Search Results (1,252)

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

Background: Vaccines that stimulate systemic and mucosal immunity to a level required to prevent SARS-CoV-2 infection and transmission are an unmet need. Highly protective hepatitis B and human papillomavirus nanoparticle vaccines highlight the potential of multivalent nanoparticle vaccine platforms to provide enhanced immunity. Here, we report the construction and characterization of self-assembling 60-subunit icosahedral nanoparticle SARS-CoV-2 vaccines using the bacterial enzyme lumazine synthase (LuS). Methods and Results: Nanoparticles displaying prefusion-stabilized SARS-CoV-2 spike ectodomains fused to the surface-exposed amino terminus of LuS were designed using structure-guided approaches. Negative stain-electron microscopy studies of purified nanoparticles were consistent with self assembly into 60-mer nanoparticles displaying 20 spike trimers. After two intramuscular doses, these purified spike-LuS nanoparticles elicited significantly higher SARS-CoV-2 neutralizing activity than spike trimers in vaccinated mice. Furthermore, intramuscular DNA priming and intranasal boosting with a SARS-CoV-2 LuS nanoparticle vaccine stimulated mucosal IgA responses. Conclusion: These data identify LuS nanoparticles as highly immunogenic SARS-CoV-2 vaccine candidates and support the further development of this platform against SARS-CoV-2 and its emerging variants. Full article

The scientific community’s interest in natural compounds with antiviral properties has considerably increased after the emergence of the severe acute respiratory syndrome coronavirus (SARS-CoV-2), especially for their potential use in the treatment of the COVID-19 infection. From this perspective, bovine coronavirus (BCoV), member of the genus β-CoV, represents a valuable virus model to study human β-CoVs, bypassing the risks of handling highly pathogenic and contagious viruses. Pimarane diterpenes are a significant group of secondary metabolites produced by phytopathogenic fungi, including several Diplodia species. Among the members of this class of natural products, sphaeropsidin A (SphA) and its analog sphaeropsidin B (SphB) are well known for their bioactivities, such as antimicrobial, insecticidal, herbicidal, and anticancer. In this study, the antiviral effects of SphA and SphB were evaluated for the first time on bovine (MDBK) cells infected with BCoV. Our findings showed that both sphaeropsidins significantly increased cell viability in infected cells. These substances also caused substantial declines in the virus yield and in the levels of the viral spike S protein. Interestingly, during the treatment, a cellular defense mechanism was detected in the downregulation of the aryl hydrocarbon receptor (AhR) signaling, which is affected by BCoV infection. We also observed that the presence of SphA and SphB determined the deacidification of the lysosomal environment in infected cells, which may be related to their antiviral activities. In addition, in silico investigations have been performed to elucidate the molecular mechanism governing the recognition of bovine AhR (bAhR) by Sphs. Molecular docking studies revealed significant insights into the structural determinants driving the bAhR binding by the examined compounds. Hence, in vitro and in silico results demonstrated that SphA and SphB are promising drug candidates for the development of efficient therapies able to fight a β-CoV-like BCoV during infection. Full article

Ferritin nanocages with spherical shells carry proteins or antigens that enable their use as highly efficient nanoreactors and nanocarriers. Mimicking the surface Spike (S) receptor-binding domain (RBD) from SARS-CoV-2, ferritin nanocages induce neutralizing antibody production or block viral entry. Herein, by implementing molecular dynamics simulation, we evaluate the efficiency in the interaction pattern (active or alternative sites) of H-ferritin displaying the 24 S RBDs with host-cell-receptor or monoclonal antibodies (mAbs; B38 or VVH-72). Our constructed nanocage targeted the receptor- or antibody-binding interfaces, suggesting that mAbs demonstrate an enhanced binding affinity with the RBD, with key interactions originating from its variable heavy chain. The S RBD interactions with ACE2 and B38 involved the same binding site but led to divergent dynamic responses. In particular, both B38 chains showed that asymmetric fluctuations had a major effect on their engagement with the Spike RBD. Although the receptor increased the binding affinity of VVH-72 for the RBD, the mAb structural orientation on the nanocage remained identical to its conformation when bound to the host receptor. Overall, our findings characterize the essential pharmacophore formed by Spike RBD residues over nanocage molecules, which mediates high-affinity interactions with either binding partner. Importantly, the ferritin-displayed RBD maintained native receptor and antibody binding profiles, positioning it as a promising scaffold for pre-fusion stabilization and protective RBD vaccine design. Full article

Despite the prevalence of fucosylated IgG in plasma, specific IgGs with low core fucosylation sporadically emerge in response to virus infections and blood cell alloantigens. This low fucosylation of IgG is implicated in the pathogenesis of SARS-CoV-2 and dengue infections. In COVID-19, the presence of IgGs with low core fucosylation (afucosylated IgGs) targeting spike protein predicts disease progression to a severe form and actively mediates this progression. This study reveals that SARS-CoV-2 infection of megakaryocytes promotes the generation of pathogenic afucosylated anti-spike IgGs, leading to outcomes, such as pulmonary vascular thrombosis, acute lung injury, and mortality in FcγRIIa-transgenic mice. Platelets from mice injected with virus-infected human megakaryocytes express significant activation biomarkers, indicating a direct link between the immune response and platelet activation. Mice injected with virus-infected human megakaryocytes demonstrate an elevated rate of thrombus formation induced by FeCl₃ (4%) and a reduction in bleeding time, emphasizing the intricate interplay of viral infection, immune response, and hemostatic complications. Treatment with inhibitors targeting FcγRIIa, serotonin, or complement anaphylatoxins of mice injected with spike-expressing MKs successfully prevents observed platelet activation, thrombus formation, and bleeding abnormalities, offering potential therapeutic strategies for managing severe outcomes associated with afucosylated IgGs in COVID-19 and related disorders. Full article

The renin–angiotensin system (RAS) plays a central role in cardiovascular regulation and has gained prominence in the pathogenesis of Coronavirus Disease 2019 (COVID-19) due to the critical function of angiotensin-converting enzyme 2 (ACE2) as the entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Angiotensin IV, but not angiotensin II, has recently been reported to enhance the binding between the viral spike protein and ACE2. To investigate the virological significance of this effect, we developed a single-round infection assay using SARS-CoV-2 viral-like particles expressing the spike protein. Our results demonstrate that while angiotensin II does not affect viral infectivity across concentrations ranging from 40 nM to 400 nM, angiotensin IV enhances viral entry at a low concentration but exhibits dose-dependent inhibition at higher concentrations. These findings highlight the unique dual role of angiotensin IV in modulating SARS-CoV-2 entry. In silico molecular docking simulations indicate that angiotensin IV was predicted to associate with the S1 domain near the receptor-binding domain in the open spike conformation. Given that reported plasma concentrations of angiotensin IV range widely from 17 pM to 81 nM, these levels may be sufficient to promote, rather than inhibit, SARS-CoV-2 infection. This study identifies a novel link between RAS-derived peptides and SARS-CoV-2 infectivity, offering new insights into COVID-19 pathophysiology and informing potential therapeutic strategies. Full article

Background/Objectives: Concurrent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A and B viruses (IAV/IBV), and respiratory syncytial virus (RSV) necessitate rapid and precise differential laboratory diagnostic methods. This study aimed to evaluate the multiplex molecular diagnostic performance of the geneLEAD VIII system (Precision System Science Co., Ltd., Matsudo, Japan), a fully automated sample-to-result precision instrument, in conjunction with the VIASURE SARS-CoV-2, Flu & RSV Real Time PCR Detection Kit (CerTest Biotec, S.L., Zaragoza, Spain). Methods: The specific detection capabilities of SARS-CoV-2, IAV/IBV, and RSV genes were evaluated using virus-spiked saliva and nasal swab samples. Using saliva samples, the viral titer detection limits of geneLEAD/VIASURE and manual referent singleplex RT-qPCR assays were compared. The performance of geneLEAD/VIASURE in analyzing single- and multiple-infection models was scrutinized. The concordance between the geneLEAD/VIASURE and the manual assays was assessed. Results: The geneLEAD/VIASURE successfully detected all the virus genes in the saliva and nasal swab samples despite some differences in the Ct values. The viral titer detection limits in the saliva samples for SARS-CoV-2, IAV, IBV, and RSV using geneLEAD/VIASURE were 10⁰, ≤10⁻², 10⁰, and 10² TCID₅₀/mL, respectively, compared to ≤10⁻¹, ≤10⁰, ≤10⁰, and ≤10⁴ TCID₅₀/mL, respectively, in the manual assays. geneLEAD/VIASURE yielded similar Ct values in the single- and multiple-infection models, with some exceptions noted in the triple-infection models when low titers of RSV were spiked with high titers of the other viruses. The concordance between geneLEAD/VIASURE and the manual assays was high, with Pearson’s R² values of 0.90, 0.85, 0.92, and 0.95 for SARS-CoV-2, IAV, IBV, and RSV, respectively. Conclusions: geneLEAD/VIASURE is a reliable diagnostic tool for detecting SARS-CoV-2, IAV/IBV, and RSV in single- and multiple-infection scenarios. Full article

(1) Background: The ongoing global health threat posed by SARS-CoV-2 requires reliable and accessible diagnostic tools, especially in resource-limited settings where RT-qPCR may be impractical. This study describes the development and validation of two enzyme-linked immunosorbent assays (ELISA) designed to detect anti-SARS-CoV-2 IgG antibodies employing recombinant S1 and S2 spike protein subunits. (2) Methods: The assays were optimized and validated using serum samples from 354 RT-qPCR-confirmed hospitalized patients and 337 pre-pandemic blood donors. (3) Results: The S1-based ELISA achieved a 52.8% sensitivity and a specificity of 93.5%, with an area under the ROC curve (AUC) of 71.6%. In contrast, the S2-based ELISA demonstrated superior diagnostic performance, with a sensitivity of 63.7%, a specificity of 99.7%, and an AUC of 83.1%. Cross-reactivity analysis using sera from individuals with unrelated infectious diseases confirmed the high specificity of the S2-ELISA. Time-stratified analysis revealed that sensitivity increased with time, peaking between 15 and 21 days post-symptom onset. Compared to commercial serological assays, the S2-ELISA demonstrated comparable or improved performance, particularly in specificity and diagnostic odds ratio. (4) Conclusions: The S2-ELISA offers a robust, highly specific, and operationally simple tool for serological detection of SARS-CoV-2 infection. Its strong diagnostic performance and accessibility make it well-suited for implementation in diverse epidemiological settings, particularly where molecular testing is limited. The development of affordable, validated serological assays such as this is critical for strengthening surveillance, understanding transmission dynamics, and informing public health responses. Full article

Background: SARS-CoV-2 booster vaccination remains essential to prevent severe COVID-19, particularly in vulnerable populations such as older adults. This study evaluated the durability and dynamics of immune responses following booster vaccination(s) in >65-year-old individuals and examined their association with protection against new infections. Methods: Immune responses were evaluated at 3, 9, and 15 months post-booster, measuring SARS-CoV-2-specific IgG antibodies against spike [IgG(S)] and nucleocapsid [IgG(N)] proteins, neutralizing activity against the Omicron BA.2 variant, and cellular immunity. A subset of participants was tested before booster administration. Regression analyses examined the influence of clinical and immunological factors—including a bivalent fourth dose—on infection risk over time. Results: Booster vaccination significantly enhanced IgG(S) and neutralizing capacity, peaking at 3 months. Although a decline was observed by 9 months, responses remained above baseline. Individuals with prior SARS-CoV-2 infection exhibited higher IgG(S) levels and neutralizing titers, and significantly lower reinfection rates (15%), compared to uninfected individuals. A fourth vaccine dose further increased IgG(S) levels. While neutralizing capacity was not consistently enhanced by the fourth dose, recipients experienced a lower rate of new infections. Immune trajectory analyses revealed that breakthrough infections elicited strong humoral responses comparable to those seen in previously infected individuals, highlighting the role of hybrid immunity. Conclusions: In older adults, booster vaccination induces durable immune responses, with hybrid immunity offering enhanced protection. A fourth dose boosts antibody levels and reduces infection risk, supporting its use in this high-risk group. Continued monitoring is needed to determine the long-term effectiveness of boosters, particularly against emerging variants. Full article

Background and aim: The COVID-19 pandemic, caused by SARS-CoV-2, remains a global health crisis despite vaccination efforts, necessitating novel therapeutic strategies. Natural compounds from Syzygium aromaticum (clove), such as eugenol and β-caryophyllene, exhibit antiviral and anti-inflammatory properties, while host genetic factors, including miR-21 rs1292037 polymorphism, may influence disease susceptibility and severity. This study investigates the dual approach of targeting SARS-CoV-2 via Syzygium aromaticum phytoconstituents while assessing the role of miR-21 rs1292037 in COVID-19 pathogenesis. Methods: Firstly, molecular docking and molecular dynamics simulations were employed to assess the binding affinities of eugenol and caryophyllene against seven key SARS-CoV-2 proteins—including Spike-RBD, 3CLpro, and RdRp—using SwissDock (AutoDock Vina) and the Desmond software package, respectively. Secondly, GC-MS was used to characterize the composition of clove extract. Thirdly, pharmacokinetic profiles were predicted using in silico models. Finally, miR-21 rs1292037 genotyping was performed in 100 COVID-19 patients and 100 controls, with cytokine and coagulation markers analyzed. Results: Docking revealed strong binding of eugenol to viral Envelope Protein (−5.267 kcal/mol) and caryophyllene to RdRp (−6.200 kcal/mol). ADMET profiling indicated favorable absorption and low toxicity. Molecular dynamics simulations confirmed stable binding of methyl eugenol and caryophyllene to SARS-CoV-2 proteins, with caryophyllene–7Z4S showing the highest structural stability, highlighting its strong antiviral potential. Genotyping identified the TC genotype as prevalent in patients (52%), correlating with elevated IL-6 and D-dimer levels (p ≤ 0.01), suggesting a hyperinflammatory phenotype. Males exhibited higher ferritin and D-dimer (p < 0.0001), underscoring sex-based disparities. Conclusion: The bioactive constituents of Syzygium aromaticum exhibit strong potential as multi-target antivirals, with molecular simulations highlighting caryophyllene’s particularly stable interaction with the 7Z4S protein. Methyl eugenol also maintained consistent binding across several SARS-CoV-2 targets. Additionally, the miR-21 rs1292037 polymorphism may influence COVID-19 severity through its role in inflammatory regulation. Together, these results support the combined application of phytochemicals and genetic insights in antiviral research, pending further clinical verification. Full article

The COVID-19-related long-standing effect or Post-Acute Sequelae of COVID-19 (PASC) is often associated with NLRP3 inflammasome activation in pulmonary inflammation elicited by SARS-CoV-2 spike proteins. Spike proteins engage toll-like receptors (TLRs) in respiratory epithelial cells, leading to excessive cytokine production. Given the need for effective therapeutic strategies to mitigate spike protein-stimulated lung inflammation, we examined the anti-inflammatory properties of luteolin and ethanolic extract from Harrisonia perforata (Blanco) Merr. root. The ethanolic extract of H. perforata root (HPEE) contained a high concentration of luteolin flavonoid (143.53 ± 1.58 mg/g extract). Both HPEE (25–100 μg/mL) and luteolin (4.5–36 μM) significantly inhibited inflammation stimulated by the Wuhan (W) and Omicron (O) spike protein S1, as evidenced by a dose-dependent significant decrease in IL-6, IL-1β, and IL-18 secretion in A549 lung epithelial cells (p < 0.05). Furthermore, pretreatment with HPEE or luteolin prior to spike protein exposure (100 ng/mL) significantly, in a dose-dependent manner, repressed the inflammatory mRNA expression (p < 0.05). Mechanistic study revealed that HPEE and luteolin suppressed NLRP3 inflammasome signaling activation by reducing their machinery protein expressions. Additionally, they inhibited the ERK/JNK/p38 MAPK signaling activation, resulting in decreased inflammatory mRNA expression and cytokine release. These findings suggest that H. perforata root extract and its major flavonoid luteolin exert potent anti-inflammatory effects and may offer therapeutic potential against spike protein-induced lung inflammation. Full article

Persistent SARS-CoV-2 infections involve prolonged viral replication and immune system interactions, potentially driving viral evolution and immune escape. This study examines viral characteristics and host gene expression changes in persistent infections. The nasopharyngeal samples from four patients with persistent SARS-CoV-2 infections at Tottori University Hospital, Japan, were analyzed. Viral isolates were cultured, and infectivity was assessed using TCID₅₀ assays. To investigate host responses, RNA sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs), and Gene Ontology (GO) enrichment analysis mapped affected biological pathways. Viral genome sequencing detected mutations associated with prolonged infection. The results showed significant infectivity differences between early- and late-phase infection. Gene expression analysis revealed a strong early phase of pro-inflammatory response (IL6, TNF, IL1B, CXCL10) followed by immune suppression. GO enrichment analysis highlighted inflammation and cytokine-mediated immune pathways. Genomic sequencing identified mutations in ORF1ab and the spike (S) protein, potentially aiding immune escape. The findings underscore that SARS-CoV-2 adapts during persistent infections, altering infectivity and immune responses. These highlight the need for continued monitoring of prolonged infections to mitigate immune escape and viral evolution. Full article

Background/Objectives: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2. Methods: MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell–cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody–antigen interface. Results: MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure. Conclusions: Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development. Full article

Background: Cell-penetrating peptides cross cell membrane barriers while carrying cargoes in a functional form. Our work identified two novel lung-targeting peptides, S7A and R11A. Here, we present studies on biodistribution, the cell types targeted, and an in vitro proof of application. Methods: Studies were performed in human bronchial epithelial cells (HBECs) with and without various endocytic inhibitors, and coincubation with fluorescently labeled transferrin or endocytic markers. Cyclic R11A (cR11A) was conjugated to siRNA duplexes and anti-viral activity against SARS-CoV-2 was tested. Biodistribution studies were performed by injecting wild-type mice with fluorescently labeled peptides, and various circulation times were allowed for, as well as cross-staining of lung sections or isolated single cells with various cellular markers, followed by fluorescence-activated cell sorting or confocal microscopy. Results: cR11A showed peak uptake in 15 min, with the highest uptake in airway epithelial type II (ATII) cells, followed by p63+ basal cells and ionocytes. Cyclization increased transduction efficiencies ~100-fold. Endocytosis studies showed a decrease in peptide uptake by pre-treatment with Pitstop2 but not Amiloride or Nystatin. Endocytic marker Lamp1 showed colocalization at the earliest time point, with the escape of the peptide from endocytic vesicles later. cR11A conjugated to ant-spike and anti-envelop proteins showed anti-viral effects with an EC₉₀ of 0.6 μM and 1.0 µM, respectively. Conclusions: We have identified a novel peptide, cR11A, that targets ATII, basal cells, and ionocytes, the cyclization of which increased transduction efficiency in vitro and in vivo. The uptake mechanism appears to be via clathrin-mediated endocytosis with escape from endocytic vesicles. cR11A can act as a vector to deliver anti-viral siRNA to epithelial cells. Full article

Multiple lines of evidence suggest that endothelial dysfunction is a key player in the pathogenesis of COVID-19, with cytokine storm as one of the main primary causes. Among the mechanisms underlying endothelial damage, clinical findings identify alterations in arginine metabolism, as patients with severe COVID-19 exhibit lower levels of nitric oxide synthase (eNOS) and upregulated arginase. In this study, we investigated, in human endothelial cells (HUVECs), the effect of conditioned medium from macrophages activated with SARS-CoV-2 Spike protein (CM_S1) on arginine metabolism. The results indicate that CM_S1 causes a marked decrease in eNOS and an increase in arginase, along with a greater intracellular arginine content and the induction of the CAT2 transporter. These effects are ascribable to the inflammatory mediators released by macrophages in CM_S1, mainly TNFα and IL-1β. Since infliximab, an antibody targeting TNFα, and baricitinib, an inhibitor of the JAK/STAT pathway, correct the observed imbalance between eNOS and arginase, our findings suggest the potential efficacy of a combined therapy to counteract endothelial dysfunction in COVID-19. Full article