Filamentous Phage Particles Research Articles

The pComb3 Phagemid Family of Phage Display Vectors.

A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.

Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR.

Gene V protein (gVp) of the bacteriophages of the Ff family is a non-specific single-stranded DNA (ssDNA) binding protein. gVp binds to viral DNA during phage replication inside host Escherichia coli cells, thereby blocking further replication and signaling the assembly of new phage particles. gVp is a dimer in solution and in crystal form. A structural model of the complex between gVp and ssDNA was obtained via docking the free gVp to structures of short ssDNA segments and via the detection of residues involved in DNA binding in solution. Using solid-state NMR, we characterized structural features of the gVp in complex with full-length viral ssDNA. We show that gVp binds ssDNA with an average distance of 5.5 Å between the amino acid residues of the protein and the phosphate backbone of the DNA. Torsion angle predictions and chemical shift perturbations indicate that there were considerable structural changes throughout the protein upon complexation with ssDNA, with the most significant variations occurring at the ssDNA binding loop and the C-terminus. Our data suggests that the structure of gVp in complex with ssDNA differs significantly from the structure of gVp in the free form, presumably to allow for cooperative binding of dimers to form the filamentous phage particle.

Vaccination with cathepsin L phage-exposed mimotopes, single or in combination, reduce size, fluke burden, egg production and viability in sheep experimentally infected with Fasciola hepatica.

Fascioliasis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. In this study, fluke burdens, liver fluke size and biomass, faecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep vaccinated with peptide mimotopes of cathepsin L and infected with metacercariae. A total of 25 sheep were allocated randomly into five groups of five animals each, and experimental groups were immunised with 1×1013 filamentous phage particles of cathepsin L1 (CL1) (TPWKDKQ), CL2 (YGSCFLR) and mixtures of CL1+CL2 mimotopes, in combination with Quil A adjuvant, and wild-type M13KE phage in a two-vaccination scheme on weeks 0 and 4. The control group received phosphate-buffered saline. All groups were challenged with 300 metacercariae two weeks after the last immunisation and euthanised 16weeks later. The CL1 vaccine was estimated to provide 57.58% protection compared with the control group; no effect was observed in animals immunised with CL2 and CL1+CL2 (33.14% and 11.63%, respectively). However, animals receiving CL2 had a significant reduction in parasite egg output. Vaccinated animals showed a significant reduction in fluke length and width and wet weights. In the CL1 group, there was a significant reduction in the total biomass of parasites recovered. Egg development was divided into seven stages: dead, empty, unembryonated, cell division, eyespot, hatched and hatching. The highest percentage of developmental stages was detected for vaccinated sheep administered CL1+CL2 with cell division, and the lowest percentage was observed in the hatching stage. Furthermore, a significant difference in all developmental stages was observed between vaccinated animals and the control group (P<0.01). The levels of anti-phage total IgG in immune sera increased significantly at four weeks after immunisation and were always significantly higher for cathepsin L vaccine group than in the challenged control group. Total IgG was inversely and significantly correlated with worm burden in the CL1 group.

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

Characterization of MDAΦ, a temperate filamentous bacteriophage of Neisseria meningitidis.

The mechanism by which Neisseria meningitidis becomes invasive is not well understood. Comparative genomics identified the presence of an 8 kb island in strains belonging to invasive clonal complexes. This island was designated MDA for meningococcal disease associated. MDA is highly conserved among meningococcal isolates and its analysis revealed a genomic organization similar to that of a filamentous prophage such as CTXΦ of Vibrio cholerae. Subsequent molecular investigations showed that the MDA island has indeed the characteristics of a filamentous prophage, which can enter into a productive cycle and is secreted using the type IV pilus (tfp) secretin PilQ. At least three genes of the prophage are necessary for the formation of the replicative cytoplasmic form (orf1, orf2 and orf9). Immunolabelling of the phage with antibodies against the major capsid protein, ORF4, confirmed that filamentous particles, about 1200 nm long, covered with ORF4 are present at the bacterial surface forming bundles in some places and interacting with pili. The MDA bacteriophage is able to infect different N. meningitidis strains, using the type IV pili as a receptor via an interaction with the adsorption protein ORF6. Altogether, these data demonstrate that the MDA island encodes a functional prophage able to produce infectious filamentous phage particles.

Modulation of extracellular matrix in cancer is associated with enhanced tumor cell targeting by bacteriophage vectors.

BackgroundGene therapy has been an attractive paradigm for cancer treatment. However, cancer gene therapy has been challenged by the inherent limitation of vectors that are able to deliver therapeutic genes to tumors specifically and efficiently following systemic administration. Bacteriophage (phage) are viruses that have shown promise for targeted systemic gene delivery. Yet, they are considered poor vectors for gene transfer. Recently, we generated a tumor-targeted phage named adeno-associated virus/phage (AAVP), which is a filamentous phage particle whose genome contains the adeno-associated virus genome. Its effectiveness in delivering therapeutic genes to tumors specifically both in vitro and in vivo has been shown in numerous studies. Despite being a clinically useful vector, a multitude of barriers impede gene transduction to tumor cells. We hypothesized that one such factor is the tumor extracellular matrix (ECM).MethodsWe used a number of tumor cell lines from different species and histological types in 2D monolayers or 3D multicellular tumor spheroid (MCTS) models. To assess whether the ECM is a barrier to tumor cell targeting by AAVP, we depleted the ECM using collagenase, hyaluronidase, or combination of both. We employed multiple techniques to investigate and quantify the effect of ECM depletion on ECM composition (including collagen type I, hyaluronic acid, fibronectin and laminin), and how AAVP adsorption, internalisation, gene expression and therapeutic efficacy are subsequently affected. Data were analyzed using a student’s t test when comparing two groups or one-way ANOVA and post hoc Tukey tests when using more than two groups.ResultsWe demonstrate that collagenase and hyaluronidase-mediated degradation of tumor ECM affects the composition of collagen, hyaluronic acid and fibronectin. Consequently, AAVP diffusion, internalisation, gene expression and tumor cell killing were enhanced after enzymatic treatment. Our data suggest that enhancement of gene transfer by the AAVP is solely attributed to ECM depletion. We provide substantial evidence that ECM modulation is relevant in clinically applicable settings by using 3D MCTS, which simulates in vivo environments more accurately.ConclusionOur findings suggest that ECM depletion is an effective strategy to enhance the efficiency of viral vector-guided gene therapy.

Tracking chemical reactions on the surface of filamentous phage using mass spectrometry

Chemical modification of phage libraries has allowed the in vitro evolution of ligands having properties not provided by natural polypeptides. The development of novel and more diverse chemical reactions on phage was hampered by the lack of analytical methods to efficiently monitor the reaction products on the more than 10 000 kDa large filamentous phage particles. Herein, we present a strategy to detect chemically modified peptides on phage based on enzymatic release of peptide from phage and mass spectrometry analysis.

“Breeding” Diagnostic Antibodies for Higher Assay Performance: A 250-Fold Affinity-Matured Antibody Mutant Targeting a Small Biomarker

Conventional immunization-based antibodies are not always applicable to monitor trace amounts of clinical biomarkers due to insufficient antigen-binding affinity. The development of in vitro affinity maturation for native antibodies has paved the way to solve this problem, but there has been minimal success in obtaining improved antibody mutants for small molecule biomarkers (haptens). We here describe a "molecular breeding" approach, in which a practical anti-hapten antibody mutant has successfully been generated from a prototype antibody with poor binding properties. Random point mutations were introduced by error-prone polymerase chain reaction in the V(H) and V(L) genes of a second-generation mutant of a single-chain Fv fragment (scFv) that binds estradiol-17β (E2), whose parent mouse Fab fragment showed poor affinity for E2 (Ka = 5.2 × 10(7) M(-1)). The resulting scFv gene library was expressed on filamentous phage particles. Phage clones with strong E2 binding were isolated with dissociation-independent methods using newly developed reagents. The binding characteristics and clinical applicability of the soluble scFvs prepared from the selected clones were examined in a competitive enzyme-linked immunosorbent assay (ELISA). We finally obtained a third-generation scFv mutant, scFv#m3-a18 (11 amino acid substitutions), which showed ~250-fold greater Ka (1.3 × 10(10) M(-1)) than the parent Fab, and yielded sensitive ELISA dose-response curves for E2 (limit of detection < 0.5 pg/assay) with practical specificity. The average human serum E2 levels, determined after acetonitrile extraction, were compatible with reported reference ranges. The present results will prompt a new era for preparing diagnostic reagents.

Effects of short elastin‐like peptides on filamentous particles and their transition behavior

While elastin-like polypeptides and peptides (ELPs) have been used for various stimulus-responsive applications, details of their switching remain unclear. We therefore constructed a novel series of filamentous phage particles displaying a high surface density of short ELPs. The surface display of ELPs did not disrupt either particle shape or dimensions, and the resulting ELP-phage particles were colloidally stable over several weeks. However, in spite of a saturating surface density, macroscopic aggregation of ELP-phages cannot be triggered in water. To investigate the underlying mechanisms we examined conformational changes in the secondary structure of the phage proteins by circular dichroism and tryptophan fluorescence, which indicate partial protein unfolding in ELP-phage particles. To gain further insight into the ELP itself, analogous "free" ELP peptides were synthesized and characterized. Circular dichroism of these peptides shows the onset of β-type conformations with increasing temperature, consistent with the accepted view of the microscopic transition that underlies the inverse phase behavior of ELPs. Increased guest residue hydrophobicity was found to depress the microscopic transition temperature of the peptides, also consistent with a previously proposed intramolecular hydrogen-bonding mechanism. Importantly, our results indicate that although the nanoscale presentation state can suppress macroscopic aggregation of ELPs, microscopic transitions of the ELP can still occur. Given the growing use of ELPs within supra-molecular scaffolds, such effects are important design considerations for future applications.

In vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines

BackgroundTargeted drug-carrying phage nanomedicines are a new class of nanomedicines that combines biological and chemical components into a modular nanometric drug delivery system. The core of the system is a filamentous phage particle that is produced in the bacterial host Escherichia coli. Target specificity is provided by a targeting moiety, usually an antibody that is displayed on the tip of the phage particle. A large drug payload is chemically conjugated to the protein coat of the phage via a chemically or genetically engineered linker that provides for controlled release of the drug after the particle homed to the target cell. Recently we have shown that targeted drug-carrying phage nanomedicines can be used to eradicate pathogenic bacteria and cultured tumor cells with great potentiation over the activity of the free untargeted drug. We have also shown that poorly water soluble drugs can be efficiently conjugated to the phage coat by applying hydrophilic aminoglycosides as branched solubility-enhancing linkers.ResultsWith an intention to move to animal experimentation of efficacy, we tested anti-bacterial drug-carrying phage nanomedicines for toxicity and immunogenicity and blood pharmacokinetics upon injection into mice. Here we show that anti-bacterial drug-carrying phage nanomedicines that carry the antibiotic chloramphenicol conjugated via an aminoglycoside linker are non-toxic to mice and are greatly reduced in immunogenicity in comparison to native phage particles or particles to which the drug is conjugated directly and are cleared from the blood more slowly in comparison to native phage particles.ConclusionOur results suggest that aminoglycosides may serve as branched solubility enhancing linkers for drug conjugation that also provide for a better safety profile of the targeted nanomedicine.

Selection of full‐length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence‐activated cell sorting screening

Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process.

Evaluation of humoral and cellular immune responses against HSV-1 using genetic immunization by filamentous phage particles: A comparative approach to conventional DNA vaccine

Phage display is based on expressing peptides as a fusion to one of the phage coat proteins. To date, many vaccine researches have been conducted to display immunogenic peptides or mimotopes of various pathogens and tumors on the surface of filamentous bacteriophages. In recent years as a new approach to application of phages, recombinant bacteriophage lambda particles were used as DNA delivery vehicles to mammalian cells. In this study, recombinant filamentous phage whole particles were used for vaccination of mice. BALB/c mice were inoculated with filamentous phage particles containing expression cassette of Herpes simplex virus 1 (HSV-1) glycoprotein D that has essential roles in the virus attachment and entry. Both humoral and cellular immune responses were measured in the immunized mice and compared to conventional DNA vaccination. A dose–response relationship was observed in both arms of immune responses induced by recombinant filamentous phage inoculation. The results were similar to those from DNA vaccination. Filamentous phages can be considered as suitable alternative candidate vaccines because of easier and more cost-effective production and purification over plasmid DNA or bacteriophage lambda particles.

“Cleavable” hapten–biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments

Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten–biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten–biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17β [E 2], cortisol, and 17α-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E 2–biotin conjugate was used to generate anti-E 2 single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V H domain of a mouse anti-E 2 antibody, and these products were expressed as phagemid particles that were reacted with the E 2–biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.

Identification of a peptide sequence that improves transport of macromolecules across the intestinal mucosal barrier targeting goblet cells

In this study, we demonstrated that the CSKSSDYQC-peptide ligand which was identified from a random phage-peptide library through an in vivo phage display technique with rats could prominently improve the transport efficiency of macromolecules, such as large filamentous phage particles (M13 bacteriophage), across the intestinal mucosal barrier. Synthetic CSKSSDYQC-peptide ligands significantly inhibited the binding of phage P1 encoding CSKSSDYQC-peptide ligands to the intestinal mucosal tissue and immunohistochemical analysis showed that the CSKSSDYQC-peptide ligands could be transported across the intestinal mucosal barrier via goblet cells as their specific gateway. Thus, we inferred that CSKSSDYQC-peptide ligand might have a specific receptor on the goblet cells and transported from intestinal lumen to systemic circulation by transcytosis mechanism. These results suggest that CSKSSDYQC-ligand could be a promising tool for development of an efficient oral delivery system for macromolecular therapeutics in the carrier-drug conjugate strategy.

A Novel Filamentous Phage from the Deep-Sea Bacterium Shewanella piezotolerans WP3 Is Induced at Low Temperature

Active filamentous phage particles were isolated from the deep-sea bacterium Shewanella piezotolerans WP3. A putative single-stranded DNA binding protein of the phage was found to be overexpressed at 4 degrees C compared to its expression at 25 degrees C by two-dimensional polyacrylamide gel electrophoresis. Reverse transcription quantitative PCR further revealed that the key genes of the SW1 phage were significantly induced at low temperature.

A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus

Yersinia pestis, the plague bacillus, has an exceptional pathogenicity but the factors responsible for its extreme virulence are still unknown. A genome comparison with its less virulent ancestor Yersinia pseudotuberculosis identified a few Y. pestis-specific regions acquired after their divergence. One of them potentially encodes a prophage (YpfPhi), similar to filamentous phages associated with virulence in other pathogens. We show here that YpfPhi forms filamentous phage particles infectious for other Y. pestis isolates. Although it was previously suggested that YpfPhi is restricted to the Orientalis branch, our results indicate that it was acquired by the Y. pestis ancestor. In Antiqua and Medievalis strains, YpfPhi genome forms an unstable episome whereas in Orientalis isolates it is stably integrated as tandem repeats. Deletion of the YpfPhi genome does not affect Y. pestis ability to colonize and block the flea proventriculus, but results in an alteration of Y. pestis pathogenicity in mice. Our results show that transformation of Y. pestis from a classical enteropathogen to the highly virulent plague bacillus was accompanied by the acquisition of an unstable filamentous phage. Continued maintenance of YpfPhi despite its high in vitro instability suggests that it confers selective advantages to Y. pestis under natural conditions.

Whole Genome Phage Display Selects for Proline-rich Boi Polypeptides against Bem1p

Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second "src homology region 3'' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1([Asn142-Ile551]), which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained approximately 7.7 x 10(7) independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules.

Gold-Phage “Nuggets” Seem Promising as Tissue Homing Devices

Particles containing metallic gold and genetically engineered filamentous M13-based phage particles appear promising as vehicles for imaging tissues that are affected by cancer, inflammation, or other defects, and have the potential to deliver drugs or stem cells to injured tissues with amazing specificity, according to researchers at the University of Texas M.D. Anderson Cancer Center in Houston. They report their findings in the January 31, 2006, issue of Proceedings of the National Academy of Sciences.

Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments

It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class I-peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were co-localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross-presentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH(4)Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.

Purification of Filamentous Bacteriophage for Phage Display using Size-Exclusion Chromatography

Phage display has proved itself as a powerful and convenient approach for rapid sampling of billions of protein-aceous structures against a ligand of interest. The most widespread system uses display of proteins and peptides as fusions to coat proteins III or VIII of the M13/fd phage. Efficient screening of phage display library requires high purity of the input phage particles. Double precipitation by polyeth-ylene glycol (PEG) with an average molecular weight of 8000 Da (PEG-8000) is a standard technique for purification of filamentous bacterio-phages (1,2). Certain contaminants could still remain in the PEG-purified phage preparation. Although efficient removal of contaminating RNases from bacteriophage preparation using treatment by ionic detergent followed by ultracentrifugation in CsCl gradient was reported (3), this method is costly, time-consuming, and is not applicable if the native state of the phage-displayed protein or peptide is affected by ionic detergents and high salt. Size-exclusion chromatography (SEC) has been used in preparation of pure eukaryotic viruses (4,5) and bacteriophage λ (6). We assumed that SEC can be the method of choice for efficient purification of filamentous phages under mild conditions. In this work we describe a rapid and simple technique for chromatographic purifi-cation of filamentous phage particles in Sephacryl™ S-500 resin (Amersham Biosciences, Piscataway, NJ, USA).The purity of phage preparations obtained after double PEG precipi-tation was assayed by electrophoresis in Tris-tricine (7). Phage particles displayed the single-chain variable fragment (scFv) of the MRL-4 anti-DNA monoclonal antibody (MAb) (8) fused to the C-terminal portion of the pIII minor phage coat protein (δpIII). Production of the scFv-δpIII fusion cloned into the pTScDisp phagemid (GenBank