Search Results (434)

The changes in charge distribution caused by mutations in the spike protein may play a crucial role in balancing infectivity and immune evasion during the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To explore how charge increments in spike protein variants influence viral evolution, a statistical analysis was conducted on 57 SARS-CoV-2 variants, examining relationships between charge distribution, lineage divergence, angiotensin-converting enzyme 2 (ACE2) affinity, immune evasion, and receptor-binding domain (RBD) expression. A phylogenetic tree was also reconstructed using only the charge properties of mutation sites. Results indicated that with increasing lineage divergence, overall positive charge initially rose sharply and then more gradually. Partitioning the spike protein into three domains—the RBD, the N-terminal flanking region (B-RBD), and the C-terminal flanking region (A-RBD)—revealed distinct patterns: positive charge increased in the RBD and A-RBD, whereas the B-RBD accumulated negative charge. Charge increments were negatively associated with ACE2 affinity and RBD expression but positively correlated with immune evasion. The k-mer-based tree derived from charge-reduced sequences showed a topology consistent with the whole-genome tree. These findings suggest that charge distribution in spike proteins is closely linked to viral evolution, with the opposing trends in the RBD and B-RBD potentially reflecting a balance between infectivity and immune escape. Full article

The ongoing evolution of SARS-CoV-2 has given rise to variants with enhanced transmissibility and pathogenicity, many of which harbor mutations in the receptor-binding domain (RBD) of the viral spike protein. These mutations often confer increased viral fitness and immune evasion by modulating interactions with the human ACE2 receptor (hACE2) and escaping neutralizing antibodies. Accurate prediction of the functional consequences of such mutations—particularly their effects on receptor binding and antibody escape—is critical for assessing the public health threat posed by emerging variants. In this study, we apply a Mutation-dependent Biomacromolecular Quantitative Structure–Activity Relationship (MB-QSAR) framework to quantitatively model the biochemical phenotypes of RBD variants. Trained on comprehensive deep mutational scanning (DMS) datasets, our models exhibit strong predictive performance, achieving correlation coefficients (r²) exceeding 0.8 for hACE2 binding affinity and 0.7 for antibody neutralization escape. Importantly, the MB-QSAR approach generalizes well to multi-mutant variants and currently circulating lineages. Structural analysis based on model-derived interaction profiles offers mechanistic insights into key RBD–ACE2 and RBD–antibody interfaces, helping the rational design of broadly protective vaccines and therapeutics. This work establishes MB-QSAR as a rapid, accurate, and interpretable tool for the prediction of protein–protein interaction and forecasting viral adaptation, thereby facilitating early risk assessment of novel SARS-CoV-2 variants. Full article

The COVID-19 pandemic, caused by SARS-CoV-2, has profoundly affected global health and the economy. The emergence of variants with spike mutations, particularly within the receptor-binding domain (RBD), has reduced the efficacy of many neutralizing antibodies (nAbs), and recent variants, including KP.3 and other circulating strains, show partial escape from infection- or vaccine-induced immunity. To overcome this, developing broad-spectrum nAbs that target the conserved S2 subunit of the spike protein is crucial. Unlike the highly mutable RBD, the S2 region remains structurally conserved, providing a promising foundation for universal protection. Deeper insight into S2 structure and function, together with advances in bispecific antibody design, could facilitate the development of next-generation therapeutics resilient to viral evolution. This review examines the structural evolution of the SARS-CoV-2 spike, focusing on the therapeutic potential of S2-targeting antibodies and strategies to overcome antibody resistance. Full article

The SARS-CoV-2 spike protein, through its receptor binding domain (S1-RBD), binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell membrane, leading to viral infection. Several mutations in S1-RBD in SARS-CoV-2 variants are known to enhance infection through an increased affinity for ACE2. While many reports are available describing the SARS-CoV-2 infection mechanism, there is a dearth of studies towards understanding the initial interaction of the S1-RBD with ACE2 on living host cells and the role of endocytosis and cytoskeleton in the process. Here, we reconstituted the interaction between S1-RBD- and ACE2-expressing host cells in a hybrid live cell-supported lipid bilayer (SLB) platform enabling live monitoring of the interaction between S1-RBD on SLBs and the ACE2 receptor on living cells and showed that cells depleted Omicron S1-RBD from SLB corrals, likely through endocytosis. Specifically, interaction of living host cells with S1-RBD-functionalized SLB substrates resulted in the enrichment of S1-RBD and ACE2 at the cell–SLB interface. Interaction of host cells with wild type (WT), Omicron, and Omicron Revertant S1-RBD functionalized on micron-scale SLB corrals, which mimic viral membranes but are flat, also resulted in their enrichment. However, cells interacting with Omicron S1-RBD revealed a depletion of the protein from many corrals, which was generally not observed with the WT S1-RBD and was reduced with the Omicron Revertant, which contains the Q493R mutation reversion, S1-RBD. Further, S1-RBD depletion coincided with the localization of the early endosomal marker EEA1. Importantly, treatment of cells with the clathrin inhibitor, pitstop 2, but not the myosin II inhibitor, blebbistatin, significantly reduced Omicron S1-RBD depletion. Collectively, these observations suggest that the SARS-CoV-2 Omicron variant has evolved, through mutations in its S1-RBD, to take advantage of the cellular endocytic pathway for enhanced infection, which is not observed with the parental SARS-CoV-2 and appears to be lost in the Omicron Revertant variant. Additionally, these results underscore the significance of the hybrid live cell–SLB platform in studying SARS-CoV-2 S1-RBD-ACE2 interaction and the potential impact of mutations in the S1-RBD on adapting to a specific cellular entry mechanism. Full article

Testing medical countermeasures for SARS-CoV-2 transmission using vertebrates can be hindered by legislation regulating animal experimentation, high costs, and ethical concerns. To overcome these challenges, we propose a new Caenorhabditis elegans strain that constitutively expresses the human angiotensin-converting enzyme 2 receptor (ACE2). This resulted in significant impairment of reproduction and a defect in pharyngeal function compared to wild-type (WT) worms. SARS-CoV-2 infection was simulated by treating worms with the receptor-binding domain (RBD) of the spike protein, which caused dose-dependent and time-dependent pharyngeal impairment in ACE2 worms but not in WT worms. The toxicity of RBD was prevented by administering an anti-human ACE2 antibody, demonstrating that interactions with the ACE2 receptor are essential. The ACE2-expressing worm strain was further used for pharmacological research with Raloxifene. In vitro, 1–3 μM of Raloxifene reduced the entry of lentiviral particles carrying the Wuhan variant and B.1.1.7 UK and B.1.1.529 Omicron strains into HEK293-ACE2, in addition to particles expressing N501Y-mutated or P681H-mutated spike proteins. Raloxifene (0.1–1 μM) completely counteracted RBD toxicity in ACE2 worms, indicating that this strain offers a cost-effective in vivo screening platform for molecules with effects involving interactions with the ACE2 receptor. Full article

The breach of an interspecies barrier by RNA viruses has facilitated the emergence of lethal hCoVs, particularly SARS-CoV-2, resulting in significant socioeconomic setbacks and public health risks globally in recent years. Moreover, the high evolutionary plasticity of hCoVs has led to the continuous emergence of diverse variants, complicating clinical management and public health responses. Studying the evolutionary trajectory of hCoVs, which provides a molecular roadmap for understanding viruses’ adaptation, tissue tropism, spread, virulence, and immune evasion, is crucial for addressing the challenges of zoonotic spillover of viruses. Tracing the evolutionary trajectory of lethal hCoVs provides essential genomic insights required for risk stratification, variant/sub-variant classification, preparedness for outbreaks and pandemics, and the identification of critical viral elements for vaccine and therapeutic development. Therefore, this review examines the evolutionary landscape of the three known lethal hCoVs, presenting a focused narrative on SARS-CoV-2 variants under monitoring (VUMs) as of May 2025. Using advanced bioinformatics approaches and data visualization, the review highlights key spike protein substitutions, particularly within the receptor-binding domain (RBD), which drive transmissibility, immune escape, and potential resistance to therapeutics. The article highlights the importance of real-time genomic surveillance and intervention strategies in mitigating emerging variant/sub-variant risks within the ongoing COVID-19 landscape. Full article

The COVID-19 pandemic, driven by SARS-CoV-2, continues to challenge global health due to emerging variants and the potential risk posed by related sarbecoviruses. Neutralizing antibodies targeting the spike (S) glycoprotein, particularly the receptor-binding domain (RBD), play a crucial role in viral neutralization and vaccine design. Although broadly neutralizing anti-RBD antibodies have been identified, the nature of cross-reactive humoral responses induced by natural infection with ancestral SARS-CoV-2 strains remains incompletely understood. Here, we isolated 105 S-specific monoclonal antibodies (mAbs) from individuals recovered from prototype SARS-CoV-2 infection. Of these, 30 mAbs cross-recognized SARS-CoV-1, including 25 RBD-directed mAbs, of which 12 displayed cross-neutralizing activity against both viruses. Among them, mAb 12C2 potently neutralized SARS-CoV-1 and multiple SARS-CoV-2 variants, likely through mechanisms that include inhibition of membrane fusion and potential destabilization of the S trimer. Cryo-electron microscopy revealed that 12C2 engages the outer face of the RBD, overlapping with the epitope recognized by the broadly neutralizing antibody S309 derived from SARS-CoV-1 convalescent. Collectively, these findings demonstrate that ancestral SARS-CoV-2 infection can elicit robust cross-neutralizing antibody responses and provide valuable insights for the design of broadly protective antibodies and vaccines. Full article

Heparan sulfate proteoglycans serve as initial attachment sites for several viruses and bacteria. Recent studies suggest that SARS-CoV-2 similarly exploits these glycosaminoglycans, facilitating conformational changes in the spike protein that promote the interaction between the receptor-binding domain (S1-RBD) and the cellular angiotensin-converting enzyme 2 receptor (ACE2), thereby triggering the virus internalization process. The molecular details that drive this process, particularly the co-receptor role of heparan sulfate (HS), remain incompletely understood. The interaction between an HS hexasaccharide (hexa) and the N343 glycosylated S1-RBD of the wild-type (WT) and Omicron variant of SARS-CoV-2 was investigated. The conformational properties of hexa with these S1-RBDs in unbound and bound states are explored using multiple independent MD simulations; the protein binding epitope of hexa, as well as the details of its interaction with S1-RBD of the Omicron variant, are characterized by comparing experimental and theoretical ¹H STD NMR signals. This investigation identifies the role played by the glycosyl moiety at N343 in potentially affecting this interaction in both WT and Omicron S1-RBD, explaining the observed low specificity and multi-modal nature of the interaction between HS oligosaccharides and these S1-RBDs. Full article

The COVID-19 pandemic has prompted the scientific community to develop new weapons against the SARS-CoV-2 spike protein. The study of its mutations is important to understand how it interacts with human receptors and how to prevent a future pandemic. In this study, four mutations of the Omega variant, along with those from the SARS-CoV-1 and MERS variants, were analyzed in complex with the angiotensin-converting enzyme 2 (ACE2) receptor. In silico studies were carried out to demonstrate that these mutations affect the interaction with the compounds under investigation. The ligands studied are heterocyclic compounds previously considered as potential inhibitors. Our results show that these compounds interact well with the spike protein and provide insights into how mutations, especially in the RBD region, can lead to perturbations in ligand–protein interactions. Full article

Glycosylation plays a pivotal role in regulating the functions and immunogenicity of antigens. Targeting the receptor-binding domain (RBD) of the spike protein (S protein) of SARS-CoV-2, we examined the impact of different glycoforms on RBD antigen immunogenicity and the underlying mechanisms. IgG-specific antibody titers and pseudovirus neutralization were compared in mice immunized with RBD antigens bearing different glycoforms, which were prepared using glycoengineering-capable Pichia pastoris and mammalian cell expression systems with distinct glycosylation pathways. The glycosylation impacted the surface charges of the RBD antigen, and influenced its adsorption onto alum. This may further lead to variations in the antigen’s immunogenicity. The high-mannose variant of the RBD antigen (H-MAN/RBD) expressed in wild-type Pichia pastoris induced significantly higher IgG-specific antibody titers and pseudovirus neutralization activity compared with the complex RBD variant (Complex/RBD) expressed in mammalian cells (293F) or glycoengineering-capable Pichia pastoris. The rate of H-MAN/RBD adsorption onto aluminum hydroxide (alum) adjuvant was significantly higher than that of Complex/RBD. It was assumed that H-MAN/RBD might carry more negative charges because of its phosphomannose-modified surfaces, leading to a higher rate of adsorption onto the positively charged alum and enhancing the immune response. To assess the impact of phosphomannose modification on antigen immunogenicity, a yeast strain was engineered to prepare a low-mannose RBD antigen (L-MAN/RBD); additionally, a yeast strain was constructed to generate a low-phosphomannose-modified RBD antigen (L-MAN-P/RBD). In conclusion, phosphomannose modification substantially enhanced the immunogenicity of RBD by altering the surface charges of the RBD antigen and facilitating its adsorption onto alum. These findings offer novel insights and strategies for vaccine design and immunotherapeutic approaches. Full article

The rapid evolution of SARS-CoV-2 has underscored the need for a detailed understanding of antibody binding mechanisms to combat immune evasion by emerging variants. In this study, we investigated the interactions between Class I neutralizing antibodies—BD55-1205, BD-604, OMI-42, P5S-1H1, and P5S-2B10—and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using multiscale modeling, which combined molecular simulations with the ensemble-based mutational scanning of the binding interfaces and binding free energy computations. A central theme emerging from this work is that the unique binding strength and resilience to immune escape of the BD55-1205 antibody are determined by leveraging a broad epitope footprint and distributed hotspot architecture, additionally supported by backbone-mediated specific interactions, which are less sensitive to amino acid substitutions and together enable exceptional tolerance to mutational escape. In contrast, BD-604 and OMI-42 exhibit localized binding modes with strong dependence on side-chain interactions, rendering them particularly vulnerable to escape mutations at K417N, L455M, F456L and A475V. Similarly, P5S-1H1 and P5S-2B10 display intermediate behavior—effective in some contexts but increasingly susceptible to antigenic drift due to narrower epitope coverage and concentrated hotspots. Our computational predictions show strong agreement with experimental deep mutational scanning data, validating the accuracy of the models and reinforcing the value of binding hotspot mapping in predicting antibody vulnerability. This work highlights that neutralization breadth and durability are not solely dictated by epitope location, but also by how binding energy is distributed across the interface. The results provide atomistic insight into mechanisms driving resilience to immune escape for broadly neutralizing antibodies targeting the ACE2 binding interface—which stems from cumulative effects of structural diversity in binding contacts, redundancy in interaction patterns and reduced vulnerability to mutation-prone positions. Full article

Glycyrrhizic acid (GA) and its derivatives have been reported to have potent pharmacological effects against viral infections, including SARS-CoV and SARS-CoV-2. However, their antiviral mechanisms against coronaviruses are not fully understood. In this study, we found that diammonium glycyrrhizinate (DG) can effectively reduce infections of several human coronaviruses, including HCoV-OC43, HCoV-229E, and SARS-CoV-2, as well as newly emerged variants, with EC₅₀ values ranging from 115 to 391 μg/mL being recorded. Time-of-addition and pseudotype virus infection studies indicated that DG treatment dramatically inhibits the process of virus entry into cells. Furthermore, we demonstrated that DG broadly binds to the RBD of human coronaviruses, thereby blocking spike-mediated cellular entry, by using TR-FRET-based receptor-binding domain (RBD)-ACE2 interaction assay, capillary electrophoresis (CE), and surface plasmon resonance (SPR) assay. In support of this notion, studies of molecular docking and amino acid mutation showed that DG may directly bind to a conserved hydrophobic pocket of the RBD of coronaviruses. Importantly, intranasal administration of DG had a significant protective effect against viral infection in a HCoV-OC43 mouse model. Finally, we found that combinations of DG and other coronavirus inhibitors exhibited antiviral synergy. In summary, our studies strongly reveal that DG exerts broad-spectrum antiviral activity against human coronaviruses by interrupting spike-mediated cellular entry, demonstrating the pharmacological feasibility of using DG as a candidate for alternative treatment and prevention of coronavirus infection. Full article

Background/Objectives: Rapid development of vaccines against SARS-CoV-2 was pivotal to controlling the COVID-19 pandemic. The emergency also provided a rare opportunity to test novel vaccine platforms such as mRNA in large clinical trials. Most of the early vaccines used SARS-CoV-2 Spike protein as the target antigen. Nevertheless, subsequent studies have shown that Receptor Binding Domain (RBD) of Spike also can yield efficacious vaccines, and we previously demonstrated that chemical conjugation of RBD to a carrier protein, EcoCRM^®, enhanced antibody responses and induced strong virus neutralization activity in mice. Methods: Here, we compared the immunogenicity of this conjugate to that of an approved mRNA vaccine from Pfizer/BioNTech in rhesus macaques over a period of nine months. Results: AS01-adjuvanted RBD conjugate induced a similar or better antibody response, receptor binding inhibition, and virus neutralization activity against different variants of SARS-CoV-2, compared to mRNA. IgG subclass profiles induced by conjugate and mRNA vaccines were initially dominated by IgG1 and IgG3 then switched to IgG2 and IgG4 dominant profiles during the subsequent six-month period. Polyclonal immune sera from the conjugate and mRNA had similar antibody avidity at multiple time points. Conclusions: In summary, antibody responses in rhesus macaques induced by the RBD-EcoCRM conjugate and the Spike mRNA vaccine are very similar. These results demonstrate the potential for the RBD-EcoCRM conjugate as a vaccine against SARS-CoV-2. Full article

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune evasion underscores the urgent need for broad-spectrum antiviral therapeutics. In this study, we strategically engineered a novel dual-targeting peptide inhibitor, R1L25HR2, by conjugating the receptor-binding domain (RBD)-targeting peptide R1 with the heptad repeat 1 (HR1)-targeting peptide HR2 through an optimized 25-mer flexible linker (GGGGS)5, aiming to simultaneously block viral attachment and membrane fusion. R1L25HR2 potently and broadly inhibits the infection of SARS-CoV-2 and its emerging variants, including recent circulating strains JN.1 and KP.2, with IC₅₀ values ranging from 5.3 to 253.5 nM, which is significantly more effective than HR2 and R1 alone. Mechanistically, R1L25HR2 inhibits viral attachment and membrane fusion by binding to both RBD and HR1 with low nanomolar affinity. These results highlight the innovative strategy of dual-targeting the RBD and HR1 domains as an effective approach to overcome viral resistance and achieve broad-spectrum antiviral activity. Full article

Background/Objectives: To evaluate how immune responses compare among ethnic groups approximately 2 years after receiving a third dose of COVID-19 vaccine (BNT162b2, mRNA-1273, ChAdOx1or BBIBP-CorV), we tested T cell responses and Spike-specific RBD-antibody titer, and neutralized antibody titer levels utilizing Spectral Flow cytometry, ELISA, and SARS-CoV-2 pseudotyped-based neutralization assays, respectively. Methods: Forty-four individuals from January–December 2023 were identified within the cohort and were classified into different ethnic backgrounds; Black (N = 13), Asian (N = 14), Caucasian (N = 17). We recognize that the “Asian” group includes diverse subpopulations with distinct genetic and environmental backgrounds, which could not be further stratified due to sample-size limitations. Spike-specific AIM+, CD4+, and CD8+ T cell responses were assessed and evaluated against SARS-CoV-2 variants, including the ancestral Wuhan, Delta, and multiple Omicron subvariants (B1.1529, BA2.86, BA.4/5, and XBB.1). Alongside we tested the RBD-IgG and neutralizing antibody titers against the ancestral Wuhan. Spearman’s correlation analysis was utilized to determine corelative relationships among the AIM+ and CD4+ T cell responses, as well as the RBD-IgG and neutralizing antibody titers. Results: Our results show robust and comparable RBD-IgG and neutralizing antibody titers across all groups, with a significant positive correlation between these two measurements. Significant differences were observed in T-cell activation, with Asian participants exhibiting lower frequencies of Spike-specific CD4+ T cells against SARS-CoV-2 Omicron subvariants and higher frequencies of cytokine-producing CD4+ T cells (TNF-α, IFN-γ, and IL-2) as compared to the Caucasian group. Breakthrough infection status was not fully controlled and may influence these findings. Conclusion: Despite a small sample size and potential confounding by natural infections within our long-time-span sampling, our data suggest persistent cellular and humoral immunity 2 years after vaccination across ethnicities, with notable differences in T cell activation and cytokine profile. These preliminary observations highlight the need for larger, more detailed studies that consider intra-ethnic diversity and hybrid immunity to better understand ethnic differences in COVID-19 vaccine responses. Full article