ABSTRACT
Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity is recovered in the presence of heparin sulfate, which targets the 'N354 pocket' to ease restrictions of conformational transition resulting in a 'RBD-up' state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms.
ABSTRACT
Spike-protein-based pseudotyped viruses were used to evaluate vaccines during the COVID-19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus-like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS-COV-1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild-type VLP pseudotyped virus over 100-fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS-CoV-2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze-thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra- and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high-throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS-CoV-2 based on VLP pseudotyped virus.
ABSTRACT
Neutralizing antibodies targeting the spike (S) protein of SARS-CoV-2, elicited either by natural infection or vaccination, are crucial for protection against the virus. Nonetheless, the emergence of viral escape mutants presents ongoing challenges by contributing to breakthrough infections. To define the evolution trajectory of SARS-CoV-2 within the immune population, we co-incubated replication-competent rVSV/SARS-CoV-2/GFP chimeric viruses with sera from COVID-19 convalescents. Our findings revealed that the E484D mutation contributes to increased viral resistant against both convalescent and vaccinated sera, while the L1265R/H1271Y double mutation enhanced viral infectivity in 293T-hACE2 and Vero cells. These findings suggest that under the selective pressure of polyclonal antibodies, SARS-CoV-2 has the potential to accumulate mutations that facilitate either immune evasion or greater infectivity, facilitating its adaption to neutralizing antibody responses. Although the mutations identified in this study currently exhibit low prevalence in the circulating SARS-CoV-2 populations, the continuous and meticulous surveillance of viral mutations remains crucial. Moreover, there is an urgent necessity to develop next-generation antibody therapeutics and vaccines that target diverse, less mutation-prone antigenic sites to ensure more comprehensive and durable immune protection against SARS-CoV-2.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Mutation , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , SARS-CoV-2/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Humans , COVID-19/immunology , COVID-19/virology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/immunology , Antibodies, Viral/blood , Animals , Chlorocebus aethiops , Vero Cells , Immune Evasion , HEK293 CellsABSTRACT
The recent emergence of a SARS-CoV-2 saltation variant, BA.2.87.1, which features 65 spike mutations relative to BA.2, has attracted worldwide attention. In this study, we elucidate the antigenic characteristics and immune evasion capability of BA.2.87.1. Our findings reveal that BA.2.87.1 is more susceptible to XBB-induced humoral immunity compared to JN.1. Notably, BA.2.87.1 lacks critical escaping mutations in the receptor binding domain (RBD) thus allowing various classes of neutralizing antibodies (NAbs) that were escaped by XBB or BA.2.86 subvariants to neutralize BA.2.87.1, although the deletions in the N-terminal domain (NTD), specifically 15-23del and 136-146del, compensate for the resistance to humoral immunity. Interestingly, several neutralizing antibody drugs have been found to restore their efficacy against BA.2.87.1, including SA58, REGN-10933 and COV2-2196. Hence, our results suggest that BA.2.87.1 may not become widespread until it acquires multiple RBD mutations to achieve sufficient immune evasion comparable to that of JN.1.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Immune Evasion , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , SARS-CoV-2/immunology , SARS-CoV-2/genetics , Antibodies, Neutralizing/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , COVID-19/immunology , COVID-19/virology , Antibodies, Viral/immunology , Humans , Mutation , Animals , Antigens, Viral/immunology , Antigens, Viral/genetics , Immunity, HumoralABSTRACT
The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of different variants of concerns with immune evasion that have been prevalent over the past three years. Nanobodies, the functional variable regions of camelid heavy-chain-only antibodies, have garnered interest in developing neutralizing antibodies due to their smaller size, structural stability, ease of production, high affinity, and low immunogenicity, among other characteristics. In this work, we describe an integrated proteomics platform for the high-throughput screening of nanobodies against different SARS-CoV-2 spike variants. To demonstrate this platform, we immunized a camel with subunit 1 (S1) of the wild-type spike protein and constructed a nanobody phage library. The binding and neutralizing activities of the nanobodies against 72 spike variants were then measured, resulting in the identification of two nanobodies (C-282 and C-39) with broad neutralizing activity against six non-Omicron variants (D614G, Alpha, Beta, Gamma, Delta, Kappa) and five Omicron variants (BA.1-5). Their neutralizing capability was validated using in vitro pseudovirus-based neutralization assays. All these results demonstrate the utility of our proteomics platform to identify new nanobodies with broad neutralizing capability and to develop a treatment for patients with SARS-CoV-2 variant infection in the future.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Camelus , Proteomics , SARS-CoV-2 , Single-Domain Antibodies , Spike Glycoprotein, Coronavirus , SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , Single-Domain Antibodies/immunology , Single-Domain Antibodies/chemistry , Proteomics/methods , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Animals , Humans , COVID-19/immunology , COVID-19/virology , Antibodies, Viral/immunology , Neutralization TestsABSTRACT
Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3-â³env backbone plasmid HpaI and truncating the C-terminal 21 amino acids of the SARS-CoV-2 spike protein (S), high-titer SARS-CoV-2-Sdel21-AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96-well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS-CoV-2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development.
ABSTRACT
SARS-CoV-2 breakthrough infections in vaccinated individuals underscore the threat posed by continuous mutating variants, such as Omicron, to vaccine-induced immunity. This necessitates the search for broad-spectrum immunogens capable of countering infections from such variants. This study evaluates the immunogenicity relationship among SARS-CoV-2 variants, from D614G to XBB, through Guinea pig vaccination, covering D614G, Alpha, Beta, Gamma, Delta, BA.1, BA.2, BA.2.75, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB, employing three immunization strategies: three-dose monovalent immunogens, three-dose bivalent immunogens, and a two-dose vaccination with D614G followed by a booster immunization with a variant strain immunogen. Three distinct immunogenicity clusters were identified: D614G, Alpha, Beta, Gamma, and Delta as cluster 1, BA.1, BA.2, and BA.2.75 as cluster 2, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB as cluster 3. Broad-spectrum protection could be achieved through a combined immunization strategy using bivalent immunogens or D614G and XBB, or two initial D614G vaccinations followed by two XBB boosters. A comparison of neutralizing antibody levels induced by XBB boosting and equivalent dosing of D614G and XBB revealed that the XBB booster produced higher antibody levels. The study suggests that vaccine antigen selection should focus on the antigenic alterations among variants, eliminating the need for updating vaccine components for each variant.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Animals , Guinea Pigs , SARS-CoV-2/genetics , COVID-19/prevention & control , Antibodies, Neutralizing , Cluster Analysis , Vaccines, Combined , Antibodies, ViralABSTRACT
The continuing emergence of SARS-CoV-2 variants highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by vaccination based on the ancestral (hereafter referred to as WT) strain would compromise the antibody response to Omicron-based boosters1-5. Vaccination strategies to counter immune imprinting are critically needed. Here we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. In mice, the efficacy of single Omicron boosting is heavily limited when using variants that are antigenically distinct from WT-such as the XBB variant-and this concerning situation could be mitigated by a second Omicron booster. Similarly, in humans, repeated Omicron infections could alleviate WT vaccination-induced immune imprinting and generate broad neutralization responses in both plasma and nasal mucosa. Notably, deep mutational scanning-based epitope characterization of 781 receptor-binding domain (RBD)-targeting monoclonal antibodies isolated from repeated Omicron infection revealed that double Omicron exposure could induce a large proportion of matured Omicron-specific antibodies that have distinct RBD epitopes to WT-induced antibodies. Consequently, immune imprinting was largely mitigated, and the bias towards non-neutralizing epitopes observed in single Omicron exposures was restored. On the basis of the deep mutational scanning profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated that these mutations could further boost the immune-evasion capability of XBB.1.5 while maintaining high ACE2-binding affinity. Our findings suggest that the WT component should be abandoned when updating COVID-19 vaccines, and individuals without prior Omicron exposure should receive two updated vaccine boosters.
Subject(s)
Antibodies, Viral , COVID-19 Vaccines , COVID-19 , Immunization, Secondary , Immunologic Memory , SARS-CoV-2 , Animals , Humans , Mice , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Epitopes, B-Lymphocyte/immunology , Immunologic Memory/immunology , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/immunology , MutationSubject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Antibodies, Viral , Antibodies, NeutralizingABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) XBB lineages have achieved dominance worldwide and keep on evolving. Convergent evolution of XBB lineages on the receptor-binding domain (RBD) L455F and F456L is observed, resulting in variants with substantial growth advantages, such as EG.5, FL.1.5.1, XBB.1.5.70, and HK.3. Here, we show that neutralizing antibody (NAb) evasion drives the convergent evolution of F456L, while the epistatic shift caused by F456L enables the subsequent convergence of L455F through ACE2 binding enhancement and further immune evasion. L455F and F456L evade RBD-targeting Class 1 public NAbs, reducing the neutralization efficacy of XBB breakthrough infection (BTI) and reinfection convalescent plasma. Importantly, L455F single substitution significantly dampens receptor binding; however, the combination of L455F and F456L forms an adjacent residue flipping, which leads to enhanced NAbs resistance and ACE2 binding affinity. The perturbed receptor-binding mode leads to the exceptional ACE2 binding and NAb evasion, as revealed by structural analyses. Our results indicate the evolution flexibility contributed by epistasis cannot be underestimated, and the evolution potential of SARS-CoV-2 RBD remains high.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Humans , Angiotensin-Converting Enzyme 2/genetics , SARS-CoV-2/genetics , COVID-19/genetics , COVID-19 Serotherapy , Antibodies, NeutralizingABSTRACT
ABSTRACTThe global outbreak of COVID-19 has caused a severe threat to human health; therefore, simple, high-throughput neutralization assays are desirable for developing vaccines and drugs against COVID-19. In this study, a high-titre SARS-CoV-2 pseudovirus was successfully packaged by truncating the C-terminus of the SARS-CoV-2 spike protein by 21 amino acids and infecting 293â T cells that had been stably transfected with the angiotensin-converting enzyme 2 (ACE2) receptor and furin (named AF cells), to establish a simple, high-throughput, and automated 384-well plate neutralization assay. The method was optimized for cell amount, virus inoculation, incubation time, and detection time. The automated assay showed good sensitivity, accuracy, reproducibility, Z' factor, and a good correlation with the live virus neutralization assay. The high-throughput approach would make it available for the SARS-CoV-2 neutralization test in large-scale clinical trials and seroepidemiological surveys which would aid the accelerated vaccine development and evaluation.
Subject(s)
COVID-19 , Vesicular Stomatitis , Animals , Humans , SARS-CoV-2/genetics , Antibodies, Neutralizing , Reproducibility of Results , Viral Pseudotyping , Antibodies, Viral , Spike Glycoprotein, Coronavirus , Vesicular stomatitis Indiana virus/genetics , Neutralization Tests/methodsABSTRACT
From December 2022 to January 2023, SARS-CoV-2 infections caused by BA.5 and BF.7 subvariants of B.1.1.529 (Omicron) spread in China. It is urgently needed to evaluate the protective immune responses in the infected individuals against the current circulating variants to predict the future potential infection waves, such as the BQ.1.1, XBB.1.5, and CH1.1 variants. In this study, we constructed a panel of pseudotyped viruses for SARS-CoV-2 for the past and current circulating variants, including D614G, Delta, BA.1, BA.5, BF.7, BQ.1.1, XBB.1.5 and CH.1.1. We investigated the neutralization sensitivity of these pseudotyped viruses to sera from individuals who had BA.5 or BF.7 breakthrough infections in the infection wave of last December in China. The mean neutralization ID50 against infected variants BA.5 and BF.7 are 533 and 444, respectively. The highest neutralizing antibody level was observed when tested against the D614G strain, with the ID50 of 742, which is about 1.52-folds higher than that against the BA.5/BF.7 variant. The ID50 for BA.1, Delta, and BQ.1.1 pseudotyped viruses were about 2-3 folds lower when compared to BA.5/BF.7. The neutralization activities of these serum samples against XBB.1.5 and CH.1.1 decreased 7.39-folds and 15.25-folds when compared to that against BA.5/BF.7. The immune escape capacity of these two variants might predict new infection waves in future when the neutralizing antibody levels decrease furtherly.
Subject(s)
Breakthrough Infections , COVID-19 , Humans , SARS-CoV-2/genetics , Antibodies, Neutralizing , China/epidemiology , Antibodies, ViralABSTRACT
The state-of-the-art SARS-CoV-2 detection methods include qRT-PCR and antibody-based lateral flow assay (LFA) point-of-care tests. Despite the high sensitivity and selectivity, qRT-PCR is slow, expensive and needs well-trained operators. On the other extreme, LFA suffers from low sensitivity albeit its fast detection speed, low detection cost and ease of use. Therefore, the continuing COVID-19 pandemic calls for a SARS-CoV-2 detection method that is rapid, convenient and cost-effective without compromise in sensitivity. Here we provide a proof-of-principle demonstration of an optimized aptamer-based nanointerferometer that enables rapid and amplification-free detection of SARS-CoV-2 spike protein-coated pseudovirus directly from human saliva with the limit of detection (LOD) of about 400 copies per mL. This LOD is on par with that of qRT-PCR, making it 1000 to 100,000-fold more sensitive than commercial LFA tests. Using various combinations of negative selections during the screens for the aptamer targeting the receptor binding domain of the spike protein of SARS-CoV-2, we isolated two aptamers that can distinguish the Omicron and Delta variants. Integrating these two aptamers with LFA strips or the nanointerferometer sensors allows both detection and differentiation of the Omicron and Delta variants which has the potential to realize rapid triage of patients infected different SARS-CoV-2 variants.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Pandemics , OligonucleotidesABSTRACT
Objective: Little is known about the measurement invariance (MI) of resilience instruments in cancer care. This study was designed to examine MI of 10-Item Resilience Scale (RS-SC-10) in Americans and Chinese with cancer using propensity score-based multidimensional item response theory (MIRT) analysis. Methods: A sample of 924 patients were enrolled in the Be Resilient to Cancer trial involving 1 hospital in America and 3 hospitals in China. Data were collected from the RS-SC-10 and Hospital Anxiety and Depression Scale. Propensity score matching and MIRT were performed to evaluate Differential Item Function. Integrated Discrimination Improvement and Net Reclassification Improvement were used to indirectly estimate the MI through incremental prediction ability of MIRT-based score over total score. Results: RS-SC-10 retained 10 items with monotonous thresholds and its original two-factor structure. Nonuniform Differential Item Function was recognized in Item 4 (P â= â0.0011, Δ%ß1 â= â4.15%) and Item 8 (P â= â0.0017, Δ%ß1 â= â5.99%). Net Reclassification Improvement ranged from 9.04% to 35.01%, and Integrated Discrimination Improvement ranged from 8.82% to 20.60%. Conclusions: Although partial MI has been identified between Americans and Chinese, RS-SC-10 remains a critical indicator to emotional distress in cancer care.
ABSTRACT
Continuous evolution of Omicron has led to a rapid and simultaneous emergence of numerous variants that display growth advantages over BA.5 (ref. 1). Despite their divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots. The driving force and destination of such sudden convergent evolution and its effect on humoral immunity remain unclear. Here we demonstrate that these convergent mutations can cause evasion of neutralizing antibody drugs and convalescent plasma, including those from BA.5 breakthrough infection, while maintaining sufficient ACE2-binding capability. BQ.1.1.10 (BQ.1.1 + Y144del), BA.4.6.3, XBB and CH.1.1 are the most antibody-evasive strains tested. To delineate the origin of the convergent evolution, we determined the escape mutation profiles and neutralization activity of monoclonal antibodies isolated from individuals who had BA.2 and BA.5 breakthrough infections2,3. Owing to humoral immune imprinting, BA.2 and especially BA.5 breakthrough infection reduced the diversity of the neutralizing antibody binding sites and increased proportions of non-neutralizing antibody clones, which, in turn, focused humoral immune pressure and promoted convergent evolution in the RBD. Moreover, we show that the convergent RBD mutations could be accurately inferred by deep mutational scanning profiles4,5, and the evolution trends of BA.2.75 and BA.5 subvariants could be well foreseen through constructed convergent pseudovirus mutants. These results suggest that current herd immunity and BA.5 vaccine boosters may not efficiently prevent the infection of Omicron convergent variants.
Subject(s)
Antibodies, Viral , Antigenic Drift and Shift , COVID-19 , Evolution, Molecular , Immunity, Humoral , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Humans , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Breakthrough Infections/immunology , Breakthrough Infections/virology , COVID-19/immunology , COVID-19/virology , COVID-19 Serotherapy , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Protein Domains/genetics , Protein Domains/immunology , Antigenic Drift and Shift/immunology , MutationABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages have escaped most receptor-binding domain (RBD)-targeting therapeutic neutralizing antibodies (NAbs), which proves that previous NAb drug screening strategies are deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd-immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following these criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS-CoV-2-vaccinated SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities.
Subject(s)
COVID-19 , Severe acute respiratory syndrome-related coronavirus , Humans , SARS-CoV-2 , Broadly Neutralizing Antibodies , Combined Antibody Therapeutics , Antibodies, Neutralizing , Epitopes , Antibodies, ViralABSTRACT
Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a growth advantage over circulating BA.2.38, BA.2.76, and BA.5 in India. However, the underlying mechanisms for enhanced infectivity, especially compared with BA.5, remain unclear. Here, we show that BA.2.75 exhibits substantially higher affinity for host receptor angiotensin-converting enzyme 2 (ACE2) than BA.5 and other variants. Structural analyses of BA.2.75 spike shows its decreased thermostability and increased frequency of the receptor binding domain (RBD) in the "up" conformation under acidic conditions, suggesting enhanced low-pH-endosomal cell entry. Relative to BA.4/BA.5, BA.2.75 exhibits reduced evasion of humoral immunity from BA.1/BA.2 breakthrough-infection convalescent plasma but greater evasion of Delta breakthrough-infection convalescent plasma. BA.5 breakthrough-infection plasma also exhibits weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75's distinct neutralizing antibody (NAb) escape pattern. Antibody therapeutics Evusheld and Bebtelovimab remain effective against BA.2.75. These results suggest BA.2.75 may prevail after BA.4/BA.5, and its increased receptor-binding capability could support further immune-evasive mutations.