Search Results (2,077)

Background/Objectives: The progression of prostate cancer to castration-resistant prostate cancer (CRPC) is often driven by constitutively active androgen receptor splice variants, such as AR-V7, which evade conventional androgen-deprivation therapies. This study aimed to develop a high-throughput, mechanism-based screening platform to identify natural-product-derived AR-V7 inhibitors from the NCI Program for Natural Product Discovery (NPNPD) library. Methods: A subset of prefractionated NPNPD samples was screened using a CRISPR-edited 22Rv1 cell line expressing endogenous AR-V7 fused to a HiBiT luminescent tag, enabling quantification of AR-V7 levels. Fractions that reduced the HiBiT signal were further evaluated in 22Rv1 and C4-2B CRPC cells and counter-screened in non-malignant RWPE-1 cells. Compounds demonstrating at least 90% inhibition in CRPC cells with no more than 10% toxicity in RWPE-1 cells underwent dose–response analysis, Western blotting, quantitative PCR, and subfractionation to isolate and characterize active constituents. Results: Of the 704 prefractionated samples, five fractions met stringent activity and selectivity criteria, with three consistently suppressing full-length androgen receptor (AR), AR-V7, and prostate-specific antigen (PSA). Primary screening results in HiBiT-22Rv1 cells were validated by secondary dose–response and Western blot assays, and hits were further prioritized to ensure toxicity remained at or below 10% in RWPE-1 cells. HiBiT-guided screening of 66 subfractions identified seven that robustly downregulated AR signaling, with reduced AR-V7 levels correlating with decreased CRPC cell viability. Chemical characterization revealed two active butanolides, isolitsealiicolide C and isolinderanolide B, which reduced AR and AR-V7 protein and mRNA levels, decreased PSA, downregulated Bcl-2, and induced cleaved PARP, consistent with apoptotic cell death in AR-positive CRPC models. Conclusions: This integrated high-throughput workflow efficiently identifies AR-V7–targeted natural products from complex libraries and highlights isolitsealiicolide C and isolinderanolide B as promising scaffolds for overcoming androgen receptor–driven resistance in CRPC. Full article

Foot-and-mouth disease virus serotype O remains a global threat, particularly the O/CATHAY topotype in East and Southeast Asia. Because timely access to emerging field isolates can be limited, reverse genetics provides a flexible approach for generating viruses representing selected circulating strains for vaccine matching and efficacy evaluation. Here, we generated O/CATH/APQA-V18 and evaluated its suitability for vaccine assessment in pigs. A contemporary Vietnamese O/CATHAY strain was selected based on viral protein-1 phylogenetic analysis, and its P1 capsid-coding region was incorporated into an O1 Manisa backbone using reverse genetics. The recombinant virus was characterized by real-time reverse transcription polymerase chain reaction, transmission electron microscopy, and sequence analysis. Two-dimensional virus neutralization tests using sera against O/PanAsia-2 and O/Primorsky yielded antigenic relationship values of 0.04 and 0.11, respectively, indicating poor antigenic matching. In vivo, O/CATH/APQA-V18 induced clinical signs in unvaccinated pigs, confirming its infectivity and pathogenicity. Vaccinated pigs had low neutralizing antibody titers against O/CATH/APQA-V18 before challenge, and only 25% were protected. These findings support O/CATH/APQA-V18 as a genetically defined and reproducible experimental virus for vaccine matching and protective efficacy evaluation against contemporary O/CATHAY viruses. Full article

Vespa basalis Smith venom (VBsV) causes painful and potentially severe systemic reactions, yet the relationships among its composition, bioactivity, and cytotoxicity remain poorly understood. Proteomic and metabolomic profiling was combined with ultrafiltration and gel filtration to characterize VBsV and obtain >3 kDa, operationally defined 700–3000 Da, and <700 Da fractions. The effect on LPS-induced NO production was evaluated by nitric oxide (NO) release in LPS-stimulated RAW264.7 cells, and cytotoxicity was assessed in tumor and THLE-2 liver cells. Five venom allergen proteins were identified, including two phospholipase A1 isoforms, hyaluronidase A, antigen 5, and venom dipeptidyl peptidase IV. Targeted profiling also detected several neurotransmitters and energy metabolites, including 5-hydroxytryptamine, L-glutamate, histamine, γ-aminobutyric acid, inosine, and adenosine. The 700–3000 Da fraction exhibited the strongest NO-suppressive and tumor-cell-viability effects among the tested fractions. The 700–3000 Da fraction suppressed NO release (IC₅₀, approximately 23 μg/mL) and reduced tumor-cell viability, while minimally affecting THLE-2 cells at 10–80 μg/mL; by contrast, the >3 kDa fraction showed greater THLE-2 cytotoxicity. These findings identify the 700–3000 Da fraction as a priority for direct compositional characterization and isolation of the responsible molecules. Full article

Background/Objectives: Respiratory pathogens such as influenza virus and SARS-CoV-2 pose major threats to global public health. Current vaccines provide limited mucosal protection against respiratory infection, while vaccine-induced immunity can wane over time. Intranasal vaccination has emerged as a promising approach to induce mucosal immunity, however relatively low immunogenicity and limited durability remain major challenges. Various strategies have therefore been explored to improve intranasal vaccine efficacy, including mucosal adjuvants and alternative vaccine platforms. In this study, we evaluated the magnitude and durability of systemic and mucosal immune responses induced by an adenoviral vector vaccine. Methods: Six-week-old female C57BL/6 mice were immunized intranasally or intramuscularly with a single dose of an adenoviral vector vaccine and compared with mice receiving one or two doses of an intranasal CpG-adjuvanted protein vaccine. Humoral immune responses in serum and BALF, as well as cellular immune responses in the mLNs, lungs, and blood, were evaluated. We further assessed Ova-specific CD8 T cells with a lung-resident phenotype by class I MHC-peptide tetramer staining combined with intravascular labeling, allowing their direct ex vivo identification. Results: A single dose of intranasal adenoviral vector vaccination elicited potent serum IgG and BALF IgA responses, together with antigen-specific CD8 T cell responses in the blood, mLNs, and lungs. Notably, these systemic and respiratory mucosal immune responses were sustained for up to 4 months after vaccination, with lung-resident CD8 T cell responses detected at this time point. In contrast, intramuscular adenoviral vector vaccination predominantly induced systemic immunity. Intranasal CpG-adjuvanted protein vaccination induced weaker responses after a single dose, while booster vaccination enhanced systemic antibody and cellular responses but showed limited induction of mucosal immunity. Conclusions: A single intranasal dose of Ad5-Ova induced systemic and respiratory mucosal immune responses comparable to those observed after two-dose Ova+CpG vaccination, while maintaining these responses for up to 4 months. These findings suggest adenoviral vectors as a promising platform for inducing long-lasting respiratory mucosal immunity. Full article

Background/Objectives: Adjuvanted influenza vaccines are critical to enhance immune responses in high-risk populations, such as the elderly. While initial studies relied on a two-vial system with antigen and adjuvant mixing prior to immunization, this study evaluated the feasibility, immunogenicity, and 12-month stability of a one-vial formulation combining a Trivalent Influenza Vaccine (TIV) with the locally produced squalene-based oil-in-water emulsion IB160. Methods: Formulations were evaluated by testing TIV:IB160 (v/v) ratios (1:1, 2:1, 3:1) in an animal model, measuring HI antibodies as the primary immunogenicity metric, complemented by qualitative strain-specific total IgG screening by ELISA. One-vial and two-vial strategies were also compared regarding immune responses. Formulations stabilities were evaluated at 6.0 °C ± 2.0 °C in one-and two-vial systems by monitoring physicochemical features and their antigenic responses, measuring hemagglutinin content of the formulated vaccines, and long-term HI antibody responses. Results: The 2:1 and 1:1 (v/v) formulation ratios (15 µg TIV:IB160) induced significantly higher HI titers across all tested strains (A/H3N2/Thailand, A/H1N1/Victoria, and B/Austria) when compared to non-adjuvanted TIV or adjuvanted TIV in the 3:1 ratio. Both the one and two-vial strategies showed significantly higher HI antibody responses than the non-adjuvanted control group (p < 0.05), with no significant differences observed between the delivery formats. Indeed, the one-vial formulation with 1:1 and 2:1 ratios remained stable for 12 months, preserving TIV antigenicity, IB160 emulsion integrity, and robust immunogenicity. Conclusions: One- and two-vial formulations are viable, stable, and highly immunogenic alternatives for adjuvanted TIV in 2:1 and 1:1 (v/v) formulation ratios (15 µg TIV:IB160). One-vial format simplifies immunization logistics, minimizes operational errors, and is more indicated for routine use, while the two-vial system remains a versatile asset for emergency responses. Full article

Objectives: The SARS-CoV-2 pandemic caused major morbidity, mortality, and economic disruption, highlighting the need to better understand zoonotic spillover risks posed by coronaviruses. We investigated whether archived human sera from forested Guinea contained evidence of prior exposure to coronaviruses antigenically related to known pathogenic human coronaviruses. Methods: Archived sera collected in 2017–2018 from wildlife hunter communities in Guinea were analysed using serological assays against spike glycoproteins from SARS-CoV, SARS-CoV-2, MERS-CoV, and seasonal human coronaviruses. Binding responses to receptor binding domains were also assessed, together with spatial and subgroup analyses. Results: Pre-pandemic sera showed IgG cross-reactivity to SARS-CoV, SARS-CoV-2, and MERS-CoV spike antigens. A distinct subgroup demonstrated strong binding to both SARS-CoV and MERS-CoV receptor binding domains. Conclusions: These findings highlight the utility of sero-epidemiology in identifying potential zoonotic spillover. However, further investigation is needed to identify these viruses and determine their homology to known pathogenic coronaviruses. Full article

Rabbit monoclonal antibodies (RabMAbs) are valuable for biomedical research and diagnostic applications because of their high affinity, specificity, and broad epitope recognition; here, we established an integrated phenotype-linked workflow for rapid RabMAb discovery using serum-derived polyclonal mouse IgG as a proof-of-concept model antigen. Three Big-Eared White rabbits were immunized in parallel, and the rabbit exhibiting the highest serum endpoint titer was selected for the complete downstream single-B-cell discovery workflow. Approximately 5 × 10⁵ activated B cells were subjected to polydisperse oblate dispersion system (POD)-based screening, yielding 13,266 antigen-positive POD events. Following recovery and 10× Genomics single-cell V(D)J sequencing, 4294 B-cell barcodes yielded valid/interpretable V(D)J data, of which 2099 contained at least one complete, productive, and translatable heavy-chain/light-chain pair, generating 2199 functional VH/VL pairing records. AbFinder™-assisted prioritization generated a computationally recommended pool of 158 candidates, and the five highest-ranked VH/VL pairs within this pool were selected for recombinant expression and validation. All five yielded antigen-reactive RabMAbs with an endpoint ELISA titer of 1:256,000 and BLI-derived apparent K_D values ranging from 4.19 × 10⁻¹⁰ to 9.79 × 10⁻⁹ M. The antibodies showed differential concentration-dependent reactivity toward mouse IgG1, IgG2a, IgG2b, and IgG3 preparations, weak reactivity toward human IgG, and clone-dependent reactivity toward rat IgG. The workflow from spleen collection to functional validation was completed within approximately three weeks. Because only five prioritized candidates from one selected responder rabbit were evaluated, the 5/5 validation outcome should not be interpreted as an overall platform hit rate or definitive validation of the prioritization algorithm. These findings support the feasibility of this workflow for research-grade and diagnostic antibody discovery, while broader evaluation will require larger candidate cohorts, independent biological validation, and additional antigen classes. Full article

Background/Objectives: Live attenuated mumps virus (MuV) vaccine strains have significantly reduced disease incidence since their introduction in the 1960s; however, a recent resurgence of outbreaks has led to calls for the development of new vaccines to overcome what appears to be reduced effectiveness linked to waning immunity and the emergence of strains antigenically mismatched to vaccine strains. A major obstacle to the development of newer live, attenuated MuV vaccines is ensuring their safety, particularly given the virus’s neurotropic properties. Indeed, several vaccine strains licensed for use outside the US have proven to be insufficiently attenuated (such as the Urabe AM9 vaccine strain) and have caused aseptic meningitis in recipients, despite efforts to test these strains for neurotoxicity pre-licensure. Historically, attenuation was achieved empirically, and pre-clinical testing for neurovirulence safety has proven unreliable. Despite efforts in recent years, the genetic basis of attenuation of MuV vaccines remains inadequately understood. The objective of this study was to elucidate the genetic basis of neurovirulence of the Urabe AM9 vaccine strain. Methods: To this end, we generated a series of chimeric viruses in which genes of the highly attenuated Jeryl Lynn (JL) vaccine strain were exchanged with corresponding genes from the Urabe AM9 vaccine strain. The resulting chimeric viruses were tested for neurovirulence in a rat model and characterized for replication in vitro and in vivo. Using a multi-tiered approach consisting of independent rescue and analysis of two to three viruses per cDNA construct, possible off-target effects of identified single-nucleotide heterogeneities were mitigated. Results: All viruses were shown to be replication-competent in Vero cells, but differences in replication efficiencies and virus-induced neurotoxicity were observed in the in vivo model. In a rat neuronal cell line, the Urabe AM9 M and HN genes had opposite effects on the growth of chimeric JL- and Urabe AM9-based viruses. Conclusions: The results presented herein indicate that MuV neuroattenuation is mediated by the concerted action of multiple genes, with the matrix (M) gene exerting a dominant effect. These findings contribute to a growing body of evidence that informs the rational design of next-generation live-attenuated mumps virus vaccines. Full article

Loss of renal function is associated with premature aging of the immune system; however, earlier studies on this topic enrolled patients with reduced GFR and did not distinguish between those with or without proteinuria. Clinical observations suggest that proteinuria alone might also impair immune defense. It is thus an open question whether patients with a urinary albumin-to-creatinine ratio > 300 mg/g (severe albuminuria) independent of their eGFR have an inflammatory risk profile and a disturbed adaptive immune response. This cross-sectional study included 25 patients with severe albuminuria (uACR > 300 mg/g; P-Group), 19 patients with an eGFR < 60 mL/min 1.73 m2 and a uACR < 300 mg/g (G-Group), and 30 patients with adequate kidney function (C-group). All three cohorts have a similar cardiovascular background with arterial hypertension and coronary artery disease. PBMCs from the patients were challenged with the superantigen Staphylococcal enterotoxin B (SEB) as well as with CMV- and SARS-CoV-2-specific antigens. Monocyte subsets and CD86 and HLA-DR expression were determined through flow cytometry. Transcripts of CD28 and IFN-α and -γ were measured by qPCR. Compared to those in the C-group, patients in the G-group showed a higher polyclonal SEB response and had significantly elevated circulating numbers of inflammatory monocytes (subset CD14++CD16+). CD28 transcripts were decreased in the P-group and G-group compared to the C-group, reaching significance for the G-Group. The CMV- and SARS-CoV-2-specific immune response, as measured by the frequency of CD4+69+137+ T and CD8+69+137+ T cells, was comparable in all three groups. Severe albuminuria per se does not alter the antigen-specific immune response; however, patients with reduced GFR, but not those with proteinuria, show inflammatory and polyclonal immune activation. Full article

Background/Objectives: Maternal antibodies can inhibit vaccine-specific humoral responses in early life, leaving infants at increased risk for severe disease for vaccine-preventable infections. In the case of SARS-CoV-2, infants under the age of 3 months represented most child hospitalizations, yet there is no approved vaccine for children under the age of 6 months. There is a clear need for effective immunization in early life to prevent infant morbidity and mortality. Here, we established a mouse model to define how maternally derived antibodies shape early-life responses to mRNA vaccination. Methods: Adult female mice were immunized with PBS or 5 mcg of the SARS-CoV-2 mRNA-1273 vaccine via intramuscular injection and paired with a male. Pups from subsequent litters were immunized with PBS or 5 mcg of mRNA-1273 vaccine via intramuscular injection. Peripheral blood and spleens were collected at time points post-immunization. We measured vaccine-elicited anti-Spike IgG in mouse pups exposed or unexposed to vaccine-specific maternal IgG. Results: Spike-specific maternal IgG is detectable at high levels immediately after pup immunization or mock immunization; however, in mock immunized pups, it wanes by three weeks post-pup immunization. Pups born to immunized dams developed Spike specific IgG comparable to pups born to naïve dams. IgG subclass analyses distinguished passively acquired antibodies from vaccine-induced responses. Despite robust binding antibody responses, neutralizing activity against D614G pseudovirus was heterogeneous and did not scale proportionally with IgG titers, showing qualitative differences in early-life humoral immunity. Splenic Spike-specific B cell frequencies and T follicular helper (Tfh) cell responses were detectable in vaccinated pups irrespective of maternal immunization status, with Tfh cell frequencies peaking at day 7 post-immunization in both groups. Conclusions: Using SARS-CoV-2 as a model pathogen, we found that early-life mRNA vaccination can elicit humoral immune responses in the presence of maternal antibodies. Furthermore, the presence of maternal antibody did not inhibit the development of antigen-specific B cells or Tfh cells in the spleen. Our findings support the potential of extending vaccination strategies into early infancy and provide a framework for optimizing mRNA-based vaccine timing and design in the context of maternal immunity. Full article

Canine urothelial carcinoma (UC) is commonly managed with non-steroidal anti-inflammatory drugs (NSAIDs), alone or combined with cytotoxic chemotherapy. Evidence supporting subcutaneous keyhole limpet hemocyanin (KLH) in canine UC is lacking. We describe two client-owned dogs with presumptive lower urinary tract UC managed with NSAIDs, KLH, and selenium after cytotoxic chemotherapy was declined. Case 1 was an 11-year-7-month-old castrated male Maltese with bladder-neck/proximal-urethral thickening. A positive veterinary bladder tumor antigen test, compatible serial imaging, and low-cellularity urine cytology showing suspected urothelial hyperplasia with dysplasia supported clinical suspicion; however, BRAF and BRAF-PLUS assays were negative and histopathology was unavailable. Case 2 was an 11-year-old neutered male Poodle with a BRAF V595E-positive urethrovesical-junction lesion on imaging, without histopathologic confirmation. Both dogs received NSAIDs, Immucothel^® (1 mg subcutaneously), intravenous sodium selenite (200 μg/kg), and oral selenium (100 μg/dog q24h). Serial CT and ultrasonography documented persistent localized abnormalities over prolonged follow-up, but measurements obtained by different modalities were not treated as directly comparable. Adverse events were assessed retrospectively and not prospectively graded using VCOG-CTCAE criteria. Because the diagnoses were presumptive and the interventions were concurrent and uncontrolled, the independent contribution, efficacy, and safety of KLH cannot be determined. These cases are descriptive and hypothesis-generating. Full article

Feline bocavirus (FBoV) is an emerging enteric virus associated with gastrointestinal diseases in cats and has attracted increasing attention in feline health. However, no commercial vaccine is currently available for the prevention and control of FBoV infection. VP2 is the major capsid protein of FBoV and represents a promising target for vaccine development. In this study, the FBoV VP2 protein was expressed using the insect baculovirus expression system. The purified VP2 protein self-assembled into virus-like particles (VLPs), which were formulated with Alum, ISA 206, or GEL 02 adjuvants to prepare VP2 VLP vaccines. The immunogenicity, cellular immune responses, antigen uptake, biodistribution, germinal center responses, and safety of the vaccines were evaluated in BALB/c mice. The results showed that all VP2 VLP vaccine formulations induced VP2-specific IgG antibodies and neutralizing antibodies, promoted B- and T-lymphocyte activation, enhanced dendritic cell maturation, and stimulated germinal center-related immune responses. Among the tested formulations, VP2+GEL 02 induced the strongest immune responses and showed favorable safety in mice. These findings demonstrate that FBoV VP2 exhibits favorable immunogenicity and represents a promising vaccine antigen for further development against feline bocavirus. Full article

Population immune pressure from vaccination and prior infection continues to drive the evolution of SARS-CoV-2. Systematic characterization of RBD mutations under complex immune backgrounds is essential for understanding viral adaptation and evolutionary trajectories. Here, we applied a deep mutational scanning (DMS) to comprehensively map the neutralization escape landscape of the Omicron variant JN.1 and its descendant lineage XEC, under immune pressure from individuals who experienced Omicron breakthrough infections following three doses of inactivated vaccines. A neutralization escape map for the single amino acid substitutions in the RBD of JN.1 or XEC was generated, and the escape efficiency of each mutation was determined. The results show that RBD escape mutations are hierarchically organized: low-intensity signals are widespread, whereas high-intensity escape is confined to a few key sites. These escape mutations are not confined solely to the receptor-binding motif (RBM) but are broadly distributed across the entire RBD. Many escape sites could accommodate multiple amino acid substitutions. Integration of DMS data with genomic surveillance of circulating variants from 2024 to 2025 revealed significant overlap between experimentally identified escape sites and mutations observed in natural isolates. This overlap increased substantially in 2025, with site concordance rising from 27.17% and 26.81% to 45.09% and 47.10% for JN.1 and XEC, respectively. The natural prevalence of these escape mutations is further shaped by factors such as receptor-binding affinity, protein stability, and epistatic interactions. Overall, our findings suggest that SARS-CoV-2 antigenic evolution follows the pattern of multiple pathways within a constrained space, providing new insights into the adaptive mechanisms of Omicron-derived variants under hybrid immune pressure. Full article

Bovine adenovirus (BAdV)-based vectors, particularly those derived from bovine adenovirus 3 (BAdV-3), are emerging non-human adenoviral platforms for veterinary vaccine development. Adenoviral vectors are attractive for vaccination because they mediate efficient transgene expression, remain largely episomal, and induce robust innate and adaptive immune responses. However, widely used human adenoviral vectors, especially human adenovirus 5 (HAdV-5), may be compromised by pre-existing anti-vector immunity, supporting the development of rare human serotypes and non-human adenoviral alternatives. BAdV-3 is the best-characterized BAdV for recombinant vector engineering and has been used to express heterologous antigens from bovine herpesvirus-1, bovine respiratory syncytial virus, influenza virus, and Mycobacterium tuberculosis. Available evidence indicates that BAdV-based vectors can induce humoral, cellular, and mucosal immune responses and support intranasal antigen delivery. Protective efficacy has been demonstrated in selected experimental models, including mice and ferrets for influenza and tuberculosis vaccine candidates, whereas cattle challenge evidence remains more limited and is primarily represented by BHV-1 gD-expressing BAdV-3 vectors. These features make BAdV vectors relevant to bovine respiratory disease control, livestock vaccination, and One Health-oriented vaccine preparedness. Nevertheless, the platform remains at an early developmental stage. Key gaps include limited target-species efficacy data, pre-existing BAdV immunity in cattle, vector shedding, recombination risk, genetic stability, producer cell optimization, manufacturing scalability, and regulatory feasibility. Full article

Background/Objectives: Particulate vaccine formulations require delivery systems and immunostimulatory components that support antigen-presenting cell interaction while maintaining formulation stability and cytocompatibility. This study examines Guanine-α-D-Fructose (GDF), a guanine–fructose small-molecule immunostimulatory candidate, incorporated into poly(lactic acid-co-glycolic acid) (PLGA) microparticles and benchmarked in vitro against established adjuvant formulations containing Alum or AddaVax. Methods: GDF-loaded microparticles were prepared and characterized for particle size, morphology, surface charge, entrapment efficiency, and release behavior. Their in vitro immunostimulatory activity was evaluated using murine dendritic cells. To explore formulation compatibility, GDF MPs were tested in combination with multiple particulate antigen formulations, including measles, gonorrhea, SARS-CoV-2, influenza A (H3N2), and Zika. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was utilized to measure cell viability, while nitric oxide generation was measured with the Griess assay. Dendritic cell activation-associated surface marker expression was analyzed by flow cytometry using MHC I, MHC II, CD80, and CD40. Results: The particles showed consistent spherical morphology, sustained GDF release, and favorable cytocompatibility at concentrations up to 250 µg/mL, with reduced viability at higher concentrations. GDF MPs increased nitric oxide production and activation-associated marker expression, with responses benchmarked against Alum and AddaVax and comparable in several assay conditions. Increased autophagosome-associated fluorescence was also observed, suggesting modulation of autophagy-related cellular activity; however, direct antigen-processing or antigen-presentation assays are still required. Conclusions: Overall, these findings support GDF-loaded PLGA microparticles as an in vitro immunostimulatory particulate formulation suitable for further mechanistic and in vivo evaluation in vaccine-related systems. Full article