Bacteriophages as tools for vaccine and drug development

Modern vaccinology is dealing with the most complicated cases of human diseases and veterinary medicine, since effective vaccines against pathogens and diseases sensitive to control by the immune system of the host have already been developed during the last few decades. Although vaccination is considered as the most radical means for preventive or therapeutic interventions, including the complete eradication of certain pathogens/diseases, the actual general success rate in the field of vaccines is low and in sharp contrast with the huge financial and human resources invested in vaccine development. There are no effective vaccines against tuberculosis, leprosy, HIV, hepatitis C virus and most parasitic diseases [1]. This is a result of the gap between vaccine development efforts and detailed and systematic knowledge regarding the complex network of interactions of the immune system with pathogens. Moreover, there are undoubtedly many yet unknown components of the immune system involved in protection, which are not considered for vaccine strategies. Consequently, a purely empirical approach dominates in modern vaccine development strategies, converting the field of vaccine study into a crossroad of endless models, preclinical and clinical experimentations in animals and humans. Perhaps the most representative example of vaccine failure is the case of the HIV/AIDS vaccine, where enormous efforts are focused on the development of new adjuvants, stimulatory and vaccine-enhancing molecules, while correct immunogens capable of inducing protective immune responses are not yet defined. Thus, there is a clear need for novel vaccine generation and delivery approaches based on nonconventional design platforms.

Bacteriophages, or phages, are bacterial viruses and can be found in water (including drinking water), soil, plants, animals and humans. Phage display is a powerful research tool and, perhaps, is the most innovative technologic development in molecular biology in the last 10 years. This simple methodology relies on expression of fusion peptides or proteins on the bacteriophage surface, while the DNA encoding them is packaged into the fusion-displaying phage genome. The most frequently used display systems are based on M13 filamentous phage (more recently also phage λ was used), which permits the generation of very large random peptides, antibody fragments (scFv and Fab), cDNA and genomic DNA phage-displayed libraries with the complexities of up to 10¹¹, as well as the display of functional protein domains such as enzymes, hormones and DNA-, RNA- or any other ligand-binding molecules [2]. An integral part of this technology are successive rounds of selection – so-called biopanning – permitting the isolation of high-affinity target-specific peptides/proteins. For example, phage display allowed the generation of monoclonal scFv and Fab antibody fragments with greater than natural affinity of fentomolar range against various ligands and, hence, these kinds of molecules are the most rapidly expanding class of drugs for the treatment of human diseases. The mapping of antigenic determinants recognized by monoclonal or polyclonal disease- or pathogen-specific antibodies using phage display is the only experimental tool permitting the identification of linear mimotopes of conformational epitopes. The phages are easy to manage, they resist heat and many organic solvents, chemical or other stresses and, importantly, the particles are highly immunogenic and do not require adjuvant. Furthermore, as particulate antigens, phage can access both major histocompatibility complex class I and II pathways, and are thus capable of inducing humoral and cellular immune responses. Both lytic and filamentous bacteriophages have been used in vaccine and drug development in different ways, ranging from identification of organ/tissue-targeting peptides by phage display to the application of phages as vaccine carriers. Thus, in vivo biopanning with a filamentous phage-display peptide library, carried out first in mice then in a cancer patient (a B-cell malignancy), resulted in identification of peptide motifs that localized to different organs; this may have broad implications for the development of targeted therapies [3]. In the study, disconnection of the patient from a life-support system followed short-term intravenous infusion of the phage library into the patient and multiple representative tissue biopsies were carried out. The effectiveness of bacteriophages as DNA vaccine delivery vehicles was demonstrated using either filamentous phage [4] or more recently, phage λ [5]. Although there is a single published report describing immunization of HIV-infected patients with phage phiX174 for the evaluation of lymphocyte function in vivo[6], the use of lytic bacteriophages discovered over 85 years ago as antibacterial therapeutic agents in humans was common in several European countries and in the USA for decades before the antibiotic era [7]. Due to the increasing prevalence of antibiotic-resistant microbes worldwide, there is an interest and an urgent need for the development of antibacterial phages; today, several companies are working with naturally occurring as well as genetically modified phage to combat drug-resistant bacteria [7]. Due to concerns that mass lysis of phages can be a problem for phage medicine (the issue is under debate), phages carrying molecules that kill the bacteria without lysis have been developed. Conversely, some drawbacks in the application of phages in vivo, such as rapid removal of the phages from the body and the presence of toxins in phage preparations can be overcome by isolating long-circulating variants of phage using a serial passage technique and by further purifying phage preparations to diminish toxin levels, respectively. Interestingly, in a recent study describing minimal toxicity from administration of a phage-random peptide library in mice, there is a statement that based on these preclinical data the US Food and Drug Administration has approved the implementation of human clinical trials with this technique [8], although the author could not find official confirmation of it. Conversely, the data regarding the first human filamentous phage vaccine trial with a small group of multiple myeloma patients can be found on the APALEXO Biotechnologie GmbH website [101] and demonstrates that phage vaccination can induce tumor-specific immune responses with the potential to exert beneficial effects on patients. In general, many phage companies plan to commercialize phage products for agricultural applications, where regulations are less stringent.

There are numerous reports regarding the application of phages as immunogens, vaccines or therapeutic agents in small animal models or in veterinary medicine [4]. The only reported recombinant bacteriophage-based vaccine for nonmodel animals was developed by the author’s research team and was used to vaccinate pigs against cysticercosis caused by Taenia solium, the causant of neurocysticercosis in humans and a common parasitic disease of the CNS worldwide [9]. Large-scale field vaccination trials in pigs are underway, with promising preliminary data. The lytic phages are promising candidates for agricultural applications, such as food processing and in livestock as antibacterial agents [7].

There are other not yet well- explored points in the vaccine field where phages can make an important contribution such as: in the generation of small molecules from landscape phage libraries as substitute antibodies [10]; delivery of anti-cocaine scFv displayed on filamentous phages to the CNS of rodents [11], which offers a novel approach for the treatment of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases in humans; genome-scale epitope-screening approaches and small molecule-based drug discovery applying combinatorial random peptide or gene/genome-based phage libraries. The diversity of applications and the success of phage display systems are due to it’s simplicity and flexibility, along with the possibilities of very cheap large-scale production of phage particles by recovering them from infected bacterial culture supernatants as nearly 100% homogenous preparations free of cellular components. The cost-effectiveness is an important issue, since the cost of vaccine/drug development now exceeds US$800 million, which leaves the majority of vaccine developers simply out of the race and thus the phage technology offers some alternatives in this regard.

While there is no guarantee that phage display will resolve all problems in vaccine and drug development, it is offering, at least qualitatively, new tactics and strategies for vaccine discovery and development.