Phage Clones Research Articles

Target-specific peptides for BK virus agnoprotein identified through phage display screening: advancing antiviral therapeutics

BK virus is implicated in polyomavirus-associated nephropathy (PVAN) and hemorrhagic cystitis, particularly in kidney transplant recipients, affecting the functionality of the transplanted kidney and posing a risk of graft loss. Despite these challenges, specific antiviral drugs targeting BK virus remain elusive. Agnoprotein, a small, positively charged protein encoded by the BK virus late gene, functions in the assembly, maturation, and release of the virus. Consequently, agnoprotein emerges as a promising target for potential anti-BK virus drugs. Utilizing phage display technology, we conducted screening to identify specific binding peptides against the agnoprotein. The primary objective of screening binding peptides is to utilize them to disrupt the virus’s life cycle, impeding its replication and transmission, thereby achieving antiviral effects. In the current experimental study, a combination of phage 7 peptide libraries and 12 peptide libraries was employed for screening purposes. Following four rounds of screening, seven positive phages demonstrating the ability to bind Agnoprotein were successfully isolated. Following ELISA validation, it was observed that the optical density (OD) values for Agnoprotein binding of the seven positive clones significantly exceeded three times the value of the negative control (NC). Subsequent analysis identified one 7-peptide and six 12-peptides within the binding peptides. Moreover, OD values of dodecapeptide phage clones bound to agnoprotein were generally higher than those of heptapeptide phage clones.In conclusion, our study demonstrates the successful identification of specific binding peptides against agnoprotein, a crucial component in the BK virus life cycle.

Feasibility of Ex Vivo Ligandomics.

We developed ligandomics for the in vivo profiling of vascular ligands in mice, discovering secretogranin III (Scg3) as a novel angiogenic factor that selectively binds to retinal vessels of diabetic but not healthy mice. This discovery led to the development of anti-Scg3 therapy for ocular vasculopathies. However, in vivo ligandomics requires intracardial perfusion to remove unbound phage clones, limiting its use to vascular endothelial cells (ECs). To extend ligandomics to non-vascular cells, we investigated ex vivo ligandomics. We isolated ECs and retinal ganglion cells (RGCs) from diabetic and healthy mouse retinas by immunopanning. We quantified the binding of clonal phages displaying Scg3 and vascular endothelial growth factor (VEGF), confirming that their binding patterns to isolated diabetic versus healthy ECs matched in vivo patterns. Additionally, Scg3 and VEGF binding to isolated RGCs reflected their in vivo activity. These results support the feasibility of ex vivo ligandomics. We further mapped ligands binding to immunopanned diabetic and healthy ECs and RGCs by ligandomics, confirming that Scg3 was enriched with selective binding to diabetic ECs but not healthy ECs or diabetic/healthy RGCs. These findings demonstrate the feasibility of ex vivo ligandomics, which can be broadly applied to various cell types, tissues, diseases, and species.

Phage-ELISA for ultrasensitive detection of Salmonella enteritidis.

The M13 phage carries approximately 5 copies of the pIII protein, each of which is capable of displaying a single-chain variable fragment (scFv) that targets a specific antigen. This feature enables the M13 phage to be widely employed in the construction of scFv libraries, thereby facilitating the identification of antibodies with high specificity and affinity for target antigens. In this study, mice were immunized three times with Salmonella enteritidis (strain C50041) to induce diverse antibodies. The variable region sequences were subsequently amplified by PCR using genome extracted from the mice's splenic cells and fused to the pIII protein to construct the scFv phage display library (C50041-M13-scFv). Through biopanning with the C50041-M13-scFv library, a phage clone (C50041-scFv-4) exhibiting high affinity for the target bacteria was successfully obtained. Moreover, the scFv antibody (scFv-4) derived from C50041-scFv-4 was expressed in a prokaryotic expression system and validated to possess high specificity and affinity for C50041 through in vitro adsorption assays. Additionally, a phage-ELISA method was established: initially, bacteria were immobilized on the bottom surface of a 96-well plate. Next, the positive clone C50041-scFv-4 was introduced to specifically bind to the host cells. Finally, horseradish peroxidase (HRP)-conjugated anti-pVIII antibodies were used to detect the pVIII proteins of the bound phage clones. Owing to the capacity of multiple C50041-scFv-4 probes to simultaneously bind to a single target Salmonella and each phage clone's ability to accommodate hundreds of HRP-labeled antibodies, the proposed phage-ELISA demonstrated remarkable sensitivity (104 CFU mL-1) for detecting Salmonella enteritidis samples. This sensitivity surpasses that of traditional ELISA by one order of magnitude in this study. Our phage-ELISA technology exhibits broad applicability across various biological species and provides an improved and robust platform for pathogen detection including bacteria and viruses.

Efficient Selection of the 2,4-Dichlorophenoxyacetic Acid Nanobody Gene from the Phage Library Constructed with Sorted Specific Cells and Expression in Plants to Confer Herbicide Resistance.

Immunomodulation in biotechnological processes requires an adequate level of specific, high-affinity recombinant antibodies expressed in the affected cells. Here, we report a new strategy to obtain a sensitive nanobody against the 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide. Unlike the previous methods that used total peripheral blood lymphocytes, specific peripheral lymphocytes stained with a fluorescein isothiocyanate-labeled 2,4-D coating antigen were isolated via fluorescence-activated cell sorting and used for phage display library construction. This strategy significantly reduced interference of anticarrier protein nanobody phages to small-molecule nanobody development and required only two cycles of panning to obtain the desired positive clones. Nb4-11, one of the most sensitive phage clones, showed good sensitivity and specificity against 2,4-D. Compared with previously reported 2,4-D nanobodies, the sequence of Nb4-11 exhibited completely different complementarity-determining regions (CDRs). The half-maximum inhibition concentration of the Nb4-11-based ic-ELISA was 29.3 ± 1.9 ng/mL. The Nb4-11 gene was subsequently transferred into Arabidopsis thaliana to confer herbicide resistance, and homozygous transgenic T3 lines were obtained. Stable expression of the 2,4-D nanobody in the model plant was confirmed via PCR, ic-ELISA, and Western blot analysis. In the dose-response bioassay, the transgenic T3 lines were resistant to 2 g of 2,4-D ai/ha. This work offers a new way to sort specific peripheral lymphocytes, efficiently develop nanobodies against small molecules, and create a novel mechanism for herbicide resistance based on the expression of nanobodies in plants.

(Invited) Engineering of Electrochemical Interface for Highly Sensitive Biomolecular Detection: From Macro to Small Molecules

Current diagnostic methods for detection of various cells or biomarker proteins take a long time to achieve reliable outcomes and does not recognize the association between genetic variation and diversity by using nucleic acid or antibody as common recognition element. Whole antibodies are large in size and relatively expensive to produce in mammalian cells. In contrast, small affinity peptides can be created synthetically in a reliable and cost-effective manner. Thus, they can be used in a wider variety of sensing paradigms including peptide-based electrochemical sensors for high-throughput capture and detection of biomarkers in a miniaturized device format.In this talk, we will be showing a peptide-based electrochemical biosensor that could detect virus or biomarker protein. The identified peptide-displaying phage clones and phage-free synthetic peptides were characterized using enzyme-linked immunosorbent assays and electrochemical analysis. To create more advanced sensor system, we fabricated novel functional electrodes, for example, nickel oxide (NiO)–reduced graphene oxide (rGO)/MXene, TA-PEI-graphene oxide and other nanocomposites. It was observed that developed sensing systems were capable of highly sensitive and specific detection of their corresponding target molecules with lower detection limit, better binding constant and recovery. More detailed results of protein detection will be also presenting in this talk.

Ultra-Selective and Sensitive Fluorescent Chemosensor Based on Phage Display-Derived Peptide with an N-Terminal Cu(II)-Binding Motif.

Copper, along with gold, was among the first metals that humans employed. Thus, the copper pollution of the world's water resources is escalating, posing a significant threat to human health and aquatic ecosystems. It is crucial to develop detection technology that is both low-cost and feasible, as well as ultra-selective and sensitive. This study explored the use of the NH2-Xxx-His motif-derived peptide from phage display technology for ultra-selective Cu2+ detection. Various Cu-binding M13 phage clones were isolated, and their affinity and cross-reactivity for different metal ions were determined. A detailed analysis of the amino acid sequence of the unique Cu-binding peptides was employed. For the development of an optical chemosensor, a peptide with an NH2-Xxx-His motif was selected. The dansyl group was incorporated during solid-phase peptide synthesis, and fluorescence detection assays were employed. The efficacy of the Cu2+-binding peptide was verified through spectroscopic measurements. In summary, we developed a highly selective and sensitive fluorescent chemosensor for Cu2+ detection based on a peptide sequence from a phage display library that carries the N-terminal Xxx-His motif.

Measuring carbohydrate recognition profile of lectins on live cells using liquid glycan array (LiGA).

Glycans constitute a significant fraction of biomolecular diversity on cellular surfaces across all kingdoms of life. As the structure of glycans is not directly encoded by the organism's DNA, it is impossible to use high-throughput DNA technologies to study the role of cellular glycosylation or to understand how glycocalyx is recognized by glycan-binding proteins (GBPs). To address this gap, we recently described a liquid glycan array (LiGA) platform that allows profiling of glycan-GBP interactions on the surface of live cells in vitro and in vivo using next-generation sequencing. LiGA is a library of DNA-barcoded bacteriophages, where each clonal bacteriophage displays 5-1,500 copies of a glycan and the distinct DNA barcode inside each bacteriophage clone encodes the structure and density of the displayed glycans. Deep sequencing of the glycophages associated with live cells yields a glycan-binding profile of GBPs expressed on the surface of cells. This protocol provides detailed instructions for how to use LiGA to probe cell surface receptors and includes information on the preparation of glycophages, analysis by MALDI-TOF mass spectrometry, the assembly of a LiGA library and its deep sequencing. Using this protocol, we measure glycan-binding profiles of the immunomodulatory sialic acid-binding immunoglobulin-like lectins‑1, -2, -6, -7 and -9 expressed on the surface of different cell types. Compared with existing methods that require complex specialist equipment, this method allows users with basic molecular biology expertise to measure the precise glycan-binding profile of GBPs on the surface of any cell type expressing exogenous GBP within 2-3 d.

Evaluation of Reproductive Histology Response of Adult Fasciola hepatica in Goats Vaccinated with Cathepsin L Phage-Exposed Mimotopes.

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 1013 phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis' gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output.

Antibody fragments targeting the extracellular domain of follicular stimulating hormone receptor for contraception in male dogs and cats

The increased LH levels resulting from the absence of negative feedback after castration has been linked to long-term health issues. A need exists for an alternative contraceptive agent that functions without interfering the LH pathways. This study aimed to develop antibody fragments against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and evaluate its effects on Sertoli cell functions. Phage clones against the extracellular domain of dog and cat FSHr selected from an antibody fragment phagemid library were analyzed for binding kinetics by surface plasmon resonance. Sertoli cells were isolated from testes of adult animals (five dogs and five cats). Efficacy test was performed by treating Sertoli cell cultures (SCCs) with anti-FSHr antibody fragments compared with untreated in triplicates. Expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB) and vascular endothelial growth factor A (VEGFA) mRNA in SCCs were quantified by RT-qPCR. The results demonstrated that the molecular weight of the purified dog and cat anti-FSHr antibody fragment was 25 kDa and 15 kDa, respectively. Based on protein molecular weight, the antibody fragment of dogs and cats was therefore, so-called single-chain variable fragments (scFv) and nanobody (nb), respectively. The binding affinity with dissociation constant (KD) was 2.32 × 10−7 M and 2.83 × 10−9 M for dog and cat anti-FSHr antibody fragments, respectively. The cross-binding kinetic interactions between the dog anti-FSHr scFv and the cat ECD of FSHr could not be fitted to the curves to determine the binding kinetics. However, the cross-binding affinity KD between the cat anti-FSHr nb and the dog ECD FSHr was 1.75 × 10−4 M. The mRNA expression of ABP, IHBB and VEGFA in SCCs was less (P < 0.05) in both dogs (12.26, 4.07 and 5.11 folds, respectively) and cats (39.53, 14.07 and 20.29 folds, respectively) treated with anti-FSHr antibody fragments, indicating the Sertoli cell functions were suppressed. In conclusion, this study demonstrated the establishment of species-specific antibody fragments against FSHr in SCCs for dogs and cats. The fragment proteins illustrate potential to be developed as non-surgical contraceptive agent targeting FSHr in companion animals.

Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology.

Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

Immunotherapy Combining Mimotopes Selected by Phage Display Plus Amphotericin B Is Effective for Treatment Against Visceral Leishmaniasis.

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.

Neutralizing anti-diphtheria toxin scFv produced by phage display.

Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. The size of the constructed scFv library was calculated to be 1.3×106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107M-1. This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

Artificial periosteum promotes bone regeneration through synergistic immune regulation of aligned fibers and BMSC-recruiting phages

Given the crucial role of periosteum in bone repair, the use of artificial periosteum to induce spontaneous bone healing instead of using bone substitutes has become a potential strategy. Also, the proper transition from pro-inflammatory signals to anti-inflammatory signals is pivotal for achieving optimal repair outcomes. Hence, we designed an artificial periosteum loaded with a filamentous bacteriophage clone named P11, featuring an aligned fiber morphology. P11 endowed the artificial periosteum with the capacity to recruit bone marrow mesenchymal stem cells (BMSCs). The artificial periosteum also regulated the immune microenvironment at the bone injury site through the synergistic effects of biochemical factors and topography. Specifically, the inclusion of P11 preserved inflammatory signaling in macrophages and additionally facilitated the migration of BMSCs. Subsequently, aligned fibers stimulated macrophages, inducing alterations in cytoskeletal and metabolic activities, resulting in the polarization into the M2 phenotype. This progression encouraged the osteogenic differentiation of BMSCs and promoted vascularization. In vivo experiments showed that the new bone generated in the AP group exhibited the most efficient healing pattern. Overall, the integration of biochemical factors with topographical considerations for sequential immunomodulation during bone repair indicates a promising approach for artificial periosteum development. Statement of significanceThe appropriate transition of macrophages from a pro-inflammatory to an anti-inflammatory phenotype is pivotal for achieving optimal bone repair outcomes. Hence, we designed an artificial periosteum featuring an aligned fiber morphology and loaded with specific phage clones. The artificial periosteum not only fostered the recruitment of BMSCs but also achieved sequential regulation of the immune microenvironment through the synergistic effects of biochemical factors and topography, and improved the effect of bone repair. This study indicates that the integration of biochemical factors with topographical considerations for sequential immunomodulation during bone repair is a promising approach for artificial periosteum development.

Abstract 59: Triple negative breast cancer (TNBC) targeting CAR-T cell constructs with phage display-derived peptides

Abstract Introduction: Triple Negative Breast Cancer (TNBC) poses treatment challenges due to the absence of effective targeted therapies. CAR-T cell therapy, successful in hematological cancers, is now being investigated for solid tumors. Finding new CAR-T targets is vital to overcome TNBC's therapeutic constraints. Methods: In this study, a Biopanning phage display strategy employing a library of random peptides was utilized to identify potential targets for CAR-T cell therapy against TNBC. Through this approach, Heat Shock Protein 60 (HSP60) emerged as a promising candidate due to its binding affinity to the selected phage clones. Subsequent validation of HSP60 surface expression on TNBC cell lines in comparison to normal cells was conducted using flow cytometry. Concurrently, patient survival data were gathered and analyzed to evaluate the clinical significance of HSP60 expression in TNBC. The identified peptides demonstrating high specificity and affinity for HSP60 on the surface of TNBC cells were chosen for the development of a third-generation CAR-T model, incorporating various optimizations and innovations. Results: The Biopanning strategy successfully identified HSP60 as a prominent target for CAR-T cell therapy in TNBC. Flow cytometry analysis confirmed significantly elevated surface expression of HSP60 on TNBC cell lines compared to their normal counterparts, substantiating its potential as an accessible antigen for targeted therapy. Patient survival analysis linked elevated HSP60 levels with poorer prognosis in TNBC, highlighting the protein's clinical relevance. Peptides showing high specificity and binding to TNBC's HSP60 were integrated into a meticulously engineered third-gen CAR-T cell model. This design aimed to boost the CAR-T's precision in targeting and eliminating HSP60-expressing TNBC cells. Iterative optimizations ensured the construct's accuracy, laying the groundwork for a potent, tailored TNBC immunotherapy. Discussion: The discovery of HSP60 as a prime target for CAR-T cell therapy in TNBC is a pivotal advancement. Its distinct presence on TNBC cell surfaces, confirmed via flow cytometry, highlights its suitability for targeted immunotherapy. The correlation between elevated HSP60 levels and poorer prognosis in TNBC patients underscores its value as both a biomarker and a therapeutic focus. The peptides' exceptional specificity and strong binding to HSP60 validate their use in crafting a third-generation CAR-T cell model. Their integration and careful refinements in CAR-T design aim to optimize efficacy while reducing off-target effects, laying a robust foundation for tailored TNBC immunotherapy. Preclinical evaluations are imperative to thoroughly assess the safety and effectiveness of this innovative CAR-T approach. Citation Format: Muhammad G. Khodary, Alehegne W. Yirsaw, Yumna A. Elsobky, Reshma Gurung, Jessie Jaynes, Temesgen Samuel, Maninder Sandey, Deepa Bedi. Triple negative breast cancer (TNBC) targeting CAR-T cell constructs with phage display-derived peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 59.

Absence of specific autoantibodies in patients with narcolepsy type 1 as indicated by an unbiased random peptide-displayed phage screening.

Narcolepsy type 1 (NT1) is an enigmatic sleep disorder characterized by the selective loss of neurons producing orexin (also named hypocretin) in the lateral hypothalamus. Although NT1 is believed to be an autoimmune disease, the orexinergic neuron-specific antigens targeted by the pathogenic immune response remain elusive. In this study, we evaluated the differential binding capacity of various peptides to serum immunoglobin G from patients with NT1 and other hypersomnolence complaints (OHCs). These peptides were selected using an unbiased phage display technology or based on their significant presence in the serum of NT1 patients as identified from previous studies. Although the subtractive biopanning strategy successfully enriched phage clones with high reactivity against NT1 serum IgG, the 101 randomly selected individual phage clones could not differentiate the sera from NT1 and OHC. Compared to the OHC control group, serum from several NT1 patients exhibited increased reactivity to the 12-mer peptides derived from TRBV7, BCL-6, NRXN1, RXRG, HCRT, and RTN4 proteins, although not statistically significant. Collectively, employing both unbiased and targeted methodologies, we were unable to detect the presence of specific autoantibodies in our NT1 patient cohort. This further supports the hypothesis that the autoimmune response in NT1 patients likely stems primarily from T cell-mediated immunity rather than humoral immunity.

Development and characterization of a novel nanobody with SRMV neutralizing activity

Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 1010 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.

Chimeric peptides targeting the receptor-binding domain of SARS-CoV-2 variants inhibit ACE2 interaction.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is pivotal in facilitating viral entry and serves as a major target for vaccine development and therapeutics. Despite undergoing mutations aimed at evading host immunity, certain regions within the RBD remain conserved. This study aimed to identify peptides capable of interacting with these conserved regions of the RBD across various variants and assess their neutralization potential. The PhD-12 phage display library underwent screening to identify phages binding to the RBD. Selected phage clones were examined for binding to the RBD of multiple variants, including 2019-nCoV, Delta (B.1.617.2), Omicron (B.1.1.529), and XBB. Peptides, expressed as chimeric constructs, were tested for their binding to the RBD, the Omicron trimeric S, inactivated SARS-CoV-2 virus, and neutralizing activity. The binding sites were analyzed using Molecular Docking. Two selected phage clones displayed peptides binding to the RBD of multiple variants. Chimeric T hioredoxin-peptides (Trx-RB9 and Trx-RB10) exhibited binding to both inactivated SARS-CoV-2 and the Omicron trimeric S, with half-maximum effective concentrations (EC50 ) values of 111.9 and 360.2 nM, respectively. Molecular docking revealed distinct binding sites within the RBD of the Omicron trimeric S for both Trx-RB9 and Trx-RB10. A mixture of Trx-RB9 and Trx-RB10 inhibited 78% of the binding of recombinant human ACE2 to the Omicron trimeric S. The chimeric Trx-RB9 and Trx-RB10 peptides bind to the RBD of SARS-CoV-2 variants and inhibit the binding of ACE2 to the RBD of the Omicron trimeric S.

Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining.

Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation.

Screening, Expression and Identification of Nanobody Against Monkeypox Virus A35R.

The monkeypox (Mpox) virus epidemic presents a significant risk to global public health security. A35R, a crucial constituent of EEV, plays a pivotal role in virus transmission, serves as a vital target for vaccine development, and has potential for serological detection. Currently, there is a dearth of research on nanobodies targeting A35R. The purpose of this study is to identify specific nanobodies target A35R, so as to provide new antibody candidates for Mpox vaccine development and diagnostic kit development. Three nanobodies specific to the monkeypox virus protein A35R were screened from a naïve phage display library. After four rounds of panning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA). Further, the nanobody fusion protein was constructed in pNFCG1-IgG1-Fc vector and expressed in HEK293F cells and purified by affinity chromatography. The specificity and affinity of the nanobodies were identified by ELISA. The binding kinetics of the VHH antibody to A35R were assessed via employment of a bio-layer interferometry (BLI) apparatus, thereby determining the nanobodies affinity. The three purified nanobodies showed specific high-affinity binding MPXV A35R, of them, VHH-1 had the best antigen binding affinity (EC50 = 0.010 ug/mL). In addition, VHH-1 on Protein A biosensor can bind Mpox virus A35R, with an affinity constant of 54 nM as determined in BLI assay. In sum, we has obtained three nanobody strains against Mpox virus A35R with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of Mpox virus.

Development and characterization of phage display-derived anti-toxin antibodies neutralizing TcdA and TcdB of Clostridioides difficile.

TcdA and TcdB are known as the major virulence attributes of Clostridioides difficile. Hence, neutralizing the TcdA and TcdB activities can be considered as an efficient therapeutic approach against C. difficile infection (CDI). In this work, we utilized phage display technique to select single-chain fragment variable (scFv) fragments as recombinant antibodies displayed on the surface of phages, which specifically target native TcdA, or TcdB (nTcdA and nTcdB), and their recombinant C-terminal combined repetitive oligopeptide (CROP) domains (rTcdA and rTcdB). After three rounds of biopanning, abundance of phage clones displaying high reactivity with TcdA or TcdB was quantified through enzyme-linked immunosorbent assay (ELISA). Furthermore, selected scFvs were characterized by cell viability and neutralization assays. The gene expression of immunological markers, IL-8 and TNF-α, was examined in treated Caco-2 cells by RT-qPCR. The epitopes of neutralizing scFvs were also identified by molecular docking. Totally, 18 scFv antibodies (seven for TcdA and 11 for TcdB) were identified by ELISA. Among selected scFvs, two clones for TcdA (rA-C2, A-C9) and three clones for TcdB (rB-B4, B-F5, B-F11) exhibited the highest neutralizing activity in Caco-2 and Vero cells. Moreover, the cocktail of anti-TcdA and anti-TcdB antibodies notably decreased the mRNA expression of TNF-α and IL-8 in Caco-2 cells. Molecular docking revealed that the interaction between scFv and toxin was mostly restricted to CROP domain of TcdA or TcdB. Our results collectively provided more insights for the development of neutralizing scFvs against C. difficile toxins using phage display. Further research is needed to meticulously evaluate the potential of scFvs as an alternative treatment for CDI using animal models and clinical trials.IMPORTANCETargeting the major toxins of Clostridioides difficile by neutralizing antibodies is a novel therapeutic approach for CDI. Here, we report a panel of new anti-TcdA (rA-C2, A-C9) and anti-TcdB (rB-B4, B-F5, and B-F11) recombinant antibody fragments (scFvs) isolated from Tomlinson I and J libraries using phage display technique. These scFv antibodies were capable of neutralizing their respective toxin and showed promise as potential therapeutics against TcdA and TcdB of C. difficile in different in vitro models. In addition, in silico analysis showed that at least two neutralization mechanisms, including inhibiting cell surface binding of toxins and inhibiting toxin internalization can be proposed for the isolated scFvs in this work. These findings provide more insights for the applicability of specific scFvs toward C. difficile toxins at in vitro level. However, further research is required to evaluate the potential application of these scFvs as therapeutic agents for CDI treatment in clinical setting.