Search Results (114)

Background/Objectives: Extraintestinal pathogenic Escherichia coli (ExPEC) is a major pathogen that causes septicemia, meningitis, and polyserositis in pigs. The increasing prevalence of antimicrobial resistance and the diverse serotypes of ExPEC highlight the urgent need for broadly protective vaccines. Methods and Results: In this study, an OmpC epitope vaccine based on the T4 phage display system was developed and evaluated. Two B-cell epitopes (OmpC-1 and OmpC-2) were identified by bioinformatic analysis and displayed on recombinant T4 phages. Immunization induced strong antigen-specific IgG responses, with the OmpC-1-T4 group showing significantly higher antibody titers than the OmpC protein group. In the O11 serotype PCN033 challenge model, survival rates reached 100% in the OmpC-1-T4 group, 60% in the OmpC-2-T4 group, and approximately 80% in the OmpC protein group. In the O18 serotype 2103 challenge model, both recombinant phage groups had survival rates of approximately 60%, whereas all the mice in the OmpC protein group died within three days. OmpC-1-T4 immunization also significantly reduced bacterial loads in lung and brain tissues after PCN033 infection and decreased TNF-α and IL-6 expression in lung tissues, accompanied by reduced inflammatory infiltration and tissue damage. Conclusions: Overall, the T4 phage-displayed OmpC epitope vaccine induced strong humoral immunity and provided protection against different ExPEC serotypes. Among the candidates, OmpC-1-T4 showed superior immune protection, bacterial clearance, and inflammation control, supporting its potential as a vaccine candidate against porcine ExPEC infection. Full article

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (K_D = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, K_D = 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems. Full article

Background/Objectives: Spotty liver disease (SLD), caused by Campylobacter hepaticus, is an emerging disease that leads to substantial production losses in the egg industry. The shift toward antibiotic-free and cage-free production systems has further intensified the impact of SLD. The current control measures largely rely on autogenous killed vaccines; however, their use is constrained by the slow and fastidious growth of C. hepaticus and inconsistent efficacy. To overcome these limitations, this study aimed to identify immunogenic mimotopes as vaccine candidates and express them on the surface of an avian pathogenic Escherichia coli (APEC) vector. Methods: To identify immunogenic mimotopes, Ph.D.-12 phage display peptide library was screened using the hyperimmune serum raised against killed whole-cell C. hepaticus in specific pathogen-free chickens. Subsequently, the outer membrane protein C (OmpC) of E. coli was used as a scaffold for constructing a surface display library. A single restriction site, PstI, located in the seventh external loop of OmpC, was strategically utilized to insert each 12-amino-acid mimotope with a six-histidine (6xHis) tag sequence at its N-terminus, generating ompC + mimotope fusion constructs. These constructs were cloned into the inducible expression vector pTrc and electroporated into an E. coli DH5α ∆ompC strain, which lacked ompC. The surface expression of the mimotopes was confirmed in vitro. The verified ompC + mimotope constructs were subsequently subcloned into the pYA3422 constitutive expression vector and electroporated into the APEC PSUO78 ∆aroA ∆asd vaccine vector strain. A chicken vaccination–challenge trial was conducted using nine groups of chickens, including an unvaccinated challenged control and an unvaccinated–unchallenged negative control. Each experimental group received a mixture of two recombinant E. coli strains carrying different mimotopes at a dose of 1 × 10⁹ CFU, which were administered orally twice at 16 and 18 weeks of age. Results: Fourteen immunogenic mimotopes corresponding to 13 different C. hepaticus proteins were identified as potential vaccine candidates. The expression of these mimotopes on the surface of the E. coli was successfully demonstrated using the OmpC-mediated surface display system. Of the 14 mimotopes tested, two flagellar-related peptides and one major outer membrane protein (MOMP)-derived peptide elicited significant immune responses and conferred protection against the C. hepaticus challenge. Conclusions: We successfully developed a functional E. coli surface display system that was capable of expressing 12-amino-acid mimotopes of C. hepaticus, providing a robust platform for evaluating vaccine candidates against SLD. Immunogenicity and efficacy studies in chickens demonstrated that three identified mimotopes conferred protection against C. hepaticus colonization of the bile and liver. Future in vivo investigations are necessary to develop and evaluate the immunogenicity and protective efficacy of a multivalent mimotope vaccine consisting of three identified mimotopes against both C. hepaticus and APEC, utilizing the ΔaroA Δasd APEC PSU078 strain as the vaccine vector. Full article

Background/Objectives: Bacteriophage-based display has been utilized for a variety of purposes, such as to assemble protein libraries and conduct biopanning. We have created a modified lambda (λ) bacteriophage platform, ideal for the display and delivery of proteins. Our system utilizes counter-selection recombineering for versatile modification, temperature-sensitive induction for timely lysate production, and an arabinose-inducible mechanism for high-titer, stable yield. Here, we investigated the ability of this specialized λ phage display platform to stimulate highly specific antibodies in mice against the displayed cancer-variant cell-surface receptor EGFRvIII, demonstrating its potential in cancer immunotherapy and broader vaccine development. Methods: λ display immunogenicity was explored by generating fusion proteins between the λ head protein D and a 13-mer peptide from the N terminus of glioblastoma variant cell-surface receptor, EGFRvIII. The 13-mer peptide was fused to either the N or C terminus of the λD protein while λ remained a dormant lysogen in the bacterial host chromosome. Recombinant phage lysates were then generated with ~420 displayed fusion proteins per phage particle. Mice were injected with purified recombinant λ phage without an adjuvant via both intraperitoneal and intramuscular routes, and sera harvested at various timepoints were profiled for immunogenicity. Results: Analysis of serum samples by ELISA and Western blotting demonstrated the ability of the λD~EGFRvIII phage display, especially in the C-terminal fusion construction, to elicit a robust anti-EGFRvIII humoral response by either injection route. Notably, the antibody response was highly specific to EGFRvIII without exhibiting cross-reactivity to wild-type EGFR. Conclusions: The data generated in this study demonstrate the λ system’s immunotherapeutic potential as a high-titer, stable, self-adjuvanting vector for the stimulation of robust antibody titers with defined specificity. Full article

The Ralstonia solanacearum species complex (RSSC) is a globally significant plant pathogenic bacterium. Given the lack of effective chemical controls, phage therapy has emerged as a promising biocontrol alternative. While combining phages with antibiotics can counteract phage resistance, RSSC may still evolve concurrent resistance to both agents. However, the fitness consequences and underlying mechanisms of such resistance remain unclear. In this study, a novel RSSC phage was isolated to experimentally investigate the trade-offs between resistance and virulence in evolved strains. Compared to the wild-type, phage-resistant, antibiotic-resistant, and dual-resistant mutants showed no significant differences in growth rate, exopolysaccharide and lipopolysaccharide production. However, their motility, soil survival, and biofilm formation were significantly impaired, with the most severe decline observed in the dual-resistant mutants. Furthermore, phage-resistant strains exhibited enhanced antibiotic resistance, while antibiotic-resistant strains displayed cross-resistance. The antibiotic resistance gene bla_OXA-249 was upregulated only in antibiotic-resistant strains. In phage-resistant bacteria, the abortive infection system was activated. A reduction in bacterial cell numbers post-infection indicated that phage resistance limits phage propagation via a “suicidal” mechanism. These findings reveal that resistance evolution in RSSC carries substantial fitness costs and highlight phage steering as a novel strategy for designing phage agents. Full article

This study demonstrates the feasibility of using a T7 phage display platform to deliver a library of tick antigens as a vaccine to disrupt tick feeding in cattle. Cattle were vaccinated at three-week intervals via intradermal and intramuscular routes with a cocktail of male and female Amblyomma americanum T7 phage display cDNA libraries, with and without adjuvant. ELISA and Western blot analyses confirmed that vaccinated cattle mounted immune responses directed against phage-displayed tick proteins rather than the T7 phage backbone. Vaccine-induced antibodies recognized both native tick salivary gland proteins and selected recombinant salivary proteins, indicating effective antigen presentation and biologically relevant immunity with binding to native tick saliva proteins. The adjuvanted formulation elicited significantly stronger immune responses than phage-only immunization. Immunized cattle exhibited robust immune memory, evidenced by a pronounced anamnestic response following tick infestation. This immunity translated into measurable anti-tick effects, including reduced tick feeding efficiency and blood ingestion. Tick reproductive success was severely compromised, with larval hatching declining from 54% in ticks fed on control cattle to 4% in ticks fed on immunized cattle. This study establishes a practical and scalable T7 phage-displayed whole-tick antigen platform capable of inducing durable anti-tick immunity in cattle. Full article

Background/Objectives: The SARS-CoV-2 virus frequently undergoes mutations to evade the human immune system. Vaccines for new strains are developed each season, and an identification test confirming the specific strain is essential for vaccine quality control, as stated by the U.S. Food and Drug Administration. However, a shorter timeline of antibody discovery was required to adjust vaccine development schedules. Therefore, anti-SARS-CoV-2 strain-specific, single-domain antibodies (sdAbs) for SARS-CoV-2 vaccines were discovered using alpaca synthetic libraries without animal immunization. Methods: A synthetic sdAb library was developed based on conserved alpaca sdAb frameworks, with a degree of freedom in the three complementarity-determining regions. Specific and high-affinity sdAb clones were selected from the library by one ribosomal display round, followed by two phage display selections using a biotinylated strain-specific SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as bait and non-biotinylated RBD variants to block. The sdAbs clones were applied to the identification test using Western blotting. The binding epitopes were determined by hydrogen–deuterium exchange mass spectrometry. Results: Five clones of XBB.1.5 and two clones of JN.1-specific sdAbs were discovered. Anti-JN.1 sdAb clone 1B9 detected JN.1 vaccine products but no other previously produced vaccine strains, Wuhan, BA.5 and XBB.1.5, by WB for vaccine identification test. Four binding epitopes for anti-JN.1 sdAb clone 1B9 were identified, including the L455S mutation, a critical amino acid to evade neutralizing antibodies for the JN.1 strain. Conclusions: Anti-XBB.1.5 and JN.1-specific sdAbs were discovered from a synthetic single-domain antibody library within 8–9 weeks, and these sdAbs were applied to vaccine identification testing. Full article

Background/Objectives: Nature has evolved millions of venom-derived peptides with diverse biological functions, a substantial fraction of which target complex membrane proteins such as G-protein-coupled receptors and ion channels. Many of these peptides are stabilized by multiple disulfide bonds, endowing them with exceptional structural stability and favorable pharmacological properties. Methods: Leveraging this natural diversity, we developed a robust venom peptide therapeutics discovery system built on phage display technology and constructed a library using approximately 482 venom-derived scaffolds. The library design was guided by a machine learning (ML) model capable of predicting mutation-tolerant residues that preserve peptide foldability, maximizing structural integrity and sequence diversity. Results: The resulting VCX library was evaluated through screening against four diverse targets (CD47, DLL3, IL33, and P2X7R), yielding strong binders for all four, a success rate of 100%. Furthermore, by integrating high-throughput recombinant expression of thioredoxin–venom fusion proteins along with ML-assisted affinity maturation, we rapidly identified potential leads for DLL3 binders. Conclusions: This venom-based discovery platform offers significant advantages in both functionality and developability compared with conventional peptide discovery approaches. By combining natural structural diversity, ML-guided design, and recombinant expression, it enables efficient identification of “antibody-like” binders with molecular weights much smaller than those of antibodies. Consequently, it provides a powerful strategy for developing next-generation peptide therapeutics targeting challenging protein–protein interactions and complex membrane proteins. Full article

Background/Objectives. Antibiotic resistance is an escalating global health concern, highlighting the need for innovative antibacterial strategies beyond traditional drugs. GroEL, a highly conserved bacterial chaperonin essential for protein folding and stress tolerance, represents a promising but underexplored therapeutic target. This study aimed to identify short peptides capable of binding GroEL monomers and potentially altering their function, with the long-term goal of disrupting bacterial survival mechanisms. Methods. A phage display screening of a 12-mer peptide library was performed against purified GroEL monomers, yielding five candidate peptides (G1–G5). Their interactions with GroEL were analyzed through molecular docking and molecular dynamics simulations using three-dimensional GroEL structures (1MNF, 1XCK, 8S32). Stability of binding and interaction profiles were assessed through molecular dynamics-based analyses and MM/GBSA free energy calculations. Results. Peptides G4 and G5 displayed the most stable and energetically favorable interactions, with G4–8S32 showing the strongest binding (−116.68 kcal/mol). These peptides localized near inter-subunit interfaces, suggesting potential interference with GroEL oligomerization or allosteric transitions, which are critical for its biological function. Conclusions. Our findings demonstrate that short peptides can stably bind GroEL and potentially modulate its activity. Peptides G4 and G5 represent at our knowledge the first promising scaffolds for developing a novel class of peptide-based antibacterial agents targeting conserved chaperonin systems. This work introduces a new avenue that warrants further experimental validation. Full article

Mimotope-based immunoassays offer an eco-friendly alternative to chemically synthesized antigens for the quantitative analysis of small molecules, but their use for practical on-site and high-throughput residue monitoring remains limited. Herein, we report the selection, production, and application of a phage display–derived mimotope targeting an anti-uniconazole monoclonal antibody (UCZ-mAb), with the aim of developing two complementary immunoassays that enable sensitive, eco-friendly detection of UCZ residues in agricultural samples. A 12-mer phage-displayed peptide library was screened to identify UCZ-specific mimotopes, and a selected sequence was genetically fused to SpyTag and expressed in Escherichia coli to generate a SpyTagged mimotope. Leveraging the SpyCatcher/SpyTag self-assembly system, the SpyTagged mimotope was directionally conjugated onto SpyCatcher-functionalized time-resolved fluorescence beads (TRFBs) and subsequently used as a signal-labeled competitive antigen in a lateral flow immunoassay (LFIA) designed for rapid on-site screening. In parallel, a wash-free magnetic separation immunoassay (MSIA) suitable for green, high-throughput screening in routine laboratories was established using self-assembled mimotope-TRFB probes. The LFIA and MSIA exhibited half-maximal inhibitory concentrations (IC₅₀) of 3.70–6.72 μg/kg and 16.4–18.3 μg/kg, respectively, in real samples. Spiked-sample recoveries ranged from 91.1 to 107.8% for LFIA and 92.6–115.7% for MSIA, demonstrating acceptable accuracy and precision. These results indicate that the SpyTagged mimotope–based LFIA and MSIA provide complementary, reliable, and sensitive platforms for on-site screening and high-throughput monitoring of UCZ residues in agricultural samples, while avoiding the drawbacks associated with traditional chemical antigen synthesis. Full article

Background: Antibodies that cross the blood–brain barrier (BBB) by targeting receptor-mediated transport (RMT) systems can allow efficient drug delivery to the central nervous system (CNS). In order to improve brain uptake of antibodies, their binding properties have been engineered, but it is not always clear what antibody properties dictate BBB transport efficiency. In this study, we therefore developed and employed an in vitro phenotypic screen and a quantitative transcytosis assay in an attempt to identify improved variants of a previously identified BBB transcytosing antibody known as 46.1. Methods: First, a random mutagenic 46.1 antibody phage display library was screened for improved transcytosis through a human induced pluripotent stem cell (iPSC)-derived BBB model. These screens yielded antibody variants that enriched over multiple screening rounds; however, when produced as soluble antibodies, the variants did not display improved in vitro transcytosis over the wild-type (WT) 46.1 antibody. As a second strategy, we performed a targeted histidine point mutation of a solvent-exposed residue in each complementarity-determining region (CDR) and evaluated the in vitro transcytosis capacity of the variants. Results and Conclusions: In this way, we identified a 46.1 variant, R162H, with modestly improved in vitro transcytosis properties. These results show that the iPSC-derived BBB screening insights and evaluation strategies presented here could facilitate the engineering and optimization of lead antibodies for CNS delivery. Full article

The focus of this study was to explore phage display systems employing bacteriophage lambda (λ) gene fusions to its capsid decoration protein gpD as reagent tools for tackling disease. The biological activity of gpD-fusions was examined by testing for the retained antimicrobial toxicity of cathelicidins or defensins fused to gpD. Our previous finding that only COOH fusions of either cathelicidins or defensins to gpD were toxigenic was expanded to show that only the reduced form of fused defensin antimicrobial polypeptides was found to be toxigenic. Compared in review are gene-fusion lytic display systems (where the fusion-display gene is integrated within the viral genome) with a surrogate system, employed herein, that exogenously provides the fusion-display protein for addition to phage capsid. It is easily possible to produce fully coated lambda display particles (LDP) serving as single epitope vaccines (SEV), or antimicrobials, or to produce partially coated LDP without any complex bacteriophage genetic engineering, making the system available to all. The potential to build vaccine vector phage particles (LDNAP) comprising essentially sheathed DNA vaccines encapsulated within an environmentally protective capsid is described. LDNAP are produced by introducing a cassette into the phage genome either by phage–plasmid recombination or cloning. The cassette carries a high-level eukaryotic expression promoter driving transcription of the vaccine candidate gene and is devoid of plasmid resistance elements. Full article

Virus-like particles (VLPs) formed as a result of self-assembly of viral capsid proteins are widely used as a platform for antigen presentation in vaccine development. However, since the inclusion of a foreign peptide into the capsid protein can alter its spatial structure and interfere with VLP assembly, such insertions are usually limited to short peptides. In this study, we have demonstrated the potential of capsid protein (CP) of single-stranded RNA phage PQ465 to present long peptides using green fluorescent protein (GFP) as a model. GFP was genetically linked to either the N- or C-terminus of PQ465 CP. Hybrid proteins were expressed in Escherichia coli and Nicotiana benthamiana plants. Spherical virus-like particles (~35 nm according to transmission electron microscopy) were successfully formed by both N- and C-terminal fusions expressed in E. coli, and by plant-produced CP with GFP fused to the C-terminus. ELISA revealed that GFP in VLPs was accessible for specific antibodies suggesting that it is exposed on the surface of PQ465-GFP particles. VLPs carrying GFP were recognized by anti-CP antibodies with less efficiency than VLPs formed by empty CP, which indicates shielding of the CP core in PQ465-GFP particles. Therefore, PQ465 CP can be used as a chimeric VLP platform for the display of relatively large protein antigens, which can operate in bacterial and plant expression systems. 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

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development. Full article