Mutations In Receptor-binding Domain Research Articles

Preventing future zoonosis: SARS-CoV-2 mutations enhance human–animal cross-transmission

The COVID-19 pandemic has driven substantial evolution of the SARS-CoV-2 virus, yielding subvariants that exhibit enhanced infectiousness in humans. However, this adaptive advantage may not universally extend to zoonotic transmission. In this work, we hypothesize that viral adaptations favoring animal hosts do not necessarily correlate with increased human infectivity. In addition, we consider the potential for gain-of-function mutations that could facilitate the virus’s rapid evolution in humans following adaptation in animal hosts. Specifically, we identify the SARS-CoV-2 receptor-binding domain (RBD) mutations that enhance human–animal cross-transmission. To this end, we construct a multitask deep learning model, MT-TopLap trained on multiple deep mutational scanning datasets, to accurately predict the binding free energy changes upon mutation for the RBD to ACE2 of various species, including humans, cats, bats, deer, and hamsters. By analyzing these changes, we identified key RBD mutations such as Q498H in SARS-CoV-2 and R493K in the BA.2 variant that are likely to increase the potential for human–animal cross-transmission.

Bebtelovimab-bound SARS-CoV-2 RBD mutants: resistance profiling and validation with escape mutations, clinical results, and viral genome sequences.

The dynamic evolution of SARS-CoV-2 variants necessitates ongoing advancements in therapeutic strategies. Despite the promise of monoclonal antibody (mAb) therapies like bebtelovimab, concerns persist regarding resistance mutations, particularly single-to-multipoint mutations in the receptor-binding domain (RBD). Our study addresses this by employing interface-guided computational protein design to predict potential bebtelovimab-resistance mutations. Through extensive physicochemical analysis, mutational preferences, precision-recall metrics, protein-protein docking, and energetic analyses, combined with all-atom, and coarse-grained molecular dynamics (MD) simulations, we elucidated the structural-dynamics-binding features of the bebtelovimab-RBD complexes. Identification of susceptible RBD residues under positive selection pressure, coupled with validation against bebtelovimab-escape mutations, clinically reported resistance mutations, and viral genomic sequences enhances the translational significance of our findings and contributes to a better understanding of the resistance mechanisms of SARS-CoV-2.

S2 Peptide-Conjugated SARS-CoV-2 Virus-like Particles Provide Broad Protection against SARS-CoV-2 Variants of Concern.

Approved COVID-19 vaccines primarily induce neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. However, the emergence of variants of concern with RBD mutations poses challenges to vaccine efficacy. This study aimed to design a next-generation vaccine that provides broader protection against diverse coronaviruses, focusing on glycan-free S2 peptides as vaccine candidates to overcome the low immunogenicity of the S2 domain due to the N-linked glycans on the S antigen stalk, which can mask S2 antibody responses. Glycan-free S2 peptides were synthesized and attached to SARS-CoV-2 virus-like particles (VLPs) lacking the S antigen. Humoral and cellular immune responses were analyzed after the second booster immunization in BALB/c mice. Enzyme-linked immunosorbent assay revealed the reactivity of sera against SARS-CoV-2 variants, and pseudovirus neutralization assay confirmed neutralizing activities. Among the S2 peptide-conjugated VLPs, the S2.3 (N1135-K1157) and S2.5 (A1174-L1193) peptide-VLP conjugates effectively induced S2-specific serum immunoglobulins. These antisera showed high reactivity against SARS-CoV-2 variant S proteins and effectively inhibited pseudoviral infections. S2 peptide-conjugated VLPs activated SARS-CoV-2 VLP-specific T-cells. The SARS-CoV-2 vaccine incorporating conserved S2 peptides and CoV-2 VLPs shows promise as a universal vaccine capable of generating neutralizing antibodies and T-cell responses against SARS-CoV-2 variants.

RBD mutations at the residues K417, E484, N501 reduced immunoreactivity with antisera from vaccinated and COVID-19 recovered patients.

It is unclear whether induced spike protein-specific antibodies due to infections with SARS-CoV-2 or to the prototypic Wuhan isolate-based vaccination can immune-react with the emerging variants of SARS-CoV-2. The main objective of the study was to measure the immunoreactivity of induced antibodies postvaccination with Covishield™ (ChAdOx1 nCoV-19 coronavirus vaccines) or infections with SARS-CoV-2 by using selected peptides of the spike protein of wild type and variants of SARS-CoV-2. Thirty patients who had recovered from SARS-CoV-2 infections and 30 individuals vaccinated with both doses of Covishield™ were recruited for the study. Venous blood samples (5 mL) were collected at a single time point from patients within 3-4 weeks of recovery from SARS-CoV-2 infections or receiving both doses of Covishield™ vaccines. The serum levels of total immunoglobulin were measured in both study groups. A total of 12 peptides of 10 to 24 amino acids length spanning to the receptor-binding domain (RBD) of wild type of SARS-CoV-2 and their variants were synthesized. The serum levels of immune-reactive antibodies were measured using these peptides. The serum levels of total antibodies were found to be significantly (p<0.001) higher in the vaccinated individuals as compared to COVID-19 recovered patients. Our study reported that the mutations in the RBD at the residues K417, E484, and N501 have been associated with reduced immunoreactivity with anti-sera of vaccinated people and COVID-19 recovered patients. The amino acid substitutions at the RBD of SARS-CoV-2 have been associated with a higher potential to escape the humoral immune response.

Exploring the mutation landscape of SARS-CoV-2 Variant JN.1

Although Covid-19 is no longer a global pandemic, the spread of SARS-CoV-2 variants, currently led by Omicron and its subvariant, remains prevalent worldwide and causes health issues. In mid-2023, a descendant of BA.2, BA.2.86, carries more than 30 mutations in the Receptor Binding Domain (RBD), helping this variant transmit to create thousands of new COVID-19 cases in many countries worldwide. JN.1 was determined as a descendant of BA.2.86 by WHO because of the mutation profile in spike protein. JN.1 transmission is currently in Europe, the United States and other countries. Therefore, examining unique mutations in RBD of JN.1 to bind with hACE2 plays an important role in discovering the infectious efficiency of this variant. Our study shows that JN.1 has a lower affinity for hACE2 than the ancestral variant BA.2.86. In particular, L455S mutation reduces binding free energy by decreasing Van der Waals interaction energy. We also provide new insights into the significance of E484K in the RBD of JN.1, helping to bind stable with hACE2. Collectively, we suggest that residues positively charged in the RBD of JN.1 play the most important role in the interaction mechanism with hACE2. Future new variants should emphasize positively charged amino acid substitutions in the RBD.

Co-Mutations and Possible Variation Tendency of the Spike RBD and Membrane Protein in SARS-CoV-2 by Machine Learning.

Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2 variants capable of breakthrough infections have attracted global attention. These variants have significant mutations in the receptor-binding domain (RBD) of the spike protein and the membrane (M) protein, which may imply an enhanced ability to evade immune responses. In this study, an examination of co-mutations within the spike RBD and their potential correlation with mutations in the M protein was conducted. The EVmutation method was utilized to analyze the distribution of the mutations to elucidate the relationship between the mutations in the spike RBD and the alterations in the M protein. Additionally, the Sequence-to-Sequence Transformer Model (S2STM) was employed to establish mapping between the amino acid sequences of the spike RBD and M proteins, offering a novel and efficient approach for streamlined sequence analysis and the exploration of their interrelationship. Certain mutations in the spike RBD, G339D-S373P-S375F and Q493R-Q498R-Y505, are associated with a heightened propensity for inducing mutations at specific sites within the M protein, especially sites 3 and 19/63. These results shed light on the concept of mutational synergy between the spike RBD and M proteins, illuminating a potential mechanism that could be driving the evolution of SARS-CoV-2.

Antigenicity assessment of SARS-CoV-2 saltation variant BA.2.87.1

ABSTRACT 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.

Mutations in the SARS-CoV-2 spike receptor binding domain and their delicate balance between ACE2 affinity and antibody evasion.

Intensive selection pressure constrains the evolutionary trajectory of SARS-CoV-2 genomes and results in various novel variants with distinct mutation profiles. Point mutations, particularly those within the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein, lead to the functional alteration in both receptor engagement and monoclonal antibody (mAb) recognition. Here, we review the data of the RBD point mutations possessed by major SARS-CoV-2 variants and discuss their individual effects on ACE2 affinity and immune evasion. Many single amino acid substitutions within RBD epitopes crucial for the antibody evasion capacity may conversely weaken ACE2 binding affinity. However, this weakened effect could be largely compensated by specific epistatic mutations, such as N501Y, thus maintaining the overall ACE2 affinity for the spike protein of all major variants. The predominant direction of SARS-CoV-2 evolution lies neither in promoting ACE2 affinity nor evading mAb neutralization but in maintaining a delicate balance between these two dimensions. Together, this review interprets how RBD mutations efficiently resist antibody neutralization and meanwhile how the affinity between ACE2 and spike protein is maintained, emphasizing the significance of comprehensive assessment of spike mutations.

Using big sequencing data to identify chronic SARS-Coronavirus-2 infections

The evolution of SARS-Coronavirus-2 (SARS-CoV-2) has been characterized by the periodic emergence of highly divergent variants. One leading hypothesis suggests these variants may have emerged during chronic infections of immunocompromised individuals, but limited data from these cases hinders comprehensive analyses. Here, we harnessed millions of SARS-CoV-2 genomes to identify potential chronic infections and used language models (LM) to infer chronic-associated mutations. First, we mined the SARS-CoV-2 phylogeny and identified chronic-like clades with identical metadata (location, age, and sex) spanning over 21 days, suggesting a prolonged infection. We inferred 271 chronic-like clades, which exhibited characteristics similar to confirmed chronic infections. Chronic-associated mutations were often high-fitness immune-evasive mutations located in the spike receptor-binding domain (RBD), yet a minority were unique to chronic infections and absent in global settings. The probability of observing high-fitness RBD mutations was 10-20 times higher in chronic infections than in global transmission chains. The majority of RBD mutations in BA.1/BA.2 chronic-like clades bore predictive value, i.e., went on to display global success. Finally, we used our LM to infer hundreds of additional chronic-like clades in the absence of metadata. Our approach allows mining extensive sequencing data and providing insights into future evolutionary patterns of SARS-CoV-2.

Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

Extraordinary Titer and Broad Anti-SARS-CoV-2 Neutralization Induced by Stabilized RBD Nanoparticles from Strain BA.5.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 μg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of β-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

Impact of BA.1, BA.2, and BA.4/BA.5 Omicron mutations on therapeutic monoclonal antibodies

The emergence of Omicron SARS-CoV-2 subvariants (BA.1, BA.2, BA.4, and BA.5), with an unprecedented number of mutations in their receptor-binding domain (RBD) of the spike-protein, has fueled a resurgence of COVID-19 infections, posing a major challenge to the efficacy of existing vaccines and monoclonal antibody (mAb) therapeutics. We conducted a systematic molecular dynamics (MD) simulation to investigate how the RBD mutations of these subvariants affect the interactions with broad mAbs including AstraZeneca (COV2-2196 and COV2-2130), Brii Biosciences (BRII-196), Celltrion (CT-P59), Eli Lilly (LY-CoV555 and LY-CoV016), Regeneron (REGN10933 and REGN10987), Vir Biotechnology (S309), and S2X259. Our results show a complete loss of binding for COV2-2196, BRII-196, CT-P59, and LY-CoV555 with all Omicron RBDs. Additionally, REGN10987 totally loses its binding with BA.1, but retains a partial binding with BA.2 and BA.4/5. The binding reduction is significant for LY-CoV016 and REGN10933 but moderate for COV2-2130. S309 and S2X259 retain their binding with BA.1 but exhibit decreased binding with other subvariants. We introduce a mutational escape map for each mAb to identify the key RBD sites and the corresponding critical mutations. Overall, our findings suggest that the majority of therapeutic mAbs have diminished or missing activity against Omicron subvariants, indicating the urgent need for a new therapeutic mAb with a better design.

Effects of tea, catechins and catechin derivatives on Omicron subvariants of SARS-CoV-2

The Omicron subvariants of SARS-CoV-2 have multiple mutations in the S-proteins and show high transmissibility. We previously reported that tea catechin (−)-epigallocatechin gallate (EGCG) and its derivatives including theaflavin-3,3’-di-O-digallate (TFDG) strongly inactivated the conventional SARS-CoV-2 by binding to the receptor binding domain (RBD) of the S-protein. Here we show that Omicron subvariants were effectively inactivated by green tea, Matcha, and black tea. EGCG and TFDG strongly suppressed infectivity of BA.1 and XE subvariants, while effect on BA.2.75 was weaker. Neutralization assay showed that EGCG and TFDG inhibited interaction between BA.1 RBD and ACE2. In silico analyses suggested that N460K, G446S and F490S mutations in RBDs crucially influenced the binding of EGCG/TFDG to the RBDs. Healthy volunteers consumed a candy containing green tea or black tea, and saliva collected from them immediately after the candy consumption significantly decreased BA.1 virus infectivity in vitro. These results indicate specific amino acid substitutions in RBDs that crucially influence the binding of EGCG/TFDG to the RBDs and different susceptibility of each Omicron subvariant to EGCG/TFDG. The study may suggest molecular basis for potential usefulness of these compounds in suppression of mutant viruses that could emerge in the future and cause next pandemic.

A multi-tier computational screening framework to effectively search the mutational space of SARS-CoV-2 receptor binding motif to identify mutants with enhanced ACE2 binding abilities.

SARS-CoV-2 gained crucial mutations at the receptor binding domain (RBD) that often changed the course of the pandemic leading to new waves with increased case fatality. Variants are observed with enhanced transmission and immune invasion abilities. Thus, predicting future variants with enhanced transmission ability is a problem of utmost research interest. Here, we have developed a multi-tier exhaustive SARS-CoV-2 mutation screening platform combining MM/GBSA, extensive molecular dynamics simulations, and steered molecular dynamics to identify RBD mutants with enhanced ACE2 binding capability. We have identified four RBM mutations (F490K, S494K, G504F, and the P499L) with significantly higher ACE2 binding abilities than wild-type RBD. Compared to wild-type RBD, they all form stable complexes with more hydrogen bonds and salt-bridge interactions with ACE2. Our simulation data suggest that these mutations allosterically alter the packing of the RBM interface of the RBD-ACE2 complex. As a result, the rupture force required to break the RBD-ACE2 contacts is significantly higher for these mutants.

Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis

BackgroundThe SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages.MethodsThe presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing.ResultsAll genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages.ConclusionThe developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Omicron Coronavirus: pH-Dependent Electrostatic Potential and Energy of Association of Spike Protein to ACE2 Receptor.

The association of the S-protein of the SARS-CoV-2 beta coronavirus to ACE2 receptors of the human epithelial cells determines its contagiousness and pathogenicity. We computed the pH-dependent electric potential on the surface of the interacting globular proteins and pH-dependent Gibbs free energy at the association of the wild-type strain and the omicron variant. The calculated isoelectric points of the ACE2 receptor (pI 5.4) and the S-protein in trimeric form (pI 7.3, wild type), (pI 7.8, omicron variant), experimentally verified by isoelectric focusing, show that at pH 6-7, the S1-ACE2 association is conditioned by electrostatic attraction of the oppositely charged receptor and viral protein. The comparison of the local electrostatic potentials of the omicron variant and the wild-type strain shows that the point mutations alter the electrostatic potential in a relatively small area on the surface of the receptor-binding domain (RBD) of the S1 subunit. The appearance of seven charge-changing point mutations in RBD (equivalent to three additional positive charges) leads to a stronger S1-ACE2 association at pH 5.5 (typical for the respiratory tract) and a weaker one at pH 7.4 (characteristic of the blood plasma); this reveals the reason for the higher contagiousness but lower pathogenicity of the omicron variant in comparison to the wild-type strain.

Antibody Fc-binding profiles and ACE2 affinity to SARS-CoV-2 RBD variants

Emerging SARS-CoV-2 variants, notably Omicron, continue to remain a formidable challenge to worldwide public health. The SARS-CoV-2 receptor-binding domain (RBD) is a hotspot for mutations, reflecting its critical role at the ACE2 interface during viral entry. Here, we comprehensively investigated the impact of RBD mutations, including 5 variants of concern (VOC) or interest—including Omicron (BA.2)—and 33 common point mutations, both on IgG recognition and ACE2-binding inhibition, as well as FcγRIIa- and FcγRIIIa-binding antibodies, in plasma from two-dose BNT162b2-vaccine recipients and mild-COVID-19 convalescent subjects obtained during the first wave using a custom-designed bead-based 39-plex array. IgG-recognition and FcγR-binding antibodies were decreased against the RBD of Beta and Omicron, as well as point mutation G446S, found in several Omicron sub-variants as compared to wild type. Notably, while there was a profound decrease in ACE2 inhibition against Omicron, FcγR-binding antibodies were less affected, suggesting that Fc functional antibody responses may be better retained against the RBD of Omicron in comparison to neutralization. Furthermore, while measurement of RBD–ACE2-binding affinity via biolayer interferometry showed that all VOC RBDs have enhanced affinity to human ACE2, we demonstrate that human ACE2 polymorphisms, E35K (rs1348114695) has reduced affinity to VOCs, while K26R (rs4646116) and S19P (rs73635825) have increased binding kinetics to the RBD of VOCs, potentially affecting virus–host interaction and, thereby, host susceptibility. Collectively, our findings provide in-depth coverage of the impact of RBD mutations on key facets of host–virus interactions.

Free Energy Perturbation Calculations of Mutation Effects on SARS-CoV-2 RBD::ACE2 Binding Affinity

The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in Omicron variants and offer a physical explanation for the underlying mechanism. Overall, our results suggest that despite the significant computational cost, FEP calculations may offer an effective strategy to understand the effects of interfacial mutations on protein–protein binding affinities and, hence, in a variety of practical applications such as the optimization of neutralizing antibodies.

A recombinant spike-XBB.1.5 protein vaccine induces broad-spectrum immune responses against XBB.1.5-included Omicron variants of SARS-CoV-2.

The XBB.1.5 subvariant has drawn great attention owing to its exceptionality in immune evasion and transmissibility. Therefore, it is essential to develop a universally protective coronavirus disease 2019 vaccine against various strains of Omicron, especially XBB.1.5. In this study, we evaluated and compared the immune responses induced by six different spike protein vaccines targeting the ancestral or various Omicron strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in mice. We found that spike-wild-type immunization induced high titers of neutralizing antibodies (NAbs) against ancestral SARS-CoV-2. However, its activity in neutralizing Omicron subvariants decreased sharply as the number of mutations in receptor-binding domain (RBD) of these viruses increased. Spike-BA.5, spike-BF.7, and spike-BQ.1.1 vaccines induced strong NAbs against BA.5, BF.7, BQ.1, and BQ.1.1 viruses but were poor in protecting against XBB and XBB.1.5, which have more RBD mutations. In sharp contrast, spike-XBB.1.5 vaccination can activate strong and broadly protective immune responses against XBB.1.5 and other common subvariants of Omicron. By performing correlation analysis, we found that the NAbs titers were negatively correlated with the number of RBD mutations in the Omicron subvariants. Vaccines with more RBD mutations can effectively overcome the immune resistance caused by the accumulation of RBD mutations, making spike-XBB.1.5 the most promising vaccine candidate against universal Omicron variants.

Deep Structural Analysis of Myriads of Omicron Sub-Variants Revealed Hotspot for Vaccine Escape Immunity.

The emergence of the Omicron variant has reinforced the importance of continued SARS-CoV-2 evolution and its possible impact on vaccine effectiveness. Specifically, mutations in the receptor-binding domain (RBD) are critical to comprehend the flexibility and dynamicity of the viral interaction with the human agniotensin-converting enzyme 2 (hACE2) receptor. To this end, we have applied a string of deep structural and genetic analysis tools to map the substitution patterns in the S protein of major Omicron sub-variants (n = 51) with a primary focus on the RBD mutations. This head-to-head comparison of Omicron sub-variants revealed multiple simultaneous mutations that are attributed to antibody escape, and increased affinity and binding to hACE2. Our deep mapping of the substitution matrix indicated a high level of diversity at the N-terminal and RBD domains compared with other regions of the S protein, highlighting the importance of these two domains in a matched vaccination approach. Structural mapping identified highly variable mutations in the up confirmation of the S protein and at sites that critically define the function of the S protein in the virus pathobiology. These substitutional trends offer support in tracking mutations along the evolutionary trajectories of SAR-CoV-2. Collectively, the findings highlight critical areas of mutations across the major Omicron sub-variants and propose several hotspots in the S proteins of SARS-CoV-2 sub-variants to train the future design and development of COVID-19 vaccines.