSV/SARS-CoV-2/GFP) (Schmidt et al., 2020). Notably, rVSV/SARS-CoV-2/GFP replicates rapidly and to high-titers (107 to 108 PFU/ml within 48 hr), mimics the SARS-CoV-2 requirement for ACE-2 as a receptor, and is neutralized by COVID19 convalescent plasma and SARS-CoV-2 S-specific human monoclonal antibodies (Schmidt et al., 2020). The replication of rVSV/SARS-CoV-2/GFP can be readily monitored and measured by GFP fluorescence and the absence of proof-reading activity in the viral polymerase (VSV-L) results in the generation of virus stocks with greater diversity than authentic SARS-CoV-2, for an equivalent viral population size. These features facilitate experiments to investigate the ability S protein variants to escape antibody neutralization. We used two adapted, high-titer variants of rVSV/SARS-CoV-2/GFP, (namely rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1) (Schmidt et al., 2020) in attempts to derive antibody-resistant mutants. Virus populations containing 1 × 106 infectious particles were generated following three passages to generated sequence diversity. On the third passage, cells were infected at an MOI of ~ 0.5 and progeny harvested after as short a time as possible so as to minimize phenotypic mixing in the viral population and to maximize the concordance between the genome sequence and the S protein sequence represented in a given virion particle. Because the mutation rate of VSV is ~ 10−4 to 10−5/base per replication cycle (Steinhauer and Holland, 1986; Steinhauer et al., 1989; Combe and Sanjuán, 2014), we estimated that this procedure should generate a large fraction of the possible replication-competent mutants within a population size of 1 × 106. The viral populations were then incubated with antibodies to neutralize susceptible variants (Figure 1A). For monoclonal antibodies, viral populations were incubated with antibodies at 5 μg/ml or 10 μg/ml, (~1000 to 10,000 x IC50) so as to minimize the number of infection events by antibody sensitive variants, and enable rapid selection of the most resistant rVSV/SARS-CoV-2/GFP variants from the starting population. For plasma samples, the possibility existed that multiple different antibody specificities could be present, that might interfere with the outgrowth of rVSV/SARS-CoV-2/GFP variants that were resistant to the most prevalent or potent antibodies in the plasma. Therefore, in these selection experiments, viruses were incubated with a range of plasma dilutions (see materials and methods). Neutralized viral populations were then applied to 293T/ACE2(B) cells (Schmidt et al., 2020), which support robust rVSV/SARS-CoV-2/GFP replication, and incubated for 48 hr. We used three potent human monoclonal antibodies C121, C135, and C144 (Robbiani et al., 2020), that are candidates for clinical development (Table 1). In addition, we used four convalescent plasma samples, three of which were from the same donors from which C121, C135, and C144, were obtained (Robbiani et al., 2020; Table 1). Two of these plasmas (COV-47 and COV-72) were potently neutralizing while the third (COV-107) had low neutralizing activity. A fourth convalescent plasma sample (COV-NY) was potently neutralizing but did not have a corresponding monoclonal antibody (Table 1). Figure 1 Download asset Open asset Selection of SARS-CoV-2 S mutations that confer antibody resistance. (A) Outline of serial passage experiments with replication-competent VSV derivatives encoding the SARS-CoV-2 S envelope glycoprotein and a GFP reporter (rVSV/SARS-CoV-2/GFP) in 293T/ACE2(B) cells in the presence of neutralizing antibodies or plasma. Each passage experiment was performed twice (once each with rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1.) (B) Representative images of 293T/ACE2(B) cells infected with 1 × 106 PFU of rVSV/SARS-CoV-2/GFP in the presence or absence of 10 μg/ml of the monoclonal antibody C121. (C) Expanded view of the boxed areas showing individual plaques of putatively antibody-resistant viruses. Table 1 Plasma and monoclonal antibodies used in this study. DonorPlasma NT50 (rVSV-SARSCoV2/GFP)Plasma NT50 HIV/CCNGnLucMonoclonal antibodyCOV-4766228016C144COV-7262747982C135COV-107122334C121COV-NY126147505ND Infection with rVSV/SARS-CoV-2/GFP in the presence of the monoclonal antibodies C121 or C144 reduced the number of infectious units from 106 to a few hundred, as estimated by the frequency of GFP-positive cells (Figure 1B) a reduction of > 1000 fold. C135 reduced infection by ~ 100 fold. Imaging of wells infected with rVSV/SARS-CoV-2/GFP in the presence of C121 or C144 revealed a small number of foci (~10 to 20/well), that suggested viral spread following initial infection (Figure 1B). In the case of C135, a greater number of GFP-positive cells were detected, obscuring the visualization of focal viral spread following initial infection. Aliquots of supernatants from these passage-1 (p1) cultures were collected 48 hr after infection, diluted in the same concentrations of monoclonal antibodies that were initially employed, and used to infect fresh (p2) cultures (Figure 1A). For p2 cultures, almost all cells became infected within 48 hr, suggesting the possible outgrowth of monoclonal antibody escape variants that were present in the original viral populations. For selection in the presence of plasma, p1 supernatants were harvested at 48 hr after infection in the presence of the highest concentrations of plasma that permitted infection of reasonable numbers (approximately 10%) of cells. Then, p2 cultures were established using p1 supernatants, diluted in the same concentrations of plasma used in p1. This approach led to clear 'escape' for the COV-NY plasma with prolific viral growth in p2 as evidenced by a large increase in the number of GFP-positive cells. For COV-47, COV-72, and CO107, plasma clearly retained at least some inhibitory activity in p2. Thereafter, p3 cultures and p4 cultures were established for COV-47, COV-72, and COV-107 plasmas at 5-fold higher concentrations of plasma than were used in p1 and p2 cultures (Figure 1A). RNA was extracted from p2 supernatants (monoclonal antibodies and COV-NY plasma) as well as later passages for the COV-47, COV-72, and COV-107 plasma selections. Sequences encoding either the RBD or the complete S protein were amplified using PCR and analyzed by Sanger and/or Illumina sequencing. For all three monoclonal antibodies and two of the four plasmas, sequence analyses revealed clear evidence for selection, with similar or identical mutants emerging in the presence of monoclonal antibodies or plasma in both rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 cultures (Figure 2A–D, Figure 2—figure supplement 1A,B, Figure 2—figure supplement 2A,B, Table 2). In the case of C121, mutations E484K and Q493K/R within the RBD were present at high frequencies in both p2 selected populations, with mutation at a proximal position (F490L) present in one p2 population (Figure 2A, Table 2). Viruses passaged in the presence of monoclonal antibody C144 also had mutations at positions E484 and Q493, but not at F490 (Figure 2C, Table 2). In contrast, virus populations passaged in the presence of monoclonal antibody C135 lacked mutations at E484 or Q493, and instead had mutations R346K/S/L and N440K at high frequency (Figure 2C, Table 2). Figure 2 with 2 supplements see all Download asset Open asset Analysis of S-encoding sequences following rVSV/SARS-CoV-2/GFP replication in the presence of neutralizing monoclonal antibodies. (A–D) Graphs depict the S codon position (X-axis) and the frequency of non-synonymous substitutions (Y-axis) following the second passage (p2) of rVSV/SARS-CoV-2/GFP on 293T/ACE2(B) cells in the absence of antibody or plasma (A), or in the presence of 10 μg/ml C121 (B), C135 (C) or C144 (D). Results are shown for both rVSV/SARS-CoV-2/GFP variants (One replicate each for rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 - the frequency of 1D7 mutations is plotted as circles and 2E1 mutations as triangles). Table 2 Mutations occurring at high frequency during rVSV/SARS-CoV-2 passage in the presence of neutralizing antibodies or plasma. Mutant frequencyMutationp2p3p4Monoclonal antibodiesC121E484K*0.50, 0.39–†–F490L0.23Q493K0.12, 0.45C135N440K0.31, 0.30––R346S0.30, 0.17R346K0.22, 0.53R346M0.16C144E484K0.44, 0.18––Q493K0.31, 0.39Q493R0.17, 0.37PlasmasCOV47N148S0.16, 0.140.29, 0.300.72, 0.14K150R0.100.18K150E0.040.160.4K150T0.22K150Q0.160.22S151P0.10.180.2COV-NYK444R0.20,0.19––K444N0.14K444Q0.33V445E0.18 *Values represent the decimal frequency with which each mutation occurs ass assessed by NGS, two values indicate occurrences in both rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 cultures, single values indicate occurrence in only one culture. † –, not done. Mutations at specific positions were enriched in viruses passaged in the presence of convalescent plasma, in two out of four cases (Figure 2—figure supplement 1A,B, Figure 2—figure supplement 2A,B, Table 2). Specifically, virus populations passaged in the presence of COV-NY plasma had mutations within RBD encoding sequence (K444R/N/Q and V445E) that were abundant at p2 (Figure 2—figure supplement 2A,B, Table 2). Conversely, mutations outside the RBD, specifically at N148S, K150R/E/T/Q and S151P in the N-terminal domain (NTD) were present at modest frequency in COV-47 p2 cultures and became more abundant at p3 and p4 (Figure 2—figure supplement 1A,B, Table 2). Replication in the presence of COV72 or COV107 plasma did not lead to the clear emergence of escape mutations, suggesting that the neutralization by these plasmas was not due to one dominant antibody specificity. In the case of COV107, the failure of escape mutants to emerge may simply be due to the lack of potency of that plasma (Table 1). However, in the case of COV-72, combinations of antibodies may be responsible for the potent neutralizing properties of the plasma in that case. Isolation and characterization of rVSV/SARS-CoV-2/GFP antibody escape mutants Based on the aforementioned analyses, supernatants from C121, C144, and C135 and COV-NY plasma p2 cultures, or COV47 p4 cultures, contained mixtures of putative rVSV/SARS-CoV-2/GFP neutralization escape mutants. To isolate individual mutants, the supernatants were serially diluted and individual viral foci isolated by limiting dilution in 96-well plates. Numerous individual rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 derivatives were harvested from wells containing a single virus plaque, expanded on 293T/ACE2(B) cells, then RNA was extracted and subjected to Sanger-sequencing (Figure 3—figure supplement 1). This process verified the purity of the individual rVSV/SARS-CoV-2/GFP variants and yielded a number of viral mutants for further analysis (Figure 3—figure supplement 1). These plaque-purified viral mutants all encoded single amino-acid substitutions in S-coding sequences that corresponded to variants found at varying frequencies (determined by Illumina sequencing) in the antibody-selected viral populations. Notably, each of the isolated rVSV/SARS-CoV-2/GFP mutants replicated with similar kinetics to the parental rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 viruses (Figure 3A), suggesting that the mutations that emerged during replication in the presence of monoclonal antibodies or plasma did not confer a substantial loss of fitness, at least in the context of rVSV/SARS-CoV-2/GFP. Moreover, for mutants in RBD sequences that arose in the C121, C135, C144, and COV-NY cultures, each of the viral mutants retained approximately equivalent sensitivity to neutralization by an ACE2-Fc fusion protein, suggesting little or no change in interaction with ACE2 (Figure 3B). Figure 3 with 1 supplement see all Download asset Open asset Characterization of mutant rVSV/SARS-CoV-2/GFP derivatives. (A) Replication of plaque-purified rVSV/SARS-CoV-2/GFP bearing individual S amino-acid substitutions that arose during passage with the indicated antibody or plasma. 293T/ACE2cl.22 cells were inoculated with equivalent doses of parental or mutant rVSV/SARS-CoV-2/GFP isolates. Supernatant was collected at the indicated times after inoculation and number of infectious units present therein was determined on 293T/ACE2cl.22 cells. The mean of two independent experiments is plotted. One set of WT controls run concurrently with the mutants are replotted in the upper and lower left panels, A different set of WT controls run concurrently with the mutants is shown in the lower right panel (B) Infection 293T/ACE2cl.22 cells by rVSV/SARS-CoV-2/GFP encoding the indicated S protein mutations in the presence of increasing amounts of a chimeric ACE2-Fc molecule. Infection was quantified by FACS. Mean of two independent experiments is plotted. The WT controls are replotted in each panel. We next determined the sensitivity of the isolated RBD mutants to neutralization by the three monoclonal antibodies. The E484K and Q493R mutants that emerged during replication in the presence of C121 or C144, both caused apparently complete, or near complete, resistance to both antibodies (IC50 > 10 μg/ml, Figure 4A,B). However, both of these mutants retained full sensitivity (IC50 < 10 ng/ml) to C135. Conversely, the R346S and N440K mutants that emerged during replication in the presence of C135 were resistant to C135, but retained full sensitivity to both C121 and C144 (Figure 4A,B). The K444N, K444T, V445G, V445E, and V445L mutants that arose during replication in the presence of COV-NY plasma conferred partial resistance to C135, with IC50 values ranging from 25 to 700 ng/ml, but these mutants retained full sensitivity to both C121 and C144 (Figure 4A,B). The spatial distribution of these resistance-conferring mutations on the SARS-CoV-2 S RBD surface suggested the existence of both distinct and partly overlapping neutralizing epitopes on the RBD (Figure 4C). The C121 and C144 neutralizing epitopes appear to be similar, and include E484 and Q493, while a clearly distinct conformational epitope seems to be recognized by C135, that includes R346 and N440 residues. Antibodies that constitute at least part of the neutralizing activity evident in COV-NY plasma appear to recognize an epitope that includes and K444 and V445. The ability of mutations at these residues to confer partial resistance to C135 is consistent with their spatial proximity to the C135 conformational epitope (Figure 4C). Figure 4 Download asset Open asset of rVSV/SARS-CoV-2/GFP RBD mutants by monoclonal antibodies. (A) of neutralization resistance of rVSV/SARS-CoV-2/GFP mutants that were isolated following passage in the presence of antibodies. 293T/ACE2cl.22 cells were inoculated with WT or mutant rVSV/SARS-CoV-2/GFP in the presence of increasing of each monoclonal and infection quantified by hr Mean and from two of two independent (B) of rVSV/SARS-CoV-2/GFP mutants isolated following replication in the presence of antibodies. Mean IC50 values were for each antibody combination in two independent (C) of neutralization resistance-conferring of the RBD et al., 2020) with positions that are by mutations were during replication in the presence of each monoclonal antibody or COV-NY plasma To whether neutralization escape mutations conferred loss of to the monoclonal antibodies, we et al., 2020), at their with (Figure The were incubated in with the monoclonal antibodies, were using protein and the of was measured using (Figure As C121, C135, and C144 monoclonal antibodies all the WT (Figure The E484K and Q493R complete, or near complete loss of to C121 and C144 antibodies but retained WT levels of to C135 (Figure Conversely, the R346S and N440K mutants complete loss of to C135, but retained WT levels of to C121 and The and mutants retained near WT levels of to all three antibodies, partial resistance to C135 (Figure the loss of of these mutants for C135 was sufficient to partial neutralization resistance but to in the Figure 5 Download asset Open asset of to monoclonal antibodies by neutralization escape mutants. (A) of the in which is to the of a The fusion protein is incubated with antibodies and using protein (B) quantified following of the indicated WT or mutant fusion proteins with the indicated antibodies and Mean of three replicates at each Analysis of mutants that were isolated from the virus population that emerged during rVSV/SARS-CoV-2/GFP replication in the presence of COV-47 plasma N148S, revealed that these mutants specific resistance to COV-47 plasma. Indeed, the COV-47 plasma NT50 for these mutants was reduced by to (Figure This that the antibody or antibodies responsible for of neutralizing activity in COV-47 plasma target an epitope that includes the potent monoclonal antibody isolated from COV-47 targets the in the epitope in sensitivity to plasmas (Figure that different epitopes are targeted by plasmas from the Figure Download asset Open asset of rVSV/SARS-CoV-2/GFP RBD mutants by convalescent plasma. of rVSV/SARS-CoV-2/GFP mutants isolated following replication in the presence COV-47 plasma (A) or COV-NY plasma 293T/ACE2cl.22 cells were inoculated with WT or mutant rVSV/SARS-CoV-2/GFP in the presence of increasing amounts of the indicated plasma, and infection quantified by hr Mean of two of two independent experiments (C) Plasma neutralization of rVSV/SARS-CoV-2/GFP mutants isolated following replication in the presence of monoclonal antibodies or convalescent plasma. Mean NT50 values were for each combination from two independent The viral population that emerged during replication in COV-NY plasma yielded mutants or and V445G, or Each of these mutations conferred substantial resistance to neutralization by COV-NY plasma, with ~ 10 to reduction in NT50 (Figure the dominant neutralizing activity in COV-NY plasma is represented by an antibody or antibodies an RBD epitope that includes K444 and V445. As was the case with COV-47 resistant mutants, viruses encoding the mutations conferring resistance to COV-NY plasma retained almost full sensitivity to neutralization by plasmas (Figure the mutations that conferred complete or near complete resistance to the potent RBD-specific monoclonal antibodies C144, C135, and C121 conferred little or no resistance to neutralization by plasma from the same or individuals (Figure These RBD-specific antibodies represent the most potent monoclonal antibodies isolated from COV-47, COV-72, and but the of plasma sensitivity by the monoclonal antibody-resistant mutants suggests that these antibodies little to the neutralization activity of plasma from the same This is consistent with the that cells these antibodies are (Robbiani et al., 2020), and with the results of the selection experiments in which rVSV/SARS-CoV-2/GFP replication in the presence of COV-47, COV-72, and COV-107 plasma did not for mutations that to the neutralization epitopes targeted by the monoclonal antibodies obtained from these individuals (Figure 2—figure supplement 1A,B, Figure 2—figure supplement Table 2). analysis of this set of monoclonal antibodies and plasmas that potent neutralization can be conferred by antibodies that target SARS-CoV-2 epitopes. Moreover, the most potently neutralizing antibodies generated in a given COVID19 convalescent individual may in only a way to the neutralizing antibody response in that same individual (see occurrence of RBD mutations The aforementioned neutralizing antibody escape mutations were generated during in replication of a recombinant virus. However, as monoclonal antibodies are for therapeutic and prophylactic

Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational attributes of the molecular complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike protein. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provides structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.

Background Accumulating evidence suggests that the receptor binding domain (RBD) of the SARS-CoV-2 Omicron variant has several times more binding affinity to the human angiotensin-converting enzyme 2 (ACE2) receptor compared to the RBD of the original covid-19 strain This increased binding affinity of Omicron variant is responsible for its increased internalization and infectivity.Methods In the present study, the impact of Coronil, a tri-herbal formulation of extracts from Withania somnifera, Tinospora cordifolia, and Ocimum sanctum on the binding properties of Omicron SARS-CoV-2 variant spike proteins (S proteins) was investigated. Compositional analysis of Coronil was performed by the Prominence-XR UHPLC system. The ELISA-based ACE2 binding inhibition assay was performed to delineate the effect of Coronil on the interaction between human ACE2 receptor and different Omicron variant spike proteins such as BA.4/BA5, XBB, BA.2.75.2, BA4.6/BF.7, BA.2.75.2, BQ.1.1, and a recently found spike protein variant JN.1 which is thought to emerge from BA.2.86.Results Coronil showed a dose-dependent inhibitory effect on the interactions between ACE2 and receptor binding domains (RBD) of all variants of spike proteins evaluated in this study including the recently emerged, highly transmissible variant spike protein JN.1. Although, Coronil significantly reduced the binding percentage in almost all the variant spike proteins, the maximum inhibition was achieved against BA.4/BA.5 where it inhibited the S protein – ACE2 interaction even at a low concentration of 3 µg/ml (16.6%). This binding inhibition was further increased to 60.3 and 84.6% at 100 and 300 µg/ml respectively.Conclusions This capability of Coronil to inhibit the binding of spike protein variants with ACE2 receptor may interfere with viral binding and internalization resulting in reduced infectivity of these Omicron spike protein variants. Overall, our data underscores the potential of Coronil in combating the various newly emerged Omicron spike protein variants. These findings may provide a basis for further studies of Coronil for its clinical effectiveness against these Omicron variants.

SARS-CoV-2 pandemic has become a major threat to human healthcare and world economy. Due to the rapid spreading and deadly nature of infection, we are in a situation to develop quick therapeutics to combat SARS-CoV-2. In this study, we have adopted a multi-level scoring approach to identify multi-targeting potency of bioactive compounds in selected medicinal plants and compared its efficacy with two reference drugs, Nafamostat and Acalabrutinib which are under clinical trials to treat SARS-CoV-2. In particular, we employ molecular docking and implicit solvent free energy calculations (as implemented in the Molecular Mechanics -Generalized Born Surface Area approach) and QM fragmentation approach for validating the potency of bioactive compounds from the selected medicinal plants against four different viral targets and one human receptor (Angiotensin-converting enzyme 2 -ACE-2) which facilitates the SARS-CoV-2entry into the cell. The protein targets considered for the study are viral 3CL main protease (3CLpro), papain-like protease (PLpro), RNA dependent RNA polymerase (RdRp), and viral spike protein-human hACE-2 complex (Spike:hACE2)including human protein target (hACE-2). Herein, thereliable multi-level scoring approach was used to validate the mechanism behind the multi-targeting potency of selected phytochemicals from medicinal plants. The present study evidenced that the phytochemicals Chebulagic acid, Stigmosterol, Repandusinic acid and Geranin exhibited efficient inhibitory activity against PLpro while Chebulagic acid was highly active against 3CLpro. Chebulagic acid andGeranin also showed excellent target specific activity against RdRp.Luteolin, Quercetin, Chrysoeriol and Repandusinic acid inhibited the interaction of viral spike protein with human ACE-2 receptor. Moreover Piperlonguminine and Piperine displayed significant inhibitory activity against human ACE-2 receptor. Therefore, the identified compounds namely Chebulagic acid, Geranin and Repandusinic acid can serve as potent multi-targeting phytomedicine for treating COVID-19

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

Differential susceptibility of macrophages to serotype II feline coronaviruses correlates with differences in the viral spike protein

Graphic abstractThe recent emergence of novel coronavirus (SARS-CoV-2) has been a major threat to human society, as the challenge of finding suitable drug or vaccine is not met till date. With increasing morbidity and mortality, the need for novel drug candidates is under great demand. The investigations are progressing towards COVID-19 therapeutics. Among the various strategies employed, the use of repurposed drugs is competing along with novel drug inventions. Based on the therapeutic significance, the chemical constituents from the extract of Tinospora cordifolia belonging to various classes like alkaloids, lignans, steroids and terpenoids are investigated as potential drug candidates for COVID-19. The inhibition potential of the proposed compounds against viral spike protein and human receptor ACE2 were evaluated by computational molecular modeling (Auto dock), along with their ADME/T properties. Prior to docking, the initial geometry of the compounds were optimized by Density functional theory (DFT) method employing B3LYP hybrid functional and 6–311 + + G (d,p) basis set. The results of molecular docking and ADME/T studies have revealed 6 constituents as potential drug candidates that can inhibit the binding of SARS-CoV-2 spike protein with the human receptor ACE2 protein. The narrowed down list of constituents from Tinospora cordifolia paved way for further tuning their ability to inhibit COVID-19 by modifying the chemical structures and by employing computational geometry optimization and docking methods.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13337-021-00666-7.

SARS‐CoV‐2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS‐CoV‐2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS‐CoV‐2 viral proteins. Here, we show that the nucleocapsid of SARS‐CoV‐2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS‐CoV‐2‐infected monocytes show enhanced cellular interleukin‐1β (IL‐1β) expression, but reduced IL‐1β secretion. While SARS‐CoV‐2 infection promotes activation of the NLRP3 inflammasome and caspase‐1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS‐CoV‐2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase‐1. These insights into how SARS‐CoV‐2 antagonizes cellular inflammatory responses may open new avenues for treating COVID‐19 in the future.

We performed a comprehensive structural analysis of the conformational space of several spike (S) protein variants using molecular dynamics (MD) simulations. Specifically, we examined four well-known variants (Delta, BA.1, XBB.1.5, and JN.1) alongside the wild-type (WT) form of SARS-CoV-2. The conformational states of each variant were characterized by analyzing their distributions within a selected space of collective variables (CVs), such as inter-domain distances between the receptor-binding domain (RBD) and the N-terminal domain (NTD). Our primary focus was to identify conformational states relevant to potential structural transitions and to determine the set of native contacts (NCs) that stabilize these conformations. The results reveal that genetically more distant variants, such as XBB.1.5, BA.1, and JN.1, tend to adopt more compact conformational states compared to the WT. Additionally, these variants exhibit novel NC profiles, characterized by an increased number of specific contacts distributed among ionic, polar, and nonpolar residues. We further analyzed the impact of specific mutations, including T478K, N500Y, and Y504H. These mutations not only enhance interactions with the human host receptor but also alter inter-chain stability by introducing additional NCs compared to the WT. Consequently, these mutations may influence the accessibility of certain protein regions to neutralizing antibodies. Overall, these findings contribute to a deeper understanding of the structural and functional variations among S protein variants.

The ACE2 Receptor for COVID-19 Entry Is Expressed on the Surface of Hematopoietic Stem/Progenitor Cells and Endothelial Progenitors As Well As Their Precursor Cells and Becomes Activated in Nlrp3 Inflammasome-Dependent Manner By Virus Spike Protein - a Potential Pathway Leading to a “Cytokine Storm”