Mutations In Receptor-binding Domain Research Articles

Epitope Classification and RBD Binding Properties of Neutralizing Antibodies Against SARS-CoV-2 Variants of Concern.

Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2) causes coronavirus disease 2019 (COVID19) that is responsible for short and long-term disease, as well as death, in susceptible hosts. The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein binds to cell surface angiotensin converting enzyme type-II (ACE2) to initiate viral attachment and ultimately viral pathogenesis. The SARS-CoV-2 S RBD is a major target of neutralizing antibodies (NAbs) that block RBD - ACE2 interactions. In this report, NAb-RBD binding epitopes in the protein databank were classified as C1, C1D, C2, C3, or C4, using a RBD binding profile (BP), based on NAb-specific RBD buried surface area and used to predict the binding epitopes of a series of uncharacterized NAbs. Naturally occurring SARS-CoV-2 RBD sequence variation was also quantified to predict NAb binding sensitivities to the RBD-variants. NAb and ACE2 binding studies confirmed the NAb classifications and determined whether the RBD variants enhanced ACE2 binding to promote viral infectivity, and/or disrupted NAb binding to evade the host immune response. Of 9 single RBD mutants evaluated, K417T, E484K, and N501Y disrupted binding of 65% of the NAbs evaluated, consistent with the assignment of the SARS-CoV-2 P.1 Japan/Brazil strain as a variant of concern (VoC). RBD variants E484K and N501Y exhibited ACE2 binding equivalent to a Wuhan-1 reference SARS-CoV-2 RBD. While slightly less disruptive to NAb binding, L452R enhanced ACE2 binding affinity. Thus, the L452R mutant, associated with the SARS-CoV-2 California VoC (B.1.427/B.1.429-California), has evolved to enhance ACE2 binding, while simultaneously disrupting C1 and C2 NAb classes. The analysis also identified a non-overlapping antibody pair (1213H7 and 1215D1) that bound to all SARS-CoV-2 RBD variants evaluated, representing an excellent therapeutic option for treatment of SARS-CoV-2 WT and VoC strains.

Nasal delivery of an IgM offers broad protection from SARS-CoV-2 variants.

Resistance represents a major challenge for antibody-based therapy for COVID-191-4. Here we engineered an immunoglobulinM (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulinG (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044mg per kg body weight confers prophylactic efficacy and a single dose at 0.4mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.

A Bioluminescent Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and In Vitro.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus–host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus–cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.

Immune response to SARS-CoV-2 variants of concern in vaccinated individuals

SARS-CoV-2 is evolving with mutations in the receptor binding domain (RBD) being of particular concern. It is important to know how much cross-protection is offered between strains following vaccination or infection. Here, we obtain serum and saliva samples from groups of vaccinated (Pfizer BNT-162b2), infected and uninfected individuals and characterize the antibody response to RBD mutant strains. Vaccinated individuals have a robust humoral response after the second dose and have high IgG antibody titers in the saliva. Antibody responses however show considerable differences in binding to RBD mutants of emerging variants of concern and substantial reduction in RBD binding and neutralization is observed against a patient-isolated South African variant. Taken together our data reinforce the importance of the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies and high antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant further highlights the importance of surveillance strategies to detect new variants and targeting these in future vaccines.

Monoclonal Antibodies for Early Treatment of COVID-19 in a World of Evolving SARS-CoV-2 Mutations and Variants.

Monoclonal antibodies targeting the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 spike protein are important outpatient treatment options in coronavirus disease 2019 to mitigate progression of disease and prevent hospitalization. The impact of different RBD mutations on the efficacy of the available monoclonal antibodies and processes for incorporating this impact into treatment algorithms are ill defined. Herein, we synthesize the data surrounding the impact of key RBD mutations on the efficacy of US Food and Drug Administration Emergency Use Authorized monoclonal antibodies and describe our approach at Michigan Medicine at monitoring mutation frequency in circulating virus and developing an algorithm that incorporates these data into outpatient treatment pathways.

In silico binding profile characterization of SARS-CoV-2 spike protein and its mutants bound to human ACE2 receptor.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), a novel coronavirus, has brought an unprecedented pandemic to the world and affected over 64 million people. The virus infects human using its spike glycoprotein mediated by a crucial area, receptor-binding domain (RBD), to bind to the human ACE2 (hACE2) receptor. Mutations on RBD have been observed in different countries and classified into nine types: A435S, D364Y, G476S, N354D/D364Y, R408I, V341I, V367F, V483A and W436R. Employing molecular dynamics (MD) simulation, we investigated dynamics and structures of the complexes of the prototype and mutant types of SARS-CoV-2 spike RBDs and hACE2. We then probed binding free energies of the prototype and mutant types of RBD with hACE2 protein by using an end-point molecular mechanics Poisson Boltzmann surface area (MM-PBSA) method. According to the result of MM-PBSA binding free energy calculations, we found that V367F and N354D/D364Y mutant types showed enhanced binding affinities with hACE2 compared to the prototype. Our computational protocols were validated by the successful prediction of relative binding free energies between prototype and three mutants: N354D/D364Y, V367F and W436R. Thus, this study provides a reliable computational protocol to fast assess the existing and emerging RBD mutations. More importantly, the binding hotspots identified by using the molecular mechanics generalized Born surface area (MM-GBSA) free energy decomposition approach can guide the rational design of small molecule drugs or vaccines free of drug resistance, to interfere with or eradicate spike-hACE2 binding.

Vaccine-escape and fast-growing mutations in the United Kingdom, the United States, Singapore, Spain, India, and other COVID-19-devastated countries

Recently, the SARS-CoV-2 variants from the United Kingdom (UK), South Africa, and Brazil have received much attention for their increased infectivity, potentially high virulence, and possible threats to existing vaccines and antibody therapies. The question remains if there are other more infectious variants transmitted around the world. We carry out a large-scale study of 506,768 SARS-CoV-2 genome isolates from patients to identify many other rapidly growing mutations on the spike (S) protein receptor-binding domain (RBD). We reveal that essentially all 100 most observed mutations strengthen the binding between the RBD and the host angiotensin-converting enzyme 2 (ACE2), indicating the virus evolves toward more infectious variants. In particular, we discover new fast-growing RBD mutations N439K, S477N, S477R, and N501T that also enhance the RBD and ACE2 binding. We further unveil that mutation N501Y involved in United Kingdom (UK), South Africa, and Brazil variants may moderately weaken the binding between the RBD and many known antibodies, while mutations E484K and K417N found in South Africa and Brazilian variants, L452R and E484Q found in India variants, can potentially disrupt the binding between the RBD and many known antibodies. Among these RBD mutations, L452R is also now known as part of the California variant B.1.427. Finally, we hypothesize that RBD mutations that can simultaneously make SARS-CoV-2 more infectious and disrupt the existing antibodies, called vaccine escape mutations, will pose an imminent threat to the current crop of vaccines. A list of most likely vaccine escape mutations is given, including S494P, Q493L, K417N, F490S, F486L, R403K, E484K, L452R, K417T, F490L, E484Q, and A475S. Mutation T478K appears to make the Mexico variant B.1.1.222 the most infectious one. Our comprehensive genetic analysis and protein-protein binding study show that the genetic evolution of SARS-CoV-2 on the RBD, which may be regulated by host gene editing, viral proofreading, random genetic drift, and natural selection, gives rise to more infectious variants that will potentially compromise existing vaccines and antibody therapies.

Mutation Signatures and In Silico Docking of Novel SARS-CoV-2 Variants of Concern.

One year since the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China, several variants of concern (VOC) have appeared around the world, with some variants seeming to pose a greater thread to public health due to enhanced transmissibility or infectivity. This study provides a framework for molecular characterization of novel VOC and investigates the effect of mutations on the binding affinity of the receptor-binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2) using in silico approach. Notable nonsynonymous mutations in RBD of VOC include the E484K and K417N/T that can be seen in South African and Brazilian variants, and N501Y and D614G that can be seen in all VOC. Phylogenetic analyses demonstrated that although the UK-VOC and the BR-VOC fell in the clade GR, they have different mutation signatures, implying an independent evolutionary pathway. The same is true about SA-VOC and COH-VOC felling in clade GH, but different mutation signatures. Combining molecular interaction modeling and the free energy of binding (FEB) calculations for VOC, it can be assumed that the mutation N501Y has the highest binding affinity in RBD for all VOC, followed by E484K (only for BR-VOC), which favors the formation of a stable complex. However, mutations at the residue K417N/T are shown to reduce the binding affinity. Once vaccination has started, there will be selective pressure that would be in favor of the emergence of novel variants capable of escaping the immune system. Therefore, genomic surveillance should be enhanced to find and monitor new emerging SARS-CoV-2 variants before they become a public health concern.

In silico Identification of a Viral Surface Glycoprotein Site Suitable for the Development of Low Molecular Weight Inhibitors for Various Variants of the SARS-CoV-2

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is a new coronavirus that today has an extremely significant impact on both global economy and society as a whole, due to its pandemic status and risk of complications. Therefore, understanding the molecular features of the interaction of receptor binding domain (RBD), which determines most of the dangerous properties of this pathogen, with human angiotensin-converting enzyme 2 (hACE2) is an important step in the process of developing a successful strategy to combat SARS-CoV-2. In addition, given the significant rate of accumulation of mutations in RBD, it makes sense to consider its different variants. Goal. Identification of a pocket potentially suitable for the search for low molecular weight inhibitors of interaction of different variants of SARS-CoV-2 RBD and hACE2. Methods. The initial structure of different variants of the RBD/hACE2 complex was obtained from Protein Data Bank (PDB). Separate RBD variants were isolated from the same data. To obtain the Y453F mutant, variant P.1 was mutagenized in PyMol 1.8. The construction of the system, which included the resulting associate or individual protein, solvent and physiological concentration of sodium chloride, was performed using CHARMM-GUI (graphical user interface for CHARMM) tools according to the standard protocol for glycoproteins. The actual simulation and balancing of the system was performed in GROMACS (GROningen MAchine for Chemical Simulation) version 2019.6 for 50 ns. Results. The interface of RBD/hACE2 interaction is formed by amino acids Q24, D30, H34, E35, E37, Y41, Y83, K353, D355 and R393 – for hACE2 and K417, Y453, F486, N487, Y489, Q493, Q498, T500, N501, Y505 – for RBD. However, it is heterogeneous and can be divided into two subinterfaces, and each includes its own pool of interactions: hACE2 Q24/Y83 + RBD N487, hACE2 H34 + RBD Y453, hACE2 E35 + RBD Q493 and hACE2 D30 + RBD K417 – for N- terminal relative to H1 hACE2 subinterface and hACE2 E37/R393 + RBD Y505, hACE2 K353 + RBD Q498/G502 and hACE2 D355 + RBD T500 – for C-terminal. According to the considered N501Y mutation, changes are observed in the mentioned interaction patterns – hydrogen bonds of hACE2 Q42 + RBD Q498, hACE2 K31 + RBD Q493 and hACE2 K31 + RBD F490 are formed, and hACE2 H34 + RBD Y453 is lost. Similar aberrations, except for the hydrogen bond with F490, are observed in the case of the N501Y + Y453F variant. Despite significant changes in the pool of interactions, the gross number of hydrogen bonds for the complexes of all three variants is relatively stable and ranges from 9 to 10. Between the defined interaction subinterfaces for all considered variants of RBD are characterized by the presence of a pocket, which is formed by residues R403, Y453, Q493, S494, Y495, G496, F497, Q498, N501 and Y505 conditionally original variant. According to the results of the molecular dynamics simulation, the Y453F replacement has little effect on the overall topology of the cavity, but sufficiently reduces the polarity of the pocket part of its localization, which leads to the impossibility of forming any polar interactions. In contrast, N501Y, due to the larger size of the tyrosine radical and the presence of parahydroxyl, forms two equivalent mutually exclusive hydrogen bonds with the carbonyls of the peptide groups G496 and Y495. Additional stabilization of the Y501 is provided by interplanar stacking with the Y505. In addition to the anchored position in ~ 25% of the trajectory there is another “open” conformation Y501. At which the radical of this tyrosine does not interact with the rest of the protein. Conclusions. 1) The interface of interaction of SARS-CoV-2 RBD with hACE2 is not continuous and it can be conditionally divided into two subiterfaces: N-terminal and C-terminal. Each is characterized by its own pattern of connections and changes according to the RBD N501Y and Y453F replacements we have considered. However, despite the presence of significant molecular rearrangements caused by N501Y and Y453F, the total number of hydrogen bonds is relatively the same for all mutants. 2) Between the identified interaction subinterfaces, SARS-CoV-2 RBD contains caveola, which due to its location may be potentially suitable for finding promising candidates for drugs aimed at inhibiting the interaction of this protein with hACE2. In this case, the substitutions of N501Y and Y453F have a significant impact on the topology of a particular pocket and can potentially modify the activity of inhibitors directed to this area.

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

SummaryMonoclonal antibodies and antibody cocktails are a promising therapeutic and prophylaxis for coronavirus disease 2019 (COVID-19). However, ongoing evolution of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) can render monoclonal antibodies ineffective. Here, we completely map all of the mutations to the SARS-CoV-2 spike receptor-binding domain (RBD) that escape binding by a leading monoclonal antibody, LY-CoV555, and its cocktail combination with LY-CoV016. Individual mutations that escape binding by each antibody are combined in the circulating B.1.351 and P.1 SARS-CoV-2 lineages (E484K escapes LY-CoV555, K417N/T escapes LY-CoV016). In addition, the L452R mutation in the B.1.429 lineage escapes LY-CoV555. Furthermore, we identify single amino acid changes that escape the combined LY-CoV555+LY-CoV016 cocktail. We suggest that future efforts diversify the epitopes targeted by antibodies and antibody cocktails to make them more resilient to the antigenic evolution of SARS-CoV-2.

Multivalent Display of SARS-CoV-2 Spike (RBD Domain) of COVID-19 to Nanomaterial, Protein Ferritin Nanocages.

SARS-CoV-2, or COVID-19, has a devastating effect on our society, both in terms of quality of life and death rates; hence, there is an urgent need for developing safe and effective therapeutics against SARS-CoV-2. The most promising strategy to fight against this deadly virus is to develop an effective vaccine. Internalization of SARS-CoV-2 into the human host cell mainly occurs through the binding of the coronavirus spike protein (a trimeric surface glycoprotein) to the human angiotensin-converting enzyme 2 (ACE2) receptor. The spike-ACE2 protein–protein interaction is mediated through the receptor-binding domain (RBD) of the spike protein. Mutations in the spike RBD can significantly alter interactions with the ACE2 host receptor. Due to its important role in virus transmission, the spike RBD is considered to be one of the key molecular targets for vaccine development. In this study, a spike RBD-based subunit vaccine was designed by utilizing a ferritin protein nanocage as a scaffold. Several fusion protein constructs were designed in silico by connecting the spike RBD via a synthetic linker (different sizes) to different ferritin subunits (H-ferritin and L-ferritin). The stability and the dynamics of the engineered nanocage constructs were tested by extensive molecular dynamics simulation (MDS). Based on our MDS analysis, a five amino acid-based short linker (S-Linker) was the most effective for displaying the spike RBD over the surface of ferritin. The behavior of the spike RBD binding regions from the designed chimeric nanocages with the ACE2 receptor was highlighted. These data propose an effective multivalent synthetic nanocage, which might form the basis for new vaccine therapeutics designed against viruses such as SARS-CoV-2.

Structural stability predictions and molecular dynamics simulations of RBD and HR1 mutations associated with SARS-CoV-2 spike glycoprotein

The COVID-19 pandemic is caused by human transmission and infection of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). There is no trusted drug against the virus; hence, efforts are on discovering novel inhibitors for the virus. The entry of a SARS-CoV-2 virus particle into a host cell is initiated by its spike glycoprotein and host Angiotensin-Converting Enzyme 2 (ACE2) receptor interaction. Spike glycoprotein domains, namely, the Receptor Binding Domain (RBD) and Heptad Repeat (HR) domains, are essential for this activity. We have studied the impact of mutations such as A348T, N354D, D364Y, G476S, V483A, S494D in the RBD (319–591), and S939F, S940T, T941A, S943P (912–984) in the HR1 domains of spike glycoprotein. Summarily, we utilized the computational screening algorithms to rank the deleterious, damaging and disease-associated spike glycoprotein mutations. Subsequently, to understand the changes in conformation, flexibility and function of the spike glycoprotein mutants, Molecular Dynamics (MD) simulations were performed. The computational predictions and analysis of the MD trajectories suggest that the RBD and HR1 mutations induce significant phenotypic effects on the pre-binding spike glycoprotein structure, which are presumably consequential to its binding to the receptor and provides lead to design inhibitors against the binding. Communicated by Ramaswamy H. Sarma

Dynamics of binding ability prediction between spike protein and human ACE2 reveals the adaptive strategy of SARS-CoV-2 in humans

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus causing the COVID-19 pandemic in 2020. High adaptive plasticity on the spike protein of SASR-CoV-2 enables it to transmit across different host species. In the present study, we collected 2092 high-quality genome sequences of SARS-CoV-2 from 160 regions in over 50 countries and reconstructed their phylogeny. We also analyzed the polymorphic interaction between spike protein and human ACE2 (hACE2). Phylogenetic analysis of SARS-CoV-2 suggests that SARS-CoV-2 is probably originated from a recombination event on the spike protein between a bat coronavirus and a pangolin coronavirus that endows it humans infectivity. Compared with other regions in the S gene of SARS-CoV-2, the direct-binding sites of the receptor-binding domain (RBD) is more conserved. We focused on 3,860 amino acid mutations in spike protein RBD (T333-C525) of SARS-CoV-2 and simulated their differential stability and binding affinity to hACE2 (S19-D615). The results indicate no preference for SARS-CoV-2 infectivity on people of different ethnic groups. The variants in the spike protein of SARS-CoV-2 may also be a good indicator demonstrating the transmission route of SARS-CoV-2 from its natural reservoir to human hosts.

Evolution of antibody immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) has infected 78million individuals and is responsible for over 1.7million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory Bcells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87individuals assessed at 1.3 and 6.2months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory Bcells remains unchanged at 6.2months after infection. Memory Bcells display clonal turnover after 6.2months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4months after the onset of coronavirus disease2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14individuals. We conclude that the memory Bcell response to SARS-CoV-2 evolves between 1.3 and 6.2months after infection in a manner that is consistent with antigen persistence.

Toll-Like Receptor Agonist R848 Protects Mice from Lethal SARS-CoV-2 Infections

Background: An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Methods: We generated a mouse-adapted strain SARS-CoV-2 by serial passages in the lung of BALB/c mice. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments demonstrated that two mutations in RBD significantly increased its binding affinity towards mouse ACE2. Significantly, TLR7/8 agonist Resiquimod block SARS-CoV-2 in vitro and in vivo. Findings: We adapted a wild-type SARS-CoV-2 by serial passages in the lung of BALB/c mice. The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, The TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge, demonstrating this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. Interpretation: We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies, especially in determining the immunopathological consequences of any intervention. This study also verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo.Funding Statement: This research was funded by Emergency Science and Technology Project of Hubei Province（2020FCA046）and Independent Science and Technology Innovation Fund of Huazhong Agricultural University in 2020 (2662020PY002).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The animal experiments were approved by the Research Ethics Committee, Huazhong Agricultural University, Hubei, China (HZAUMO-2020-0007). All the animal experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the Research Ethics Committee, Huazhong Agricultural University, Hubei, China.

Recurrent Emergence and Transmission of a SARS-CoV-2 Spike Deletion H69/V70

SARS-CoV-2 Spike amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six-nucleotide out of frame deletion in the S gene, which results in loss of two amino acids: H69 and V70. We show that 𝚫H69/V70 and other common NTD deletions occur at loop structures in RNA where polymerase activity is often compromised. In addition, the same two amino acid deletion is also observed in a set of closely related pangolin sarbecoviruses, but not in the bat sarbecovirus RaTG13. We report that in human infections 𝚫H69/V70 often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F, and in the latter two cases has followed the RBD mutation suggesting an epistatic interaction. One of the +.-|.-|.-| N501Y lineages, B.1.1.7, has undergone rapid expansion and includes eight other S gene mutations: RBD (N501Y and A570D), S1 (𝚫H69/V70 and 𝚫144/145) and S2 (P681H, T716I, S982A and D1118H). In vitro, we show that 𝚫H69/V70 does not impact serum neutralisation across multiple convalescent sera. However, the 𝚫H69/V70 does increase infectivity of Spike and is able to compensate for infectivity defects induced by RBD mutations N501Y, N439K and Y453F. In addition replacement of H69 and V70 in B.1.1.7 Spike substantially reduces its infectivity. Mechanistically, we demonstrate that 𝚫H69/V70 is associated with two fold increased Spike incorporation into virions, though it does not increase cell-cell fusion. Based on our data 𝚫H69/V70 increases Spike infectivity, likely acting as a permissive mutation that allows acquisition of otherwise deleterious immune escape mutations. Enhanced surveillance for the 𝚫H69/V70 deletion with and without RBD mutations should be considered as a global priority not only as a marker for the B.1.1.7 variant, but potentially also for other emerging variants of concern.Funding Statement: RKG is supported by a Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA). COG-UK is supported by funding from the Medical Research Council (MRC) & UK&ch & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute. SAK is supported by the Bill and Melinda Gates Foundation via PANGEA grant: OPP1175094. DLR is funded by the MRC (MC UU 1201412). WH is funded by the MRC (MR/R024758/1).We thank Dr James Voss for the kind gift of HeLa cells stably expressing ACE2. SL is funded by Medical Research Council MC_UU_12014/12. This study was also partly funded by Rosetrees Trust. Work in the AML lab is supported by NIHR Biomedical Research Centre Grant RCAG 817.Declaration of Interests: RKG has received consulting fees from UMOVIS lab, Gilead Sciences and ViiV Healthcare, and a research grant from InvisiSmart Technologies. On behalf of COG-UK, there are no other interests to declare.Ethics Approval Statement: Ethical approval for use of serum samples. Controls with COVID-19 were enrolled to the NIHR BioResource Centre Cambridge under ethics review board (17/EE/0025).

Facing the challenge of viral mutations in the age of pandemic: Developing highly potent, broad-spectrum, and safe COVID-19 vaccines and therapeutics.

Facing the challenge of viral mutations in the age of pandemic: Developing highly potent, broad-spectrum, and safe COVID-19 vaccines and therapeutics.

Epistasis at the SARS-CoV-2 RBD Interface and the Propitiously Boring Implications for Vaccine Escape

Emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlaps the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Epistasis among RBD mutations can increase the risk of vaccine escape. We compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for wild type (WT), Gamma and Delta variants using Rosetta. Epistasis at the RBD surface appears to be limited and the effects of most multiple mutations are additive. For Delta variant, epistasis weakly stabilizes NAb interaction relative to ACE2, whereas in Gamma, epistasis more substantially destabilizes NAb interaction. Thus, the repertoire of potential escape mutations for Delta is not substantially different from that of WT, whereas Gamma poses a moderately greater risk of vaccine escape.Funding: Intramural Research Program of the National Institutes of Health of the USA (National Library of Medicine), to E. V. KooninDeclaration of Interests: The declare they have no competing interests.

Cross-Species Tropism and Antigenic Landscapes of Circulating SARS-CoV-2 Variants

Mutations in the SARS-CoV-2 spike receptor-binding domain (RBD) may alter the viral host tropism and affect the activities of neutralizing antibodies (nAbs). Here, we investigated 153 RBD mutants and 10 globally circulating VOC/VOI variants for their cross-species infection potentials and sensitivities to nAbs. Several RBD mutations strengthened viral infectivity in cells-expressing ACE2 orthologs of non-human animals, particularly those less susceptible to the ancestral strain. Antigenic profiles revealed the mutations surrounding aa 439-448 and aa 484 were more likely to cause neutralization resistance. Strikingly, the enhanced cross-species infection potentials to the mouse and ferret, instead of the neutralization-escape scores of the mutations, accounted for the positive correlation with the current cumulative prevalence of mutations in humans. Our results provided prospective landscapes for the cross-species transmission potentials and antigenic drift of circulating spike mutants and highlighted the possible spillover of variants to originally-insusceptible animals in associating with their quick spread worldwide. Funding: This study was supported by the National Natural Science Foundation of China 81902057 (to Y.L.Z.), U1905205 (to Q.Y.), and 81871316 (to Q.Y.) and the CAMS Innovation Fund for Medical Sciences (No. 2019RU022 to N.X.). Declaration of Interests: None to declare. Ethics Approval Statement: The study was approved (number: SPHIRB-202101) by the institutional review board of the School of Public Health of Xiamen University following the Declaration of Helsinki, and written informed consent was obtained.Animal studies were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, and were conducted under the approval of the Institutional Animal Care and Use Committee of Xiamen University (mouse experiments) or JOINN Laboratories, Inc. (monkey experiments).

Thermodynamics and kinetics in antibody resistance of the 501Y.V2 SARS-CoV-2 variant.

Understanding the thermodynamics and kinetics of the binding process of an antibody to the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein is very important for the development of COVID-19 vaccines. In particular, it is essential to understand how the binding mechanism may change under the effects of RBD mutations. In this context, we have demonstrated that the South African variant (B1.351 or 501Y.V2) can resist the neutralizing antibody (NAb). Three substitutions in the RBD including K417N, E484K, and N501Y alter the free energy landscape, binding pose, binding free energy, binding kinetics, hydrogen bonding, nonbonded contacts, and unbinding pathway of RBD + NAb complexes. The low binding affinity of NAb to 501Y.V2 RBD confirms the antibody resistance of the South African variant. Moreover, the fragment of NAb + RBD can be used as an affordable model to investigate changes in the binding process between the mutated RBD and antibodies.