Search Results (3,265)

IgG Fc binding protein (FcGBP) is a mucin-like protein that binds strongly to IgG and IgG–antigen complexes in intestinal mucus. FcGBP presence and its altered expression levels in meconium accumulating in the fetal intestine and amniotic fluid flowing in the intestine may provide new knowledge of the mechanisms responsible for the immune adaptation of the fetus to extrauterine life. FcGBP concentrations were measured by ELISA in the first-pass meconium and amniotic fluid samples collected from 120 healthy neonates delivered by either vaginal birth (n = 35) or cesarean section (n = 85) at 36 to 41 weeks gestation. The meconium FcGBP concentrations (405.78 ± 145.22 ng/g) decreased (r = −0.241, p = 0.007) over the course of 36 to 41 weeks gestation, but there were no significant changes (p > 0.05) in the amniotic fluid FcGBP (135.70 ± 35.83 ng/mL) in the same period. Both meconium and amniotic fluid FcGBP concentrations were higher (p < 0.05) in neonates delivered by cesarean section. Decreases in the meconium FcGBP concentrations correlated (r = −0.37, p = 0.027) with the gestational age in neonates delivered by vaginal birth but not in those delivered by cesarean section (p > 0.05). No association was found between the FcGBP concentrations in meconium and amniotic fluid and the birth weight (p > 0.05). With the development of the mucosal immune system in the fetal intestine over the course of the third trimester of gestation, the meconium FcGBP concentrations decrease. Increased FcGBP concentrations measured in the meconium and amniotic fluid of neonates delivered by cesarean section may possibly indicate altered intestinal mucosal function. Intrauterine growth is not associated with the intestinal mucosal barrier maturation involving FcGBP. Full article

Freshwater salinization, an escalating global environmental stressor, poses a significant threat to freshwater biodiversity, including fish communities. This study investigates the grass carp (Ctenopharyngodon idellus), a species with the highest aquaculture output in China, to elucidate the molecular underpinnings of its physiological adaptations to fluctuating salinity gradients. We used high-throughput mRNA sequencing and differential gene expression profiling to analyze transcriptional dynamics in intestinal and kidney tissues of grass carp exposed to heterogeneous salinity stressors. Concurrent serum biochemical analyses showed salinity stress significantly increased Na⁺, Cl⁻, and osmolarity, while decreasing lactate and glucose. Salinity stress exerted a profound impact on the global transcriptomic landscape of grass carp. A substantial number of co-regulated differentially expressed genes (DEGs) in kidney and intestinal tissues were enriched in immune and metabolic pathways. Specifically, genes associated with antigen processing and presentation (e.g., cd4-1, calr3b) and apoptosis (e.g., caspase17, pik3ca) exhibited upregulated expression, whereas genes involved in gluconeogenesis/glycolysis (e.g., hk2, pck2) were downregulated. KEGG pathway enrichment analyses revealed that metabolic and cellular structural pathways were predominantly enriched in intestinal tissues, while kidney tissues showed preferential enrichment of immune and apoptotic pathways. Rigorous validation of RNA-seq data via qPCR confirmed the robustness and cross-platform consistency of the findings. This study investigated the core transcriptional and physiological mechanisms regulating grass carp’s response to salinity stress, providing a theoretical foundation for research into grass carp’s resistance to salinity stress and the development of salt-tolerant varieties. Full article

Background: It is still challenging to develop effective vaccines against tumorigenic human gammaherpesviruses such as Epstein–Barr virus (EBV). A major obstacle is the lack of a small animal model that reproduces the natural infection course of human gammaherpesviruses to allow for proper assessment of vaccine efficacy. Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of wild rodents and laboratory mice and therefore can be used as a surrogate for human gammaherpesviruses to evaluate vaccination strategies. Methods: In this study, two mRNA vaccine candidates were generated, one encoding a fusion protein of the MHV68 gH with the gL (gHgL-mRNA) and the other expressing the MHV68 gB protein (gB-mRNA). The immunogenicity and protective efficacy of the mRNA vaccine candidates were evaluated in a mouse model of MHV68 infection. Results: The gHgL-mRNA but not the gB-mRNA candidate vaccine was able to induce neutralizing antibodies in mice, whereas both vaccines could elicit antigen-specific T-cell responses. Following MHV68 intranasal inoculation, complete blocking of the establishment of viral latency was observed in some mice immunized with individual gHgL-mRNA or gB-mRNA vaccines. Notably, co-immunization with the two mRNA vaccines appeared to be more effective than individual vaccines, achieving sterile immunity in 50% of the vaccinated mice. Conclusions: This study demonstrates that immunization with mRNA platform-based subunit vaccines is indeed capable of preventing MHV68 latent infection, thus validating a safe and efficacious vaccination strategy that may be applicable to human gammaherpesviruses. Full article

Prostate cancer is among the most prevalent malignancies in men worldwide, underscoring the need for early and accurate diagnostic tools. This study presents a proof-of-concept and pilot clinical validation of a novel bioelectric impedance-based biosensor for the detection of prostate-specific antigen (PSA) in human serum. The system integrates Molecular Identification through Membrane Engineering (MIME) with the xCELLigence real-time cell analysis platform, employing Vero cells electroinserted with anti-PSA antibodies. Optimization experiments identified 15,000 cells/well as the optimal configuration for impedance response. The biosensor exhibited specific, concentration-dependent changes in impedance upon exposure to PSA standard solutions and demonstrated significant differentiation between PSA-positive and PSA-negative human serum samples relative to the clinical threshold of 4 ng/mL. The biosensor offered rapid results within one minute, unlike standard immunoradiometric assay (IRMA), while showing strong diagnostic agreement. The system’s specificity, sensitivity, and reproducibility support its potential for integration into point-of-care screening workflows. This bioelectric assay represents one of the fastest PSA detection approaches reported to date and offers a promising solution for reducing overdiagnosis while improving clinical decision-making and patient outcomes. Full article

Avian influenza A viruses (IAVs) pose a persistent threat to the poultry industry, causing substantial economic losses. Although traditional vaccines have helped reduce the disease burden, they typically rely on multivalent antigens, emphasize humoral immunity, and require intensive production. This study aimed to establish a genetically matched host–cell system to evaluate antigen-specific immune responses and identify conserved CD8+ T cell epitopes in avian influenza viruses. To this end, we developed an MHC class I genotype (B21)-matched host (Lohmann VALO SPF chicken) and cell vector (DF-1 cell line) model. DF-1 cells were engineered to express the hemagglutinin (HA) gene of clade 2.3.4.4b H5N1 either transiently or stably, and to stably express the matrix 1 (M1) and nucleoprotein (NP) genes of A/chicken/South Korea/SL20/2020 (H9N2, Y280-lineage). Following prime-boost immunization with HA-expressing DF-1 cells, only live cells induced strong hemagglutination inhibition (HI) and virus-neutralizing (VN) antibody titers in haplotype-matched chickens. Importantly, immunization with DF-1 cells transiently expressing NP induced stronger IFN-γ production than those expressing M1, demonstrating the platform’s potential for differentiating antigen-specific cellular responses. CD8+ T cell epitope mapping by mass spectrometry identified one distinct MHC class I-bound peptide from each of the HA-, M1-, and NP-expressing DF-1 cell lines. Notably, the identified HA epitope was conserved in 97.6% of H5-subtype IAVs, and the NP epitope in 98.5% of pan-subtype IAVs. These findings highlight the platform’s utility for antigen dissection and rational vaccine design. While limited by MHC compatibility, this approach enables identification of naturally presented epitopes and provides insight into conserved, functionally constrained viral targets. Full article

Triple-negative breast cancer (TNBC) is a clinically and molecularly heterogeneous subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. In this study, tumor specimens from 104 TNBC patients were analyzed to characterize molecular and clinicopathological features and to assess the expression and therapeutic potential of four key surface markers: epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), tissue factor (TF), and trophoblast cell surface antigen (TROP2). Multiplex immunofluorescence (mIF) demonstrated elevated EGFR and TROP2 expression in the majority of samples. Significant positive correlations were observed between EGFR and TF, as well as between TROP2 and both TF and EpCAM. Expression analyses revealed increased EGFR and TF levels with advancing tumor stage, whereas EpCAM expression declined in advanced-stage tumors. TROP2 and TF expression were significantly elevated in higher-grade tumors. Additionally, EGFR and EpCAM levels were significantly higher in patients with elevated Ki-67 indices. Binding specificity assays using single-chain variable fragment (scFv-SNAP) fusion proteins confirmed robust targeting efficacy, particularly for EGFR and TROP2. These findings underscore the therapeutic relevance of EGFR and TROP2 as potential biomarkers and targets in TNBC. Full article

Since carbohydrate antigen 19-9 (CA 19-9) is a significant biomarker for the clinical diagnosis and treatment of pancreatic cancer, a sensitive sandwich-type immunosensor was proposed with an epoxy functionalized covalent organic framework (EP-COF_TTA-DHTA) as the antibody carrier and an electroactive COF_{TTA-2,6-NA(OH)2} as the signal amplification probe for the sensitive detection of CA 19-9. The flexible covalent linkage between the epoxy-functionalized EP-COF_TTA-DHTA and the antibodies was employed to improve the dynamics of the antigen–antibody interaction significantly. Meanwhile, AuNPs@COF_{TTA-2,6-NA(OH)2} with abundant electroactive sites enhanced the current response of the immunoreaction significantly. After optimizing the incubation time and concentration of the antibody, CA 19-9 was quantitatively detected by differential pulse voltammetry (DPV) based on the sensitive sandwich-type immunosensor with a low detection limit of 0.0003 U/mL and a wide linear range of 0.0009–100 U/mL. The electrochemical immunosensor exhibits high specificity, stability and repeatability, and it provides a feasible and efficient method for the pathologic analysis and treatment of tumor markers. Full article

In a previous study, eight chronically HBV-infected nucleos (t)ide analog (NA)-naïve patients began receiving entecavir (ETV) concomitant with a single ARC-520 HBV siRNA injection. This single dose of ARC-520 (SD) was followed by 6–8 months of ETV alone before the patients received 4–9 monthly doses of ARC-520, the multi-dose (MD) period, while continuing ETV. Quantities of HBV DNA, RNA, and antigens were measured from serum and a liver biopsy collected ~30 months after the last MD from five patients. All full-length HBV transcripts from the livers were characterized. Viral parameters and HBV transcripts from patients were compared to these measurements collected at multiple points in ARC-520 + ETV-treated chronically HBV-infected chimpanzees. Multiple forms of HBx mRNA were observed, and these differed between chimpanzees and patients. Products of cccDNA were greatly decreased in patients who were previously highly viremic and HBeAg+, although a biopsied patient had similar amounts of cccDNA to the highly viremic HBeAg+ chimpanzees. The comparison of all HBV transcripts and cccDNA levels between patients and chimpanzees demonstrate the transcriptional silencing of cccDNA following the siRNA treatment of patients but not the chimpanzees that received a different treatment regimen. Results from this small study suggest that continued NA treatment during and between periods of HBV antigen re-expression post-siRNA treatment enhanced viral parameter reductions. Full article

Sepsis is a fetal disease that requires a clear diagnostic biomarker for timely antibiotic treatment. Recent research has identified a pyroptosis-inducing epitope known as P5-5 in tetranectin (TN), a plasma protein produced by monocytes. Previously, we produced a 12F1 monoclonal antibody against the P5-5 and discovered that it could not only diagnose the presence but also monitor the progress of sepsis in the clinic. In the current study, we further investigated the structure site of the P5-5 and the recognition mechanism between the 12F1 mAb and the P5-5 epitope. To this end, 10 amino acids (NDALYEYLRQ) in the P5-5 were individually mutated to alanine, and their binding to the mAb was tested to confirm the most significant antigenic recognition sites. In the meanwhile, the spatial conformation of 12F1 mAb variable regions was modeled, and the molecular recognition mechanisms in detail of the mAb to the P5-5 epitope were further studied by molecular docking. Following epitope prediction and experimental verification, we demonstrated that the motif “DALYEYL” in the epitope sequence position 2−8 of TN-P5-5 is the major binding region for mAb recognition, in which two residues (4L and 8L) were essential for the interaction between the P5-5 epitope and the 12F1 mAb. Therefore, our study greatly narrowed down the previously reported motif from ten to seven amino acids and identified two Leu as critical contact residues. Finally, a single-chain variable fragment (scFv) from the 12F1 hybridoma was constructed, and it was confirmed that the identified motif and residues are prerequisites for the strong binding between P5-5 and 12F1. Altogether, the data of the present work could serve as a theoretic guide for the clinical design of biosynthetic drugs by artificial intelligence to treat sepsis. Full article

Myxoma virus (MYXV), a rabbit-specific poxvirus and non-pathogenic in humans and mice, is an excellent candidate oncolytic virus for cancer therapy. MYXV also has immunotherapeutic benefits. In ovarian cancer (OC), immunosuppressive tumor-associated macrophages (TAMs) are key to inhibiting antitumor immunity while hindering therapeutic benefit by chemotherapy and dendritic cell (DC) vaccine. Because MYXV favors binding/entry of macrophages/monocytes, we examined the therapeutic potential of MYXV against TAMs. We found previously that a replication-defective MYXV with targeted deletion of an essential gene, M062R, designated ΔM062R MYXV, activated both the host DNA sensing pathway and the SAMD9 pathway. Treatment with ΔM062R confers therapeutic benefit comparable to that of wild-type replicating MYXV in preclinical models. Here we found that ΔM062R MYXV, when integrated with cisplatin and DC immunotherapy, further improved treatment benefit, likely through promoting tumor antigen-specific T cell function. Moreover, we also tested ΔM062R MYXV in targeting human immunosuppressive TAMs from OC patient ascites in a co-culture system. We found that ΔM062R treatment subverted the immunosuppressive properties of TAMs and elevated the avidity of cytokine production in tumor antigen-specific CD4⁺ T cells. Overall, ΔM062R presents a promising immunotherapeutic platform as a beneficial adjuvant to chemotherapy and DC vaccine. Full article

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

Background/Objectives: Porcine circovirus type 2 (PCV2) infection causes porcine circovirus disease (PCVD), a global immunosuppressive disease in pigs. Its clinical manifestations include post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS), which cause significant economic losses to the swine industry. The Cap protein, which is the major protective antigen of PCV2, can self-assemble to form virus-like particles (VLPs) in the insect baculovirus expression system. Few studies have compared the expression of Cap proteins in different baculovirus expression systems. Methods: In this study, we compared two commonly commercialized baculovirus construction systems with the Cap protein expression in various insect cells. Results: The results demonstrate that the flashBAC system expressed the Cap protein at higher levels than the Bac-to-Bac system. Notably, when expressing four copies of the Cap protein, the flashBAC system achieved the highest protein yield in High Five cells, where it reached 432 μg/mL at 5 days post-infection (dpi) with 27 °C cultivation. Animal experiments confirmed that the purified Cap protein effectively induced specific antibody production in mice and swine. Conclusions: This study provides critical data for optimizing the production of the PCV2 Cap protein, which is of great significance for reducing the production cost of PCV2 vaccines and improving the industrial production efficiency. Full article

Background: Understanding the duration and quality of immune memory following SARS-CoV-2 infection and vaccination is critical for informing public health strategies and vaccine development. While waning antibody levels have raised concerns about long-term protection, the persistence of memory B cells (MBCs) and T cells plays a vital role in sustaining immunity. Materials and Methods: We conducted a longitudinal prospective study over 12 months, enrolling 285 participants in total, either after natural infection or vaccination with BNT162b2 or mRNA-1273. Peripheral blood samples were collected at four defined time points (baseline, 1–2 months, 6–7 months, and 12–13 months after vaccination or disease onset). Immune responses were assessed through serological assays quantifying anti-RBD IgG and neutralizing antibodies, B-ELISPOT, and multiparameter flow cytometry for S1-specific memory B cells. Results: Both mRNA vaccines induced robust B cell and antibody responses, exceeding those observed after natural infection. Memory B cell frequencies peaked at 6 months and declined by 12 months, but remained above the baseline. The mRNA-1273 vaccine elicited stronger and more durable humoral and memory B-cell-mediated immunity compared to BNT162b2, likely influenced by its higher mRNA dose and longer prime-boost interval. Class-switched memory B cells and S1-specific B cells were significantly expanded in vaccine recipients. Natural infection induced more heterogeneous immune memory. Conclusions: Both mRNA vaccination and natural SARS-CoV-2 infection induce a comparable expansion of memory B cell subsets, reflecting a consistent pattern of humoral immune responses across all studied groups. These findings highlight the importance of vaccination in generating sustained immunological memory and suggest that the vaccine platform and dosage influence the magnitude and durability of immune responses against SARS-CoV-2. Full article

Background/Objectives: An initial COVID-19 candidate vaccine containing a purified ancestral SARS-CoV-2 spike antigen was characterized with an ELISA using recombinant monoclonal antibodies (mAbs) generated against this variant. Upon the emergence of a new Beta (B.1.351) spike variant early in the pandemic, the assessment of a bivalent vaccine containing ancestral and Beta spike antigens began. Due to accelerated project timelines, mAbs generated specifically against the Beta spike antigen were not available at the time to address assay development and vaccine testing requirements. Methods: Using only the initial mAb panel raised against the ancestral spike antigen, an epitope-blocking ELISA strategy was developed to independently measure Beta spike antigen in bivalent vaccine formulations. To facilitate this, epitope profiling of spike antigens from both ancestral and Beta variants was performed with biolayer interferometry and hydrogen–deuterium exchange mass spectrometry using the original panel of mAbs. Results: The resulting blocking ELISA was precise and specific for the Beta spike antigen and detected the expected amount of this antigen in bivalent vaccine formulations. The specific amount of ancestral spike protein in the bivalent vaccine was also confirmed using the original ELISA developed at the onset of the pandemic. Conclusions: This epitope-blocking strategy helped to overcome key reagent availability issues and could be applied to other projects involving related proteins. Full article

Background/Objectives: Liquid biopsy is a minimally invasive alternative to tissue biopsy and is used to obtain information about a disease from a blood sample or other body fluids. In the context of cancer, circulating tumor cells (CTC) can be used as biomarkers to determine the nature of the tumor, its stage of progression, and the efficiency of the administered therapy through monitoring. However, the low concentration of CTCs in blood (1–10 cells/mL) is a challenge for their isolation. Therefore, a minimally invasive medical device (BMProbe™) was developed that isolates CTCs via antigen–antibody binding directly from the bloodstream. Current investigations focus on the process of detaching bound cells from the BMProbe™ surface for cell cultivation and subsequent drug testing to enable personalized therapy planning. Methods: This article presents two approaches for detaching LNCaP cells from anti-EpCAM coated BMProbes™: enzymatic detachment using TrypLE™ and detachment through enzymatic pretreatment with supplementary flow-induced shear stress. The additional shear stress is intended to increase the detachment efficiency. To determine the flow rate required to gently detach the cells, a computational fluid dynamics (CFD) simulation was carried out. Results: The experimental test results demonstrate that 91% of the bound cells can be detached enzymatically within 10 min. Based on the simulation, a maximum flow rate of 47.76 mL/min was defined in the flow detachment system, causing an average shear stress of 8.4 Pa at the probe edges. The additional flow treatment did not increase the CTC detachment efficiency. Conclusions: It is feasible that the detachment efficiency can be further increased by a longer enzymatic incubation time or higher shear stress. The influence on the integrity and viability of cells must, however, be considered. Full article