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Bacteriophages as tools for vaccine and drug development

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.
Expert Rev. Vaccines 4(1), 57 (2005)

Karen Manoutcharian Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, CP 04510, D.F., Mexico karman@servidor.unam.mx

Modern vaccinology is dealing with the most in animals and humans. Perhaps the most reprecomplicated cases of human diseases and veteri- sentative example of vaccine failure is the case of nary medicine, since effective vaccines against the HIV/AIDS vaccine, where enormous efforts pathogens and diseases sensitive to control by the are focused on the development of new adjuimmune system of the host have already been vants, stimulatory and vaccine-enhancing moledeveloped during the last few decades. Although cules, while correct immunogens capable of vaccination is considered as the most radical inducing protective immune responses are not means for preventive or therapeutic interven- yet defined. Thus, there is a clear need for novel tions, including the complete eradication of cer- vaccine generation and delivery approaches based tain pathogens/diseases, the actual general suc- on nonconventional design platforms. Bacteriophages, or phages, are bacterial cess rate in the field of vaccines is low and in sharp contrast with the huge financial and viruses and can be found in water (including human resources invested in vaccine develop- drinking water), soil, plants, animals and ment. There are no effecThe phages are easy to humans. Phage display is a powerful research tool tive vaccines against tubermanage, they resist heat and, perhaps, is the most culosis, leprosy, HIV, technologic hepatitis C virus and most and many organic solvents, innovative parasitic diseases [1]. This is chemical or other stresses development in molecular biology in the last a result of the gap between and, importantly, the 10 years. This simple vaccine development particles are highly efforts and detailed and immunogenic and do not methodology relies on expression of fusion pepsystematic knowledge require adjuvant. tides or proteins on the regarding the complex network of interactions of the immune system with bacteriophage surface, while the DNA encoding pathogens. Moreover, there are undoubtedly them is packaged into the fusion-displaying many yet unknown components of the immune phage genome. The most frequently used dissystem involved in protection, which are not play systems are based on M13 filamentous considered for vaccine strategies. Consequently, a phage (more recently also phage was used), purely empirical approach dominates in modern which permits the generation of very large ranvaccine development strategies, converting the dom peptides, antibody fragments (scFv and field of vaccine study into a crossroad of endless Fab), cDNA and genomic DNA phage-dismodels, preclinical and clinical experimentations played libraries with the complexities of up

10.1586/14760584.4.1.5

2005 Future Drugs Ltd

ISSN 1476-0584

Manoutcharian

to 1011, as well as the display of functional protein domains such by further purifying phage preparations to diminish toxin levels, as enzymes, hormones and DNA-, RNA- or any other ligand- respectively. Interestingly, in a recent study describing minimal binding molecules [2]. An integral part of this technology are suc- toxicity from administration of a phage-random peptide library cessive rounds of selection so-called biopanning permitting in mice, there is a statement that based on these preclinical data the isolation of high-affinity target-specific peptides/proteins. For the US Food and Drug Administration has approved the impleexample, phage display allowed the generation of monoclonal mentation of human clinical trials with this technique [8], scFv and Fab antibody fragments with greater than natural affin- although the author could not find official confirmation of it. ity of fentomolar range against various ligands and, hence, these Conversely, the data regarding the first human filamentous phage kinds of molecules are the most rapidly expanding class of drugs vaccine trial with a small group of multiple myeloma patients can for the treatment of human diseases. The mapping of antigenic be found on the APALEXO Biotechnologie GmbH website [101] determinants recognized by monoclonal or polyclonal disease- or and demonstrates that phage vaccination can induce tumor-spepathogen-specific antibodies using phage display is the only cific immune responses with the potential to exert beneficial experimental tool permitting the identification of linear mimo- effects on patients. In general, many phage companies plan to topes of conformational epitopes. The phages are easy to man- commercialize phage products for agricultural applications, age, they resist heat and many organic solvents, chemical or other where regulations are less stringent. stresses and, importantly, the particles are highly immunogenic There are numerous reports regarding the application of and do not require adjuvant. Furthermore, as particulate anti- phages as immunogens, vaccines or therapeutic agents in gens, phage can access both major histocompatibility complex small animal models or in veterinary medicine [4]. The only class I and II pathways, and are thus capable of inducing humoral reported recombinant bacteriophage-based vaccine for nonand cellular immune responses. Both lytic and filamentous bacte- model animals was developed by the authors research team riophages have been used in vaccine and drug development in and was used to vaccinate pigs against cysticercosis caused different ways, ranging from identification of organ/tissue-target- by Taenia solium, the causant of neurocysticercosis in ing peptides by phage display to the application of phages as vac- humans and a common parasitic disease of the CNS worldcine carriers. Thus, in vivo biopanning with a filamentous phage- wide [9]. Large-scale field vaccination trials in pigs are underdisplay peptide library, carried out first in mice then in a cancer way, with promising preliminary data. The lytic phages are patient (a B-cell malignancy), resulted in identification of pep- promising candidates for agricultural applications, such as tide motifs that localized to different food processing and in livestock as While there is no guarantee that antibacterial agents [7]. organs; this may have broad implicaphage display will resolve all tions for the development of targeted There are other not yet wellproblems in vaccine and drug explored points in the vaccine field therapies [3]. In the study, disconnection of the patient from a life-support development, it is offering, at least where phages can make an important contribution such as: in the system followed short-term intravequalitatively, new tactics and nous infusion of the phage library into strategies for vaccine discovery generation of small molecules from landscape phage libraries as substithe patient and multiple representative and development. tute antibodies [10]; delivery of antitissue biopsies were carried out. The effectiveness of bacteriophages as DNA vaccine delivery vehicles cocaine scFv displayed on filamentous phages to the CNS of was demonstrated using either filamentous phage [4] or more rodents [11], which offers a novel approach for the treatment of recently, phage [5]. Although there is a single published report neurodegenerative disorders such as Alzheimers and Parkindescribing immunization of HIV-infected patients with sons diseases in humans; genome-scale epitope-screening phage phiX174 for the evaluation of lymphocyte function in vivo approaches and small molecule-based drug discovery applying combinatorial random peptide or gene/genome-based phage [6], the use of lytic bacteriophages discovered over 85 years ago as antibacterial therapeutic agents in humans was common in sev- libraries. The diversity of applications and the success of eral European countries and in the USA for decades before the phage display systems are due to its simplicity and flexibility, antibiotic era [7]. Due to the increasing prevalence of antibiotic- along with the possibilities of very cheap large-scale producresistant microbes worldwide, there is an interest and an urgent tion of phage particles by recovering them from infected bacneed for the development of antibacterial phages; today, several terial culture supernatants as nearly 100% homogenous prepacompanies are working with naturally occurring as well as geneti- rations free of cellular components. The cost-effectiveness is cally modified phage to combat drug-resistant bacteria [7]. Due to an important issue, since the cost of vaccine/drug developconcerns that mass lysis of phages can be a problem for phage ment now exceeds US$800 million, which leaves the majority medicine (the issue is under debate), phages carrying molecules of vaccine developers simply out of the race and thus the that kill the bacteria without lysis have been developed. Con- phage technology offers some alternatives in this regard. While there is no guarantee that phage display will resolve versely, some drawbacks in the application of phages in vivo, such as rapid removal of the phages from the body and the presence of all problems in vaccine and drug development, it is offering, toxins in phage preparations can be overcome by isolating long- at least qualitatively, new tactics and strategies for vaccine circulating variants of phage using a serial passage technique and discovery and development.

Expert Rev. Vaccines 4(1), (2005)

Bacteriophages as tools for vaccine & drug development

References
1

Zinkernagel RM. Immunity, immunopathology and vaccines against HIV? Vaccine 20, 19131917 (2002). Benhar I. Biotechnological applications of phage and cell display. Biotechnol. Adv. 19, 133 (2004). Arap W, Kolonin MG, Trepel M et al. Steps toward mapping the human vasculature by phage display. Nature Med. 8(2), 121127 (2002). Manoutcharian K, Gevorkian G, Cano A, Almagro JC. Phage-displayed biomolecules as preventive and therapeutic agents. Curr. Pharmac. Biotech. 2, 217223 (2001). Clark JR, March JB. Bacterial viruses as human vaccines? Expert Rev. Vaccines 3(4), 463476 (2004).
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Fogelman I, Davey V, Ochs HD et al. Evaluation of CD4+ T-cell function in vivo in HIV-infected patients as measured by bacteriophage phi 174 immunization. J. Infect. Dis. 182, 435441 (2000). Thiel K. Old dogma, new tricks 21st century phage therapy. Nature Biotech. 22(1), 3136 (2004). Krag DN, Fuller SP, Oligino L et al. Phagedisplayed random peptide libraries in mice: toxicity after serial panning. Cancer Chemother. Pharmacol. 50(4), 325332 (2002). Manoutcharian K, Daz-Orea A, Gevorkian G et al. Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis. Vet. Immunol. Immunopath. 99, 1124 (2004).

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Petrenko VA, Smith GP. Phage from landscape libraries as substitute antibodies. Prot. Engin. 13(8), 589592 (2000). Carrera MRA, Kaufmann GF, Mee JM et al. Treating cocaine with viruses. Proc. Natl Acad. Sci. USA 101(28), 1041610421 (2004).

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Website
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APALEXO Biotechnologie GmbH www.apalexo.com (Accessed January, 2005)

Affiliation
Karen Manoutcharian Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico CP 04510, D.F. karman@servidor.unam.mx

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