therapeutic and prophylactic applications of bacteriophage

Therapeutic and Prophylactic Applicationsof Bacteriophage Components in ModernMedicine

Sankar Adhya1, Carl R. Merril2, and Biswajit Biswas3

1National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208922National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 208923Department of Genomics, Biological Defense Research Directorate, Naval Medical Research Center,Fort Detrick, Maryland 21702

Correspondence: [email protected]

As the interactions of phage with mammalian innate and adaptive immune systems are betterdelineated and with our ability to recognize and eliminate toxins and other potentiallyharmful phage gene products, the potential of phage therapies is now being realized. Earlyefforts to use phage therapeutically were hampered by inadequate phage purification andlimited knowledge of phage–bacterial and phage–human relations. However, although useof phage as an antibacterial therapy in countries that require controlled clinical studies hasbeen hampered by the high costs of patient trials, their use as vaccines and the use of phagecomponents such as lysolytic enzymes or lysozymes has progressed to the point of commer-cial applications. Recent studies concerning the intimate associations between mammalianhosts and bacterial and phage microbiomes should hasten this progress.

The human superorganism is made up ofeukaryotic and prokaryotic cells, in which

the prokaryotic cells far outnumber the eukary-otic cells. Given our current limited under-standing of ourselves as super organisms it isremarkable that we have achieved successes, al-beit limited, in developing therapies for infec-tious diseases. This was especially true in the19th and 20th centuries when specific strainsof bacteria were first being discovered and insome cases associated with animal or humandisease states.

The therapeutic advances realized at thattime were based on limited observations and

heroic empirical efforts, such as the discoveryof the antisyphilitic compound Salvarsan or 606(in reference to the hundreds of compoundsscreened) by S. Hata in Paul Ehrlich’s laboratoryin the early 1900s (Sneade 2005).

Given the state of knowledge at the begin-ning of the 20th century concerning microbiol-ogy and the immune defense mechanisms, thediscovery of bacteriophage (phage) viruses thatkill specific strains of bacteria was welcomed.However, soon after their discovery a numberof issues arose, including a controversy as towhether the bacteriophages were self-replicatingparticles or a lytic enzymatic activity activated

Editors: Pascale Cossart and Stanley Maloy

Additional Perspectives on Bacterial Pathogenesis available at www.perspectivesinmedicine.org

Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a012518

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in the bacteria. One of the pioneers associatedwith their discovery, Felix d’Herelle, consultedwith Albert Einstein who reassured d’Herellethat his experiments designed to prove thatthey were self-replicating particles were valid(Summers 1999). Perhaps more serious werethe problems that arose owing to a lack of appre-ciation of the narrow host range of most phagestrains, so that a phage isolated from an infectedpatient did not appear to be effective in treatinganother patient with a seemingly similar infec-tion. In addition, there was a lack of knowledgeof bacterial toxins and methods to purify phagepreparations so that they were not contami-nated with such toxins. These problems cou-pled with inadequately designed animal andclinical experiments were some of the factorsthat led, in the United States, to the developmentof a government agency to oversee therapeuticdevelopments and claims (Merril et al. 2003,2006).

In addition to these early problems, the dis-covery of the broad host range antibiotics—be-ginning with penicillin—more than 85 yearsago, resulted in the virtual abandonment of ef-forts to continue in the development of thephage as a therapeutic antibacterial agent in theUnited States. However, the increasing incidenceof antibiotic-resistant bacterial strains, particu-larly the bacterial strains commonly found inhospital settings, has prompted a reexaminationof the potentials of phage antibacterial therapies.In addition, there is now an increased awarenessofclinical problems associatedwith theextendedbacterial host range of most commonly used an-tibiotics. Although an extended bacterial hostrange was one of the criteria used in searchingforclinically relevant antibiotics, as this propertypartially alleviated the need to accurately iden-tify infecting bacterial species before therapywas initiated, it is now recognized that suchextended host range antibiotics often result incollateral damage to the natural human micro-biome (Jernberg et al. 2010). Even before themedical community recognized the extent ofthis problem, it was clear that individuals treatedsuccessfully for one species of infectious agentwould often succumb to an infection by anothersecondary foreign bacterial strain.

As most phage strains have a narrow bacte-rial host range, they are expected to cause min-imal perturbations of the normal human mi-crobiome when they are used to treat bacterialinfections. Also, as their mechanisms of anti-bacterial activity of phage are not related tothe mode of action of the antibiotic therapies,it should be possible to find effective phagetherapies for the antibiotic-resistant disease-as-sociated bacterial strains.

In addition our ability to develop moleculartools that permit manipulations of phage ge-nomes and our knowledge of the microbiologyand physiology of phage along with someknowledge of their interactions with the humanimmune system, they are beginning to be adapt-ed to serve as vaccines. The phage strains arealso being used to produce antibacterial pro-teins that are encoded in their genomes. By ma-nipulating the promoters for these genes, it ispossible to produce antibacterial proteins suchas the phage-based lysozymes in quantity. Theclinical applications of phage, including the de-velopment of phage-based vaccines and phageproducts are now beginning to enter commer-cial development.

PHAGE ANTIBACTERIAL THERAPY

One of the discoverers of phage, Felix d’Herelle,clearly recognized the clinical potential of phageas an antibacterial agent (Summers 1999). Theability of phage to kill bacteria and in additionto replicate exponentially suggests some of thepotential advantages of phage antibacterialtherapy. Although d’Herelle’s reported success-es in his first clinical applications of antibacte-rial phage therapies, others had difficultieswhen they tried to scale up the therapies tolarger and/or different patient populations(Merril et al. 2003, 2006). Despite these prob-lems, numerous phage strains were isolatedand used clinically in Europe, the Middle East,and Asia (Alisky et al. 1998; Sulakvelidze et al.2001). Unfortunately, most of the phage data inthese studies are presented in a qualitative man-ner with inadequate clinical details and con-trols.

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Potential Problems for Use of Phage asAntibacterial Therapy

Oral and topical antibacterial applications ofphage should present few problems other thanan occasional inflammation owing to an im-mune response or a reaction to bacterial by-products that have not been sufficiently reducedin concentration during the phage preparation.However, system applications require greatercare in reducing bacterial contaminations in-cluding endo and exotoxins. In addition, phagepharmacokinetic data is rudimentary at best, asmany phage strains have not been administeredsystemically to animals and almost none havebeen studied carefully in humans. Animal stud-ies have clearly shown that most phage are takenup by the liver with some trapping by the spleen(Merril et al. 2003). In some cases, it has beenpossible to select phage strains that can remainin the circulation, and remain available for inter-action with possible systemic bacterial infectiousagents by genetic selection methods (Merril et al.1996; Vitiello et al. 2005). In a similar manner itshould be possible to select phage strains thathave a reduced immunogenicity. Reduced im-munogenicity, to minimize adverse reactionsto phage therapy, may be also accomplished bydeveloping phage strains displaying epitopesthat match surface proteins on normal humanblood cells. In this regard it might be useful todevelop a “platform” phage that is free of toxinand antibiotic resistance genes, but with the abil-ity to stay in the circulation longer, and withreduced immunogenicity. Such a platformphage could be engineered so that it could beeasily modified forenhancementof itshost rangefor specific bacterial strains. For example, thiscould be accomplished with a phage in whichthe tail proteins could be specified in a mannersimilar to that found in coliphage K1-5. Thisphage is a “dual” specificity phage that encodestwo different tail proteins allowing it to attackand replicate on both K1 and K5 strains of Es-cherichia coli (Scholl et al. 2001). Alternativelyone could use a site-specific recombination sys-tem that permits the phage to switch betweenalternative tail fiber proteins, or by using a re-verse transcriptase controlled tropism switch to

generate alternate tail fibers (Sandmeier 1994;Liu et al. 2002). Phage strains considered fortherapeutic purposes should be screened for tox-ins, antibiotic resistance, and genes that increasebacterial pathogenicity (Merril et al. 2003). Withthe growth of the genomic data banks and thedevelopment of bioinformatics, tools such asscreens will be become more effective (Pauwelset al. 2009; Verheust et al. 2010).

Animal Models for Treatment of InfectiousDiseases

At the beginning of international hostilities inthe 1940s, the U.S. Department of Defensesponsored carefully designed phage therapy ex-periments for animal models of infectious dis-eases. Despite the encouraging results obtainedin these experiments this approach was virtuallyabandoned in the United States following thediscovery and development of broad-spectrumantibiotics (Merril et al. 2003, 2006). However,with the advent of antibiotic-resistant patho-genic bacterial strains, research on the potentialof phage therapy has been rejuvenated. In addi-tion, with the expanded knowledge of phagemolecular biology and interactions with mam-malian immune systems it is possible to genet-ically engineer phage that might be more effica-cious than the wild types found in nature. Thispotential was shown in the development of longcirculating phage strains that could stay in thecirculatory system longer than the laboratorystrains from which they were derived. Therehave been a number of successful demonstra-tions of the effectiveness of phage in animalmodels of bacterial infectious diseases (e.g.,see Dubos et al. 1943; Smith and Huggins1982; Merril et al. 1996; Biswas et al. 2002;Bull et al. 2002; Gu et al. 2012) including arecent success in the treatment of rats using ananimal model system for sepsis and meningitiscaused by S242, a fatal neonatal meningitisE. coli strain (Pouillot et al. 2012).

Clinical Studies

One of the first problems encountered by theinfectious disease physician is to determine

Phage Therapeutics and Prophylaxis

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what agent would offer an efficacious treatmentfor the patient’s disease. Although the narrowhost range of phage strains would appear tooffer an impediment for the clinical adaptationof phage therapy, it should be possible to ad-dress this problem by developing libraries ofclinically relevant phage strains carrying markergenes. The use of such strains could provideboth an identification of the infecting bacterialstrains along with information as to whichstrains of phage might offer an effective therapyfor the patient’s infection (Merril et al. 2003,2006). One of the major problems confrontingphage therapy is the need and cost of adequateclinical trials. This is of course a problem for anynew antibacterial therapy. The early clinical ap-plications often lacked the new standard dou-ble-blind statistically valid experimental designs(Carlton 1999). The years of experience devotedto developing an understanding of the molecu-lar biology, physiology, and methods for the pu-rification of phage should be an asset in helpingto design clinical applications and evaluations.Clinical trials needed to meet the full require-ments of the U.S. Food and Drug Administra-tion (FDA) for therapeutic phage have not yetbeen fulfilled for any of the phage strains thathave been shown to be effective in animal mod-els of infectious diseases. Such trials are expen-sive; however, given the need for such agents,for the treatment of antibiotic-resistant bacte-rial infectious organisms, approval should begained for some in the not-too-distant future.

USE OF PHAGE COMPONENTS

As Prophylactic Agents

Despite reasonable success of phage therapy indiagnosis and treatment of bacterial infectionsby bacteriophages as discussed above, parallelresearch has been progressing in the use of bac-teriophage components mostly as prophylaxis.Lysozymes, originally discovered by AlexanderFleming (Fleming 1922), are ubiquitous in na-ture, being associated with phage in mammals.Lysozymes have been used for decades as a pro-phylactic agent to kill Gram-positive bacteriabecause of their ability to degrade cell walls

from outside by hydrolysis of one of the fourmajor bonds in the peptidoglycan, which resultsin hypotonic bursting of the inner membraneand leaking of intracellular components andthereby causing cell death. Lysozymes from dif-ferent sources including phage have been usedas an adduct in the brewing industry and inyogurt and other milk products. As discussedbelow, phage-encoded lysozymes are of twokinds: (1) endolysin, which is made by phageduring phage lytic growth after infection of bac-terial host, and which lyses the cell wall frominside to help release phage progeny particles;and (2) phage tail-associated murein lytic en-zymes (TAME), which can hydrolyze cell wallbonds from outside after phage adsorption tothe host.

Brewing industries face problems with theirfermentation systems, as airborne bacteria fre-quently contaminate them. Bacterial con-tamination can make a beer turbid, aromatic,odious, or ropy. The most common contamina-tions are lactic-acid-producing Gram-positivebacteria (Lactobacillus and Pediococcus) andacetic-acid-producing Gram-negative bacteria(Acetobacter). The spoilage comes from thefact that acetic acid and lactic acids, among oth-er by-products, are made by bacteria from sugar.Some measured amount of specific strains oflactic-acid-producing bacteria are allowed dur-ing fermentation to keep the pH low, thus facil-itating beer production as well as lowering acid-ity by “malolactic acid” production. In winemaking, unwanted contaminations are routine-ly taken care of by sanitation practices. In fact,direct lysozyme addition acts as a preservativefor storage of foods like yogurt, tofu, cheese, andsake (Larson 2005).

More recently, the addition of phage endo-lysins has been shown to protect against a broadspectrum of Gram-positive bacteria. If micro-bial contamination is envisioned, lysozyme isused to kill the lactic acid bacteria. In fact, ly-sozyme can be added to the final product. De-pending on factors like temperature, pH, etc.,lysozyme starts working immediately after ad-dition and kills bacteria at once. Lysozyme inbeer remains 50% active for at least a 6-monthperiod. Lysozyme present in beer has minimal

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effects on the physical and sensory properties ofthe beer (Feeney and Nagy 1952; Daeschel et al.1991; Landschoot 2005; Waite and Daeschel2007).

As Curative Agents

More recently, however, lysozymes are being de-veloped for treatment of mammalian infections.Endolysin, used by phage to release progenyparticles from bacterial hosts, acts by breakingup the cell from inside. This occurs after anotherphage product, called holin, creates pores in theinner membrane through which lysozymes canpass through the membrane and reach the cellwall (Wang et al. 2003). However, peptidogly-can-degrading enzymes have the ability to di-gest bacterial cell walls. The phage-encoded en-dolysins precisely do that except in a species- orgenus-specific manner (Nelson et al. 2001; Fis-chetti 2010). Phage-encoded endolysin usuallycan lyse from outside only the correspondinghost. Recently, cell wall lysis-mediated killingactivity of phage-encoded pure lysozymes hasbeen successfully used to its maximal level by,among others, Fischetti and colleagues, andLoessner and colleagues. Like the current surgein bacteriophage therapy, development of endo-lysin-mediated therapy of infection was moti-vated by the emergence of antibiotic-resistantbacteria in clinics. Endolysins create holes inthe cell from outside by peptidoglycan digestionand expansion of the inner cytoplasmic mem-brane and subsequent hypotonic lysis.

Most human infections begin at the muco-sal membranes followed by their colonization,which are usually a reservoir of many pathogens.Very few anti-infective agents are known thatprevent mucosal colonization. Nonetheless, ifone can reduce the bacterial load of the mucousmembranes in the community, in hospitals, andin nursing homes, the incidence of the diseaseswould very likely reduce. Precisely, endolysinsare expected to be very effective in such cases.Specific endolysins have now been identified tovery effectively kill Gram-positive bacteria (Nel-son et al. 2001; Loeffler et al. 2003). Interesting-ly, the endolysins bind very tightly to their cellwall substrates and thus and do not have a turn-

over number requiring multiple molecules tobind and hydrolyze several cell wall bonds tomake effective cell wall lysis (Loessner et al.2002; Jervis et al. 2005). An oral colonizationanimal model has been developed with Strep-tococcus pyogenes, a nasal model with pneumo-cocci, and a vaginal model with group B strep-tococci. These studies showed, for example, thatnanogram quantities of endolysin kill S. pyogenes106-fold in 2–4 h after lysozyme treatments. Allcurrent success in dealing with endolysin-medi-ated cell killing are only with Gram-positivebacteria simply because the enzyme when addedfrom outside can access the cell wall peptidogly-can. Gram-negative bacteria, however, have anouter membrane that sterically interferes withlysozyme action thus making lysozyme-mediat-ed cell killing generally ineffective. However, re-cently, a structurally engineered phage lysozymecontaining the FyuA-binding domain of pesti-cin fused to the amino terminus of T4 lysozymehas given encouraging results against Gram-negative pathogens (Lukacik et al. 2012).

Immunity and Resistance

Both in bacteriophage therapy and in directlysozyme (of phage or any other source) therapy,it has always been feared that such foreign ob-jects or proteins when used systematically woulddevelop neutralizing antibodies and thus hindertheir antibacterial action in the future. However,it has been found that highly immune serumslows down but does not block bacteriolyticactivities of pneumococcal-specific endolysins(Loeffler et al. 2001). Another concern of endo-lysin treatment was its short circulation timeafter administration (Loeffler et al. 2001). Butthis issue did not affect the treatment because ofthe very rapid action time of the lysozyme mol-ecules. Both in antibiotic and phage therapy,another major concern is the phenomenon ofthe increase of resistant pathogenic strains. In-terestingly, every attempt to generate resistantmutants against endolysins in the laboratorysetup has so far failed (Loeffler et al. 2001). Al-though this gives more credence to the potentialof lysozyme therapy, we note that most anti-biotic-resistance elements that have infiltrated



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into antibiotic therapy are because of bacteria-acquiring resistance elements from natural en-vironments by lateral gene transfer.

Use of Lysozyme for Treating BacterialContamination in Plant Cell Cultures

Even egg white lysozyme has been successfullyused to reduce Bacillus circulans and Sphingomo-nas paucimobilis infection of in vitro shoot cul-tures quince and hybrid rootstock (Marino et al.2003). Although lysozyme did not have a nega-tive effect on shoot growth, under optimal con-ditions it was effective in eliminating B. circulansin quince shoot and hybrid rootstock cultures,and has a bacteriostatic effect on S. paucimobilis.These results suggest that lysozyme may be ableto replace antibiotic treatments of in vitro shootcultures although it may not be effective againstevery plant bacterial infection.

Use of Bacteriophage Tail-Associated LyticEnzymes as Antibacterial Therapeutic Agents

It has been known for a while that peptido-glycan-degrading murein hydrolases cleave bac-terial cell walls very efficiently in a species-specific manner. Thus, they have the potentialof being therapeutic agents against specificpathogens. Recently, this idea has been success-fully exploited. These hydrolases are differentfrom the phage-encoded endolysins, which aremade and used by phage to come out of hostcells by cell wall degradation from inside as dis-cussed above. Unlike the endolysins the mureinhydrolases are present at the tip of tails of phagevirions. It is believed that the tail-attachedhydrolases help DNA injection after phage ad-sorption. If a bacterial cell is infected by phageparticles at very high multiplicities, then thecell lyses before phage DNA starts replication.It has been called “lysis from without” (Del-bruck 1940). These tail hydrolases are more orless ubiquitous among tailed phages. Moti-vated by the emergence of drug-resistant hu-man pathogen Staphylococcus aureus, Paul etal. (2011) identified the muralytic activity ofthe tail of the broad host range staphylococcal-specific phage, called phage K. These investiga-

tors showed the efficacy of the K-encoded puri-fied hydrolase as a bacteriocidal agent in cellculture. The efficacy of this protein was furtherenhanced by making a hybrid protein compris-ing the catalytic domain of the hydrolase andthe staphylococcal cell wall binding domain ofa bacteriocin called lysostaphin (Baba andSchneewind 1998) to generate a protein calledP128. The bacteriolytic activity of P128 wasmore than two orders of magnitude higherthan the catalytic domain of the hydrolasealone because of increased substrate specificity.The hybrid has already been tested in experi-mental nasal colonization of methicillin-resis-tant S. aureus (MRSA) in experimental rats; theprotein has been shown extremely effective indecolonizing the animals. Thus it is an excellentprospective therapeutic against infection.

PHAGE-MEDIATED VACCINE

Prevention of infectious diseases by direct inoc-ulation of a minute quantity of pathogens wasshown in China during the 15th century, wherehealthy children were inoculated with a minutequantity of exudates collected from the pustuleof smallpox-infected persons. It was observedthat the process of this crude inoculation tech-nique protected the children from future epi-demics of smallpox (Tizard 1984; Needham1999). Despite the fact that the science behindthe treatment of infectious diseases throughvaccination was first shown by Edward Jennermore than three centuries ago (Riedel 2005) andthat use of vaccine in treatment of diseases iswidespread now, there are still problems andlimitations in preparing vaccines by traditionalmeans. This set the stage to explore otherapproaches for producing better vaccines (Bur-din et al. 2004; O’Hagan and Rappuoli 2004).The advancement of recombinant DNA tech-nology led us to produce vaccine candidatesthat are recombinant protein molecules display-ing subunits of pathogens. However, such vac-cine candidates lack many immunogenic fea-tures of the original pathogens (Petrovsky andAguilar 2004). Although many of these recom-binant vaccines contain succession of immuno-dominant epitopes, they fail to stimulate the

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production of neutralizing antibodies. To over-come this situation, currently many of theserecombinant vaccines are mixed with adjuvantsto enhance immunostimulatory effects. Unfor-tunately, the adjuvants that have been tried areeither questionable or the process is time con-suming and thus not an ideal method of devel-oping vaccines for rapidly evolving diseases likeswine flu (Barrett and Stanberry 2008).

Previously immunostimulatory effects ofsynthetic-immunodominant peptides (epi-topes) of antigen and recombinant viruses ornucleotide acid-based (DNA) vaccines wereevaluated through various clinical trials. Resultsof these trials indicated that these vaccines werelacking an effective delivery system for properactivation of the immune system (Petrovsky andAguilar 2004). These activation deficiencieswere reflected in the inability of the vaccinesto elicit both humoral and cell-mediated im-munity against targeted pathogens. Thus, devel-opment of a proper delivery system for any typeof subunit or DNAvaccine is of paramount im-portance (Burdin et al. 2004).

Vaccinologists are now evaluating bacterio-phages (phages) as vaccine delivery agents, andthe idea is gaining popularity owing to the in-herited character of phages to stimulate botharms of the immune system (humoral and cell-mediated immunity).

The ability of phage to deliver gene(s) tomammalian cells was discovered in studies inwhich lambda (l) phage carrying a galactosetransferase gene was shown capable of inducinggalactose transferase enzyme activity in humanfibroblast cells from a child lacking the capacityto make the Gal-transferase enzyme (Merrilet al. 1971). This ability of phage to deliver genesto human cells was later independently con-firmed with a phage carrying the b-galactosi-dase gene in human cells deficient in that en-zyme activity (Horst et al. 1975).

A concern that was expressed concerningthese experiments is that the DNA used to cor-rect the genetic defects in the eukaryotic cells isbacterial or prokaryotic. However, it has nowbeen clearly shown that prokaryotic genes,such as the genes in a segment of the Ti plasmidof the bacteria Agrobacterium tumefaciens can

function in eukaryotes. These genes have beenshown to be capable of transforming eukaryoticcells in plants and they are the cause of the com-monly observed plant crown gall tumors. Inaddition, the bacterial Ti plasmid genes havealso been shown to be capable of affecting yeast,filamentous fungi, cultivated mushrooms, andhuman cells in culture (Lacroix et al. 2006).

Currently, two separate approaches of anti-gen delivery systems are attempted with phagevaccines. These systems are designated as “phagedisplay vaccines” and “phage DNA vaccines”(Clark and March 2004a). Broadly, phage dis-play vaccines are mainly referred for producingimmunogenic phage particles that display for-eign antigen on phage surface. This generally isachieved by expressing antigens as fusion prod-ucts of one of the major surface proteins ofphage virion; phage DNAvaccines, on the otherhand, are produced by incorporation of foreignantigen genes in phage genome under the con-trol of strong eukaryotic promoters. In the lattercase, phage acts as a passive carrier to transfer theforeign DNA in mammalian cells where the an-tigen gene is expressed. These two methods areoften combined to produce phages that carry aforeign antigen gene along with a display proteinor peptide on the surface. The display proteinsor peptides are generally selected for their spe-cific binding affinity of antigen-presenting cells.Thus, such phage constructs pose the additionalability of delivering the antigen gene directly tothe immunoreactive cells like dendritic cells,Kupffer cells, etc. (Zanghi et al. 2007).

Phage Display Vaccine

A filamentous phage M13 display system wasfirst used for expression of antibody fragmentsand their affinity maturation (Benhar 2001).Although M13 is successful in several cases indisplaying melanoma-specific tumor antigen toproduce cancer vaccines effective in reducingtumor growth in animal models (Fang et al.2005), the M13 display system has limitations(Gupta et al. 2003). The system is not efficient indisplaying larger proteins when using the highcopy coat protein gp VIII. The other coat pro-tein, gpIII, which can effectively display foreign



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proteins or peptides, has a low copy number inthe M13 virion (Smith 1985).

Soc and Hoc, two of the virion proteins ofphage T4, are more effective major display plat-forms for foreign antigens. Unlike M13, T4 isable to display peptides of various sizes andsequences without much problem. Hoc andSoc are also highly immunogenic and thereforeact as good adjuvants. Thus, T4 phage is con-sidered as a better display platform than fila-mentous phages (Li et al. 2006a). Currently,several vaccines for infectious diseases are pre-pared by using the T4 phage display system,which has shown promising results in animalmodels (Jiang et al. 1997).

T7 is another phage capable of displayingproteins and peptides including antigens. Fu-sion products are generated by cloning the cod-ing sequences for the peptides and proteins at thecarboxyl end of the capsid protein, 10B (Lind-ner et al. 2011). Lewis lung cancer vaccine pre-pared by T7 phage display of vascular endothe-lial growth factor (VEGF) has been successfullyused to break immunologic tolerance. Wheninjected in mice, T7-VEGF display producedstrong immunogenic response against cancercells and inhibited cancer growth (Li et al.2006b). The comparative aspects of differentphages for antigen display are shown in Table 1.

Recently, the common cloning vector phagelambda (l) is also gaining popularity as a phagedisplay platform. The complex cloning methodof l phage display is considerably simplified by

using a Cre-loxP site-specific recombinationprocess to develop a l phage display systemthat facilitates rapid high efficiency cloning offoreign DNA in l genome (Gupta et al. 2003).The method allows displaying foreign genesfused to l capsid protein gene D; nearly about450 copies of foreign antigen can display as Dfusion on l surface (Gupta et al. 2003). Theability of l to display multiple copies of thesame antigen on the surface of a single phageparticle is more effective in eliciting a strongimmune response than low copy display (Clarkand March 2004a). Production of successful ldisplay antigen to produce neutralizing anti-bodies against infectious porcine circovirus in-fection has been shown by Hayes et al. (2010),using gpD fusion antigen on phage l. Activa-tion of immune system by phage display vaccineis described in Figure 1.

Phage DNA Vaccine

Successful delivery and expression of foreigngenes in eukaryotic cells by phage-mediatedgene transfer is also in common use now. Theuse of strong eukaryotic promoters has extend-ed the capacity of phage to deliver foreign anti-gen genes to mammalian cells. Generally forproduction of vaccine, the antigen genes underthe control of a eukaryotic promoter are clonedinside of a nonessential region of phage ge-nome. When injected in a mammalian system,this phage vaccine acting as a DNA vaccine can

Table 1. List of phages and their virion proteins, which are used for display

Vector phage

Phage virion proteins

for fusion display Copy number of display Size of display shown

M13 gpVIII �2700 Small peptide (6–8 amino acids)gpIII �5 Large protein with reduced viability

T4 Soc �960 Large protein (up to 837 amino acids)Hoc �160 Protein (up to 183 amino acids)

T7 10A �415 Protein (40–50 amino acids)10B �1 Protein (up to 1200 amino acids)

Lambda (l) gpD �420 Large protein (1024 amino acids)

Data were taken from Smith (1985), Markland et al. (1991), Mikawa et al. (1996), Ren et al. (1996, 1997), Ren and Black

(1998), Iwasaki et al. (2000), Savinov and Austin (2001), Sche et al. (2001), Gupta et al. (2003), Pacheco et al. (2006), and Li

et al. (2007).

The above phages are used for presentation of displayed antigens to mammalian cells; the l system was also developed as a

DNA delivery platform to antigen-presenting cells (APC).

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induce potent immune response by expressingforeign antigen inside of APCs (Fig. 2) (Clarkand March 2006). Several l-based DNA vac-cines for infectious diseases have been preparedthat have shown promising results in animalmodels (Clark and March 2004b; March et al.2004).

Advantages of Phage-Mediated VaccineDelivery

Recent global surveillance indicates that infec-tious microorganisms are emerging at an alarm-ing rate (Barrett and Stanberry 2008). There isalso a significant concern about the potential ofgenerating highly virulent microorganisms us-ing genetic engineering and synthetic biologytechniques. The outbreak caused by these typesof natural or man-made variants can spreadrapidly and has to be stopped before diseases

become epidemic. The quarantine and rapidvaccination against targeted pathogens is theonly way to prevent epidemics during massivedisease outbreaks. One of the best advantages ofphage vaccine is that it can be produced inex-pensively and deployed rapidly during diseaseoutbreaks.

Utilizing advanced knowledge in bioinfor-matics, gene sequencing, and phage genetics, itis possible to create and manufacture vaccines inrecord time. Genomic and proteomic data ofany old or newly emerging pathogenic bacteriaor virus can be scanned using existing algo-rithms and database management systems toidentify novel antigenic motifs that can becloned into phage genome to produce phagevaccine. The resulting phage vaccine can prop-agate rapidly using bacteriological media anddoes not need cell culture or a chicken-egg-based system for manufacturing sufficient quan-

Naïve T cell

APCNaïve T cell

Cytotoxic T cell

Perforin

IFN-γ

IL-2

IL-4, IL-5

Th1 effector cell

Th2 effector cell5

4

2

36

1

Ig secretion

CD8

TCR

TCR

CD4

MHCI

MHCII

B cell

Plasmacell

Figure 1. Immune activation of mammalian system with phage display antigens. (1) Particulate nature of phageactivates the antigen-presenting cells (APC), which process the antigens for immune presentation. (2) Presen-tation of processed antigens by major histocompatibility complexes (MHC) class I molecules to CD8 T cells,which leads to T-cell activation. (3) Antigens are presented by MHC class II molecules to CD4 T cell, which inturn activates Th1 and Th2 effector cells. (4) Th1 cells generate cytotoxic T-cell (CTL) responses and help produceinterferon g (IFN-g). (5) Th2 cells activate B cells to make antibodies. (6) Direct activation of B cells by phagevaccines also leads to massive antibody response. T-cell receptors are denoted by TCR.



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tities of vaccine to protect against a new patho-gen; such preparations can be made in weeksrather than months. The phage particles arenonpathogenic to humans and the resultantvaccines do not require adjuvants. By usingphage vaccine, all relevant information can bedelivered to the immune system to allow for animmune response that includes antibody pro-duction and the development of immunity toa pathogen (Fig. 1). The particulate nature ofphage vaccine attracts APCs that engulf, pro-cess, and present phage-mediated antigensthrough MHCs (class I and class II pathways)(Manoutcharian et al. 1999; Gaubin et al. 2003)to evoke both cell-mediated and humoral im-munities (Fig. 1). It was expected that phage as

an extracellular antigen will only activate anti-body production through MHC class II biaspathway, but it has been shown that phage as aparticulate antigen can access the MHC I path-way through cross priming, which activates acellular immune response (Gaubin et al.2003). In addition, the immunostimulatory un-methylated CpG motifs of the phage genomeare recognized by the innate immune cellsthrough Toll-like receptors (TLRs) (Krieg et al.1995; Mason et al. 2005), which further enhanceimmunity. Immunostimulatory effect of phagevaccination through oral route has been evalu-ated by Clark et al. They have succeeded in mak-ing a phage DNA vaccine against Yersina pestisby cloning V-antigen in phage l genome. Upon

Antigen gene fused tophage coat protein gene (red and black)

Phage coatprotein gene (black)

Chemical coupler (red)

Phage coatprotein (blue)

Antigen (green)

Assembled on phage

head in vitro

Antigen gene (red)Antigen gene fused to phage coat protein gene in vivo

Antigen chemically coupled to phage coat protein in vivo

Eukaryotic promoter antigen gene cassette

cloned into phage chromosome

Purifie

d an

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Phage DNA vaccine

Eukaryotic promoter antigen gene cassette

cloned into phage chromosome

Antigen gene fused to phage coat protein gene in vivo

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3

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1

5Eukaryotic promoter antigengene cassette (green)

Antigen

Pathogen

Figure 2. Schematic representations of several phage-mediated vaccine delivery systems. (1) Production of virus-like particles (VLPs) by adding purified phage capsid proteins fused to antigens during assembly of phageparticles in vitro. (2) Antigens are attached by chemical conjugation on preassembled phage head. (3) Antigensare displayed on phage surface by fusion of antigen genes with phage capsid protein genes. (4) Phage as a DNAdelivery vehicle where antigen genes are cloned in phage genome under the control of eukaryotic promoters. (5)Phage for DNAvaccine, where phage carries antigen genes under the control of eukaryotic promoters. The phagealso displays foreign proteins on its surface as fusion of phage capsid proteins. This protein targets the phage toantigen-presenting cells (APC).

S. Adhya et al.


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oral administration, it produced a significantimmune response in mouse (Clark and March2004b). Others have also explored the oral de-livery route of phage vaccination, and showedantigen-specific response (Delmastro et al.1997; Zuercher et al. 2000). These results indi-cate the possibility of oral delivery of phage vac-cine in developing countries.

In summary, phage particles pose severalintrinsic characters that make them lucrativefor developing vaccine-delivery platforms. Thenatural stability of phage particles provides easyprocessing and cheaper manufacturing capabil-ity for large-scale production of vaccines. Theparticulate nature of phage makes them easy topurify by simple centrifugation steps, which arefar easier and cheaper than the methods appliedfor purifying soluble recombinant protein vac-cines. Additionally, phage itself acts as a strongadjuvant and thus enhances excellent immuneresponse against any antigens present alongwith phage (Frenkel et al. 2000; Manoutcharianet al. 2004). In fact, the response against phage-displayed antigens can break immunologicaltolerance against self-proteins and thus is anideal vehicle for delivering cancer antigens(Fang et al. 2005).

PERSPECTIVES

The ever-increasing list of antibiotic-resistantbacterial strains and newly emerging viruses isforcing a reexamination of alternate modes oftherapies for infectious diseases. Of these, thephage and their molecular components andproducts offer a number of new therapeutic al-ternatives. Although the development of phage-based applications will require new directions,there are advantages over the prior art of treat-ing infectious disease. A useful aspect is the abil-ity to develop therapies for bacterial infectionthat minimally perturb the human micro-biome. As recent research of the microbiomehas shown, even small alterations can have ef-fects that range from alterations in the percep-tion of hunger to increased susceptibility to le-thal infections by foreign bacterial strains. Theseadvances in our ability to work with phage ex-tend from the treatment of human infections to

increasing the efficiency of industrial processes,particularly those that rely on fermentation. Thedevelopment of phage-based delivery of anti-gens and genes will result in new vaccines forprotection from numerous diseases and newtherapeutic approaches to both infectious andnoninfectious diseases, such as some forms ofcancer. The newly developed genomic sequenc-ing methods along with other high-throughputtechnologies including cell sorters and multi-well metabolic monitoring robots should resultin the rapid development of the needed infor-mation for phage-based therapies.

REFERENCES

Alisky J, Iczkowski K, Rapoport A, Troitsky N. 1998. Bacte-riophages show promise as antimicrobial agents. J Infect36: 5–15.

Baba T, Schneewind O. 1998. Instruments of microbial war-fare: Bacteriocin synthesis, toxicity and immunity. TrendsMicrobiol 6: 66–71.

Barrett ADT, Stanberry LR. 2008. Vaccines for biodefenceand emerging and neglected diseases. Academic, Waltham,MA.

Benhar I. 2001. Biotechnological applications of phage andcell display. Biotechnol Adv 19: 1–33.

Biswas B, Adhya S, Washart P, Paul B, Trostel AN, Powell B,Carlton R, Merril CR. 2002. Bacteriophage therapy res-cues mice bacteremic from a clinical isolate of vanco-mycin-resistant Enterococcus faecium. Infect Immun 70:204–210.

Bull JJ, Levin BR, DeRouin T, Walker N, Bloch CA. 2002.Dynamics of success and failure in phage and antibi-otic therapy in experimental infections. BMC Microbiol2: 35.

Burdin N, Guy B, Moingeon P. 2004. Immunological foun-dations to the quest for new vaccine adjuvants. BioDrugs18: 79–93.

Carlton RM. 1999. Phage therapy: Past history and futureprospects. Arch Immunol Ther Exp 47: 267–274.

Clark JR, March JB. 2004a. Bacterial viruses as human vac-cines? Expert Rev Vaccines 3: 463–476.

Clark JR, March JB. 2004b. Bacteriophage-mediated nucleicacid immunisation. FEMS Immunol Med Microbiol 40:21–26.

Clark JR, March JB. 2006. Bacteriophages and biotechnol-ogy: Vaccines, gene therapy and antibacterials. TrendsBiotechnol 24: 212–218.

Daeschel MA, Jung DS, Watson BT. 1991. Controlling winemalolactic fermentation with nisin and nisin-resistantstrains of Leuconostoc oenos. Appl Environ Microbiol 57:601–603.

Delbruck M. 1940. The growth of bacteriophage and lysis ofthe host. J Gen Physiol 23: 643–660.

Delmastro P, Meola A, Monaci P, Cortese R, Galfre G. 1997.Immunogenicity of filamentous phage displaying pep-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



tide mimotopes after oral administration. Vaccine 15:1276–1285.

Dubos RJ, Straus JH, Pierce C. 1943. The multiplication ofbacteriophage in vivo and its protective effect against anexperimental infection with Shigella dysenteriae. J ExpMed 78: 161–168.

Fang J, Wang G, Yang Q, Song J, Wang Y, Wang L. 2005. Thepotential of phage display virions expressing malignanttumor specific antigen MAGE-A1 epitope in murinemodel. Vaccine 23: 4860–4866.

Feeney RE, Nagy DA. 1952. The antibacterial activity of theegg white protein conalbumin. J Bacteriol 64: 629–643.

Fischetti VA. 2010. Bacteriophage endolysins: A novel anti-infective to control Gram-positive pathogens. Int J MedMicrobiol 300: 357–362.

Fleming A. 1922. On a remarkable bacteriolytic elementfound in tissues and secretions. P Roy Soc London B 93:306–317.

Frenkel D, Katz O, Solomon B. 2000. Immunization againstAlzheimer’s b-amyloid plaques via EFRH phage admin-istration. Proc Natl Acad Sci 97: 11455–11459.

Gaubin M, Fanutti C, Mishal Z, Durrbach A, De BerardinisP, Sartorius R, Del Pozzo G, Guardiola J, Perham RN,Piatier-Tonneau D. 2003. Processing of filamentous bac-teriophage virions in antigen-presenting cells targetsboth HLA class I and class II peptide loading compart-ments. DNA Cell Biol 22: 11–18.

Gu J, Liu X, Li Y, Han W, Lei L, Yang Y, Zhao H, Gao Y, Song J,Lu R, et al. 2012. A method for generation phage cocktailwith great therapeutic potential. PloS ONE 7: e31698.

Gupta A, Onda M, Pastan I, Adhya S, Chaudhary VK. 2003.High-density functional display of proteins on bacterio-phage l. J Mol Biol 334: 241–254.

Hayes S, Gamage LN, Hayes C. 2010. Dual expression systemfor assembling phage l display particle (LDP) vaccine toporcine Circovirus 2 (PCV2). Vaccine 28: 6789–6799.

Horst J, Kluge JF, Beyreuther K, Gerok W. 1975. Gene trans-fer to human cells: Transducing phagel plac gene expres-sion in GM1-gangliosidosis fibroblasts. Proc Natl AcadSci 72: 3531–3535.

Iwasaki K, Trus BL, Wingfield PT, Cheng N, Campusano G,Rao VB, Steven AC. 2000. Molecular architecture of bac-teriophage T4 capsid: Vertex structure and bimodal bind-ing of the stabilizing accessory protein, Soc. Virology 271:321–333.

Jernberg C, Lofmark S, Edlund C, Jansson JK. 2010. Long-term impacts of antibiotic exposure on the human intes-tinal microbiota. Microbiology 156: 3216–3223.

Jervis EJ, Guarna MM, Doheny JG, Haynes CA, Kilburn DG.2005. Dynamic localization and persistent stimulation offactor-dependent cells by a stem cell factor/cellulosebinding domain fusion protein. Biotechnol Bioeng 91:314–324.

Jiang J, AbuShilbayeh L, Rao VB. 1997. Display of a PorApeptide from Neisseria meningitidis on the bacteriophageT4 capsid surface. Infect Immun 65: 4770–4777.

Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA,Teasdale R, Koretzky GA, Klinman DM. 1995. Cpg motifsin bacterial-DNA trigger direct B-cell activation. Nature374: 546–549.

Lacroix B, Tzfira T, Vainstein A, Citovsky V. 2006. A case ofpromiscuity: Agrobacterium’s endless hunt for new part-ners. Trends Genet 22: 29–37.

Landschoot AV. 2005. Antibacterial properties of hen eggwhite lysozyme against beer spoilage bacteria and effectof lysozyme on yeast fermentation. Cerevisia 32: 219–225.

Larson AE. 2005. Lysozyme in antimicrobial in food. Taylorand Francis, London.

Li Q, Shivachandra SB, Leppla SH, Rao VB. 2006a. Bacter-iophage T4 capsid: A unique platform for efficient surfaceassembly of macromolecular complexes. J Mol Biol 363:577–588.

Li XH, Tang L, Liu D, Sun HM, Zhou CC, Tan LS, Wang LP,Zhang PD, Zhang SQ. 2006b. Antitumor effect of recom-binant T7 phage vaccine expressing xenogenic vascularendothelial growth factor on Lewis lung cancer in mice.Ai Zheng 25: 1221–1226.

Li Q, Shivachandra SB, Zhang Z, Rao VB. 2007. Assembly ofthe small outer capsid protein, Soc, on bacteriophage T4:A novel system for high density display of multiple largeanthrax toxins and foreign proteins on phage capsid. JMol Biol 370: 1006–1019.

Lindner T, Kolmar H, Haberkorn U, Mier W. 2011. DNAlibraries for the construction of phage libraries: Statisticaland structural requirements and synthetic methods. Mol-ecules 16: 1625–1641.

Liu M, Deora R, Doulatov SR, Gingery M, Eiserling FA,Preston A, Maskell DJ, Simons RW, Cotter PA, ParkhillJ, et al. 2002. Reverse transcriptase-mediated tropismswitching in Bordetella bacteriophage. Science 295: 2091–2094.

Loeffler JM, Nelson D, Fischetti VA. 2001. Rapid killing ofStreptococcus pneumoniae with a bacteriophage cell wallhydrolase. Science 294: 2170–2172.

Loeffler JM, Djurkovic S, Fischetti VA. 2003. Phage lyticenzyme Cpl-1 as a novel antimicrobial for pneumococcalbacteremia. Infect Immun 71: 6199–6204.

Loessner MJ, Kramer K, Ebel F, Scherer S. 2002. C-terminaldomains of Listeria monocytogenes bacteriophage mureinhydrolases determine specific recognition and high-affin-ity binding to bacterial cell wall carbohydrates. Mol Mi-crobiol 44: 335–349.

Lukacik P, Barnard TJ, Keller PW, Chaturvedi KS, Seddiki N,Fairman JW, Noinaj N, Kirby TL, Henderson JP, StevenAC, et al. 2012. Proc Natl Acad Sci 109: 9857–9862.

Manoutcharian K, Terrazas LI, Gevorkian G, Acero G, Pe-trossian P, Rodriguez M, Govezensky T. 1999. Phage-dis-played T-cell epitope grafted into immunoglobulinheavy-chain complementarity-determining regions: Aneffective vaccine design tested in murine cysticercosis.Infect Immun 67: 4764–4770.

Manoutcharian K, Diaz-Orea A, Gevorkian G, Fragoso G,Acero G, Gonzalez E, De Aluja A, Villalobos N, Gomez-Conde E, Sciutto E. 2004. Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cys-ticercosis. Vet Immunol Immunopathol 99: 11–24.

March JB, Clark JR, Jepson CD. 2004. Genetic immunisa-tion against hepatitis B using whole bacteriophage l par-ticles. Vaccine 22: 1666–1671.

S. Adhya et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Marino G, Ferrarini V, Giardini S, Biavati B. 2003. Use oflysozyme for treatment of bacterial contamination in invitro shoot cultures of fruit plants. In Vitro Cell Dev Biol39: 327–331.

Markland W, Roberts BL, Saxena MJ, Guterman SK, LadnerRC. 1991. Design, construction and function of a multi-copy display vector using fusions to the major coat pro-tein of bacteriophage M13. Gene 109: 13–19.

Mason KA, Ariga H, Neal R, Valdecanas D, Hunter N, KriegAM, Whisnant JK, Milas L. 2005. Targeting toll-like re-ceptor 9 with CpG oligodeoxynucleotides enhances tu-mor response to fractionated radiotherapy. Clin CancerRes 11: 361–369.

Merril CR, Geier MR, Petricciani JC. 1971. Bacterial virusgene expression in human cells. Nature 233: 398–400.

Merril CR, Biswas B, Carlton R, Jensen NC, Creed GJ, ZulloS, Adhya S. 1996. Long-circulating bacteriophage as an-tibacterial agents. Proc Natl Acad Sci 93: 3188–3192.

Merril CR, Scholl D, Adhya SL. 2003. The prospect for bac-teriophage therapy in Western medicine. Nat Rev DrugDiscov 2: 489–497.

Merril CR, Scholl D, Adhya S. 2006. The bacteriophage. Ox-ford University Press, London.

Mikawa YG, Maruyama IN, Brenner S. 1996. Surface displayof proteins on bacteriophage l heads. J Mol Biol 262:21–30.

Needham J. 1999. Science and civilisation in China. Cam-bridge University Press, Cambridge.

Nelson D, Loomis L, Fischetti VA. 2001. Prevention andelimination of upper respiratory colonization of miceby group A streptococci by using a bacteriophage lyticenzyme. Proc Natl Acad Sci 98: 4107–4112.

O’Hagan DT, Rappuoli R. 2004. Novel approaches to vac-cine delivery. Pharm Res 21: 1519–1530.

Pacheco S, Gomez I, Sato R, Bravo A, Soberon M. 2006.Functional display of Bacillus thuringiensis Cry1Ac toxinon T7 phage. J Invertebr Pathol 92: 45–49.

Paul VD, Sundarrajan S, Rajagopalan SS, Hariharan S, Kem-pashanaiah N, Padmanabhan S, Sriram B, Ramachan-dran J. 2011. Lysis-deficient phages as novel therapeuticagents for controlling bacterial infection. BMC Microbiol11: 195.

Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-GalloK, Verheust C, Debyser Z, Herman P. 2009. State-of-the-art lentiviral vectors for research use: Risk assessment andbiosafety recommendations. Curr Gene Ther 9: 459–474.

Petrovsky N, Aguilar JC. 2004. Vaccine adjuvants: Currentstate and future trends. Immunol Cell Biol 82: 488–496.

Pouillot F, Chomton M, Blois H, Courroux C, Noelig J, BidetP, Bingen E, Bonacorsi S. 2012. Efficacy of bacteriophagetherapy in experimental sepsis and meningitis caused bya clone O25b:H4-ST131 Escherichia coli strain producingCTX-M-15. Antimicrob Agents Chemother 56: 3568–3575.

Ren Z, Black LW. 1998. Phage T4 SOC and HOC display ofbiologically active, full-length proteins on the viral cap-sid. Gene 215: 439–444.

Ren ZJ, Lewis GK, Wingfield PT, Locke EG, Steven AC, BlackLW. 1996. Phage display of intact domains at high copynumber: A system based on SOC, the small outer capsidprotein of bacteriophage T4. Protein Sci 5: 1833–1843.

Ren ZJ, Baumann RG, Black LW. 1997. Cloning of linearDNAs in vivo by overexpressed T4 DNA ligase: Construc-tion of a T4 phage hoc gene display vector. Gene 195:303–311.

Riedel S. 2005. Edward Jenner and the history of smallpoxand vaccination. Proc (Bayl Univ Med Cent) 18: 21–25.

Sandmeier H. 1994. Acquisition and rearrangement of se-quence motifs in the evolution of bacteriophage tail fi-bres. Mol Microbiol 12: 343–350.

Savinov SN, Austin DJ. 2001. The cloning of human genesusing cDNA phage display and small-molecule chemicalprobes. Comb Chem High T Scr 4: 593–597.

Sche PP, McKenzie KM, White JD, Austin DJ. 2001. Corri-gendum to: “Display cloning: Functional identificationof natural product receptors using cDNA-phage display”[Chemistry & Biology 6 (1999) 707–716]. Chem Biol 8:399–400.

Scholl D, Rogers S, Adhya S, Merril CR. 2001. BacteriophageK1–5 encodes two different tail fiber proteins, allowing itto infect and replicate on both K1 and K5 strains ofEscherichia coli. J Virol 75: 2509–2515.

Smith GP. 1985. Filamentous fusion phage: Novel expres-sion vectors that display cloned antigens on the virionsurface. Science 228: 1315–1317.

Smith HW, Huggins MB. 1982. Successful treatment of ex-perimental Escherichia coli infections in mice usingphage: Its general superiority over antibiotics. J Gen Mi-crobiol 128: 307–318.

Sneade W. 2005. Drug discovery: A history. Wiley-Inter-science, Hoboken, NJ.

Sulakvelidze A, Alavidze Z, Morris JG Jr. 2001. Bacterio-phage therapy. Antimicrob Agents Chemother 45: 649–659.

Summers WC. 1999. Felix dHerelle and the origins ofmolecular biology. Yale University Press, New Haven andLondon.

Tizard IR. 1984. Immunology: An introduction. CengageLearning, Stamford, CT.

Verheust C, Pauwels K, Mahillon JD, Helinski R, Herman P.2010. Contained use of bacteriophages: Risk assessmentand biosafety recommendations. Appl Biosafety 15: 32–44.

Vitiello CL, Merril CR, Adhya S. 2005. An amino acid sub-stitution in a capsid protein enhances phage survival inmouse circulatory system more than a 1000-fold. VirusRes 114: 101–103.

Waite JG, Daeschel MA. 2007. Contribution of wine com-ponents to inactivation of food-borne pathogens. J FoodSci 72: M286–M291.

Wang IN, Deaton J, Young R. 2003. Sizing the holin lesionwith an endolysin-b-galactosidase fusion. J Bacteriol 185:779–787.

Zanghi CN, Sapinoro R, Bradel-Tretheway B, Dewhurst S.2007. A tractable method for simultaneous modificationsto the head and tail of bacteriophage l and its applicationto enhancing phage-mediated gene delivery. Nucleic Ac-ids Res 35: e59.

Zuercher AW, Miescher SM, Vogel M, Rudolf MP, StadlerMB, Stadler BM. 2000. Oral anti-IgE immunization withepitope-displaying phage. Eur J Immunol 30: 128–135.



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2014; doi: 10.1101/cshperspect.a012518Cold Spring Harb Perspect Med Sankar Adhya, Carl R. Merril and Biswajit Biswas Components in Modern MedicineTherapeutic and Prophylactic Applications of Bacteriophage

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PathogenesisVaccines, Reverse Vaccinology, and Bacterial

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Jérémy Manry and Lluis Quintana-Murci

Strategies Persistent InfectionSalmonella and Helicobacter

Denise M. Monack

Host Specificity of Bacterial PathogensAndreas Bäumler and Ferric C. Fang

Helicobacter pyloriIsland of PathogenicitycagEchoes of a Distant Past: The

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Epithelial Cells Invasion of IntestinalShigellaThe Inside Story of

Nathalie Carayol and Guy Tran Van Nhieu

RNA-Mediated Regulation in Pathogenic Bacteria

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