M13 Phage Research Articles

M13 bacteriophage as biometric component for orderly assembly of dynamic light scattering immunosensor

The ordered assembly of nanostructure is an effective strategy used to manipulate the hydrodynamic diameter (DH) of nanoparticles. Herein, a versatile dynamic light scattering (DLS) immunosensing platform is presented to sensitively detect small molecules and biomacromolecules by using the M13 phage as the building module to order the assembly of gold nanoflowers and gold-coated magnetic nanoparticles, respectively. After the directional assembly of M13 phage, the DH of the probes was significantly increased due to its larger filamentous structure, thus improving the detection sensitivity of the DLS immunosensor. The designed M13 assembled DLS immunosensor with competitive and sandwich formats showed high sensitivities for ochratoxin A and alpha-fetoprotein in real corn and undiluted serum samples, with the detection limits of 1.37 and 57 pg/mL, respectively. These values are approximately 15.8 and 164.9 times lower than those of traditional phage-based enzyme-linked immunosorbent assays. Collectively, this work provides a promising strategy to manipulate the DH of nanoparticles by highly evolved biomaterials such as engineered M13 phages and opens upon a new direction for developing DLS immunosensors to detect various targets by the fusion expression of special peptide or nanobody on the pIII or pVIII protein of M13 phage.

Exploiting spatial dimensions to enable parallelized continuous directed evolution

Current strategies to improve the throughput of continuous directed evolution technologies often involve complex mechanical fluid‐controlling system or robotic platforms, which limits their popularization and application in general laboratories. Inspired by our previous study on bacterial range expansion, in this study, we report a system termed SPACE for rapid and extensively parallelizable evolution of biomolecules by introducing spatial dimensions into the landmark phage‐assisted continuous evolution system. Specifically, M13 phages and chemotactic Escherichia coli cells were closely inoculated onto a semisolid agar. The phages came into contact with the expanding front of the bacterial range, and then comigrated with the bacteria. This system leverages competition over space, wherein evolutionary progress is closely associated with the production of spatial patterns, allowing the emergence of improved or new protein functions. In a prototypical problem, SPACE remarkably simplified the process and evolved the promoter recognition of T7 RNA polymerase (RNAP) to a library of 96 random sequences in parallel. These results establish SPACE as a simple, easy to implement, and massively parallelizable platform for continuous directed evolution in general laboratories.

Hybrid M13 bacteriophage-based vaccine platform for personalized cancer immunotherapy

Although cancer vaccines exhibit great advances in the field of immunotherapy, developing an efficient vaccine platform for personalized tumor immunotherapy is still a major challenge. Here we demonstrate that a bioactive vaccine platform (HMP@Ag) fabricated with hybrid M13 phage and personal tumor antigens can facilitate delivery of antigens into lymph nodes and activate antigen-presenting cells (APCs) through the Toll-like receptor 9 (TLR9) signaling pathway, which boosts both innate and adaptive immune response. As an adjuvant platform, hybrid M13 phages can deliver various tumor-specific antigens through simple adsorption to support the current development of personalized vaccines for cancers. Notably, the HMP@Ag vaccine not only prevented the tumors, but also delayed the tumor growth in established (subcutaneous and orthotopic) and metastatic tumor-bearing models while synergy with immune checkpoint blockade (ICB) therapy. Moreover, HMP@Ag triggered a robust neoantigen-based specific immune response in tumor-specific mutation models. In a clinically relevant surgery model, using autologous cell membrane from primary tumors-based HMP@Ag cooperation with ICB dramatically inhibited the post-operation recurrence, and elicited a long-term immune memory effect simultaneously. These findings imply that the M13 phage represents a powerful tool to develop a bio-activated hybrid platform for personalized therapy.

Biomimetic virus-based soft niche for ischemic diseases

The essential therapeutic cues provided by a nanofibrous arginine-glycine-aspartic acid-engineered M13 phage were exploited as extracellular matrix (ECM)-mimicking niches, contributing to de novo soft tissue niche engineering. The interplay of biomimetic phage cues with surrounding organ tissues was identified, and cells were implanted between tissues to achieve an appropriate soft tissue niche that enables the proper functioning of the implanted stem cells at the injured site. With the polyacrylamide (PA) hydrogel mimicking the soft tissue organ stiffness ranges, it was found that biochemical and topological cues in conjunction with the ∼1–2 kPa elastic and mechanical cues of engineered phage nanofibers in soft tissues efficiently enhance the desired response of implanted stem cells. This phage cue with angiogenic and antioxidant functions overcomes the pathological environment to support implanted cells and surrounding soft tissues at the ischemic site, thereby successfully decreasing myogenic degeneration, minimizing fibrosis, and enhancing blood vessel regeneration with M2 macrophage polarization by improving the survival of the implanted endothelial progenitor cells (EPC) in an ischemic mouse model. These biomimetic phage nanofiber cues are considerably supportive of cell therapy, as they establish promising therapeutic extracellular de novo soft tissue niches for curing ischemic diseases.

HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer.

Simple SummaryThe high incidence and death rates of breast cancer make the development of new therapies an urgent need. The introduction into the clinic of the anti-HER2 monoclonal antibody trastuzumab considerably improved the overall survival and time-to-disease progression of patients with HER2-positive breast cancer. However, many patients do not benefit from it because of resistance to therapy. Cancer vaccines, by inducing into the patient an anti-cancer specific immunity, might represent an alternative immunotherapeutic approach, but despite promises, so far no anti-HER2 cancer vaccine has been approved for human use. In this study, we propose therapeutic phage-based vaccines, against HER2 and its aggressive isoform Δ16HER2, able to elicit a protective immunity and potentially capable of preventing relapse in HER2-positive breast cancer patients, even in those who develop trastuzumab resistance.The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient’s immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.

Expanding the chemical diversity of M13 bacteriophage.

Bacteriophage M13 virions are very stable nanoparticles that can be modified by chemical and genetic methods. The capsid proteins can be functionalized in a variety of chemical reactions without loss of particle integrity. In addition, Genetic Code Expansion (GCE) permits the introduction of non-canonical amino acids (ncAAs) into displayed peptides and proteins. The incorporation of ncAAs into phage libraries has led to the discovery of high-affinity binders with low nanomolar dissociation constant (KD) values that can potentially serve as inhibitors. This article reviews how bioconjugation and the incorporation of ncAAs during translation have expanded the chemistry of peptides and proteins displayed by M13 virions for a variety of purposes.

Development of a human antibody that exhibits antagonistic activity toward CC chemokine receptor 7.

BackgroundCC chemokine receptor 7 (CCR7) is a member of G-protein-coupled receptor family and mediates chemotactic migration of immune cells and different cancer cells induced via chemokine (C–C motif) ligand 19 (CCL19) or chemokine (C–C motif) ligand 21 (CCL21). Hence, the identification of blockade antibodies against CCR7 could lead to the development of therapeutics targeting metastatic cancer.MethodsCCR7 was purified and stabilized in its active conformation, and antibodies specific to purified CCR7 were screened from the synthetic M13 phage library displaying humanized scFvs. The in vitro characterization of selected scFvs identified two scFvs that exhibited CCL19-competitive binding to CCR7. IgG4’s harboring selected scFv sequences were characterized for binding activity in CCR7+ cells, inhibitory activity toward CCR7-dependent cAMP attenuation, and the CCL19 or CCL21-dependent migration of CCR7+ cells.ResultsAntibodies specifically binding to purified CCR7 and CCR7+ cells were isolated and characterized. Two antibodies, IgG4(6RG11) and IgG4(72C7), showed ligand-dependent competitive binding to CCR7 with KD values of 40 nM and 50 nM, respectively. Particularly, IgG4(6RG11) showed antagonistic activity against CCR7, whereas both antibodies significantly blocked the ligand-induced migration and invasion activity of CCR7+ cancer cells.ConclusionsTwo antibody clones were successfully identified from a synthetic scFv-displaying phage library using purified recombinant CCR7 as an antigen. Antibodies specifically bound to the surface of CCR7+ cells and blocked CCR7+ cell migration. Particularly, 6RG11 showed antagonist activity against CCR7-dependent cAMP attenuation.

An Efficient UV-C Disinfection Approach and Biological Assessment Strategy for Microphones

Hygiene is a basic necessity to prevent infections, and though it is regarded as vital in general, its importance has been stressed again during the pandemic. Microbes may spread through touch and aerosols and thereby find their way from host to host. Cleaning and disinfection of possibly contaminated surfaces prevents microbial spread, thus reducing potential illnesses. One item that is used by several people in a way that promotes close contact by touch and aerosol formation is the microphone. A microphone is a complex piece of equipment with respect to shape and various materials used to fabricate it and, hence, its disinfection is challenging. A new device has been developed to efficiently sterilize microphones by using UV-C and a biological assessment has been done to identify its efficacy and translatability. For this investigation, a contamination procedure was developed by using M13 bacteriophage as a model to illustrate the effectiveness of the disinfection. The susceptibility to UV-C irradiation of M13 in solution was compared to that of the PR8 H1N1 influenza virus, which has a similar UV-C susceptibility as SARS-CoV-2. It was found that 10 min of UV-C treatment reduced the percentage of infectious M13 by 99.3% based on whole microphone inoculation and disinfection. UV-C susceptibility of M13 and influenza in suspension were found to be very similar, indicating that the microphone sterilization method and device function are highly useful and broadly applicable.

Phage-free production of artificial ssDNA with Escherichia coli.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L-1 of ssDNA in a stirred-tank bioreactor, thus realizing up to a 40-fold increase in maximal product concentration over previous scalable phage-free ssDNA production methods.

Phage Particles of Controlled Length and Genome for In Vivo Targeted Glioblastoma Imaging and Therapeutic Delivery.

M13 bacteriophage (phage) are versatile, genetically tunable nanocarriers that have been recently adapted for use as diagnostic and therapeutic platforms. Applying p3 capsid chlorotoxin fusion with the "inho" circular single-stranded DNA (cssDNA) gene packaging system, we produced miniature chlorotoxin inho (CTX-inho) phage particles with a minimum length of 50 nm that can target intracranial orthotopic patient-derived GBM22 glioblastoma tumors in the brains of mice. Systemically administered indocyanine green conjugated CTX-inho phage accumulated in brain tumors, facilitating shortwave infrared detection. Furthermore, we show that our inho phage can carry cssDNA that are transcriptionally active when delivered to GBM22 glioma cells in vitro. The ability to modulate the capsid display, surface loading, phage length, and cssDNA gene content makes the recombinant M13 phage particle an ideal delivery platform.

A new method for quantitative analysis of M13 bacteriophage by atomic force microscopy

Quantitative analysis is essential for virus research, especially in determining the virus titer. The classical method plaque assay is time-consuming, complex, and difficult for the phages that cannot form apparent plaque on the solid medium. In order to realize rapid and effective detection, a new method combining atomic force microscopy (AFM) observation and mathematical calculation is established. In this research, M13 phages with an appropriate dilution ratio were observed and counted by AFM. Based on the counting results, the titer of M13 phages can be calculated simply through mathematical substitution. Instead of cultivating overnight in plaque assay, this new method can be implemented within a few hours. Moreover, it is a method that can achieve visualization for titer determination and have the potential to determine the phages that fail to form apparent plaque, which is significant in virus quantitative assessment.

Biomaterial actuator of M13 bacteriophage in dynamically tunable plasmonic coupling structure

Nano-gap plasmonic nanostructures like nanoparticle on a metallic film (NPOM) exhibit enhanced near-field properties because of surface plasmon coupling. Utilizing metallic NPs shape, size, material choice, and dielectric film-induced nano-gap (or hot-spots), it is possible to exploit potential applications in sensors. To achieve dynamic sensing, the application of tunable nano-gaps in response to external stimuli has been attempted using biopolymers. M13 bacteriophages are viable candidates as nano-gap materials owing to their widely adjustable physicochemical properties by genetic engineering technology. Here, we demonstrate a collective light-scattering spectrum of the surface-enhanced plasmonic resonance characterized by the surface morphology of the employed M13 bacteriophage thin film. Our NPOM (Ag nanocube/M13 bacteriophage film/Au film) nanostructure revealed differences in experimental and simulated scattering peak wavelength shift when nano-gap size(s) are varied. An AFM (Atomic Force Microscopy) surface morphology analysis of the M13 bacteriophage film showed its non-uniform thickness distribution explaining the differences in scattering peak wavelength properties. We confirmed that the M13 bacteriophage performs well as a sensor based on static and dynamic moisture exposure. Benefitting from the adaptability and sensitivity of the M13 biomaterial, our gap plasmonic coupling structure can be used for sensing VOCs as a biosensor.

Phage‐Derived Oncolytic Viruses with 3C from Seneca Valley Virus for Targeted Therapy of Cervical Cancer

AbstractCancer gene therapy based on various gene delivery vectors has some potential but also has obvious disadvantages. In this study, a new M13 phage‐based oncolytic virus is constructed that carried the RGD peptides to target tumor cells and the 3C gene of Seneca Valley virus (SVV) preceded by a eukaryotic initial transcriptional region (ITR) to transcribe an oncolytic protein to kill tumor cells. Recombinant virus particles of 1200 nm in length are obtained in large quantities by transfecting the recombinant M13 phage plasmid into the host BL2738 and are investigated in vitro in tumor cells and in vivo in tumor‐bearing mice to evaluate their antitumor effect. The experiments using Hela cells confirm that the engineered M13 phage can target and enter Hela cells, and express the SVV 3C protein, resulting in apoptosis of target cells by upregulating the expression of caspase 3. Furthermore, the results of experiments in vivo also show that the recombinant phage significantly inhibits the enhanced tumor volume in nude mice compared to the control groups. The M13 phage may be engineered to fuse with a variety of oncolytic proteins to inhibit the growth of tumor cells in the future, providing a promising phage‐based targeted oncolytic reagent.

Characterization of rabbit polyclonal antibody against camel recombinant nanobodies.

Nanobodies (Nbs) are recombinant single-domain fragments derived from camelids’ heavy-chain antibodies (HCAbs). Nanobodies are increasingly used in numerous biotechnological and medical applications because of their high stability, solubility, and yield. However, one major obstacle prohibiting Nb expansion is the affordability of specific detector antibodies for their final revelation. In this work, the production of a specific anti-Nb antibody as a general detector for camel antibodies, conventional cIgG, and HCAb, and their derived Nbs was sought. Thus, a T7 promoter plasmid was constructed and used to highly express six different Nbs that were used in a successful rabbit immunization. Affinity-purified rabbit anti-Nb rIgG was able to detect immobilized or antigen-bound Nbs via enzyme-linked immunosorbent assay, and its performance was comparable to that of a commercial anti-6× His antibody. Its capacities in dosing impure Nbs, detecting Nbs displayed on M13 phages, and revealing denatured Nbs in immune blotting were all proven. As expected, and because of shared epitopes, rabbit anti-Nb cross-reacted with cIgG, HCAbs, and 6× His-tagged proteins, and the percentage of each fraction within anti-Nb rIgG was determined. Anti-Nb is a promising tool for the checkpoints throughout the recombinant Nb technology.

Alternative use of phage display: phage M13 can remain viable in the intestines of poultry without causing damage

Phage display (PD) is a tool for developing new molecules to control pathogens. Peptides selected by PD are commonly synthesised and tested, but the use of phage M13 displaying the selected peptides as a direct biding in the intestinal tract has not yet been tested. This study evaluated whether phage M13 can remain viable in the chicken gastrointestinal tract and whether it causes injury or humoral immune response. We inoculated phage M13 or E. coli ER2738 (ECR) infected with M13 into birds at different ages. We found the virus in faeces at 5 or 13 days after inoculation, just when it infected the ECR. The presence of phage M13 or ECR did not result in gut injuries and had no impacts on weight gain and bird health. Furthermore, the levels of IgY were similar in all treatments, which indicates that the virus can be used in chicken until 42 days without being recognised by the immune system. This work provides a scientific basis for the use of PD as a tool in numerous applications to control different pathogens.

LUNG CANCER DIAGNOSIS BY DEEP LEARNING-BASED DNA-DERIVED BACTERIOPHAGE NOSE ANALYSIS OF EXHALED GAS

TYPE: Abstract TOPIC: Lung Cancer PURPOSE: Lung cancer has a high mortality rate, however an early diagnosis can contribute to a favourable prognosis. Here, we demonstrate the development of a DNA-derived bacteriophage nose (D2pNose) based on genetically engineered phage to diagnose lung cancer by using deep learning model of exhaled gas. METHODS: Exhaled breath was collected in a controlled way from individuals with lung cancer (n=31) and from healthy subjects (n=31). The D2pNose is an array of phage coated proteins of the M13 bacteriophage generated with 20 types of amino acid. Each 20 type sensors consisted of red-, green-, and blue-colored M13 bacteriophage films. The color change profiles of D2pNose were appeared when various volatile organic compounds are exposed to the same density. In this study, colorimetric dataset of exhaled gas obtained from two groups were randomly shuffled and sequentially divided into training and validation set without any pretreatment steps. Diagnostic performance of dataset was then assessed by using 5-fold cross validation of convolutional neural network model. RESULTS: The deep learning model was trained with D2pNose colorimetric datasets of exhaled gas derived from two groups and could classify them with an accuracy of 75.15% and a classification success rate of 87.1% for lung cancer based on raw human breathing without any pretreatment. CONCLUSIONS: These study results suggest that the combination of D2pNose analysis of exhaled gas and deep learning could be a reliable method for lung cancer screening without any pretreatment. CLINICAL IMPLICATIONS: Analysis of exhaled gas and deep learning method could be a lung cancer screening method. DISCLOSURE: Nothing to declare. KEYWORD: lung cancer

Development of an electrochemical immunosensor for the determination of molinate by using phages labeled with CdS nanocrystals as a novel strategy to signal amplification

Research on immunoassays and immunosensors is currently focused mainly on the development of strategies to amplify the resulting signal. In this sense, the development of new hapten tracers that allow simpler, more sensitive, and more reliable immunoassays is desirable. Therefore, we present the development of an electrochemical immunosensor using viral particles (phages) bound with dozens of CdS nanocrystals (CdS NCs) as an outperforming alternative to conventional competitive hapten conjugates. This allows us to obtain an multiplicator effect in signal obtained. Therefore, as proof of concept, an electrochemical immunosensor was developed for the detection of the molinate in river water samples, based on a noncompetitive immunoassay using recombinant M13 phage particles labeled with CdS NCs (CdS NCs-phage). The phage particles have on its surface peptides that recognize the molinate/antibody immune complex. Molinate determination was performed by square wave anodic stripping voltammetry of Cd2+ release from CdS NCs. The limit of detection and the midpoint sensitivity obtained were 34 pg mL-1 and 0.43 ng mL-1, respectively. In addition, other very good analytical parameters were obtained, where percent coefficient of variation (%CV) between 5 % and 25 %, and recovery percentages close to 99 % were found. Results obtained were compared with those determined by HPLC-UV visible, obtaining an excellent correlation between both methods. This electrochemical immunosensor was successfully used to analyze molinate in river water samples without pre-treatment, obtaining excellent recoveries.

The M13 Phage Assembly Machine Has a Membrane-Spanning Oligomeric Ring Structure.

Bacteriophage M13 assembles its progeny particles in the inner membrane of the host. The major component of the assembly machine is G1p and together with G11p it generates an oligomeric structure with a pore-like inner cavity and an ATP hydrolysing domain. This allows the formation of the phage filament, which assembles multiple copies of the membrane-inserted major coat protein G8p around the extruding single-stranded circular DNA. The phage filament then passes through the G4p secretin that is localized in the outer membrane. Presumably, the inner membrane G1p/G11p and the outer G4p form a common complex. To unravel the structural details of the M13 assembly machine, we purified G1p from infected E. coli cells. The protein was overproduced together with G11p and solubilized from the membrane as a multimeric complex with a size of about 320 kDa. The complex revealed a pore-like structure with an outer diameter of about 12 nm, matching the dimensions of the outer membrane G4p secretin. The function of the M13 assembly machine for phage generation and secretion is discussed.

Three-Dimensional Plasmonic Nanocluster-Driven Light-Matter Interaction for Photoluminescence Enhancement and Picomolar-Level Biosensing.

Plasmonic nanoparticle clusters promise to support unique engineered electromagnetic responses at optical frequencies, realizing a new concept of devices for nanophotonic applications. However, the technological challenges associated with the fabrication of three-dimensional nanoparticle clusters with programmed compositions remain unresolved. Here, we present a novel strategy for realizing heterogeneous structures that enable efficient near-field coupling between the plasmonic modes of gold nanoparticles and various other nanomaterials via a simple three-dimensional coassembly process. Quantum dots embedded in the plasmonic structures display ∼56 meV of a blue shift in the emission spectrum. The decay enhancement factor increases as the total contribution of radiative and nonradiative plasmonic modes increases. Furthermore, we demonstrate an ultracompact diagnostic platform to detect M13 viruses and their mutations from femtoliter volume, sub-100 pM analytes. This platform could pave the way toward an effective diagnosis of diverse pathogens, which is in high demand for handling pandemic situations.

Near-field electrospinning of polymer/phage whispering gallery mode microfiber resonators for label-free biosensing

Whispering gallery mode (WGM) resonators have attracted attention as optical biosensors due to their capacity for rapid label-free detection. Separately, the M13 bacteriophage has been investigated as an alternative biorecognition element. In this work, a novel polymer WGM biosensor functionalized with filamentous virus biocapture agents was fabricated using near-field electrospinning (NFES) in a single step. Polyvinyl alcohol (PVA) was mixed with streptavidin-binding M13 bacteriophage and rhodamine 6 G in aqueous solution for blend electrospinning of active cavity microfiber-based resonators. Confocal fluorescence microscopy revealed alignment of M13 bioreceptors along the fiber axis, while x-ray photoelectron spectroscopy (XPS) and streptavidin-conjugated nanoparticle binding studies indicated that bioactive M13 bacteriophage were on the fiber surface. Dye-doped, crosslinked PVA/M13 fibers supported moderate quality (Q) factor first order WGM resonances within their cross-sections that exhibited a spectral shift in response to streptavidin binding. Composite PVA/M13 microfibers demonstrated specific label-free biosensing of streptavidin with a theoretical limit of detection of 3 nM and a maximum surface coverage of 21 ± 5%. These results demonstrate the potential of NFES to fabricate WGM resonators from PVA/M13 microfibers and use them as a platform for rapid, sensitive label-free sensing.