Jessica Nicastro

Bacteriophage lambda display systems: developments and applications

Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique formation of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future applications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control.

Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.

Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage

As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub‐populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins.

Lambda phage nanoparticles displaying HER2-derived E75 peptide induce effective E75-CD8+ T response

We have investigated the in vitro immunogenicity and in vivo prophylactic and therapeutic potential of lambda (λ) phage particles displaying the E75 peptide (derived from HER2 protein) in an implantable TUBO breast tumor model of BALB/c mice. The mice were immunized with the E75-displaying phage (λF7-gpD::E75) every 2-week intervals over a 6-week period, and the generated immune responses were studied. Results showed in vitro induction of immune responses by the λF7 (gpD::E75) construct compared to the control λF7 and buffer groups. In the in vivo prophylactic study, all the control and vaccinated mice groups developed tumors. However, in the therapeutic experiments, we observed a significant difference in tumor size at days 14–36 for mice immunized with λF7 (gpD::E75) compared to control groups (P < 0.05). Moreover, the survival time prolonged in mice immunized with λF7 (gpD::E75). The discrepancy between the results obtained from the in vitro and in vivo studies may have been a result of the induction of Foxp3 CD4+CD25+ which has been previously reported to hamper effective T cell functionality. In conclusion, we observed a significant immune stimulatory response in the in vitro study, while in vivo, the vaccine was not able to exert significant tumor inhibitory effects. We suggest that the presence of Foxp3+ CD4+CD25+ cells may have impaired the anti-tumor response in mice challenged in vivo with the TUBO xenograft tumor.

Specific Systems for Imaging

Microscopy allows for the characterization of small objects invisible to the naked eye, a technique that, since its conception, has played a key role in the development across nearly every field of science and technology. Given the nanometer size of the materials explored in the field of nanotechnology, the contributions of modern microscopes that can visualize these materials are indispensable, and the ever-improving technology is paramount to the future success of the field. This chapter will focus on four fundamental areas of microscopy used in the field of nanotechnology including fluorescence microscopy (Sect. 3.1), particle tracking and photoactivated localization microscopy (Sect. 3.2), quantum dots and fluorescence resonance energy transfer (Sect. 3.3), and cellular MRI and PET labeling (Sect. 3.4). The functionality, as well as the current and recommended usage of each given imaging system, will be discussed.

The viral approach to breast cancer immunotherapy: ARAB et al.

Despite years of intensive research, breast cancer remains the leading cause of death in women worldwide. New technologies including oncolytic virus therapies, virus, and phage display are among the most powerful and advanced methods that have emerged in recent years with potential applications in cancer prevention and treatment. Oncolytic virus therapy is an interesting strategy for cancer treatment. Presently, a number of viruses from different virus families are under laboratory and clinical investigation as oncolytic therapeutics. Oncolytic viruses (OVs) have been shown to be able to induce and initiate a systemic antitumor immune response. The possibility of application of a multimodal therapy using a combination of the OV therapy with immune checkpoint inhibitors and cancer antigen vaccination holds a great promise in the future of cancer immunotherapy. Display of immunologic peptides on bacterial viruses (bacteriophages) is also increasingly being considered as a new and strong cancer vaccine delivery strategy. In phage display immunotherapy, a peptide or protein antigen is presented by genetic fusions to the phage coat proteins, and the phage construct formulation acts as a protective or preventive vaccine against cancer. In our laboratory, we have recently tested a few peptides (E75, AE37, and GP2) derived from HER2/neu proto‐oncogene as vaccine delivery modalities for the treatment of TUBO breast cancer xenograft tumors of BALB/c mice. Here, in this paper, we discuss the latest advancements in the applications of OVs and bacterial viruses display systems as new and advanced modalities in cancer immune therapeutics.

Phage-based nanomedicines as new immune therapeutic agents for breast cancer

Despite years of investigation, breast cancer remains a major cause of death worldwide. Phage display is a powerful molecular method in which peptide and protein libraries can be displayed via genetic fusions on the surface of phages. This approach has tremendous potential for biomedical applications and has already facilitated the discovery of specific antibodies, specific antigens, and peptides with potential roles in the diagnosis and treatment of malignancies including breast cancer. In this review, we discuss the new and the latest advancements in the applications of the phage display technique in the provision of immune therapeutics for breast cancer.

Phage-Mediated Immunomodulation

While the natural hosts for bacteriophages are bacteria, there is growing evidence for the ability of phage to interact with mammalian cells, particularly with those of the human immune system. These interactions typically encompass two main features: (i) phage immunogenicity, or ability of phages to induce specific immune responses; and (ii) phage immunomodulation, which can be defined as the ability of phages to modify the immune system in both innate and adaptive responses. The aim of this chapter is to explore the interactions between phages and the immune system, and more specifically the implications of these interactions in the development of novel medical applications.

Phage Device Coatings

The presence of biofilms and their associated antimicrobial resistance provides a challenge to various industries where new and affective device coating strategies are required. Bacteriophages have the natural capacity to act as antibacterials and have been used extensively for this purpose, including in device coatings, since the beginning of the 20th century. This Chapter explores the emerging industry of phage-coated medical devices. An extensive review on the biology and challenges behind biofilm formations, including the contributors to biofilm resistance and current antimicrobial strategies will be covered. Alternative medical device coating strategies will also be explored, including the benefits, challenges, and promise of phage-based device coatings as bioactive agents.

Bacteriophages Functionalized for Gene Delivery and the Targeting of Gene Networks

Bacteriophages (phages) offer many potential and existing applications to biotechnology, including their modification and use as protein/gene carriers. Phages possess many intrinsic physicochemical attributes that make them excellent candidates for use in gene therapy. In this chapter we will explore how phages have been employed in gene delivery as well as their future utility in this exciting medical application.