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.     

Experimental Evidence for Enhanced Receptor Binding by Rapidly Spreading SARS-CoV-2 Variants

Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 7-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation only slightly enhances the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 2-fold weaker than N501Y. However, the recently emerging double mutant E484K/N501Y binds even stronger than N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.     

SARS-CoV-2 VOCs,Mutational diversity and clinical outcome: Are they modulating drug efficacy by altered binding strength?

The global COVID-19 pandemic continues due to emerging Severe Acute Respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC). Here, we performed comprehensive analysis of in-house sequenced SARS-CoV-2 genome mutations dynamics in the patients infected with the VOCs - Delta and Omicron, within Recovered and Mortality patients. Statistical analysis highlighted significant mutations - T4685A, N4992N, and G5063S in RdRp; T19R in NTD spike; K444N and N532H in RBD spike, associated with Delta mortality. Mutations, T19I in NTD spike, Q493R and N440K in the RBD spike were significantly associated with Omicron mortality. We performed molecular docking for possible effect of significant mutations on the binding of Remdesivir. We found that Remdesivir showed less binding efficacy with the mutant Spike protein of both Delta and Omicron mortality compared to recovered patients. This indicates that mortality associated mutations could have a modulatory effect on drug binding which could be associated with disease outcome.     

A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the “class 3” epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.     

新型冠状病毒基因组序列的网络平台与基因分型

新型冠状病毒(SARS-CoV-2)是引起2019新型冠状病毒肺炎(COVID-19)的病原体,目前COVID-19仍在世界范围内大规模流行。随着学者对SARS-CoV-2研究的不断深入,以及各大数据库的序列资源共享,一些学者开发了SARS-CoV-2相关序列分析网络在线平台,并发表了对SARS-CoV-2基因分型、命名的规则。"GISAID"是目前SARS-CoV-2基因组序列共享和储存最大的数据库,"Nextstrain"和"CoV-GLUE"是国际最常用的SARS-CoV-2序列分析平台。目前有四种比较通用的SARS-CoV-2的基因分型方法,在本文中分别简称为:"中国分型法"、"Pangolin分型法"、"GISAID分型法"和"Nextstrain分型法"。这四种分型方法的定义不尽相同,但又有相似之处。本综述对目前SARS-CoV-2在线分析网络平台和不同的基因分型方法进行了较为系统的介绍、对比和总结。     

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002–2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.     

SARS-CoV-2 beta variant substitutions alter spike glycoprotein receptor binding domain structure and stability

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic have visited a terrible cost on the world in the forms of disease, death, and economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 have seemingly brought within reach the end of the pandemic. However, the virus has acquired mutations. and emerging variants of concern are more infectious and reduce the efficacy of existing vaccines. Although promising efforts to combat these variants are underway, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain of three amino-acid substitutions found in several variants of concern, including alpha (B.1.1.7), beta (B.1.351), and gamma (P.1). We found that these substitutions alter the receptor-binding domain structure, stability, and ability to bind to angiotensin converting enzyme 2, in such a way as to possibly have opposing and compensatory effects. These findings provide new insights into how these variants of concern may have been selected for infectivity while maintaining the structure and stability of the receptor binding domain.

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in late 2019 and its subsequent spread around the world have caused the deadliest airborne pandemic in the United States, recently surpassing the 1918 influenza pandemic nearly a century ago (1). The international scientific community has risen to the challenge of combating SARS-CoV-2 and COVID-19. The year 2020 ended with the fastest development of vaccine candidates, starting with the genetic sequence of the virus being reported (2) to human trials of novel mRNA-based vaccines within 3 months. Now, there are three SARS-CoV-2 vaccines approved for use within the United States and many more next-generation and pan-coronavirus vaccines currently in development. These advances have made substantial contributions to the control of the COVID-19 pandemic within the United States. Despite multiple manufacturers receiving emergency use authorization and an unprecedented vaccination campaign, significant challenges remain including uncertainty regarding durability, vaccination hesitancy, limited access to healthcare among disadvantaged persons, as well as the continued emergence of variants of concern (VOC). Our ultimate success in quelling this pandemic may lie in our ability, not only to characterize new variants, but also to be able to predict the emergence of new variants. Such advances will require an increased understanding of evolutionary pressures and constraints on viral variation.Three SARS-CoV-2 lineages, the alpha variant lineage B.1.1.7 (or 501Y.V1) first identified within the United Kingdom, the beta variant lineage B.1.351 (or 501Y.V2) identified in South Africa, and the gamma variant lineage P.1 (or 501Y.V3) identified in Brazil, have been demonstrated to possess increased infectivity (3) and in the case, beta and gamma exhibit reduced neutralization by antibodies reacting with the cognate regions of the spike protein within the original Wuhan strain of SARS-CoV-2 (4, 5, 6). The alpha variant possesses the N501Y substitution within the spike glycoprotein receptor-binding domain (RBD) which has been shown to enhance binding to angiotensin converting enzyme 2 (ACE2), the entry receptor for SARS-CoV-2 (7, 8, 9). The beta and gamma variants possess N501Y as well as substitutions at two other sites within the RBD, E484K, and K417N in beta and K417T in gamma (10). These RBD substitutions present in the spike protein of the B.1.351 and P.1 variants have been shown to reduce the binding and neutralization of mRNA vaccine-induced antibodies as well as potent human monoclonal antibodies (11).The consequences of the K417N, E484K, and N501Y substitutions on RBD-ACE2 interactions have also been examined, with the increased infectivity of the alpha variant resulting from the enhanced binding to ACE2 when the RBD N501Y substitution is present (9). The E484K substitution has been shown to enhance ACE2 binding (12) and reduce the efficacy of neutralizing antibodies (13). A recent study examined the effects of the K417N substitution on ACE2 binding and antibody interactions using molecular dynamics and found that K417N disrupts RBD-ACE2 interactions, as well as interactions with a monoclonal antibody (14). However, the effects of these substitutions on the structure of the RBD itself have not been examined. Based on the nature of these substitutions, including residue changes in charge or polar to nonpolar substitutions, we hypothesized that the K417N, E484K, and N501Y substitutions alter the RBD structure and stability as well as ACE2 binding interactions. We studied those changes in single-substitution RBD variants as well as in the RBD containing all three substitutions using molecular dynamics and biophysical approaches. Our data suggest that these VOC substitutions significantly alter RBD structure and stability, with consequences for ACE2 binding and proteolytic susceptibility, having potentially opposing consequences for the fitness of new variants. These findings have implications for viral evolution and the design of subunit vaccine candidates.     

In silico mutagenesis of human ACE2 with S protein and translational efficiency explain SARS-CoV-2 infectivity in different species

The coronavirus disease COVID-19 constitutes the most severe pandemic of the last decades having caused more than 1 million deaths worldwide. The SARS-CoV-2 virus recognizes the angiotensin converting enzyme 2 (ACE2) on the surface of human cells through its spike protein. It has been reported that the coronavirus can mildly infect cats, and ferrets, and perhaps dogs while not pigs, mice, chicken and ducks. Differences in viral infectivity among different species or individuals could be due to amino acid differences at key positions of the host proteins that interact with the virus, the immune response, expression levels of host proteins and translation efficiency of the viral proteins among other factors. Here, first we have addressed the importance that sequence variants of different animal species, human individuals and virus isolates have on the interaction between the RBD domain of the SARS-CoV-2 spike S protein and human angiotensin converting enzyme 2 (ACE2). Second, we have looked at viral translation efficiency by using the tRNA adaptation index. We find that integration of both interaction energy with ACE2 and translational efficiency explains animal infectivity. Humans are the top species in which SARS-CoV-2 is both efficiently translated as well as optimally interacting with ACE2. We have found some viral mutations that increase affinity for hACE and some hACE2 variants affecting ACE2 stability and virus binding. These variants suggest that different sensitivities to coronavirus infection in humans could arise in some cases from allelic variability affecting ACE2 stability and virus binding.     

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mutational Pattern in the Fourth Pandemic Phase in Greece

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%).     

Genomic and epidemiological characteristics of SARS-CoV-2 in Africa

Since late 2019, the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, has rapidly evolved to become a global pandemic. Each country was affected but with a varying number of infected cases and mortality rates. Africa was hit late by the pandemic but the number of cases rose sharply. In this study, we investigated 224 SARS-CoV-2 genome sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) in the early part of the outbreak, of which 69 were from Africa. We analyzed a total of 550 mutations by comparing them with the reference SARS-CoV-2 sequence from Wuhan. We classified the mutations observed based on country and region, and afterwards analyzed common and unique mutations on the African continent as a whole. Correlation analyses showed that the duo variants ORF1ab/RdRp 4715L and S protein 614G variants, which are strongly linked to fatality rate, were not significantly and positively correlated with fatality rates (r = -0.03757, P = 0.5331 and r = -0.2876, P = 0.6389, respectively), although increased number of cases correlated with number of deaths (r = 0.997, P = 0.0002). Furthermore, most cases in Africa were mainly imported from American and European countries, except one isolate with no mutation and was similar to the original isolate from Wuhan. Moreover, unique mutations specific to countries were identified in the early phase of the outbreak but these mutations were not regional-specific. There were common mutations in all isolates across the continent as well as similar isolate-specific mutations in different regions. Our findings suggest that mutation is rapid in SARS-CoV-2 in Africa and although these mutations spread across the continent, the duo variants could not possibly be the sole cause of COVID-19 deaths in Africa in the early phase of the outbreak.     

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.     

Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both “up” and “down” configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.     

Biophysical Fitness Landscape of the SARS-CoV-2 Delta Variant Receptor Binding Domain

Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased mortality among infected people. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single as well as the double mutations on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than increased receptor binding, is the main biophysical parameter that determined the fitness landscape of the Delta variant RBD.     

Virus-receptor interactions of SARS-CoV-2 spikereceptor-binding domain and human neuropilin-1 b1 domain

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wahan, China and it causes disease which is known as COVID-19. This infection spreads everywhere in the world, and it leads to an enormous number of death among individuals. The mystery issue about SARS-CoV-2 that appears not to have functions of a hemagglutinin and neuraminidase like other coronaviruses. Angiotensin-converting enzyme 2 (ACE2) is the main surface receptor for entering SARS-CoV-2 into the host cell. This entry process is mediated by binding the SARS-CoV-2 spike receptor-binding domain (RBD) to ACE2. Recently, researchers discover a new receptor responsible for the SARS-CoV-2 entry which is neuropilin-1 (NRP1). So, this work provides afford a knowledge of how the initial interaction between SARS-CoV-2 spike RBD and NRP1 b1 domain may occur. Understanding this interaction would be very necessary for drug design against SARS-CoV-2.     

Receptor-binding domain of SARS-CoV-2 spike protein efficiently inhibits SARS-CoV-2 infection and attachment to mouse lung

COVID-19, caused by a novel coronavirus, SARS-CoV-2, poses a serious global threat. It was first reported in 2019 in China and has now dramatically spread across the world. It is crucial to develop therapeutics to mitigate severe disease and viral spread. The receptor-binding domains (RBDs) in the spike protein of SARS-CoV and MERS-CoV have shown anti-viral activity in previous reports suggesting that this domain has high potential for development as therapeutics. To evaluate the potential antiviral activity of recombinant SARS-CoV-2 RBD proteins, we determined the RBD residues of SARS-CoV-2 using a homology search with RBD of SARS-CoV. For efficient expression and purification, the signal peptide of spike protein was identified and used to generate constructs expressing recombinant RBD proteins. Highly purified RBD protein fused with the Fc domain of human IgG showed potent anti-viral efficacy, which was better than that of a protein fused with a histidine tag. Intranasally pre-administrated RBD protein also inhibited the attachment of SARS-COV-2 to mouse lungs. These findings indicate that RBD protein could be used for the prevention and treatment of SARS-CoV-2 infection.     

Facing the wrath of enigmatic mutations: a review on the emergence of severe acute respiratory syndrome coronavirus 2 variants amid coronavirus disease-19 pandemic

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging respiratory virus responsible for the ongoing coronavirus disease 19 (COVID-19) pandemic. More than a year into this pandemic, the COVID-19 fatigue is still escalating and takes hold of the entire world population. Driven by the ongoing geographical expansion and upcoming mutations, the COVID-19 pandemic has taken a new shape in the form of emerging SARS-CoV-2 variants. These mutations in the viral spike (S) protein enhance the virulence of SARS-CoV-2 variants by improving viral infectivity, transmissibility and immune evasion abilities. Such variants have resulted in cluster outbreaks and fresh infection waves in various parts of the world with increased disease severity and poor clinical outcomes. Hence, the variants of SARS-CoV-2 pose a threat to human health and public safety. This review enlists the most recent updates regarding the presently characterized variants of SARS-CoV-2 recognized by the global regulatory health authorities (WHO, CDC). Based on the slender literature on SARS-CoV-2 variants, we collate information on the biological implications of these mutations on virus pathology. We also shed light on the efficacy of therapeutics and COVID-19 vaccines against the emerging SARS-CoV-2 variants.     

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.     

Revealing the Threat of Emerging SARS-CoV-2 Mutations to Antibody Therapies

The ongoing massive vaccination and the development of effective intervention offer the long-awaited hope to end the global rage of the COVID-19 pandemic. However, the rapidly growing SARS-CoV-2 variants might compromise existing vaccines and monoclonal antibody (mAb) therapies. Although there are valuable experimental studies about the potential threats from emerging variants, the results are limited to a handful of mutations and Eli Lilly and Regeneron mAbs. The potential threats from frequently occurring mutations on the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) to many mAbs in clinical trials are largely unknown. We fill the gap by developing a topology-based deep learning strategy that is validated with tens of thousands of experimental data points. We analyze 796,759 genome isolates from patients to identify 606 non-degenerate RBD mutations and investigate their impacts on 16 mAbs in clinical trials. Our findings, which are highly consistent with existing experimental results about Alpha, Beta, Gamma, Delta, Epsilon, and Kappa variants shed light on potential threats of 100 most observed mutations to mAbs not only from Eli Lilly and Regeneron but also from Celltrion and Rockefeller University that are in clinical trials. We unveil, for the first time, that high-frequency mutations R346K/S, N439K, G446V, L455F, V483F/A, F486L, F490L/S, Q493L, and S494P might compromise some of mAbs in clinical trials. Our study gives rise to a general perspective about how mutations will affect current vaccines.     

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.     

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.