Editorial

Karen ManoutcharianInstituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, CP 04510, D.F., Mexicokarman@servidor.unam.mx

10.1586/14760584.4.1.5 © 2005 Future Drugs Ltd ISSN 1476-0584 5

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), 5–7 (2005)

Modern vaccinology is dealing with the mostcomplicated cases of human diseases and veteri-nary medicine, since effective vaccines againstpathogens and diseases sensitive to control by theimmune system of the host have already beendeveloped during the last few decades. Althoughvaccination is considered as the most radicalmeans for preventive or therapeutic interven-tions, including the complete eradication of cer-tain pathogens/diseases, the actual general suc-cess rate in the field of vaccines is low and insharp contrast with the huge financial andhuman resources invested in vaccine develop-ment. There are no effec-tive vaccines against tuber-culosis, leprosy, HIV,hepatitis C virus and mostparasitic diseases [1]. This isa result of the gap betweenvaccine developmentefforts and detailed andsystematic knowledgeregarding the complex net-work of interactions of the immune system withpathogens. Moreover, there are undoubtedlymany yet unknown components of the immunesystem involved in protection, which are notconsidered for vaccine strategies. Consequently, apurely empirical approach dominates in modernvaccine development strategies, converting thefield of vaccine study into a crossroad of endlessmodels, preclinical and clinical experimentations

in animals and humans. Perhaps the most repre-sentative example of vaccine failure is the case ofthe HIV/AIDS vaccine, where enormous effortsare focused on the development of new adju-vants, stimulatory and vaccine-enhancing mole-cules, while correct immunogens capable ofinducing protective immune responses are notyet defined. Thus, there is a clear need for novelvaccine generation and delivery approaches basedon nonconventional design platforms.

Bacteriophages, or phages, are bacterialviruses and can be found in water (includingdrinking water), soil, plants, animals and

humans. Phage display is apowerful research tooland, perhaps, is the mostinnovative technologicdevelopment in molecularbiology in the last10 years. This simplemethodology relies onexpression of fusion pep-tides or proteins on the

bacteriophage surface, while the DNA encodingthem is packaged into the fusion-displayingphage genome. The most frequently used dis-play systems are based on M13 filamentousphage (more recently also phage λ was used),which permits the generation of very large ran-dom peptides, antibody fragments (scFv andFab), cDNA and genomic DNA phage-dis-played libraries with the complexities of up

‘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.’

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Manoutcharian

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

to 1011, as well as the display of functional protein domains suchas enzymes, hormones and DNA-, RNA- or any other ligand-binding molecules [2]. An integral part of this technology are suc-cessive rounds of selection – so-called biopanning – permittingthe isolation of high-affinity target-specific peptides/proteins. Forexample, phage display allowed the generation of monoclonalscFv and Fab antibody fragments with greater than natural affin-ity of fentomolar range against various ligands and, hence, thesekinds of molecules are the most rapidly expanding class of drugsfor the treatment of human diseases. The mapping of antigenicdeterminants recognized by monoclonal or polyclonal disease- orpathogen-specific antibodies using phage display is the onlyexperimental tool permitting the identification of linear mimo-topes of conformational epitopes. The phages are easy to man-age, they resist heat and many organic solvents, chemical or otherstresses and, importantly, the particles are highly immunogenicand do not require adjuvant. Furthermore, as particulate anti-gens, phage can access both major histocompatibility complexclass I and II pathways, and are thus capable of inducing humoraland cellular immune responses. Both lytic and filamentous bacte-riophages have been used in vaccine and drug development indifferent ways, ranging from identification of organ/tissue-target-ing peptides by phage display to the application of phages as vac-cine carriers. Thus, in vivo biopanning with a filamentous phage-display peptide library, carried out first in mice then in a cancerpatient (a B-cell malignancy), resulted in identification of pep-tide motifs that localized to differentorgans; this may have broad implica-tions for the development of targetedtherapies [3]. In the study, disconnec-tion of the patient from a life-supportsystem followed short-term intrave-nous infusion of the phage library intothe patient and multiple representativetissue biopsies were carried out. Theeffectiveness of bacteriophages as DNA vaccine delivery vehicleswas demonstrated using either filamentous phage [4] or morerecently, phage λ [5]. Although there is a single published reportdescribing immunization of HIV-infected patients withphage phiX174 for the evaluation of lymphocyte function in vivo[6], the use of lytic bacteriophages discovered over 85 years ago asantibacterial therapeutic agents in humans was common in sev-eral European countries and in the USA for decades before theantibiotic era [7]. Due to the increasing prevalence of antibiotic-resistant microbes worldwide, there is an interest and an urgentneed for the development of antibacterial phages; today, severalcompanies are working with naturally occurring as well as geneti-cally modified phage to combat drug-resistant bacteria [7]. Due toconcerns that mass lysis of phages can be a problem for phagemedicine (the issue is under debate), phages carrying moleculesthat kill the bacteria without lysis have been developed. Con-versely, some drawbacks in the application of phages in vivo, suchas rapid removal of the phages from the body and the presence oftoxins 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 minimaltoxicity from administration of a phage-random peptide libraryin mice, there is a statement that based on these preclinical datathe US Food and Drug Administration has approved the imple-mentation 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 phagevaccine trial with a small group of multiple myeloma patients canbe found on the APALEXO Biotechnologie GmbH website [101]

and demonstrates that phage vaccination can induce tumor-spe-cific immune responses with the potential to exert beneficialeffects on patients. In general, many phage companies plan tocommercialize phage products for agricultural applications,where regulations are less stringent.

There are numerous reports regarding the application ofphages as immunogens, vaccines or therapeutic agents insmall animal models or in veterinary medicine [4]. The onlyreported recombinant bacteriophage-based vaccine for non-model animals was developed by the author’s research teamand was used to vaccinate pigs against cysticercosis causedby Taenia solium, the causant of neurocysticercosis inhumans and a common parasitic disease of the CNS world-wide [9]. Large-scale field vaccination trials in pigs are under-way, with promising preliminary data. The lytic phages arepromising candidates for agricultural applications, such as

food processing and in livestock asantibacterial agents [7].

There are other not yet well-explored points in the vaccine fieldwhere phages can make an impor-tant contribution such as: in thegeneration of small molecules fromlandscape phage libraries as substi-tute antibodies [10]; delivery of anti-

cocaine scFv displayed on filamentous phages to the CNS ofrodents [11], which offers a novel approach for the treatment ofneurodegenerative disorders such as Alzheimer’s and Parkin-son’s diseases in humans; genome-scale epitope-screeningapproaches and small molecule-based drug discovery applyingcombinatorial random peptide or gene/genome-based phagelibraries. The diversity of applications and the success ofphage display systems are due to it’s simplicity and flexibility,along with the possibilities of very cheap large-scale produc-tion of phage particles by recovering them from infected bac-terial culture supernatants as nearly 100% homogenous prepa-rations free of cellular components. The cost-effectiveness isan important issue, since the cost of vaccine/drug develop-ment now exceeds US$800 million, which leaves the majorityof vaccine developers simply out of the race and thus thephage technology offers some alternatives in this regard.

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

‘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.’

Bacteriophages as tools for vaccine & drug development

www.future-drugs.com 7

References

1 Zinkernagel RM. Immunity, immunopathology and vaccines against HIV? Vaccine 20, 1913–1917 (2002).

2 Benhar I. Biotechnological applications of phage and cell display. Biotechnol. Adv. 19, 1–33 (2004).

3 Arap W, Kolonin MG, Trepel M et al. Steps toward mapping the human vasculature by phage display. Nature Med. 8(2), 121–127 (2002).

4 Manoutcharian K, Gevorkian G, Cano A, Almagro JC. Phage-displayed biomolecules as preventive and therapeutic agents. Curr. Pharmac. Biotech. 2, 217–223 (2001).

5 Clark JR, March JB. Bacterial viruses as human vaccines? Expert Rev. Vaccines 3(4), 463–476 (2004).

6 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, 435–441 (2000).

7 Thiel K. Old dogma, new tricks – 21st century phage therapy. Nature Biotech. 22(1), 31–36 (2004).

8 Krag DN, Fuller SP, Oligino L et al. Phage-displayed random peptide libraries in mice: toxicity after serial panning. Cancer Chemother. Pharmacol. 50(4), 325–332 (2002).

9 Manoutcharian K, Díaz-Orea A, Gevorkian G et al. Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis. Vet. Immunol. Immunopath. 99, 11–24 (2004).

10 Petrenko VA, Smith GP. Phage from landscape libraries as substitute antibodies. Prot. Engin. 13(8), 589–592 (2000).

11 Carrera MRA, Kaufmann GF, Mee JM et al. Treating cocaine with viruses. Proc. Natl Acad. Sci. USA 101(28), 10416–10421 (2004).

Website

101 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