Search Results (276)

This study developed novel immunostimulatory hydrogels composed of aldehyde-modified Arnebia euchroma polysaccharide (oxidized ARP, O-ARP) and gelatin (GE) for oligonucleotide delivery. Structural characterization of three O-ARP derivatives with varying degrees of oxidation confirmed reduced molecular weight, a preserved yet altered molar ratio of monosaccharide composition, and triple-helical conformation. Hydrogels (GE-O1, GE-O2, GE-O3) were formed via a Schiff base reaction between aldehyde and amino groups. Molecular dynamics simulations showed that O-ARP and gelatin can form a stable three-dimensional (3D) network via hydrogen bonding and van der Waals interactions. In vitro studies demonstrated that both the O1-ARP derivative and GE-O1 hydrogel significantly improved RAW 264.7 macrophage viability, phagocytosis, NO production, and pro-inflammatory cytokine secretion, including IL-6, IL-1β, and TNF-α. The cationic GE-O1 hydrogel efficiently loaded anionic CpG oligonucleotides (CpG-ODN 1862) via electrostatic interaction, forming GE-O1-CpG complexes and promoting cellular uptake. Importantly, the GE-O1-CpG complex exhibited superior immunomodulatory effects compared with either GE-O1 or CpG alone, indicating a synergistic dual immunostimulatory response. In vivo studies confirmed the biosafety of GE-O1-CpG. Using OVA as a model antigen, GE-O1-CpG/OVA enhanced both humoral and cellular immune responses. These findings support GE-O1-CpG hydrogels as potential system for combined immunomodulation and nucleotide delivery. Full article

Parvovirus infection causes severe diseases in both feline and canine species. It primarily affects adult cats and dogs but poses a higher risk to kittens and puppies. This virus is highly contagious and is easily transmitted through contaminated food, shared shelter environments, as well as the hands and clothing of people. The recovered species may continue to shed parvovirus in their feces for an extended period, leading to severe environmental contamination. There is no universal vaccine available that protects dogs and cats against parvovirus infections. The main goal of this study is to design a pan-parvovirus multiepitope-based vaccine that could be administered to dogs and cats. We utilized AI-machine learning-incorporated server tools such as IEDB and NetMHCpan to predict B-cell and T-cell epitopes. VaxiJen and ToxinPred were used to analyze immune characteristic features and docking with feline alleles using the HADDOCK server. Following this, the immune response and stability of the vaccine construct were confirmed with disulfide engineering, normal mode analysis, and molecular docking performed with toll-like receptors of both feline and canine (TLR4 and TLR5), and molecular dynamics simulation was performed for 10 ns. The triggered immune response was determined with immuno-simulation (ImmSim), and their activity in a biological environment was reinforced with in silico cloning. The B-cell epitopes (NS1-9, NS2-4, VP1-12 and VP2-9) predicted with the IEDB database were subjected to antigenicity prediction. MHC class I and IFN prediction and MHC class II and IL-4 prediction were performed with IEDB and NetMHCpan. The T-cell epitopes showed high binding affinities with the feline alleles. The final vaccine was designed by combining the top-ranked B-cell epitopes and T-cell epitopes, filtered for high antigenicity, non-allergic, non-toxic, and good solubility, and with the better binding affinity score of the structural and non-structural proteins (NS1, NS2, VP1, and VP2) of feline and canine parvoviruses through linkers and adjuvants. The disulfide bond prediction and normal mode analysis showed that our vaccine construct is stable and flexible. The molecular docking analysis was performed between the designed vaccine epitopes and the TLRs (TLR4–feline and TLR5–canine) with Biovia Discovery Studio using Zdock; it showed better binding interactions with a value of 22.26 (Zdock score), −47.409 (Zrank score) for feline and 16.54 (Zdock score), −134.295 (Zrank score) for canine. A pan-multi-epitope-based vaccine based on the two structural and non-structural proteins (NS1, NS2, VP1, and VP2) was designed and constructed to provide dual protection against parvovirus in both feline and canine species. The molecular docking and molecular dynamics simulation analysis showed higher binding affinities and stable conformations with canine (TLR5) and feline (TLR4) toll-like receptors. Although computational analysis supports the prediction of top-ranked epitopes and their immunogenic properties with greater precision, further experimental validation is required before they can be used against these viruses. Full article

Influenza A virus (IAV) remains a major threat to global public health because of its capacity for antigenic drift, reassortment, zoonotic transmission, and pandemic emergence. Viral transcription and genome replication are carried out by the influenza virus RNA-dependent RNA polymerase (FluPol), a heterotrimeric complex composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA), which functions together with nucleoprotein (NP) within viral ribonucleoprotein complexes (vRNPs). FluPol activity is regulated not only by viral determinants and host cofactors but also by diverse post-translational modifications. Among these, ubiquitination has emerged as a particularly versatile regulatory mechanism because it can control protein stability, polymerase assembly, subunit interactions, conformational dynamics, NP–RNA interactions, and innate immune signaling. Depending on the modified substrate, ubiquitin linkage type, acceptor residue, and responsible E3 ligase or deubiquitinase, ubiquitination may either restrict IAV replication or be exploited by the virus to enhance polymerase function and vRNP activity. This review summarizes recent advances in ubiquitination-mediated regulation of FluPol and NP, focusing on the responsible E3 ubiquitin ligases, deubiquitinases, ubiquitination sites, ubiquitin-chain types, and host restriction mechanisms. We further discuss the crosstalk between ubiquitination and other post-translational modifications, highlight unresolved mechanistic questions, and evaluate the therapeutic potential and challenges of targeting ubiquitin-dependent pathways for antiviral intervention. Collectively, this review provides a conceptual framework for understanding how ubiquitination shapes IAV replication and identifies E3 ligases and DUBs as potential targets for host-directed antiviral strategies. Full article

Aqueous two-phase systems (ATPSs) provide a versatile, fully aqueous platform for probing solute–water interactions and protein structure. This review first surveys the diversity and phase behavior of biphasic aqueous systems formed by polymers and salts. We describe how phase diagrams characterize ATPS formation and composition and how both polymer chemistry and salt identity, rather than molecular size alone, govern phase separation by modulating the solvent properties of water. Building on a modified binodal model, we show that phase separation and solute partitioning can be understood in terms of changes in aqueous solvent dipolarity/polarizability, hydrogen-bond donor/acceptor properties, hydrophobicity, and electrostatics, quantified via solvatochromic probes and homologous solute series. These measurements underpin solvent interaction analysis (SIA), in which the partition coefficients of small molecules and proteins across panels of ATPSs are used to generate “structural signatures” that sensitively report on amino acid substitutions, conformational changes, aggregation, ligand binding, osmolyte effects, and post-translational modifications, independent of protein size. We discuss how SIA can be implemented in vial-, plate-, and microfluidic formats and combined with diverse analytical readouts (HPLC, MS, colorimetric assays, and immunoassays), and we contrast this structure-focused approach with conventional concentration-only proteomic and biomarker strategies. Particular emphasis is placed on structure-based biomarker discovery, where disease-relevant shifts in proteoform distributions—especially glycosylation changes—are often more informative than bulk protein levels and where SIA can complement or simplify complex glycomics and top-down proteomics workflows. As a case study, we describe the recently FDA-approved IsoPSA assay, which applies SIA principles to prostate-specific antigen by measuring cancer-associated structural alterations in circulating PSA via its partition behavior in a proprietary ATPS. IsoPSA generates a single index that discriminates between high-grade prostate cancer and benign and low-grade conditions. Prospective, longitudinal, and MRI-integrated clinical studies demonstrate that IsoPSA improves pre-biopsy risk stratification, reduces unnecessary biopsies, and provides robust negative and positive predictive values within the PSA “gray zone.” Collectively, the data support aqueous solvent interaction analysis as a broadly applicable, mechanistically grounded technology for protein characterization, drug–protein interaction studies, and structure-centric biomarker development, exemplified by the clinical translation of IsoPSA. Full article

The HIV-1 Env glycoprotein mediates both cell-free and cell-to-cell viral transmission and represents the primary target for protective humoral immune responses. Studies examining antibody neutralization of cell-free and cell-to-cell HIV transmission have found that cell-to-cell transmission is more resistant to neutralization. This resistance may be explained in part by antigenically distinct Env populations on virions and infected cells. Cell-surface Env may be more heterogeneous due to variations in cleavage, glycosylation, and conformational state. Nevertheless, the mechanisms that maintain antigenically distinct Env populations at the cell surface and on virions remain unclear, despite virion assembly occurring at the plasma membrane. In this focused review, we consider how Env endocytosis and recycling influence Env incorporation into virions and antibody recognition. We further consider how Env cleavage may influence trafficking and endocytic fate. Given the central role of Env’s cytoplasmic tail in engaging endosomal trafficking pathways, we review emerging structural models of the CT and discuss how its organization, symmetry, and conformational flexibility may contribute to Env trafficking and intracellular sorting. We also discuss how heterogeneous Env populations may influence antibody susceptibility. Finally, we review therapeutic strategies, including combinatorial antibodies and small-molecule Env modulators, that may enhance antibody recognition of infected cells and virions. Full article

Inflammatory and oxidative stress within the pancreatic islet microenvironment can alter insulin-derived peptides and generate neoepitopes that may reshape autoreactive T cell recognition in type 1 diabetes (T1D). One such modification, C19S, represents a cysteine-to-serine substitution at position 19 of the insulin B-chain and has recently been identified among human leukocyte antigen class II (HLA-II)-associated insulin neoepitopes recognized by autoreactive CD4+ T cells. Although the biological relevance of C19S has been determined, the molecular features that may distinguish C19S-specific T cell receptor (TCR) engagement from native insulin recognition remain incompletely defined. Here, we used comparative protein–protein docking, molecular dynamics (MD) simulations, interface-contact analysis, conformational landscape analysis, and binding-energy calculations to examine TCR engagement of human leukocyte antigen DQ8 (HLA-DQ8) presenting either native insulin peptide or the corresponding C19S insulin peptide. Initial modeling indicated that both peptide-HLA-DQ8 complexes were compatible with TCR-bound ternary complex formation. However, the C19S-containing complex was predicted to exhibit altered peptide-centered dynamics, changes in peptide backbone presentation, and reorganization of both TCR-peptide and TCR-HLA-DQ8 contacts. Comparative molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) and molecular mechanics generalized Born surface area (MM/GBSA) analyses further suggested a distinct calculated energetic profile under the applied modeling conditions for the C19S-containing complex, with residue-level decomposition localizing energetic differences to selected interface hotspots. Together, these findings provide a molecular framework for generating hypotheses about how C19S may reshape the HLA-DQ8-presented insulin recognition surface, with implications for future experimental studies of autoreactive CD4+ T cell recognition and antigen-specific tolerogenic strategies in T1D. Full article

Classical Major Histocompatibility Complex Class I (MHC-I) molecules are traditionally viewed as stable peptide-presenting structures expressed on the surface of all nucleated cells. Their expression by professional antigen-presenting dendritic cells (DCs) enables CD8+ T-cell activation, differentiation, and immune surveillance. However, accumulating evidence indicates that cell-surface MHC-I molecules exist in three major conformational states: (1) β2m-associated, peptide-loaded conformers that originate in the endoplasmic reticulum and pass through the Golgi apparatus after binding proteasome-generated cytosolic peptides (hereafter referred to as closed conformers); (2) β2m-free, peptide-empty conformers that arise following β2m dissociation from closed conformers either at the plasma membrane or after internalization and recycling (hereafter referred to as open conformers); and (3) β2m-associated, peptide-empty conformers that represent an intermediate state between closed and open conformers. Here, we propose a conceptual framework, supported by computational predictors of intrinsically disordered regions, in which transitions between closed and open MHC-I conformers are coupled to intracellular regulatory processes, including post-translational modifications of conserved motifs, intracellular trafficking, and signaling. Although direct experimental evidence linking these processes remains limited, we integrate independent observations into a working model that may guide future investigations into MHC-I-mediated cell–cell communication in both immune and non-immune contexts, in health and disease. For clarity, in this article we define “open conformers” as structurally competent, β2m-free, and peptide-deficient MHC-I molecules. Full article

In November 2024, a highly mutated descendant of the Omicron BA.3 subvariant, designated BA.3.2, emerged in South Africa carrying 39 spike mutations, two large N-terminal domain (NTD) deletions and a novel four-amino acid insertion. A key feature of BA.3.2 is extensive NTD remodeling, including a major deletion spanning residues 135–148 affecting the β-hairpin region and contributing to the loss of most of the N1 loop. This study compares the evolutionary dynamics and structural features of BA.3.2 with BA.3. Phylodynamic analyses show that BA.3 underwent early demographic stability followed by a decline in genetic diversity, consistent with limited circulation, whereas BA.3.2 displays recent emergence and a progressive reduction in effective population size without rapid expansion. Selection analyses indicate BA.3 evolution is mainly driven by changes in the receptor-binding domain, while BA.3.2 shows dispersed signals across spike regions, including codon 1162. Structural and molecular dynamic analyses reveal increased flexibility and a broader conformational landscape in the BA.3.2 NTD, driven by the deletion and resulting loss of stabilizing interactions. Overall, BA.3.2 follows a distinct evolutionary trajectory characterized by antigenic remodeling of the spike NTD, underlining the need for continued surveillance of emerging SARS-CoV-2 descendant lineages. Full article

Porcine diarrheagenic Escherichia coli strains exhibit heterogeneous virulence profiles involving multiple factors, complicating broad-spectrum serological diagnosis. Here, we designed a multiepitope fusion antigen, MEAET, containing predicted B-cell epitopes from six major virulence factors, K88/F4, F18, LT, Stx2e, HlyA, and Tir, and developed an indirect ELISA for broad antibody detection. Structural modeling predicted a well-folded conformation with multiple discontinuous B-cell epitopes, and molecular docking suggested favorable interactions between the C-terminal dendritic cell-targeting peptide and the porcine immune receptor SLA1. Immunization of piglets with purified recombinant MEAET generated hyperimmune serum with a titer exceeding 1:102,400, which specifically recognized all six individual antigens by Western blot. The optimized ELISA demonstrated satisfactory repeatability (intra-assay CV ≤ 4.26%) and reproducibility (inter-assay CV ≤ 5.57%), showed no detectable cross-reactivity with antibodies against other tested porcine pathogens, and exhibited good analytical sensitivity, with positive sera detectable at dilutions up to 1:3200. The optimized ELISA showed good repeatability and reproducibility, with intra-assay and inter-assay coefficients of variation of ≤4.26% and ≤5.57%, respectively, no detectable cross-reactivity with the tested heterologous controls, and good analytical sensitivity, with positive sera remaining detectable at dilutions up to 1:3200. The S/P ratio cut-off was 0.203. In field sera, positivity rates were 74.5% (35/47) in diarrheic pigs and 34.0% (36/106) in asymptomatic pigs. These primary results indicate that the MEAET-based indirect ELISA represents a promising tool for serological detection of antibodies against multiple virulence factors of diarrheagenic E. coli in pigs and for field surveillance in swine herds. Full article

Rhodococcus equi (R. equi) primarily induces fatal pulmonary and extrapulmonary pyogenic granulomatous infections in foals, imposing substantial economic burdens on the equine industry. The emergence and spread of multidrug-resistant (MDR) R. equi have led to a therapeutic impasse in clinical settings. Although vaccination is a proven strategy against MDR pathogens, no commercial vaccine is currently available for R. equi. In this study, we employed a bioinformatics approach to systematically identify and prioritize antigenic epitopes derived from R. equi for multi-epitope vaccine design. Using ABCPred, NetMHCpan EL, and IEDB servers, 27 MHC-I and 9 MHC-II epitopes were selected from five previously validated R. equi vaccine candidates: ABC transporter, PBD2, NlpC/P60, Esterase, and M23. These epitopes were coupled with distinct peptide linkers to construct six multi-epitope vaccine constructs, designated V1–V6. The physicochemical properties, antigenicity, immunogenicity, and toxicity of the six vaccine constructs were analyzed, and the V3 and V4 constructs were ultimately selected. Using the HDOCK and Gromacs tools, the intermolecular interactions, binding affinity, and thermal stability of the V3 and V4 constructs with the equine MHC molecules EQCA-I and EQCA-II were evaluated. The results confirm that V3 and V4 exhibit strong binding affinity to EQCA-I and EQCA-II, with stable conformations following binding, indicating theoretical potential to induce humoral and cellular immunity in foals. Recombinant plasmids for V3 and V4 were constructed, and the V3 and V4 proteins were successfully prepared, confirming the feasibility of prokaryotic expression for these vaccine constructs. Immunization assays in SPF BALB/c mice showed that the multi-epitope vaccines elicited robust antigen-specific IgG antibody responses, reflecting preliminary humoral immunogenicity. However, these murine data have translational limitations, as they cannot fully represent equine immune responses. The findings establish a crucial theoretical foundation for the advancement of vaccines targeting R. equi while offering a reference for the design of vaccines against other drug-resistant microbial pathogens. Full article

Porcine epidemic diarrhea virus (PEDV) continues to undergo genetic evolution and remains a major etiological agent of enteric disease in swine, causing significant economic losses worldwide. This study investigated the molecular epidemiology and genetic characteristics of PEDV circulating in Chongqing, China, between 2022 and 2024. A total of 296 diarrheic piglet samples collected from nine regions were screened using RT-qPCR, of which 48.31% (143/296) tested positive for PEDV. A subset of positive samples was subjected to S gene amplification and sequencing, yielding 15 complete sequences. Phylogenetic analysis revealed that all sequenced strains clustered within the G2c lineage and showed high nucleotide similarity (93.37–94.09%) to the classical CV777 strain. Recombination analysis indicated potential recombination events among field strains involving S-INDEL and G2b-like parental lineages, although these findings are based on a limited number of sequences. Sequence analysis identified multiple amino acid substitutions within the COE antigenic region, while other neutralizing epitopes (SS2, SS6, and 2C10) remained largely conserved. In addition, variation in predicted N-glycosylation sites was observed among some strains. Structural modelling suggested that these changes may influence spike protein conformation and antigenic properties; however, these interpretations are based on in silico analysis and require experimental validation. Overall, the findings indicate ongoing genetic evolution of PEDV in Chongqing and suggest circulation of G2c-associated variants in diarrheic piglets. However, given the limited and non-random nature of sequencing, these results may not fully represent the broader viral population. Continued large-scale molecular surveillance and functional studies are needed to better understand PEDV evolution and to support the development of improved control strategies and vaccines. Full article

Nipah virus (NiV) is a highly pathogenic zoonotic pathogen. Since its discovery in 1998, recurrent epidemics have occurred in South and Southeast Asia, with a case fatality rate ranging from 40% to 100%. The outbreak in West Bengal, India in early 2026 has once again highlighted its severe threat to public health. To date, no licensed human vaccines or specific therapeutics against NiV are available worldwide. This review systematically summarizes the breakthroughs in antigen design for NiV vaccines, with a focus on conformational stabilization of prefusion F (pre-F) protein, chimeric G/F antigens, and multivalent nanoparticle strategies. In addition, we comparatively analyze the clinical progress of mainstream vaccine platforms, including viral vectors, mRNA and subunit vaccines. Given the sporadic nature and high mortality of NiV infection, the conventional licensing pathway relying on large-scale phase III clinical trials faces substantial practical obstacles. Accordingly, this article discusses adaptive adjustments in regulatory science. We propose several strategies to accelerate the clinical translation and emergency stockpiling of NiV vaccine candidates, including establishing unified correlates of protection thresholds, coordinating multinational regulatory resources, and optimizing the implementation of Animal Rule. Full article

Dengue virus (DENV), which comprises four antigenically distinct serotypes (DENV-1 to DENV-4), remains a major global public health concern and continues to expand geographically; however, the structural features of the viral genome remain incompletely understood. Although G-quadruplexes (G4s) have previously been reported in coding regions of DENV, their presence within the 3′ untranslated region (3′ UTR) has not been experimentally characterized. Here, we focused on selected guanine-rich motifs within the 3′ UTRs of DENV-1 to DENV-4 and investigated their ability to form RNA G4 structures. Using bioinformatic analysis, we identified comparable G-rich regions in the 3′ UTRs of the four serotypes, with serotype-dependent differences in conservation. We then examined the propensity of the selected putative quadruplex-forming sequences (PQSs) to adopt G4 structures using circular dichroism spectroscopy, UV melting analysis, ¹H NMR spectroscopy, ligand-binding analysis, and reverse transcription stop (RT-stop) assays. Our results provided in vitro evidence that the 3′ UTR oligonucleotides from DENV-1 to DENV-4 are capable of forming ligand-responsive G4 structures, with serotype-dependent differences in conservation, stability, and conformational homogeneity. In addition, reverse transcription (RT)-stop analysis revealed ligand-dependent arrest at the corresponding PQS sites in the presence of the G4 ligand 6OTD, which stabilizes G4 structures. These findings suggest the DENV 3′ UTR as an additional source of ligand-responsive RNA G4-forming elements and support future studies on their possible roles in DENV RNA regulation. Full article

Macrophages are highly plastic innate immune cells that adopt context-dependent phenotypes along a continuum, integrating developmental origin with local microenvironmental cues rather than conforming to discrete M1/M2 states. This review delineates the molecular circuits shaping macrophage identity—TLR/cytokine signaling, microRNA networks, metabolic rewiring, and epigenetic mechanisms including histone lactylation—and traces how circulating monocyte subsets contribute to tissue macrophage diversity. We examine macrophage plasticity across a broad disease spectrum—oncology, autoimmune and rheumatic diseases, inflammatory bowel disease, infectious diseases, metabolic disorders, and neurological conditions—showing that the pathogenic phenotype is strikingly context-dependent: for instance, M2-like tumor-associated macrophages promote immune evasion in solid tumors, whereas M1-skewed programs drive tissue damage in autoimmunity. Soluble markers (sCD163, sCD14, soluble mannose receptor) are emerging biomarkers of disease activity and prognosis. High-dimensional flow cytometry and mass cytometry (CyTOF) bridge molecular biology and clinical phenotyping, enabling integrated readouts of surface phenotype, intracellular signaling, and metabolic state. Therapeutic strategies discussed include selective tumor-associated macrophage (TAM) reprogramming, chimeric antigen receptor (CAR)-M cell therapies, and biomaterial-based platforms. Future priorities encompass spatially resolved multi-omics, epigenetic and metabolic targeting, and macrophage-centered vaccine approaches. Standardized cytometry panels will be essential for biomarker-guided stratification and context-specific interventions. Full article

Porcine enteric coronaviruses (PECs), including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), remain major causes of neonatal diarrhea, dehydration, mortality, and economic loss in swine production. Despite substantial progress in vaccine development, durable field protection is still inconsistent. In this narrative review, this narrative review synthesizes current knowledge on PEC vaccine design from three connected perspectives: antigenic breadth, mucosal immunity, and translational evaluation. The economic and virological context of PEC vaccine development is first summarized, including the recurrent production burden of PECs, coronavirus genome organization, structural proteins, and the central role of the spike protein in receptor engagement, membrane fusion, and neutralizing antibody induction. Key issues are then discussed, including how spike diversity, conformational stability, epitope accessibility, glycan shielding, and antigen matching influence protective breadth; why intestinal secretory IgA, mucosal immune-cell trafficking, local memory responses, and lactogenic immunity should be prioritized as biologically relevant endpoints; and how delivery route, adjuvant selection, and platform design shape response quality. Current evidence on recombinant protein, viral-vectored, nanoparticle, virus-like particle, probiotic, plant-derived, and mRNA-based approaches is compared with attention to both promise and current evidentiary and translational limitations. The available literature suggests that future progress in PEC vaccinology is likely to depend less on platform novelty alone than on integrated vaccine designs that align antigen selection, mucosal delivery, maternal–neonatal protection, heterologous challenge, manufacturability, and field applicability. Full article