Search Results (1,921)

Introduction/Background: Rift Valley fever virus (RVFV) and peste des petits ruminants virus (PPRV) are significant pathogens affecting small ruminants, causing substantial economic losses in the affected regions. The development of effective vaccines against both viruses is crucial for disease control. Recombinant viruses expressing heterologous antigens have shown promise as multivalent vaccine candidates. Unlike conventional PPRV vaccines, our recombinant RVFV-vectored vaccines offer a novel dual-protection strategy against RVF and PPR, combining safety, immunogenicity, and a DIVA strategy. Methods: Recombinant RVFVs (ZH548 strain) were generated to express either the hemagglutinin (H) or fusion (F) proteins from the PPRV strain Nigeria 75/1. The stability of these recombinant viruses was assessed through consecutive passages in cell culture. Immunogenicity studies were carried out in both mice and sheep to assess the induction of cellular and humoral immune responses capable of providing protection against RVFV and PPRV. These studies included intracellular cytokine staining (ICS), IFN-γ ELISAs, standard ELISAs for antibody detection, and virus neutralization assays. Results: The recombinant RVFVs expressing PPRV H or F proteins demonstrated stability in cell culture, maintaining high viral titers and consistent transgene expression over four passages. Immunization of mice resulted in the production of serum antibodies capable of neutralizing both RVFV and PPRV in vitro as well as cell-mediated immune responses specific to PPRV and RVFV antigens. In mice vaccinated with a high dose (10⁵ pfu), RVFV neutralizing titers reached ≥1:160 and PPRV neutralizing titers ranged from 1:40 to 1:80 by day 30 post-immunization. In sheep, neutralizing antibody titers against RVFV exceeded 1:160 as early as 2 days post-inoculation, while PPRV-specific neutralization titers reached up to 1:80 by day 21 in responsive individuals. In mice, administration of rZH548ΔNSs:F_PPRV elicited a detectable CD8+ IFNγ+ T-cell response against PPRV, with levels ranging from 1.29% to 1.56% for the low and high doses, respectively. In sheep, rZH548ΔNSs:F_PPRV also induced a robust IFNγ production against PPRV at 14 and 21 days post-infection (dpi). Conclusions: The successful generation and characterization of recombinant RVFVs expressing PPRV antigens demonstrate the potential of using rationally attenuated RVFV as a vector for multivalent vaccine development. Notably, the strategy proved more effective for the recombinant virus expressing the F protein, as it consistently induced more robust cellular and humoral immune responses. These results suggest that this approach could be a viable strategy for simultaneous immunization against Rift Valley fever and other prevalent ruminant diseases, such as peste des petits ruminants. Even though challenge studies were not performed in target species, the strong immune response observed supports including them in future studies. Full article

Porcine circovirus type 3 (PCV3), initially identified in the United States in 2016, is associated with multisystemic inflammation, myocarditis, reproductive failure in sows, and growth retardation in piglets, posing a significant economic threat to the swine industry. In this study, prokaryotic-expressed recombinant PCV3 Cap protein was used to immunize mice and rabbits. A monoclonal antibody (mAb 4G1) was generated through hybridoma technology, targeting a novel linear epitope (³⁷DYYDKK⁴²) within the first β-sheet of the Cap structure. This epitope exhibits high conservation (99.35%, 1239/1247) based on sequence alignment analysis, and residues 39 and 42 are critical residues affecting mAb binding. Subsequently, using rabbit polyclonal antibody (pAb) as the capture antibody and mAb 4G1 as the detection antibody, a double antibody sandwich ELISA (DAS-ELISA) method was developed. The assay demonstrates a cut-off value of 0.271, a detection limit for positive pig serum is 1:800, and shows no cross-reactivity with other swine pathogens. Intra- and inter-assay coefficients of variation were <10%, with a linear detection range for Cap protein down to 3.4 ng/mL. The coincidence rate between the DAS-ELISA and qPCR was 93.33% (70/75) for PCV3 detection in serum, with a kappa value of 0.837. This study establishes a simple, sensitive, and operationally efficient DAS-ELISA and provides a reference for monitoring PCV3 infection in swine herds. Full article

In the production of recombinant antibody/Fc-fusion proteins using mammalian cells, many aggregates often form alongside the target proteins, particularly with bispecific antibodies. To ensure the safety of biological products, it is essential to control the amount of aggregates within a specific range. A traditional downstream process typically involves using Protein A (ProA) resin to capture the target antibody, followed by two polishing steps to ensure purity; for instance, using an anion exchange chromatography (AEX) in flow-through mode and a cation exchange chromatography (CEX) in binding–elution mode. In this study, we choose a Dual Action Fab (DAF), which can bind two antigens and is prone to aggregation when expression in CHO (Chinese Hamster Ovary) cells. We introduce hydrophobic interaction membrane chromatography (HIMC) operating in flow-through mode, which enhances production efficiency while reducing costs and the risks associated with column packing. We evaluated the impact of the operating buffer system, as well as the pH and conductivity of the loading samples, on aggregate removal using HIMC. Additionally, we investigated the mechanism of aggregate binding and found that loading conditions had a limited impact on this process. Overall, our findings indicate that employing HIMC can achieve a 20% reduction in aggregate levels. These results demonstrate that HIMC in flow-through mode is an effective and robust approach for reducing aggregates during antibody purification. Full article

Background: B7-H3, an immunoregulatory protein of the B7 family, has been associated with both anti-cancer immunity and tumor promotion, with its expression commonly correlated with poor prognosis. Although it is frequently expressed across cancers, its heterogeneity may limit the effectiveness of B7-H3-targeted therapies. Consequently, a sensitive and non-invasive method is needed to assess B7-H3 expression for patient selection and stratification. Single-domain antibody fragments (sdAbs) offer a promising platform for developing such a diagnostic tool. Methods: To generate B7-H3 sdAbs, two Ilamas were immunized with the recombinant human B7-H3 protein. Positive clones were selected through Phage biopanning and characterized for thermal stability, binding specificity, and affinity to human and murine B7-H3 proteins. Selected sdAbs were radiolabeled with Technetium-99m (^99mTc) and evaluated for B7-H3 detection in two xenograft tumor models using micro-SPECT/CT imaging and dissection studies. Results: Sixteen purified sdAbs bound specifically to recombinant B7-H3 proteins and cells expressing native B7-H3 antigens, with nanomolar affinities. The four best-performing sdAbs bound promiscuously to tested mouse and human B7-H3 isoforms. Lead sdAb C51 labeled with ^99mTc displayed specific accumulation across two human B7-H3⁺ tumor models, achieving high contrast with a tumor-to-blood ratio of up to 10 ± 3.16, and a tumor uptake of up to 4.96 ± 1.4%IA/g at 1.5 h post injection. Conclusions: The lead sdAb enabled rapid, specific, and non-invasive imaging of human B7-H3⁺ tumors. Its isoform promiscuity supports broad applicability across cancers expressing different human B7-H3 isoforms. These results support further development for clinical translation to enable patient selection and improved B7-H3-targeted therapies. Full article

Virus inactivation exhibits varying disinfection kinetics due to structural or genomic differences. Standard post-disinfection assessment relies on observing cytopathic effects in inoculated cell cultures, which are limited by sensitivity, availability, cost, and turnaround time. This study explores nucleic acid aptamers as molecular sensors to differentiate between intact and post-disinfection virus particles. To discover aptamers, 12 cycles of an automated SELEX (Systematic Evolution of Ligands by Exponential Enrichment) experiment were performed using recombinant (r)-VP2 protein of canine parvovirus (CPV). Enrichment of single stranded (ss) DNA binders was evaluated by sequencing the enriched libraries. The most abundant sequences were tested for binding with coated rVP2 and CPV (intact and treated with heat and peracetic acid (PAA) disinfectant) followed by detection using PCR. Binding specificity was assessed using intact and heat-treated feline panleukopenia virus (FPV) and porcine parvovirus (PPV). Sequencing of the DNA libraries from selection cycle 6 and cycle 12 products showed individual sequence enrichment with maximum frequencies of 2.14% and 8.65%, respectively. The top three abundant sequences from each cycle confirmed rVP2 binding. In the case of CPV, only heat-treated and PAA-treated CPV showed binding to the candidate sequences. However, reduced binding to the CPV-specific antibody was observed for rVP2 and treated CPV compared to intact CPV. No apparent binding of the tested sequences was observed for FPV and PPV. Aptamers binding to denatured but not intact CPV demonstrate the potential to distinguish between the two states, providing a basis for developing a molecular assay to assess disinfection efficacy. Full article

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite responsible for toxoplasmosis, a disease with significant health implications for humans and animals. The surface antigen 1 (SAG1) of T. gondii is a major immunodominant protein that facilitates host cell invasion, making it an ideal target for diagnostic and therapeutic interventions. Immunoglobulin Y (IgY), the primary antibody in avian species, offers unique advantages over mammalian IgG, including easier animal care, lower costs, high-yield production, and potential passive immunization. Objectives: This study aimed to induce, purify, and characterize IgY antibodies targeting T. gondii SAG1 from hen egg yolks. Methods: The coding region of the mature portion of T. gondii SAG1 was amplified by PCR, cloned into the pET32a(+) vector for heterologous expression in E. coli. The recombinant SAG1 (rSAG1) was purified by affinity chromatography and used to immunize hens. IgY was extracted from egg yolks using PEG. SDS-PAGE and spectrophotometry were used to evaluate purity and concentration. By ELISA, Western blot, and flow cytometry, the specificity of IgY was assessed against recombinant and endogenous, native, and denatured SAG1. Results: Purified IgY demonstrated strong recognition of both recombinant and native SAG1 in ELISA and Western blot, and against T. gondii tachyzoites by flow cytometry. Conclusions: SAG1-specific IgY was produced in a pure form; it could be helpful in research, diagnosis, and treatment at low costs on a larger production scale, with minimal animal harm. Full article

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the global swine industry. Gilt immunization using modified live virus (MLV) vaccines is crucial for herd stability, but it is complicated by frequent mixed infections of PRRSV strains on farm. This study monitored the administration of tylvalosin during a PRRSV-2 MLV (TJM) immunization program, focusing on viral dynamics and immune responses in gilts naturally exposed to co-circulating classical (GD240101) and highly pathogenic like (HP-PRRSV-like, GD240102) PRRSV strains. Methods: The animal study was approved by the Laboratory Animal Ethical Committee of China Agricultural University. One hundred gilts were randomized into control and tylvalosin groups (n = 50/group). All received the TJM MLV vaccination. The tylvalosin group received tylvalosin tartrate premix cyclically in-feed for three cycles. Serum and saliva samples were collected periodically. PRRSV RNA (RT-qPCR) and specific antibodies (ELISA) were assessed. Viral population dynamics (relative abundance, mutation, recombination of TJM, GD240101, and GD240102) were monitored via next-generation sequencing (NGS) on a pooled PRRSV-positive sample. Results: In this field trial where tylvalosin was used, a shorter duration of PRRSV viremia and saliva shedding was observed to compare with controls. NGS analysis showed accelerated vaccine strain (TJM) clearance in the tylvalosin group (by week 3 vs. week 9 in control). Field strain dynamics were also altered, showing a faster decline in the tylvalosin group. Antibody response uniformity was altered, with lower coefficient of variation (CV) for PRRSV and CSFV observed following tylvalosin usage. Conclusions: In gilts receiving tylvalosin for the management of bacterial pathogens during a PRRSV MLV immunization program, it was associated with accelerated viral clearance and enhanced systemic immune response uniformity under mixed-infection field conditions. NGS provides invaluable data for dissecting these complex viral dynamics. Crucially, these findings describe a biological drug–host–virus interaction and should not be interpreted as an endorsement for the prophylactic use of antimicrobials. In alignment with global antimicrobial stewardship principles, tylvalosin should be reserved for the therapeutic treatment of diagnosed bacterial diseases to mitigate the risk of promoting resistance. Full article

Despite growing recognition of the role of the gut microbiome in host health and in modulating pathogen activity, the dynamic and reciprocal relationship between enteric viruses and the gut microbial ecosystem remains insufficiently defined and requires further exploration. This comprehensive review examines the bidirectional interplay between the gut microbiome and enteric viral infections by addressing (i) viruses associated with gastrointestinal alterations, (ii) how enteric viral infections alter the composition and function of the gut microbiome, (iii) how the gut microbiome modulates viral infectivity and host susceptibility, and (iv) current microbial-based approaches for preventing or treating enteric viral infections. Gastrointestinal viral infections induce gut microbiome dysbiosis, marked by reductions in beneficial bacteria and increases in potentially pathogenic populations. Specific gut microorganisms can modulate host susceptibility, with certain bacterial genera increasing or decreasing infection risk and disease severity. Pattern recognition receptors in the intestinal epithelium detect microbial signals and trigger antimicrobial peptides, mucus, and interferon responses to control viral replication while maintaining tolerance to commensal bacteria. The gut microbiome can indirectly facilitate viral infections by creating a tolerogenic environment, suppressing antiviral antibody responses, and modulating interferon signaling, or directly enhance viral replication by stabilizing virions, promoting host cell attachment, and facilitating coinfection and viral recombination. In turn, commensal gut bacteria can inhibit viral entry, enhance host antiviral responses, and strengthen mucosal barrier function, contributing to protection against gastrointestinal viral infections. Probiotics and fecal microbiota transplantation constitute potential microbial-based therapeutics that support antiviral defenses, preserve epithelial integrity, and restore microbial balance. In conclusion, the role of the gut microbiome in modulating enteric viral infections represents a promising area of future investigation. Therefore, integrating microbiome insights with virology and immunology could enable predictive and personalized strategies for prevention and treatment. Full article

The CA125 epitope within the MUC16 tandem repeat region is detected via the CA125 II test for ovarian cancer surveillance. This test utilizes the M11 and OC125 antibodies. A revised model of MUC16 with 19 tandem repeats has recently been identified, including splice variants that exclude entire repeats. Additionally, OC125 has exhibited gaps in coverage of the tandem repeat region. To identify antibodies that bind more repeats and are suitable for spliceoform detection, more antibodies must be characterized using the revised model. This study characterized the binding of two M11-like and two OC125-like antibodies against the updated tandem repeat numbering system. 16 individual tandem repeats were expressed and purified. Binding interactions between each of the antibodies and recombinant repeats were examined by indirect enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). The M11-like antibodies displayed different binding patterns when compared to each other, while the two OC125-like antibodies exhibited similar binding patterns. M11-like clone M77161 bound to all 16 repeats tested, indicating that it may be suitable for accurate detection of CA125. These findings demonstrate how different antibodies vary in their binding to CA125, contributing to ongoing development of improved clinical and research tools for ovarian cancer. Full article

Brucellosis, a zoonotic infection caused by the intracellular pathogen Brucella, leads to chronic multi-organ damage. Currently, rapid, accurate, and sensitive diagnostic technologies are crucial for the prevention and control of brucellosis. This study describes the development of a chemiluminescent immunoassay (Bru-CLIA) for sheep and bovine brucellosis antibody detection, utilizing Brucella abortus strain A19 lipopolysaccharide-coated magnetic particles (LPS-MPs) as the serum antigen and acridinium ester-labeled recombinant streptococcal protein G (AE-SPG) for signal generation. After optimizing the assay’s parameters, the Bru-CLIA demonstrated a sensitivity of approximately 1 IU/mL and 2 IU/mL for detecting sheep and bovine brucellosis, respectively. No cross-reactivity was observed with sera from animals immunized with Escherichia coli O157:H7, Mycobacterium tuberculosis, Vibrio cholerae, Legionella, Salmonella, Foot and Mouth Disease virus types O and A, Bovine viral diarrhea virus, Sheep contagious pleuropneumonia, Goat pox virus, or Peste des Petits Ruminants virus, indicating strong specificity. The testing of 81 sheep serum samples and 96 bovine serum samples revealed that Bru-CLIA showed 87.65% and 93.75% concordance with the ID-VET commercial kits for sheep and bovine brucellosis detection, respectively. These results demonstrate that Bru-CLIA offers high specificity, sensitivity, repeatability, and reliability, making it a viable rapid diagnostic tool for the epidemiological surveillance of brucellosis. Full article

Background/Objectives: Calsequestrin (CSQ) is a calcium-binding protein that is highly soluble and can serve as a solubility-enhancing fusion tag in recombinant protein expression. Its unique property of calcium-induced precipitation followed by EDTA-mediated resolubilization enables efficient purification. However, the broader application of CSQ-tagged proteins in research have been hampered by the lack of reliable anti-CSQ detection reagents. This study aimed to develop single-domain antibodies (sdAbs) against CSQ for use in diverse immunoassays and cell-based analyses. Methods: Single-domain antibodies were selected from phage-displayed chicken VH libraries generated from CSQ-immunized chickens. After biopanning, CSQ-specific VH sdAb clones were isolated and expressed as VH–human kappa light chain constant region (VH-Cκ) fusion proteins in E. coli. The PE06 clone was chosen for further characterization and conjugated to horseradish peroxidase (HRP) and Alexa Fluor 647 for assay applications. Results: PE06 VH-Cκ fusion protein demonstrated specific binding to CSQ-tagged proteins and enabled reliable detection in enzyme-linked immunosorbent assay (ELISA), immunoblotting, and flow cytometry. These results validated its utility as a chemically defined detection reagent for CSQ fusion proteins expressed in E. coli. Conclusions: This study establishes a CSQ-specific chicken VH sdAb as a versatile detection tool for CSQ-tagged proteins. The approach expands the utility of CSQ as a protein fusion tag and enables the development of recombinant antibodies fused with CSQ, such as scFv-CSQ constructs, for broad application in research and assay systems. Full article

Background: Gold nanoparticles (AuNPs) are a promising platform for vaccine antigen delivery due to their ability to stimulate both innate and adaptive immune responses. These effects depend strongly on physicochemical properties such as size, polydispersity, morphology, and surface charge, which are in turn determined by the synthesis method. While amino acids are often used as capping agents for AuNPs, their direct use as both reducing and stabilizing agents has been rarely investigated. Objectives: This study aimed to establish an ultrasound-assisted method for synthesizing AuNPs using amino compounds as both reducing and stabilizing agents, and assess their physicochemical characteristics, antigen-binding capacity, and immunogenicity. Methods: AuNPs were synthesized using L-cysteine, L-arginine, and cysteamine as dual reducing/stabilizing agents under ultrasonic conditions. The nanoparticles were combined with a recombinant receptor-binding domain (RBD) of SARS-CoV-2 and evaluated in mice for their ability to induce antibody responses. Results: The synthesized AuNPs exhibited hydrodynamic diameters ranging from 6.3 to 12.4 nm and zeta potentials from −40.5 to +36.5 mV, depending on the amino compound used. All formulations elicited robust anti-RBD IgG responses, but virus neutralization activity varied significantly. Notably, AuNP–arginine induced the strongest neutralizing response despite lower adsorption capacity and stability, suggesting that epitope preservation and antigen presentation quality were more decisive than antigen density. Conclusions: These findings underscore the importance of nanoparticle design in optimizing antigen presentation and highlight the potential of amino compound-synthesized AuNPs as effective antigen delivery vehicles for future vaccine development. Full article

Background: The accurate evaluation of anti-ABO antibodies is essential for risk stratification in ABO-incompatible (ABOi) transplantation. Historically, hemagglutination-based titration has been the cornerstone of such an assessment; however, different tools are being evaluated in this context. In recent years, several novel methods have been reported. Methods: A narrative review was conducted using PubMed, Scopus, and Google Scholar, focusing on recent studies evaluating anti-ABO antibody measurement techniques in the context of ABOi organ transplantation. Results: In addition to the conventional tube method, techniques such as column agglutination technology, flow cytometry, and enzyme-linked immunosorbent assay are utilized for anti-ABO antibody assessment. However, any particular technique, significant interinstitutional and interoperator variabilities have been reported due to differences in the detailed protocols and the inherently subjective nature of some techniques. Moreover, these assays are based on the antibody binding to ABO antigens expressed on red blood cells, which might not accurately reflect the clinical context of organ transplantation. In recent years, technological advances have enabled the development of novel assays evaluating antibody responses specifically against the ABO antigens expressed on vascular endothelial cells. These include glycan microarrays, which differentiate responses by ABO antigen subtypes, and CD31-based microarrays, wherein recombinant CD31 proteins expressing ABO antigens are immobilized. These approaches are applied to assess clinically relevant anti-ABO antibodies in the context of ABOi organ transplantation. Conclusions: The objective evaluation of antibody titers against ABO antigens on vascular endothelial cells might not only enable a more accurate risk assessment but also facilitate meaningful comparisons between institutions. Full article

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important therapeutic cytokine. Methylotrophic yeasts such as Komagataella phaffii and Ogataea parapolymorpha are attractive hosts for recombinant protein production. In this study, these yeasts were engineered to produce GM-CSF in both secreted and cell-surface-anchored forms. Secreted GM-CSF accumulated to tens of milligrams per liter in culture supernatants following induction. Fluorescent antibody labeling confirmed that yeast strains expressing surface-displayed GM-CSF exhibited markedly increased fluorescence compared to parental strains. The highest signal was observed in K. phaffii and further validated by immunofluorescence microscopy. Functional assays demonstrated that K. phaffii cells displaying GM-CSF stimulated TF-1 cell proliferation 1.41-fold compared to control yeast lacking GM-CSF. These results confirm both the successful surface display and preserved biological activity of the cytokine. This work demonstrates the feasibility of engineering K. phaffii to present active human GM-CSF on the cell surface. Surface display was efficient and yielded biologically functional cytokine, as shown by fluorescence-based quantification and TF-1 proliferation assays. The study highlights yeast surface display as a promising platform for delivering therapeutic cytokines without requiring purification steps. Full article

Background: Rabies remains a fatal zoonotic disease, necessitating effective and affordable vaccines. While current vaccines are effective, they require multiple doses and may not induce long-lasting immunity in all settings. The rabies virus glycoprotein (RABV-G) is the principal antigen responsible for eliciting virus-neutralizing antibodies, but its recombinant monomeric forms often suffer from poor immunogenicity due to misfolding and aggregation. Methods: A recombinant trimeric RABV-G ectodomain (rRABV-G-XVIII) was engineered by fusing it to a human collagen XVIII-derived trimerization domain. The protein was expressed in E. coli, purified under denaturing conditions, and refolded. Trimer formation was verified using size-exclusion chromatography. Mice were immunized with rRABV-G-XVIII, with or without adjuvant, and compared to a monomeric form (rRABV-GE). Antigen-specific antibody responses were measured by ELISA, neutralizing activity was assessed, and protective efficacy was evaluated via intracerebral challenge with the CVS-27 rabies strain. Results: rRABV-G-XVIII formed stable trimers and induced strong humoral immune responses, with high ELISA titers and virus-neutralizing activity comparable to an inactivated rabies vaccine. Mice immunized with rRABV-GE showed lower antibody responses and partial protection, which improved with adjuvant. All rRABV-G-XVIII-immunized mice were fully protected against rabies challenge, independent of adjuvant use. Conclusions: Stabilization of RABV-G in its native trimeric conformation markedly improves immunogenicity and protective efficacy. This approach offers a promising strategy for the development of rabies subunit vac-cines with simplified formulations and potential for cost-effective production in bacterial systems. Full article