Search Results (846)

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/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD can enhance antigen presentation to improve the potency and breadth of immune responses. Though RBDs directly fused to a self-assembling scaffold can be delivered as messenger RNA (mRNA) formulated with lipid nanoparticles (LNPs), reports of SARS-CoV-2 vaccine candidates that combine these approaches remain scarce. Methods: Here, we designed RBD fused to AP205 or TIP60 self-assembling nanoparticles following a search of available structures focused on several scaffold properties. RBD-AP205 and RBD-TIP60 were tested for antigenicity following transfection and for immunogenicity and neutralization potency when delivered as mRNA in mice, with RBD-ferritin as a direct comparator. Results: All scaffolded RBD constructs were readily secreted to transfection supernatant and showed antigenicity in ELISA, though clear heterogeneity in assembly was observed. RBD-AP205 and RBD-TIP60 also exhibited robust antibody binding and neutralization titers in mice that were comparable to those elicited by RBD-ferritin or a full-length membrane-bound spike. Conclusions: These data suggest that AP205 and TIP60 can present RBD as effectively as ferritin and induce similar immune responses. By describing additional scaffolds for multimeric display that accommodate mRNA delivery platforms, this work can provide new tools for future vaccine design efforts. 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

Background/Objectives: The ongoing evolution of SARS-CoV-2 has highlighted the limitations of parenteral vaccines in preventing viral transmission, largely due to their failure to elicit robust mucosal immunity. Methods: Here, we evaluated an intranasal (IN) vaccine formulation consisting of recombinant receptor-binding domain (RBD) adsorbed onto human probiotic Bacillus subtilis DG101 spores. Results: In BALB/c mice, IN spore-RBD immunization induced strong systemic and mucosal humoral responses, including elevated specific IgG, IgM, and IgA levels in serum, bronchoalveolar lavage fluid (BALF), nasal-associated lymphoid tissue (NALT), and saliva. It further promoted mucosal B cell and T cell memory, along with a Th1/Tc1-skewed T cell response, characterized by increased IFN-γ-expressing CD4⁺ and CD8⁺ T cells in the lungs. Conclusions: All in all, these findings highlight the potential of intranasal vaccines adjuvanted with probiotic B. subtilis spores in inducing sterilizing immunity and limiting SARS-CoV-2 transmission. 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: Mucosal immunity plays a pivotal role in preventing infections with SARS-CoV-2. While COVID-19 mRNA vaccines induce robust systemic immune responses in patients with inflammatory bowel disease (IBD), little is known about their efficacy in the mucosal immune compartment. In this sub-investigation of the ongoing STAR-SIGN study, we present the first analysis of mucosal immunity elicited by XBB.1.5 mRNA vaccines in immunocompromised patients with IBD. Methods: IgG and IgA antibodies targeting the receptor-binding domain of the SARS-CoV-2 JN.1 variant were quantified longitudinally in the saliva of IBD patients using the multiplex immunoassay MultiCoV-Ab. Antibody levels were quantified before and 2–4 weeks after vaccination with XBB.1.5 mRNA vaccines. All patients previously received three doses with original COVID-19 vaccines. Results: Mucosal IgG antibodies were readily induced by XBB.1.5 mRNA vaccines (p = 0.0013 comparing pre- and post-vaccination levels). However, mucosal IgA levels were comparable before and after vaccination (p = 0.8233). Consequently, mucosal IgG and IgA antibody levels correlated only moderately before and after immunization (pre-vaccination: r = 0.5294; p = 0.0239; post-vaccination: r = 0.4863; p = 0.0407). Contrary to a previous report in healthy individuals, vaccination did not induce serum IgA in patients with IBD (p = 0.5841 comparing pre- and post-vaccination levels). These data suggest that COVID-19 mRNA vaccines fail to elicit mucosal IgA in patients with IBD. Conclusions: Since mucosal IgA plays a pivotal role in infection control, the lack of IgA induction indicates that patients lack sufficient protection against SARS-CoV-2 infections which warrants the development of mucosal COVID-19 vaccines. Full article

Background/Objectives: Neuropilin-1 (NRP1) is a key co-receptor for SARS-CoV-2, complementing the ACE2 receptor. Several investigations have documented highly conserved sequences in this receptor, supporting the implication of NRP1 as a key mediator in SARS-CoV-2 cellular entry mechanisms. Methods: To investigate this hypothesis, we examined 104,737 SARS-CoV-2 genome fastas from GISAID genomic data, corresponding to isolates collected between 2020 and 2025 in Mexico. Specifically, we focused on the RRAR motif, a known furin-binding site for NRP-1 and the binding site for ACE2 with the spike protein. Our analysis revealed high conservation (>98%) of the RRAR domain compared to a rapidly diminishing ACE2-binding domain. A complementary analysis, using Data from Gene Expression Omnibus (GEO, GSE150316), showed that NRP1 expression in lung tissue remains relatively stable, whereas ACE2 displayed high inter-individual variability and lower abundance compared to NRP1. Based on this evidence, we designed two humans–rats NRP1 siRNAs that were tested in vivo using a melittin-induced lung injury model. Results: The RT-PCR assays confirmed an effective NRP1 knockdown, and the siRNA-treated group showed a significant reduction in the lesions severity. These findings highlight NRP1 as a stable and relevant therapeutic target and suggest the protective potential of siRNA-mediated gene silencing. Conclusions: The evidence presented here supports the rational design of NRP1-directed therapies for multiple circulating SARS-CoV-2 variants in Mexico. Full article

The Bacillus Calmette-Guérin (BCG) vaccine confers broad, non-specific immunity that may bolster defenses against respiratory viruses. While NOD2 (nucleotide-binding oligomerization domain-containing protein 2)-driven pathways are central to innate immune responses, the contribution of surface receptor modulation on monocytes to shaping these responses remains underexplored. We analyzed whole-blood cultures from BCG-vaccinated Polish children, stratified by serostatus to SARS-CoV-2 and RSV, and stimulated for 48 h with live BCG, purified viral antigens, or both. RT-qPCR quantified mRNA levels of NOD2 and key cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF), while flow cytometry assessed CD14, HLA-DR, CD11b, and CD206 expression. Co-stimulation with BCG + RSV elicited the strongest transcriptional response, notably a 2–4-fold upregulation of NOD2, IL-1β, and IL-6 versus RSV alone. In SARS-CoV-2(+) donors, RSV alone induced higher NOD2 expression than BCG or BCG + RSV, while IL-2 peaked following BCG + SARS-CoV-2. Across conditions, NOD2 positively correlated with IL-4 and IL-6 but negatively correlated with IL-1β in SARS-CoV-2 cultures. Viral antigens increased CD14 and HLA-DR on monocytes, suggesting activation; CD206 rose only in dual-seropositive children. Our findings indicate that BCG stimulation affects pediatric antiviral immunity through NOD2-related cytokine production and induction of a CD14⁺HLA-DR⁺ phenotype, supporting its potential role in boosting innate defenses against respiratory pathogens. Full article

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG_2a and IgG₁ after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity. 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

The diversity of structural types of carrageenans (CRGs)—sulfated polysaccharides of red algae—determines their different biological activities. The different types of CRGs (kappa, lambda, kappa/beta-CRGs) were isolated from the red algae of the Pacific coast. Molecular docking was performed to determine potential interactions of CRGs with the receptor-binding domain (RBD) of SARS-CoV-2 and its cellular receptor—angiotensin—converting enzyme type 2 (ACE2). CRGs interacted with ACE2 and RBD via hydrogen bonding and ionic interactions. The strongest binding affinity of CRGs and ACE2 was observed for kappa-CRG. Molecular docking was confirmed by results studying the effects of CRGs against SARS-CoV-2 in vitro. The ability of CRGs, as well as the complex CRG with sea urchin echinochrome (Ech), to inhibit SARS-CoV-2 replication in Vero E6 cells was studied using cytopathic effect (CPE) inhibition and RT-PCR assays. The simultaneous treatment of cells with CRGs and the virus revealed that kappa-CRG exhibited the most significant antiviral effect among all the polysaccharides, with a selective index (SI) of 33. The kappa-CRG/Ech complex exhibited the highest virucidal effect on SARS-CoV-2 particles with an SI above 70 (more than two times higher than that of CRG and Ech) and reduced viral RNA levels by 45% (IC = 45%). Our results illustrate that CRGs and kappa-CRG/Ech complex can act as protective agents against SARS-CoV-2. 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/Objectives: Vaccine immunogenicity is often suboptimal in vulnerable populations such as the elderly, infants, and individuals in low- and middle-income countries. One contributing factor may be pre-existing immunomodulatory conditions, including helminth infections. This study investigates the impact of Fasciola hepatica (F. hepatica) derived molecules on the early humoral response to the COVID-19 mRNA vaccine Comirnaty^® in a mouse model. Methods: BALB/c mice were pretreated with a F. hepatica protein extract (FH) or complete Freund’s adjuvant (CFA) prior to vaccination. Cytokine production and antibody responses were assessed at 0, 14, and 21 days post-vaccination (dpv) through serum analysis and ex vivo splenocyte stimulation with the SARS-CoV-2 receptor-binding domain (RBD) or LPS. Results: At 0 dpv, FH-treated mice showed increased serum IL-10, while CFA treatment induced IL-12. FH- but not CFA-treated splenocytes secreted IL-10 upon RBD or LPS stimulation. At 21 dpv, FH-treated mice lacked IFN-γ production but maintained IL-10 and showed elevated IL-4, consistent with a Th2-skewed profile. Although total anti-RBD IgG levels were similar between groups, FH-treated mice exhibited reduced IgG avidity and a higher IgG1/IgG2 ratio. CFA-treated mice showed delayed avidity maturation. Conclusions: Prior exposure to F. hepatica antigens can modulate the early immune response to Comirnaty^®, affecting both cellular activation and antibody quality. This altered response may reflect a reduced early protective capacity of the vaccine, which might need to be considered when designing or evaluating vaccination strategies using mRNA vaccines in helminth-endemic regions. Full article

Aim. This study aimed to assess the SARS-CoV-2 herd immunity in the Republic of Armenia (RA) by late 2022. Materials and Methods. A randomized study was conducted from 28 November to 2 December (2022) by the Saint Petersburg Pasteur Institute (Russia) in collaboration with the National Center for Disease Control and Prevention (Armenia). This study was approved by the ethics committees at both organizations. A volunteer cohort (N = 2974) was formed and grouped by participant age, region, or activity. Antibodies (Abs) to viral nucleocapsid antigen (Nc) and receptor-binding domain (RBD) in plasma were determined by ELISA. The statistical significance of differences was calculated using a p < 0.05 threshold, unless noted. Results. At the end of 2022, estimated SARS-CoV-2 seroprevalence (Nc and/or RBD Abs) among the Armenian population was 99% (95%CI: 98.5–99.3). It was evenly distributed throughout the cohort without any significant differences by age, region, or activity. Volunteers with low (32–124 BAU/mL) or medium (125–332 BAU/mL) anti-Nc Ab levels prevailed: 32.4% (95%CI: 30.7–34.1) and 25.5% (95% CI: 24.0–27.1), respectively. Regarding anti-RBD Abs, maximum levels (>450 BAU/mL) were detected in 40% of children. The share of individuals with high anti-RBD Abs levels increased with age, reaching 65% among those aged 70⁺ years. The important contribution to the formation of herd immunity to coronavirus infection was made by vaccination in the preceding period (1 April 2021 to 1 May 2022). The contribution from individuals with post-vaccination immunity was estimated to be above 80%. Hybrid immunity, formed after vaccination of those who had earlier experienced COVID-19, was characterized by greater effectiveness than post-vaccination immunity alone. Conclusions. Within the context of mass prophylactic vaccination, effective herd immunity to SARS-CoV-2 was formed, which helped to stop epidemic spread in the Republic. 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