Amin Addetia et al.
Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail
Cell, May 2023; doi.org/10.1016/j.celrep.2023.112621
Abstract
Continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is eroding antibody responses elicited by prior vaccination and infection. The SARS-CoV-2 receptor-binding domain (RBD) E406W mutation abrogates neutralization mediated by the REGEN-COV therapeutic monoclonal antibody (mAb) COVID-19 cocktail and the AZD1061 (COV2-2130) mAb. Here, we show that this mutation remodels the receptor-binding site allosterically, thereby altering the epitopes recognized by these three mAbs and vaccine-elicited neutralizing antibodies while remaining functional. Our results demonstrate the spectacular structural and functional plasticity of the SARS-CoV-2 RBD, which is continuously evolving in emerging SARS-CoV-2 variants, including currently circulating strains that are accumulating mutations in the antigenic sites remodeled by the E406W substitution.
Manon Ragonnet-Cronin et al.
Generation of SARS-CoV-2 escape mutations by monoclonal antibody therapy
Nature, June 2023; doi.org/10.1038/s41467-023-37826-w
Abstract
COVID-19 patients at risk of severe disease may be treated with neutralising monoclonal antibodies (mAbs). To minimise virus escape from neutralisation these are administered as combinations e.g. casirivimab+imdevimab or, for antibodies targeting relatively conserved regions, individually e.g. sotrovimab. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to detect emerging drug resistance in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the antibody epitopes and for casirivimab+imdevimab multiple mutations are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.
AminAddetia et al.
Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail
Cell, May 2023; doi.org/10.1016/j.celrep.2023.112621
Abstract
Continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is eroding antibody responses elicited by prior vaccination and infection. The SARS-CoV-2 receptor-binding domain (RBD) E406W mutation abrogates neutralization mediated by the REGEN-COV therapeutic monoclonal antibody (mAb) COVID-19 cocktail and the AZD1061 (COV2-2130) mAb. Here, we show that this mutation remodels the receptor-binding site allosterically, thereby altering the epitopes recognized by these three mAbs and vaccine-elicited neutralizing antibodies while remaining functional. Our results demonstrate the spectacular structural and functional plasticity of the SARS-CoV-2 RBD, which is continuously evolving in emerging SARS-CoV-2 variants, including currently circulating strains that are accumulating mutations in the antigenic sites remodeled by the E406W substitution.
Bin Ju et al
Omicron BQ.1.1 and XBB.1 unprecedentedly escape broadly neutralizing antibodies elicited by prototype vaccination
Cell, May 2023; doi.org/10.1016/j.celrep.2023.112532
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have seriously attacked the antibody barrier established by natural infection and/or vaccination, especially the recently emerged BQ.1.1 and XBB.1. However, crucial mechanisms underlying the virus escape and the broad neutralization remain elusive. Here, we present a panoramic analysis of broadly neutralizing activity and binding epitopes of 75 monoclonal antibodies isolated from prototype inactivated vaccinees. Nearly all neutralizing antibodies (nAbs) partly or totally lose their neutralization against BQ.1.1 and XBB.1. We report a broad nAb, VacBB-551, that effectively neutralizes all tested subvariants including BA.2.75, BQ.1.1, and XBB.1. We determine the cryoelectron microscopy (cryo-EM) structure of VacBB-551 complexed with the BA.2 spike and perform detailed functional verification to reveal the molecular basis of N460K and F486V/S mutations mediating the partial escape of BA.2.75, BQ.1.1, and XBB.1 from the neutralization of VacBB-551. Overall, BQ.1.1 and XBB.1 raised the alarm over SARS-CoV-2 evolution with unprecedented antibody evasion from broad nAbs elicited by prototype vaccination.
TomokazuTamura et al.
Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants
Nature, May 2023; doi.org/10.1038/s41467-023-38435-3
Abstract
In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.
Yuri Furusawa et al.
In SARS-CoV-2 delta variants, Spike-P681R and D950N promote membrane fusion, Spike-P681R enhances spike cleavage, but neither substitution affects pathogenicity in hamsters
eBioMedicine, April 2023; doi.org/10.1016/j.ebiom.2023.104561
Abstract
The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized.
Methods
We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics in vitro and in vivo.
Findings
S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3 cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected.
Interpretation
Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity.
JumpeiIto et al.
Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant
Nature, May 2023; doi.org/10.1038/s41467-023-38188-z
Abstract
In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.
Taha Y. Taha et al.
Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6
Nature, April 2023; doi.org/10.1038/s41467-023-37787-0
Abstract
Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectioussubgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.
Katrin Hufnagel et al.
Discovery and systematic assessment of early biomarkers that predict progression to severe COVID-19 disease
Nature, April 2023; doi.org/10.1038/s43856-023-00283-z
Abstract
The clinical course of COVID-19 patients ranges from asymptomatic infection, via mild and moderate illness, to severe disease and even fatal outcome. Biomarkers which enable an early prediction of the severity of COVID-19 progression, would be enormously beneficial to guide patient care and early intervention prior to hospitalization.
Methods
Here we describe the identification of plasma protein biomarkers using an antibody microarray-based approach in order to predict a severe cause of a COVID-19 disease already in an early phase of SARS-CoV-2 infection. To this end, plasma samples from two independent cohorts were analyzed by antibody microarrays targeting up to 998 different proteins.
Results
In total, we identified 11 promising protein biomarker candidates to predict disease severity during an early phase of COVID-19 infection coherently in both analyzed cohorts. A set of four (S100A8/A9, TSP1, FINC, IFNL1), and two sets of three proteins (S100A8/A9, TSP1, ERBB2 and S100A8/A9, TSP1, IFNL1) were selected using machine learning as multimarker panels with sufficient accuracy for the implementation in a prognostic test.
Conclusions
Using these biomarkers, patients at high risk of developing a severe or critical disease may be selected for treatment with specialized therapeutic options such as neutralizing antibodies orantivirals. Early therapy through early stratification may not only have a positive impact on the outcome ofindividual COVID-19 patients but could additionally prevent hospitals from being overwhelmed in potential future pandemic situations.
Muhammad Suleman et al.
Structural insights into the effect of mutations in the spike protein of SARS-CoV-2 on the binding with human furin protein
Cell, March 2023; doi.org/10.1016/j.heliyon.2023.e15083
Abstract
The SARS COV-2 and its variants are spreading around the world at an alarming speed, due to its higher transmissibility and the conformational changes caused by mutations. The resulting COVID-19 pandemic has imposed severe health consequences on human health. Several countries of the world including Pakistan have studied its genome extensively and provided productive findings. In the current study, the mCSM, DynaMut2, and I-Mutant servers were used to analyze the effect of identified mutations on the structural stability of spike protein however, the molecular docking and simulations approaches were used to evaluate the dynamics of the bonding network between the wild-type and mutant spike proteins with furin. We addressed the mutational modifications that have occurred in the spike protein of SARS-COV-2 that were found in 215 Pakistani's isolates of COVID-19 patients to study the influence of mutations on the stability of the protein and its interaction with the host cell. We found 7 single amino acid substitute mutations in various domains that reside in spike protein. The H49Y, N74K, G181V, and G446V were found in the S1 domain while the D614A, V622F, and Q677H mutations were found in the central helices of the spike protein. Based on the observation, G181V, G446V, D614A, and V622F mutants were found highly destabilizing and responsible for structural perturbation. Protein-protein docking and molecular simulation analysis with that of furin have predicted that all the mutants enhanced the binding efficiency however, the V622F mutant has greatly altered the binding capacity which is further verified by the KD value (7.1 E−14) and therefore may enhance the spike protein cleavage by Furin and increase the rate of infectivity by SARS-CoV-2. On the other hand, the total binding energy for each complex was calculated which revealed −50.57 kcal/mol for the wild type, for G181V −52.69 kcal/mol, for G446V −56.44 kcal/mol, for D614A −59.78 kcal/mol while for V622F the TBE was calculated to be −85.84 kcal/mol. Overall, the current finding shows that these mutations have increased the binding of Furin for spike protein and shows that D614A and V622F have significant effects on the binding and infectivity.
HuiminGuo et al.
Additional mutations based on Omicron BA.2.75 mediate its further evasion from broadly neutralizing antibodies
Cell, February 2023; doi.org/10.1016/j.isci.2023.106283
Abstract
SARS-CoV-2 Omicron BA.2.75 subvariant has evolved to a series of progeny variants carrying several additional mutations in the receptor-binding domain (RBD). Here, we investigated whether and how these single mutations based on BA.2.75 affect the neutralization of currently available anti-RBD monoclonal antibodies (mAbs) with well-defined structural information. Approximately 34% of mAbs maintained effective neutralizing activities against BA.2.75, consistent with that against BA.2, BA.4/5, and BA.2.12.1. Single additional R346T, K356T, L452R, or F486S mutations further facilitated BA.2.75-related progeny variants to escape from broadly neutralizing antibodies (bnAbs) at different degree. Only LY-CoV1404 (bebtelovimab) displayed a first-class neutralization potency and breadth against all tested Omicron subvariants. Overall, these data make a clear connection between virus escape and antibody recognizing antigenic epitopes, which facilitate to develop next-generation universal bnAbs against emerging SARS-CoV-2 variants.
Dadonaite B et al.
A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike
CELL, February 2023; doi.org/10.1016/j.cell.2023.02.001
Abstract
A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7000 distinct amino-acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.
Pastorino C. et al.
Determinants of Spike infectivity, processing, and neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2
Cell, July 2022; doi.org/10.1016/j.chom.2022.07.006
Abstract
SARS-CoV-2 Omicron rapidly outcompeted other variants and currently dominates the COVID-19 pandemic.Its enhanced transmission and immune evasion are thought to be driven by numerous mutations in the OmicronSpike protein. Here, we systematically introduced BA.1 and/or BA.2 Omicron Spike mutations into theancestral Spike protein and examined the impacts on Spike function, processing, and susceptibility toneutralization. Individual mutations of S371F/L, S375F, and T376A in the ACE2-receptor-binding domainas well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes to G339D, D614G,N764K, and L981F moderately enhanced it. Most mutations in the N-terminal region and receptor-bindingdomain reduced the sensitivity of the Spike protein to neutralization by sera from individuals vaccinatedwith the BNT162b2 vaccine and by therapeutic antibodies. Our results represent a systematic functional analysisof Omicron Spike adaptations that have allowed this SARS-CoV-2 variant to dominate the currentpandemic.
Gilbert P.B. et al.
A Covid-19 Milestone Attained — A Correlate of Protection for Vaccines
NEJM, December 2022; doi/full/10.1056/NEJMp2211314
Abstract
The rapid identification of a correlate of protection (CoP) for Covid-19 vaccines — on the basis of several harmonized randomized phase 3 trials using common validated assays — constitutes an important success in vaccinology. A CoP is an immune marker that can be used to reliably predict a vaccine’s level of efficacy in preventing a clinically relevant outcome. The level of this marker is measured shortly (2 to 4 weeks) after completion of the vaccination regimen and provides an actionable basis for decisions such as regulatory approval of an efficacious vaccine for a new population that was not included in the pivotal randomized phase 3 trials, or approval of a refined version of a vaccine that was previously shown to be efficacious.
Once established, a CoP can be used as the primary end point for provisional or full approval of a vaccine for a specific use, if a clinical immunobridging study confirms that high enough levels of the CoP are achieved. For example, the Food and Drug Administration (FDA) extended approval of the mRNA-1273 (Moderna) and BNT162b2 (Pfizer–BioNTech) Covid vaccines from older to younger age groups on the basis of a comparison of neutralizing antibody titers. Moreover, FDA guidance and a European Medicines Agency declaration from the International Coalition of Medicines Regulatory Authorities recommended that approval of new vaccine strains and booster doses be based on clinical immunobridging studies showing noninferiority or superiority with respect to a CoP end point. Other applications of a CoP include ensuring vaccine consistency from lot to lot, supporting recommendations for coadministration with other vaccines, and determination of appropriate expiration dates.
Confusion about CoPs is understandable, given the myriad complicated issues involved in identifying them and the fact that different uses for CoPs require different validation measures. Evidence that a marker is a CoP is generally derived from five main sources: natural history studies that correlate infection-induced immune responses with outcomes; vaccine-challenge studies in animals or humans; studies that experimentally manipulate the immune marker to directly assess mechanistic causation (e.g., by administering various vaccine doses or using passive antibody transfer); efficacy trials that quantify the relationship between vaccine efficacy and the level of the immune marker in individual vaccine recipients; and meta-analyses of series of efficacy trials that correlate vaccine efficacy with the mean immune-marker level.
Correlation between Covid-19 Vaccine Efficacy and Neutralizing Antibody Titers.
Strong evidence has been generated from all five of these sources for both serum anti-spike IgG concentration and anti–SARS-CoV-2 neutralizing antibody titer as CoPs for vaccines against symptomatic Covid-19; for brevity, we focus here on the neutralizing antibody titer. Meta-analyses have established high correlations between the standardized mean titer and vaccine efficacy, and the neutralizing antibody titer has consistently been shown to be a mechanistic CoP in challenge studies in nonhuman primates. The U.S. government’s COVID-19 Vaccine Correlates of Protection Program assessed CoPs in phase 3 trials of four vaccines: COVE for mRNA-1273,1 ENSEMBLE for Ad26.COV2.S,2 PREVENT-19 for NVX-CoV2373,3 AZD1222 (United States/Chile/Peru) for ChAdOx1 nCoV-19, and COV002 (United Kingdom) also for ChAdOx1 nCoV-19.4 Vaccine efficacy always markedly increased with the titer (see graphs).
Both binding and neutralizing antibodies have been accepted as CoPs by regulators and have provided very high value for vaccine research, development, and use for more than a dozen vaccines against diverse viral or bacterial diseases. Large studies have generated robust evidence that these antibody markers are CoPs for Covid-19 vaccines — indeed, more evidence than is available for many CoPs for other types of vaccines. The FDA has accepted the titer of neutralizing antibodies against likely circulating strains as a CoP for multiple Covid-19 vaccines. Many open questions remain, given that this CoP was identified in trials involving people who had not previously been infected with SARS-CoV-2 and who received intramuscular, spike-only vaccines and were then exposed to pre-delta viruses. Nevertheless, while pursuing the next milestones — identifying CoPs for new viral variants, for new populations including previously infected people, for new vaccine classes, and for various aspects of Covid-19 disease (e.g., symptom types, durations, and severities) — we should acknowledge that neutralizing antibodies are the current CoP for vaccine efficacy, which merits use for near-term decisions about vaccines.
Heilmann E. et al.
SARS-CoV-2 3CLpro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376
Science, October 2022; doi/10.1126/scitranslmed.abq7360
Abstract
Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CLpro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CLpro, and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CLpro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.
Yuyong Zhou et al.
Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system
Science, December 2022; doi/10.1126/sciadv.add7197
Abstract
The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.
